RU2728430C2 - Il-8-binding antibodies and use thereof - Google Patents

Abstract

FIELD: biochemistry.SUBSTANCE: invention relates to an antibody which specifically binds IL-8. Also disclosed is an isolated nucleic acid encoding said antibody, an expression vector comprising said nucleic acid, a host cell comprising said vector, a pharmaceutical composition, containing said antibody is also disclosed. Disclosed is a method of producing said antibody, a method of treating a patient, a method of inhibiting angiogenesis and a method of inhibiting neutrophil migration, using said antibody, use of said antibody for preparing a pharmaceutical composition.EFFECT: invention provides effective treatment of disorders characterized by IL-8 excess.15 cl, 40 dwg, 42 tbl, 23 ex

Description

Cross-reference to related claims

This patent application claims priority from Japanese Patent Application No. 2015-185254, filed in Japan on September 18, 2015. Its entire contents are incorporated herein by reference.

The technical field to which the invention relates

One of the objects of the invention, not limiting its scope, are antibodies to IL-8, pharmaceutical compositions containing these antibodies, nucleic acids encoding the antibodies, and host cells containing nucleic acids. Also provided are methods for the preparation and use of antibodies to IL-8 and a pharmaceutical composition for the treatment of, for example, IL-8-associated disorders.

Background of the invention

Antibodies are gaining attention as pharmaceuticals due to their high plasma stability and few side effects. A variety of IgG-type therapeutic antibodies are commercially available, and many therapeutic antibodies are currently in development (Reichert et al., Nat. Biotechnol. 23, 2005, pp. 1073-1078 (NPL1); Pavlou et al., Eur J. Pharm Biopharm 59 (3) 2005 pp 389-396 (NPL2)). At the same time, various methods have been developed to create second-generation therapeutic antibodies; including methods to increase effector function, antigen binding ability, pharmacokinetic characteristics or stability, and reduce the risk of immunogenicity (Kim et al., Mol. Cells. 20 (1), 2005, pp. 17-29 (NPL3)). The dosage of therapeutic antibodies is typically very high, and therefore the development of therapeutic antibodies is hindered by factors such as the complexity of preparing subcutaneous compositions and the high cost of manufacturing. Methods to improve the pharmacokinetic characteristics, pharmacodynamic characteristics, and antigen binding capacity of therapeutic antibodies provide ways to reduce the dose and cost of producing therapeutic antibodies.

Substitution of amino acid residues in the constant region is one of the methods for improving the pharmacokinetic characteristics of antibodies (Hinton et al., J. Immunol. 176 (1), 2006, pp. 346-356 (NPL4); Ghetie et al., Nat. Biotechnol. 15 (7), 1997, pp. 637-640) (NPL5). Affinity maturation technology has been proposed as a method for increasing the antigen-neutralizing ability of an antibody (Rajpal et al., Proc. Natl. Acad. Sci. USA 102 (24), 2005, pp. 8466-8471 (NPL6); Wu et al., J. Mol. Biol. 368, 2007, p. 652 ((NPL7)), and it can provide an increase in antigen binding activity by introducing mutation (s) of amino acid residue (s) in the CDRs and / or framework regions of the antibody variable domain. antibodies can increase the biological activity of the antibody in vitro or reduce the dose and can also increase the effectiveness in vivo (in the body) (Wu et al., J. Mol. Biol. 368, 2007, pp. 652-665 (NPL8)) ...

The amount of antigen that can be neutralized by one antibody molecule depends on the affinity of the antibody for the antigen; and therefore, it is possible to neutralize the antigen with a small amount of antibody by increasing the affinity. The affinity of an antibody for an antigen can be increased by various conventional methods known in the art (see, for example, Rajpal et al., Proc. Natl. Acad. Sci. USA 102 (24), 2005, pp. 8466-8471 (NPL6)). In addition, it is theoretically possible to neutralize one antigen molecule (2 antigen molecules when the antibody is bivalent) with one antibody molecule, if it can covalently bind to the antigen by increasing the affinity to infinity. However, one of the currently existing limitations in the development of therapeutic antibodies is that one antibody molecule, as a rule, binds and neutralizes only one antigen molecule (2 antigen molecules when the antibody is bivalent). It has now been reported that the use of an antibody that binds to an antigen in a pH dependent manner (hereinafter referred to as a "pH dependent antibody" or "pH bindable antibody") makes it possible to bind a single antibody molecule with multiple antigen molecules and their neutralization (see, for example, WO 2009/125825 (PTL1); Igawa et al., Nat. Biotechnol. 28, 2010, pp. 1203-1207 (NPL9)). The pH-dependent antibody is characterized by strong binding to antigen under neutral pH conditions in plasma and is separated by dissociation from the antigen under conditions of acidic pH in the endosome of the cell. After separation from the antigen, the antibody is recycled into the plasma by the FcRn, and after that it becomes free, which allows it to bind to another antibody molecule and neutralize it; and thus, one pH-dependent antibody can re-bind to and neutralize multiple antigen molecules.

It has now been established that the ability of an antibody to recycle can be provided by the difference in the concentration of calcium ions (Ca) between plasma and endosome and the use of an antibody that has the ability to interact with the antigen, which manifests itself in dependence on calcium (in the present description, below it is referred to as "dependent from the concentration of calcium antibody ") WO 2012/073992 (PTL2)). (Hereinafter, the pH-dependent antibody and "calcium ion concentration-dependent antibody" are referred to collectively as "pH / Ca concentration-dependent antibody").

By binding to FcRn, IgG-type antibodies have a long retention time in plasma. Binding between IgG-type antibody and FcRn is strong under acidic pH conditions (eg, pH 5.8), but binding is virtually non-existent under neutral pH conditions (eg, pH 7.4). The IgG-type antibody is absorbed by cells nonspecifically and returns to the cell surface by binding to FcRn in the endosome under conditions of acidic pH in the endosome. IgG is then separated by dissociation from the FcRn under neutral plasma pH conditions.

It has been found that a pH-dependent antibody modified to increase its ability to bind to FcRn under neutral pH conditions has the ability to re-bind to antigen molecules and eliminate them from plasma; and the introduction of the specified antibody allows you to eliminate the antigen from the plasma (WO 2011/122011 (PTL3)). According to the above publication, a pH-dependent antibody modified to increase its ability to bind to FcRn under neutral pH conditions (e.g., pH 7.4) can also further accelerate antigen elimination as compared to a pH-dependent antibody that contains an Fc region native IgG-type antibody (WO 2011/122011 (PTL3)).

Moreover, when mutations are introduced into the Fc region of an IgG-type antibody to eliminate its ability to bind to FcRn under acidic pH conditions, it no longer has the ability to recycle from the endosome into plasma, which significantly reduces the retention time of the antibody in plasma. In this regard, a method for increasing the ability to bind to FcRn under conditions of acidic pH is described as a method for increasing the retention time in plasma of an IgG-type antibody. The introduction of amino acid modifications into the Fc region of an IgG-type antibody to increase the ability to bind to FcRn under acidic pH conditions can increase the efficiency of endosome to plasma recycling, which leads to an increase in plasma retention time. For example, it has been found that modifications M252Y / S254T / T256E (YTE; Dall'Acqua et al., J. Biol. Chem. 281, 2006, pp. 23514-235249 (NPL10)), M428L / N434S (LS; Zalevsky et al. ., Nat. Biotechnol. 28, 2010, pp. 157-159 (NPL11)) and N434H (Zheng et al., Clin. Pharm. & Ther. 89 (2), 2011, pp. 283-290 (NPL12)) lead to an increased half-life of the antibody compared to native IgG1.

However, in addition to the problem of immunogenicity or a high rate of aggregation, which can be increased for an antibody containing a specified variant of the Fc region, the ability of which to bind to FcRn increases under neutral pH conditions or under conditions of acidic pH, it is also described to increase the binding of anti-drug agent (hereinafter referred to as “preexisting ADA”) (eg, rheumatoid factor) that was present in the patient's body prior to the administration of the therapeutic antibody (WO 2013/046722 (PTL4), WO 2013/046704 (PTL5)). WO 2013/046704 (PTL5) describes that a variant of the Fc region containing specific mutations (namely, modifications of two Q438R / S440E residues according to the EU numbering) has an increased ability to bind to FcRn under acidic pH conditions, and also demonstrated a significant decreased binding to rheumatoid factor compared to unmodified native Fc. However, WO 2013/046704 (PTL5) does not specifically demonstrate that this variant Fc region has an extended plasma retention time compared to an antibody with a native Fc region.

Thus, there is a need for safer and more preferred Fc region variants with further increased plasma retention times that do not bind to preexisting ADA.

Antibody-mediated cell-dependent cytotoxicity (hereinafter referred to as "ADCC", complement-dependent cytotoxicity (hereinafter referred to as "CDC"), antibody-dependent cellular phagocytosis (ADCP), which is phagocytosis of target cells mediated by the IgG antibody -type are described as the effector functions of an IgG-type antibody For an IgG-type antibody to exhibit ADCC activity or ADCP activity, the Fc region of an IgG-type antibody must bind to an antibody receptor present on the surface of an effector cell, such as a killer cell , natural killer cell or activated macrophage (referred to as "Fcγ receptor", "FcgR", "Fc gamma receptor" or "FcγR" in the context of section A. The isoforms FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa and FcγRIIIb as proteins of the FcγR family and their corresponding allotypes (Jefferis et al., Immunol. Lett. 82, 2002, pp. 57-65 (NPL13)) ... The balance of the respective affinities of the antibody for the activating receptor, which is FcγRIa, FcγRIIa, FcγRIIIa or FcγRIIIb, and the inhibitory receptor, which is FcγRIIb, is an important element in optimizing the effector functions of the antibody.

To date, various methods have been described for increasing or improving the activity of a therapeutic antibody against an antigen. For example, the ability of an antibody to bind to activating FcγR (s) plays an important role in the cytotoxicity of an antibody, and therefore antibodies have been developed that target a membrane-type antigen and have increased cytotoxicity as a result of increased binding to activating FcγR (s) (see, for example, WO 2000 / 042072 (PTL6); WO 2006/019447 (PTL7); Lazar et al., Proc. Nat. Acad. Sci. USA. 103, 2006, pp. 4005-4010 (NPL14); Shinkawa et al., J. Biol Chem 278, 2003, pp 3466-3473 (NPL15); Clynes et al., Proc Natl Acad Sci USA 95, 1998, 652-656 (NPL16); Clynes et al. Nat. Med. 6, 2000, pp. 443-446 (NPL17)). Similarly, the activity of binding to the inhibitory FcγR receptor (in humans, FcγRIIb) plays an important role in immunosuppressive activity, agonistic activity, and therefore, a study was carried out of antibodies with increased binding activity to inhibitory FcγR, the target of which is a membrane-type antigen (Li et al., Proc Nat Acad Sci USA 109 (27) 2012 10966-10971 (NPL18)). In addition, investigated, primarily with regard to side effects, the effect of binding to FcγR antibodies that bind to soluble antigen (Scappaticci et al., J. Natl. Cancer Inst. 99 (16), 2007, pp. 1232-1239 (NPL19 )). For example, when an antibody with increased ability to bind to FcγRIIb is used as a drug, a reduced risk of developing antibodies to the drug can be expected (Desai et al., J. Immunol. 178 (10), 2007, pp. 6217-6226 ( NPL20)).

In recent years, it has been described that the introduction of amino acid modifications into the Fc region of an IgG-type antibody in order to increase the activity of an antibody targeting a soluble antigen in relation to binding to activating (s) and / or inhibiting FcγR can further accelerate the elimination of antigen from serum ( WO 2012/115241 (PTL8), WO 2013/047752 (PTL9), WO 2013/125667 (PTL10), WO 2014/030728 (PTL11)). In addition, a variant of the Fc region was identified, which practically does not differ in its FcγRIIb-binding activity from the Fc-region of the native IgG-type antibody, but has a reduced activity of binding to other activating FcγR (WO 2014/163101 (PTL12)).

The plasma retention time of soluble antigen is very short compared to an antibody that has an FcRn-mediated recycling mechanism, and therefore, soluble antigen may have an increased plasma retention time and increased plasma concentration as a result of binding to an antibody that has such a recycling mechanism (e.g. with an antibody that does not have the characteristics of a pH / Ca concentration dependent antibody). Thus, for example, when a soluble antigen in plasma has several types of physiological functions, then even if one of the types of physiological functions is blocked by binding to the antibody, the concentration in the plasma of the antigen can worsen pathogenic symptoms due to other physiological functions as a result of an increased time. retention in plasma and / or increased concentration of antigen due to / due to binding to the antibody. In this case, in addition to the method based on the use of the above examples of modifications to increase the ability of an antibody to eliminate antigen, for example, a method based on the use of the formation of a multivalent immune complex consisting of several pH / Ca concentration dependent antibodies and several antigens has been proposed, and increasing binding to FcRn, FcγR, complement component receptor (WO 2013/081143 (PTL13)).

It is described that even when the Fc region is not modified, then by modifying the amino acid (s) residue (s) by changing the charge of said amino acid (s) residue (s), which (s) can ) to be exposed on the surface of the variable region of the antibody, in order to increase or decrease the isoelectric point (pI) of the antibody, it is possible to control the half-life of the antibody in the blood regardless of the type of target antigen or antibody and without a significant decrease in the antigen-binding activity of the antibody (WO 2007/114319 (PTL14 ): methods for replacing amino acids mainly in FR; WO 2009/041643 (PTL15): methods for replacing amino acids mainly in CDRs). These documents demonstrate that it is possible to prolong the plasma half-life of an antibody by lowering the pI of the antibody and, conversely, shorten the half-life of an antibody in plasma by increasing the pI of the antibody.

Regarding the modification of the charge of amino acid residues in the constant region of an antibody, data have been published that the uptake of antigen by cells can be enhanced by modifying the charge of a specific amino acid residue (s), primarily in the CH3 domain, to increase the pI of the antibody and, in addition, it is described that the specified modification preferably does not affect the binding to FcRn (WO 2014/145159 (PTL16)). It was also found that modification of the charge of amino acid residues in the constant region (mainly in the CH1 domain) of an antibody, carried out in order to reduce the pI, can prolong the half-life of an antibody in plasma and, in combination with mutations of amino acid residues, carried out in order to increase binding to FcRn, can enhance binding to FcRn and prolong the half-life of the antibody in plasma (WO 2012/016227 (PTL17)).

However, when these modification techniques designed to increase or decrease the pI of an antibody are combined with techniques other than modification techniques to increase or decrease binding to FcRn or FcγR, it remains unclear whether this effect will lengthen the plasma retention time of the antibody or enhance elimination. antigen from plasma.

The extracellular matrix (ECM) is a structure that coats cells in vivo and consists mainly of glycoproteins such as collagen, proteoglycan, fibronectin, and laminin. The role of the ECM in vivo is to create a microenvironment for cells to ensure their survival, and the ECM is also important for the implementation of various cellular functions such as cell proliferation and cell adhesion.

It has been found that ECM can participate in the in vivo kinetics of proteins introduced into a living organism. The blood concentration of the VEGF-Trap molecule, which is a fusion protein of the VEGF receptor and Fc, was assessed when administered subcutaneously (Holash et al., Proc. Natl. Acad. Sci., 99 (17), 2002, pp. 11393 -11398 (NPL21)). The plasma concentration of the subcutaneously administered VEGF-Trap molecule, which has a high pI, was found to be low and therefore its bioavailability was low. The modified VEGF-Trap molecule, the pI of which was lowered by amino acid substitutions, had a higher plasma concentration, and its bioavailability was improved. In addition, the change in bioavailability correlates with the strength of binding to the ECM, and thus it is clear that the bioavailability of the VEGF-Trap molecule when administered subcutaneously depends on the strength of its binding to the ECM in the subcutaneous region.

WO 2012/093704 (PTL18) discloses that there is an inverse correlation between antibody binding to the ECM and plasma retention time, and therefore antibody molecules that do not bind to the ECM have better plasma retention times compared to antibodies that contact the ECM.

Thus, methods for reducing binding to the extracellular matrix have been described in order to improve the bioavailability of the protein in vivo and increase the retention time in plasma. In contrast, the benefits of enhancing antibody binding to ECM have not been determined to date.

Human IL-8 (interleukin 8) is a member of the chemokine family of 72 or 77 amino acid residues. The term "chemokine" is a generic name for a family of proteins with a molecular weight of 8-12 kDa and containing 4 cysteine residues, which form intermolecular disulfide bonds. Chemokines are subdivided into CC-chemokine, CXC-chemokine, C-chemokine, CA3C-chemokine based on the location of the cysteine. IL-8 is classified as a CXC chemokine and is also referred to as CXCL8.

IL-8 exists in solution in monomeric and homodimeric forms. The IL-8 monomer contains antiparallel β-strands and has a structure in which the C-terminus of the α-helix crosses and covers the β-strands. The IL-8 monomer in the 72-amino acid form of IL-8 contains two disulfide cross-links between cysteine 7 and cysteine 34 and between cysteine 9 and cysteine 50. IL-8 homodimers are stabilized by non-covalent bonds between the β-strands of the two monomers, because homodimers lack covalent binding between molecules.

IL-8 expression is induced in various cells, such as peripheral blood monocytes, tissue macrophages, NK cells, fibroblasts, and vascular endothelial cells, in response to stimulation with proinflammatory cytokines (Russo et al., Exp. Rev. Clin. Immunol. 10 ( 5), 2014, pp. 593-619 (NPL22)).

Chemokines are generally undetectable or only weakly detectable in healthy tissue, but high levels are found in areas of inflammation, and they are involved in the manifestation of inflammation by facilitating leukocyte infiltration in the area of inflammation in the tissue. IL-8 is a pro-inflammatory chemokine that is known to activate neutrophils, enhance expression of cell adhesion molecules, and increase neutrophil adhesion to vascular endothelial cells. IL-8 also has chemotactic neutrophilic activity, and IL-8, produced in injured tissue, facilitates chemotaxis of neutrophils attached to vascular endothelial cells into tissue and induces inflammation along with neutrophil infiltration. It is also known that IL-8 is an effective angiogenic factor for endothelial cells and is involved in enhancing tumor angiogenesis.

Inflammatory diseases associated with elevated (eg, excessive) levels of IL-8 include inflammatory skin diseases such as inflammatory keratosis (eg, psoriasis), atopic dermatitis, contact dermatitis; chronic inflammatory disorders, including autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus (SLE), and Behcet's disease; inflammatory bowel diseases such as Crohn's disease and ulcerative colitis; inflammatory liver diseases such as hepatitis B, hepatitis C, alcoholic hepatitis, and drug-induced allergic hepatitis; inflammatory kidney disease such as glomerulonephritis; inflammatory respiratory diseases such as bronchitis and asthma; inflammatory chronic vascular diseases such as atherosclerosis; multiple sclerosis, oral ulcer, chorditis and inflammation associated with the use of artificial organs and / or artificial blood vessels. Elevated (eg, excessive) levels of IL-8 are also associated with malignant tumors such as ovarian cancer, lung cancer, prostate cancer, stomach cancer, breast cancer, melanoma, head and neck cancers, and kidney cancer; with infection-related sepsis; cystic fibrosis; and lung fibrosis (see, eg, Russo et al., Exp. Rev. Clin. Immunol. 10 (5), 2014, pp. 593-619 (NPL22), the publication is incorporated herein by reference in its entirety).

For some of these diseases, human antibodies to IL-8 with high affinity have been developed in the form of pharmaceutical compositions (Desai et al., J. Immunol. 178 (10), 2007, pp. 6217-6226 (NPL23)), however, they until they go on sale. To date, only one pharmaceutical composition is available containing an anti-IL-8 antibody, which is an anti-IL-8 antibody, which is intended for the treatment of psoriasis as an external therapy. New antibodies to IL-8 are being developed to treat disease.

List of links

Patent Literature

[PTL1] WO 2009/125825.

[PTL2] WO 2012/073992.

[PTL3] WO 2011/122011.

[PTL4] WO 2013/046722.

[PTL5] WO 2013/046704.

[PTL6] WO 2000/042072.

[PTL7] WO 2006/019447.

[PTL8] WO 2012/115241.

[PTL9] WO 2013/047752.

[PTL10] WO 2013/125667.

[PTL11] WO 2014/030728.

[PTL12] WO 2014/163101.

[PTL13] WO 2013/081143.

[PTL14] WO 2007/114319.

[PTL15] WO 2009/041643.

[PTL16] WO 2014/145159.

[PTL17] WO 2012/016227.

[PTL18] WO 2012/093704.

Non-patent literature

[NPL1] Reichert et al. Nat. Biotechnol. 23, 2005, cc. 1073-1078.

[NPL2] Pavlou et al., Eur. J. Pharm. Biopharm. 59 (3), 2005, pp. 389-396.

[NPL3] Kim et al. Mol. Cells. 20 (1), 2005, pp. 17-29.

[NPL4] Hinton et al., J. Immunol. 176 (1), 2006, pp. 346-356.

[NPL5] Ghetie et al., Nat. Biotechnol. 15 (7), 1997, cc. 637-640.

[NPL6] Rajpal et al. Proc. Natl. Acad. Sci. USA 102 (24), 2005, cc. 8466-8471.

[NPL7] Wu et al. J. Mol. Biol. 368, 2007, p. 652.

[NPL8] Wu et al. J. Mol. Biol. 368, 2007, cc. 652-665.

[NPL9] Igawa et al., Nat. Biotechnol. 28, 2010, cc. 1203-1207.

[NPL10] Dall'Acqua et al., J. Biol. Chem. 281, 2006, cc. 23514-235249.

[NPL11] Zalevsky et al., Nat. Biotechnol. 28, 2010, cc. 157-159.

[NPL12] Zheng et al., Clin. Pharm. & Ther. 89 (2), 2011, pp. 283-290.

[NPL13] Jefferis et al. Immunol. Lett. 82, 2002, pp. 57-65.

[NPL14] Lazar et al. Proc. Nat. Acad. Sci. USA. 103, 2006, cc. 4005-4010.

[NPL15] Shinkawa et al., J. Biol. Chem. 278, 2003, cc. 3466-3473.

[NPL16] Clynes et al. Proc. Natl. Acad. Sci. USA 95, 1998, cc. 652-656.

[NPL17] Clynes et al., Nat. Med. 6, 2000, cc. 443-446.

[NPL18] Li et al. Proc. Nat. Acad. Sci. USA. 109 (27), 2012, pp. 10966-10971.

[NPL19] Scappaticci et al., J. Natl. Cancer Inst. 99 (16), 2007, pp. 1232-1239.

[NPL20] Desai et al. J. Immunol. 178 (10), 2007, pp. 6217-6226.

[NPL21] Holash et al. Proc. Natl. Acad. Sci. 99 (17) 2002 cc. 11393-11398.

[NPL22] Russo et al. Exp. Rev. Clin. Immunol. 10 (5), 2014, pp. 593-619.

[NPL23] Desai et al. J. Immunol. 178 (10), 2007, pp. 6217-6226.

Summary of the invention

In one non-limiting aspect of the invention, the embodiments of Section A of the description include, but are not limited to, molecules with improved pharmacokinetic properties over antibodies, such as ion concentration-dependent antigen binding properties that improve plasma half-life and / or antigen clearance from plasma.

In one non-limiting aspect of the invention, the embodiments of section B of the description are, but are not limited to, safer and more preferred Fc region variants that have an increased half-life and a reduced ability to bind to preexisting antibodies to drugs (ADA).

In one non-limiting aspect of the invention, the embodiments described in Section B of the specification are, but are not limited to, anti-IL-8 antibodies that have a pH-dependent binding affinity for IL-8. An additional embodiment of the invention are anti-IL-8 antibodies that have the ability to rapidly eliminate IL-8 when administered to an individual when compared to a reference antibody. Another embodiment of the invention referred to in Section B of the specification are anti-IL-8 antibodies that have the ability to stably retain their IL-8 neutralizing activity when administered to an individual. In some embodiments, anti-IL-8 antibodies have reduced immunogenicity. Additional embodiments of the invention specified in section B of the description are methods of making and using the above anti-IL-8 antibodies. Another non-limiting alternative object of the invention, which is indicated in section B of the description, are novel anti-IL-8 antibodies that can be incorporated into a pharmaceutical composition.

One of the non-limiting aspects of the invention, which is indicated in section A of the present description, is the unexpectedly established fact that the ability of an ion concentration-dependent antibody (which is an antibody containing an ion-concentration-dependent antigen-binding domain ("antigen-binding domain , the antigen-binding activity of which varies depending on the conditions of ion concentration ")) to eliminate the antigen from the plasma can be increased by modifying at least one of the amino acid residues exposed on the antibody surface in order to increase the isoelectric point (pI). According to another non-limiting aspect of the invention, it has been found that an ion concentration-dependent antibody with an increased pI can also increase its ability to bind to the extracellular matrix. Thus, without going into a specific theory, when creating the invention, it was found that the elimination of antigen from plasma can be increased by increasing the binding of the antibody to the extracellular matrix.

According to one of the non-limiting aspects of the invention, which is indicated in section B of the present description, when creating the invention, a thorough study was carried out with respect to safe and more preferred variants of the Fc region that do not have the ability to bind antibodies to drugs (preexisting ADA) and which could additionally have improved plasma retention times. As a result, when creating the invention, it was unexpectedly found that variants of the Fc region containing the substitution of amino acid at position 434 according to EU numbering by Ala (A) and specific mutations of two residues (such as Q438R / S440E according to EU numbering) in the form of a combination of mutations amino acid residues are preferred to maintain a significant reduction in binding to rheumatoid factor, while ensuring antibody retention in plasma.

According to one of the non-limiting aspects of the invention, which is indicated in section B of the present description, during the creation of the invention several pH-dependent anti-IL-8 antibodies (anti-IL-8 antibodies that bind to IL-8 depending on the pH ). By validating the results using various methods, the invention identified pH-dependent antibodies to IL-8, which have an effect on the rapid elimination of IL-8 in comparison with a reference antibody when administered to an individual. Certain embodiments of the invention referred to in Section B of the specification relate to pH-dependent antibodies in IL-8 that have the ability to stably maintain their IL-8 neutralizing activity. In other embodiments, the pH dependent IL-8 antibodies have reduced immunogenicity and very high levels of expression.

In addition, in section B of the description, the successful preparation of antibodies to IL-8, which contains an Fc region, the binding affinity to FcRn of which at acidic pH increases compared to the binding affinity of the native Fc region to FcRn, is described. Alternatively, anti-IL-8 antibodies have been successfully produced containing an Fc region, the binding affinity of which to preexisting ADA is reduced compared to the binding affinity of the native Fc region to preexisting ADA. Alternatively, anti-IL-8 antibodies have been successfully produced with an Fc region, the plasma half-life of which is increased relative to the plasma half-life of the native Fc region. Alternatively, the invention has successfully produced pH-dependent antibodies to IL-8 containing an Fc region, the binding affinity of which to effector receptors is reduced compared to the binding affinity of the native Fc region to effector receptors. Other objects of the invention are identified during the creation of the invention nucleic acids encoding the above antibodies to IL-8. Another object of the invention is also the hosts obtained during the creation of the invention, which contain the aforementioned nucleic acids. Another object of the invention is a method for the production of the aforementioned anti-IL-8 antibodies, developed during the creation of the invention, which comprises culturing the aforementioned host. Another aspect of the invention is a method of facilitating the elimination of IL-8 from an individual as compared to elimination using a reference antibody, which is a method of facilitating the elimination of IL-8 from an individual, which comprises administering the aforementioned anti-IL-8 antibodies to the individual.

One of the embodiments of the invention specified in section A of the description are (but not limited to):

[1] an antibody containing an antigen-binding domain, the antigen-binding activity of which varies depending on the conditions of ion concentration, in which the isoelectric point (pI) is increased by modifying at least one amino acid residue that can be displayed on the surface of the antibody;

[2] the antibody according to [1], where the antigen is a soluble antigen;

[3] the antibody according to item [1] or item [2], in which the antigen-binding domain is a domain, the antigen-binding activity of which is higher under conditions of high ion concentration than under conditions of low ion concentration;

[4] an antibody according to one of paragraphs. [1] - [3], where the ion concentration is the hydrogen ion concentration (pH) or the calcium ion concentration;

[5] the antibody according to [4], for which the ratio of KD in the acidic pH range to the KD in the neutral pH range, KD (acidic pH range) / KD (neutral pH range), with respect to the antigen is 2 or higher;

[6] an antibody according to one of paragraphs. [1] - [5], in which at least one amino acid residue is replaced by histidine in the antigen-binding domain or at least one histidine is inserted into the antigen-binding domain;

[7] an antibody according to one of paragraphs. [1] - [6], which can enhance the elimination of antigen from plasma compared to the antibody before modification;

[8] an antibody according to one of paragraphs. [1] - [7], in which the binding activity of the extracellular matrix is increased compared to the antibody before modification;

[9] an antibody according to one of paragraphs. [1] - [8], in which the modification of the amino acid residue is a substitution of the amino acid residue;

[10] an antibody according to one of paragraphs. [1] - [9], in which the amino acid residue modification is selected from the group consisting of:

(a) replacing a negatively charged amino acid residue with an uncharged amino acid residue;

(b) replacing a negatively charged amino acid residue with a positively charged amino acid residue;

and

(c) replacing an uncharged amino acid residue with a positively charged amino acid residue;

[11] an antibody according to one of paragraphs. [1] - [10], where the antibody contains a variable region and / or constant region and the modification of an amino acid residue is a modification of an amino acid residue in the variable region and / or constant region;

[12] an antibody according to [11], in which the variable region comprises hypervariable region (s) (CDR) and / or framework (s) region (s) (FR);

[13] the antibody according to [12], wherein the variable region comprises a heavy chain variable region and / or a light chain variable region, and at least one amino acid residue is modified at a position in a CDR or an FR selected from the group consisting of :

(a) positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 18, 19, 23, 25, 26, 39, 41, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 77, 81, 82, 82a, 82b, 83, 84, 85, 86, 105, 108, 110 and 112 in the FR of the heavy chain variable region;

(b) positions 31, 61, 62, 63, 64, 65 97 in the CDR of the variable region of the heavy chain;

(c) positions 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 37, 38, 39, 41, 42, 43, 45, 46, 49, 57, 60, 63, 65, 66, 68, 69, 70, 74, 76, 77, 79, 80, 81, 85, 100, 103, 105, 106, 107 and 108 in the FR of the light chain variable region; and

(d) positions 24, 25, 26, 27, 52, 53, 54, 55 and 56 in the CDR of the light chain variable region, according to Cabot's numbering;

[14] the antibody according to [13], in which at least one amino acid residue is modified at a position in a CDR or FR selected from the group consisting of:

(a) position 8, 10, 12, 13, 15, 16, 18, 23, 39, 41, 43, 44, 77, 82, 82a, 82b, 83, 84, 85 and 105 in the FR of the heavy chain variable region;

(b) position 31, 61, 62, 63, 64, 65 and 97 in the CDR of the variable region of the heavy chain;

(c) position 16, 18, 37, 41, 42, 45, 65, 69, 74, 76, 77, 79 and 107 in the FR of the light chain variable region; and

(d) position 24, 25, 26, 27, 52, 53, 54, 55 and 56 in the CDR of the variable region of the light chain;

[15] an antibody according to one of paragraphs. [11] - [14], in which at least one amino acid residue is modified at a position in the constant region selected from the group consisting of positions 196, 253, 254, 256, 258, 278, 280, 281, 282, 285, 286, 307, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434 and 443, according to the EU numbering;

[16] the antibody according to [15], in which at least one amino acid residue is modified at a position in the constant region selected from the group consisting of positions 254, 258, 281, 282, 285, 309, 311, 315, 327 , 330, 342, 343, 345, 356, 358, 359, 361, 362, 384, 385, 386, 387, 389, 399, 400, 401, 402, 413, 418, 419, 421, 433, 434 and 443 ;

[17] the antibody according to [16], in which at least one amino acid residue is modified at a position in the constant region selected from the group consisting of positions 282, 309, 311, 315, 342, 343, 384, 399, 401 , 402 and 413, according to EU numbering;

[18] an antibody according to one of paragraphs. [1] - [17], in which the constant region has binding activity against the Fc gamma receptor (FcγR) and in which the FcγR-binding activity at neutral pH is increased compared to the activity of the reference antibody, which contains the constant region of native IgG ;

[19] the antibody according to [18], where FcγR is FcγRIIb;

[20] an antibody according to one of paragraphs. [1] - [17], in which the constant region has a binding activity against one or more activating FcγR selected from the group consisting of FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb and FcγRIIa and with respect to FcγRIIb and FcγRIIb-binding activity or increased, and the binding activity against activating FcγR is reduced in comparison with the activities of the reference antibody, which differs only in that it contains the constant region of native IgG;

[21] an antibody according to one of paragraphs. [1] - [20], in which the constant region has FcRn-binding activity and in which the FcRn-binding activity under neutral pH conditions (for example, pH 7.4) is increased compared to the activity of the reference antibody, which differs only in that what contains the constant region of native IgG;

[22] an antibody according to one of paragraphs. [1] - [21], which is a multispecific antibody that binds to at least two antigens;

[23] an antibody according to one of paragraphs. [1] - [22], where the antibody is an IgG-type antibody;

[24] a pharmaceutical composition containing an antibody according to one of paragraphs. [1] - [23];

[25] a pharmaceutical composition according to [24], designed to enhance the elimination of antigen from plasma;

[26] a pharmaceutical composition according to item [24] or item [25], designed to enhance binding of an antibody to an extracellular matrix;

[27] a nucleic acid encoding an antibody according to one of paragraphs. [1] - [23];

[28] a vector containing the nucleic acid according to [27];

[29] a host cell containing the vector according to [28];

[30] a method for producing an antibody containing an antigen-binding domain, the antigen-binding activity of which varies depending on the conditions of ion concentration, where the method includes culturing a host cell according to [29] and collecting the antibody from the cell culture;

[30A] a method for producing an antibody containing an antigen binding domain, the antigen binding activity of which varies depending on ion concentration conditions, wherein the method comprises modifying at least one amino acid residue that can be exposed on the surface of the antibody to increase the isoelectric point (pI);

[30B] the method according to [30A], wherein at least one amino acid residue is modified

(I) at a position in a CDR or FR selected from the group consisting of: (a) positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 18, 19, 23, 25, 26, 39 , 41, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 77, 81, 82, 82a, 82b, 83, 84, 85, 86, 105, 108, 110 and 112 in FR variable region of the heavy chain; (b) positions 31, 61, 62, 63, 64, 65 and 97 in the CDR of the variable region of the heavy chain; (c) positions 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 37, 38, 39, 41, 42, 43, 45, 46, 49, 57, 60, 63, 65, 66, 68, 69, 70, 74, 76, 77, 79, 80, 81, 85, 100, 103, 105, 106, 107 and 108 in the FR of the light chain variable region; and (d) positions 24, 25, 26, 27, 52, 53, 54, 55 and 56 in the CDR of the variable region of the light chain, according to Cabot numbering; or

(Ii) at a position in the constant region selected from the group consisting of positions 196, 253, 254, 256, 258, 278, 280, 281, 282, 285, 286, 307, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434 and 443 according to EU numbering;

[31] the method according to item [30A] or item [30B], in which the modification of the amino acid residue contains a modification selected from the group consisting of:

(a) replacing a negatively charged amino acid residue with an uncharged amino acid residue;

(b) replacing a negatively charged amino acid residue with a positively charged amino acid residue;

(c) replacing an uncharged amino acid residue with a positively charged amino acid residue; and

(d) substitution or insertion of histidine in the CDR or FR.

[32] the method according to one of paragraphs. [30] or [30A] - [30B], which further includes one or more of the following steps:

(a) selection of an antibody that can enhance the elimination of antigen from plasma;

(b) selection of antibodies with increased activity of binding to the extracellular matrix;

(c) selecting an antibody with increased FcγR-binding activity under neutral pH conditions (eg, pH 7.4);

(d) selecting an antibody with increased FcγRIIb-binding activity under neutral pH conditions (eg, pH 7.4);

(e) selecting an antibody with preserved or increased FcγRIIb binding activity and decreased binding activity against one or more activating FcγRs, preferably selected from the group consisting of FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb, and FcγRIIa;

(f) selecting an antibody with increased FcRn binding activity under neutral pH conditions (eg, pH 7.4);

(g) selection of antibodies with an increased isoelectric point (pI);

(h) confirmation of the isoelectric point (pI) of the collected antibody and subsequent selection of the antibody with an increased isoelectric point (pI); and

(i) selecting an antibody whose antigen-binding activity changes or increases depending on the conditions of ion concentration;

compared with a reference antibody;

An alternative embodiment of the invention specified in section A of the description is (but not limited to)

[A1] an antibody having a constant region in which at least one amino acid residue selected from the group of modification sites identical to the modification sites from the group indicated in [15] or [16] is modified in the constant region;

[A2] an antibody according to [A1], which further has a heavy chain variable region and / or a light chain variable region, in which the variable region has CDRs and / or FR regions, and in which at least one amino acid residue, selected from the group of modification sites identical to the modification sites from the group specified in clause [13] or clause [14], modified into CDR and / or FR;

[A3] an antibody having a constant region in which at least one amino acid residue selected from the group of modification sites identical to the modification sites from the group specified in [15] or [16] is modified in the constant region to increase pI;

[A4] an antibody according to [A3], which further has a heavy chain variable region and / or a light chain variable region, in which the variable region has a CDR region (s) and / or FR region (s), and in which at least one amino acid residue selected from the group of modification sites identical to the modification sites from the group indicated in item [13] or item [14] is modified in CDR and / or FR;

[A5] an antibody containing an antigen-binding domain, the antigen-binding activity of which varies depending on the ion concentration conditions, where the antibody has a constant region, and in which at least one amino acid residue selected from the group of modification sites identical to the modification sites from the group specified in item [15] or item [16], modified in the constant region;

[A6] an antibody according to [A5], which further has a heavy chain variable region and / or a light chain variable region, in which the variable region has a CDR region (s) and / or FR region (s), and in which at least one amino acid residue selected from the group of modification sites identical to the modification sites from the group indicated in item [13] or item [14] is modified in CDR and / or FR;

[A7] the use of an antibody according to one of paragraphs. [1] - [23] and [A1] - [A6] for the preparation of a medicinal product intended to enhance the elimination of antigen from plasma;

[A8] the use of an antibody according to one of paragraphs. [1] - [23] and [A1] - [A6] for the preparation of a medicament intended to increase binding to the extracellular matrix;

[A9] the use of antibodies according to one of paragraphs. [1] - [23] and [A1] - [A6] for the elimination of antigen from plasma; and

[A10] the use of an antibody according to one of paragraphs. [1] - [23] and [A1] - [A6] to enhance binding to the extracellular matrix;

[A11] an antibody obtained by the method according to one of paragraphs. [30], [30A], [30B], [31], [32].

Various embodiments of the invention referred to in section A of the description are combinations of one or more of the elements specified above in any of [1] - [30], [30A], [30B], [31], [32] and [A1] - [A11], in part or in whole, if the specified combination is not technically impossible according to generally accepted technical knowledge known in this field. For example, in some embodiments of the invention described in section A of the description, there is a method for producing a modified antibody containing an antigen binding domain that enhances the elimination of antigen from plasma compared to the antibody before modification, where the method comprises:

(a) modification of at least one amino acid residue that can be displayed on the surface of an antibody, present:

(I) at a position in a CDR or FR selected from the group consisting of: (a) positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 18, 19, 23, 25, 26, 39 , 41, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 77, 81, 82, 82a, 82b, 83, 84, 85, 86, 105, 108, 110 and 112 in FR variable region of the heavy chain; (b) positions 31, 61, 62, 63, 64, 65 and 97 in the CDR of the variable region of the heavy chain; (c) positions 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 37, 38, 39, 41, 42, 43, 45, 46, 49, 57, 60, 63, 65, 66, 68, 69, 70, 74, 76, 77, 79, 80, 81, 85, 100, 103, 105, 106, 107 and 108 in the FR of the light chain variable region; and (d) positions 24, 25, 26, 27, 52, 53, 54, 55 and 56 in the CDR of the variable region of the light chain, according to Cabot numbering; or

(Ii) at a position in the constant region selected from the group consisting of positions 196, 253, 254, 256, 258, 278, 280, 281, 282, 285, 286, 307, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434 and 443 according to EU numbering;

(b) modification of the antigen-binding domain so that the obtained antigen-binding activity changes depending on the conditions of ion concentration, in which the steps indicated in subparagraphs (a) and (b) can be performed simultaneously or sequentially;

(c) culturing a host cell to express the nucleic acid encoding the modified antibody; and

(d) collecting the modified antibody from the host cell culture.

In other embodiments, the method optionally further comprises one or more of the following steps:

(e) selection of an antibody that can enhance the elimination of antigen from plasma;

(f) selection of antibodies with increased activity of binding to the extracellular matrix;

(g) selection of antibodies with increased FcγR-binding activity under neutral pH conditions (eg, pH 7.4);

(h) selection of antibodies with increased FcγRIIb-binding activity under neutral pH conditions (eg, pH 7.4);

(i) selecting an antibody with preserved or increased FcγRIIb binding activity and decreased binding activity against one or more activating FcγRs, preferably selected from the group consisting of FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb, and FcγRIIa;

(j) selecting an antibody with increased FcRn binding activity under neutral pH conditions (eg, pH 7.4);

(k) selection of an antibody with an increased isoelectric point (pI);

(m) confirmation of the isoelectric point (pI) of the collected antibody and subsequent selection of the antibody with an increased isoelectric point (pI); and

(m) selection of an antibody, the antigen-binding activity of which changes or increases depending on the conditions of ion concentration;

compared to the antibody before modification.

Another embodiment of the invention indicated in section A of the description is (but not limited to):

[D1] a method for producing a modified antibody with a prolonged or reduced half-life in plasma compared to the half-life before antibody modification, wherein the method comprises:

(a) modifying the nucleic acid encoding the antibody prior to modification to change the charge of at least one amino acid residue at a position selected from the group consisting of positions 196, 253, 254, 256, 258, 278, 280, 281, 282, 285, 286, 307, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434 and 443 according to EU numbering;

(b) culturing a host cell to express the nucleic acid; and

(c) collecting the antibody from the host cell culture; or

[D2] a method of prolonging or decreasing the plasma half-life of an antibody, wherein the method comprises modifying at least one amino acid residue at a position selected from the group consisting of positions 196, 253, 254, 256, 258, 278, 280, 281, 282 , 285, 286, 307, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 389, 399, 400 , 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434 and 443, according to the EU numbering.

One of the embodiments of the invention indicated in section B of the description is, for example (but not limited to):

[33] a variant of the Fc region containing an FcRn binding domain, where the FcRn binding domain contains Ala at position 434; Glu, Arg, Ser or Lys at position 438; and Glu, Asp or Gln at position 440 according to EU numbering;

[34] a variant of the Fc region according to [33], where the FcRn-binding domain contains Ala at position 434; Arg or Lys at position 438; and Glu or Asp at position 440 according to EU numbering;

[35] a variant of the Fc region according to [33] or [34] wherein the FcRn binding domain contains Ile or Leu at position 428; and / or Ile, Leu, Val, Thr or Phe at position 436 according to EU numbering;

[36] a variant Fc region according to [35], where the FcRn-binding domain contains Leu at position 428; and / or Val or Thr at position 436 according to EU numbering;

[37] a variant of the Fc-region according to one of claims. [33] - [36], where the FcRn-binding domain contains a combination of amino acid substitutions selected from the group consisting of: N434A / Q438R / S440E; N434A / Q438R / S440D; N434A / Q438K / S440E; N434A / Q438K / S440D; N434A / Y436T / Q438R / S440E; N434A / Y436T / Q438R / S440D; N434A / Y436T / Q438K / S440E; N434A / Y436T / Q438K / S440D; N434A / Y436V / Q438R / S440E; N434A / Y436V / Q438R / S440D; N434A / Y436V / Q438K / S440E; N434A / Y436V / Q438K / S440D; N434A / R435H / F436T / Q438R / S440E; N434A / R435H / F436T / Q438R / S440D; N434A / R435H / F436T / Q438K / S440E; N434A / R435H / F436T / Q438K / S440D; N434A / R435H / F436V / Q438R / S440E; N434A / R435H / F436V / Q438R / S440D; N434A / R435H / F436V / Q438K / S440E; N434A / R435H / F436V / Q438K / S440D; M428L / N434A / Q438R / S440E; M428L / N434A / Q438R / S440D; M428L / N434A / Q438K / S440E; M428L / N434A / Q438K / S440D; M428L / N434A / Y436T / Q438R / S440E; M428L / N434A / Y436T / Q438R / S440D; M428L / N434A / Y436T / Q438K / S440E; M428L / N434A / Y436T / Q438K / S440D; M428L / N434A / Y436V / Q438R / S440E; M428L / N434A / Y436V / Q438R / S440D; M428L / N434A / Y436V / Q438K / S440E; M428L / N434A / Y436V / Q438K / S440D; L235R / G236R / S239K / M428L / N434A / Y436T / Q438R / S440EH L235R / G236R / A327G / A330S / P331S / M428L / N434A / Y436T / Q438R / S440E, according to EU-numbering;

[38] a variant of the Fc region according to [37], wherein the FcRn binding domain contains a combination of amino acid substitutions selected from the group consisting of:

N434A / Q438R / S440E; N434A / Y436T / Q438R / S440E; N434A / Y436V / Q438R / S440E; M428L / N434A / Q438R / S440E; M428L / N434A / Y436T / Q438R / S440E; M428L / N434A / Y436V / Q438R / S440E; L235R / G236R / S239K / M428L / N434A / Y436T / Q438R / S440E and L235R / G236R / A327G / A330S / P331S / M428L / N434A / Y436T / Q438R / S440E, according to EU numbering;

[39] a variant of the Fc-region according to one of claims. [33] - [38], the FcRn-binding activity of which under conditions of acidic pH (for example, pH 5.8) is increased compared to the activity of the Fc-region of native IgG;

[40] a variant of the Fc-region according to one of claims. [33] - [39], the binding activity of which in relation to antibodies to the drug medium (ADA) does not significantly increase under conditions of neutral pH compared to the activity of the Fc-region of native IgG;

[41] a variant Fc region according to [40], wherein the anti-drug antibody (ADA) is rheumatoid factor (RF);

[42] a variant of the Fc-region according to one of claims. [33] - [41], in which the plasma clearance (CL) is decreased, the plasma retention time is increased, or the plasma half-life (t1 / 2) is increased compared to the parameters of the Fc region of native IgG;

[43] a variant of the Fc-region according to one of claims. [33] - [42], in which the retention time in plasma is increased compared to the reference variant of the Fc region, containing a combination of amino acid substitutions N434Y / Y436V / Q438R / S440E according to EU numbering;

[44] an antibody containing a variant Fc region according to one of paragraphs. [33] - [43];

[45] the antibody according to [44], wherein the antibody is an IgG-type antibody;

[46] a pharmaceutical composition comprising an antibody according to [44] or [45];

[47] the pharmaceutical composition according to [46], designed to increase the retention time of the antibody in plasma;

[48] a nucleic acid encoding a variant Fc region according to one of paragraphs. [33] - [43] or the antibody according to item [44] or item [45];

[49] a vector containing the nucleic acid according to [48];

[50] a host cell containing the vector according to [49];

[51] a method for producing an Fc region variant containing an FcRn binding domain or an antibody containing a variant, which comprises culturing a host cell according to [50] and then collecting a variant Fc region or an antibody containing a variant from the cell culture ;

[52] the method according to [51], which further optionally includes one or more steps selected from the group consisting of the following steps:

(a) selecting a variant of the Fc region with increased FcRn binding activity under conditions of acidic pH compared to the activity of the Fc region of native IgG;

(b) selection of a variant of the Fc region, the binding activity of which against antibodies to drug (ADA) is not significantly increased under conditions of neutral pH compared to the activity of the Fc region of native IgG;

(c) selecting a variant of the Fc region with an increased retention time in plasma compared with the retention time of the Fc region of native IgG; and

(d) selecting an antibody containing a variant Fc region that can enhance the elimination of antigen from plasma as compared to a reference antibody containing the Fc region of native IgG; and

[53] a method for producing an Fc region variant containing an FcRn binding domain or an antibody that contains a variant, wherein the method comprises an amino acid change that results in an Fc region variant or an antibody containing a variant containing Ala at position 434 ; Glu, Arg, Ser or Lys at position 438; and Glu, Asp or Gln at position 440 according to EU numbering.

One of the embodiments of the invention indicated in section B of the description is, for example (but not limited to):

[B1] the application of a variant of the Fc-region according to one of claims. [33] - [43] or antibodies according to item [44] or item [45] for the preparation of a medicament intended to increase the retention time in plasma;

[B2] application of a variant of the Fc-region according to one of paragraphs. [33] - [43] or antibodies according to [44] or [45] for the preparation of a medicament designed to insignificantly increase the binding activity of an anti-drug antibody (ADA) at a neutral pH compared to the Fc region native IgG;

[B3] the application of a variant of the Fc-region according to one of claims. [33] - [43] or antibodies according to item [44] or item [45] to increase the retention time in plasma;

[B4] the application of a variant of the Fc-region according to one of claims. [33] - [43] or antibodies according to [44] or [45] to insignificantly increase the binding activity with anti-drug antibody (ADA) under neutral pH conditions compared to the Fc region of native IgG; and

[B5] a variant of the Fc region or an antibody containing a variant obtained by the method according to one of paragraphs. [51], [52] and [53].

Various embodiments of the invention indicated in section B of the description are combinations of one or more of the elements specified in any of paragraphs. [33] - [53] and [Bl] - [B5], in part or in whole, if the specified combination is not technically impossible according to the generally accepted technical knowledge known in this field. For example, some embodiments of the invention identified in section B of the description are a variant Fc region containing an FcRn binding domain, where the FcRn binding domain may comprise:

(a) Ala at position 434; Glu, Arg, Ser or Lys at position 438; and Glu, Asp or Gln at position 440 according to EU numbering;

(b) Ala at position 434; Arg or Lys at position 438; and Glu or r Asp at position 440 according to EU numbering;

(c) Ile or Leu at position 428; Ala at position 434; Ile, Leu, Val, Thr, or Phe at position 436; Glu, Arg, Ser or Lys at position 438; and Glu, Asp or Gln at position 440 according to EU numbering;

(d) Ile or Leu at position 428; Ala at position 434; Ile, Leu, Val, Thr, or Phe at position 436; Arg or Lys at position 438; and Glu or Asp at position 440 according to EU numbering;

(e) Leu at position 428; Ala at position 434; Val or Thr at position 436; Glu, Arg, Ser or Lys at position 438; and Glu, Asp or Gln at position 440 according to EU numbering; or

(e) Leu at position 428; Ala at position 434; Val or Thr at position 436; Arg or Lys at position 438; and Glu or Asp at position 440 according to EU numbering.

One of the embodiments of the invention mentioned in section B of the description is, for example (but not limited to):

[54] an isolated anti-IL-8 antibody that binds to human IL-8, which contains at least one amino acid (s) substitution (s) in at least one of the regions indicated below in subparagraphs (a) - (e) , and which binds to IL-8 in a pH-dependent manner:

(a) an HVR-H1 containing the amino acid sequence of SEQ ID NO: 67;

(b) an HVR-H2 containing the amino acid sequence of SEQ ID NO: 68;

(c) an HVR-H3 containing the amino acid sequence of SEQ ID NO: 69;

(d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 70

(e) an HVR-L2 containing the amino acid sequence of SEQ ID NO: 71; and

(f) an HVR-L3 containing the amino acid sequence of SEQ ID NO: 72;

[55] the antibody to IL-8 according to [54], which contains the amino acid substitutions of tyrosine at position 9 of the amino acid sequence of SEQ ID NO: 68, arginine at position 11 of the amino acid sequence of SEQ ID NO: 68 and tyrosine at position 3 of the amino acid sequence of SEQ ID NO: 69;

[56] the anti-IL-8 antibody according to [54] or [55], which further comprises amino acid substitutions for alanine at position 6 of the amino acid sequence of SEQ ID NO: 68 and glycine at position 8 of the amino acid sequence of SEQ ID NO: 68;

[57] an antibody to IL-8 according to one of paragraphs. [54] - [56], which contains amino acid substitutions for asparagine at position 1 of the amino acid sequence of SEQ ID NO: 71, leucine at position 5 of the amino acid sequence of SEQ ID NO: 71 and glutamine at position 1 of the amino acid sequence of SEQ ID NO: 72;

[58] an antibody to IL-8 according to one of paragraphs. [54] - [57], which contains (a) an HVR-H1 containing the amino acid sequence of SEQ ID NO: 67, (b) an HVR-H2 containing the amino acid sequence of SEQ ID NO: 73 and (c) an HVR-H3 containing the amino acid sequence of SEQ ID NO: 74;

[59] an antibody to IL-8 according to one of paragraphs. [54] - [58], which contains (a) an HVR-L1 containing the amino acid sequence of SEQ ID NO: 70, (b) an HVR-L2 containing the amino acid sequence of SEQ ID NO: 75 and (c) an HVR-L3 containing the amino acid sequence of SEQ ID NO: 76;

[60] an antibody to IL-8 according to one of paragraphs. [54] - [59], which contains the variable region of the heavy chain having SEQ ID NO: 78, and the variable region of the light chain having SEQ ID NO: 79;

[61] an antibody to IL-8 according to one of paragraphs. [54] - [60], which contains an Fc-region having at least one property selected from the properties specified in subparagraphs (a) - (e) below:

(a) increased binding affinity of the Fc region to FcRn compared to the binding affinity to FcRn of the native Fc region at acidic pH;

(b) reduced binding affinity of the Fc region to pre-present ADA compared to the binding affinity of native Fc to pre-present ADA;

(c) an extended plasma half-life of the Fc region compared to the plasma half-life of the native Fc region;

(d) decreased plasma clearance of the Fc region compared to the plasma clearance of the native Fc region; and

(e) reduced binding affinity of the Fc region to the effector receptor compared to the binding affinity of the native Fc region to the effector receptor; and

(f) increased binding to extracellular matrix.

[62] the anti-IL-8 antibody according to [61], in which the Fc region contains amino acid (s) substitution (s) at one or more positions selected from the group consisting of positions 235, 236, 239, 327, 330, 331, 428, 434, 436, 438 and 440, according to EU numbering;

[63] the antibody to IL-8 according to [62], in which the Fc region contains one or more amino acid (s) substitution (s) selected from the group consisting of L235R, G236R, S239K, A327G, A330S, P331S , M428L, N434A, Y436T, Q438R and S440E;

[64] the anti-IL-8 antibody according to [63], in which the Fc region contains amino acid substitutions L235R, G236R, S239K, M428L, N434A, Y436T, Q438R and S440E;

[65] an anti-IL-8 antibody according to [63], in which the Fc region contains amino acid substitutions L235R, G236R, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R and S440E;

[66] an antibody to IL-8, which contains a heavy chain containing the amino acid sequence of SEQ ID NO: 81, and a light chain containing the amino acid sequence of SEQ ID NO: 82;

[67] an antibody to IL-8, which contains a heavy chain containing the amino acid sequence of SEQ ID NO: 80, and a light chain containing the amino acid sequence of SEQ ID NO: 82;

[68] an isolated nucleic acid encoding an anti-IL-8 antibody according to one of paragraphs. [54] - [67];

[69] a vector containing the nucleic acid according to [68];

[70] a host cell containing the vector according to [69];

[71] a method for producing antibodies to IL-8, which comprises culturing a host according to [70];

[72] a method for producing an antibody to IL-8 according to [71], which comprises isolating an antibody from a culture supernatant;

[73] a pharmaceutical composition containing an anti-IL-8 antibody according to one of claims. [54] - [67] and a pharmaceutically acceptable carrier;

[74] an antibody to IL-8 according to one of paragraphs. [54] - [67], intended for use in a pharmaceutical composition;

[75] an antibody to IL-8 according to one of paragraphs. [54] - [67], intended for use in the treatment of a disorder characterized by an excess of IL-8;

[76] the use of antibodies to IL-8 according to one of paragraphs. [54] - [67] for the preparation of a pharmaceutical composition for treating a disorder characterized by an excess of IL-8;

[77] a method of treating a patient who has a disorder characterized by an excess of IL-8, comprising administering to the individual an anti-IL-8 antibody according to one of claims. [54] - [67];

[78] a method for enhancing the elimination of IL-8 from an individual's body, comprising administering to the individual an anti-IL-8 antibody according to one of paragraphs. [54] - [67];

[79] a pharmaceutical composition containing an antibody according to one of paragraphs. [54] - [67], where the antibody binds to IL-8 and binds to the extracellular matrix; and

[80] a method for producing an antibody to IL-8, which contains a variable region with a pH-dependent binding activity to IL-8, wherein the method comprises:

(a) assessing the binding of anti-IL-8 antibody to the extracellular matrix,

(b) selection of antibodies to IL-8 with a strong ability to bind to the extracellular matrix,

(c) culturing a host that contains a vector containing a nucleic acid that encodes an antibody, and

(d) isolating the antibody from the culture solution.

An alternative embodiment of the invention indicated in section B of the description is:

[B1] the use of antibodies to IL-8 according to one of paragraphs. [54] - [67] and [B26] - [B31] for the preparation of a pharmaceutical composition designed to suppress the accumulation of IL-8, which has biological activity;

[B2] the use of antibodies to IL-8 according to one of paragraphs. [54] - [67] and [B26] - [B31] to suppress the accumulation of IL-8, which has biological activity;

[B3] the use of antibodies to IL-8 according to one of paragraphs. [54] - [67] and [B26] - [B31] for preparing a pharmaceutical composition for inhibiting angiogenesis;

[B4] the use of antibodies to IL-8 according to one of paragraphs. [54] - [67] and [B26] - [B31] for inhibiting angiogenesis;

[B5] the use of antibodies to IL-8 according to one of paragraphs. [54] - [67] and [B26] - [B31] for preparing a pharmaceutical composition for inhibiting the facilitation of neutrophil migration;

[B6] the use of antibodies to IL-8 according to one of paragraphs. [54] - [67] and [B26] - [B31] to inhibit the facilitation of neutrophil migration;

[B7] an anti-IL-8 antibody according to one of paragraphs. [54] - [67] and [B26] - [B31], intended for use to suppress the accumulation of IL-8, which has biological activity;

[B8] a method for inhibiting the accumulation of IL-8, which has biological activity, wherein the method comprises administering to the subject an anti-IL-8 antibody according to one of paragraphs. [54] - [67] and [B26] - [B31];

[B9] a pharmaceutical composition designed to suppress the accumulation of IL-8, which has biological activity, containing an antibody to IL-8 according to one of paragraphs. [54] - [67] and [B26] - [B31];

[B10] an anti-IL-8 antibody according to one of paragraphs. [54] - [67] and [B26] - [B31] for use in inhibiting angiogenesis;

[B11] a method of inhibiting angiogenesis in an individual, wherein the method comprises administering to the individual an anti-IL-8 antibody according to one of claims. [54] - [67] and [B26] - [B31];

[B12] a pharmaceutical composition for inhibiting angiogenesis, comprising an anti-IL-8 antibody according to one of claims. [54] - [67] and [B26] - [B31];

[B13] an anti-IL-8 antibody according to one of paragraphs. [54] - [67] and [B26] - [B31], for use in inhibiting the facilitation of neutrophil migration;

[B14] a method of inhibiting the facilitation of neutrophil migration in an individual, wherein the method comprises administering to the individual an anti-IL-8 antibody according to one of paragraphs. [54] - [67] and [B26] - [B31];

[B15] a pharmaceutical composition for inhibiting the facilitation of neutrophil migration, comprising an anti-IL-8 antibody according to one of claims. [54] - [67] and [B26] - [B31];

[B16] an anti-IL-8 antibody according to one of paragraphs. [54] - [67] and [B26] - [B31] for use in the treatment of a disorder characterized by an excess of IL-8;

[B17] the use of an antibody according to one of paragraphs. [54] - [67] and [B26] - [B31] for preparing a pharmaceutical composition for treating a disorder characterized by an excess of IL-8;

[B18] the use of an antibody according to one of paragraphs. [54] - [67] and [B26] - [B31] for the treatment of a disorder characterized by an excess of IL-8;

[B19] a method for treating a disorder characterized by an excess of IL-8, wherein the method comprises administering to an individual an anti-IL-8 antibody according to one of claims. [54] - [67] and [B26] - [B31];

[B20] a pharmaceutical composition for treating a disorder characterized by an excess of IL-8, which contains an anti-IL-8 antibody according to one of claims. [54] - [67] and [B26] - [B31];

[B21] an anti-IL-8 antibody according to one of paragraphs. [54] - [67] and [B26] - [B31], intended for use to enhance the elimination of IL-8;

[B22] the use of antibodies to IL-8 according to one of paragraphs. [54] - [67] and [B26] - [B31] for the preparation of a pharmaceutical composition intended to enhance the elimination of IL-8;

[B23] the use of antibodies to IL-8 according to one of paragraphs. [54] - [67] and [B26] - [B31] to enhance the elimination of IL-8;

[B24] a method of enhancing the elimination of IL-8 in an individual, wherein the method comprises administering to the individual an anti-IL-8 antibody according to one of paragraphs. [54] - [67] and [B26] - [B31]; and

[B25] a pharmaceutical composition intended to enhance the elimination of IL-8, which contains an antibody to IL-8 according to one of paragraphs. [54] - [67] and [B26] - [B31].

[B26] An anti-IL-8 antibody in which the Fc region contains amino acid (s) substitution (s) at one or more positions selected from the group consisting of positions 235, 236, 239, 327, 330, 331, 428, 434, 436, 438 and 440, according to EU numbering.

[B27] An anti-IL-8 antibody according to [B26], which comprises an Fc region having at least one property selected from the properties specified in subparagraphs (a) - (e) below:

(a) increased binding affinity of the Fc region to FcRn compared to the binding affinity to FcRn of the native Fc region at acidic pH;

(b) reduced binding affinity of the Fc region to pre-present ADA compared to the binding affinity of native Fc to pre-present ADA;

(c) an extended plasma half-life of the Fc region compared to the plasma half-life of the native Fc region;

(d) decreased plasma clearance of the Fc region compared to the plasma clearance of the native Fc region; and

(e) reduced binding affinity of the Fc region to the effector receptor compared to the binding affinity of the native Fc region to the effector receptor; and

(f) increased binding to extracellular matrix.

[B28] An anti-IL-8 antibody according to [B26] or [B27], containing an Fc region that contains one or more amino acid substitution (s) selected from the group consisting of L235R , G236R, S239K, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R and S440E, according to EU numbering.

[B29] The IL-8 antibody according to [C28], comprising an Fc region that contains one or more amino acid (s) substitution (s) selected from the group consisting of (a) L235R, G236R, S239K, M428L, N434A, Y436T, Q438R and S440E; or (b) L235R, G236R, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R and S440E, according to EU numbering.

[B30] An anti-IL-8 antibody according to [B26], which contains a heavy chain containing the amino acid sequence of SEQ ID NO: 81 and a light chain containing the amino acid sequence of SEQ ID NO: 82.

[B31] The anti-IL-8 antibody according to [B26], which comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 80 and a light chain containing the amino acid sequence of SEQ ID NO: 82.

[B32] Isolated nucleic acid encoding an anti-IL-8 antibody according to one of paragraphs. [B26] - [B31].

[B33] The vector containing the nucleic acid according to [B32].

[B34] A host cell containing the vector according to [B33].

[B35] A method for producing an antibody to IL-8, which comprises culturing a host cell according to [B34].

[B36] The method of obtaining antibodies to IL-8 according to one of paragraphs. [B26] - [B31], which comprises isolating an antibody from a cell culture supernatant;

[B37] A pharmaceutical composition containing an anti-IL-8 antibody according to one of paragraphs. [B26] - [B31] and a pharmaceutically acceptable carrier.

[B38] A method of treating a patient who has a disorder characterized by an excess of IL-8, comprising administering to the individual an anti-IL-8 antibody according to one of claims. [B26] - [B31].

[B39] A method of enhancing the elimination of IL-8 from an individual, comprising administering to the individual an anti-IL-8 antibody according to one of claims. [B26] - [B31].

[B40] A method of inhibiting IL-8, wherein the method comprises contacting an anti-IL 8 antibody according to one of claims. [54] - [67] and [B26] - [B31] with IL-8.

[B41] The method of [B40], wherein the method results in inhibition of the biological activity of IL-8.

Various embodiments of the invention identified in section B of the description are combinations of one or more of the elements specified in any of paragraphs. [54] - [80] and [B1] - [B41], in part or in whole, if the specified combination is not technically impossible according to generally accepted technical knowledge known in this field.

Brief Description of Drawings

The drawings show:

in fig. 1 - Changes in plasma concentration of human IL-6 receptor in transgenic human FcRn mice, which was injected with a human IL-6 receptor binding antibody that binds to the human IL-6 receptor in a pH-dependent manner, and whose constant region is a region of native IgG1 (Low_pI-IgG1), or with an antibody with an increased pI variable region of the antibody (High_pI-IgG1);

in fig. 2 - Changes in the plasma concentration of human IL-6 receptor in transgenic human FcRn mice, which was injected individually with a human IL-6 receptor binding antibody, which binds to the human IL-6 receptor in a pH-dependent manner, and in which the ability to bind to FcRn under neutral pH conditions (Low_pI-F939), and to antibodies with increased pI variable region of the antibody (Middle_pI-F939, High_pI-F939);

in fig. 3: Changes in plasma concentration of human IL-6 receptor in human FcRn transgenic mice, which were injected individually with a human IL-6 receptor binding antibody that binds to the human IL-6 receptor in a pH-dependent manner and in which FcγR- binding activity is increased under conditions of neutral pH (Low_pI-F1180), and with antibodies with an increased pI variable region of the antibody (Middle_pI-F1180, High_pI-F1180);

in fig. 4 - Changes in plasma concentration of human IL-6 receptor in transgenic human FcRn mice, in which the plasma concentration of human IL-6 receptor is maintained in a stable state, which was individually administered with a human IL-6 receptor binding antibody that binds with human IL-6 receptor in a pH-dependent manner, and in which the constant region is a region of native IgG1 (Low_pI-IgG1), an antibody that contains a variant of the Fc region in which the Fc region of the antibody has increased FcRn binding activity in conditions of neutral pH (Low_pI-F11), and with antibodies with an increased pI variable region of these antibodies (High_pI-IgG1, High_pI-F11);

in fig. 5 - the level of binding of the extracellular matrix of each of the three types of antibodies with different pI, which bind to the human IL-6 receptor in a pH-dependent manner (Low_pI-IgG1, Middle_pI-IgG1 and Highpl-IgG1), and two types of antibodies with different pI, which do not bind to the human IL-6 receptor in a pH-dependent manner (Low_pI (NPH) -IgG1 and High_pI (NPH) -IgG1). "NPH" in the context of the above description A means "independent of pH";

in fig. 6 - relative values of the level of binding of human FcγRIIb (according to BIACORE®) of antibodies that contain a variant Fc region, each of which pI is increased by modifying the amino acid residue in the constant region of the Ab1H-P600 antibody, which binds to IgE in a pH-dependent manner, when taking the value for Ab1H-P600 as 1.00;

in fig. 7 - relative values of the rate at which antibodies containing a variant Fc region, each of which pI is increased by modifying an amino acid residue in the constant region of Ab1H-P600, are absorbed by cells of an hFcγRIIb expressing cell line, when evaluating that the value for Ab1H-P600 taken as 1.00;

in fig. 8 shows the level of binding of Fv4-IgG1 having the Fc region of native human IgG1 with rheumatoid factor in the serum of each of the patients suffering from rheumatoid arthritis (RA);

in fig. 9 - the level of binding of Fv4-YTE, having a variant of the Fc region with increased FcRn binding activity, with rheumatoid factor in the serum of each of the RA patients;

in fig. 10 - the level of binding of Fv4-LS, having a variant of the Fc region with increased FcRn binding activity, with rheumatoid factor in the serum of each of the RA patients;

in fig. 11 shows the level of binding of Fv4-N434H, having a variant of the Fc region with increased FcRn binding activity, with rheumatoid factor in the serum of each of the RA patients;

in fig. 12 shows the level of binding of Fv4-F1847m having a variant Fc region with increased FcRn binding activity to rheumatoid factor in the serum of each RA patient;

in fig. 13 shows the level of binding of Fv4-F1848m, having a variant Fc region with increased FcRn binding activity, to rheumatoid factor in the serum of each of the RA patients;

in fig. 14 shows the level of binding of Fv4-F1888m having a variant Fc region with increased FcRn binding activity to rheumatoid factor in the serum of each of the RA patients;

in fig. 15 - the level of binding of Fv4-F1889m having a variant Fc region with increased FcRn-binding activity, with rheumatoid factor in the serum of each of the RA patients;

in fig. 16 shows the level of binding of Fv4-F1927m having a variant Fc region with increased FcRn binding activity to rheumatoid factor in the serum of each of the RA patients;

in fig. 17 - the level of binding of Fv4-F1168m having a variant Fc region with increased FcRn-binding activity, with rheumatoid factor in the serum of each of the RA patients;

in fig. 18 - average binding values of Fv4-IgG1, which has the Fc region of native human IgG1, and each of antibodies containing a new variant Fc region, in which the Fc region is a variant Fc region with increased binding activity for each FcRn, with rheumatoid factor in the serum of RA patients;

in fig. 19 - changes in plasma concentration in cynomolgus monkeys of each antibody to human IgE when administered with OHB-IgG1, which is an antibody to human IgE and has the Fc region of native human IgG1, and with each of the antibodies containing a new variant of the Fc region, in each Fc region is a variant Fc region with increased binding activity for FcRn (OHB-LS, OHB-N434A, OHB-F1847m, OHB-F1848m, OHB-F1886m, OHB-F1889m and OHB-F1927m);

in fig. 20 - Changes in the plasma concentration of antibodies to human IL-6 receptor in transgenic human FcRn mice when administered with Fv4-IgG1, which is an antibody to human IL-6 receptor and has the Fc region of native human IgG1, or with Fv4 -F1718, which is an antibody with increased FcRn binding activity at acidic pH;

in fig. 21 - sensograms characterizing binding to IL-8 H998 / L63 and Hr9 at pH 7.4 and pH 5.8, obtained using Biacore analysis;

in fig. 22 - changes in the concentration of human IL-8 in the plasma of mice after the introduction of H998 / L63 or H89 / L118 to mice at a dose of 2 mg / kg in a mixture with human IL-8;

in fig. 23 shows changes in plasma concentration of human IL-8 in mice when H89 / L118 was administered to mice at a dose of 2 mg / kg or 8 mg / kg mixed with human IL-8;

in fig. 24 - changes in the concentration of human IL-8 in the plasma of mice after the introduction of H89 / L118 or H553 / L118 to mice at a dose of 2 mg / kg or 8 mg / kg in a mixture with human IL-8;

in fig. 25A: Changes in the relative indices of antibody concentration-dependent plasma chemiluminescence using Hr9, H89 / L118 or H553 / L118 antibodies prior to conservation;

in fig. 25B - changes in the relative indices of antibody-dependent plasma chemiluminescence when using antibodies Hr9, H89 / L118 or H553 / L118 after conservation for 1 week;

in fig. 25B — Changes in the relative values of antibody concentration-dependent plasma chemiluminescence using Hr9, H89 / L118, or H553 / L118 antibodies after 2 weeks of conservation;

in fig. 26 — Predicted ADA incidence for each of anti-IL-8 antibodies (hWS4, Hr9, H89 / L118, H496 / L118, or H553 / L118) and predicted ADA incidence for other previously present therapeutic antibodies, as estimated using EpiMatrix;

in fig. 27 - Predicted ADA incidence for each anti-IL-8 antibody (H496 / L118, H496v1 / L118, H496v2 / L118, H496v3 / L118, H1004 / L118, or H1004 / L395) and predicted ADA incidence for other pre-existing therapeutic antibodies estimated using EpiMatrix;

in fig. 28A: Changes in the relative values of antibody concentration-dependent plasma chemiluminescence using Hr9, H89 / L118 or H1009 / L395-F1886s antibodies prior to conservation;

in fig. 28B - changes in the relative indices of antibody-dependent plasma chemiluminescence when using antibodies Hr9, H89 / L118 or H1009 / L395-F1886s after conservation for 1 week;

in fig. 28B — Changes in the relative values of antibody concentration-dependent plasma chemiluminescence using Hr9, H89 / L118, or H1009 / L395-F1886s antibodies after 2 weeks of storage;

in fig. 29: Changes in plasma concentration of human IL-8 in mice upon administration to mice of each of H1009 / L395, H553 / L118 and H998 / L63 mixed with human IL-8;

in fig. 30 - the level of binding to the extracellular matrix when adding Hr9, H89 / L118 or H1009 / L395 to the cellular matrix, alone or in a mixture with human IL-8;

in fig. 31 - changes in the concentration of antibodies in the plasma of mice with the introduction of an antibody having a variable region H1009 / L395 and an Fc region that does not bind to FcRn (F1942m), when administered alone or in a mixture with human IL-8 transgenic in human FcRn mice;

in fig. 32 — Predicted ADA incidence for H1009 / L395 and H1004 / L395 and predicted ADA incidence for other pre-existing therapeutic antibodies estimated using EpiMatrix;

in fig. 33: Changes in the concentration of the corresponding antibody to human IL-8 in the plasma of cynomolgus monkeys upon administration of H89 / L118-IgG1 having the variable region H89 / L118 and the Fc region of native human IgG1, and each antibody that has a variant of the Fc region with increased FcRn binding activity (H89 / L118-F1168m, H89 / L118-F1847m, H89 / L118-F1848m, H89 / L118-F1886m, H89 / L118-F1889m and H89 / L118-F1927m);

in fig. 34 - binding of antibodies that have a variable region H1009 / L395 and whose Fc region is a variant (F1886m, F1886s or F1974m), with each of the FcγR;

in fig. 35: Changes in plasma concentration of human IL-8 in mice upon administration of anti-IL-8 antibody to human FcRn transgenic mice mixed with human IL-8. In this case, the anti-IL-8 antibody was H1009 / L395-IgG1 (2 mg / kg), which contains the variable region H1009 / L395 and the Fc region of native human IgG1, or H1009 / L395-F1886s (2, 5 or 10 mg / kg), which contains the variable region H1009 / L395 and a modified Fc region;

in fig. 36 - Changes in the concentration of antibody in the plasma of cynomolgus monkeys upon administration of Hr9-IgG1 or H89 / L118-IgG1, which both contain the Fc region of native human IgG1 or H1009 / L395-F1886s or H1009 / L395-F1974m, which both contain a modified Fc region ;

in fig. 37 - time profile of the concentration of IgE in plasma when using some antibodies to IgE in mice C57BL6J, depending on the modification of the variable region of the antibody;

in fig. 38 (FIGS. 38A-38D) are Octet sensograms of 25 [twenty-five] pH-dependent and / or calcium-dependent antigen-binding clones selected;

fig. 38B is a continuation of FIG. 38A;

fig. 38B is a continuation of FIG. 38B;

fig. 38D is a continuation of FIG. 38B;

in fig. 39 - the time profile of the concentration of C5 in plasma when using some bispecific antibodies to C5 in mice C57BL6J, depending on the modification of the variable region of the antibody;

in fig. 40 - time profile of plasma IgE concentration when using some antibodies to IgE in C57BL6J mice, depending on the modification of the constant region of the antibody.

Description of embodiments of the invention

Detailed description of the invention

The following are non-limiting embodiments of the invention specified in sections A, B and C of the description of the invention. All embodiments of the invention, which are described in the examples below, are presented for the purpose of a correct understanding of their description, which is also given in the section "Detailed description of the invention", without restrictions due to patent practice, decrees, regulations or other rules that could lead to a narrower interpretation of the content presented in the examples in countries in which it is intended to obtain a patent right based on this patent application.

Section A descriptions or section B descriptions

Some of the embodiments of the invention specified in section A of the description are antibodies containing an antigen-binding domain, the antigen-binding activity of which changes depending on the conditions of ion concentration, in which the isoelectric point (pI) is increased by modifying at least one amino acid residue that can be exposed on the surface of the antibody (in the context of Section A, the descriptions are also referred to as "concentration-dependent antibodies with increased pI"; and antigen-binding domains of antibodies are also referred to as "concentration-dependent antigen-binding domains with increased pI"). The invention is based on the unexpectedly established fact that the elimination of antigen from plasma can be facilitated by a concentration-dependent antibody, the isoelectric point (pI) of which has been increased by modifying at least one amino acid residue that can be exposed on the surface of the antibody ( for example, when the antibody is administered in vivo); and the fact that the binding of the antibody to the extracellular matrix can be increased by a concentration-dependent antibody with an increased (increased) pI. The invention is, in particular, also based on the surprisingly discovered fact that the said beneficial effect depends on a combination of two different factors: an ion concentration-dependent antigen-binding domain or an antibody ion concentration-dependent; and an antibody in which the pI is increased by modifying at least one amino acid residue that can be exposed on the surface (in the context of section A of the description, also referred to as "an antibody with an increased pI";) and an antibody in which the pI is lowered (reduced) by modifying at least one amino acid residue that can be exposed on the surface (in the context of section A of the description, referred to as "pI-reduced antibody"). Thus, the invention is of the type of pioneering invention that can provide significant technological improvement in the field of technology (eg, in the field of medicine) to which section A of the description belongs.

For example, an object of the invention is an antibody that contains an antigen binding domain and the pI of which is increased by modifying at least one amino acid residue that can be displayed on the surface of the antibody, which can be further modified so that the antigen binding activity of the antigen binding domain changes depending on the ion concentration conditions, which also falls within the scope of Section A of the specification (in the context of Section A of the specification, said antibody is also referred to as "ion concentration-dependent antibody with increased pI").

For example, an object of the invention is an antibody containing an ion concentration-dependent antigen-binding domain, in which at least one amino acid residue that can be displayed on the surface of the antibody has a charge different from the charge of at least one amino acid residue in the corresponding position (s) ( y) in an antibody before modification (native antibody (for example, a native antibody in the form of Ig, preferably a native IgG type antibody) or in a reference antibody or parent antibody (for example, an antibody before modification or an antibody before or during library construction, etc.) and the total pI of the antibody is increased, which also falls within the scope of Section A of the present description (such an antibody is also referred to as "ion concentration-dependent antibody with increased pI" in the context of Section A of the present description).

For example, an object of the invention is an antibody containing an ion concentration-dependent antigen-binding domain, the pI of which is increased by modifying at least one amino acid residue that can be displayed on the surface of the antibody prior to modification (for example, a native Ig antibody, preferably a native IgG- type) or in a reference antibody or parent antibody (for example, an antibody before modification, or an antibody before or during library construction, or the like)), which also falls within the scope of Section A of the present description (such an antibody is also referred to as "dependent on concentration of ions of an antibody with an increased pI "in the context of Section A of the present description).

For example, an object of the invention is an antibody comprising an ion concentration-dependent antigen binding domain in which at least one amino acid residue that can be displayed on the surface of an antibody has been modified to increase the pI of an antibody, which also falls within the scope of Section A of the present disclosure (such an antibody also denoted as "concentration-dependent antibody with increased pI" in the context of section A of the present description).

In the context of sections A and B of the present description, "amino acids" includes not only naturally occurring amino acids, but also non-naturally occurring amino acids. In the context of sections A and B of the present description, amino acids or amino acid residues can be designated using either one-letter (eg, A) or three-letter codes (eg, Ala), or both (eg, Ala (A)).

In the context of sections A and B of this description, "amino acid modification", "amino acid residue modification" or their equivalents means (but are not limited to) chemical modification of one or more (for example, 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10 or more than 10) of specific amino acids (residues) in the amino acid sequence of the antibody within the molecule or the addition, deletion, substitution or insertion of one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) amino acids in the amino acid sequence of the antibody. Addition, deletion, substitution or insertion of amino acids can be carried out at the level of the nucleic acid encoding the amino acid sequence, for example, by site-directed mutagenesis (Kunkel et al., Proc. Natl. Acad. Sci. USA 82, 1985, pp. 488-492) or by elongation PCR; by affinity maturation of antibodies or using a chain shuffling technique for heavy or light chains of an antibody; or by panning-based antigen selection using phage display libraries (Smith et al., Methods Enzymol. 217, 1993, pp. 228-257); and they can be carried out individually or in appropriate combinations. The specified amino acid modification is carried out (but not limited to only this) by replacing one or more amino acid residues in the amino acid sequence of the antibody with another amino acid (individually). Addition, deletion, substitution, or insertion of an amino acid and modification of the amino acid sequence by humanization or chimerization can be accomplished by methods known in the art. A change or modification of an amino acid (residue), such as the addition, deletion, substitution or insertion of an amino acid, can also be carried out in the variable region of an antibody or in the constant region of an antibody, which can be used to produce recombinant antibodies as antibodies falling within the scope of section A or B descriptions.

In one of the embodiments of the invention, which falls within the scope of sections A and B of the present description, the term "substitution of amino acids (residues)" refers to substitutions for other amino acids (residues) and can be created by changing the characteristics that are indicated below in subparagraphs (a ) - (c): (a) the structure of the polypeptide framework in the area of the fold or spiral structure; (b) electrical charge or hydrophobicity at the target site; or (c) the size of the side chain.

Amino acid residues are classified into the following groups based on general side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu and He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn and Gln; (3) acidic: Asp and Glu; (4) basic: His, Lys and Arg; (5) residues that affect chain orientation: Gly and Pro; and (6) aromatic: Trp, Tyr and Phe.

Substitution of amino acid residues in each group is referred to as a conservative substitution, and substitution of amino acid residues between groups is referred to as a non-conservative substitution. Substitution of amino acid residues can be a conservative substitution, a non-conservative substitution, or a combination thereof. Suitable methods can be used to replace amino acids with amino acids other than naturally occurring amino acids (Wang et al., Annu. Rev. Biophys. Biomol. Struct. 35, 2006, pp. 225-249; Forster et al., Proc. Natl Acad Sci USA 100 (11) 2003 6353-6357). You can use, for example, a cell-free translation system containing tRNA, in which a non-naturally occurring amino acid is fused with a suppressor tRNA that suppresses amber mutation, complementary to the UAG codon (amber codon), which is a stop codon (Clover Direct (Protein Express)).

In the context of these sections A and B of the present description, it is understood that the structure of an "antigen" is not limited to a particular structure, as long as the antigen includes an epitope that binds to the antibody. The antigen can be an inorganic or organic substance. Antigens can be any ligands, including various cytokines such as interleukins, chemokines, and cell growth factors. Alternatively, the subject of the invention is, for example, receptors which are present in a soluble form or which have been modified to become soluble in biological fluids such as plasma, which can also be used as antigens. Examples of such soluble receptors include, but are not limited to, the soluble IL-6 receptor described by Mullberg et al., J. Immunol. 152 (10), 1994, pp. 4958-4968. In addition, antigens can be monovalent (eg, soluble IL-6 receptor) or multivalent (eg, IgE).

In one embodiment of the invention, antigens that can bind to the antibody described in sections A and B of the description are preferably soluble antigens present in biological fluids (for example, biological fluids illustrated in WO 2013/125667, preferably in plasma interstitial fluid, lymph fluid, ascites fluid, or pleural fluid) of individuals (in the context of sections A and B of this description, individuals who need to be administered (who need to be treated) with an antibody, which can actually be any animal, for example, humans, mice, etc.) etc.); however, the antigens can also be membrane antigens.

In the context of sections A and B of the present description, "prolonging the plasma half-life" or "shortening the plasma half-life" of the target molecule (which may be an antigen or antibody) or their equivalents can also be denoted more specifically, using in addition to the parameter "Plasma half-life" (t1 / 2) any other parameter such as mean plasma retention time, plasma clearance (CL) and area on the concentration-time curve (AUC) (Pharmacokinetics: Enshuniyoru Rikai (Understanding through practice) practice)) Nanzando). These parameters can be specifically assessed, for example, by performing non-compartmental analysis according to the protocol supplied with the WinNonlin in vivo kinetic analysis software (Pharsight). Those of ordinary skill in the art will appreciate that these parameters tend to be correlated with one another.

In the context of sections A and B of the present description, the term "epitope" refers to an antigenic determinant in an antigen and means a region on an antigen to which the antigen binding domain of an antibody binds. Thus, an epitope can be characterized, for example, based on its structure. Alternatively, an epitope can be characterized by the antigen-binding activity of an antibody that recognizes the epitope. When the antigen is a peptide or polypeptide, the epitope can be characterized by amino acid residues that form the epitope. Alternatively, the epitope can be a sugar chain, the epitope can be characterized based on the specific structure of the sugar chain. The antigen-binding domain specified in sections A and B of the description can bind to a single epitope or to different epitopes on an antigen.

A linear epitope can be a primary amino acid sequence. Such a linear epitope typically contains at least three, and usually at least five, for example 8-10 amino acids or 6-20 amino acids, as a unique sequence.

In a conformational epitope, as a rule, the amino acids forming the epitope are not present as a contiguous primary sequence. An antibody can recognize a conformational epitope in the three-dimensional structure of a peptide or protein. Methods for determining epitope conformation include, but are not limited to, X-ray crystallography, 2D nuclear magnetic resonance, site-specific spin labeling, and electron paramagnetic resonance (Epitope Mapping Protocols in: Methods in Molecular Biology, ed. Morris, vol. 66, 1996).

In the context of sections A and B of this description, the term "antibody" is not limited to a specific antibody and is used in its broadest sense if it has the ability to bind to a target antigen. Examples of antibodies include, but are not limited to, well-known canonical antibodies (e.g., native immunoglobulins (abbreviated as "Ig")) and molecules and variants derived therefrom, e.g. Fab, Fab ', F (ab') ₂ , dimeric antibodies (diabodi), ScFv (Holliger et al., Proc. Natl. Acad. Sci. USA 90, 1993, pp. 6444-6448 (1993); EP 404.097; WO 93/11161; Peer et al., Nature Nanotechnology 2, 2007, pp. 751-760), low molecular weight antibodies (minibodi) (Orita et al., Blood 105, 2005, pp. 562-566), scaffold proteins, antibodies with one arm (including all variants of antibodies with one arm disclosed in WO 2005/063816), multispecific antibodies (e.g., bispecific antibodies: antibodies that have specificity for two different epitopes, including antibodies that recognize different antigens and antibodies that recognize different epitopes of the same antigen). In the context of sections A and B of the present description, "bispecific antibodies" can be obtained (but not limited to), for example, in the form of antibody molecules that share a common L-chain, described in WO 2005/035756, or using the method described in WO 2008/119353, in which two general types of antibodies having IgG4-like constant regions are mixed to provide an exchange reaction between the two types of these antibodies (one of ordinary skill in the art is known as "Fab arm swap"). In an alternative embodiment, the antibodies may have a structure in which the heavy chain variable region and the light chain variable region are linked together to form a single chain (eg, sc (Fv) ₂ ). Alternatively, they can be antibody-like molecules (e.g., scFv-Fc) obtained by ligating an Fc region (a constant region lacking a CH1 domain) with an scFv (or sc (Fv) ₂ ), with variable the heavy chain (VH) region is ligated to the light chain variable region (VL). Multispecific antibodies consisting of scFv-Fc have the structure (scFv) ₂ -Fc in which the first and second polypeptides are VH1-linker-VL1-Fc and VH2-linker-VL2-Fc structures, respectively. Alternatively, they can be antibody-like molecules in which a single-domain antibody is linked to an Fc region (Marvin et al., Curr. Opin. Drug Discov. Devel. 9 (2), 2006, pp. 184-193) containing fusion proteins with Fc (for example, immunoadhesin) (US 2013/0171138), their functional fragments, functionally equivalent substances and their variants modified with a sugar chain. As used herein, the term "native IgG (eg, native IgG1)" refers to polypeptides that contain the same amino acid sequence as naturally occurring IgG (eg, native IgG1) and which belong to the class of antibodies encoded primarily the genome of immunoglobulin gamma. Native IgG may contain spontaneous mutations and the like.

Typically, if an antibody has a structure that is substantially the same or similar to that of native IgG, then the Y-shaped structure of the four chains (two heavy chain polypeptides and two light chain polypeptides) may represent the base structure. Typically, the heavy chain and light chain can be linked to each other via a disulfide bond (SS bond) and form a heterodimer. These heterodimers can be connected to each other through a disulfide bond and form a Y-shaped heterotetramer. Two heavy chains or two light chains can be identical or different from each other.

For example, an IgG-type antibody can be cleaved into two Fab units (region) and one Fc unit (region) by cleavage with papain, which cleaves the hinge region (which is referred to as "hinge" in the context of sections A and B of this description), in which the heavy the Fab region chain is linked to the Fc region. Typically, the Fab region contains an antigen binding domain. Since phagocytes, such as leukocytes and macrophages, have receptors that have the ability to bind to the Fc region (Fc receptors), they can recognize antibodies bound to the antigen through Fc receptors and carry out their phagocytosis (opsonization). Moreover, the Fc region is involved in mediating immune responses, such as ADCC or CDC, and has an effector function, inducing a response after antibody binding to antigens. It is known that the effector function of an antibody varies with the type of immunoglobulin (isotype). The Fc region of the IgG class may be a region extending, for example, from cysteine at position 226 or from proline at position 230 (EU numbering) to the C-terminus; however, the Fc region is not limited to these. The Fc region can be obtained by partial cleavage of a monoclonal antibody in the form of IgG1, IgG2, IgG3, or IgG4 or other antibodies with a protease such as pepsin, followed by elution of the adsorbed fractions from a Protein A or Protein G column.

In the context of sections A and B herein, the positions of amino acid residues in the variable region (CDR and / or FR) of an antibody are designated according to Cabot, while the positions of amino acid residues in the constant region or Fc region are indicated according to EU numbering based on amino acid positions. according to Cabot (Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991).

In the context of sections A and B of this description, the term "library" can refer to molecules (populations), such as a variety of antibodies with sequence variability, in which the corresponding sequences may be the same or different from each other; many fusion polypeptides containing antibodies; or nucleic acids or polynucleotides encoding said amino acid sequences, as detailed in WO 2013/125667 (eg, paragraphs 0121-0125). The library may contain, for example, at least 10 ⁴ antibody molecules, more preferably at least 10 ⁵ antibody molecules, even more preferably at least 10 ⁶ antibody molecules, most preferably at least 10 or more antibody molecules. The library can be phage libraries. The phrase "primarily consists of" means that antibodies, which may have different antigen-binding activities, constitute a subset of numerous independent clones with different sequences in the library. In one embodiment of the invention, immune libraries that are constructed from antibody genes derived from lymphocytes from animals immunized with a specific antigen, patients with infection, humans with elevated blood antibody titers as a result of vaccination, or patients with cancer or autoimmune disease can appropriately used as randomized variable region libraries. In an alternative embodiment of the invention, naive libraries containing naive sequences (antibody sequences without deviation in the antibody spectrum) that are constructed from antibody genes derived from lymphocytes of healthy individuals can also be suitably used as randomized variable region libraries (Gejima et al., Human Antibodies 11, 2002, pp. 121-129); Cardoso et al., Scand. J. Immunol. 51, 2000, pp. 337-344). Amino acid sequences containing naive sequences can be sequences derived from these naive libraries. In an alternative embodiment, synthetic libraries in which a CDR sequence from a genomic DNA V gene or a reshaped functional V gene has been replaced with a set of synthetic oligonucleotides containing a sequence that encodes a codon set of the appropriate length can also be suitably used as randomized variable region libraries ... In this case, it is also possible to substitute only the heavy chain CDR3 sequence since sequence variations are found in the CDR3 gene. A standard way of obtaining a variety of amino acids in the variable region of an antibody can be used to increase the variation in amino acid residues at positions that can be displayed on the surface of an antibody.

In one embodiment of the invention, in which the antibodies referred to in section A or B of the description have, for example, a structure substantially the same or similar to that of native Ig antibodies, they typically have variable regions ("V regions" ) [heavy chain variable region ("VH region") and light chain variable region ("VL region")] and constant regions ("C regions") ["heavy chain constant region (" CH region ") and constant region of the light chain ("CL region")]. The CH-region is further divided into three regions: CH1-CH3. Typically, the heavy chain Fab region contains the VH region and CH1, and typically the heavy chain Fc region contains CH2 and CH3. Typically, the hinge region is located between CH1 and CH2. In addition, the variable region typically contains hypervariable regions ("CDR") and framework regions ("FR"). Typically, the VH region and VL region each contain three CDRs (CDR1, CDR2, and CDR3) and four FRs (FR1, FR2, FR3, and FR4). Typically, six CDRs in the variable regions of the heavy chain and light chain interact and form the antigen-binding domain of an antibody. On the other hand, if only one CDR is present, although its antigen binding affinity is known to be lower than when six CDRs are present, then it still retains the ability to recognize and bind antigen.

Ig antibodies are classified into several classes (isotypes) based on structural differences in the constant regions. In many mammals, based on structural differences in the constant region, five classes of immunoglobulins are identified: IgG, IgA, IgM, IgD and IgE. In addition, in humans, IgG has four subclasses: IgG1, IgG2, IgG3, and IgG4; a IgA has two subclasses: IgA1 and IgA2. The heavy chain is classified as γ-chain, μ-chain, α-chain, δ-chain and ε-chain according to the differences in the constant region, and based on these differences, there are five classes (isotypes) of immunoglobulins: IgG, IgM, IgA, IgD and IgE. On the other hand, two types of light chains are known: λ-chain and κ-chain, and all immunoglobulins have either of these two types of chains.

In one embodiment of the invention, in which the antibodies specified in section A or B of the description have a heavy chain, for example, the heavy chain can be any of the chains selected from γ-chain, μ-chain, α-chain, δ-chain and ε-chain, or can be obtained from one of them, and when the antibody specified in sections A and B of the description has a light chain, the light chain can be, for example, either a κ-chain or a λ-chain, or can be obtained from any of them. In addition, in the context of sections A and B of this description, an antibody can refer to any isotype (e.g., IgG, IgM, IgA, IgD, or IgE) and any subclass (e.g., human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2; murine IgG1, IgG2a, IgG2b and IgG3) or can be obtained from any of them (but not limited to them).

In the context of sections A and B of this description, the "antigen binding domain" can be of any structure as long as it binds to the antigen of interest. These domains may include, for example, the variable regions of the heavy chains and light chains of an antibody (eg, 1-6 CDR); a module of about 35 amino acids, designated the A domain, which is included in Avimer, a cell membrane protein present in the body (WO 2004/044011 and WO 2005/040229); Adnectin containing the 10Fn3 domain that binds to a protein in the fibronectin glycoprotein expressed on the cell membrane (WO 2002/032925); an affibody having a framework of an IgG-binding domain consisting of a three-helical bundle comprising 58 amino acids of the protein (WO 1995/001937); engineered ankyrin repeat (DARPin) proteins, which are a region exposed on the molecular surface of an ankyrin repeat (AR) that has a structure in which a 33 amino acid bend subunit is refolded, two antiparallel helices and a loop (WO 2002 / 020565); anticalins and other molecules, which are a four-loop region supporting one side of a centrally coiled barrel-like structure of eight antiparallel chains that are highly conserved among lipocaine molecules such as neutrophil gelatinase associated lipocaine (NGAL) (WO 2003/029462); and a concave region formed by a parallel strand structure within a horseshoe-shaped structure formed by folded repeats, which are composed of a leucine-rich repeat (LRR) module of a variable lymphocyte receptor (VLR) that does not have an immunoglobulin structure and is used in the acquired resistance system in jawless vertebrates, such as lampreys and mixins (WO 2008/016854). Preferred antigen binding domains referred to in section A or B of the description may be domains having the heavy chain and light chain variable regions of an IgG-type antibody, and more specifically, ScFv, single chain antibodies, Fv, scFv ₂ (single chain FV ₂ ), Fab and F (ab ') ₂ .

In one of the embodiments of the invention referred to in section A of the description, "ion concentration" is not limited to a particular type of ion and refers to the concentration of hydrogen ions (pH) or concentration of metal ions. As used herein, metal ions are Group I ions other than hydrogen, such as alkali metals and copper group elements, Group II elements such as alkaline earth metals and zinc group elements, Group III elements excluding boron, Group IV elements excluding carbon and silicon, Group VIII elements such as iron group and platinum group elements, elements belonging to subgroup A of Groups V, VI and VII, and elements representing metals such as antimony, bismuth and polonium. Metal atoms have the ability to release valence electrons to become cations. This phenomenon is referred to as ionization tendency. It is believed that metals with a strong tendency to ionize are reactive.

In one of the embodiments of the invention referred to in section A of the description, the preferred metal ions may be calcium ions, as described in detail in WO 2012/073992 and WO 2013/125667.

In one embodiment of the invention referred to in Section A of the specification, the "ion concentration condition (s)" may be a condition (s) affecting the difference in biological behavior of an ion concentration dependent antibody between low ion concentration and high concentration of ions. In addition, the term “antigen binding activity varies depending on the conditions of ion concentration” can mean that the antigen binding activity of the ion concentration dependent antigen binding domain or the ion concentration dependent antibody specified in section A or B varies depending on whether the concentration is ions low or high. Such cases include, for example, but are not limited to, cases with higher (stronger) or less strong (weaker) antigen-binding activity at a high ion concentration than at a low ion concentration.

In one of the embodiments of the invention referred to in section A of the description, the ion concentration can be a hydrogen ion concentration (pH) or a calcium ion concentration. When the ion concentration is the concentration of hydrogen ions (pH), the ion concentration-dependent antigen-binding domain is also referred to as the "pH-dependent antigen-binding domain", and when the ion concentration is the concentration of calcium ions, it can also be referred to as the "concentration-dependent calcium ions antigen-binding domain ".

In one embodiment, in the context of section A of the description, concentration-dependent antigen-binding domains, concentration-dependent antigen-binding domains, concentration-dependent antigen-binding domains with an increased pI, and concentration-dependent antigen binding domains with an increased pI can be obtained from libraries consisting mainly of from antibodies that differ in sequence (have variability), and antigen-binding domains of which contain at least one amino acid residue, which causes a change in the antigen-binding activity of the antigen-binding domain or antibody, depending on the conditions of ion concentration. Antigen binding domains can preferably be located in the variable region of the light chain (which can be modified) and / or the variable region of the heavy chain (which can be modified). In addition, to create a library, these variable regions of the light chain or heavy chain can be combined with the variable regions of the heavy chain or light chain, designed as a library of randomized sequences of variable regions. When the ion concentration is the concentration of hydrogen or calcium ions, examples of libraries include, but are not limited to, for example, libraries in which the heavy chain variable regions, designed as a randomized variable region sequence library, are combined with the light chain variable region sequences , in which the amino acid residue (s) in the germline sequence such as SEQ ID NO: 1 (Vk1), SEQ ID NO: 2 (Vk2), SEQ ID NO: 3 (Vk3) or SEQ ID NO: 4 (Vk4) is (s) replaced by at least one amino acid residue that can alter the antigen binding activity depending on the ion concentration. In addition, when the ion concentration is the concentration of calcium ions, libraries include, for example, libraries in which the variable region sequence of the heavy chain SEQ ID NO: 5 (6RL # 9-IgG1) or SEQ ID NO: 6 (6KC4-1 # 8 5-IgG1) are combined with light chain variable regions designed as a randomized sequence library of variable regions or light chain variable regions having germline sequence.

In one embodiment, when the ion concentration is a calcium ion concentration, the high calcium ion concentration is not limited to particular values, but may be a concentration selected from the range of 100 μM to 10 mM, 200 μM to 5 mM, 400 μM to 3 mM, 200 μM to 2 mM, or 400 μM to 1 mM. A concentration of from 500 μM to 2.5 mM may also be preferred due to the proximity to the plasma calcium ion concentration in vivo. The low concentration of calcium ions is not limited to specific values, but can be a concentration selected from the range of 0.1 to 30, 0.2 to 20, 0.5 to 10, or 1 to 5, or 2 to 4 μM ... A concentration selected from the range of 1 to 5 μm may also be preferred due to its proximity to the concentration of calcium ions in early endosomes in vivo.

Whether the antigen-binding activity of an antigen-binding domain or an antibody containing a domain changes depending on the conditions of the concentration of metal ions (for example, the concentration of calcium ions), can be easily determined using known methods, for example, those described herein in the context of section A of the description or described in WO 2012/073992. For example, the antigen binding activity of an antigen binding domain or an antibody containing a domain can be measured at low and high calcium ion concentrations and compared. In this case, conditions other than the concentration of calcium ions should preferably be the same. In addition, in determining the antigen binding activity, conditions other than the concentration of calcium ions can be selected appropriately from those known to one of ordinary skill in the art. Antigen binding activity can be determined, for example, in HEPES buffer conditions at 37 ° C or by BIACORE assay (GE Healthcare) or other assays.

In one of the embodiments of the invention described in the context of section A of the description, it is preferable that the antigen binding activity of a concentration-dependent antigen-binding domain, dependent on the concentration of antibody ions, dependent on the concentration of ions of an antigen-binding domain with an increased pI, or dependent on the concentration of ions of an antibody with an increased pI , was higher under conditions of high concentration of calcium ions than under conditions of low concentration of calcium ions. In this case, the ratio between antigen-binding activity under conditions of low concentration of calcium ions and antigen-binding activity under conditions of high concentration of calcium ions is not limited to a particular value; however, the KD value (dissociation constant) for the antigen under conditions of low calcium ion concentration and the KD value under conditions of high calcium concentration, i.e., KD (3 μM Ca) / KD (2 mM Ca), may preferably be 2 or higher more preferably 10 or higher, and even more preferably 40 or higher. The upper limit of KD (3 μM Ca) / KD (2 mMCa) is not limited and can be any value such as 400, 1000, or 10000.

When the antigen is a soluble antigen, the dissociation constant (KD) can be used as a measure of antigen binding activity. However, if the antigen is a membrane antigen, then the apparent dissociation constant (KD) can be used. The dissociation constant (KD) and the apparent dissociation constant (KD) can be determined by known methods, for example, using BIACORE analysis (GE healthcare), a Scatchard plot or a flow cytometer.

Alternatively, for example, the dissociation reaction rate constant (kd) can also be used as another indicator characterizing the ratio of antigen-binding activities. When the rate constant of the dissociation reaction (kd) is used instead of the dissociation constant (KD) as an indicator characterizing the ratio of antigen-binding activities, the value of the ratio of the rate constant of the dissociation reaction (kd) under conditions of low concentration of calcium ions and the rate constant of the dissociation reaction (kd) under conditions high concentration of calcium ions, i.e. kd (low calcium ion concentration conditions) / kd (low calcium ion concentration conditions) may be preferably 2 or higher, more preferably 5 or higher, even more preferably 10 or higher, and even more preferably 30 or higher. The upper limit of kd (low calcium ion concentration condition) / kd (low calcium ion concentration condition) is not limited and may be any value such as 50, 100, or 200.

When the antigen is a soluble antigen, the dissociation rate constant (kd) can be used as a measure of antigen binding activity. However, if the antigen is a membrane antigen, the apparent dissociation rate constant (kd) can be used. The dissociation reaction rate constant (kd) and the apparent dissociation rate constant (kd) can be determined using known methods, for example, using BIACORE analysis (GE healthcare) or flow cytometry.

In one embodiment, methods for producing or screening calcium ion-dependent antigen-binding domains or calcium-ion-dependent antibodies whose antigen-binding activity is higher under high calcium conditions than under low calcium ion conditions, or their libraries, are not limited to specific methods. Techniques include, for example, those described in WO 2012/073992 (eg, in paragraphs 0200-0213).

Such methods include, for example:

(a) determining the antigen-binding activity of the antigen-binding domain or antibody under conditions of low calcium ion concentration;

(b) determining the antigen-binding activity of the antigen-binding domain or antibody under conditions of high concentration of calcium ions; and

(c) selection of an antigen-binding domain or antibody, the antigen-binding activity of which under conditions of a low concentration of calcium ions is lower than the antigen-binding activity under conditions of a high concentration of calcium.

Alternatively, the method could include, for example:

(a) contacting an antigen with an antigen binding domain or antibody or with a library thereof under conditions of high concentration of calcium ions;

(b) incubating the antigen-binding domain or antibody bound to the antigen in step (a) under conditions of low calcium ion concentration; and

(c) isolation of the antigen-binding domain or antibody, separated by dissociation in step (b).

Alternatively, the method could include, for example:

(a) contacting the antigen with an antigen-binding domain or antibody, or with a library thereof, under conditions of low calcium ion concentration;

(b) selection of an antigen-binding domain or antibody that has not bound to the antigen or has a low antigen-binding activity in step (a);

(c) enabling the antigen-binding domain or the antibody selected in step (b) to bind to the antigen under conditions of high concentration of calcium ions; and

(d) isolating the antigen-binding domain or antibody that binds to the antigen in step (c).

Alternatively, the method could include, for example:

(a) contacting an antigen-binding domain or antibody or a library thereof with a column with an immobilized antigen under conditions of high concentration of calcium ions;

(b) elution of the antigen binding domain or antibody bound to the column in step (a) from the column under conditions of low calcium concentration; and

(c) isolating the antigen-binding domain or antibody eluted in step (b).

Alternatively, the method could include, for example:

(a) allowing the antigen binding domain or antibody or their library to pass through the immobilized antigen column under conditions of low calcium ion concentration to collect the antigen binding domain or antibody eluted without binding to the column;

(b) allowing the antigen binding domain or antibody assembled in step (a) to bind to the antigen under conditions of high calcium ion concentration; and

(c) isolation of the antigen-binding domain or antibody that binds to the antigen in step (b).

Alternatively, the method could include, for example:

(a) contacting the antigen with an antigen binding domain or antibody, or with a library thereof, under conditions of high concentration of calcium ions;

(b) obtaining an antigen binding domain or antibody that binds to the antigen in step (a);

(c) incubation of the antigen-binding domain or antibody obtained in step (b) under conditions of low concentration of calcium ions; and

(d) isolation of an antigen-binding domain or antibody, the antigen-binding activity of which at stage (c) is weaker than the selection criterion at stage (b).

Each step of these different screening methods can be repeated several times, or the steps can be combined as appropriate to obtain the most stable molecules. The above conditions can preferably be selected for low and high calcium ion concentration conditions. In this way, the desired calcium ion concentration dependent domains or calcium ion concentration dependent antibodies can be obtained.

In the context of section A of the description in one embodiment of the invention, the antigen-binding domains or antibodies used as the starting product, for example, can be modified antigen-binding domains or antibodies having an increased pI as a result of modification of the charge of at least one amino acid residue that can be displayed on their surfaces. In an alternative embodiment, when amino acids that alter the binding activity of a concentration-dependent antigen binding domain are introduced into the sequence, they can be introduced in combination with a modification of the charge of at least one amino acid residue that can be exposed on the surface of the antigen binding domain or antibody, leading to an increase in pI.

Alternatively, in the context of section A of the present description, for example, you can use already existing antigen-binding domains or antibodies, already existing libraries (phage library, etc.); antibodies obtained from hybridomas created by immunizing animals or from B cells of immunized animals or their libraries; or antigen-binding domains, antibodies or libraries obtained by introducing naturally occurring or non-naturally occurring amino acid mutations therein that can chelate calcium (as described below) (e.g., libraries with increased content of calcium-chelating amino acids or libraries, into which amino acids with the ability to chelate calcium are introduced into specific sites).

In the context of section A of the description in one of the embodiments of the invention, in which the concentration of ions is the concentration of calcium ions, there is no limitation regarding the type of amino acids that alter the binding activity of the concentration-dependent antigen binding domains or ion-dependent antigen binding domains with increased pI if they can form a calcium-binding motif. For example, calcium-binding motifs are known to those skilled in the art (see, for example, Springer et al., Cell 102, 200, pp. 275-277; Kawasaki et al., Protein Prof. 2, 1995, pp. 305-490; Moncrief et al., J. Mol. Evol. 30, 1990, pp. 522-562; Chauvaux et al., Biochem. J. 265, 1990, pp.: 261-265; Bairoch et al., FEBS Lett. 269 , 1990, pp. 454-456; Davis, New Biol. 2, 1990, pp. 410-419; Schaefer et al., Genomics 25, 1995, pp. 638-643; Economou et al., EMBO J. 9, 1990, pp. 349-354; Wurzburg et al., Structure 14 (6), 2006, pp. 1049-1058). Thus, if the antigen-binding domain has an arbitrary calcium-binding motif, such as a type C lectin, eg ASGPR, CD23, MBR, or DC-SIGN, then the antigen-binding activity of the domain can be changed depending on the conditions of calcium ion concentration. Such calcium binding motifs can include, for example, in addition to those described above, a calcium binding motif included in the antigen binding domain shown in SEQ ID NO: 7 (which corresponds to "Vk5-2").

In the context of section A of the description, in one embodiment of the invention, in which the ion concentration is the concentration of calcium ions, amino acids having metal chelating activity can be used as amino acids that alter the binding activity of the ion concentration-dependent antigen-binding domains or concentration-dependent ions of antigen-binding domains with increased pI. For example, any amino acids having metal chelating activity can be suitably used as long as they can form a calcium-binding motif. In particular, these amino acids include amino acids having the ability to serve as electron donors. Amino acids preferably include, but are not limited to, Ser (S), Thr (T), Asn (N), Gln (Q), Asp (D), and Glu (E).

Localization of these amino acids having metal-chelating activity in the antigen-binding domain is not limited to specific positions. In one embodiment, the amino acids can be located at any of the positions in the heavy chain variable region and / or light chain variable region that can form an antigen binding domain. At least one amino acid residue that causes a calcium ion concentration-dependent change in the antigen-binding activity of an antibody may be contained, for example, in CDRs (one or more of CDR1, CDR2 and CDR3) and / or FR (one or more of FR1, FR2, FR3 and FR4) of the heavy chain and / or light chain. Amino acid (s) residue (s) can be placed, for example, at one or more of positions 95, 96, 100a and 101 according to Cabot numbering in the CDR3 of the heavy chain; at one or more positions 30, 31 and 32 according to Cabot numbering in the light chain CDR1; at position 50 according to Cabot numbering in the light chain CDR2; and / or at position 92 according to Cabot numbering in the light chain CDR3. These amino acid residues can be placed individually or in combination.

Troponin C, calmodulin, parvalbumin, myosin light chain, and other compounds are known to have multiple calcium-binding sites, and may have originated from a common source during molecular evolution, and in one embodiment, one or more of CDR1, CDR2, and CDR3 light chains were designed to contain their linking motifs. For the purposes described above, you can use, for example, the cadherin domain; EF-shoulder included in calmodulin; C2 domain included in protein kinase C; Gla-domain contained in the coagulation factor IX protein; C-type lectin asialoglycoprotein receptor or mannose binding receptor; The A domain of the LDL receptor; annexation; thrombospondin type-3 domain; and an EGF-like domain.

In one embodiment of the invention, in which the concentration is the concentration of hydrogen ions (pH), the concentration of protons, i.e. nuclei of a carbon atom are used as a synonym for hydrogen index (pH). When the level of activity of a hydrogen ion in aqueous solution is designated as aH ⁺ , the pH is defined as -log10aH ⁺ . When the ionic strength of the aqueous solution is low (eg, less than 10 ^-3 ), aH ^{+ is} practically equivalent to the strength of hydrogen ions. For example, the ionic product of water at 25 ° C and a pressure equal to one atmosphere is Kw = aH ⁺ × aOH = 10 ^-14 ; thus, in the case of pure water, aH ⁺ = aOH = 10 ^-7 . In this case, pH = 7 is neutral and an aqueous solution with a pH below 7 is acidic, and an aqueous solution with a pH above 7 is alkaline. Thus, hydrogen ion concentration conditions can be conditions focused on differences in the biological behavior of a pH-dependent antibody at high hydrogen ion concentration (acidic pH range) and low hydrogen ion concentration (neutral pH range) as conditions for hydrogen ion concentration, or pH conditions. For example, in the context of section A of the description, “antigen-binding activity under high hydrogen ion concentration (acidic pH range) is lower than antigen-binding activity under low hydrogen ion concentration (neutral pH range)” may mean that the antigen-binding activity of the ion concentration-dependent antigen-binding domain depending on the concentration of antibody ions, depending on the concentration of ions of the antigen-binding domain with increased pI or depending on the concentration of antibodies with increased pI is weaker at pH selected from pH from 4.0 to 6.5, preferably from pH from 4.5 to 6 , 5, more preferably from pH from 5.0 to 6.5 and even more preferably from pH from 5.5 to 6.5 than at a pH selected from pH from 6.7 to 10.0, preferably from pH from 6.7 to 9.5, more preferably from pH 7.0 to 9.0, and even more preferably from pH 7.0 to 8.0. Preferably, the above expression may mean that the antigen-binding activity at a pH close to that in early endosomes in vivo is weaker than the activity at plasma pH in vivo; and in particular, it can mean that the antigen-binding activity of an antibody, for example, at pH 5.8, is weaker than, for example, at pH 7.4.

Whether the antigen binding activity of the antigen binding domain or the antibody containing the domain changes depending on the hydrogen ion concentration conditions can be easily determined using known methods, for example, the assay methods specified in the context of section A of the description or described in WO 2009/125825. For example, the antigen binding activity of an antigen binding domain or an antibody containing a domain against an antigen of interest can be measured at low and high hydrogen ion concentrations and compared. In this case, it is preferable that other conditions other than the concentration of hydrogen ions are the same. When performing the determination of antigen binding activity, one of ordinary skill in the art can appropriately select conditions other than hydrogen ion concentration and, for example, measurements can be carried out under the conditions of using a HEPES buffer at 37 ° C or using a BIACORE assay (GE Healthcare) or etc.

In the context of section A of this description, unless specifically indicated otherwise, the term "neutral pH range" (which is also referred to as "low concentration of hydrogen ions", "high pH", "neutral pH conditions" or "neutral pH") is not limited specifically a specific meaning; however, it can preferably be selected from a pH of 6.7 to 10.0, a pH of 6.7 to 9.5, a pH of 7.0 to 9.0, or a pH of 7.0 to 8.0. The neutral pH range may preferably correspond to pH 7.4, close to the in vivo pH in plasma (blood), but for convenience of measurement, for example, pH 7.0 can be used.

In the context of section A of this description, unless specifically indicated otherwise, the term "acidic pH range" (which is also referred to as "high concentration of hydrogen ions", "low pH", "acidic pH conditions" or "acidic pH") is not limited specifically a specific meaning; however, it can preferably be selected from pH 4.0 to pH 6.5, pH 4.5 to 6.5, pH 5.0 to 6.5, or 5.5 to 6.5. The acidic pH range can preferably correspond to pH 5.8, close to the in vivo concentration of hydrogen ions in the early endosome, but for convenience of measurements, for example, pH 6.0 can be used.

In one embodiment of the invention in the context of section A of the description, when the ion concentration is the concentration of hydrogen ions, then preferably the antigen binding activity of an ion concentration dependent antigen binding domain, an antibody ion concentration dependent, an antigen binding domain ion concentration dependent, or the concentration of antibody ions with an increased pI is higher under neutral pH conditions than under acidic pH conditions. In this case, the ratio of antigen-binding activity under conditions of neutral pH and antigen-binding activity under conditions of acidic pH is not limited to a specific value; however, the value of the ratio KD (dissociation constant) for an antigen in an acidic pH to a KD in a neutral pH, i.e., KD (acidic pH range) / KD (neutral pH range), (for example, KD (pH 5.8 ) / KD (pH 7.4)) can be 2 or higher; 10 or higher; or 40 or higher. The upper limit of the KD (acidic pH range) / KD (neutral pH range) value is not limited to a particular value and can be any value such as 400, 1000, or 10000.

In an alternative embodiment, the dissociation reaction rate constant (kd) can also be used, for example, as a measure of the ratio of the above antigen binding activities. When the dissociation rate constant (kd) is used instead of the dissociation constant (KD) as an indicator characterizing the ratio of antigen-binding activities, the value of the ratio of the dissociation rate constant (kd) for the antigen under conditions of high concentration of hydrogen ions and under conditions of low concentration of hydrogen ions, those. kd (acidic pH range) / kd (neutral pH range) can be 2 or higher, 5 or higher, 10 or higher, or 30 or higher. The upper limit of kd (acidic pH range) / kd (neutral pH range) is not limited to a particular value and can be any value such as 50, 100, or 200.

When the antigen is a soluble antigen, then the dissociation rate constant (kd) can be used as a measure of antigen binding activity, and if the antigen is a membrane antigen, then the apparent dissociation rate constant (kd) can be used for this purpose. The dissociation reaction rate constant (kd) and the apparent dissociation reaction rate constant (kd) can be determined using known methods, for example, using BIACORE analysis (GE healthcare) or a flow cytometer.

In one embodiment, methods for producing or screening pH-dependent antigen-binding domains or pH-dependent antibodies whose antigen-binding activity is higher under neutral pH conditions than under acidic pH conditions, or their libraries, are not limited to specific methods. Techniques include, for example, those described in WO 20092/125825 (eg, in paragraphs 0158-0190).

Such methods include, for example:

(a) determining the antigennegative activity of the antigennegative domain or antibody under conditions of acidic pH;

(b) determining the antigen-binding activity of the antigen-binding domain or antibody under neutral pH conditions; and

(c) selection of an antigen-binding domain or antibody, the antigen-binding activity of which is lower under acidic pH conditions than under neutral pH conditions.

Alternatively, the method could include, for example:

(a) contacting an antigen with an antigen-binding domain or antibody or a library thereof under neutral pH conditions;

(b) incubating the antigen-binding domain or antibody bound to the antigen in step (a) under acidic pH conditions; and

(c) isolation of the antigen-binding domain or antibody, separated by dissociation in step (b).

Alternatively, the method could include, for example:

(a) contacting the antigen with an antigen-binding domain or antibody or a library thereof under acidic pH conditions;

(b) selection of an antigen-binding domain or antibody that has not bound to the antigen or has a low antigen-binding activity in step (a);

(c) enabling the antigen binding domain or the antibody selected in step (b) to bind to the antigen under neutral pH conditions; and

(d) isolating the antigen-binding domain or antibody that binds to the antigen in step (c).

Alternatively, the method could include, for example:

(a) contacting an antigen-binding domain or antibody or a library thereof with a column with an immobilized antigen under neutral pH conditions;

(b) elution of the antigen-binding domain or antibody bound to the column in step (a) under acidic pH conditions; and

(c) isolating the antigen-binding domain or antibody eluted in step (b).

Alternatively, the method could include, for example:

(a) allowing the antigen binding domain or antibody or their library to pass through the immobilized antigen column under acidic pH conditions to collect the antigen binding domain or antibody eluted without binding to the column;

(b) allowing the antigen binding domain or antibody assembled in step (a) to bind to the antigen under neutral pH conditions; and

(c) isolation of the antigen-binding domain or antibody that binds to the antigen in step (b).

Alternatively, the method could include, for example:

(a) contacting an antigen with an antigen-binding domain or antibody or a library thereof under neutral pH conditions;

(b) obtaining an antigen binding domain or antibody that binds to the antigen in step (a);

(c) incubating the antigen-binding domain or antibody obtained in step (b) under acidic pH conditions; and

(d) isolation of an antigen-binding domain or antibody, the antigen-binding activity of which at stage (c) is weaker than the selection criterion at stage (b).

Each step of these different screening methods can be repeated several times or the steps can be combined. The above conditions can preferably be selected for acidic and neutral pH conditions. Thus, the desired pH-dependent antigen-binding domains or pH-dependent antibodies can be obtained.

In the context of section A of the description in one embodiment of the invention, the antigen-binding domains or antibodies used as the starting product, for example, can be modified antigen-binding domains or antibodies having an increased pI as a result of modification of the charge of at least one amino acid residue that can be displayed on their surfaces. In an alternative embodiment, when amino acids that alter the binding activity of the ionic concentration-dependent antigen binding domain are introduced into the sequence, they can be introduced in combination with a modification of the charge of at least one amino acid residue that can be exposed on the surface of the antigen binding domain or antibody, resulting in to increase pI.

Alternatively, in the context of section A of the description of the present description, for example, you can use already existing antigen-binding domains or antibodies, already existing libraries (phage library, etc.); antibodies obtained from hybridomas created by immunizing animals or from B cells of immunized animals, or a library thereof; or antigen binding domains, antibodies or libraries obtained by introducing mutations into them using naturally occurring or non-naturally occurring amino acids with a side chain pKa of 4.0-8.0 (as described below), for example, libraries with increased content mutations using naturally occurring or non-naturally occurring amino acids with a pKa side chain of 4.0-8.0 or a library with introduced at specific sites mutations using naturally occurring or non-naturally occurring amino acids with a pKa side chain 4.0-8.0). Said preferred antigen binding domain may have, for example, an amino acid sequence in which at least one amino acid residue is replaced by amino acid (s) with a pKa side chain of 4.0-8.0 and / or into which (s) amino acid (s) are inserted with a side chain pKa of 4.0-8.0 as described in WO 2009/125825.

In one of the embodiments of the invention in the context of section A of the description, the site into which the mutation of amino acids with a pKa side chain of 4.0-8.0 is introduced is not limited to a specific site and the mutation can be introduced at any site if the antigen-binding activity becomes weaker in acidic pH range than in the neutral pH range (the ratio KD (acidic pH range) / KD (neutral pH range) is increased or the value of the ratio kd (acidic pH range) / kd (neutral pH range) is increased) compared to the variant before replacement or embedding. When the antigen binding domain has a variable region or CDR, the site can be within the variable region or CDR. Those of ordinary skill in the art can appropriately determine the number of substituted or inserted amino acids; and this number can be 1 or more. In addition, other r amino acids can be removed by deletion, inserted and / or substituted or modified in addition to the substitution or insertion described above. Substitution or insertion of amino acids with a pKa of the side chain of 4.0-8.0 can be performed arbitrarily using techniques such as histidine scan, in which histidine is used instead of alanine in an alanine scan known to those of ordinary skill in the art and / or antibodies in which the value of the ratio KD (acidic pH range) / KD (neutral range of pH) or the value of the ratio kd (acidic range of pH) / kd (neutral range of pH) is increased in comparison with the variant before the introduction of the mutation, can be selected among antigen-binding domains or antibodies obtained as a result of arbitrary mutation leading to the substitution or insertion of these amino acids or their libraries.

In addition, antigen-binding domains or antibodies can be preferably domains or antibodies, the antigen-binding activity of which in the neutral pH range before and after the introduction of these mutations does not significantly decrease, does not significantly decrease, is practically identical or increases; in other words, the activity of which can be maintained at a level of at least 10% or higher, preferably 50% or higher, even more preferably 80% or higher, and even more preferably 90% or higher or even higher. If the binding activity of an antigen-binding domain or antibody is reduced as a result of the substitution or insertion of amino acids with a pKa of 4.0-8.0, then the binding activity can be restored or increased, for example, by substitution, deletion, addition or insertion of one or more amino acids at sites other than those in which the above substitutions or insertions have been made.

In an alternative embodiment, amino acids with a pKa side chain of 4.0-8.0 can be placed at any position within the heavy chain and / or light chain variable regions that can form an antigen binding domain. At least one amino acid residue with a pKa side chain of 4.0-8.0 can be located, for example, in CDRs (one or more of CDR1, CDR2 and CDR3) and / or FR (one or more of FR1, FR2, FR3 and FR4) heavy chain and / or light chain. These amino acid residues include, but are not limited to, amino acid residues at one or more of positions 24, 27, 28, 31, 32 and 34 according to Cabot numbering or in the CDR1 variable region of the light chain; amino acid residues at one or more of positions 50, 51, 52, 53, 54, 55, and 56 according to Cabot numbering or in the light chain variable region CDR2; CDR2; and / or amino acid residues at one or more of positions 89, 90, 91, 92, 93, 94 and 95A according to Cabot numbering or in the CDR3 variable region of the light chain. These amino acid residues can be included individually or in combination, if the antigen-binding activity of the antibody changes depending on the conditions of the concentration of hydrogen ions.

In one embodiment, in the context of Section A of the specification, an arbitrary amino acid residue can suitably be used as an amino acid residue that alters the antigen binding activity of an antigen binding domain or antibody depending on the concentration of hydrogen ions. In particular, said amino acid residues may include residues with a side chain pKa of 4.0-8.0. Said amino acids having the ability to donate electrons may include, for example, naturally occurring amino acids such as His (H) and Glu (E), and non-naturally occurring amino acids such as histidine analogs (US 2009/0035836), m-NO ₂ -Tyr (pKa 7.45), 3,5-Br ₂ -Tyr (pKa 7.21) and 3.5-12-Tyr (pKa 7.38) (Heyl et al., Bioorg. Med Chem. 11 (17) 2003, pp. 3761-3768). You can also use amino acids with a pKa side chain of 6.0-7.0, which includes, for example, His (H).

In the context of section A of the present description, unless specifically indicated otherwise and if this is not impossible in this context, it is obvious that the isoelectric point (pI) can be determined either theoretically or experimentally isoelectric point and is also referred to as "pI".

The pI value can be determined experimentally, for example, using isoelectric focusing in combination with electrophoresis. In this case, the theoretical value of pI can be calculated, for example, using software for analyzing the gene and amino acid sequence (Genetyx, etc.).

In one embodiment of the invention, is the pI of an antibody with an increased pI or an antibody specified in section A of the description increased compared to an antibody before modification (a native antibody (e.g., a native Ig antibody, preferably a native IgG-type antibody) or a reference antibody (for example, an antibody before antibody modification or before or during library construction)) can be determined by evaluating the pharmacokinetic characteristics of the antibody in addition to or instead of the above methods using plasma, for example, mice, rats, rabbits, dogs, monkeys or human, in combination with methods such as BIACORE, cell proliferation assay, ELISA, enzyme immunoassay (EIA), radioimmunoassay (RIA), or immunofluorescence assay.

In the context of section A of the present description, "amino acid residue that can be exposed on the surface" generally means an amino acid residue located on the surface of the polypeptide that forms the antibody. An "amino acid residue localized on the surface of a polypeptide" can mean an amino acid residue whose side chain can be in contact with solvent molecules (most often water molecules). However, the side chain need not be completely in contact with the solvent molecules, and even if only a portion of the side chain is in contact with the solvent molecules, the amino acid is considered a "surface localized amino acid residue." Amino acid residues localized on the surface of a polypeptide also include amino acid residues localized near the surface of an antibody and which as a result can affect the overall electrical charge of another amino acid residue, even if the side chain of which is only partially in contact with solvent molecules. Those of ordinary skill in the art can create a homologous model of a polypeptide or antibody, for example, using commercially available software. Alternatively, methods known to those skilled in the art, such as, for example, X-ray crystallography, can be used. The amino acid residues that can be exposed on the surface are determined, for example, using the coordinates of a three-dimensional model using a computer program such as InsightII (Accelrys). Sites that can be exposed on a surface can be determined using algorithms known in the art (for example, see Lee and Richards, J. Mol. Biol. 55, 1971, pp. 379-400; Connolly, J. Appl. Cryst . 16, 1983, pp. 548-558). The sites that can be exposed on the surface can be determined using software suitable for protein modeling and information about the three-dimensional structure obtained for the antibody. Suitable software for this purpose includes, for example, the SYBYL Biopolymer Module software (Tripos Associates). When the algorithm requires a user-defined parameter "size", the "size" of the sample used in the calculations can be specified as a radius equal to approximately 1.4 Angstroms or less. In addition, methods for determining areas that can be exposed on a surface are described in Pacios (Comput. Chem. 18 (4), 1994, pp. 377-386; J. Mol. Model. 1, 1995, pp. 46-53) ... Based on the above information, suitable amino acid residues located on the surface of the polypeptide that forms the antibody can be selected.

The method for increasing the pI of a protein involves, for example, decreasing the number of amino acids with a negatively charged side chain (for example, aspartic acid and glutamic acid) and / or increasing the number of amino acids with a positively charged side chain (for example, arginine, lysine and histidine) under neutral pH conditions. Amino acid residues with a negatively charged side chain have a negative charge corresponding to -1 at a pH that is sufficiently higher than the pKa of the side chain, according to theory well known to those skilled in the art. For example, the theoretical pKa value for the side chain of aspartic acid is 3.9 and the side chain has a negative charge corresponding to -1 under neutral pH conditions (eg, in a solution with pH 7.0). Conversely, amino acids with a positively charged side chain have a positive charge corresponding to +1 at a pH that is sufficiently lower than the pKa of their side chain. For example, the theoretical pKa value for the side chain of arginine is 12.5 and the side chain has a positive charge corresponding to +1 under neutral pH conditions (eg, in a solution with pH 7.0). At the same time, it is known that amino acids whose side chains are free of charge under neutral pH conditions (for example, in a solution with pH 7.0) include 15 types of naturally occurring amino acids, i.e. alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan and tyrosine. It goes without saying that amino acids that can be used to change the pI may be non-naturally occurring amino acids.

Based on the above, using the method of increasing the pI of a protein under conditions of neutral pH (for example, in a solution with pH 7.0), it is possible to change the charge of the protein of interest by +1, for example, by replacing amino acids (residues) with uncharged side chains of aspartic acid or glutamic acid (whose side chain has a negative charge of -1) in the amino acid sequence of the protein. In addition, a change in charge by +1 can be carried out in a protein, for example, by replacing amino acids with uncharged side chains with arginine or lysine (the side chain of which has a positive charge of +1). In addition, it is possible to simultaneously change the charge of the protein by +2 by replacing arginine or lysine (whose side chain has a positive charge of +1) aspartic acid or glutamic acid (whose side chain has a negative charge of -1). Alternatively, to increase the pI of a protein, amino acids with uncharged side chains and / or preferably amino acids with positively charged side chains can be added to or inserted into the amino acid sequence of the protein or amino acids with uncharged side chains and / or preferably amino acids with negatively charged side chains present in the amino acid protein sequences can be removed by deletion. It is obvious that, for example, the N-terminal and C-terminal amino acid residues of a protein have a charge inherent in their main chain (NH ₃ ⁺ amino groups at the N-end and COO ^{- a} carbonyl group at the C-end) in addition to the charges inherent in their side chain. Thus, the pI of a protein can be increased by carrying out a specific addition, deletion, substitution or insertion in the functional groups of the main chain.

It should be obvious to those of ordinary skill in the art that the effect of a change in the net charge or pI of a protein, which involves the modification of one or more amino acids (residues) in the amino acid sequence, in order to present or increase the electrical charges of the amino acids (residues), does not depend only ( or substantially) on the antibody-forming amino acid sequences themselves or the type of target antigen, but rather depends on the type and amount of amino acid residues that are added, removed, replaced, or inserted.

Antibodies that have been modified to have an increased pI by modifying at least one amino acid residue that can be displayed on the surface of the antibody ("increased pI antibodies" or "increased pI antibodies") can be incorporated more rapidly into cells, or can enhance the elimination of antigen from plasma, as described or suggested, for example, in WO 2007/114319, WO 2009/041643, WO 2014/145159 or WO 2012/016227.

Of the several isotypes of antibodies, for example, an IgG-type antibody has a sufficiently high molecular weight that its main metabolic pathway does not involve renal excretion. It is known that an IgG-type antibody that has an Fc region as part of the molecule can be "reused" (recycled) by "rescue" via FcRn, and therefore has a long in vivo half-life. It is believed that the IgG-type antibody is metabolized mainly by the metabolic pathway in endothelial cells (He et al. J. Immunol. 160 (2), 1998, pp. 1029-1035). In particular, it is assumed that upon nonspecific penetration into endothelial cells, IgG-type antibodies are "reutilized" by binding to FcRn, and IgG-type antibodies that cannot bind are metabolized. The plasma half-life of an IgG-type antibody can be shortened when its Fc region is modified so that its FcRn binding activity is reduced. On the other hand, it has been demonstrated that the plasma half-life of an antibody with an increased pI depends on a pI with a high level of correlation, as described, for example, in WO 2007/114319 and WO 2009/041643. In particular, the plasma half-life of antibodies with an increased pI, which are described in the documents cited above, is reduced without modifying the amino acid sequence forming the Fc, which can potentially lead to the acquisition of immunogenicity, and this result suggests that the technology of increasing pI is widely used even in the case of those types of antibody molecules, the main metabolic pathway of which is renal excretion, such as scFv, Fab or Fc-containing fusion proteins.

The pH concentration in biological fluids (eg plasma) corresponds to the neutral pH range. Without going into a specific theory, it can be assumed that in biological fluids the net positive charge of an antibody with an increased pI increases due to an increase in pI, and as a result, the antibody is attracted with greater force through the physicochemical Coulomb interaction to the surface of the endothelial cell, which has a net negative charge. compared to antibodies that do not have an increased pI; through nonspecific binding, the antibody binds to it and enters the cells, which leads to a shortening of the half-life of the antibody in the plasma or an increase in the elimination of the antigen from the plasma. In addition, an increased pI of an antibody increases the penetration of the antibody (or antigen / antibody complex) into cells and / or intracellular permeability, which is believed to result in a decrease in plasma antibody concentration, a decrease in antibody bioavailability and / or a shortened plasma half-life of the antibody; and it is assumed that this phenomenon is widespread in vivo regardless of cell type, tissue type, organ type, etc. In addition, the formation of an antibody-antigen complex and its penetration into cells can be influenced not only by the pI of the antibody, but also by the pI of the antigen, reducing or increasing the penetration into cells.

In one embodiment, methods for producing and screening antibodies with an increased pI may include, for example, the methods described in WO 2007/114319 (e.g. paragraphs 0060-0087), WO 2009/041643 (e.g. paragraphs 0115), WO 2014 / 145159 and WO 2012/016227. The specified method may include, for example:

(a) modifying a nucleic acid that encodes an antibody containing at least one amino acid residue that can be displayed on the surface of the antibody, which can modify the charge of the amino acid residue (s) so that the pI of the antibody is increased;

(b) culturing the host cell to express the nucleic acid; and

(c) harvesting the antibody from the host cell culture.

Alternatively, the method may include, for example:

(a ') a modification of a nucleic acid that encodes an antibody containing at least one amino acid residue that can be displayed on the surface of the antibody, so as to modify the charge of the amino acid residue (s);

(b ') culturing the host cell to express the nucleic acid;

(c ') collecting the antibody from the host cell culture; and

(d ') (optionally with confirmation or measurement) selection of an antibody with an increased pI compared to the antibody before modification. In the context of the present description, the antibody used as the starting product, or the antibody before modification, or the reference antibody can be, for example, a concentration-dependent antibody. Alternatively, by modifying the amino acid residue (s), the amino acid (s) that alter the binding activity of the concentration-dependent antigen binding domain may also be included in the sequence.

Alternatively, the method may simply be a method comprising culturing the host cell obtained in step (b) or (b ') and collecting the antibody from the cell culture.

In an alternative embodiment, the method may be, for example, a method for producing a multispecific antibody that comprises a first polypeptide and a second polypeptide, and optionally a third polypeptide and a fourth polypeptide comprising:

(A) a modification of the nucleic acid (s) which (s) encode (s) a first polypeptide and / or a second polypeptide, and optionally a third polypeptide and / or a fourth polypeptide, any one or more of which contains at least one amino acid a residue that can be displayed on the surface of the polypeptide, modifying the charge of the amino acid residue (s) so that the pI of the antibody is increased;

(B) culturing the host cell to express the nucleic acid; and

(B) harvesting the multispecific antibody from the host cell culture.

Alternatively, the method may include, for example:

(A ') a modification of the nucleic acid (s) that (s) encode (s) a first polypeptide and / or a second polypeptide, and optionally a third polypeptide and / or a fourth polypeptide, any one or more of which contains at least one an amino acid residue that can be displayed on the surface of a polypeptide so that the charge of the amino acid residue (s) is changed;

(B ') culturing the host cell to express the nucleic acid;

(B ') collecting the multispecific antibody from the host cell culture; and

(D ') (optionally with confirmation or measurement) selection of an antibody with an increased pI compared to the antibody before modification.

In the context of the present description, the antibody used as the starting product, or the antibody before modification, or the reference antibody can be, for example, a concentration-dependent antibody. Alternatively, by modifying the amino acid residue (s), the amino acid (s) that alter (s) the binding activity of the concentration-dependent antigen binding domain may also be included in the sequence.

Alternatively, the method may simply be a method comprising culturing the host cell obtained in step (B) or (B ') and collecting the antibody from the cell culture. In this case, the polypeptides whose nucleic acid (s) are to be modified may preferably be a homomultimer of a first polypeptide, a homomultimer of a second polypeptide, or a heteromultimer of a first and second polypeptide (and optionally a homomultimer of a first polypeptide, a homomultimer of a fourth polypeptide, or a heteromultimer of a third and fourth ).

In an alternative embodiment, the method may be, for example, a method for producing a humanized or human antibody with a shortened plasma half-life, which comprises: in an antibody that contains a CDR selected from the group consisting of CDRs of human origin, CDRs from non-human animals and synthetic CDR (s); FR of human origin; and a human constant region, (I) modify at least one amino acid residue that can be exposed on the surface of at least one region selected from the group consisting of CDR, FR and constant region to obtain amino acid residue (s) , which has (s) a charge different from the charge of the amino acid residue (s) present at the corresponding position (s) prior to modification that results in an increase in the pI of the antibody.

Alternatively, the method may include, for example, the steps of, in an antibody that contains a CDR selected from the group consisting of CDRs of human origin, CDRs from non-human animals, and synthetic CDR (s); FR of human origin; and the human constant region,

(I ') modify at least one amino acid residue that can be displayed on the surface of at least one region selected from the group consisting of CDR, FR and constant region, to obtain amino acid residue (s), which (s) ) has a different charge than the amino acid residue (s) present in the corresponding position (s) prior to modification, and

(II ') selection of an antibody with an increased pI compared to the antibody before modification is performed (optionally, and).

In the context of the present description, the antibody used as the starting product, or the antibody before modification, or the reference antibody can be, for example, a concentration-dependent antibody. Alternatively, by modifying the amino acid residue (s), the amino acid (s) that alter (s) the binding activity of the concentration-dependent antigen binding domain may also be included in the sequence.

Alternatively, it is possible, for example, to use pre-existing antigen-binding domains or antibodies, pre-existing libraries (phage library, etc.); antibodies obtained from hybridomas created by immunizing animals or from B cells of immunized animals or their libraries; or antigen binding domains, antibodies or libraries with an increased pI obtained by modifying in the above antigen binding domains, antibodies or their libraries of at least one amino acid residue that can be displayed on a surface, according to, for example, any of the above described embodiments of the invention.

In one embodiment of the invention, the pI value of the antibodies specified in section A of the description may preferably be increased, for example, by at least 0.01, 0.03, 0.05, 0.1, 0.2, 0, 3, 0.4, 0.5, or more, or by at least 0.6, 0.7, 0.8, 0.9 or more, and to significantly shorten the plasma half-life of the antibody, the pI value can be increased, for example by at least 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 or more or at least 1.6, 1.7, 1.8, 1.9 , 2.0, 2.1, 2.2, 2.3, 2.4, 2.5 or more, or 3.0 or more, compared to antibodies before modification or modification (native antibodies (for example, native antibodies in Ig form, preferably native IgG-type antibodies) or reference antibodies or parental antibodies (for example, antibodies before antibody modification or before or during library construction)). One of ordinary skill in the art, depending on the purpose, can routinely determine the optimal pI value for the antibodies specified in section A of the description, taking into account the balance between pharmacological action and toxicity and, for example, the amount of antigen binding domains of antibodies or antigen pI. Without wishing to be bound by theory, it can be assumed that the antibodies described in section A of the description, according to one embodiment of the invention, have valuable properties in addition to the ability to act as a shuttle between plasma and cellular endosomes and the ability of a single antibody molecule to re-bind to multiple antigens. due to the presence of a concentration-dependent antigen binding domain, such as an increase in the net positive charge of an antibody as a result of an increase in pI, and this leads to a rapid uptake of the antibody by cells. These characteristics should shorten the half-life of the antibody in plasma, increase the activity of binding of antibodies to the extracellular matrix, or increase the elimination of the antibody from plasma. One of ordinary skill in the art can determine the optimal pI value to exhibit the beneficial effects of these characteristics.

In one of the embodiments of the invention in the context of section A of the description, compared with antibodies before modifying or changing at least one amino acid residue to increase the pI (native antibodies (for example, native antibodies in the form of Ig, preferably native IgG antibodies) or reference antibodies or parental antibodies (for example, antibodies before antibody modification or before or during library construction), which may be concentration-dependent antibodies), with concentration-dependent antibodies specified in section A of the description, with increased pI ability to eliminate antigens from plasma can increase preferably, for example, at least 1.1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3, 5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 times or more (with the introduction of antibodies in vivo) or their ability to bind to the extracellular matrix can be increased preferably, for example, by less her measure in 1.1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4 , 25, 4.5, 4.75, or 5 times or more.

In one embodiment, in the context of section A of the description, compared to antibodies prior to the introduction of the ion concentration-dependent antigen binding domain (native antibodies (e.g., native Ig antibodies, preferably native IgG antibodies) or reference antibodies or parental antibodies ( for example, antibodies before antibody modification or before or during library construction), which can be antibodies with an increased pI), in the ion concentration-dependent antibodies specified in section A of the description, with an increased pI, the ability to eliminate antigens from plasma can be increased preferentially, for example at least 1.1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 times or more (if the introduction of antibodies in vivo) or their ability to bind to the extracellular matrix can be increased preferably, for example, at least 1.1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, or 5 times or more.

According to one embodiment of the invention, assay methods used to decide whether the binding activity to the extracellular matrix of the antibodies specified in section A of the description is increased compared to antibodies before modification or alteration (native antibodies (for example, native antibodies in Ig, preferably native IgG-type antibodies) or reference antibodies or parental antibodies (for example, antibodies before antibody modification or before or during library construction), which can be antibodies with increased pI), are not limited to specific methods. For example, the analysis can be performed using an ELISA system that detects the binding between the antibody and the extracellular matrix when the antibody is added to the extracellular matrix immobilized on the plate and the labeled anti-antibody antibody is added thereto. Alternatively, as indicated in Examples 1-4 herein and in WO 2012/093704, electrochemiluminescence (ECL) can also be used, which allows the ability to bind to the extracellular matrix to be determined with high sensitivity. This method can be carried out, for example, using an ECL system in which a mixture of an antibody and a ruthenium-labeled antibody is added to an extracellular matrix immobilized on a plate and the binding between the antibodies and the extracellular matrix is measured based on ruthenium electrochemiluminescence. The concentration of the antibody to which it is added can be adjusted appropriately; the added concentration can be increased to increase the sensitivity of the extracellular matrix binding detection. These extracellular matrices can be obtained from animals or plants if they contain glycoproteins such as collagen, proteoglycan, fibronectin, laminin, entactin, fibrin and perlecan; animal derived extracellular matrices may be preferred. For example, extracellular matrices derived from animals such as humans, mice, rats, monkeys, rabbits or dogs can be used. For example, human native extracellular matrix can be used as an indicator of the pharmacodynamics of antibodies in human plasma. The conditions under which the evaluation of the binding of the antibody to the extracellular matrix is carried out may preferably be a neutral pH range (about pH 7.4), which corresponds to physiological conditions; however, conditions do not have to be in the neutral range, and binding can also be assessed in the acidic pH range (eg, about pH 6.0). Alternatively, when assessing the binding of an antibody to an extracellular matrix, the assay can be performed in the presence of the antigen molecule to which the antibody binds, and the binding activity of the antigen-antibody complex with the extracellular matrix can be determined.

In one embodiment, the antibodies described in section A of the description (to a significant extent) may retain antigen-binding activity relative to antibodies until at least one amino acid residue is modified or altered to increase the pI (native antibodies (e.g., native antibodies in the form Ig, preferably native IgG-type antibodies) or reference antibodies or parental antibodies (for example, antibodies before antibody modification or before or during library construction) In this case, "(substantially) retention of antigen-binding activity" can mean the presence of activity, constituting at least 50% or more, preferably 60% or more, more preferably 70% or 75% or more, and even more preferably 80%, 85%, 90% or 95% or more as compared to the binding activity of the antibody before modification or changes.Alternatively, the antibodies specified in section A of the description should retain binding activity b only to the extent that allows them to maintain their function when they bind to antigens, so the affinity, determined at 37 ° C under physiological conditions, may be, for example, 100 nM or less, preferably 50 nM or less, more preferably 10 nM or less, and even more preferably 1 nM or less.

In one of the embodiments of the invention, specified in section A of the description, the term "modification of at least one amino acid residue that can be displayed on the surface of an antibody" or an equivalent term may mean that one or more additions, deletions, substitutions and insertions of at least at least one amino acid residue that can be exposed on the surface of an antibody. The specified modification may preferably include the replacement of at least one amino acid residue.

Substitution of amino acid residues may include, for example, replacing amino acid residues whose side chains are free of charge with amino acid residues having a negatively charged side chain, replacing amino acid residues having a positively charged side chain with amino acid residues whose side chains have no charge, and replacing amino acid residues having a positively charged side chain with amino acid residues having a negatively charged side chain in the amino acid sequence of the antibody of interest, which can be done individually or in appropriate combinations. The insertion or addition of amino acid residues can include, for example, the insertion or addition of amino acids having no side chain and / or the insertion or addition of amino acids having a positively charged side chain into the amino acid sequence of the antibody of interest, which can be done individually or in appropriate combinations. ... Deletion of amino acid residues can include, for example, deletion of amino acid residues whose side chain has no charge and / or deletion of amino acid residues having a negatively charged side chain in the amino acid sequence of an antibody of interest, which can be done individually or in appropriate combinations.

Those of ordinary skill in the art can appropriately combine one or more of these additions, deletions, substitutions, and insertions in the amino acid sequence of an antibody of interest. A modification that can lead to a decrease in the local charge of amino acid residues is also acceptable, since only the pure pI of the antibody specified in section A of the description should be increased. For example, if necessary, antibodies whose pI has been raised (too much) can be modified to lower the pI (too small). It is also acceptable to reduce the local charge of amino acid residues as a result of modification of at least one amino acid residue, which is carried out simultaneously or at different times for other purposes (for example, to increase the stability of the antibody or reduce immunogenicity). Such antibodies include antibodies from libraries designed for specific purposes.

In one embodiment of the invention, among the amino acids (residues) used to modify at least one amino acid residue that can be displayed on the surface of an antibody, naturally occurring amino acids are classified as follows: an amino acid with a negatively charged side chain may be Glu (E ) or Asp (D); an amino acid with an uncharged side chain can be Ala (A), Asn (N), Cys (C), Gln (Q), Gly (G), His (H), Ile (I), Leu (L), Met ( M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), or Val (V); and the amino acid with a positively charged side chain may be His (H), Lys (K), or Arg (R).

As described in detail in Examples 1-4 herein, in a neutral pH solution (e.g. pH 7.0), substantially 100% lysine and arginine have a positive charge when present as a residue in an antibody, while only approximately 9% of histidine residues are positively charged when present as a residue in an antibody, and the remainder is likely to have no charge. Thus, Lys (K) or Arg (R) is preferably chosen as amino acids with a positively charged side chain.

In one embodiment, the antibodies described in section A of the description preferably have a variable region and / or a constant region. In addition, the variable region may preferably comprise a heavy chain variable region and / or a light chain variable region and / or may preferably have CDRs (e.g., one or more of CDR1, CDR2, and CDR3) and / or FR (e.g., one or more of FR1, FR2, FR3 and FR4). The constant region may preferably include a heavy chain constant region and / or a light chain constant region, and depending on the sequence and type, it may be, for example, an IgG-type constant region (preferably a human IgG1-type constant region, human IgG2-type, human IgG3-type or human IgG4-type, human κ chain constant region and human λ chain constant region). Modified versions of these constant regions can also be used.

In one embodiment, the modification of at least one amino acid residue that can be displayed on the surface of an antibody can be either a single amino acid modification or a combination of multiple amino acid modifications. A preferred method may include introducing a combination of multiple amino acid substitutions at sites where amino acids can be exposed on the surface of an antibody. In addition (but not limited to), these multiple amino acid substitutions are preferably introduced at positions that are close to each other in the three-dimensional structure. When amino acids with a positively charged side chain (such as Lys (K) or Arg (R)) must replace amino acids that can be exposed on the surface of the antibody molecule (which are preferably (but not limited to) amino acids with a negatively charged side chain (for example, Glu (E) or Asp (D)); or when previously present amino acids having a positive charge are used (for example, Lys (K) or Arg (R)), for example, one or more amino acids (which may include amino acids, immersed inside the antibody molecule, depending on the situation), which are close in the three-dimensional structure to amino acids, can also be replaced with amino acids having a positive charge, to subsequently create a dense state of local positive charge localized in close proximity in the three-dimensional structure. "In close proximity in a three-dimensional structure" is not limited to specific etno; but it may mean a state in which one or more amino acid substitutions are introduced, for example, within 20 Angstroms, preferably within 15 Angstroms, and more preferably within 10 Angstroms. Whether the amino acid substitution site of interest is exposed on the surface of the antibody molecule, or whether the amino acid substitution site is near other amino acid substitution (s) or pre-existing amino acids, can be assessed by known techniques such as X-ray crystallography.

In addition to those described above, methods for generating multiple positive charges at sites close to each other in a three-dimensional structure may include methods that use amino acids that originally had a positive charge in the constant region of native IgG. Such amino acids include, for example: arginine at positions 255, 292, 301, 344, 355 and 416 according to the EU numbering; and lysine at positions 121, 133, 147, 205, 210, 213, 214, 218, 222, 246, 248, 274, 288, 290, 317, 320, 322, 326, 334, 338, 340, 360, 370, 392, 409, 414 and 439 according to EU numbering. A plurality of positive charges can be placed close to each other in the three-dimensional structure by substituting positively charged amino acid (s) at sites in the three-dimensional structure close to said positively charged amino acids.

If the antibodies specified in section A of the description have a variable region (which can be modified), then amino acid residues that are not masked when binding to the antigen (i.e., can still be exposed on the surface) can be modified and / or the amino acid the modification may not be introduced at sites that are masked upon antigen binding, or an amino acid modification may be made that does not (substantially) inhibit antigen binding. If the amino acid residues that can be exposed on the surface of the antibody molecule are modified are present in the ion concentration-dependent binding domain, then the amino acids of the antigen-binding domain can be modified so that the modification does not (significantly) reduce the binding activity of amino acid residues that can alter the antigen-binding activity of the antibody in depending on the conditions of ion concentration (for example, residues in the calcium-binding motif or in the site of insertion of histidine and / or site with substituted histidine), or amino acid residues can be modified at sites other than sites of amino acid residues, which can alter the antigen-binding activity of the antibody, depending on on the conditions of ion concentration. On the other hand, if the amino acid residues that can be exposed on the surface of the antibody molecule, present in the ion concentration-dependent binding domain, have already been modified, then the type or position of amino acid residues that can change the antigen-binding activity of the antibody depending on the ion concentration conditions can be chosen so that the pI of the antibody does not fall below an acceptable level. If the pI of the antibody falls below an acceptable level, then the pI of the entire antibody can be reduced by modifying at least one amino acid residue that can be exposed on the surface of the antibody molecule.

High pI FR sequences can preferably be selected from, but not limited to, human germline FR sequences or region sequences that are equivalent to them, the amino acids of which can be modified in some cases.

If the antibodies referred to in section A of the description have a constant region (which may be modified) comprising an FcγR binding domain (which may be a domain that binds to any of the isoforms or any of the FcγR allotypes listed below) and / or an FcRn binding domain, the sites for modifying at least one amino acid residue that can be displayed on the surface of the constant region, if necessary, can be amino acid residues other than residues in the FcγR binding domain and / or in the FcRn binding domain. Alternatively, if the sites of modification are selected from amino acid residues in the FcγR binding domain and / or in the FcRn binding domain, it may be preferable to select sites that do not (significantly) affect the binding activity or binding affinity of FcγR and / or FcRn, or, if they can, then the sites are biologically or pharmacologically acceptable.

In one embodiment of the invention, the site of at least one amino acid residue that is modified to obtain the antibody specified in section A of the description, in which the pI is increased by modifying at least one amino acid residue that can be exposed on the surface of the variable region (which can be modified), not limited to a specific site; however, said site may be selected from the group consisting, according to Cabot numbering, of: (a) positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 18, 19, 23, 25, 26, 39, 41, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 77, 81, 82, 82a, 82b, 83, 84, 85, 86, 105, 108, 110 and 112 in the FR of the severe variable region chains; (b) positions 31, 61, 62, 63, 64, 65 and 97 in a CDR of the variable region of the heavy chain; (c) positions 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 37, 38, 39, 41, 42, 43, 45, 46, 49, 57, 60, 63, 65, 66, 68, 69, 70, 74, 76, 77, 79, 80, 81, 85, 100, 103, 105, 106, 107 and 108 in the FR of the light chain variable region; and (d) positions 24, 25, 26, 27, 52, 53, 54, 55 and 56 in the CDR of the light chain variable region, where the amino acid at each position after modification can be selected from any of the amino acids described above in terms of side chain charge such as (but not limited to) Lys (K), Arg (R), Gln (Q), Gly (G), Ser (S) or Asn (N). In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 of the above amino acid positions are modified. In some embodiments, 1-20, 1-15, 1-10, or 1-5 of the above amino acid positions are modified.

In one embodiment of the invention, from the position (s) to be modified, the next position (s) can be used to increase the pI of the antibody specified in section A in combination with other position (s), which itself (s) can (gut) have a significant effect on increasing the pI of the antibody. The specified position (s), contributing (s) to increase pI, can be selected, for example, for the variable region of the light chain from the group consisting of positions 27, 52, 56, 65 and 69 according to Cabot numbering.

In addition, there is no limitation on the site of at least one amino acid residue that is modified in the CDR and / or FR; however, such a site can be selected from the group consisting of: (a) positions 8, 10, 12, 13, 15, 16, 18, 23, 39, 41, 43, 44, 77, 82, 82a, 82b, 83, 84 , 85 and 105 in the FR of the heavy chain variable region; (b) positions 31, 61, 62, 63, 64, 65 and 97 in the CDR of the variable region of the heavy chain; (c) positions 16, 18, 37, 41, 42, 45, 65, 69, 74, 76, 77, 79 and 107 in the FR of the light chain variable region; and (d) positions 24, 25, 26, 27, 52, 53, 54, 55 and 56 in the CDR of the light chain variable region. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 of the above amino acid positions are modified. In some embodiments, 1-20, 1-15, 1-10, or 1-5 of the above amino acid positions are modified.

If the site of modification of at least one amino acid residue is selected, for example, from the group containing the above groups, then the type of amino acid after modification in the variable region of the heavy chain is, for example:

(a) 8K, 8R, 8Q, 8G, 8S, or 8N for position 8; (b) 13K, 13R, 13Q, 13G, 13S, or 13N for position 13; (c) 15K, 15R, 15Q, 15G, 15S, or 15N for position 15; (d) 16K, 16R, 16Q, 16G, 16S, or 16N for position 16; (e) 18K, 18R, 18Q, 18G, 18S, or 18N for position 18; (f) 39K, 39R, 39Q, 39G, 39S or 39N for position 39; (g) 41K, 41R, 41Q, 41G, 41S, or 41N for position 41; (h) 43K, 43R, 43Q, 43G, 43S, or 43N for position 43; (i) 44K, 44R, 44Q, 44G, 44S or 44N for position 44; (k) 63K, 63R, 63Q, 63G, 63S or 63N for position 63; (l) 64K, 64R, 64Q, 64G, 64S or 64N for position 64; (m) 77K, 77R, 77Q, 77G, 77S or 77N for position 77; (n) 82K, 82R, 82Q, 82G, 82S or 82N for position 82; (o) 82aK, 82aR, 82aQ, 82aG, 82aS, or 82aN for position 82a; (n) 82bK, 82bR, 82bQ, 82bG, 82bS or 82bN for position 82b; (p) 83K, 83R, 83Q, 83G, 83S, or 83N for position 83; (c) 84K, 84R, 84Q, 84G, 84S, or 84N for position 84; (t) 85K, 85R, 85Q, 85G, 85S or 85N for position 85; or (y) 105K, 105R, 105Q, 105G, 105S, or 105N for position 105. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 of the above amino acids are modified provisions. In some embodiments, 1-20, 1-15, 1-10, or 1-5 of the above amino acid positions are modified.

Examples of combinations of modified amino acid positions in the variable region of the heavy chain include, but are not limited to, for example:

any two or more positions selected from the group consisting of positions 16, 43, 64 and 105; any two or more positions selected from the group consisting of positions 77, 82a and 82b; provisions 77 and 85; provisions 41 and 44; positions 82a and 82b; positions 82 and 82b; positions 82b and 83; or positions 63 and 64 according to Cabot numbering, where the amino acid at each position after modification can be selected in terms of side chain charge from any of the amino acids described above, such as Lys (K), Arg (R), Gln (Q), Gly ( G), Ser (S), or Asn (N) (but not limited to).

A specific combination may be, for example, 16Q / 43R / 64K / 105Q; 77R / 82aN / 82bR; 77R / 82aG / 82bR; 77R / 82aS / 82bR; 77R / 85G; 41R / 44R; 82aN / 82bR; 82aG / 82bR; 82aS / 82bR; 82K / 82bR; 82bR / 83R; 77R / 85R; or 63R / 64K.

Similarly, the amino acid type after modification in the light chain variable region is, for example: (a) 16K, 16R, 16Q, 16G, 16S, or 16N for position 16; (b) 18K, 18R, 18Q, 18G, 18S, or 18N for position 18; (c) 24K, 24R, 24Q, 24G, 24S, or 24N for position 24; (d) 25K, 25R, 25Q, 25G, 25S, or 25N for position 25; (e) 26K, 26R, 26Q, 26G, 26S, or 26N for position 26; (f) 27K, 27R, 27Q, 27G, 27S, or 27N for position 27; (g) 37K, 37R, 37Q, 37G, 37S, or 37N for position 37; (h) 41K, 41R, 41Q, 41G, 41S, or 41N for position 41; (i) 42K, 42R, 42Q, 42G, 42S or 42N for position 42; (k) 45K, 45R, 45Q, 45G, 45S or 45N for position 45; (l) 52K, 52R, 52Q, 52G, 52S or 52N for position 52; (m) 53K, 53R, 53Q, 53G, 53S or 53N for position 53; (n) 54K, 54R, 54Q, 54G, 54S or 54N for position 54; (o) 55K, 55R, 55Q, 55G, 55S, or 55N for position 55; (n) 56K, 56R, 56Q, 56G, 56S, or 56N for position 56; (p) 65K, 65R, 65Q, 65G, 65S or 65N for position 65; (c) 69K, 69R, 69Q, 69G, 69S, or 69N for position 69; (t) 74K, 74R, 74Q, 74G, 74S or 74N for position 74; (y) 76K, 76R, 76Q, 76G, 76S, or 76N for position 76; (f) 77K, 77R, 77Q, 77G, 77S or 77N for position 77; (x) 79K, 79R, 79Q, 79G, 79S, or 79N for position 79; and (c) 107K, 107R, 107Q, 107G, 107S, or 107N for position 107. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 of the above amino acids are modified provisions. In some embodiments, 1-20, 1-15, 1-10, or 1-5 of the above amino acid positions are modified.

Examples of combinations of modified amino acid positions in the variable region of the light chain include, but are not limited to, for example: positions 24 and 27; provisions 25 and 26; provisions 41 and 42; provisions 42 and 76; provisions 52 and 56; provisions 65 and 79; provisions 74 and 77; provisions 76 and 79; any two or more positions selected from the group consisting of positions 16, 24 and 27; any two or more positions selected from the group consisting of positions 24, 27 and 37; any two or more positions selected from the group consisting of positions 25, 26 and 37; any two or more positions selected from the group consisting of positions 27, 76 and 79; any two or more positions selected from the group consisting of positions 41, 74 and 77; any two or more positions selected from the group consisting of positions 41, 76 and 79; any two or more positions selected from the group consisting of positions 24, 27, 41 and 42; any two or more positions selected from the group consisting of positions 24, 27, 52 and 56; any two or more positions selected from the group consisting of positions 24, 27, 6 and 69; any two or more positions selected from the group consisting of positions 24, 27, 74 and 77; any two or more positions selected from the group consisting of positions 24, 27, 76 and 79; any two or more positions selected from the group consisting of positions 25, 26, 52 and 56; any two or more positions selected from the group consisting of positions 25, 26, 65 and 69; any two or more positions selected from the group consisting of positions 25, 26, 76 and 79; any two or more positions selected from the group consisting of positions 27, 41, 74 and 77; any two or more positions selected from the group consisting of positions 27, 41, 76 and 79; any two or more positions selected from the group consisting of positions 52, 56, 74 and 77; any two or more positions selected from the group consisting of positions 52, 56, 76 and 79; any two or more positions selected from the group consisting of positions 65, 69, 76 and 79; any two or more positions selected from the group consisting of positions 65, 69, 74 and 77; any two or more positions selected from the group consisting of positions 18, 24, 45, 79 and 107; any two or more positions selected from the group consisting of positions 27, 52, 56, 74 and 77; any two or more positions selected from the group consisting of positions 27, 52, 56, 76 and 79; any two or more positions selected from the group consisting of positions 27, 65, 69, 74 and 77; any two or more positions selected from the group consisting of positions 27, 65, 69, 76 and 79; any two or more positions selected from the group consisting of positions 41, 52, 56, 74 and 77; any two or more positions selected from the group consisting of positions 41, 52, 56, 76 and 79; any two or more positions selected from the group consisting of positions 41, 65, 69, 74 and 77; any two or more positions selected from the group consisting of positions 41, 65, 69, 76 and 79; any two or more positions selected from the group consisting of positions 24, 27, 41, 42, 65 and 69; any two or more positions selected from the group consisting of positions 24, 27, 52, 56, 65 and 69; any two or more positions selected from the group consisting of positions 24, 27, 65, 69, 74 and 77; any two or more positions selected from the group consisting of positions 24, 27, 65, 69, 76 and 79; any two or more positions selected from the group consisting of positions 24, 27, 41, 42, 74 and 77; any two or more positions selected from the group consisting of positions 24, 27, 52, 56, 74 and 77; any two or more positions selected from the group consisting of positions 24, 27, 41, 42, 76 and 79; any two or more positions selected from the group consisting of positions 24, 27, 52, 56, 76 and 79; any two or more positions selected from the group consisting of positions 24, 27, 74, 76, 77 and 79; any two or more positions selected from the group consisting of positions 52, 56, 65, 69, 74 and 77; any two or more positions selected from the group consisting of positions 52, 56, 65, 69, 76, and 79 according to Cabot numbering, where the amino acid at each position after modification can be selected from any of the amino acids described above in terms of side chain charge such as (but not limited to) Lys (K), Arg (R), Gln (Q), Gly (G), Ser (S) or Asn (N).

A specific combination may be, for example, 24R / 27Q; 24R / 27R; 24K / 27K; 25R / 26R; 25K / 26K; 41R / 42K; 42K / 76R; 52R / 56R; 65R / 79K; 74K / 77R; 76R / 79K; 16K / 24R / 27R; 24R / 27R / 37R; 25R / 26R / 37R; 27R / 76R / 79K; 41R / 74K / 77R; 41R / 76R / 79K; 24R / 27R / 41R / 42K; 24R / 27R / 52R / 56R; 24R / 27R / 52K / 56K; 24R / 27R / 65R / 69R; 24R / 27R / 74K / 77R; 24R / 27R / 76R / 79K; 25R / 26R / 52R / 56R; 25R / 26R / 52K / 56K; 25R / 26R / 65R / 69R; 25R / 26R / 76R / 79K; 27R / 41R / 74K / 77R; 27R / 41R / 76R / 79K; 52R / 56R / 74K / 77R; 52R / 56R / 76R / 79K; 65R / 69R / 76R / 79K; 65R / 69R / 74K / 77R; 18R / 24R / 45K / 79Q / 107K; 27R / 52R / 56R / 74K / 77R; 27R / 52R / 56R / 76R / 79K; 27R / 65R / 69R / 74K / 77R; 27R / 65R / 69R / 76R / 79K; 41R / 52R / 56R / 74K / 77R; 41R / 52R / 56R / 76R / 79K; 41R / 65R / 69R / 74K / 77R; 41R / 65R / 69R / 76R / 79K; 24R / 27R / 41R / 42K / 65R / 69R; 24R / 27R / 52R / 56R / 65R / 69R; 24R / 27R / 65R / 69R / 74K / 77R; 24R / 27R / 65R / 69R / 76R / 79K; 24R / 27R / 41R / 42K / 74K / 77R; 24R / 27R / 52R / 56R / 74K / 77R; 24R / 27R / 41R / 42K / 76R / 79K; 24R / 27R / 52R / 56R / 76R / 79K; 24R / 27R / 74K / 76R / 77R / 79K; 52R / 56R / 65R / 69R / 74K / 77R; or 52R / 56R / 65R / 69R / 76R / 79K.

WO 2007/114319 or WO 2009/041643 has already explained or demonstrated on the basis of theoretical data, homology modeling or experimental methods that the effect of increasing pI as a result of modification of certain amino acid residues in the variable region depends not only (or largely) on the amino acid sequences that make up the antibody, or the type of target antigen, but rather depends on the type and number of amino acid residues replaced. It has been demonstrated that even after modification of certain amino acids, antigen binding activity against certain types of antigens is (to a large extent) maintained or, more likely to be expected by those skilled in the art, at least should be maintained.

For example, WO 2009/041643 specifically demonstrates that in the FR of the heavy chain of a humanized anti-glypican 3 antibody having SEQ ID NO: 8, preferred amino acid residue modification sites that can be exposed on the surface are positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 19, 23, 25, 26, 39, 42, 43, 44, 46, 69, 72, 73, 74, 76, 77, 82, 85, 87, 89, 90, 107, 110, 112 and 114 according to Cabot's numbering. It is also described that the amino acid residue at position 97 according to Cabot's numbering is preferred because it is exposed on the surface of almost all antibodies. WO 2009/041643 also demonstrates that amino acid residues at positions 52, 54, 62, 63, 65 and 66 in the antibody heavy chain CDRs are preferred. It has also been demonstrated that amino acid residues at positions 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 43, 44, 45, 46, 48, 49, 50, 54 are preferred. 62, 65, 68, 70, 71, 73, 74, 75, 79, 81, 82, 84, 85, 86, 90, 105, 108, 110, 111 and 112 according to Cabot numbering in FR of the light chain of the humanized anti-glypican antibody 3 having SEQ ID NO: 9. Amino acid residues at positions 24, 27, 33, 55, 59 in the light chain CDR of said antibody have also been demonstrated to be preferred. In addition, WO 2009/041643 specifically states that the amino acid residues at positions 31, 64 and 65 according to Cabot numbering in the heavy chain CDRs of the anti-human IL-6 receptor antibody having SEQ ID NO: 10 are preferred sites for modification amino acid residues that can be exposed on the surface, while maintaining antigen-binding activity. In addition, it is demonstrated that amino acid residues at positions 24, 27, 53 and 55 according to Cabot numbering in the light chain CDR of the anti-human IL-6 receptor antibody having SEQ ID NO: 11 are preferred. In addition, it has been specifically found that the amino acid residue at position 31 according to Cabot numbering in the heavy chain CDRs of an anti-human IL-6 receptor antibody having SEQ ID NO: 12 is a preferred site for modifying an amino acid residue that can be exposed on a surface, with while maintaining antigen-binding activity. It was also found that amino acid residues at positions 24, 53, 54 and 55 according to Cabot numbering in the light chain CDRs of the anti-human IL-6 receptor antibody having SEQ ID NO: 13 are preferred. WO 2009/041643 also discloses that the amino acid residues at positions 61, 62, 64 and 65 according to Cabot numbering in the heavy chain CDRs of an anti-human glypican 3 antibody having SEQ ID NO: 14 are preferred sites for modification of amino acid residues that can exposed on the surface, while maintaining antigen-binding activity. It has also been found that amino acid residues at positions 24 and 27 according to Cabot numbering in the light chain CDRs of an anti-human glypican 3 antibody having SEQ ID NO: 15 are preferred. It has also been demonstrated that amino acid residues at positions 61, 62, 64 and 65 according to Cabot numbering in the heavy chain CDRs of an anti-human IL-31 receptor antibody having SEQ ID NO: 16 are preferred sites for modification of amino acid residues that can be displayed on surface, while maintaining antigen-binding activity. WO 2009/041643 also discloses that amino acid residues at positions 24 and 54 according to Cabot numbering in the light chain CDRs of an anti-human IL-31 receptor antibody having SEQ ID NO: 17 are preferred. Similarly, WO 2007/114319 discloses that antibodies hA69-PF, hA69-p18, hA69-N97R, hB26-F123e4, hB26-p15 and hB26-PF, which are obtained by modifying the charge of one or more amino acid residues that can be exposed on the surface, were characterized by changes in pI, as demonstrated by isoelectric focusing, and had equivalent binding activity against their factor IXa or factor X antigens, compared to antibodies before modification or change. It is also described that when these antibodies were administered to mice, a high level of correlation of the pI of each antibody with their plasma clearance (CL), plasma retention time, and plasma half-life (T1 / 2) was found. WO 2007/114319 also demonstrates that amino acid residues at positions 10, 12, 23, 39, 43, 97 and 105 in the variable region are preferred as amino acid residue modification sites that can be exposed on the surface.

In an alternative or additional embodiment of the invention, for example, using X-ray crystallography techniques or homology models constructed by homologous modeling from an antibody constant region (preferably a human constant region, more preferably a human Ig-type antibody constant region and even more preferably a human antibody constant region IgG-type, but not limited to), you can identify amino acid residues that can be displayed on the surface of the constant region of the antibody, to determine the sites of modification of at least one amino acid residue in order to obtain the antibody specified in section A of the description, with an increased pI. The site of modification of at least one amino acid residue that can be displayed on the surface of the constant region is not limited to a particular site; however, the site can preferably be selected from the group consisting of: positions 196, 253, 254, 256, 257, 258, 278, 280, 281, 282, 285, 286, 306, 307, 308, 309, 311, 315, 327 , 330, 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 388, 389, 399, 400, 401, 402, 413, 415, 418, 419 , 421, 424, 430, 433, 434 and positions 443, according to EU numbering and can preferably be selected from the group consisting of: positions 254, 258, 281, 282, 285, 309, 311, 315, 327, 330, 342 , 343, 345, 356, 358, 359, 361, 362, 384, 385, 386, 387, 389, 399, 400, 401, 402, 413, 418, 419, 421, 433, 434 and 443 and preferably you can also be selected from the group consisting of: positions 282, 309, 311, 315, 342, 343, 384, 399, 401, 402 and 413, amino acids at each position after modification can be selected from amino acids described above in terms of side chain charge such such as (but not limited to) Lys (K), Arg (R), Gln (Q), or Asn (N). When the site of modification of at least one amino acid residue is selected, for example, from the group that contains the groups described above, for example, the type of amino acids after modification at each site may be as follows:

254K, 254R, 254Q or 254N at position 254; 258K, 258R, 258Q or 258N at position 258;

281K, 281R, 281Q or 281N at position 281; 282K, 282R, 282Q or 282N at position 282;

285K, 285R, 285Q or 285N at position 285; 309K, 309R, 309Q or 309N at position 309;

311K, 311R, 311Q, or 311N at position 311; 315K, 315R, 315Q, or 315N at position 315;

327K, 327R, 327Q or 327N at position 327; 330K, 330R, 330Q or 330N at position 330;

342K, 342R, 342Q, or 342N at position 342; 343K, 343R, 343Q or 343N at position 311;

345K, 345R, 345Q, or 345N at position 345; 356K, 356R, 356Q or 356N at position 356;

358K, 358R, 358Q or 358N at position 358; 359K, 359R, 359Q or 359N at position 359;

361K, 361R, 361Q or 361N at position 361; 362K, 362R, 362Q or 362N at position 362;

384K, 384R, 384Q or 384N at position 384; 385K, 385R, 385Q or 385N at position 385;

386K, 386R, 386Q or 386N at position 386; 387K, 387R, 387Q or 387N at position 387;

389K, 389R, 389Q or 389N at position 389; 399K, 399R, 399Q or 399N at position 399;

400K, 400R, 400Q or 400N at position 400; 401K, 401R, 401Q or 401N at position 401;

402K, 402R, 402Q or 402N at position 402; 413K, 413R, 413Q, or 413N at position 413;

418K, 418R, 418Q, or 418N at position 418; 419K, 419R, 419Q or 419N at position 419;

421K, 421R, 421Q, or 421N at position 421; 433K, 433R, 433Q, or 433N at position 433;

434K, 434R, 434Q, or 434N at position 434; and 443K, 443R, 443Q or 443N at position 443.

In an alternative embodiment, the site of modification of at least one amino acid residue and the type of amino acid after modification may include 345R or 345K and / or 430R, 430K, 430G or 435T according to EU numbering.

In one of the variants of the antibodies specified in section A of the description, the net pI value of the antibody can be increased by modifying at least one amino acid residue that can be displayed on the surface of the variable region (which can be modified), as described above, and at least one an amino acid residue that can be exposed on the surface of the constant region (which can be modified) as described above.

In the context of sections A and B of the description, if the antibody referred to in section A or B of the description is an IgG-type antibody or a molecule derived from it, then the constant region of the heavy chain of the antibody may contain an IgG1-type, IgG2-type, IgG3 constant region -type or IgG4-type. In section A or B of the description, the constant region of the heavy chain may be, but is not limited to, the constant region of the human heavy chain. Several human IgG allotypes are known. In particular, it has been described that there are some differences in the amino acid sequence of the constant region of human IgG between individuals (Methods Mol. Biol. 882, 2012, pp. 635-680; Sequences of proteins of immunological interest, NIH publication No. 91-3242). Examples include human IgG1 constant region (SEQ ID NO: 18), human IgG2 constant region (SEQ ID NO: 19), human IgG3 constant region (SEQ ID NO: 20), and human IgG4 constant region (SEQ ID NO: 21).

Among them, for example, for human IgG1, allotypes are known, designated as G1lm1,17 and G1m3. The allotypes differ in their amino acid sequences: G1m1,17 has aspartic acid at position 356 and leucine at position 358 according to EU numbering, and G1m3 has glutamic acid at position 356 and methionine at position 358 according to EU numbering. However, there are no data to suggest the presence of significant differences in the important functions and properties of antibodies of the indicated allotypes. Thus, it should be apparent to one of ordinary skill in the art that various assays can be performed using specific allotypes and the results are not limited to the allotypes used in the examples and the same actions are expected for any of the allotypes. In the context of sections A and B of the description of the invention, when referring to "human IgG1", "human IgG2", "human IgG3" or "human IgG4", allotypes are not limited to specific allotypes and may include all of these allotypes.

In an alternative or additional embodiment of the invention described in section A or B of the description, the constant region of the light chain of an antibody may include any constant region of the κ chain (IgK) type or λ chain type (IgL1, IgL2, IgL3, IgL6 or IgL7). The constant region of the light chain may preferably be, but is not limited to, the constant region of a human light chain. There are data such as those described in Sequences of proteins of immunological interest)), NIH Publication No. 91-3242, on several allotypic sequences associated with genetic polymorphisms of the human κ chain constant region and the human λ chain constant region. Such allotypes include, for example, human κ chain constant region (SEQ ID NO: 22) and human λ chain constant region (SEQ ID NO: 23). However, there are no data to suggest the presence of significant differences in the important functions and properties of antibodies of the indicated allotypes. Thus, it should be obvious to one of ordinary skill in the art that when referring to specific allotypes in the context of sections A and B of this description, the same actions are expected for any of the allotypes (hereinafter, in the context of this description, generally referred to as the constant region of native (human ) IgG (type)).

In addition, since the Fc region of a native IgG-type antibody is part of the constant region of a native IgG-type antibody, when the antibodies specified in section A or B of the description are, for example, IgG-type antibodies or molecules derived therefrom, then the antibodies may have an Fc region within the constant region of native IgG (IgG1-, IgG2-, IgG3- or IgG4-type) (hereinafter referred to as the Fc region of native (human) IgG (type)) ... The Fc region of native IgG may mean an Fc region composed of the same amino acid sequence as an Fc region derived from naturally occurring IgG. Specific examples of a native human IgG parental Fc region may include human IgG1 constant region Fc regions (SEQ ID NO: 18), human IgG2 constant region (SEQ ID NO: 19), human IgG3 constant region (SEQ ID NO: 20) and the constant region of human IgG4 (SEQ ID NO: 21) as described above (the Fc region of the IgG class can mean, for example, the region extending from cysteine at position 226 according to EU numbering to the C-terminus or from proline to position 230 according to EU-numbering to C-end.).

In one embodiment, the antibodies described in sections A and B of the description may include variants in which one or more modifications are made, selected from amino acid substitution, addition, deletion or insertion, in the constant region of a native (preferably human) IgG (heavy chain constant region and / or light chain constant region) or in the Fc region of native (preferably human) IgG.

The scope of section A of the description of the invention includes the data published in WO 2013/081143 that, for example, concentration-dependent antibodies that have the ability to form multivalent immune complexes with a multimeric antigen (multivalent antigen-antibody complexes), and multispecific, depending on concentration of antibody ions, or multiparatopic, depending on the concentration of antibody ions, which can form multivalent immune complexes (multivalent antigen-antibody complexes), by recognizing two or more epitopes on monomeric antigens, they can bind more strongly to FcγR, FcRn, complement receptor due to avidity ( total binding strength between several epitopes and several paratopes) through at least two or more multivalent constant regions (which can be modified) or Fc regions (which can be modified) included in the antibody molecule, and as a result That is, these antibodies can penetrate cells faster. Thus, modified in order to increase the pI by modifying at least one amino acid residue that can be displayed on the surface of the antibody, the concentration-dependent antibodies described above, which have the ability to form multivalent immune complexes with a multimeric antigen or monomeric antigens, can also be used as antibodies shown in section A of the description (depending on the concentration of antibodies with increased pI). It will be apparent to those of ordinary skill in the art that concentration-dependent antibodies with an increased pI, which can form multivalent immune complexes with a multimeric antigen or monomeric antigens, can penetrate cells more rapidly than ion concentration-dependent antibodies with an increased pI, which do not have the ability to form multivalent immune complexes. Those of ordinary skill in the art can also understand that, in one embodiment, the binding activity of the antibodies described in section A of the description to FcRn and / or FcγR may increase under neutral pH conditions, in which case the concentration-dependent antibodies with increased pI, which can form multivalent immune complexes with multimeric antigen or monomeric antigens, can penetrate cells even faster.

In one embodiment, the antibodies referred to in section A of the description may be single-arm antibodies (including all single-arm antibody variants as described in WO 2005/063816). Typically, single-arm antibodies are antibodies lacking one of the two Fab regions of the canonical IgG-type antibody and can be produced, for example, by, but not limited to, methods described in WO 2005/063816. In an IgG-type antibody (but not limited to), which has, for example, the structure of the heavy chain VH-CH1-hinge-CH2-CH3, when one of the Fab regions is cleaved at a site closer to the N-terminus than to hinge (for example, VH or CH1), then the antibody must be expressed in a form containing an additional sequence, and when one of the Fab regions is cleaved at a site closer to the C-terminus than the hinge (for example, CH2), then Fc - the area must be incomplete. Thus (but not limited to), from the standpoint of the stability of the antibody molecule, it is preferable to obtain antibodies with one arm by cleavage in the hinge region (hinge) of one of the two Fab regions of the IgG-type antibody. More preferably, the heavy chain after cleavage should be linked to the uncleaved heavy chain via an intramolecular disulfide bond. WO 2005/063816 discloses that such single-arm antibodies have increased stability compared to Fab molecules. Antibodies with increased or decreased pI can also be generated by generating said single-arm antibodies. In addition, when the ion concentration-dependent antigen-binding domain is introduced into antibodies with an increased pI, which are antibodies with one arm, the half-life of the antibody in plasma can be further shortened, the cellular uptake of the antibody can be further increased, the elimination of antigen from the plasma can be further enhanced or the affinity antibodies to the extracellular matrix can be further increased compared to antibodies with an increased pI that do not have an ion concentration-dependent antigen-binding domain.

Without wishing to be bound by any particular theory, in an embodiment in which the cell penetration enhancing effect of single-arm antibodies is expected, one may consider, but not limited to, the case in which the pI of soluble antigen is lower than that of antibodies. The net pI of a complex of antibodies and antigens can be calculated using conventional methods assuming that the complex is an individual molecule. In this case, at a lower pI of soluble antigen, there is a lower net pI of the complex; and a higher pI of soluble antigen has a higher net pI of the complex. When a canonical IgG-type antibody molecule (having two Fabs) is bound to one low pI soluble antigen, compared to binding to two low pI soluble antigens, in the latter case, the net pI of the complex will be lower. When said canonical antibody is converted to a single-arm antibody, only one antigen can bind to one antibody molecule; thus, it is possible to suppress the decrease in the pI complex as a result of binding to the second antigen. In other words, it is assumed that when the pI of the soluble antigen is lower than that of the antibody, then the conversion to a single-arm antibody allows the pI of the complex to be increased compared to the canonical antibody and thereby accelerate cell penetration.

In addition (but not limited to), when the Fab of a canonical IgG-type antibody molecule (having two Fabs) has a lower pI than Fc, then conversion to a single-arm antibody increases the net pI of a complex consisting of a single-arm antibody and antigen. In addition, when said conversion to a single-arm antibody is carried out, it is preferable from the standpoint of stability of the single-arm antibody to cleave one of the Fabs in the hinge region located at the junction between the Fab and the Fc. In this case, it can be expected that the pI can be effectively increased by choosing a site that should increase the pI of a single-arm antibody to the required degree.

Thus, it should be apparent to those of ordinary skill in the art that regardless of (or substantially regardless) of the amino acid sequence of the antibody itself and the type of soluble antigen, the pI of the antibody can be increased and, as a consequence, uptake of the antigen by cells can be accelerated by converting the antibody into an antibody with one arm based on calculating the theoretical pI of an antibody (theoretical pI Fc and theoretical pI Fab), theoretical pI of soluble antigen, and predicting the effect of differences on their theoretical pI.

In one embodiment, the antibodies described in section A or B of the description may be multispecific antibodies, and the multispecific antibody may be (but are not limited to) a bispecific antibody. The multispecific antibody can be a multispecific antibody that contains a first polypeptide and a second polypeptide. As used herein, a "multispecific antibody that comprises a first polypeptide and a second polypeptide" means an antibody that binds to at least two or more types of different antigens, or to at least two or more epitopes in the same antigen. The first polypeptide and the second polypeptides may preferably comprise a heavy chain variable region, and more preferably the variable region comprises CDRs and / or FRs. In another embodiment, the first polypeptide and the second polypeptide may preferably each contain a heavy chain constant region. In a further embodiment, the multispecific antibody can comprise a third polypeptide and a fourth polypeptide, each of which comprises a light chain variable region and preferably also a light chain constant region. In this case, the first to fourth polypeptides can be combined to produce a multispecific antibody.

In one embodiment of the invention, in which the antibodies specified in section A of the description are multispecific antibodies and the multispecific antibodies contain a heavy chain constant region, the following sequences can be used to lower their pI, for example, the following sequences can be used: an IgG2 or IgG4 sequence at position 137 ; an IgG1, IgG2 or IgG4 sequence at position 196; an IgG2 or IgG4 sequence at position 203; the IgG2 sequence at position 214; an IgG1, IgG3 or IgG4 sequence at position 217; an IgG1, IgG3 or IgG4 sequence at position 233; an IgG4 sequence at position 268; an IgG2, IgG3 or IgG4 sequence at position 274; an IgG1, IgG2 or IgG4 sequence at position 276; an IgG4 sequence at position 355; the IgG3 sequence at position 392; an IgG4 sequence at position 419; or an IgG1, IgG2 or IgG4 sequence at position 435. In this case, to increase their pI, for example, the following sequences can be used: an IgG1 or IgG3 sequence at position 137; the IgG3 sequence at position 196; an IgG1 or IgG3 sequence at position 203; an IgG1, IgG3 or IgG4 sequence at position 214; the IgG2 sequence at position 217; the IgG2 sequence at position 233; an IgG1, IgG2 or IgG3 sequence at position 268; an IgG1 sequence at position 274; the IgG3 sequence at position 276; an IgG1, IgG2 or IgG3 sequence at position 355; an IgG1, IgG2 or IgG4 sequence at position 392; an IgG1, IgG2 or IgG3 sequence at position 419; or an IgG3 sequence at position 435.

In one embodiment, in which the antibodies described in section A of the description contain two heavy chain constant regions, the pI values of the two heavy chain constant regions may be the same or different from each other. These heavy chain constant regions may be IgG1, IgG2, IgG3 and IgG4 heavy chain constant regions, which initially have different pIs. Alternatively, differences in pI between the two constant regions of the heavy chain can be introduced. Modification of the sites of at least one amino acid residue for the introduction of the specified difference in pI into the constant region can affect the position (s) indicated (s) above or position (s) selected (s), for example from the group consisting of position 137, position 196, position 203, position 214, position 217, position 233, position 268, position 274, position 276, position 297, position 355, position 392, position 419 and position 435, according to the EU numbering of the heavy chain constant region as described in WO 2009/041643. Alternatively, the amino acid residue at position 297, which is the glycosylation site, can be modified to remove the sugar chain, since removal of the sugar chain from the constant region of the heavy chain changes the pI.

In one embodiment, the antibodies described in section A or B of the description may be polyclonal antibodies or monoclonal antibodies, and mammalian derived monoclonal antibodies are preferred. Monoclonal antibodies include those produced by hybridomas or derived from host cells genetically engineered with expression vectors that carry antibody genes. The antibodies referred to in section A or B of the description may be, for example, antibodies such as chimeric antibodies, humanized antibodies, or affinity matured antibodies, or molecules derived therefrom.

In one embodiment, the antibodies described in section A or B of the description can be obtained (but not limited to) from any species of animals (for example, humans; or animals other than humans, such as mice, rats, hamsters, rabbits, monkeys , cynomolgus monkeys, rhesus monkeys, gamabrils, baboons, chimpanzees, goats, sheep, dogs, cows or camels) or any birds; and the antibodies are preferably derived from humans, monkeys, or mice.

In one embodiment, the antibodies referred to in section A or B of the description may be of the Ig type, and preferably may be of the IgG type.

In the context of sections A and B of the present description, Fc receptor (which is also referred to as "FcR") means a receptor protein that can bind to the Fc region of an immunoglobulin (antibody) or derived from it molecule, or a variant Fc region. For example, Fc receptors for IgG, IgA, IgE, and IgM are referred to as FcγR, FcαR, FcεR, and FcμR, respectively, in the context of Section A of the present disclosure. Fc receptors can be, for example, FcRn (which is also referred to as "neonatal Fc receptor) in the context of sections A and B of this description.

In the context of section A of the present description, "FcγR" can mean a receptor protein that can bind to the Fc region of an antibody in the form of IgG1, IgG2, IgG3 or IgG4 or a molecule derived therefrom, or a variant of the Fc region, and can include any one or more or all members of the family of proteins encoded primarily by the FcγR gene. In humans, this family includes (but is not limited to) Fcγ RI (CD64), including isoforms Fcγ RIa, FcγRIb, and Fcγ RIc; FcγRII (CD32), including FcγRIIa isoforms (including allotypes H131 (type H) and R131 (type R)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) and FcγRIIc; and FcγRIII (CD16), including the FcγRIIIa isoforms (including allotypes V158 and F158) and FcγRIIIb (including the FcγRIIIb-NA1 and FcγRIIIb-NA2 allotypes), and any human FcγR isoforms or FcγR allotypes that have not yet been discovered. In addition, FcγRIIb1 and FcγRIIb2 are described as splicing variants of human FcγRIIb (hFcγRIIb). In addition, a splicing variant designated FcγRIIb3 has been described (J Exp Med, 170, 1989, cc. 1369-1385). In addition to these splicing variants, human FcγRIIb includes all splicing variants registered in the NCBI, which are NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1 and NP_003992.3. In addition, hFcγRIIb includes each of the previously described gene polymorphisms as well as FcγRIIb (Li et al., Arthritis Rheum. 48, 2003, cc. 3242-3252; Kono et al., Hum. Mol. Genet. 14, 2005, cc. 2881-2892 and Kyogoju et al., Arthritis Rheum. 46, 2002, cc. 1242-1254) and each gene polymorphism that will be described in the future.

FcγR can be obtained from any organism and may include FcγR derived from, but not limited to, humans, mice, rats, rabbits, or monkeys. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), and any as yet undiscovered mouse FcγR and FcγR isoforms or allotypes. Said preferred FcγRs include, for example, human FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16), or FcγRIIIB (CD16). Since FcγR is present in the form of a membrane form in vivo, it can be used in experimental systems after being artificially converted into the corresponding soluble form.

For example, as described in WO 2014/163101, the polynucleotide sequence and amino acid sequence of FcγRI can be those shown in NM_000566.3 and NP_000557.1, respectively; the polynucleotide sequence and amino acid sequence of FcγRIIA can be those shown in BC020823.1 and AAN20823.1, respectively; the polynucleotide sequence and amino acid sequence of FcγRIIB may be those shown in BC146678.1 and AAI46679.1, respectively; the polynucleotide sequence and amino acid sequence of FcγRIIIA may be those shown in BC033678.1 and AAN33678.1, respectively; the polynucleotide sequence and amino acid sequence of FcγRIIIB can be those shown in BC128562.1 and AAI28563.1, respectively (shown in RefSeq access codes).

FcγRIIa has two gene polymorphisms in which the amino acid at position 131 of FcγRIIa is replaced with histidine (type H) or arginine (type R) (J. Exp. Med. 172, 1990, pp. 19-25).

In FcγRI (CD64), which includes FcγRIa, FcγRIb, and FcγRIc, and FcγRIII (CD16), including FcγRIIIa (including allotypes V158 and F158), an α chain that binds to the Fc region of IgG by association with a common γ chain that has ITAM (tyrosine-based immunoreceptor activating motifs) that transmit activation signals into cells. FcγRIIIb (including the allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) is a GPI-anchored protein. Moreover, the cytoplasmic domain FcγRII (CD32), which includes FcγRIIa (including the H131 and R131 allotypes) and the FcγRIIc isoforms, contains ITAM. These receptors are expressed primarily on immune cells such as macrophages, mast cells, and antigen-presenting cells. Activation signals transduced after binding of these receptors to the F-region of IgG enhance the ability of macrophages to phagocytosis, production of pro-inflammatory cytokines, degranulation of mast cells, and increased function of antigen-presenting cells. An FcγR that has the ability to transduce the activation signals described above is also referred to as activating FcγRs in the context of sections A and B of the present description.

Moreover, the cytoplasmic domain of FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) contains an ITIM (inhibitory motif based on tyrosine of an immunoreceptor), which transmits inhibitory signals. In B cells, cross-linking of FcγRIIb and B cell receptor (BCR) suppresses activation signals from BCRs, resulting in suppression of BCR antibody production. In macrophages, cross-linking of FcγRIII and FcγRIIb suppresses the ability to phagecytosis and to produce proinflammatory cytokines. An FcγR that has the ability to transduce the inhibitory signals described above is also referred to as an inhibitory Fcγ receptor in the context of sections A and B of the present description.

In the context of section A of the present description, to decide whether the binding activity of an antibody or an Fc region (variant) is increased, (practically) or decreased for different FcγRs compared to an antibody or an Fc region (a variant) before modification, one can use methods known to those of ordinary skill in the art. Such methods are not limited to specific methods and the methods described in the examples and, for example, BIACORE analysis, which uses the phenomenon of surface plasmon resonance (SPR) (Proc. Natl. Acad. Sci. USA 103 (11), 2006, pp. . 4005-4010). Alternatively, ELISA and fluorescence cell separation (FACS) can be used, as well as screening based on Amplified Luminescent Proximity Homogeneous Assay (ALPHA) .In these assays, the extracellular domain of human FcγR can be used as a soluble antigen (for example, WO 2013/047752).

To assess the binding activity between an FcγR binding domain within an antibody or Fc region (variant) and an FcγR, acidic or neutral pH conditions can be used under various pH conditions. As the temperature at which the measurements of the binding activity (binding affinity) between the FcγR binding domain and the FcγR are performed, for example, any temperature between 10 ° C and 50 ° C can be used. The preferred temperature for determining the binding activity (binding affinity) between the human FcγR-binding domain and the FcγR is, for example, 15 ° C-40 ° C. More preferably, any temperature from 20 ° C to 35 ° C can be used to determine the binding activity (binding affinity) between the FcγR binding domain and the FcγR, for example, any of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35 ° C. An example of such a temperature is (but not limited to) 25 ° C.

In one embodiment, when the antibody specified in section A or B of the description has a constant region (which can be modified), the constant region may have an Fc region or a variant Fc region (preferably a human Fc region or a variant human Fc -region) and preferably has an FcγR-binding domain falling within the scope of section A of the description, and an FcRn-binding domain falling within the scope of sections A and B of the present description.

In one embodiment, in which the antibody described in Section A of the specification has an FcγR binding activity, it may have an FcγR binding domain, preferably a human FcγR binding domain. An FcγR binding domain is not limited to a specific domain if the antibody has binding activity or affinity for FcγR at acidic pH and / or neutral pH, and it may be a domain that has the ability to directly or indirectly bind FcγR.

In one embodiment of the invention, in which the antibody specified in section A of the description has an FcγR-binding activity, it is preferred that the FcγR-binding activity of the antibody under neutral pH conditions is increased compared to the activity of a reference antibody that contains a constant region of native IgG ... To compare the FcγR binding activity of two antibodies, it is preferable (but not limited to) that the antibody specified in section A of the description, and the reference antibody that contains the constant region of native IgG, have identical amino acid sequences in regions (for example, the variable region ), preferably other than the constant region of the antibody specified in section A of the description, which has been modified in one or more amino acid residues.

In one of the embodiments of the invention, in which the antibody specified in section A of the description, has FcγR-binding activity or increased FcγR-binding activity under conditions of neutral pH (for example, pH 7.4), then, without being bound by theory, you can assume that the antibody has a combination of the following properties: the ability to shuttle between plasma and cell endosome and re-bind to several antigens in the form of one antibody molecule due to the presence of an ion concentration-dependent antigen-binding domain; the ability to quickly penetrate cells due to increased pI and increased total positive antibody charge; and the ability to quickly enter cells due to the increased FcγR-binding activity under neutral pH conditions. As a result, the plasma half-life of the antibody can be further shortened, or the binding activity of the antibody to the extracellular matrix can be further increased, or the elimination of the antigen from the plasma can be further enhanced; as a result, the antibody specified in section A of the description has valuable properties. One of ordinary skill in the art can use conventional techniques to determine the pI value of an antibody that is optimal for these properties.

In one embodiment of the invention, an FcγR-binding domain can be obtained, the FcγR-binding activity of which is higher than the activity of the Fc region or constant region of native human IgG, in which the sugar chain linked at position 297 according to EU numbering is fucose containing sugar chain, by modifying amino acid residues in the Fc region or constant region of native human IgG (see WO 2013/047752). In addition, a domain of any structure that binds to FcγR can be used as an FcγR binding domain. In this case, the FcγR binding domain can be obtained without the need to introduce an amino acid modification, and alternatively, its affinity for FcγR can be increased by introducing an additional modification. Such FcγR-binding domains can include the Fab fragment of antibodies that bind to FcγRIIIa, camelid single-domain antibodies, and antibodies in the form of single-chain Fvs, as described in Schlapschly et al., Protein Eng. Des. Sel. 22 (3), 2009 cc. 175-188, Behar et al., Protein Eng. Des. Sel. 21 (1), 2008, pp. 1-10 and Kipriyanov et al., J Immunol. 169 (1), 2002, cc. 137-144, and the FcγRI-binding cyclic peptide described by Bonetto et al., FASEB J. 23 (2), 2009, pp. 575-585. Whether the FcγR binding activity of the FcγR binding domain is higher than that of the Fc region or the constant region of native human IgG, in which the sugar chain linked at position 297, according to EU numbering, is a fucose containing sugar chain, can be assessed accordingly using methods described above.

In one of the embodiments of the invention specified in section A of the description, the original FcγR-binding domain preferably includes, for example, the Pc region of (human) IgG or constant region (human) IgG. If a variant of the original Fc region or the original constant region can bind to human Fc γR in the neutral pH range, then any Fc region or constant region can be used as the original Fc region or the original constant region. An Fc region or a constant region obtained by further modification of the original Fc region or the original constant region in which the amino acid (s) residue (s) have already been modified as compared to the Fc region or constant region can also be used, respectively, as an Fc- region or constant region specified in section A of the description. Parent Fc region or parent constant region can be understood as the polypeptide itself, a composition containing the parent Fc region or parent constant region, or an amino acid sequence corresponding to the parent Fc region or parent constant region. The original Fc region or the original constant region may include known Fc regions or known constant regions obtained by recombination techniques. The source of the original Fc region or the original constant region is not limited to a specific source and can be obtained from any animal organism other than humans or humans. In addition, the original FcγR-binding domain can be obtained from cynomlgus monkeys, marmosets, rhesus monkeys, chimpanzees or humans. The Fc starting region or the starting constant region can preferably be derived from human IgG1; however, they are not limited to a specific IgG class. This means that the Fc region of human IgG1, IgG2, IgG3 or IgG4 can be used as a suitable starting FcγR binding domain, and this also means that in the context of section A of the present description, the Fc region or constant region of an IgG class or subclass, obtained from any organism, preferably can be used as the original Fc region or the original constant region. Examples of a variant or modified form of native IgG are described in the published literature, for example, Strohl, Curr. Opin. Biotechnol. 20 (6), 2009, pp. 685-691; Presta, Curr. Opin. Immunol. 20 (4), 2008, pp. 460-470; Davis et al., Protein Eng. Des. Sel. 23 (4), 2010, pp. 195-202; in WO 2009/086320, WO 2008/092117; WO 2007/041635; and WO 2006/105338 (but not limited to).

In one embodiment of the invention, the amino acid residues of the original FcγR binding domain of the original Fc region or the original constant region may contain, for example, one or more mutations: for example, substitutions of amino acid residues that differ from residues in the original Fc region or the original constant region ; insertion of one or more amino acid residues into amino acid residues in the original Fc region or the original constant region; or deletion of one or more amino acid residues from the original Fc region or the original constant region. The amino acid sequences of the Fc regions or constant regions after modifications are preferably amino acid sequences containing at least a portion of the Fc region or constant region, which may not naturally occur. Such variants should be identical or have less than 100% sequence similarity with the original Fc regions or the original constant regions. For example, variants have an amino acid sequence identical or similar at about 75% to less than 100%, more preferably from about 80% to less than 100%, even more preferably from about 85% to less than 100%, even more preferably from about 90% to less than 100%, and even more preferably from about 95% to less than 100%, with the amino acid sequence of the original Fc region or the original constant region. In an example that does not limit the scope of the invention, at least one amino acid in the modified Fc region or constant region specified in section A of the description is different from the amino acid in the original Fc region or the original constant region.

In one embodiment, an Fc region or constant region that has FcγR binding activity in the acidic pH range and / or neutral pH range, which may be included in the antibody specified in section A of the description, can be obtained by any method. In particular, a variant of the Fc region or constant region that has FcγR binding activity in the neutral pH range can be obtained by modifying the amino acids of a human IgG-type antibody, which can be used as the original Fc region or the original constant region. Fc regions of an IgG-type antibody or constant regions of an IgG-type antibody suitable for modification may include, for example, Fc regions or constant regions of human IgG (IgG1, IgG2, IgG3 or IgG4 or variants thereof) and mutants spontaneously formed from them. For Fc regions or constant regions of antibodies in the form of human IgG1, human IgG2, human IgG3, or human IgG4, a variety of IgG allotype sequences associated with genetic polymorphism are described in "Sequences proteins of immunological interest)), NIH publication No. 91-3242 and any of these can be used in section A of the description. In particular, in the case of human IgG1, the amino acid sequence at positions 356-358 according to EU numbering can be either DEL or EEM.

In another embodiment, in the context of section A of the description, the modification of other amino acids is not limited to these, if the variants have FcγR-binding activity in the neutral pH range. The amino acid position (s) of this modification are described, for example, in: WO 2007/024249, WO 2007/021841, WO 2006/031370, WO 2000/042072, WO 2004/029207, WO 2004/099249, WO 2006/105338 , WO 2007/041635, WO 2008/092117, WO 2005/070963, WO 2006/020114, WO 2006/116260, WO 2006/023403, WO 2013/047752, WO 2006/019447, WO 2012/115241, WO 2013/125667 , WO 2014/030728, WO 2014/163101, WO 2013/118858 and WO 2014/030750.

Sites of amino acid modification in the constant region or Fc-region designed to increase FcγR-binding activity in the neutral pH range may include, for example, one or more positions selected from the group consisting of positions: 221, 222, 223, 224, 225 , 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 254, 255, 256 , 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284, 285 , 286, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 311, 313, 315, 317, 318, 320, 322 , 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 339, 376, 377, 378, 379, 380, 382, 385, 392, 396 , 421, 427, 428, 429, 434, 436 and 440, according to EU numbering in the Fc region or constant region of a human IgG type antibody described in WO 2013/047752. Modification of the specified amino acid residue can increase the FcγR-binding activity of the Fc region or the constant region of an IgG-type antibody under neutral pH conditions. WO 2013/047752 describes as preferred modifications in the constant region or Fc region of the IgG type, for example, modification of one or more amino acid residues selected from the group consisting of: amino acid at position 221 to Lys or Tyr; the amino acid at position 222 to any of Phe, Trp, Glu, and Tyr; the amino acid at position 223 to any of Phe, Trp, Glu, and Lys; the amino acid at position 224 to any of Phe, Trp, Glu, and Tyr; the amino acid at position 225 to any of Glu, Lys, and Trp; the amino acid at position 227 to any of Glu, Gly, Lys and Tyr; the amino acid at position 228 to any of Glu, Gly, Lys and Tyr; amino acids at position 230 to any of Ala, Glu, Gly, and Tyr; the amino acid at position 231 to any of Glu, Gly, Lys, Pro, and Tyr; the amino acid at position 232 to any of Glu, Gly, Lys and Tyr; the amino acid at position 233 to any of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 234 to any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 235 to any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 236 to any of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 237 to any of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 238 to any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 239 to any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp and Tyr; amino acids at position 240 to any of Ala, Ile, Met, and Thr; the amino acid at position 241 to any of Asp, Glu, Leu, Arg, Trp, and Tyr; the amino acid at position 243 to any of Glu, Leu, Gln, Arg, Trp, and Tyr; amino acids at position 244 on His; amino acids at position 245 on Ala; amino acids at position 246 to any of Asp, Glu, His and Tyr; the amino acid at position 247 to any of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val and Tyr; amino acids at position 249 to any of Glu, His, Gin, and Tyr; amino acids at position 250 on Glu or Gin; amino acids at position 251 on Phe; the amino acid at position 254 to any of Phe, Met, and Tyr; amino acids at position 255 to any of Glu, Leu, and Tyr; amino acids at position 256 to any of Ala, Met, and Pro; amino acids at position 258 to any of Asp, Glu, His, Ser, and Tyr; amino acids at position 260 to any of Asp, Glu, His and Tyr; the amino acid at position 262 to any of Ala, Glu, Phe, Ile, and Thr; the amino acid at position 263 to any of Ala, Ile, Met, and Thr; the amino acid at position 264 to any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr; the amino acid at position 265 to any of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 266 to any of Ala, Ile, Met, and Thr; the amino acid at position 267 to any of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 268 to any of Asp, Glu, Phe, Gly, He, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, and Trp; the amino acid at position 269 to any of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 270 to any of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr; the amino acid at position 271 to any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 272 to any of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; amino acids at position 273 on either Phe or Ile; the amino acid at position 274 to any of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; amino acids at position 275 on Leu or Trp; the amino acid at position 276 to any of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 278 to any of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp; amino acids at position 279 on Ala; the amino acid at position 280 to any of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, and Tyr; the amino acid at position 281 to any of Asp, Lys, Pro, and Tyr; the amino acid at position 282 to any of Glu, Gly, Lys, Pro, and Tyr; the amino acid at position 283 to any of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, and Tyr; the amino acid at position 284 to any of Asp, Glu, Leu, Asn, Thr, and Tyr; the amino acid at position 285 to any of Asp, Glu, Lys, Gln, Trp, and Tyr; the amino acid at position 286 to any of Glu, Gly, Pro, and Tyr; the amino acid at position 288 to any of Asn, Asp, Glu, and Tyr; the amino acid at position 290 to any of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, and Tyr; the amino acid at position 291 to any of Asp, Glu, Gly, His, Ile, Gin, and Thr; the amino acid at position 292 to any of Ala, Asp, Glu, Pro, Thr, and Tyr; the amino acid at position 293 to any of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 294 to any of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 295 to any of Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 296 to any of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, and Val; the amino acid at position 297 to any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 298 to any of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 299 to any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, and Tyr; the amino acid at position 300 to any of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp; amino acids at position 301 to any of Asp, Glu, His and Tyr; amino acids at position 302 to Ile; the amino acid at position 303 to any of Asp, Gly, and Tyr; the amino acid at position 304 to any of Asp, His, Leu, Asn, and Thr; amino acids at position 305 to any of Glu, Ile, Thr, and Tyr; the amino acid at position 311 to any of Ala, Asp, Asn, Thr, Val, and Tyr; amino acids at position 313 on Phe; amino acids at position 315 on Leu; the amino acid at position 317 on either Glu or Gin; the amino acid at position 318 to any of His, Leu, Asn, Pro, Gln, Arg, Thr, Val, and Tyr; the amino acid at position 320 to any of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 322 to any of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 323 to Ile; the amino acid at position 324 to any of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 325 to any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 326 to any of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 327 to any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 328 to any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 329 to any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 330 to any of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 331 to any of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 332 to any of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 333 to any of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, and Tyr; the amino acid at position 334 to any of Ala, Glu, Phe, Ile, Leu, Pro, and Thr; the amino acid at position 335 to any of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, and Tyr; the amino acid at position 336 to any of Glu, Lys and Tyr; the amino acid at position 337 to any of Glu, His, and Asn; the amino acid at position 339 to any of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, and Thr; the amino acid at position 376 on either Ala or Val; the amino acids at position 377 to either Gly or Lys; amino acids at position 378 to Asp; amino acids at position 379 on Asn; the amino acid at position 380 to any of Ala, Asn and Ser; amino acids at position 382 on either Ala or Ile; amino acids at position 385 on Glu; amino acids at position 392 on Thr; amino acids at position 396 on Leu; amino acids at position 421 on Lys; amino acids at position 427 on Asn; amino acids at position 428 on either Phe or Leu; amino acids at position 429 on Met; amino acids at position 434 on Trp; amino acids at position 436 on Ile; and the amino acid at position 440 to any of Gly, His, Ile, Leu and Tyr, according to EU numbering. The number of amino acids that can be modified is not limited, and it is possible to modify an amino acid at only one position or amino acids at two or more positions. Combinations of amino acid modifications at two or more positions are shown in Table 5 in WO 2013/047752. Modification of these amino acid residues can also be introduced, respectively, into the antibodies specified in section A of the description.

In one embodiment, the binding activity of the (FcγR binding domain) of the antibody specified in Section A of the specification against (human) FcγRs, such as one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb, may be greater than the activity (Fc region or constant region) of native IgG or a reference antibody containing the original Fc region or the original constant region. For example, the FcγR binding activity (FcγR binding domain) of the antibody specified in section A of the description may be 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 100% or more, 105% or more, preferably 110% or more, 115% or more, 120% or more, 125% or more, especially preferably 130% or more, 135% or more, 140% or more, 145% or more, 150% or more, 155% or more, 160% or more, 165% or more, 170% or more, 175% or more, 180% or more, 185% or more, 190% or more, or 195% or more compared to the FcγR-binding activity of the reference antibody, in or 2 or more, 2.5 or more, 3 or more, 3.5 or more, 4 or more, 4.5 or more, 5 or more, 7.5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more , 80 or more, 90 or more, or 100 or more times higher than the FcγR-binding activity of the reference body.

In another embodiment, the level of increase in binding activity to inhibitory FcγR (FcγRIIb-1 and / or FcγRIIb-2) (in the neutral pH range) may be higher than the level of increase in binding activity to activating FcγR (FcγRIa: FcγRIb; FcγRIc; FcγRIIIa, including allotype V158; FcγRIIIa, including allotype F158; FcγRIIIb, including allotype FcγRIIIb-NA1; FcγRIIIb, including allotype FcγRIIIb-NA2; FcγRIIa, including allotype H131; or FcγRIIa, including allotype R131).

In one embodiment, the antibody described in section A of the description may have binding activity against FcγRIIb (including FcγRIIb-1 and FcγRIIb-2).

In one embodiment, the preferred FcγR binding domains described in Section A of the description also include, for example, FcγR binding domains that bind to a particular FcγR that is greater than binding to another FcγR (FcγR binding domains with a selective FcγR -binding activity). When an antibody is used (or an Fc region as the FcγR binding domain), one antibody molecule can only bind to one FcγR molecule. Thus, one antibody molecule associated with an inhibitory FcγR state cannot bind to other activating FcγRs, and one antibody molecule associated with an activating FcγR state cannot bind to other activating FcγRs or inhibitory FcγRs.

As described above, the activating FcγR preferably includes, for example, FcγRI (CD64) such as FcγRIa, FcγRIb, or FcγRIc; and FcγRIII (CD16) such as FcγRIIIa (such as allotype V158 or F158) or FcγRIIIb (such as FcγRIIIb-NA1 or FcγRIIIb-NA2). And the inhibitory FcγR preferably includes, for example, FcγRIIb (such as FcγRIIb-1 or FcγRIIb-2).

In one embodiment, FcγR binding domains that have a higher binding activity for inhibitory FcγR than for activating FcγR can be used as a selective FcγR binding domain included in the antibody described in section A of the description. Such selective FcγR-binding domains may include, for example, FcγR-binding domains that have a higher binding activity for FcγRIIb (such as FcγRIIb-1 and / or FcγRIIb-2) than for any one or more FcγR activating domains selected from the group consisting of: FcγRI (CD64) such as FcγRIa, FcγRIb, or FcγRIc; FcγRIII (CD16) such as FcγRIIIa (such as allotype V158 or F158) or FcγRIIIb (such as FcγRIIIb-NA1 or FcγRIIIb-NA2); FcγRII (CD32) such as FcγRIIa (including the H131 or R131 allotype); HFcγRIIc.

In addition, whether an FcγR binding domain has selective binding activity can be determined by comparing the binding activity for each FcγR by performing analysis using the methods described above, for example, by comparing the value (ratio) obtained by dividing the KD value for activating FcγR by the KD value for inhibitory FcγR, more specifically by comparing the index of selective action for FcγR calculated using equation 1 below:

Equation 1:

index of selective action in relation to FcγR = KD value for activating FcγR / KD value for inhibiting FcγR

In equation 1, the KD value for activating FcγR means the KD value for one or more of: FcγRIa; FcγRIb; FcγRIc; FcγRIIIa, including allotype V158 and / or F158; FcγRIIIb, including FcγRIIIb-NA1 and / or FcγRIIIb-NA2; FcγRIIa, including the H131 and / or R131 allotype; and FcγRIIc; and the KD value for inhibiting FcγR means the KD value for FcγRIIb-1 and / or FcγRIIb-2. Activating FcγR and inhibiting FcγR for use in determining KD values can be selected from any combination thereof. For example, the value (ratio) determined by dividing the KD value for FcγRIIa, including the H131 allotype, by, but not limited to, the KD value for FcγRIIb-1 and / or FcγRIIb-2 can be applied.

For FcγR, the selective action index can be, for example: 1.2 or higher, 1.3 or higher, 1.4 or higher, 1.5 or higher, 1.6 or higher, 1.7 or higher, 1.8 or higher, 1.9 or higher, 2 or higher, 3 or higher, 5 or higher, 6 or higher, 7 or higher, 8 or higher, 9 or higher, 10 or higher, 15 or higher, 20 or higher, 25 or higher, 30 or higher, 35 or higher, 40 or higher, 45 or higher, 50 or higher, 55 or higher, 60 or higher, 65 or higher, 70 or higher, 75 or higher, 80 or higher, 85 or higher, 90 or higher, 95 or higher, 100 or higher, 110 or higher, 120 or higher, 130 or higher, 140 or higher, 150 or higher, 160 or higher, 170 or higher, 180 or higher, 190 or higher, 200 or higher, 210 or higher, 220 or higher, 230 or higher, 240 or higher, 250 or higher, 260 or higher, 270 or higher, 280 or higher, 290 or higher, 300 or higher, 310 or higher, 320 or higher, 330 or higher, 340 or higher, 350 or higher, 360 or higher, 370 or higher, 380 or higher, 390 or higher, 400 or higher, 410 or higher, 420 or higher, 430 or higher, 440 or higher, 450 or higher , 460 or above, 470 or above, 480 or above, 490 or above, 500 or above, 520 or above, 540 or above, 560 or above, 580 or above, 600 or above, 620 or above, 640 or above, 660 or above, 680 or above, 700 or above, 720 or above, 740 or above, 760 or above, 780 or above, 800 or above, 820 or above, 840 or above, 860 or above, 880 or above, 900 or above , 920 or above, 940 or above, 960 or above, 980 or above, 1000 or above, 1500 or above, 2000 or above, 2500 or above, 3000 or above, 3500 or above, 4000 or above, 4500 or above, 5000 or above, 5500 or above, 6000 or above, 6500 or above, 7000 or above, 7500 or above, 8000 or above, 8500 or above, 9000 or above, 9500 or above, 10000 or above, or 100000 or above (but not limited only to them).

In one embodiment of the invention (an antibody comprising) a variant Fc region or a variant constant region in which the amino acid at position 238 or 328 according to the EU numbering of a human IgG (IgG1, IgG2, IgG3 or IgG4) is Asp or Glu, respectively, can preferably be used in antibodies specified in section A of the description that contain a variant Fc region or a variant constant region, since, as described specifically in WO 2013/125667, WO 2012/115241 and WO 2013/047752, they have a higher binding activity with respect to FcγRIIb-1 and / or FcγRIIb-2 than with respect to FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, including allotype V158, FcγRIIIa, including allotype F158, FcγRIIIb, including allotype FcγRIIIγR-NAbIIa, including FcγRIIIγRIII allotype FcγRIIIa , including allotype H131, FcγRIIa, including allotype R131 and / or FcγRIIc. In this embodiment, the antibodies referred to in Section A of the specification have binding activity against all activating FcγRs (which, as used herein, are selected from the group consisting of FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb, FcγRIIa) and FcγRIIb, and FcγRIIb binding activity is maintained or increased and / or their binding activity to all activating FcγRs is reduced compared to a reference antibody that contains a native IgG constant region or a native IgG Fc region.

In one embodiment of the invention, with respect to antibodies specified in section A of the description that contain a variant Fc region or a variant constant region, their binding activity against FcγRIIb may be maintained or increased and their binding activity against FcγRIIa (type H) and FcγRIIa ( type R) can be reduced compared to the activity of a reference antibody having a constant region or Fc region of native IgG. These antibodies have increased selectivity for binding FcγRIIb relative to FcγRIIa.

In the context of section A of the present description, the degree to which "the binding activity for all activating FcγRs is reduced" may be (but not limited to) 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, 90% or less, 88% or less, 86% or less, 84% or less, 82% or less, 80% or less, 78% or less, 76% or less, 74% or less, 72% or less, 70% or less, 68% or less, 66% or less, 64% or less, 62% or less, 60% or less, 58% or less, 56% or less, 54% or less, 52% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, 0.05% or less, 0.01% or less, or 0.005% or less.

In the context of Section A of the present disclosure, the extent to which "FcγRIIb binding activity is maintained or increased", "FcγRIIb binding activity is maintained or increased" or "FcγRIIb binding activity is maintained or increased" may be (but is not limited to) ) 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 87% or more, 88% or more, 89% or more , 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more , 99.5% or more, 100% or more, 101% or more, 102% or more, 103% or more, 104% or more, 105% or more, 106% or more, 107% or more, 108% or more , 109% or more, 110% or more, 112% or more, 114% or more, 116% or more, 118% or more, 120% or more, 122% or more, 124% or more, 126% or more , 128% or more, 130% or more, 132% or more, 134% or more, 136% or more, 138% or you more, 140% or more, 142% or more, 144% or more, 146% or more, 148% or more, 150% or more, 155% or more, 160% or more, 165% or more, 170% or higher, 175% or higher, 180% or higher, 185% or higher, 190% or higher, 195% or higher, 2 times or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, 10,000 or more, or 100,000 times or more.

In the context of Section A of the present disclosure, the extent to which “the binding activity against FcγRIIa (type H) and FcγRIIa (type R) is reduced” or “decreased binding activity against FcγRIIa (type H) and FcγRIIa (type R)” may be (but not limited to only the specified) 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, 90% or less, 88% or less, 86% or less, 84% or less, 82% or less, 80% or less, 78% or less, 76% or less, 74% or less, 72% or less, 70% or less, 68% or less, 66% or less, 64% or less, 62% or less, 60% or less, 58% or less, 56% or less, 54% or less, 52% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, 0.05% or less, 0.01% or less, or 0.005% or less.

In the context of Section A of the present disclosure, modifications resulting in increased binding selectivity to FcγRIIb over FcγRIIa (type R) may preferably be modifications that increase the binding selectivity to FcγRIIb over FcγRIIa (type H) may more preferably be the modifications specified in WO 2013/047752, preferred amino acid substitutions for such modifications may include, for example, according to EU numbering: (a) modification by replacing Gly at position 237 with Trp; (b) modification by replacing Gly at position 237 with Phe; (c) modification by replacing Pro at position 238 with Phe; (d) modification by replacing Asn at position 325 with Met; (e) modification by replacing Ser at position 267 with Ile; (e) modification by replacing Leu at position 328 with Asp; (g) modification by replacing Ser at position 267 with Val; (h) modification by replacing Leu at position 328 with Trp; (i) modification by replacing Ser at position 267 with Gin; (j) modification by replacing Ser at position 267 with Met; (k) modification by replacing Gly at position 236 with Asp; (m) modification by replacing Ala at position 327 with Asn; (m) modification by replacing Asn at position 325 with Ser; (o) modification by replacing Leu at position 235 with Tyr; (p) modification by replacing Val at position 266 with Met; (p) modification by replacing Leu at position 328 with Tyr; (c) modification by replacing Leu at position 235 with Trp; (m) modification by replacing Leu at position 235 with Phe; (y) modification by replacing Ser at position 239 with Gly; (f) modification by replacing Ala at position 327 with Glu; (x) modification by replacing Ala at position 327 with Gly; (c) modification by replacing Pro at position 238 with Leu; (h) modification by replacing Ser at position 239 with Leu; (w) modification by replacing Leu at position 328 with Thr; (y) modification by replacing Leu at position 328 with Ser; (e) modification by replacing Leu at position 328 with Met; (aa) modification by replacing Pro at position 331 with Trp; (ab) modification by replacing Pro at position 331 with Tyr; (ab) modification by replacing Pro at position 331 with Phe; (ah) modification by replacing Ala at position 327 with Asp; (ad) modification by replacing Leu at position 328 with Phe; (ae) modification by replacing Pro at position 271 with Leu; (ah) modification by replacing Ser at position 267 with Glu; (a) modification by replacing Leu at position 328 with Ala; (ai) modification by replacing Leu at position 328 with Ile; (a) modification by replacing Leu at position 328 with Gin; (al) modification by replacing Leu at position 328 with Val; (a) modification by replacing Lys at position 326 with Trp; (an) modification by replacing Lys at position 334 with Arg; (ao) modification by replacing His at position 268 with Gly; (an) modification by replacing His at position 268 with Asn; (ar) modification by replacing Ser at position 324 with Val; (ac) modification by replacing Val at position 266 with Leu; (at) modification by replacing Pro at position 271 with Gly; (ay) modification by replacing Ile at position 332 with Phe; (af) modification by replacing Ser at position 324 with Ile; (ax) modification by replacing Glu at position 333 with Pro; (ac) modification by substituting Asp for Tyr at position 300; (ah) modification by replacing Ser at position 337 with Asp; (ah) modification by replacing Tyr at position 300 with Gin; (ah) modification by replacing Thr at position 335 with Asp; (ae) modification by replacing Ser at position 239 with Asn; (b) modification by replacing Lys at position 326 with Leu; (bb) modification by replacing Lys at position 326 with Ile; (bc) modification by replacing Ser at position 239 with Glu; (bd) modification by replacing Lys at position 326 with Phe; (bd) modification by replacing Lys at position 326 with Val; (f) modification by replacing Lys at position 326 with Tyr; (bg) modification by replacing Ser at position 267 with Asp; (bz) modification by replacing Lys at position 326 with Pro; (bi) modification by replacing Lys at position 326 with His; (bc) modification by replacing Lys at position 334 with Ala; (bl) modification by replacing Lys at position 334 with Trp; (bm) modification by replacing His at position 268 with Gin; (bn) modification by replacing Lys at position 326 with Gin; (b) modification by replacing Lys at position 326 with Glu; (bp) modification by replacing Lys at position 326 with Met; (br) modification by replacing Val at position 266 with Ile; (bc) modification by replacing Lys at position 334 with Glu; (bt) modification by replacing Tyr at position 300 with Glu; (bu) modification by replacing Lys at position 334 with Met; (bf) modification by replacing Lys at position 334 with Val; (bx) modification by replacing Lys at position 334 with Thr; (bc) modification by replacing Lys at position 334 with Ser; (bh) modification by replacing Lys at position 334 with His; (bsh) modification by replacing Lys at position 334 with Phe; (bsh) modification by replacing Lys at position 334 with Gin; (bs) modification by replacing Lys at position 334 with Pro; (va) modification by replacing Lys at position 334 with Tyr; (wb) modification by replacing Lys at position 334 with Ile; (cc) modification by replacing Gin at position 295 with Leu; (bd) modification by replacing Lys at position 334 with Leu; (c) modification by replacing Lys at position 334 with Asn; (ve) modification by replacing His at position 268 with Ala; (vg) modification by replacing Ser at position 239 with Asp; (b) modification by replacing Ser at position 267 with Ala; (vi) modification by replacing Leu at position 234 with Trp; (bc) modification by replacing Leu at position 234 with Tyr; (bl) modification by replacing Gly at position 237 with Ala; (vm) modification by replacing Gly at position 237 with Asp; (h) modification by replacing Gly at position 237 with Glu; (b) modification by replacing Gly at position 237 with Leu; (bp) modification by replacing Gly at position 237 with Met; (bp) modification by replacing Gly at position 237 with Tyr; (su) modification by replacing Ala at position 330 with Lys; (b) modification by replacing Ala at position 330 with Arg; (wu) modification by replacing Glu at position 233 with Asp; (vf) modification by replacing His at position 268 with Asp; (in) modification by replacing His at position 268 with Glu; (cc) modification by replacing Lys at position 326 with Asp; (hf) modification by replacing Lys at position 326 with Ser; (lsh) modification by replacing Lys at position 326 with Thr; (wsc) modification by replacing Val at position 323 with Ile; (ve) modification by replacing Val at position 323 with Leu; (ha) modification by replacing Val at position 323 with Met; (db) modification by replacing Tyr at position 296 with Asp; (dc) modification by replacing Lys at position 326 with Ala; (dy) modification by replacing Lys at position 326 with Asn; and (d) modification by replacing Ala at position 330 with Met.

The modifications described above can be present in only one position, or in two or more positions in combination. Alternatively, these preferred modifications may include, for example, those set forth in Tables 14-15, 17-24 and 26-28 of WO 2013/047752, for example, human constant region or human Fc region variants in which the amino acid at position 238 according to EU the β-numbering is Asp and the amino acid at position 271 according to the EU numbering is Gly in human IgG (IgG1, IgG2, IgG3 or IgG4); in addition, one or more position (s) 233, 234, 237, 264, 265, 266, 267, 268, 269, 272, 296, 326, 327, 330, 331, 332, 333 and 396 according to the EU numbering may (gut) be replaced (s). In this case, variants may include (but are not limited to) variants of a human constant region or a human Fc region that contains one or more amino acids from:

Asp at position 233, Tyr at position 234, Asp at position 237, Ile at position 264, Glu at position 265, any of Phe, Met and Leu at position 266, any of Ala, Glu, Gly and Gin at position 267, or Asp or Glu at position 268, Asp at position 269, any of Asp, Phe, Ile, Met, Asn and Gin at position 272, Asp at position 296, or Ala or Asp at position 326, Gly at position 327, or Lys or Arg at position 330, Ser at position 331, Thr at position 332, any of Thr, Lys and Arg at position 333 and any of Asp, Glu, Phe, Ile, Lys, Leu, Met, Gln, Arg and Tyr at position 396 according to EU numbering.

In an alternative embodiment, the antibodies described in section A of the descriptions containing a variant Fc region or a variant constant region may have retained or increased binding activity against FcγRIIb and reduced binding activity against FcγRIIa (type H) and FcγRIIa (type R) compared to a reference antibody containing a constant region or Fc region of native IgG. Preferred amino acid substitution sites for these variants can be described in WO 2014/030728, for example, amino acid at position 238 according to EU numbering, and at least one amino acid position selected from the group consisting of position 233, 234, 235, 237, 264 , 265, 266, 267, 268, 269, 271, 272, 274, 296, 326, 327, 330, 331, 332, 333, 334, 355, 356, 358, 396, 409 and 419, according to EU numbering.

More preferably, variants may have Asp at position 238 according to EU numbering and at least one amino acid selected from the group: Asp at position 233, Tyr at position 234, Phe at position 235, Asp at position 237, Ile at position 264, Glu at at position 265, Phe, Leu or Met at position 266, Ala, Glu, Gly or Gin at position 267, Asp, Gin or Glu at position 268, Asp at position 269, Gly at position 271, Asp, Phe, Ile, Met, Asn, Pro or Gin at position 272, Gin at position 274, Asp or Phe at position 296, Ala or Asp at position 326, Gly at position 327, Lys, Arg or Ser at position 330, Ser at position 331, Lys, Arg , Ser or Thr at position 332, Lys, Arg, Ser or Thr at position 333, Arg, Ser or Thr at position 334, Ala or Gin at position 355, Glu at position 356, Met at position 358, Ala, Asp, Glu , Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr at position 396, Arg at position 409 and Glu at position 419 according to EU numbering.

In an alternative embodiment, the antibodies described in section A of the descriptions containing a variant Fc region or a variant constant region may have retained binding activity against FcγRIIb and reduced binding activity against all activating FcγR, FcγRIIa (type R), in particular, compared to a reference antibody containing a constant region or Fc region of native IgG. Preferred amino acid substitution sites for such variants can be those described in WO 2014/163101, for example, in addition to amino acid position 238 (according to EU numbering), at least one amino acid position selected from positions 235, 237, 241, 268, 295, 296, 298, 323, 324 and 330, according to EU-numbering. More preferably, variants can have Asp at position 238 according to EU numbering and at least one amino acid selected from an amino acid from the group: Phe at position 235; Gin or Asp at position 237; Met or Leu at position 241; Pro at position 268; Met or Val at position 295; Glu, His, Asn or Asp at position 296; Ala or Met at position 298; Ile at position 323; Asn or His at position 324; and His or Tyr at position 330 according to EU numbering.

In the context of section A of the present description, the level of "retained binding activity against FcγRIIb" can be (but not limited to) 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80 % or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90 % or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5 % or more, 100% or more, 101% or more, 102% or more, 103% or more, 104% or more, 105% or more, 106% or more, 107% or more, 108% or more, 109 % or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 175% or more, or more than 2 times or more.

In the context of Section A of the present description, the above level of "reduction of binding activity for all activating FcγR, in particular FcγRIIa (type R)" may be (but not limited to) 74% or less, 72% or less, 70% or less , 68% or less, 66% or less, 64% or less, 62% or less, 60% or less, 58% or less, 56% or less, 54% or less, 52% or less, 50% or less , 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less , 3% or less, 2% or less, 1% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less , 0.05% or less, 0.01% or less, or 0.005% or less.

WO 2014/030750 also describes mouse constant region and Fc region variants. In an embodiment of the invention, the antibodies specified in section A or B of the description may contain such a variant.

In the context of sections A and B of the present description, in contrast to FcγR, which belongs to the superfamily of immunoglobulins, "FcRn", in particular human FcRn, has structural similarity to polypeptides of molecules of the major histocompatibility complex (MHC) class I and its sequence is 22% identical - 29% of the sequence of MHC class I molecules (Ghetie et al., Immunol. Today 18 (12), 1997, pp. 592-598). FcRn is expressed as a heterodimer composed of a soluble β- or light chain (β2-microglobulin) complexed with a transmembrane α- or heavy chain. Similarly to MHC, the FcRn α-chain contains three extracellular domains (α1, α2, and α3) and a short cytoplasmic domain that binds proteins to the cell surface; α1 and α2 domains interact with the FcRn-binding domain of the antibody Fc region (Raghavan et al., Immunity 1, 1994, pp. 303-315).

FcRn is expressed in the maternal placenta and yolk sac of mammals and is involved in the transfer of IgG from the mother to the fetus. In addition, in the small intestine of newborn rodents, when FcRn is expressed, it is involved in the transfer of maternal IgG through the brush border of the epithelium from consumed colostrum or milk. FcRn is expressed in a wide variety of other tissues and endothelial cell systems of various species. In adults, FcRn is also expressed in the vascular endothelium, the muscular vasculature and the sinusoidal capillaries of the liver. FcRn appears to play a role in maintaining plasma IgG concentration by binding to IgG and recycling IgG in serum. Typically, the binding of FcRn to IgG molecules is strictly pH dependent. Optimal binding is found in the acidic pH range below 7.0.

The polynucleotide and amino acid sequences of human FcRn can be obtained, for example, from the precursors that are shown in NM_004107.4 and NP_004098.1 (containing the signal sequence), respectively (RefSeq access codes are shown in parentheses).

The progenitors form complexes with human β2-microglubulin in vivo. Thus, using known recombinant expression techniques, a soluble human FcRn capable of complexing with human β2-microglubulin can be prepared for appropriate use in various experimental systems. Said soluble human FcRn can be used to evaluate antibodies or Fc region variants for their FcRn binding activity. In section A or B of the description, FcRn is not limited to a specific FcRn as long as it is in a form that can bind to an FcRn binding domain; however, the preferred FcRn may be a human FcRn.

In the context of sections A and B of the present description, if an antibody or Fc region variant has FcRn binding activity, then it may have an “FcRn binding domain”, preferably a human FcRn binding domain. An FcRn binding domain is not limited to a specific domain as long as the antibody has binding activity or affinity for FcRn at acidic pH and / or neutral pH; or may represent a domain that has the ability to directly or indirectly bind to FcRn. Such domains include, but are not limited to, the Fc regions of IgG-type immunoglobulins, albumin, albumin domain 3, anti-FcRn antibodies, anti-FcRn peptides, and anti-FcRn scaffold molecules that have the ability to directly bind to FcRn, and molecules that bind to IgG or albumin, which have the ability to indirectly bind to FcRn. Section A or B of the disclosure also provides for the use of domains that have FcRn binding activity in the acidic pH range and / or neutral pH range. If the domains initially have FcRn-binding activity in the acidic pH range and / or neutral pH range, then they can be used without additional modification. If the domains have only weak or no FcRn binding activity in the acidic pH range and / or neutral pH range, then the amino acid residues in the FcRn binding domain of an antibody or Fc region variant can be modified so that they acquire FcRn binding activity in acidic pH range and / or neutral pH range. Alternatively, the amino acids of the domains that initially have FcRn binding activity in the acidic pH range and / or neutral pH range can be modified to further increase their FcRn binding activity. FcRn binding activity in the acidic pH range and / or neutral pH range can be compared before and after amino acid modification to identify amino acid modifications of interest for the FcRn binding domains.

FcRn binding domains can preferably be regions that directly bind to FcRn. These preferred FcRn binding domains include, for example, antibody constant regions and Fc regions. However, regions that can bind to a polypeptide having FcRn binding activity, such as albumin and IgG, can indirectly bind to FcRn via albumin, IgG. Thus, FcRn binding regions can be regions that bind to a polypeptide capable of binding to albumin or IgG. To enhance the elimination of antigen from plasma, FcRn-binding domains are preferred, the FcRn-binding activity of which is higher at neutral pH, and to improve the retention time of the antibody in plasma, FcRn-binding domains are preferred, the FcRn-binding activity of which is higher at acidic pH (but not limited to only those indicated). For example, you can select FcRn-binding domains, the FcRn-binding activity of which is initially higher at neutral pH or acidic pH. Alternatively, the amino acids of the antibody or Fc region variant can be modified to impart FcRn binding activity at neutral pH or acidic pH. Alternatively, pre-existing FcRn binding activity can be increased at neutral pH or acidic pH.

In the context of Sections A and B of the present description, to decide whether the FcRn binding activity of an antibody or an Fc region (variant) is increased, (practically) maintained or decreased compared to an antibody or an Fc region (variant) before modification, one can apply known methods such as those described in the examples herein, and, for example, BIACORE, Scatchard plot and flow cytometry (see WO 2013/046722). The extracellular domain of human FcRn can be used as soluble antigen in these assays. One of ordinary skill in the art can readily select conditions other than pH to measure the FcRn binding activity of an antibody or an Fc region (variant). The analysis can be carried out, for example, under conditions of MES buffer and 37 ° C, as described in WO 2009/125825. The FcRn binding activity of an antibody or an Fc region (variant) can be assessed, for example, by introducing FcRn as an analyte on a chip with an immobilized antibody.

The FcRn binding activity of an antibody or Fc region (variant) can be estimated based on the dissociation constant (KD), the apparent dissociation constant (apparent KD), the dissociation reaction rate constant (kd), and the apparent dissociation reaction rate constant (apparent kd).

As pH conditions for measuring the binding activity between an FcRn and an FcRn binding domain included in an antibody or an Fc region (variant), acidic pH conditions or neutral pH conditions can be used. As the temperature conditions for measuring the binding activity (binding affinity) between the FcRn and the FcRn binding domain, any temperature from 10 ° C to 50 ° C can be used. To determine the binding activity (binding affinity) between FcRn and the human FcRn binding domain, a temperature of 15 ° C to 40 ° C can preferably be used. More preferably, you can use any temperature from 20 ° C to 35 ° C, such as any of the following: 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35 ° C. As an example of the specified temperature, you can use (but not limited to) 25 ° C.

In one embodiment, in which the antibodies described in section A or B of the description have FcRn binding activity, they may have an FcRn binding domain, preferably a human FcRn binding domain. An FcRn binding domain is not limited to a specific domain as long as the antibodies have binding activity or affinity for FcRn at acidic pH and / or neutral pH, and may be a domain that has the ability to directly or indirectly bind to FcRn. In one particular embodiment of the invention, it may be preferred that the antibody specified in section A or B of the description has, for example, increased FcRn binding activity under neutral pH conditions compared to a reference antibody containing a constant region of native IgG (see. WO 2013/046722). To make a comparison of the FcRn binding activity of two antibodies, it is preferable (but not limited to only indicated) that the antibody specified in section A or B of the description, and the reference antibody, which contains the constant region of native IgG, have identical amino acid sequences in regions (for example, variable region), preferably other than the constant region of the antibody specified in section A or B of the description, in which one or more amino acid residues have been modified.

In one of the embodiments of the invention, in which the antibody specified in section A of the description, has increased FcRn-binding activity under conditions of neutral pH, then, without going into a particular theory, it can be assumed that the antibody specified in section A of the description may have two or more of the following properties: the ability to shuttle between the plasma and the cell endosome and re-bind to multiple antigens in a single antibody molecule due to the presence of an ion concentration-dependent antigen-binding domain; the ability to quickly penetrate into cells due to increased pI and increased total positive antibody charge; and the ability to quickly enter cells due to increased FcRn-binding activity under neutral pH conditions. As a result, the plasma half-life of the antibody can be further shortened, or the binding activity of the antibody to the extracellular matrix can be further increased, or the elimination of antigen from the plasma can be further enhanced. One of ordinary skill in the art can use conventional techniques to determine the pI value of an antibody that is optimal for these properties.

Sections A and B of the present disclosure indicate that according to Yeung et al., J. Immunol. 182, 2009, pp. 7663-7671, the activity of native human IgG1 in relation to binding to human FcRn is characterized by a KD value of 1.7 mM in the acidic pH range (pH 6.0), and in the neutral pH range the activity is practically not measurable. Thus, to increase FcRn binding activity in the neutral pH range, it may be preferable to use as the antibody specified in section A or B of the description: an antibody or a constant region variant or an Fc region variant, which has the ability to bind human FcRn in an acidic pH range characterized by a KD of 20 μM or greater and in which the ability to bind human FcRn in the neutral pH range is comparable or stronger than that of native human IgG; preferably an antibody or constant region variant or Fc region variant in which the ability to bind human FcRn in the acidic pH range has a KD of 2.0 μM or is stronger and in which the ability to bind human FcRn in the neutral pH range has a KD value of 40 μM or is stronger; and more preferably an antibody or constant region variant or Fc region variant in which the ability to bind human FcRn in the acidic pH range is characterized by a KD of 0.5 μM or greater and in which the ability to bind human FcRn in the neutral pH range is characterized by a KD of 15 μM or is stronger. KD values can be determined using the method described by Yeung et al., J. Immunol. 182, 2009, pp. 7663-7671 (by immobilizing an antibody on a chip and adding human FcRn as an analyte).

In the context of sections A and B of the present description, as the FcRn-binding domain, a domain of any structure that binds to FcRn can be used. In this case, the FcRn binding domain can be obtained without the need for introducing an amino acid modification, or the affinity for FcRn can be increased by introducing an additional modification.

In the context of sections A and B of the present description, the original FcRn binding domain preferably includes, for example, an Fc region or constant region of (human) IgG. If a variant of the original Fc region or the original constant region can bind to human FcRn in the acidic pH range and / or neutral pH range, then any Fc region or constant region can be used as the original Fc region or the original constant region. Or an Fc region or a constant region obtained by further modification of the original Fc region or the original constant region in which the amino acid residue (s) have already been modified with respect to the Fc region or constant region can also be used, respectively, as Fc-region or constant region. The original Fc region or the original constant region may include known Fc regions obtained by recombination. The source Fc region or the original constant region, depending on the context, can mean the polypeptide itself, a composition containing the original Fc region or the original constant region, or an amino acid sequence corresponding to the original Fc region or the original constant region. The source of the original Fc region or the original constant region is not limited to a specific source and can be obtained from any animal organism other than humans or from a human organism. In addition, the original FcγR-binding domain can be obtained from cynomlgus monkeys, marmosets, rhesus monkeys, chimpanzees or humans. The Fc starting region or the starting constant region can preferably be derived from human IgG1; however, they are not limited to a specific IgG class. This means that the Fc region of human IgG1, IgG2, IgG3 or IgG4 can be used as a suitable starting FcRn binding domain, and it also means that the Fc region or constant region of an IgG class or subclass derived from any organism, preferably can be used as the original Fc-region or the original constant region. Examples of a variant or modified form of native IgG are described in the published literature, for example, Strohl, Curr. Opin. Biotechnol. 20 (6), 2009, pp. 685-691; Presta, Curr. Opin. Immunol. 20 (4), 2008, pp. 460-470; Davis et al., Protein Eng. Des. Sel. 23 (4), 2010, pp. 195-202; in WO 2009/086320, WO 2008/092117; WO 2007/041635; and WO 2006/105338.

In the context of sections A and B of the present description, amino acid residues of the original FcRn binding domain, the original Fc region or the original constant region may contain, for example, one or more mutations: for example, substitutions of amino acid residues that differ from residues in the original Fc region, or original constant region; insertion of one or more amino acid residues into amino acid residues in the original Fc region or the original constant region; or deletion of one or more amino acid residues from the original Fc region or the original constant region. The amino acid sequences of the Fc regions or constant regions after modifications may preferably be amino acid sequences containing at least a portion of the Fc region or constant region that does not occur naturally. Such variants should be identical or have less than 100% sequence similarity with the original Fc regions or the original constant regions. For example, variants have an amino acid sequence identical or similar at about 75% to less than 100%, more preferably about 80% to less than 100%, even more preferably about 85% to less than 100%, even more preferably about from 90% to less than 100%, and even more preferably from about 95% to less than 100%, of the amino acid sequence of the original Fc region or the original constant region. In an example that does not limit the scope of the invention, at least one amino acid in the modified Fc region or constant region indicated in section A or B of the description is different from the amino acid in the original Fc region or the original constant region.

In the context of sections A and B of this description, an Fc region or constant region that has FcRn binding activity in the acidic pH range and / or neutral pH range can be obtained by any method. In particular, a variant Fc region or constant region that has FcRn binding activity in an acidic pH range and / or a neutral pH range can be obtained by modifying the amino acids of a human IgG-type antibody, which can be used as a parent Fc region or a parent constant area. Fc regions of an IgG-type antibody or constant regions of an IgG-type antibody suitable for modification may include, for example, Fc regions or constant regions of human IgG (IgG1, IgG2, IgG3 or IgG4 or variants thereof) and mutants spontaneously formed from them. For Fc regions or constant regions of antibodies in the form of human IgG1, human IgG2, human IgG3 or human IgG4, a variety of IgG allotype sequences associated with genetic polymorphism are described in “Sequences of immunological interest)), NIH publication No. 91-3242, and any of them can be used in section A or B of the description. In particular, in the case of human IgGl, the amino acid sequence at positions 356-358 according to EU numbering can be either DEL or EEM.

In one of the embodiments of the invention described in section A or B of the description, the modification of other amino acids is not limited to the specified, if the variants have FcRn-binding activity in the acidic pH range and / or neutral pH range. Amino acid modification sites to increase FcRn binding activity under neutral pH conditions are described, for example, in WO 2013/046722. These sites of modification include, for example, one or more positions selected from the group consisting of: positions 221-225, 227, 228, 230, 232, 233-241, 243-252, 254-260, 262-272, 274, 276, 278-289, 291-312, 315-320, 324, 325, 327-339, 341, 343, 345, 360, 362, 370, 375-378, 380, 382, 385 to 387, 389, 396, 414, 416, 423, 424, 426-438, 440 and 442, according to EU numbering in the Fc region or constant region of a human IgG type antibody as described in WO 2013/046722. WO 2013/046722 also discloses as part of preferred modifications in the Fc region or constant region, for example, modification of one or more amino acids selected from the group consisting of: amino acid at position 256 on Pro, amino acid at position 280 on Lys, amino acids at position 339 on Thr, amino acids at position 385 on His, amino acids at position 428 on Leu and amino acids at position 434 on Trp, Tyr, Phe, Ala or His, according to EU numbering. The number of amino acids to be modified is not limited, and modification can be carried out at only one position or at two or more positions. Modification of these amino acid residues can increase the binding capacity of FcRn at the Fc region or constant region of an IgG-type antibody under neutral pH conditions. Modification of these amino acid residues can be introduced, respectively, into the antibodies specified in section A or B of the description.

In a further or alternative embodiment, the corresponding amino acid modification sites can also be used to increase the FcRn binding activity under acidic pH conditions. Of these sites of modification, one or more sites of modification that increase the binding of FcRn also in the neutral pH range can be used, respectively, in section A or B of the description. These sites of modification include, for example, those described in WO 2011/122011, WO 2013/046722, WO 2013/046704 and WO 2013/046722. The amino acid sites for the specified modification of the constant region or Fc region of a human IgG-type antibody and the types of amino acids after modification are described in Table 1 in WO 2013/046722. WO 2013/046722 also describes as particularly preferred sites of modification in the constant region or Fc region, for example, located at one or more amino acid positions selected from the group consisting of positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436 according to EU numbering. Modification of amino acid residues at these positions can also increase binding to human FcRn of the FcRn binding domain in the neutral pH range. WO 2013/046722 also discloses as part of preferred modifications in the constant region or Fc region of the IgG type, for example, replacing one or more amino acid residues selected from the group consisting of: (a) amino acid at position 237 to Met; (b) the amino acid at position 238 on Ala; (c) the amino acid at position 239 on Lys; (d) the amino acid at position 248 on Ile; (e) the amino acid at position 250 to any of Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, and Tyr; (e) the amino acid at position 252 to any of Phe, Trp and Tyr; (g) amino acids at position 254 on Thr; (h) the amino acid at position 255 on the Glu; (i) the amino acid at position 256 to any of Asp, Glu, and Gin; (j) the amino acid at position 257 to any of Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, and Val; (k) the amino acid at position 258 on His; (m) amino acids at position 265 on Ala; (m) the amino acid at position 270 on Phe; (o) the amino acid at position 286 on either Ala or Glu; (p) the amino acid at position 289 on His; (p) the amino acid at position 297 on Ala; (c) the amino acid at position 298 on Gly; (t) the amino acid at position 303 on Ala; (y) the amino acid at position 305 on Ala; (f) the amino acid at position 307 to any of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, and Tyr; (x) the amino acid at position 308 to any of Ala, Phe, Ile, Leu, Met, Pro, Gin, and Thr; (c) the amino acid at position 309 to any of Ala, Asp, Glu, Pro, and Arg; (h) the amino acid at position 311 to any of Ala, His and Ile; (y) the amino acid at position 312 either on Ala or on His; (y) the amino acid at position 314 on either Lys or Arg; (e) the amino acid at position 315 on either Ala or His; (aa) the amino acids at position 317 on Ala; (ab) the amino acid at position 325 on Gly; (ab) the amino acid at position 332 on Val; (ah) the amino acid at position 334 on Leu; (ad) amino acid at position 360 on His; (ae) the amino acids at position 376 on Ala; (ah) the amino acid at position 380 on Ala; (aza) the amino acid at position 382 on Ala; (ai) the amino acid at position 384 on Ala; (a) the amino acids at position 385 on either Asp or His; (al) the amino acid at position 386 on Pro; (a) the amino acid at position 387 on Glu; (an) the amino acid at position 389 on either Ala or Ser; (ao) the amino acids at position 424 on Ala; (ap) the amino acid at position 428 to any of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp and Tyr; (ar) the amino acid at position 433 on Lys; (ac) amino acids at position 434 on Ala, Phe, His, Ser, Trp and Tyr; and (at) the amino acid at position 436 on His; according to EU numbering. The number of amino acids that can be modified is not limited, and it is possible to modify an amino acid at only one position or amino acids at two or more positions. Combinations of amino acid modifications at two or more positions are shown in Table 2 in WO 2013/046722. Modification of these amino acid residues can also be introduced, respectively, into the antibodies specified in sections A and B of the description.

In one embodiment, the FcRn binding activity of the FcRn binding domain of the antibody specified in section A or B of the description is increased compared to the activity of a reference antibody containing the original Fc region or the original constant region of native IgG, or compared to the activity a reference antibody containing the original Fc region or the original constant region. Thus, the FcRn-binding activity of a variant of the Fc region or a variant of the constant region specified in section A or B of the description, or an antibody containing the specified variant, is higher than that of a reference antibody. This may mean that when comparing the FcRn-binding activity with the activity of the reference antibody, the activity of the antibody specified in section A or B of the description may be, for example: 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 100% or more, 105% or more, preferably 110% or more, 115% or more, 120 % or more, 125% or more, also preferably 130% or more, 135% or more, 140% or more, 145% or more, 150% or more, 155% or more, 160% or more, 165% or more , 170% or more, 175% or more, 180% or more, 185% or more, 190% or more, 195% or more, or more than 2 or more, 2.5 or more, 3 or more, 3, 5 or more, 4 or more, 4.5 or more, or 5 times or more.

In one embodiment of the invention, the amino acid sequences to be modified in the antibody specified in section A or B of the description may preferably contain human sequences (sequences present in native human derived antibodies) in order not to increase the immunogenicity of the antibody when the antibody is administered. in vivo (preferably in the human body). Alternatively, after modification, mutations can be introduced at positions other than the amino acid modification sites so that one or more FRs (FR1, FR2, FR3 and FR4) are replaced with a human sequence. Methods for replacing FRs with a human sequence are known in the art and include, but are not limited to, those described by Ono et al., Mol. Immunol. 36 (6), 1999, pp. 387-395. Humanization methods are known in the art and include, but are not limited to, those described in: Methods 36 (1), 2005, pp. 43-60).

In one embodiment, the framework sequences (also referred to as “FR sequences) of the heavy chain and / or variable region of the light chain of the antibody described in section A or B of the description may comprise human germline framework sequences. When the framework sequences are fully human germline sequences, the antibody is expressed to induce little or no immunogenic response when administered to humans (eg, to treat or prevent a particular disease).

FR sequences can preferably include, for example, fully human FR sequences, such as those shown in the V-Base (vbase.mrc-cpe.cam.ac.uk/). These FR sequences can be used respectively in section A or B of the description. Germline sequences can be categorized according to their similarity (Tomllnson et al., J. Mol. Biol. 227, 1992, pp. 776-798; Williams et al., Eur. J. Immunol. 23, 1993, pp. 1456-1461; and Cox et al., Nat. Genetics 7, 1994, pp. 162-168). Preferred germline sequences can be suitably selected from the Vκ group, which is subdivided into seven subgroups; the group Vλ, which is subdivided into ten subgroups; and group VH, which is subdivided into seven subgroups.

Fully human VH sequences may preferably include, for example, VH sequences from: VH1 subgroup (e.g., VH1-2, VH1-3, VH1-8, VH1-18, VH1-24, VH1-45, VH1-46, VH1 -58 and VH1-69); subgroups VH2 (for example, VH2-5, VH2-26 and VH2-70); subgroups VH3 (VH3-7, VH3-9, VH3-11, VH3-13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-30, VH3-33, VH3-35 , VH3-38, VH3-43, VH3-48, VH3-49, VH3-53, VH3-64, VH3-66, VH3-72, VH3-73 and VH3-74); VH4 subgroups (VH4-4, VH4-28, VH4-31, VH4-34, VH4-39, VH4-59 and VH4-61); subgroups VH5 (VH5-51); subgroups VH6 (VH6-1); or subgroups VH7 (VH7-4 and VH7-81). They are also described, for example, in Matsuda et al., J. Exp. Med. 188, 1998, pp. 1973-1975), and accordingly, ordinary specialists in this field can create antibodies based on information about these sequences. It may be preferable to use a different fully human FR sequence or sequences of regions equivalent to it.

Fully human Vκ sequences may preferably include, for example: A20, A30, L1, L4, L5, L8, L9, L11, L12, L14, L15, L18, L19, L22, L23, L24, O2, O4, O8, O12 , O14 or O18, which are classified as subgroup Vk1; A1, A2, A3, A5, A7, A17, A18, A19, A23, O1 and O11, which are classified as subgroup Vk2; A11, A27, L2, L6, L10, L16, L20 and L25, which are classified as subgroup Vk3; B3, which is classified as a subgroup Vk4; B2 (also referred to as "Vk5-2"), which is classified as a subgroup Vk5; or A10, A14 and A26, which are classified as a subgroup of Vk6 (Kawasaki et al., Eur. J. Immunol. 31, 2001, pp. 1017-1028; Hopre Seyler Biol. Chem. 374, 1993, pp. 1001-1022 ; Brensing-Kuppers et al., Gene 191, 1997, pp. 173-181).

Fully human Vλ sequences may preferably include, for example: V1-2, V1-3, V1-4, V1-5, V1-7, V1-9, V1-11, V1-13, V1-16, V1-17 , V1-18, V1-19, V1-20 and V1-22, which are classified as subgroup VL1; V2-1, V2-6, V2-7, V2-8, V2-11, V2-13, V2-14, V2-15, V2-17 and V2-19, which are classified as subgroup VL2; V3-2, V3-3 and V3-4, which are classified as subgroup VL3; V4-1, V4-2, V4-3, V4-4 and V4-6, which are classified as subgroup VL4; or V5-1, V5-2, V5-4 and V5-6, which are classified as subgroup VL5 (Kawasaki et al., Genome Res. 7, 1997, cc. 250-261).

Typically, these FR sequences differ from each other by one or more amino acid residues. These FR sequences can be used to modify the amino acid residues of antibodies. Fully human FR sequences that can be used for modification also include, for example, KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (see, for example, cited above Kabat et al., 1991; Wu et al. ., J. Exp. Med. 132, 1970, pp. 211-250).

In the context of sections A and B of the present description, the term "flexible residues" can refer to variations in amino acid residues that are present in positions with a high diversity of amino acids, where the variable regions of the light chain or heavy chain have several different amino acids compared to amino acid sequences of known and / or native antibodies or antigen binding domains. Highly diverse positions are usually located in the CDR. The data presented in Kabat, Sequences of Proteins of Immunological Interest (National Institute of Health Bethesda Md.) (1987 and 1991), can effectively determine these positions with high diversity in known and / or native antibodies. In addition, several databases on the Internet (vbase.mrc-cpe.cam.ac.uk/, bioinf.org.uk/abs/index.html) contain collections of sequences of numerous human light chains and heavy chains and their localization. Information about these sequences and locations is valuable for locating flexible residues. For example (but not limited to) when the amino acid residue at a particular position has variability, preferably 2-20, 3-19, 4-18, 5-17, 6-16, 7-15, 8-14, 9-13 or 10-12 amino acid residues, this position can be considered as characterized by (high) diversity.

In an embodiment of the invention, it should be apparent that when an antibody referred to in section A or B of the description contains all or part of the variable region of the light chain and / or variable region of the heavy chain, the antibody may contain, if necessary, one or more corresponding flexible residues. For example, a heavy chain and / or light chain variable region sequence selected to have an FR sequence that initially contains amino acid residues that alter the antigen-binding activity of an antibody depending on ion concentration conditions (hydrogen ion concentration or calcium ion concentration), can be designed so that it contains other amino acid residues in addition to the specified amino acid residues. In this case, for example, the amount and localization of flexible residues can also be determined without being limited to a specific embodiment of the invention, if the antigen-binding activity of the antibody specified in section A or B of the description varies depending on the conditions of ion concentration. In particular, the CDR sequence and / or FR sequence of the heavy chain and / or light chain may contain at least one flexible residue. For example, if the concentration of ions is the concentration of calcium ions, then flexible residues that can be introduced into the sequence of the variable region of the light chain (indicated above as Vk5-2) include, but are not limited to, one or more amino acid residues at the positions indicated in Table 1 or Table 2. Similarly, the corresponding flexible residues can be introduced, for example, in an ion concentration dependent antibody or in an antibody not having the indicated ion concentration dependence, containing all or part of the light chain variable region and / or the heavy variable region chain, in which at least one amino acid residue that can be displayed on the surface of the antibody, modified to increase the pI.

(positions indicated according to Cabot numbering)

In one embodiment, when the antibody is a humanized chimeric antibody, the pI of the chimeric antibody is increased by modifying one or more amino acid residues that can be displayed on the surface of the antibody to produce a humanized antibody as described in section A or B of the description with a shortened time plasma half-life compared to a chimeric antibody lacking the indicated modification. Modification of amino acid residues that can be displayed on the surface of a humanized antibody can be carried out prior to or simultaneously with humanizing the antibody. Alternatively, by using the humanized antibody as a starting material, amino acid residues that can be exposed on the surface can be modified to further alter the pI of the humanized antibody.

Adams et al at Cancer Immunol. Immunother. 55 (6), 2006, pp. 717-727, describe that humanized antibodies trastuzumab (antigen: HER2), bevacizumab (antigen: VEGF), and pertuzumab (antigen: HER2), which were humanized using the same FR sequences of a human antibody, turned out to be practically comparable in plasma pharmacokinetics. In particular, it can be assumed that plasma pharmacokinetics is practically comparable when humanization is performed using the same FR sequences. According to one embodiment of the invention described in section A of the description, the concentration of antigen in plasma is reduced by increasing the pI of the antibody by modifying amino acid residues that can be displayed on the surface of the antibody, in addition to the humanization step. In an alternative embodiment of the invention specified in section A or B of the description, you can use human antibodies. By modifying amino acid residues that can be displayed on the surface of a human antibody obtained from a human antibody library, using a human antibody producing mouse, a recombinant cell, etc. and increasing the pI of the human antibody, the ability of the original obtained human antibody to eliminate antigen from plasma can be increased.

In one embodiment, the antibodies described in section A of the description may contain modified sugar chains. Antibodies with modified sugar chains include, for example, antibodies with modified glycosylation (WO 99/54342), antibodies lacking fucose (WO 00/61739; WO 02/31140, WO 2006/067847; WO 2006/067913), and antibodies having sugar chains that are bisectioned with GlcNAc (WO 02/79255).

In one embodiment of the invention, the antibodies specified in section A or B of the description can be used, for example, in methods to achieve increased antitumor activity against cancer cells or in methods of enhancing the elimination of antigens harmful to the body from plasma.

An alternative embodiment of the invention referred to in section A or B of the description refers to libraries of ion concentration-dependent antigen binding domains with increased pI or concentration-dependent antibodies with increased pI as described above.

An alternative embodiment of the invention specified in section A or B of the description relates to nucleic acids (polynucleotides) encoding the above-described concentration-dependent antigen-binding domains with increased pI or depending on the concentration of antibodies with increased pI. In a specific embodiment of the invention, nucleic acids can be obtained using appropriate known methods. Specific variants thereof are described, for example, in WO 2009/125825, WO 2012/073992, WO 2011/122011, WO 2013/046722, WO 2013/046704, WO 2000/042072, WO 2006/019447, WO 2012/115241, WO 2013 / 047752, WO 2013/125667, WO 2014/030728, WO 2014/163101, WO 2013/081143, WO 2007/114319, WO 2009/041643, WO 2014/145159, WO 2012/016227 and WO 2012/093704, each of which is fully incorporated into this description by reference.

In one embodiment, the nucleic acids described in section A or B of the description may be isolated or purified nucleic acids. The nucleic acids encoding the antibodies mentioned in section A or B of the description can be any genes and can be DNA or RNA, or other nucleic acid analogs.

In the context of sections A and B of the present description, when the amino acids of the antibody are modified, the amino acid sequence of the antibody prior to modification may be the known sequence or the amino acid sequence of the newly produced antibody. For example, antibodies can be obtained from antibody libraries or by cloning nucleic acids that encode the antibody from hybridomas or B cells that produce monoclonal antibodies. Methods for obtaining nucleic acids encoding an antibody from hybridomas that can be used include: performing immunization by conventional immunization methods using the antigen of interest or cells expressing the antigen of interest as the sensitizing antigen; fusion of the resulting immune cells with known parent cells using conventional fusion techniques; screening monoclonal antibody-producing cells (hybridomas) using conventional screening methods; synthesis of cDNA variable region (V region) of the antibody using reverse transcriptase from mRNA of the resulting hybridomas; and linking cDNA to DNA encoding the constant region (C region) of the antibody of interest.

Sensitizing antigens that are used to produce nucleic acids encoding the above-described heavy chain and light chain include, but are not limited to, both complete antigens that are immunogenic and incomplete antigens, including haptens that are not immunogenic. For example, complete proteins of interest or peptides that are portions of proteins can be used. In addition, it is known that potential antigens are substances that consist of polysaccharides, nucleic acids, lipids, and other compositions. Thus, in some embodiments of the invention, the antigens for the antibodies specified in section A or B of the description are not limited to specific antigens. Antigens can be obtained, for example, by methods based on the use of baculoviruses (see, for example, WO 98/46777). Hybridomas can be obtained, for example, according to the method of G. Kohler and C. Milstein, Methods Enzymol. 73, 1981 pp. 3-46. When the immunogenicity of the antigen is low, immunization can be carried out by binding the antigen to an immunogenic macromolecule such as albumin. Alternatively, if desired, soluble antigens can be obtained by binding the antigen to other molecules. When a transmembrane molecule such as membrane antigens (eg, a receptor) is used as an antigen, a portion of the extracellular region of the membrane antigen can be used as a fragment, or a cell expressing the transmembrane molecule on their surface can be used as an immunogen.

In some embodiments, antibody-producing cells can be obtained by immunizing an animal with the appropriate sensitizing antigen described above. Alternatively, antibody-producing cells can be obtained by in vitro immunization of lymphocytes that have the ability to produce antibodies. A variety of mammals can be used for immunization and other routine antibody production procedures. Commonly used animals include rodents, lagomorphs and primates. Animals can be, for example, rodents such as mice, rats and hamsters; lagomorphs such as rabbits; and primates including monkeys such as cynomolgus monkeys, rhesus monkeys, baboons, and chimpanzees. In addition, transgenic animals carrying a spectrum of human antibody genes are also known, and these animals can be used to produce human antibodies (see, for example, WO 96/34096; Mendez et al., Nat. Genet. 15, 1997, pp. 146 -156; WO 93/12227, WO 92/03918, WO 94/02602, WO 96/34096 and WO 96/33735). Instead of using these transgenic animals, the desired human antibodies with antigen-binding activity can also be produced by, for example, sensitizing human lymphocytes in vitro with the desired antigens or cells expressing the desired antigens, and then fusing the sensitized lymphocytes with human myeloma cells such as U266 (publication Japanese Patent No. H01-59878).

Immunization of animals can be carried out, for example, by appropriate dilution and suspension of the sensitizing antigen in phosphate buffered saline (PBS), saline or other media and, if necessary, mixing with an adjuvant for emulsification; and subsequent injection intraperitoneally or subcutaneously in animals. The sensitizing antigen mixed with Freund's incomplete adjuvant can then preferably be administered several times every 4 to 21 days. The production of antibodies can be confirmed, for example, by measuring the titer of the antibody of interest in the serum of the animal.

Antibody-producing cells derived from lymphocytes or animals immunized with the desired antigen can be fused to myeloma cells to create hybridomas using conventional fusion agents (e.g., polyethylene glycol) (Goding, Monolonal Antibodies: Principles and Practice, Academic Press, 1986 , pp. 59-103). If necessary, the hybridomas are cultured and propagated and the binding specificity of antibodies produced by the hybridomas is assessed, for example, by immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA). Then, if necessary, antibody-producing hybridomas that have been determined to have specificity, affinity, or activity of interest can also be subcloned using methods such as dilution.

Nucleic acids encoding a selected antibody can be cloned from hybridomas or antibody-producing cells (sensitized lymphocytes, etc.) using probes that can specifically bind to the antibody (eg, oligonucleotides complementary to sequences that encode antibody constant regions). Alternatively, nucleic acids can be cloned from mRNA using RT-PCR. Heavy chains and light chains for use in the production of antibodies described in section A or B of the description can be obtained from antibodies, which, for example, belong to any class and subclass of Ig-type antibodies, and IgG may be preferred.

In one embodiment of the invention, nucleic acids encoding amino acid sequences that form the heavy chain (full or part thereof) and / or light chain (full or part thereof) of the antibody specified in section A or B of the description, for example, are modified using methods genetic engineering. Recombinant antibodies with artificial sequence modification, for example, to reduce heterologous antigenicity in humans, such as chimeric antibodies or humanized antibodies, can be suitably generated by, for example, modification of nucleotide residues encoding amino acid sequences associated with antibody components such as mouse antibodies, rat antibodies, rabbit antibodies, hamster antibodies, sheep antibodies, or camel antibodies. Chimeric antibodies can be obtained, for example, by ligating DNA encoding the variable region of a murine antibody with DNA encoding a constant region of a human antibody and inserting the coding sequence of the resulting ligated DNA into an expression vector, then introducing the resulting recombinant vector into a host for gene expression ... Humanized antibodies, which are also referred to as reshaped human antibodies, are antibodies in which FRs of a human antibody are linked in frame with the CDRs of an antibody isolated from a non-human mammal, such as a mouse, to form a coding sequence. The DNA sequence encoding said humanized antibody can be synthesized by overlapping extension PCR using a number of oligonucleotides as templates. Materials and experimental methods for performing PCR with overlapping extensions are described in WO 98/13388 et al. For example, DNA encoding an amino acid sequence, for example, the variable region of an antibody specified in section A or B of the description, can be obtained using PCR with overlapping extensions. using a number of oligonucleotides designed so that they have overlapping nucleotide sequences. The overlapping DNAs are then ligated in frame with DNA encoding the constant region to obtain the coding sequence. DNA ligated by the above method can then be inserted into an expression vector so that the DNA can be expressed and the resulting vector can be introduced into a host or host cell. The antibody encoded by the DNA can be expressed by growing the host or culturing the host cells. The expressed antibody can be purified, respectively, from the culture medium of the host or others (EP 239400; WO 96/02576). In addition, the FRs of the humanized antibody fused through the CDRs can be selected, for example by allowing the CDRs to form an antigen binding site suitable for the antigen. If necessary, the amino acid residues that form the FR of the variable region of the selected antibody, for example, can be modified by appropriate substitution.

In one embodiment of the invention, nucleic acid cassettes can be cloned into appropriate vectors to express the antibodies specified in section A or B of the description, or fragments thereof. Several types of vectors are available for this purpose, such as phagemid vectors. In general, phagemid vectors can contain various elements, including regulatory sequences such as promoters or signal sequences, genes for phenotype selection, replication initiation sites, and other necessary elements.

Methods for introducing the desired amino acid modifications into antibodies are known in the art. For example, libraries can be constructed by introducing at least one modified amino acid residue that can be displayed on the surface of the antibodies specified in section A or B of the description, and / or at least one amino acid that can change the antigen-binding activity of antibodies depending on concentration conditions ions. Additionally, if necessary, you can add flexible residues using the method described in Kunkel et al., Methods Enzymol. 154, 1987, pp. 367-382.

An alternative embodiment of the invention referred to in Section A of the specification are vectors containing nucleic acids that encode the above-described concentration-dependent antigen binding domain with an increased pI or the above-described concentration-dependent antibody with an increased pI. In a specific embodiment of the invention, vectors can be obtained, for example, similar to the vectors described in WO 2009/125825, WO 2012/073992, WO 2011/122011, WO 2013/046722, WO 2013/046704, WO 2000/042072, WO 2006/019447 , WO 2012/115241, WO 2013/047752, WO 2013/125667, WO 2014/030728, WO 2014/163101, WO 2013/081143, WO 2007/114319, WO 2009/041643, WO 2014/145159, WO 2012/016227 or WO 2012/093704, each of which is fully incorporated into this description by reference.

In one embodiment of the invention, nucleic acids encoding the variants described in section A or B of the description can be functionally cloned (inserted) into appropriate vectors and introduced into host cells. For example, when E. coli is used as a host, vectors include a cloning vector, a pBluescript vector (Stratagene), or any of various commercially available vectors.

In one embodiment of the invention, expression vectors are used as vectors containing the nucleic acid specified in section A or B of the description. Expression vectors can be used to provide in vitro expression of the polypeptide in E. coli, in cell cultures, or in vivo. For example, the pBEST vector (Promega) can be used for in vitro expression; vector pET (Invitrogen) for expression in E. coli; vector pME18S-FL3 (GenBank, accession no. AB009864) for expression in cell culture; and vector pME18S (Takebe et al., Mol. Cell Biol. 8, 1988, pp. 466-472) for in vivo expression. DNA can be inserted into vectors by conventional techniques, for example, by reaction in the presence of ligase using sites recognized by restriction endonucleases (see Current protocols in Molecular Biology, ed. Ausubel et al., Publish. John Wiley & Sons. Section 11.4-11.11, 1987).

An alternative embodiment of the invention referred to in section A of the specification is a host or host cells that contain a vector containing a nucleic acid that encodes the above-described concentration-dependent antigen binding domain with an increased pI or the above-described concentration-dependent antibody with an increased pI. In a specific embodiment, the host or host cells can be obtained, for example, by the methods described in WO 2009/125825, WO 2012/073992, WO 2011/122011, WO 2013/046722, WO 2013/046704, WO 2000/042072, WO 2006/019447, WO 2012/115241, WO 2013/047752, WO 2013/125667, WO 2014/030728, WO 2014/163101, WO 2013/081143, WO 2007/114319, WO 2009/041643, WO 2014/145159, WO 2012/016227 or WO 2012/093704, each of which is fully incorporated into this description by reference.

The type of host cell mentioned in section A or B of the description is not limited to a specific type, and host cells include, for example, bacterial cells such as E. coli, as well as various animal cells. Host cells can suitably be used as production systems for the production and expression of antibodies. Both eukaryotic and prokaryotic cells can be used.

Eukaryotic cells that can be used as host cells include, for example, animal cells, plant cells, and fungal cells. Examples of animal cells include mammalian cells, for example, CHO (Puck et al., J. Exp. Med. 108, 1995, pp. 945-956), COS, HEK293, 3T3, myeloma cells, BHK (baby hamster kidney cells), HeLa and Vero; amphibian cells, for example Xenopus oocyte (Valle et al., Nature 291, 1981, pp. 338-340); and insect cells such as Sf9, Sf21 and Tn5. Recombinant vectors or other vectors can be introduced into host cells, for example, using calcium phosphate-based methods, DEAE-dextran-based methods, DOTAP-liposome-based methods (Boehringer-Mannheim), electroporation, and lipofection.

Plant cells that are known to serve as a protein production system include, for example, cells derived from Nicotiana tabacum and cells derived from duckweed (Lemna minor). Calluses can be cultured from these cells to obtain the antibodies specified in section A or B of the description. Protein production systems based on fungal cells include systems based on the use of yeast cells, for example, cells of the genus Saccharomyces such as Saccharomyces cerevisiae and Schizosaccharomyces pombe; and cells of filamentous fungi, for example, of the genus Aspergillus, such as Aspergillus niger. When prokaryotic cells are used, bacterial cell-based production systems can be used. Bacterial cell-based production systems include, for example, Bacillus subtilis-based systems as well as E. coli.

To obtain the antibody specified in section A or B of the description, using the host cells, the host cells are transformed with an expression vector containing a nucleic acid that encodes the antibody specified in section A or B of the description, and cultured to express the nucleic acid. For example, when animal cells are used as hosts, culture media can include, for example, DMEM, MEM, RPMI1640 and IMDM, which can be used, respectively, in combination with the addition of a serum such as FBS or fetal bovine serum (FCS). Alternatively, cells can be cultured in serum-free medium.

Alternatively, animals or plants can be used as in vivo production systems to generate the antibodies specified in section A or B of the description. For example, the nucleic acid (s) encoding the antibody specified in section A or B of the description can be introduced into said animals or plants to produce the antibody in vivo, and the antibody can then be harvested from animals or plants.

When animals are used as the host, production systems based on mammals or insects are available. Preferred mammals include, but are not limited to, goats, pigs, sheep, mice and bulls (Vicki Glaser, SPECTRUM Biotechnology Applications, (1993)). You can also use transgenic animals.

In one example, the nucleic acid that encodes the antibody described in section A or B of the description is obtained as a fusion gene with a gene encoding a polypeptide that is a specific component of milk, such as goat β-casein. A polynucleotide fragment containing the fusion gene is then injected into the goat embryos and transplanted into a female goat. The antibody of interest can be obtained from milk produced in transgenic goats that are born from embryo goats or their offspring. Transgenic goats can be injected with appropriate hormones to increase the volume of milk containing the antibody produced by the goats (Ebert et al., Bio / Technology 12, 1994, pp. 699-702).

Insects used to produce the antibodies described in section A or B of the description include, for example, silkworms. When silkworms are used, a baculovirus with a polynucleotide encoding an antibody of interest is inserted into their genome to infect the silkworms. An antibody of interest can be obtained from the general water of the body of infected silkworms (Susumu et al., Nature 315, 1985, pp. 592-594).

When plants are used to generate the antibodies described in section A or B of the description, tobacco plants can be used. When tobacco plants are used, a recombinant vector resulting from the insertion of a polynucleotide that encodes an antibody of interest is inserted into a plant expression vector, for example pMON 530, and can be introduced into bacteria such as Agrobacterium tumefaciens. The resulting bacteria can be used to infect tobacco, for example Nicotiana tabacum (Ma et al., Eur. J. Immunol. 24, 1994, pp. 131-138) and the desired antibody can be obtained from the leaves of the infected tobacco. These modified bacteria can also be used to infect duckweed (Lemna minor) and the desired antibody can be obtained from cloned cells of the infected duckweed (Cox et al., Nat. Biotechnol. 24 (12), 2006, pp. 1591-1597).

In order to secrete an antibody that is expressed in host cells in the endoplasmic reticulum cavity, in the periplasmic space, or in the extracellular environment, suitable secretory signals can be incorporated into the polypeptide of interest. These signals may be endogenous to the antibody of interest, or may be a heterogeneous signal known in the art.

The antibody specified in section A or B of the description, obtained according to the methods described above, can be isolated from the inside or outside (for example, from media and milk) of host or host cells and purified to obtain a substantially pure and homogeneous antibody. Antibodies can be isolated and purified, for example, by appropriately selected and pooled chromatographic columns, filtration, ultrafiltration, desalting, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, and other methods. Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reversed phase chromatography, and adsorption chromatography. Said chromatography can be carried out, for example, using liquid chromatography such as HPLC and HPLC. Columns for use in affinity chromatography can be a protein A column or a protein G column. A protein A column includes, for example, Hyper D, POROS, Sepharose F.F. (Pharmacia).

The antibody can be modified, if necessary, or the peptide can be partially removed by treating the antibody with appropriate protein-modifying enzymes before or after purification of the antibody. As such protein-modifying enzymes, for example, trypsin, chymotrypsin, lysylendopeptidase, protein kinase and glucosidase can be used.

An alternative embodiment of the invention, specified in section A of the description, are methods for producing antibodies containing an antigen-binding domain, the antigen-binding activity of which varies depending on the conditions of ion concentration, which may include culturing host cells or growing hosts and collecting antibodies from cultures of these cells, materials secreted by the host, or by other routes known in the art.

One of the embodiments of the invention, specified in section A of the description, is a method of obtaining, which includes any one or more steps selected from the group consisting of: (a) selection of an antibody that can enhance the elimination of antigen from plasma; (b) selecting an antibody with increased binding activity against the extracellular matrix; (c) selecting an antibody with increased FcγR-binding activity under neutral pH conditions; (d) selecting an antibody with increased FcγRIIb binding activity under neutral pH conditions; (e) selecting an antibody with preserved or increased FcγRIIb binding activity and decreased binding activity against one or more activating FcγRs selected from the group consisting of FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb, and FcγRIIa; (e) selecting an antibody with increased FcRn binding activity under neutral pH conditions; (g) selection of antibodies with a specific pI; (h) confirming the pI of the collected antibody and then selecting the antibody with the increased pI; and (i) selecting an antibody whose antigen-binding activity changes or increases depending on the conditions of ion concentration compared to the reference antibody.

In the context of the present description, a reference antibody includes, but is not limited to, a native antibody (for example, a native Ig antibody, preferably a native IgG type antibody) and an antibody before modification (an antibody before or during library construction, for example, depending on the concentration of ions (antibody to an increase in its pI or an antibody with an increased pI to the introduction of a concentration-dependent antigen-binding domain).

After obtaining the antibodies specified in section A of the description, the formed antibodies can be investigated by analysis of the pharmacokinetics of antibodies using plasma, such as plasma of mice, rats, rabbits, dogs, monkeys, humans, to select antibodies with an increased ability to eliminate antigen from plasma compared to with a reference antibody.

Alternatively, after obtaining the antibodies described in section A of the description, the generated antibodies can be compared with a reference antibody in terms of the ability to bind to the extracellular matrix using electrochemiluminescence or other methods to select antibodies with increased ability to bind to the extracellular matrix.

Alternatively, after obtaining the antibodies specified in section A of the description, the generated antibodies can be compared with a reference antibody for binding activity to different FcγRs under neutral pH conditions using BIACORE® or other methods to select antibodies with increased binding activity against different FcγR under conditions of neutral pH. In this case, the different FcγRs may be of the FcγR type of interest, for example FcγRIIb. Similarly, antibodies can also be selected whose FcγRIIb binding activity (under neutral pH conditions) is maintained or increased, and their binding activity against one or more activating FcγRs selected and the group consisting of FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb and FcγRIIa and the like are reduced. In these cases, the FcγR may be a human FcγR.

Alternatively, after obtaining the antibodies specified in section A of the description, the resulting antibodies can be compared with a reference antibody for FcRn binding activity under neutral pH conditions using BIACORE or other known technologies to select antibodies with increased FcRn binding activity under neutral conditions. pH. In this case, the FcRn can be a human FcRn.

Alternatively, after obtaining the antibodies specified in section A of the description, the resulting antibodies can be determined by pI using isoelectric focusing or other methods to select antibodies with an increased pI compared to the reference antibody. In this case, you can select antibodies, the pI value of which is increased, for example, by at least 0.01, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4 or 0.5, or more or at least 0.6, 0.7, 0.8 or 0.9 or more; or antibodies, the pI value of which is increased by at least 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5 or more, or by at least 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 or more, or 3.0 or more.

Alternatively, after obtaining the antibodies specified in section A of the description, the generated antibodies can be compared with a reference antibody in terms of binding activity to the desired antigen under conditions of low and high ion concentration, using BIACORE or other methods to select antibodies whose antigen binding activity is changed or increases depending on the conditions of ion concentration. The ion concentration can be, for example, the concentration of hydrogen ions or the concentration of metal ions. When the ion concentration is the concentration of metal ions, it can be, for example, the concentration of calcium ions. Whether the binding activity changes or increases can be assessed by such traits as, for example: (a) altered or increased uptake of the antigen by cells; (b) altered or increased ability to bind to various antigen molecules several times; (c) an altered or increased decrease in plasma antigen concentration; or (d) altered plasma retention time of the antibody. Alternatively, any two or more of these selection methods can be combined if necessary.

An alternative embodiment of the invention, referred to in section A of the description, are methods for producing or screening antibodies that contain an antigen binding domain, the antigen binding activity of which varies depending on the conditions of ion concentration, and the pI of which is increased by modifying at least one amino acid residue that can be exposed on the surface of the antibody ("concentration-dependent antibodies with increased pI"). The methods of obtaining can be carried out, for example, by appropriate combining, if necessary, related embodiments of the invention specified in the context of section A of the present description, for example, a variant of the methods for obtaining or screening antibodies with increased pI described above, as well as a variant of methods for obtaining concentration-dependent calcium ions antigen-binding domains or calcium ion concentration-dependent antibodies, the antigen-binding activity of which is higher under conditions of high concentration of calcium ions than under conditions of low concentration of calcium ions, or their libraries described above, and / or a variant of methods for obtaining or screening pH-dependent antigen-binding domains, or pH-dependent antibodies, the antigen-binding activity of which is higher under neutral pH conditions than under acidic pH conditions, or their libraries described above.

An alternative embodiment of the invention specified in section A of the description is a method for producing or screening an antibody containing an antigen-binding domain, the ability of which to bind extracellular matrix is increased when its antigen-binding activity changes depending on the conditions of ion concentration and its pI is increased by modifying at least one an amino acid residue that can be exposed on the surface of an antibody ("concentration-dependent antibodies with an increased pI"). An increase in the pI of a concentration-dependent antibody may be included within the scope of the method. This method can be carried out, for example, by appropriately combining, if necessary, related embodiments of the invention described in the context of section A of the present description, for example, a variant of the method for obtaining or screening antibodies with an increased pI described above, as well as a variant of methods for obtaining or screening concentration-dependent ions calcium antigen-binding domains or calcium ion concentration-dependent antibodies, the antigen-binding activity of which is higher under conditions of high concentration of calcium ions than under conditions of low concentration of calcium ions, or their libraries described above, and / or a variant of methods for obtaining or screening pH-dependent antigen-binding domains or pH-dependent antibodies, the antigen-binding activity of which is higher under neutral pH conditions than under acidic pH conditions, or their libraries described above. For example, the resulting antibodies can be compared to a reference antibody in terms of their ability to bind extracellular matrix using electrochemiluminescence or other known techniques to select antibodies with increased ability to bind extracellular matrix.

In the context of the present description, a reference antibody includes, but is not limited to, a native antibody (for example, a native Ig antibody, preferably a native IgG type antibody) and an antibody before modification (an antibody before or during library construction, for example, depending on the concentration of ions (antibody to an increase in its pI or an antibody with an increased pI to the introduction of a concentration-dependent antigen-binding domain).

An alternative embodiment of the invention specified in section A of the description is a method for producing an antibody containing an antigen-binding domain, the antigen-binding activity of which varies depending on the conditions of ion concentration, where the method comprises modifying at least one amino acid residue that can be displayed on the surface of the antibody, such , which leads to an increase in the isoelectric point (pI). In some embodiments, the modification of an amino acid residue comprises a modification selected from the group consisting of: (a) replacing a negatively charged amino acid residue with an uncharged amino acid residue; (b) replacing a negatively charged amino acid residue with a positively charged amino acid residue; and (c) replacing an uncharged amino acid residue with a positively charged amino acid residue. In some embodiments, at least one modified amino acid residue is replaced with histidine. In other embodiments, the antibody comprises a variable region and / or a constant region, and the amino acid residue is modified in the variable region and / or constant region. In other embodiments of the invention, at least one amino acid residue modified according to the method is at a position in a CDR or FR selected from the group consisting of: (a) positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 18, 19, 23, 25, 26, 39, 41, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 77, 81, 82, 82a, 82b, 83, 84, 85, 86, 105, 108, 110 and 112 in the FR of the heavy chain variable region; (b) positions 31, 61, 62, 63, 64, 65, and 97 in the CDR of the heavy chain variable region; (c) positions 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 37, 38, 39, 41, 42, 43, 45, 46, 49, 57, 60, 63, 65, 66, 68, 69, 70, 74, 76, 77, 79, 80, 81, 85, 100, 103, 105, 106, 107 and 108 in the FR of the light chain variable region; and (d) positions 24, 25, 26, 27, 52, 53, 54, 55, and 56 in the CDR of the light chain variable region, according to Cabot's numbering. In further embodiments of the invention, at least one amino acid residue modified according to the method is at a position in a CDR or FR selected from the group consisting of: (a) positions 8, 10, 12, 13, 15, 16, 18, 23, 39, 41, 43, 44, 77, 82, 82a, 82b, 83, 84, 85 and 105 in the FR of the heavy chain variable region; (b) positions 31, 61, 62, 63, 64, 65 and 97 in the CDR of the variable region of the heavy chain; (c) positions 16, 18, 37, 41, 42, 45, 65, 69, 74, 76, 77, 79 and 107 in the FR of the light chain variable region; and (d) positions 24, 25, 26, 27, 52, 53, 54, 55 and 56 in the CDR of the light chain variable region. In some embodiments, the antigen is a soluble antigen. In some embodiments, the method further comprises comparing the KD of an antibody obtained according to the method for its corresponding antigen at acidic pH (eg, pH 5.8) and neutral pH (eg, pH 7.4). In additional embodiments, the method comprises selecting an antibody that has a KD (acidic pH range (e.g., pH 5.8)) / KD (neutral pH range (e.g., pH 7.4)) ratio for the antigen of 2 or greater ... In some embodiments, the method further comprises comparing the antigen-binding activity of an antibody prepared according to the method under conditions of high ion concentration (eg, hydrogen or calcium ion concentration) and low ion concentration. In other embodiments, the method further comprises selecting an antibody that has a higher antigen-binding activity at a high ion concentration (eg, 2 times) than at a low ion concentration. In some embodiments of the invention, in which the ion concentration is a calcium ion concentration, the high calcium ion concentration can be selected from a concentration ranging from 100 μM to 10 mM, from 200 μM to 5 mM, from 400 μM to 3 mM, from 200 μM and up to 2 mM, or from 400 μM and up to 1 mM. It may also be preferable to have a concentration selected from a concentration ranging from 500 μM to 2.5 mM, which is close to the plasma (blood) concentration of calcium ions in vivo. In some embodiments, the low calcium ion concentration can be selected from 0.1 μM to 30 μM, 0.2 μM to 20 μM, 0.5 μM to 10 μM, or 1 μM to 5 μM. , or from 2 μM to 4 μM. A concentration selected from 1 μM to 5 μM, which is close to the concentration of calcium ions in early endosomes in vivo, may also be preferred. In some embodiments, the lower limit of the ratio KD (low calcium ion concentration) / KX) (high calcium ion condition) (e.g., KD (3μM Ca) / KD (2 μM Ca)) is 2 or more, 10 or more or 40 or more, and the upper limit is 400 or less, 1000 or less, or 10,000 or less. In some alternative embodiments, the lower limit of the ratio kd (low calcium ion conditions) / kd (high calcium ion conditions) (e.g., kd (3 μM Ca) / kd (2 μM Ca)) is 2 or more, 5 or more , 10 or more, or 30 or more, and the upper limit is 50 or less, 100 or less, or 200 or less. In some embodiments, in which the ion concentration is a hydrogen ion concentration, a low hydrogen ion concentration (neutral pH range) can be selected from pH 6.7 to 10.0, pH 6.7 to 9.5, pH 7.0 to 9.0 or pH 7.0 to 8.0. The low concentration of hydrogen ions may preferably correspond to a pH of 7.4, which is close to the in vivo pH in plasma (blood), but for convenience of measurements, for example, pH 7.0 can be used. In some embodiments, the high hydrogen ion concentration (acidic pH range) can be selected from pH 4.0 to 6.5, pH 4.5 to 6.5, pH 5.0 to 6.5, or pH 5 .5 to 6.5. The acidic pH range may preferably correspond to a pH of 5.8, which is close to the in vivo hydrogen ion concentration in the early endosome, but pH 6.0, for example, can be used for ease of measurement. In some embodiments, the lower limit of the KD (acidic pH range) / KD (neutral pH range) ratio (e.g., KD (pH 5.8) / KD (pH 7.4)) is 2 or more, 10 or more, or 40 or more, and the upper limit is 400 or less, 1000 or less, or 10,000 or less. In some embodiments, the method further comprises comparing the elimination of the antigen from the plasma after administration of the antibody obtained according to the method with the elimination upon administration of a reference antibody that differs only in that it does not include the modification (s) introduced according to the method. In other embodiments, the method further comprises selecting an antibody obtained according to the method that enhances the elimination of antigen from plasma (eg, 2-fold) compared to an antibody that does not contain modifications introduced according to the method. In some embodiments, the method further comprises selecting an antibody obtained according to the method that has an increased ability to bind extracellular matrix as compared to an antibody that differs only in that it does not include the modification (s) introduced according to the method. In additional embodiments of the invention, the method also includes selecting an antibody obtained according to the method that has an increased ability to bind extracellular matrix (for example, 2-fold when binding to an antigen), compared to an antibody that differs only in that it does not include the modification (and ), introduced (s) according to the method. In other embodiments, the antibodies prepared according to the method (substantially) retain antigen-binding activity when compared to antibodies prior to modification or alteration of at least one amino acid residue to increase the pI (native antibodies (e.g., native Ig antibodies, preferably native IgG antibodies -type) or a reference antibody (for example, an antibody before modification or before or during library creation)). In this case, “(substantially) retains antigen binding activity” may mean having an activity of at least 50% or more, preferably 60% or more, more preferably 70% or 75% or more, and even more preferably 80%, 85%, 90% or 95% or more, compared to the binding activity of antibodies before modification or change.

An additional embodiment of the invention specified in section A of the description is a method for producing an antibody containing an antigen-binding domain, the antigen-binding activity of which varies depending on the conditions of ion concentration, where the method includes modifying at least one amino acid residue that can be displayed on the surface of the constant region of the antibody which raises the isoelectric point (pI). In some embodiments, the modification of an amino acid residue comprises a modification selected from the group consisting of: (a) replacing a negatively charged amino acid residue with an uncharged amino acid residue; (b) replacing a negatively charged amino acid residue with a positively charged amino acid residue; and (c) replacing an uncharged amino acid residue with a positively charged amino acid residue. In some embodiments, at least one modified amino acid residue is replaced with histidine. In additional embodiments, the antibody comprises a variable region and / or a constant region and the amino acid residue is modified in the variable region and / or constant region. In additional embodiments of the invention, at least one amino acid residue modified according to the method is at a position in the constant region selected from the group consisting of positions 196, 253, 254, 256, 258, 278, 280, 281, 282, 285 , 286, 307, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 389, 399, 400, 401 , 402, 413, 415, 418, 419, 421, 424, 430, 433, 434 and 443, according to the EU numbering. In additional embodiments of the invention, at least one amino acid residue modified according to the method is at a position in the constant region selected from the group consisting of positions 254, 258, 281, 282, 285, 309, 311, 315, 327, 330 , 342, 343, 345, 356, 358, 359, 361, 362, 384, 385, 386, 387, 389, 399, 400, 401, 402, 413, 418, 419, 421, 433, 434 and 443, according to EU numbering. In further additional embodiments of the invention, at least one amino acid residue modified according to the method is at a position in the constant region selected from the group consisting of positions 282, 309, 311, 315, 342, 343, 384, 399, 401, 402 and 413, according to EU numbering. In some embodiments, the method further comprises comparing the antigen-binding activity of an antibody prepared according to the method under conditions of high ion concentration (eg, hydrogen or calcium ion concentration) and low ion concentration. In additional embodiments, the method further comprises selecting an antibody that has a higher antigen-binding activity at a high ion concentration than at a low ion concentration. In some embodiments, the method comprises comparing the elimination of the antigen from the plasma after administration of an antibody obtained according to the method and after administration of a reference antibody, which differs only in that it does not include the modification (s) introduced according to the method. In additional embodiments, the method also includes selecting an antibody obtained according to the method that enhances the elimination of antigen from plasma (eg, 2-fold) compared to an antibody that does not contain the modifications introduced according to the method. In some embodiments, the method comprises comparing the binding to the extracellular matrix of an antibody obtained according to the method with an antibody that differs only in that it does not include the modification (s) introduced according to the method. In additional embodiments of the invention, the method also includes selecting an antibody obtained according to the method that has an increased ability to bind to the extracellular matrix (for example, 2-fold when binding to an antigen) compared to an antibody that differs only in that it does not include the modification (and ), introduced (s) according to the method. In some embodiments, the method comprises comparing the binding activity for the Fc-gamma receptor (FcγR) at neutral pH (eg, pH 7.4) of an antibody prepared according to the method with the activity of a reference antibody comprising a native IgG constant region. In additional embodiments of the invention, the method comprises selecting an antibody prepared according to the method that has increased FcγR binding activity at neutral pH (eg, pH 7.4) compared to the activity of a reference antibody containing a native IgG constant region. In some embodiments, the selected antibody prepared according to the method has increased FcγRIIb binding activity at neutral pH. In some embodiments, the selected antibody obtained according to the method has binding activity for one or more FcγRs, preferably selected from the group consisting of FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb, and FcγRIIa, and against FcγRIIb, and optionally FcRIIb β-binding activity is maintained or increased, and the binding activity against activating FcγRs decreases compared to the activity of the reference antibody, which differs only in that its constant region is the region of native IgG. In some embodiments, the method further comprises comparing the FcRn binding activity under neutral pH conditions of the antibody prepared according to the method with a reference antibody that differs only in that its constant region is a native IgG region. In other embodiments, the method further comprises selecting an antibody obtained according to the method that has increased FcRn binding activity under neutral pH conditions as compared to that of a reference antibody, which differs only in that its constant region is a native IgG region (e.g. , 2 times). In some embodiments, the antibodies prepared according to the method (substantially) retain antigen-binding activity when compared to antibodies prior to modification or alteration of at least one amino acid residue to increase the pI (native antibodies (e.g., native Ig antibodies, preferably native IgG antibodies -type) or reference antibodies (for example, antibodies before antibody modification or before or during library creation)). In this case, “(substantially) retains antigen binding activity” may mean having an activity of at least 50% or more, preferably 60% or more, more preferably 70% or 75% or more, and even more preferably 80%, 85%, 90% or 95% or more, compared to the binding activity of antibodies before modification or change.

In additional embodiments, the method comprises modifying at least one amino acid residue that may be exposed on the surface of the variable region and constant region of an antibody to increase the isoelectric point (pI). In other embodiments, the at least one amino acid residue modified according to the method is at the above position in the constant region. In additional embodiments of the invention, at least one amino acid residue modified according to the method is located at a position as described above in the variable region. In further embodiments of the invention, at least one amino acid residue modified according to the method is at the above position in the constant region, and at least one amino acid residue modified according to the method is at the above position in the variable region. In some embodiments, the antigen is a soluble antigen. In some embodiments, the method further comprises comparing the KD of an antibody prepared according to the method for its corresponding antigen at acidic pH (eg, pH 5.8) and neutral pH (eg, pH 7.4). In additional embodiments, the method comprises selecting an antibody that has a KD (acidic pH range (e.g., pH 5.8)) / KD (neutral pH range (e.g., pH 7.4)) for antigen of 2 or higher. In some embodiments, the method further comprises comparing the antigen-binding activity of an antibody prepared according to the method under conditions of high ion concentration (eg, hydrogen or calcium ion concentration) and low ion concentration. In other embodiments, the method further comprises selecting an antibody that has a higher antigen-binding activity at a high ion concentration than at a low ion concentration. In some embodiments of the invention, in which the ion concentration is a calcium ion concentration, the high calcium ion concentration can be selected from a concentration ranging from 100 μM to 10 mM, from 200 μM to 5 mM, from 400 μM to 3 mM, from 200 μM and up to 2 mM, or from 400 μM and up to 1 mM. A concentration selected from a concentration between 500 μM and 2.5 mM may also be preferred. In some embodiments, the low calcium ion concentration can be selected from a concentration ranging from 0.1 μM to 30 μM, from 0.2 μM to 20 μM, from 0.5 μM to 10 μM, or from 1 μM to 5 μM, or from 2 μM to 4 μM. A concentration selected from a concentration of 1 μM to 5 μM may also be preferred. In some embodiments, the lower limit of the ratio KD (low calcium ion concentration) / KD (high calcium ion condition) (e.g., KD (3μM Ca) / KD (2μM Ca)) is 2 or more, 10 or more, or 40 or more, and the upper limit is 400 or less, 1000 or less, or 10,000 or less. In some alternative embodiments, the lower limit of the ratio kd (low calcium ion conditions) / kd (high calcium ion conditions) (e.g., kd (3 μM Ca) / kd (2 μM Ca)) is 2 or more, 5 or more , 10 or more, or 30 or more, and the upper limit is 50 or less, 100 or less, or 200 or less. In some embodiments, in which the ion concentration is a hydrogen ion concentration, a low hydrogen ion concentration (neutral pH range) can be selected from pH 6.7 to 10.0, pH 6.7 to 9.5, pH 7.0 to 9.0 or pH 7.0 to 8.0. The low concentration of hydrogen ions may preferably correspond to a pH of 7.4, which is close to the in vivo pH in plasma (blood), but for convenience of measurements, for example, pH 7.0 can be used. In some embodiments, the high hydrogen ion concentration (acidic pH range) can be selected from pH 4.0 to 6.5, pH 4.5 to 6.5, pH 5.0 to 6.5, or pH 5 .5 to 6.5. The acidic pH range can, for example, correspond to pH 5.8 or 6.0. In some embodiments, the lower limit of the KD (acidic pH range) / KD (neutral pH range) ratio (e.g., KD (pH 5.8) / KD (pH 7.4)) is 2 or more, 10 or more, or 40 or more, and the upper limit is 400 or less, 1000 or less, or 10,000 or less.

In some embodiments, the method further comprises comparing the elimination of the antigen from the plasma after administration of the antibody obtained according to the method and after administration of a reference antibody that differs only in that it does not include the modification (s) introduced according to the method. In additional embodiments, the method also includes selecting an antibody obtained according to the method that enhances the elimination of antigen from plasma (eg, 2-fold) compared to an antibody that does not contain the modifications introduced according to the method. In some embodiments, the method further comprises comparing the binding to the extracellular matrix of an antibody obtained according to the method with an antibody that differs only in that it does not include the modification (s) introduced according to the method. In additional embodiments of the invention, the method also includes selecting an antibody obtained according to the method that has an increased ability to bind to the extracellular matrix (for example, 5-fold when forming a complex with an antigen) compared to an antibody that differs only in that it does not include the modification ( i) introduced (s) according to the method. In some embodiments, the method comprises comparing the binding activity for the FcraMMa receptor (FcγR) at neutral pH (eg, pH 7.4) of an antibody prepared according to the method with the activity of a reference antibody containing a native IgG constant region. In additional embodiments of the invention, the method comprises selecting an antibody prepared according to the method that has increased FcγR binding activity at neutral pH (eg, pH 7.4) compared to the activity of a reference antibody containing a native IgG constant region. In some embodiments, the selected antibody prepared according to the method has increased FcγRIIb binding activity at neutral pH. In some embodiments, the selected antibody obtained according to the method has binding activity against one or more activating FcγRs, preferably selected from the group consisting of FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb, and FcγRIIa, and against FcγRIIb, and optionally FcγRIIb-binding activity is maintained or increased, and the binding activity against activating FcγRs is reduced compared to the activity of the reference antibody, which differs only in that its constant region is a native IgG region. In some embodiments, the method further comprises comparing the FcRn binding activity under neutral pH conditions of the antibody prepared according to the method with a reference antibody that differs only in that its constant region is a native IgG region. In other embodiments, the method further comprises selecting an antibody prepared according to the method that has increased FcRn binding activity under neutral pH conditions as compared to that of a reference antibody, which differs only in that its constant region is a native IgG region (e.g. , 2 times). In additional embodiments, the antibodies obtained according to the method (substantially) retain antigen-binding activity when compared with antibodies before modifying or changing at least one amino acid residue to increase the pI (native antibodies (e.g., native Ig antibodies, preferably native IgG antibodies -type) or reference antibodies (for example, antibodies before antibody modification or before or during library creation)). In this case, “(substantially) retains antigen binding activity” may mean having an activity of at least 50% or more, preferably 60% or more, more preferably 70% or 75% or more, and even more preferably 80%, 85%, 90% or 95% or more, compared to the binding activity of antibodies before modification or change.

An alternative embodiment of the invention referred to in Section A of the specification is an antibody obtained by the method for producing or screening antibodies described in Section A above.

An alternative embodiment of the invention referred to in section A of the description is a composition or pharmaceutical composition containing the antibody specified in section A of the description above. In one embodiment of the invention, the pharmaceutical composition specified in section A of the description may be a pharmaceutical composition designed to accelerate the elimination of antigen from a biological fluid (preferably plasma, etc.) of individuals and / or to increase binding of the extracellular matrix (when the antibody specified in section A of the description, is administered (applied) to an individual (preferably in vivo)). The pharmaceutical composition specified in section A of the description may optionally contain a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutical compositions" can generally refer to agents intended for use in the treatment, prevention, diagnosis, or analysis of diseases.

Compositions or pharmaceutical compositions mentioned in section A of the description can be prepared accordingly. In some embodiments, they can be used parenterally, for example, in the form of a sterile solution or suspension for injection in water or any other pharmaceutically acceptable liquid. The compositions can be suitably formulated in unit dosage form according to the required standard good pharmaceutical practice by combining, respectively, with pharmaceutically acceptable carriers or media. These pharmaceutically acceptable carriers or media include, but are not limited to, sterile water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavors, excipients, fillers, preservatives, and binding agents. The amount of active ingredient in the compositions can be adjusted so that the dose is within an acceptable predetermined range.

In some embodiments, the compositions or pharmaceutical compositions described in Section A of the description may be administered parenterally. Compositions or pharmaceutical compositions can be suitably formulated, for example, as an injectable, transnasal, transpulmonary, or transdermal composition. The compositions or pharmaceutical compositions can be administered systemically or locally, for example, by intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection.

Some embodiments of the invention are antibodies, the pI of which is increased by modifying at least one amino acid residue that can be exposed on the surface (antibodies with an increased pI); methods of producing said antibodies, or using these antibodies to enhance elimination of antigen from plasma (when the antibodies are administered to individuals in vivo). It should be obvious that for such embodiments, the scope of Section A of the present specification and the contents of additional examples may be applied as appropriate. Other embodiments of the invention are antibodies, the pI of which is lowered by modifying at least one amino acid residue that can be exposed on the surface ("antibodies with lowered pI"); methods of producing said antibodies, or using such antibodies to improve plasma retention time (when the antibodies are administered to individuals in vivo). During the creation of the invention, it was found that cellular internalization of antibodies can be increased by increasing the pI by introducing specific amino acid mutations at specific sites in the amino acid sequence of the constant region. It will be apparent to those of ordinary skill in the art that the plasma retention time of an antibody can be prolonged as the pI is lowered to suppress cellular internalization of the antibody by introducing amino acids with a different side chain charge into the sites described above. It should be obvious that the scope of Section A of the present description and the content of additional examples provided in the present description can be applied accordingly in such embodiments of the invention.

One of the embodiments of the invention is a method for producing a modified antibody, the half-life in plasma of which is prolonged or shortened compared to the half-life before the modification of the antibody, where the method includes: (a) modifying the nucleic acid that encodes the antibody before modification, to change the charge at least at least one amino acid residue located at a position selected from the group consisting of positions 196, 253, 254, 256, 257, 258, 278, 280, 281, 282, 285, 286, 306, 307, 308, 309, 311 , 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 388, 389, 399, 400, 401, 402, 413, 415 , 418, 419, 421, 424, 430, 433, 434 and 443, according to the EU numbering; (b) culturing a host cell to express the modified nucleic acid and to produce an antibody; and (c) collecting the resulting antibody from the host cell culture.

An additional embodiment of the invention is a method of prolonging or shortening the plasma half-life of an antibody, wherein the method comprises modifying at least one amino acid residue located at a position selected from the group consisting of position 196, 253, 254, 256, 257, 258, 278, 280, 281, 282, 285, 286, 306, 307, 308, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 388, 389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434 and 443 according to the EU numbering.

These methods may further include determining whether the plasma half-life of the harvested and / or modified antibody is prolonged or shortened compared to the half-life prior to antibody modification.

Amino acid substitution (s) can be used to change the charge. In some embodiments, the substituted amino acid residue (s) may be selected from the group consisting of, but not limited to, amino acid residues of groups (a) and (b) below: (a) Glu (E ) and Asp (D); and (b) Lys (K), Arg (R) and His (H).

In some embodiments, the antibody may be an Ig-type antibody, such as an IgG-type antibody. In some embodiments, the antibody can be a chimeric antibody, a humanized antibody, or a human antibody. In some embodiments, the antibody may be a multispecific antibody, such as a bispecific antibody.

Section B descriptions

The embodiments of the invention mentioned in section B of the description include, but are not limited to, variants of the Fc region, their uses and methods of preparation.

In the context of sections A and B of the present description, "variant Fc region" may mean, for example, an Fc region modified relative to the Fc region of a native IgG type antibody by replacing at least one amino acid with another amino acid, or may refer to an Fc -region, modified relative to the specified variant of the Fc-region by additional replacement of at least one amino acid with another amino acid. As used herein, variants of such an Fc region include not only Fc regions into which an amino acid modification has been introduced, but also Fc regions containing the same amino acid sequence as the above Fc region.

An alternative embodiment of the invention, referred to in section B of the description, are Fc region variants containing an FcRn binding domain that contains Ala at position 434; any of Glu, Arg, Ser, and Lys at position 438; and any of Glu, Asp and Gln at position 440 according to EU numbering (in the context of section B of the present description, said variant Fc region is also referred to as "new variant Fc region" for purposes of clarity).

In practice, the Fc region variants described in section B of the description can be incorporated into virtually any antibody (eg, multispecific antibodies such as bispecific antibodies), regardless of the type of target antigen. For example, anti-factor IXa / factor X bispecific antibodies can be generated using the Fc region variants described in Example 20 (e.g., containing F8M-F1847mv [F8M-F1847mv1 (SEQ ID NO: 323) and F8M-F1847mv2 (SEQ ID NO: 324) as heavy chains and F8ML (SEQ ID NO: 325) as light chain]; F8M-F1868mv [F8M-F1868mv1 (SEQ ID NO: 326) and F8M-F1868mv2 (SEQ ID NO: 327) as heavy chains and F8ML (SEQ ID NO: 325) as light chain]; and F8M-F1927mv [F8M-F1927mv1 (SEQ ID NO: 328) and F8M-F1927mv2 (SEQ ID NO: 329) as heavy chains and F8ML (SEQ ID NO: 325) as light chain]).

As described above, WO 2013/046704 discloses such Fc region variants into which a mutation is introduced to increase their FcRn binding activity under acidic conditions in combination with a specific mutation (a representative example of which is the mutation of two Q438R / S440E residues according to EU numbering ), which are characterized by a significantly reduced binding to rheumatoid factor. However, WO 2013/046704 does not disclose that Fc region variants whose binding to rheumatoid factor is reduced by the Q438R / S440E modification exhibit an extended plasma retention time compared to antibodies with a native Fc region. Thus, there is a need for safer and more valuable Fc region variants with improved plasma retention times, but which do not bind to previously present ADA. The invention has developed safer and more valuable Fc region variants with improved plasma retention times, but which do not bind to anti-drug antibodies (preexisting ADA, etc.). In particular, it was surprisingly found and for the first time described that variants of the Fc region, which contain combined mutations of amino acid residues, leading to the substitution of Ala (A) amino acids at position 434 according to EU numbering, and a specific mutation of two residues (a representative example is Q438R / S440E) are preferred for prolonging the retention time of the antibody in plasma while maintaining a significantly reduced ability to bind to rheumatoid factor.

Thus, the new Fc region variants described in section B of the present description have advantages and unexpected improved properties over the Fc region variants described in WO 2013/046704, which is fully incorporated into this description by reference.

One of the embodiments of the invention specified in section B of the description are new combinations of amino acid substitutions in the FcRn-binding domain that increase the FcRn-binding activity of antibodies in the acidic pH range and in the neutral pH range, in particular in the acidic pH range.

In one embodiment, the Fc region variant specified in section B of the description contains Ala at position 434; any of Glu, Arg, Ser, and Lys at position 438; and any of Glu, Asp and Gin at position 440 according to EU numbering; and more preferably contains Ala at position 434; or Arg or Lys at position 438; and either Glu or Asp at position 440 according to EU numbering. Preferably, the Fc region variant specified in section B of the description further comprises either Ile or Leu at position 428 and / or any of Ile, Leu, Val, Thr and Phe at position 436 according to EU numbering. More preferably, the Fc region variant contains Leu at position 428 and / or either Val or Thr at position 436 according to EU numbering.

In one embodiment, the Fc region variant specified in Section B of the specification may be a native Ig Fc region variant, and more preferably an IgG-type native antibody Fc region variant (IgG1-, IgG2-, IgG3- or IgG4-type). The native Fc region is described in particular in the context of sections A and B of the present description. More specifically, in section B, a native Fc region may mean an unmodified or naturally occurring Fc region, and preferably an unmodified or naturally occurring Fc region of a native antibody as Ig, in the Fc region of which amino acid residues remain unmodified. ... The source of the Fc region of the antibody can be an Ig, such as IgM or IgG, for example, human IgG1, IgG2, IgG3, or IgG4. In one embodiment, the source may be human IgG1. The term "(reference) antibody" containing a native Fc region may refer to an antibody containing an unmodified or naturally occurring Fc region.

Positions 428, 434, 438 and 440 are common to the Fc regions of all native human IgG1, IgG2, IgG3 and IgG4 antibodies. However, at position 436 in the Fc region, native human IgG1, IgG2, and IgG4 antibodies carry Tyr (Y), and native human IgG3-type antibodies have Phe (F). On the other hand, Stapleton et al., Nature Comm., 2011, p. 599 disclosed that human IgG3 allotypes that contain the amino acid substitution R435H according to EU numbering have a human plasma half-life comparable to IgG1. Hence, it was suggested that plasma retention times could be improved by increasing FcRn binding under acidic conditions by introducing the amino acid substitution R435H in combination with the amino acid substitution at position 436.

WO 2013/046704 also specifically discloses substitutions of two amino acid residues Q438R / S440E, Q438R / S440D, Q438K / S440E and Q438K / S440D according to EU numbering, which result in a significant decrease in binding to rheumatoid factor when combined with an amino acid substitution that increases FcRn binding in acidic conditions.

Thus, in a preferred embodiment, the FcRn binding domain of the Fc region variant specified in Section B of the description may comprise a substituted amino acid combination selected from the group consisting of: (a) N434A / Q438R / S440E; (b) N434A / Q438R / S440D; (c) N434A / Q438K / S440E; (d) N434A / Q438K / S440D; (e) N434A / Y436T / Q438R / S440E; (f) N434A / Y436T / Q438R / S440D; (g) N434A / Y436T / Q438K / S440E; (h) N434A / Y436T / Q438K / S440D; (i) N434A / Y436V / Q438R / S440E; (j) N434A / Y436V / Q438R / S440D; (l) N434A / Y436V / Q438K / S440E; (m) N434A / Y436V / Q438K / S440D; (m) N434A / R435H / F436T / Q438R / S440E; (o) N434A / R435H / F436T / Q438R / S440D; (n) N434A / R435H / F436T / Q438K / S440E; (p) N434A / R435H / F436T / Q438K / S440D; (c) N434A / R435H / F436V / Q438R / S440E; (t) N434A / R435H / F436V / Q438R / S440D; (y) N434A / R435H / F436V / Q438K / S440E; (f) N434A / R435H / F436V / Q438K / S440D; (x) M428L / N434A / Q438R / S440E; (c) M428L / N434A / Q438R / S440D; (h) M428L / N434A / Q438K / S440E; (w) M428L / N434A / Q438K / S440D; (u) M428L / N434A / Y436T / Q438R / S440E; (e) M428L / N434A / Y436T / Q438R / S440D; (aa) M428L / N434A / Y436T / Q438K / S440E; (ab) M428L / N434A / Y436T / Q438K / S440D; (ab) M428L / N434A / Y436V / Q438R / S440E; (ah) M428L / N434A / Y436V / Q438R / S440D; (hell) M428L / N434A / Y436V / Q438K / S440E; (ae) M428L / N434A / Y436V / Q438K / S440D; (ah) L235R / G236R / S239K / M428L / N434A / Y436T / Q438R / S440E; and (az) L235R / G236R / A327G / A330S / P331S / M428L / N434A / Y436T / Q438R / S440E, according to EU numbering.

In a further preferred embodiment, the FcRn binding domain of the Fc region variant specified in section B of the description may contain a combination of substituted amino acids selected from the group consisting of: (a) N434A / Q438R / S440E; (b) N434A / Y436T / Q438R / S440E; (c) N434A / Y436V / Q438R / S440E; (d) M428L / N434A / Q438R / S440E; (e) M428L / N434A / Y436T / Q438R / S440E; (f) M428L / N434A / Y436V / Q438R / S440E; (g) L235R / G236R / S239K / M428L / N434A / Y436T / Q438R / S440E; and (h) L235R / G236R / A327G / A330S / P331S / M428L / N434A / Y436T / Q438R / S440E, according to EU numbering.

In one embodiment, it is preferred that the FcRn binding activity of the Fc region variant described in Section B of the description is increased under acidic pH conditions compared to the Fc region of native IgG.

An increase in the FcRn binding activity (binding affinity) of an FcRn binding domain in the pH range may correspond to an increase in the measured FcRn binding activity (binding affinity) over the measured FcRn binding activity (binding affinity) of the native FcRn binding domain. In this case, the value of the ratio KD (native Fc region) / KD (variant Fc region specified in section B of the description), which characterizes the difference in binding activity (binding affinity), may be at least 1.5, 2, 3 , 4, 5, 10, 15, 20, 50, 70, 80, 100, 500 or 1000 times. This increase can take place in the acidic pH range and / or in the neutral pH range; however, an increase in the acidic pH range may be preferable from the standpoint of the mechanism of action indicated in section B of the description.

In some embodiments, the FcRn binding activity (for example, at pH 6.0 and 25 ° C) of the Fc region variant of section B of the description, the FcRn binding activity of which increases in the acidic pH range to a greater extent than Fc- regions of native IgG, for example, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 75, 100, 200, 500, 1000 times or more. In some embodiments, the increased FcRn binding activity of the Fc region variant in the acidic pH range may be greater than the FcRn binding activity of the native IgG Fc region by at least 5 or at least 10 times.

Manipulation of the FcRn binding domain by introducing amino acid substitutions can sometimes reduce antibody stability (WO 2007/092772). Proteins with insufficient stability tend to form aggregates readily during storage, and the stability of pharmaceutical proteins is very important in the manufacture of pharmaceuticals. Thus, the decrease in stability associated with substitutions in the Fc region can lead to difficulties in the development of stable antibody preparations (WO 2007/092772).

The purity of pharmaceutical proteins with respect to monomers and high molecular weight products is important for pharmaceutical development. After purification with protein A, wild-type IgG1 does not contain significant amounts of high molecular weight products, while manipulation of the FcRn binding domain, consisting in the introduction of substitutions, can lead to the formation of a large number of high molecular weight products. In this case, these high molecular weight products may need to be removed from the drug substance using purification steps.

Amino acid substitutions in antibodies can lead to negative consequences, such as increased immunogenicity of therapeutic antibodies, which in turn can cause a cytokine storm and / or the production of anti-drug antibodies (ADA). The clinical utility and efficacy of therapeutic antibodies can be limited by ADA because they interfere with the efficacy and pharmacokinetics of therapeutic antibodies and sometimes cause serious side effects. The immunogenicity of therapeutic antibodies is influenced by a number of factors, and the presence of effector T cell epitopes is one such factor. Likewise, the presence of pre-existing antibodies against the therapeutic antibody can also lead to problems. An example of such a pre-existing antibody is rheumatoid factor (RF), an autoantibody (an antibody directed against its own protein) to the Fc region of an antibody (i.e., IgG). Rheumatoid factor has been found particularly in patients with systemic lupus erythematosus (SLE) or rheumatoid arthritis. In arthritic patients, RF and IgG are linked to form immune complexes involved in the development of the disease. A humanized anti-CD4 IgG1 antibody with the Asn434His mutation has now been described to provide significant binding to rheumatoid factor (Zheng et al., Clin. Pharmacol. Ther. 89 (2), 2011, pp. 283-290). Detailed study confirmed that the Asn434His mutation in human IgG1 increases the binding of rheumatoid factor to the Fc region of the antibody compared to the parental human IgG1.

RF is an anti-human IgG polyclonal autoantibody. The RF epitope in the human IgG sequence varies between clones, the RF epitope is likely to be located in the CH2 / CH3 interface as well as in the CH3 domain, which may overlap with the FcRn binding epitope. Thus, mutations leading to an increase in FcRn-binding activity at neutral pH, apparently, can also increase the binding activity to specific RF clones.

In the context of section B of the description, the term "anti-drug antibody" or "ADA" can refer to an endogenous antibody that has the ability to bind to an epitope located on the therapeutic antibody, and as a result can bind to the therapeutic antibody. The term "preexisting drug antibody" or "preexisting ADA" can refer to an antibody to a drug that is present and detectable in the patient's blood prior to administration of the therapeutic antibody to the patient. In some embodiments, the preexisting ADA is a human antibody. In other embodiments, the preexisting ADA is rheumatoid factor.

The binding activity of an Fc region (variant) of an antibody against preexisting ADA can, for example, be characterized by an electrochemiluminescence (ECL) response at acidic pH and / or at neutral pH. ECL analysis is described, for example, in Moxness et al., Clin Chem. 51, 2005, pp. 1983-1985 and Example 6. Assays can be performed, for example, under the conditions of using the MES buffer and 37 ° C. The antigen binding activity of antibodies can be determined, for example, using the BIACORE® assay.

The binding activity against preexisting ADA can be assessed at any temperature from 10 ° C to 50 ° C. In some embodiments, the binding activity (binding affinity) of a human Fc region to a human pre-present ADA is determined at 15 ° C to 40 ° C, eg, 20 ° C to 25 ° C or 25 ° C. In a further embodiment, the interaction between human preexisting ADA and human Fc region is measured at pH 7.4 (or pH 7.0) and 25 ° C.

In the context of Section B of the present disclosure, the expression “binding activity against (pre-present) ADA is significantly increased” or an equivalent expression may mean that the measured ability to bind (pre-present) ADA (binding affinity) (i.e., KD) of an Fc region variant specified in section B of the description, or the antibody containing it is increased, for example, in 0.55, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1, 3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2 or 2.3 times or more, compared to the measured binding capacity ( preexisting) ADA (binding affinity) of a reference Fc region or a reference antibody containing a reference Fc region. This increase in binding activity for preexisting ADA can be found in an individual patient or in a group of patients.

In one embodiment, in the context of Section B of the specification, the term "patients" or "patient" may include, but is not limited to, all people suffering from a disease who are being treated with a therapeutic antibody. Patients can be people with an autoimmune disease such as arthritis or systemic lupus erythematosus (SLE). Arthritis can include rheumatoid arthritis.

In one of the embodiments of the invention referred to in section B of the description, the term "binding activity against a pre-existing ADA is significantly increased in an individual patient" may mean that the binding activity of an antibody containing a variant Fc region (e.g., a therapeutic antibody) against a previously present ADA measured in a patient is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60%, or more than the binding activity of the reference antibody against the previously present ADA. Alternatively, this may mean that the ECL response of the antibody is preferably greater than 250, or at least 500, or at least 1000, or even at least 2000. Preferably, the increase may be an increase relative to a reference antibody whose ECL response is less than 500 or 250. In particular, an ECL response characterizing the binding activity of a reference antibody to a previously present ADA and said binding activity of an antibody having a variant Fc region preferably ranges from less than 250 to at least 250, from less than 250 to at least 500, from less than 500 to 500 or more, from less than 500 to 1000 or more, or from less than 500 to at least 2000, but not limited to them.

In one embodiment of the invention, the expression "binding activity against preexisting ADA is increased" may mean that in a group of patients, the measured proportion of patients who have an ECL response of at least 500 (preferably at least 250) for an antibody containing a variant of the Fc region with (a) increased binding activity against FcRn at acidic pH and (b) increased binding activity against preexisting ADA at neutral pH, increased compared to the proportion of patients who have an ECL response of at least 500 (preferably at least 250 or more), relative to the reference antibody, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50 %, compared with the proportion of patients who give an ECL response to a reference antibody.

In one of the embodiments of the invention referred to in section B of the description, the expression "binding activity against preexisting ADA is reduced" may mean that the measured binding activity (i.e., KD or ECL response) of an antibody containing a variant Fc region, reduced in comparison with the activity of the reference antibody. This decrease can be found in an individual patient or in a group of patients. The expression "The affinity of an antibody containing a variant Fc region for preexisting ADA at neutral pH is significantly reduced in each patient." reference antibodies against preexisting ADA, measured at neutral pH, for example, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

Alternatively, the expression "the binding activity of an antibody containing a variant Fc region towards preexisting ADA is significantly reduced in an individual patient" may, for example, mean that the ECL response to the antibody used, which should be, for example, 500 or more (preferably , 1000 or more, or 2000 or more), when measured was less than 500, preferably less than 250 compared to the ECL response to the reference antibody.

In a preferred embodiment, the Fc region variants described in section B of the description and antibodies containing them have low binding activity against preexisting ADA at neutral pH. In particular, it is preferable that the binding activity of antibodies containing the Fc region variants specified in section B of the description with respect to the previously present ADA at neutral pH was lower or not significantly increased compared to the binding activity of the reference antibody containing the Fc region native IgG, against preexisting ADA at neutral pH (eg, pH 7.4). The expression "binding activity (binding affinity) for preexisting ADA is low or the affinity is at baseline" may mean that an individual patient has an ECL response of less than 500 or less than 250 (but not limited to). The expression "binding activity against preexisting ADA is low in a group of patients" can mean, for example, that the ECL response is less than 500 in 90%, preferably in 95% and more preferably in 98% of patients in the group.

It may be preferable to select variants of the Fc-region specified in section B of the description, or antibodies containing them, the binding activity of which against (preexisting) ADA in plasma at neutral pH does not significantly increase and whose FcRn-binding activity at neutral pH and / or at acidic pH increased. Preferably, the FcRn binding activity at acidic pH (eg, pH 5.8) is increased. In one embodiment, the Fc region variants preferably do not have significantly increased ADA binding activity under neutral pH conditions (e.g., pH 7.4) compared to the native IgG Fc region, and the ADA may be a preexisting ADA, preferably rheumatoid factor (RF).

In one embodiment, it may be preferable that the Fc region variants described in Section B of the description have increased FcRn binding activity under acidic pH conditions compared to the Fc region of native IgG, and as a result exhibit reduced plasma clearance (CL ), prolonged plasma retention time or prolonged plasma half-life (t1 / 2). The correlation between these parameters is known in the art.

In one embodiment, it may be preferred that the Fc region variants described in Section B of the description have increased FcRn binding activity under acidic pH conditions, but not significantly increased ADA binding activity under neutral pH conditions as compared to Fc. -region of native IgG, and they were characterized by reduced plasma clearance (CL), prolonged plasma retention time or prolonged plasma half-life (t1 / 2). The ADA may represent a pre-existing ADA, preferably rheumatoid factor (RF).

In one embodiment, the Fc region variants specified in section B of the description are advantageous because their retention time in plasma is improved compared to the reference Fc region variant containing the combination of amino acid substitutions N434Y / Y436V / Q438R / S440E according to EU - numbering.

Examples 5-7 describe a comparison of the plasma retention times of two Fc region variants: the F1718 Fc region variant (Fc region with mutations introduced at four sites: N434Y / Y436V / Q438R / S440E) described in WO 2013/046704, and a new variant of the Fc region F1848m (introduction of mutations at four sites: N434A / Y436V / Q438R / S440E). The amino acid mutations for the two variants of the Fc region differ only in that at position 434, according to EU numbering, the introduced amino acid mutation is Y (tyrosine) in the case of F1718 and A (alanine) in the case of F1848m. However, compared to native IgG1, F 1848m has an improved plasma retention time, while F1718 does not have such an improved plasma retention time (see example (7-2)). Thus, the Fc region variants described in section B of the description may advantageously have improved plasma retention times compared to the reference Fc region variants containing the combination of amino acid substitutions N434Y / Y436V / Q438R / S440E. The results of the experiments described in Examples (5-2) and (7-3) demonstrate that among the various Fc region variants, F1847m, F1886m, F1889m and F1927m further improved plasma retention time relative to F1848m. Thus, it should be apparent to those of ordinary skill in the art that the Fc region variants mentioned in section B of the description containing F1847m, F1886m, F1889m or F1927m, as well as F1848m, have improved plasma retention times compared to the reference Fc variants. - areas containing substitutions N434Y / Y436V / Q438R / S440E.

FcγR or complement protein binding can also have adverse effects (eg, inappropriate platelet activation). Fc region variants that do not bind to effector receptors such as the FcγRIIa receptor may be safer and / or more advantageous. In some embodiments, the Fc region variants described in section B of the description have only weak binding activity for effector receptors or do not bind to effector receptors. Examples of effector receptors include activating FcγR, in particular FcγRI, FcγRII, and FcγRIII. FcγRI includes FcγRIa, FcγRIb and FcγRIc and their subtypes. FcγRII includes FcγRIIa (having two allotypes: R131 and H131) and FcγRIIb. FcγRIII includes FcγRIIIa (which has two allotypes: V158hF158) and FcγRIIIb (which has two allotypes: FcγRIIIb-NA1 and FcγRIIIb-NA2). Antibodies that have only weak binding activity to effector receptors or do not bind to effector receptors include, for example, antibodies containing a silent Fc region and antibodies that lack an Fc region (e.g. Fab, F (ab) ' ₂ , scFv, sc (Fv) ₂ and dimeric antibodies).

Examples of Fc regions that have only weak binding activity for effector receptors or do not bind to effector receptors are described, for example, in Strohl et al., Curr. Op. Biotech. 20 (6), 2009, pp. 685-691, and specifically include, for example, deglycosylated Fc regions (N297A and N297Q) and silent Fc regions resulting from manipulation of Fc regions to render them silent effector functions (or to suppress immunity) (IgG1-L234A / L235A, IgG1-H268Q / A330S / P331S, IgG1-C226S / C229S, IgG1-C226S / C229S / E233P / L234V / L235A, IgG1-L234F / L235E / P331S, IgG2-V234A / G-H2LQ / A330 / A331S, IgG4-L235A / G237A / E318A and IgG4-L236E). WO 2008/092117 describes antibodies containing a silent Fc region that includes the substitution G236R / L328R, L235G / G236R, N325A / L328R or N325L / L328R according to EU numbering. WO 2000/042072 describes antibodies containing a silent Fc region that includes substitutions at one or more positions EU233 (position 233 according to EU numbering), EU234, EU235 and EU237. WO 2009/011941 discloses antibodies comprising a silent Fc region lacking EU231-EU238 residues. Davis et al., J. Rheum. 34 (11), 2007, pp. 2204-2210, antibodies with a silent Fc region containing substitutions C220S / C226S / C229S / P238S are described. Shields et al., J. Biol. Chem. 276 (9), 2001, pp. 6591-6604 describe antibodies containing a silent Fc region that includes the D265A substitution. Modifications to these amino acid residues can also be appropriately introduced into the Fc region variants specified in section B of the description.

The term "weak binding to effector receptors" can mean that the binding activity to effector receptors is, for example, 95% or less, preferably 90% or less, 85% or less, 80% or less, 75% or less, more preferably 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, relative to native IgG or an antibody containing the Fc region of native IgG.

"Silent Fc region" means a variant of the Fc region containing one or more amino acid substitutions, insertions, additions, deletions and other modifications that reduce binding to effector receptors compared to the native Fc region. Since the binding activity on effector receptors can be significantly reduced, such silent Fc regions can no longer bind to effector receptors. Silent Fc regions may include, for example, Fc regions containing amino acid substitutions at one or more positions selected from the group consisting of: EU234, EU235, EU236, EU237, EU238, EU239, EU265, EU266, EU267, EU269, EU270, EU271, EU295, EU296, EU297, EU298, EU300, EU324, EU325, EU327, EU328, EU329, EU331 and EU332. Modifications to these amino acid residues can also be appropriately introduced into the Fc region variants specified in section B of the description.

In further embodiments of the invention, the silent Fc region has a substitution at one or more positions selected from the group consisting of: EU234, EU235, EU236, EU237, EU238, EU239, EU265, EU266, EU267, EU269, EU270, EU271, EU295 , EU296, EU297, EU298, EU300, EU324, EU325, EU327, EU328, EU329, EU331 and EU332, and preferably a group that consists of: EU235, EU237, EU238, EU239, EU270, EU298, EU325 and EU329, where the replacement is amino acid residue is selected from the list below:

The amino acid at position EU234 is preferably replaced with an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Met, Phe, Pro, Ser and Thr.

The amino acid at position EU235 is preferably replaced with an amino acid selected from the group consisting of Ala, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Pro, Ser, Thr, Val and Arg.

The amino acid at position EU236 is preferably replaced with an amino acid selected from the group consisting of Arg, Asn, Gln, His, Leu, Lys, Met, Phe, Pro and Tyr.

The amino acid at position EU237 is preferably replaced with an amino acid selected from the group consisting of Ala, Asn, Asp, Gln, Glu, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Val, Tyr and Arg.

The amino acid at position EU238 is preferably replaced with an amino acid selected from the group consisting of Ala, Asn, Gln, Glu, Gly, His, Ile, Lys, Thr, Trp and Arg.

The amino acid at position EU239 is preferably replaced with an amino acid selected from the group consisting of Gln, His, Lys, Phe, Pro, Trp, Tyr and Arg.

The amino acid at position EU265 is preferably replaced with an amino acid selected from the group consisting of Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr and Val.

The amino acid at position EU266 is preferably replaced with an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Phe, Pro, Ser, Thr, Trp and Tyr.

The amino acid at position EU267 is preferably replaced with an amino acid selected from the group consisting of Arg, His, Lys, Phe, Pro, Trp and Tyr.

The amino acid at position EU269 is preferably replaced with an amino acid selected from the group consisting of Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val.

The amino acid at position EU270 is preferably replaced with an amino acid selected from the group consisting of Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val.

The amino acid at position EU271 is preferably replaced with an amino acid selected from the group consisting of Arg, His, Phe, Ser, Thr, Trp and Tyr.

The amino acid at position EU295 is preferably replaced with an amino acid selected from the group consisting of Arg, Asn, Asp, Gly, His, Phe, Ser, Trp and Tyr.

The amino acid at position EU296 is preferably replaced with an amino acid selected from the group consisting of Arg, Gly, Lys and Pro.

The amino acid at position EU297 is preferably replaced by Ala.

The amino acid at position EU298 is preferably replaced with an amino acid selected from the group consisting of Arg, Gly, Lys, Pro, Trp and Tyr.

The amino acid at position EU300 is preferably replaced with an amino acid selected from the group consisting of Arg, Lys and Pro.

The amino acid at position EU324 is preferably replaced with either Lys or Pro.

The amino acid at position EU325 is preferably replaced with an amino acid selected from the group consisting of Ala, Arg, Gly, His, Ile, Lys, Phe, Pro, Thr, Trp, Tyr and Val.

The amino acid at position EU327 is preferably replaced with an amino acid selected from the group consisting of Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val.

The amino acid at position EU328 is preferably replaced with an amino acid selected from the group consisting of Arg, Asn, Gly, His, Lys and Pro.

The amino acid at position EU329 is preferably replaced with an amino acid selected from the group consisting of Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, Val and Arg.

The amino acid at position EU330 is preferably replaced with either Pro or Ser.

The amino acid at position EU331 is preferably replaced with an amino acid selected from the group consisting of Arg, Gly and Lys.

The amino acid at position EU332 is preferably replaced with an amino acid selected from the group consisting of Arg, Lys and Pro.

The silent Fc region may preferably contain a substitution for either Lys or Arg in EU235, substitution for either Lys or Arg in EU237, substitution for either Lys or Arg in EU238, substitution for either Lys or Arg in EU239 , a substitution for Phe in EU270, substitution for Gly in EU298, substitution for Gly in EU325, or substitution for either Lys or Arg in EU329. More preferably, the silent Fc region may contain an arginine substitution in EU235 or a lysine substitution in EU239. Even more preferably, the silent Fc region may contain the L235R / S239K substitutions. Modifications to these amino acid residues can also be appropriately introduced into the Fc region variants specified in section B of the description.

In one embodiment of the invention, antibodies containing the variant Fc region specified in section B of the description have only weak ability to bind complement protein or do not bind to complement proteins. In some embodiments, the complement protein is C1q. In some embodiments, weak complement protein binding refers to the ability to bind a complement protein that is 10-fold or more, 50 or more, or 100-fold or more less than that of a native IgG complement protein or antibody containing a native IgG Fc region. The ability to bind the Fc region complement protein can be reduced by amino acid modifications such as amino acid substitution, insertion, addition, or deletion.

In one embodiment, the Fc region variants described in section B of the description, or antibodies containing the Fc region variants, can be evaluated for (human) FcRn binding activity in the neutral pH range and / or in the acidic pH range by one and the same by the same method described above.

In one embodiment of the invention, a method for modifying antibody constant regions to obtain the Fc region variants described in section B of the description may be based, for example, on evaluating several constant region isotypes (IgG1, IgG2, IgG3 and IgG4) to select isotypes having reduced antigen-binding activity in the acidic pH range and / or having a reduced rate of dissociation in the acidic pH range. An alternative method can be based on the introduction of amino acid substitutions in the amino acid sequence of the native IgG isotype to reduce antigen-binding activity in an acidic pH range (eg, pH 5.8) and / or to increase the rate of dissociation in an acidic pH range. The sequence of the hinge region of the constant region of an antibody varies greatly depending on the isotypes (IgG1, IgG2, IgG3 and IgG4), and differences in the amino acid sequence of the hinge region can have a significant impact on antigen binding activity. Thus, isotypes with reduced antigen-binding activity in the acidic pH range and / or increased dissociation rate in the acidic pH range can be selected by selecting suitable isotypes depending on the type of antigen or epitope. In addition, since differences in the amino acid sequence of the hinge region can have a significant effect on antigen-binding activity, amino acid substitutions in amino acid sequences of native isotypes can be localized in the hinge region.

An alternative embodiment of the invention referred to in section B of the description is the use of an antibody containing the above-described variant of the Fc region specified in section B to accelerate the release of an antibody that has been internalized into cells in antigen-bound form out of cells in free from antigen form. As used herein, “release of an antibody that has been internalized into cells in antigen-bound form, out of cells in antigen-free form” does not necessarily mean that an antibody that has been internalized in cells in antigen-bound form is completely released out of cells in antigen-free form. It is acceptable if the proportion of antibodies released in antigen-free form outside the cells is increased relative to antibodies before their FcRn-binding domain is modified (for example, before the FcRn-binding activity of the antibody increases in an acidic pH range). It is preferred that the antibody released out of the cells retains its antigen binding activity.

The term “ability to eliminate antigen from plasma”, or equivalent term, can refer to the ability to eliminate antigen from plasma when an antibody is administered or secreted in vivo. Thus, the term “the ability of an antibody to eliminate antigen from plasma is increased” may mean that after administration of the antibody, for example, the rate of antigen elimination from plasma is increased as compared to the rate before its FcRn binding domain is modified. An increase in the ability of an antibody to eliminate antigen from plasma can be assessed, for example, by administering a soluble antigen and antibody in vivo and measuring the concentration of soluble antigen in plasma after administration. The soluble antigen can be antibody-bound or antibody-free antigen, and its concentrations can be defined as "plasma concentration of antibody-bound antigen" and "plasma concentration of antibody-free antigen", respectively. The latter term is synonymous with plasma concentration of free antigen. The term "total plasma antigen concentration" can refer to the sum of the concentration of an antibody-bound antigen and the concentration of an antibody-free antigen.

An alternative embodiment of the invention referred to in section B of the description is a method of prolonging the retention time in plasma of an antibody containing the Fc region variant specified in section B of the description. Native human IgG can bind to FcRn from non-human animals. For example, since native human IgG can bind to murine FcRn more strongly than to human FcRn (Ober et al., Intl. Immunol. 13 (12), 2001, pp. 1551-1559), antibodies can be administered to mice to evaluate the properties of antibodies ... Alternatively, for example, mice that disrupt their own FcRn gene and instead are introduced into the human FcRn gene, which express it as a transgene (Roopenian et al. Meth. Mol. Biol. 602, 2010, pp. 93-104 (2010) can also be used to evaluate antibodies.

In the context of sections A and B of the present disclosure, the plasma concentration of free antigen not bound to the antibody, or the ratio of the concentration of free antigen to the total antigen concentration, can be determined (e.g., Ng et al., Pharm. Res. 23 (1), 2006, pp. . 95-103). Alternatively, when an antigen has a particular function in vivo, whether the antigen is bound to an antibody that neutralizes the function of the antigen (antagonist molecule) can be determined by assessing whether the function of the antigen is neutralized. Whether antigen function is neutralized can be determined by measuring, in particular, an in vivo marker reflecting antigen function. Whether an antigen is associated with an antibody that activates the function of the antigen (agonist molecule) can be assessed by measuring, in particular, an in vivo marker reflecting the function of the antigen.

No particular limitation is imposed on measurements such as determination of plasma concentration of free antigen, determination of the ratio of plasma amount of free antigen to total plasma amount of antigen, and in vivo marker assessment; however, measurements should preferably be made after a certain period of time after the administration of the antibody. In the context of section B, the descriptions "after a certain period of time after the administration of the antibody" is not limited to a specific period, and the period may be determined accordingly by those of ordinary skill in the art depending on the properties of the administered antibody, etc., and includes, for example, 1 day, 3 days, 7 days, 14 days, or 28 days after antibody injection. As used herein, the term "plasma antigen concentration" can refer to either "total plasma antigen concentration" which is the sum of the concentration of antibody-bound antigen and the concentration of the antibody-free antigen, or "plasma free antigen concentration" is the concentration of antibody-free antigen.

The molar ratio of antigen to antibody can be calculated using the formula: C = A / B, in which the value A denotes the molar concentration of antigen at each time point, the value B denotes the molar concentration of the antibody at each time point, and the value C denotes the ratio of the molar concentration of antigen to antibody molar concentration (antigen / antibody molar ratio) at each time point.

A lower C value indicates a more efficient elimination of the antigen by the antibody, and a higher C value indicates a less efficient elimination of the antigen by the antibody.

In some aspects of the invention, when the antibody specified in section B of the description is administered, the antigen / antibody molar ratio is reduced by 2, 5, 10, 20, 50, 100, 200, 500, or 1000 times or more compared to the introduction of a reference antibody containing Fc region of native human IgG as human FcRn binding domain.

Reduction in total plasma antigen concentration or antigen / antibody molar ratio can be assessed using methods known in the art such as those described in Examples 6, 8 and 13 of WO 2011/122011. More specifically, they can be assessed based on either the antigen-antibody co-injection model or the steady state antigen infusion model using human FcRn transgenic mouse strain 32 or 276 (Jackson Laboratories, Methods Mol. Biol. 602, 2010, cc. 93-104), when the antibody of interest specified in section B of the description does not cross-react with the murine copy antigen. When the antibody cross-reacts with a mouse copy, then the assessment can be performed by simple injection of the antibody into human FcRn transgenic mice line 32 or 276 (Jackson Laboratories). In a co-injection model, mice are injected with a mixture of antibody and antigen. In a steady state antigen infusion model, an infusion pump filled with antigen solution is implanted in mice until a constant antigen concentration is reached, and then the antibody is injected into the mice. All experimental mice are administered the same dose. Plasma total antigen concentration, plasma free antigen concentration and plasma antibody concentration can be measured at desired time points.

To assess the long-term effects of the antibody referred to in section B of the description, the total concentration or concentration of free antigen in plasma or the molar ratio of antigen / antibody can be measured at 2, 4, 67, 14, 28, 56 or 84 days after administration. In other words, to evaluate the properties of an antibody, the plasma antigen concentration can be determined over an extended period of time by measuring the plasma total or free antigen concentration or the antigen / antibody molar ratio two, four, seven, 14, 28, 56, or 84 days after administration. antibodies. Whether the concentration of antigen in plasma or the molar ratio of antigen / antibody by the antibody decreases, can be determined by evaluating the indicated decreases at one or more of the above time points.

To assess the short-term effects of the antibody referred to in section B of the description, the total or plasma free antigen concentration or antigen / antibody molar ratio may be measured 15 min, 1 h, 2 h, 4 h, 8 h, 12 h, or 24 h after introduction. In other words, to evaluate the properties of an antibody, the plasma antigen concentration can be determined over a short period of time by measuring the plasma total or free antigen concentration or the antigen / antibody molar ratio at 2, 4, 7, 14, 28, 56 days or 84 days after the introduction of antibodies. In other words, to evaluate the properties of an antibody, the plasma antigen concentration can be determined over a short period of time by measuring the total concentration or concentration of free antigen in plasma or the molar ratio of antigen / antibody after 15 minutes, 1 hour, 2 hours, 4 hours, 8 hours. 12 hours or 24 hours after administration. If the retention time in human plasma is difficult to determine, it can be predicted based on the retention time in the plasma of mice (for example, normal mice, transgenic mice expressing human antigen, or transgenic mice expressing human FcRn) or monkeys (for example, cynomolgus monkeys).

An alternative embodiment of the invention, referred to in section B of the description, is an antibody containing a variant Fc region specified in section B of the description above. Various variants of the antibodies specified in sections A and B of the present description may be acceptable, without contradicting the known technical knowledge and, if they are appropriate for this context.

In one embodiment of the invention, antibodies containing the variant Fc region specified in section B of the description can be used as therapeutic antibodies to treat human patients with autoimmune diseases, graft rejection (graft-versus-host reaction), and other inflammatory diseases. or allergic diseases as described in WO 2013/046704.

In one embodiment of the invention, antibodies containing the variant Fc region specified in section B of the description may have modified sugar chains. Antibodies with modified sugar chains may include, for example, antibodies with modified glycosylation (WO 99/54342), antibodies with fucose deficiency (WO 00/61739, WO 02/31140, WO 2006/067847, WO 2006/067913) and antibodies having sugar chains that are bisectioned with GlcNAc (WO 02/79255). In one embodiment, the antibodies may be dglycosylated. In some embodiments, the antibodies comprise, for example, mutations at the heavy chain glycosylation site to inhibit glycosylation at that location, as described in WO 2005/03175. These non-glycosylated antibodies can be obtained by modifying the heavy chain glycosylation site, i. E. by introducing the substitution N297Q or N297A according to EU numbering and expression of the proteins in suitable host cells.

An alternative embodiment of the invention specified in section B of the description is a composition or pharmaceutical composition comprising an antibody containing the specified variant of the Fc region. Acceptable may be the various compositions or pharmaceutical compositions specified in sections A and B of the present description, without contradicting the known technical knowledge and, if they fit the given context. These compositions can be used to increase the plasma retention time (in individuals who are administered (which are treated) with the antibody specified in section B of the description).

An alternative embodiment of the invention mentioned in section B of the description are nucleic acids encoding a variant Fc region, or antibodies containing a variant Fc region. Various variants of the nucleic acids specified in sections A and B of the present description, without contradicting the known technical knowledge and, if they are appropriate for this context, may be acceptable. Alternatively, in section B of the description, vectors containing nucleic acids are indicated. Various variations of them as specified in sections A and B of this description may be acceptable if they do not contradict the known technical knowledge and, if they fit the given context. Alternatively, in section B of the description, hosts or host cells containing vectors are indicated. Various variations of them as specified in sections A and B of this description may be acceptable if they do not contradict the known technical knowledge and, if they fit the given context.

An alternative embodiment of the invention described in section B of the description are methods for producing a variant Fc region containing an FcRn binding domain, or an antibody containing a variant Fc region, which include culturing the host cells described above, or growing the hosts described above and collecting the variant Fc region or antibody containing the variant Fc region from the cell culture, products secreted from the host. In this case, section B of the description may include methods of preparation, which optionally also include one or more of the following steps: (a) selection of a variant of the Fc region with increased FcRn binding activity under conditions of acidic pH compared to the activity of the Fc region of native IgG; (b) selecting a variant of the Fc region, the binding activity of which against the (previously present) ADA is not significantly increased under neutral pH conditions compared to the activity of the Fc region of native IgG; (c) selecting a variant of the Fc region with an increased retention time in plasma compared with the retention time of the Fc region of native IgG; and (d) selecting an antibody containing a variant Fc region that can enhance the elimination of antigen from plasma as compared to a reference antibody that contains the Fc region of native IgG.

From the standpoint of assessing the retention time in plasma of the Fc region variant specified in section B, it may be preferable (but not limited to only specified) that an antibody containing the Fc region variant obtained in section B of the description, and a "reference antibody containing The "native IgG Fc region" that was compared were identical to each other with the exception of the Fc region. The FcRn can be a human FcRn.

For example, after obtaining an antibody containing a variant Fc region specified in section B of the description, the antibody can be compared with a reference antibody that contains the Fc region of native IgG for FcRn binding activity under conditions of acidic pH (for example, pH 5, 8) using BIACORE® or other known techniques to select an Fc region variant or antibody containing an Fc region variant whose FcRn binding activity is increased under acidic pH conditions.

Alternatively, for example, after obtaining an antibody containing a variant Fc region specified in section B of the description, the antibody can be compared with a reference antibody that contains the Fc region of native IgG for ADA binding activity under neutral pH conditions using electrochemiluminescence (ECL) or known techniques for selecting an Fc region variant or antibody containing an Fc region variant whose ADA binding activity is not significantly increased under neutral pH conditions.

Alternatively, for example, after obtaining an antibody containing a variant Fc region specified in section B of the description, the antibody can be compared with a reference antibody that contains the Fc region of native IgG, by performing pharmacokinetic analyzes of the antibody using plasma, for example, mice, rats, rabbits, dogs, monkeys, or humans to select for Fc region variants or antibodies containing the Fc region variant that have demonstrated improved plasma retention times in individuals.

Alternatively, for example, after obtaining an antibody containing a variant Fc region specified in section B of the description, the antibody can be compared with a reference antibody that contains the Fc region of native IgG, by performing pharmacokinetic analyzes of the antibody using plasma, for example, mice, rats, rabbits, dogs, monkeys or humans to select for Fc region variants or antibodies containing an Fc region variant having an increased ability to eliminate antigen from plasma.

Alternatively, for example, the selection methods described above can be suitably combined if necessary.

One of the embodiments of the invention, specified in section B of the description, is a method of obtaining a variant of the Fc region containing the FcRn binding domain, or an antibody containing a variant, where the method includes such an amino acid change that the resulting variant of the Fc region or an antibody containing the variant include Ala at position 434; Glu, Arg, Ser or Lys at position 438; and Glu, Asp or Gln at position 440 according to EU numbering. In an additional embodiment, said method comprises replacing amino acids such that the resulting Fc region variant or antibody containing the variant further comprises Ile or Leu at position 428 and / or Ile, Leu, Val, Thr or Phe at position 436, according to EU numbering. In another embodiment, the amino acids are changed such that the resulting Fc region variant or antibody containing the variant obtained according to the method further includes Leu at position 428 and / or Val or Thr at position 436 according to EU numbering.

One of the embodiments of the invention specified in section B of the description is a method of obtaining a variant of the Fc region containing an FcRn binding domain, or an antibody containing a variant, where the method comprises replacing amino acids so that the resulting variant of the Fc region or an antibody containing variant, contained Ala at position 434; Arg or Lys at position 438; and Glu or Asp at position 440 according to EU numbering. In an additional embodiment of the invention, said method comprises replacing amino acids such that the resulting Fc region variant or antibody containing the variant obtained according to the method additionally contains Ile or Leu at position 428 and / or Ile, Leu, Val, Thr, or Phe at position 436 according to EU numbering. In a further embodiment, the amino acids are changed so that the resulting Fc region variant or antibody containing the variant obtained according to the method additionally contains Leu at position 428 and / or Val or Thr at position 436 according to EU numbering.

In one embodiment, the method comprises replacing all amino acids at positions 434, 438 and 440 with Ala; Glu, Arg, Ser, or Lys; and Glu, Asp or Gln, respectively. In an additional embodiment of the invention, said method comprises replacing amino acids such that the resulting Fc region variant or antibody containing the variant obtained according to the method additionally contains Ile or Leu at position 428 and / or Ile, Leu, Val, Thr or Phe at position 436 according to EU numbering. In a further embodiment, the amino acids are changed so that the resulting Fc region variant or antibody containing the variant obtained according to the method additionally contains Leu at position 428 and / or Val or Thr at position 436 according to EU numbering.

An alternative embodiment of the invention specified in section B of the description is a variant Fc region or an antibody containing a variant Fc region obtained using any of the methods of preparation specified in section B above.

An alternative embodiment of the invention referred to in section B of the description are methods of reducing the binding activity against (preexisting) ADA antibodies comprising a variant Fc region with increased FcRn binding activity at acidic pH; and methods for preparing Fc region variants with increased FcRn binding activity at acidic pH (e.g., pH 5.8) and reduced binding activity against pre-existing ADA, which include: (a) preparing an antibody containing an Fc region (variant) , FcRn-binding activity of which at acidic pH is increased in comparison with the reference antibody; and (b) introduction into the Fc region (according to EU numbering) (I) an amino acid substitution with Ala at position 434; (Ii) an amino acid substitution for any of Glu, Arg, Ser and Lys at position 438; and (III) an amino acid change to any of Glu, Asp, and Gln at position 440, (IV) optionally an amino acid change to Ile or Leu at position 428; and / or (V) optionally an amino acid substitution with any of Ile, Leu, Val, Thr, and Phe at position 436.

In some embodiments, the Fc domain (variant) specified in step (a) is preferably the Fc domain (variant) of human IgG. In addition, in order to increase the FcRn binding activity at acidic pH and reduce the binding activity against (previously present) ADA in a neutral pH range (e.g., pH 7.4), the Fc region (variant) must contain a combination of substituted amino acids selected from a group consisting of: (a) N434A / Q438R / S440E; (b) N434A / Q438R / S440D; (c) N434A / Q438K / S440E; (d) N434A / Q438K / S440D; (e) N434A / Y436T / Q438R / S440E; (f) N434A / Y436T / Q438R / S440D; (g) N434A / Y436T / Q438K / S440E; (h) N434A / Y436T / Q438K / S440D; (i) N434A / Y436V / Q438R / S440E; (j) N434A / Y436V / Q438R / S440D; (l) N434A / Y436V / Q438K / S440E; (m) N434A / Y436V / Q438K / S440D; (m) N434A / R435H / F436T / Q438R / S440E; (o) N434A / R435H / F436T / Q438R / S440D; (n) N434A / R435H / F436T / Q438K / S440E; (p) N434A / R435H / F436T / Q438K / S440D; (c) N434A / R435H / F436V / Q438R / S440E; (t) N434A / R435H / F436V / Q438R / S440D; (y) N434A / R435H / F436V / Q438K / S440E; (f) N434A / R435H / F436V / Q438K / S440D; (x) M428L / N434A / Q438R / S440E; (c) M428L / N434A / Q438R / S440D; (h) M428L / N434A / Q438K / S440E; (w) M428L / N434A / Q438K / S440D; (u) M428L / N434A / Y436T / Q438R / S440E; (e) M428L / N434A / Y436T7Q438R / S440D; (aa) M428L / N434A / Y436T / Q438K / S440E; (ab) M428L / N434A / Y436T / Q438K / S440D; (ab) M428L / N434A / Y436V / Q438R / S440E; (ah) M428L / N434A / Y436V / Q438R / S440D; (hell) M428L / N434A / Y436V / Q438K / S440E; (ae) M428L / N434A / Y436V / Q438K / S440D; (ah) L235R / G236R / S239K / M428L / N434A / Y436T / Q438R / S440E; and (az) L235R / G236R / A327G / A330S / P331S / M428L / N434A / Y436T / Q438R / S440E.

The methods may optionally also include: (c) evaluating whether the binding activity on the (previously present) ADA of the antibody containing the resulting Fc region variant is decreased compared to the binding activity of the reference antibody.

Alternatively, the methods can be used to enhance the release of antibody, which has been internalized into cells in antigen-bound form, out of cells in antigen-free form, without significantly increasing the binding activity of the antibody against (previously present) ADA at neutral pH.

Section B Description

Section B of the disclosure relates to antibodies to IL-8, nucleic acids that encode antibodies, pharmaceutical compositions containing the antibodies, methods for producing antibodies, and uses of the antibodies for the treatment of IL-8 related diseases, as detailed below. The meaning of the concepts below in the present description applies to the entire section B of the description, and they do not contradict the technical knowledge known to those of ordinary skill in the art, as well as embodiments of the invention known to them or generally accepted in the art.

I. Definitions falling within the scope of section B of the description

In the context of Section B of this description, "acidic pH" means a pH that can be selected, for example, from a pH of 4.0 to 6.5. In one embodiment, acidic pH means (but is not limited to) pH 4.0, pH 4.1, pH 4.2, pH 4.3, pH 4.4, pH 4.5, pH 4.6 , pH 4.7, pH 4.8, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6 , pH 5.7, pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4 or pH 6.5. In a specific embodiment, acidic pH refers to pH 5.8.

In the context of section B of the present description, "neutral pH" means a pH that can be selected, for example, from pH from 6.7 to 10.0. In one embodiment, pH neutral means (but is not limited to) pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3 , pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3 , pH 8.4, pH 8.5, pH 8.6, pH 8.7, pH 8.8, pH 8.9, pH 9.0, pH 9.5 or pH 10.0. In a specific embodiment, pH neutral refers to pH 7.4.

The term "IL-8" in the context of Section B refers to any native IL-8 derived from any vertebrate animal, primate (eg, humans, cynomolgus monkeys, rhesus monkeys) and other mammals (eg, dogs and rabbits), unless specifically otherwise indicated. The term "IL-8" refers to full-length IL-8, unprocessed IL-8, and any form of IL-8 resulting from processing in a cell. The term "IL-8" also refers to derivatives of native IL-8, for example, splicing variants or allelic variants. An example of the amino acid sequence of human IL-8 is shown in SEQ ID NO: 66.

The terms "antibody to IL-8" and "antibody that binds to IL-8" refer to an antibody that has the ability to bind to IL-8 with an affinity sufficient for the antibody to be useful as a diagnostic and / or therapeutic an agent that targets IL-8.

In one embodiment, the anti-IL-8 antibody binding to an unrelated non-IL-8 protein is less than about 10% of the antibody binding to IL-8.

The term "affinity" in the context of section B of the description refers to the total strength of all non-covalent interactions between one binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to the intrinsic binding affinity of a molecule reflecting a 1: 1 interaction between components of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can generally be characterized using the dissociation constant (KD). Affinity can be assessed using conventional methods known in the art, including those presented in section B of this description.

In some embodiments, an antibody that binds to IL-8 may have a dissociation constant (KD) of, for example, ≤1000 nM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0, 01 nM or ≤0.001 nM (for example, 10 ^-8 M or less, 10 ^-8 M to 10 ^-13 M, 10 ^-9 M to 10 ^-13 M).

In the context of Section B of the present description, the term "antibody" is used in its broadest sense and refers to various structures of antibodies, including (but not limited to) monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies) and antibody fragments, when provided that they have the desired antigen-binding activity.

An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of a reference antibody to its antigen by, for example, 50%, 60%, 70%, or 80% or more; conversely, the reference antibody blocks the binding of the antibody to its antigen by, for example, 50%, 60%, 70%, or 80% or more. An example of a competitive analysis that can be used is provided herein (but not limited to).

The term "chimeric" antibody refers to an antibody in which part of the heavy and / or light chain is from a particular source or species, and the remainder of the heavy and / or light chain is from another source or other species.

The term "humanized" antibody refers to a chimeric antibody that contains amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, a humanized antibody may consist of substantially at least one, and generally two, variable regions in which all or substantially all of the HVRs (e.g., CDRs) correspond to regions of a non-human antibody, and all or substantially all of the FRs correspond to regions of a human antibodies. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody.

The term "monoclonal antibody" in the context of section B of the present description refers to an antibody obtained from a population of substantially homogeneous antibodies, i. E. individual antibodies in the population are identical and / or bind to the same epitope, with the exception of possible antibody variants, for example, containing mutations that occur naturally or arising during the production of a monoclonal antibody preparation, these variants are usually present in minor amounts. Unlike polyclonal antibody preparations, which typically include different antibodies to different determinants (epitopes), each monoclonal antibody from a monoclonal antibody preparation is directed against a single antigen determinant. Thus, the adjective "monoclonal" defines an antibody characteristic by characterizing it as derived from a substantially homogeneous population of antibodies, rather than engineered to meet the requirements for antibody production by any particular method. For example, monoclonal antibodies that can be used according to section B of the present description can be created using a variety of technologies, including, but not limited to, the hybridoma method, recombinant DNA methods, phage display methods, and methods based on the use of transgenic animals that contain all or a portion of the human immunoglobulin loci, these methods, and other exemplary methods for producing monoclonal antibodies are provided herein.

In the context of Section B of the present description, the term "native antibody" refers to naturally occurring immunoglobulin molecules with various structures. In one embodiment, the native IgG antibody is, for example, a heterotetrameric glycoprotein with a molecular weight of about 150,000 Da, consisting of two identical light chains and two identical heavy chains linked by disulfide bridges. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also referred to as the variable heavy domain or the variable domain of the heavy chain, followed by three constant domains (CH1, CH2 and CH3). Likewise, from the N-terminus to the C-terminus, each light chain has a variable region (VL), which is also called a variable light domain or a light chain variable domain, followed by a light chain constant domain (CL). The light chain of an antibody, depending on the amino acid sequence of its constant domain, can belong to one of two types, which are designated kappa (κ) and lambda (λ). Said constant domains as used in Section B of the description include domains of any of the indicated allotypes (alleles) or any subclass / isotype. The heavy chain constant region includes (but is not limited to) the constant region of a native IgG-type antibody (IgG1, IgG2, IgG3, and IgG4). Known IgG1 alleles include, for example, IGHG1 * 01, IGHG1 * 02, IGHG1 * 03, IGHG1 * 04, and IGHG1 * 05 (see imgt.org), and any of these can be used as a native human IgG1 sequence. The constant domain sequence can be obtained from one allele or subclass / isotype, or from multiple alleles or subclasses / isotypes. In particular, these antibodies include, but are not limited to, an antibody whose CH1 is derived from IGHG1 * 01, and CH2 and CH3 are derived from IGHG1 * 02 and IGHG1 * 01, respectively.

The term "effector functions" in the context of section B of the description refers to those types of biological activities that are associated with the Fc region of an antibody, which vary depending on the isotype of the antibody. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-mediated cell-dependent cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (eg, B-cell receptor); and, but not limited to, B-cell activation.

The term "Fc region" in the context of Section B of this disclosure is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The concept includes native Fc regions and Fc region variants. Native Fc region refers to the Fc region of a native antibody.

In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (residues 446-447) of the Fc region may or may not be present. Unless otherwise indicated, in the context of Section B of the present description, the numbering of amino acid residues in the Fc region or constant region corresponds to the EU numbering system, which is also referred to as the EU index, described by Kabat E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

The term "framework" or "FR" in the context of section B of the present description means residues of the variable domain, other than residues of the hypervariable region (HVR). The FR of a variable domain usually consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences typically have the following location in the VH (or VL): FR1-H1 (L1) -FR2-H2 (L2) -FR3-H3 (L3) -FR4.

"Human consensus framework" in the context of section B of the present description is a framework that includes the most common amino acid residues in selected sequences of the VL or VH frameworks of human immunoglobulin. Typically, the selection of human immunoglobulin VL or VH sequences is made from a subset of variable domain sequences. Typically, a subset of sequences is the subset described by Kabat E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

In one embodiment, the VL subgroup is the κI subgroup according to Kabat et al., Supra. In one embodiment of the invention with respect to VH, the subgroup is subgroup III according to Kabat et al., Supra.

For the purposes of Section B of the present description, "acceptor human framework" means a framework that contains the amino acid sequence of a framework (VL) or (VH) derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may have the same amino acid sequence or may contain existing substitutions in the amino acid sequence. In some embodiments, the number of amino acid substitutions is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the sequence of the human VL acceptor framework is identical to the sequence of the VL framework of a human immunoglobulin or the sequence of a human consensus framework.

The term "variable region" or "variable domain" in the context of Section B of the present description refers to the domain of the heavy or light chain of an antibody that is involved in the binding of an antibody to an antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs) (see, for example, Kindt and et al., Kuby Immunology, 6th ed., WH Freeman and Co., 2007, p. 91). A single VH or VL domain may be sufficient to provide antigen binding specificity. In addition, antibodies that bind to a particular antigen can be isolated using the VH or VL domain from the antibody that binds to the antigen to screen a library of complementary VL or VH domains, respectively (see, for example, Portolano et al. , J. Immunol. 150, 1993, pp. 880-887; Clarkson et al., Nature 352, 1991, pp. 624-628).

The term "hypervariable region" or "HVR" in the context of section B of the present description refers to each of the regions of the variable domain of an antibody, the sequences of which are hypervariable ("complementarity determining regions" or "CDR") and / or form loops of a certain structure ("hypervariable loops "), And / or contain residues in contact with the antigen (" contacts with the antigen "). Typically, antibodies contain six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3).

Examples of HVRs include (but are not limited to): (a) hypervariable loops including amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53- 55 (H2) and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196, 1987, pp. 901-917); (6) CDRs including amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2) and 95-102 (H3) ( Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991); (c) regions of contact with the antigen, including amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2) and 93-101 ( H3) (MacCallum et al., J. Mol. Biol. 262, 1996, pp. 732-745); and (d) combinations of residues indicated in subparagraphs (a), (b) and / or (c), including amino acid residues HVR 46-56 (L2), 47-56 (L2), 48-56 (L2), 49 -56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3) and 94-102 (H3).

Unless otherwise indicated, HVR and other residues in variable regions (eg, residues in FR) are numbered according to Kabat et al., Supra.

An "individual" in the context of Section B of this specification is a mammal. Mammals include, but are not limited to, domesticated animals (such as cows, sheep, cats, dogs, and horses), primates (such as humans and non-human primates, such as monkeys), rabbits and rodents (such as mice and rats ). In some embodiments, the individual or subject is a human.

An "isolated" antibody in the context of Section B of this specification is an antibody that is separated from a component of its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99% as determined by, for example, electrophoretic analyzes (such as SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic analyzes (such as, for example, such as ion exchange chromatography or reversed phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al. J. Chrom. B 848, 2007, pp. 79-87.

An "isolated" nucleic acid in the context of Section B of the present disclosure is a nucleic acid molecule that is separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained in a cell, which normally includes a nucleic acid molecule, but in which the nucleic acid molecule is present outside the chromosome, or in which its location on the chromosome is different from its naturally occurring location on the chromosome.

The term "isolated nucleic acid encoding an anti-IL-8 antibody" in the context of section B of this description refers to one or more nucleic acid molecules encoding the heavy and light chains of an anti-IL-8 antibody (or fragments thereof), including the specified molecule (s) (s) in the same vector or in different vectors, and said nucleic acid molecule (s) (s) present at one or more positions in the host cell.

In the context of Section B of the present description, the terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of said cells. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and the offspring derived therefrom, regardless of the number of passages. The offspring may not be completely identical in nucleic acid composition to the parent cell, but may contain mutations. It is within the scope of the invention to include mutant progeny that have the same function or biological activity as found by screening or selection in the original transformed cell.

The term "vector" in the context of section B of the present description refers to a nucleic acid molecule that has the ability to multiply another nucleic acid to which it is associated. The concept includes a vector in the form of a self-replicating nucleic acid structure, as well as a vector included in the genome of a host cell into which it has been introduced. Certain vectors have the ability to control the expression of nucleic acids to which they are operably linked. These vectors in the context of the present description are referred to as "expression vectors".

In the context of Section B of this description, the term “package insert” refers to instructions that are usually included in commercial packaging of therapeutic products containing information about indications, use, dose, route of administration, combination therapy, contraindications and / or precautions that are associated with the use of these therapeutic products.

In the context of Section B herein, "percent (%) amino acid sequence identity" relative to a polypeptide reference sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the polypeptide reference sequence, after sequence alignment and, if necessary, introducing gaps to achieve maximum percentage of sequence identity, and not considering any conservative substitutions as a component in assessing sequence identity. Comparative analysis for the purpose of determining percent amino acid sequence identity can be performed in various ways known in the art using, for example, publicly available computer programs such as the BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) program. Those of skill in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the entire length of the sequences being compared. However, for the purposes of the present invention,% amino acid sequence identity values are obtained using a computer program to compare ALIGN-2 sequences. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code was provided in the U.S. User Documentation Kit. Copyright Office, Washington D.C., 20559, where it is registered as the U.S. Copyright Registration No. TXU510087. ALIGN-2 is publicly available from Genentech, Inc., South San Francisco, NY. California, or it can be compiled from source. ALIGN-2 can be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All parameters required for sequence comparison are set by the ALIGN-2 program and should not be changed.

In situations in which ALIGN-2 is used to compare amino acid sequences, the% amino acid sequence identity of a given amino acid sequence A versus, with, or relative to a given amino acid sequence B (which can alternatively be referred to as a given amino acid sequence A that has or contains a specific % amino acid sequence identity compared to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 × X / Y fraction, where X is the number of amino acid residues determined to be identical when evaluated using the ALIGN-2 sequence comparative analysis program , where the program compares A and B, and where Y denotes the total number of amino acid residues in B. It should be obvious that if the length of the amino acid sequence of A is not equal to the length of the amino acid sequence of B, then% the amino acid sequence identity of A with respect to B cannot be equal to the% identity of the amino acid sequence of B with respect to A. Unless otherwise specified, all% amino acid sequence identity values specified in Section B of this description were obtained using the ALIGN- computer program described in the previous paragraph. 2.

In the context of section B of the present description, the term "pharmaceutical composition" refers to a preparation that is in such a form that it provides the biological activity of the active substance included in its composition, which should be effective, and which does not contain additional components that have unacceptable toxicity to the individual to enter the song.

"Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition, other than the active ingredient, that is non-toxic to an individual. Pharmaceutically acceptable carriers include, but are not limited to, a buffer, excipient, stabilizer, or preservative.

In the context of Section B of the present description, the term "treatment" (and its grammatical variations such as "to treat" or "the treatment process") refers to clinical intervention with the aim of changing the natural course of the disease in the individual to be treated, and it can be carried out either to prevent , or during the development of clinical pathology. The required treatment actions are (but are not limited to) preventing the onset or recurrence of the disease, relieving symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastases, reducing the rate of development of the disease, alleviating or temporarily alleviating the disease state, and remission or improving prognosis. In some embodiments, the antibody described in Section B can be used to slow the progression of a disease or disorder.

In the context of Section B of the present description, the term "effective amount" of an antibody or pharmaceutical composition means an amount effective in doses and for a period of time necessary to achieve the desired therapeutic or prophylactic result.

II. Compositions and methods

One of the embodiments of the invention mentioned in section B of the description is based on the applicability of antibodies to IL-8, which have a pH-dependent affinity for IL-8, in the form of pharmaceutical compositions. The antibodies mentioned in Section B of the specification are used, for example, to diagnose or treat diseases in which IL-8 is present in excess.

A. Examples of antibodies to IL-8

One of the embodiments of the invention described in section B of the description is an antibody to IL-8, which has a pH-dependent affinity for IL-8.

One of the embodiments of the invention described in section B of the description is an antibody to IL-8 having a pH-dependent affinity for IL-8, which contains a sequence with at least one, two, three, four, five, six, seven or eight amino acid (s) substitution (s) in the following amino acid sequences: (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 67; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 68; (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 69; (d) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 70; (e) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 71; and (e) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 72.

Another embodiment of the invention referred to in Section B of the specification is an anti-IL-8 antibody having a pH-dependent affinity for IL-8 that contains at least one amino acid substitution (s) in at least one of the following amino acid sequences: (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 67; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 68; (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 69; (d) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 70; (e) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 71; and (e) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 72.

Unless otherwise specified, amino acids may be substituted with any other amino acid. In one embodiment, the anti-IL-8 antibody specified in Section B of the specification contains one or more amino acid substitution (s) at position (s) selected from the group consisting of the following positions: aspartic acid at position 1 in SEQ ID NO: 67; (b) tyrosine at position 2 in

sequences SEQ ID NO: 67; (c) tyrosine at position 3 in

sequences SEQ ID NO: 67; (d) leucine at position 4 in

sequences SEQ ID NO: 67; (e) serine at position 5 in

sequences SEQ ID NO: 67; (f) leucine at position 1 in

sequences SEQ ID NO: 68; (g) isoleucine at position 2 in

sequences SEQ ID NO: 68; (h) arginine at position 3 in

sequences SEQ ID NO: 68; (i) asparagine at position 4 in

sequences SEQ ID NO: 68; (j) lysine at position 5 in

sequences SEQ ID NO: 68; (l) alanine at position 6 in

sequences SEQ ID NO: 68; (m) asparagine at position 7 in

sequences SEQ ID NO: 68; (n) glycine at position 8 in

sequences SEQ ID NO: 68; (o) tyrosine at position 9 in

sequences SEQ ID NO: 68; (p) threonine at position 10 in

sequences SEQ ID NO: 68; (p) arginine at position 11 in

sequences SEQ ID NO: 68; (c) glutamic acid at position 12 in

sequences SEQ ID NO: 68; (t) tyrosine at position 13 in

sequences SEQ ID NO: 68; (y) serine at position 14 in

sequences SEQ ID NO: 68; (f) alanine at position 15 in

sequences SEQ ID NO: 68; (x) serine at position 16 in

sequences SEQ ID NO: 68; (c) valine in position 17 in

sequences SEQ ID NO: 68; (h) lysine at position 18 in

sequences SEQ ID NO: 68; (w) glycine at position 19 in

sequences SEQ ID NO: 68; (y) glutamic acid at position 1 in

sequences SEQ ID NO: 69; (e) asparagine at position 2 in

sequences SEQ ID NO: 69; (aa) tyrosine at position 3 in

sequences SEQ ID NO: 69; (ab) arginine at position 4 in

sequences SEQ ID NO: 69; (ab) tyrosine at position 5 in

sequences SEQ ID NO: 69; (ah) aspartic acid at position 6 in

sequences SEQ ID NO: 69; (hell) valine at position 7 in

sequences SEQ ID NO: 69; (ae) glutamic acid at position 8 in

sequences SEQ ID NO: 69; (already) leucine at position 9 in

sequences SEQ ID NO: 69; (az) alanine at position 10 in

sequences SEQ ID NO: 69; (ai) tyrosine at position 11 in

sequences SEQ ID NO: 69; (ak) arginine at position 1 in

sequences SEQ ID NO: 70; (al) alanine at position 2 in

sequences SEQ ID NO: 70; (am) serine at position 3 in

sequences SEQ ID NO: 70; (an) glutamic acid at position 4 in

sequences SEQ ID NO: 70; (ao) isoleucine at position 5 in

sequences SEQ ID NO: 70; (an) isoleucine at position 6 in

sequences SEQ ID NO: 70; (ar) tyrosine at position 7 in

sequences SEQ ID NO: 70; (ac) serine at position 8 in

sequences SEQ ID NO: 70; (at) tyrosine at position 9 in

sequences SEQ ID NO: 70; (ay) leucine at position 10 in

sequences SEQ ID NO: 70; (af) alanine at position 11 in

sequences SEQ ID NO: 70; (ax) asparagine at position 1 in

sequences SEQ ID NO: 71; (ac) alanine at position 2 in

sequences SEQ ID NO: 71; (ah) lysine at position 3 in

sequences SEQ ID NO: 71; (ah) threonine at position 4 in

sequences SEQ ID NO: 71; (ach) leucine at position 5 in

sequences SEQ ID NO: 71; (ae) alanine at position 6 in

sequences SEQ ID NO: 71; (bа) aspartic acid at position 7 in

sequences SEQ ID NO: 71; (bb) glutamine at position 1 in

sequences SEQ ID NO: 72; (bc) histidine at position 2 in

sequences SEQ ID NO: 72; (bd) histidine at position 3 in

sequences SEQ ID NO: 72; (bd) phenylalanine at position 4 in

sequences SEQ ID NO: 72; (de) glycine at position 5 in

sequences SEQ ID NO: 72; (bg) phenylalanine at position 6 in

sequences SEQ ID NO: 72; (bz) proline at position 7 in

sequences SEQ ID NO: 72; (bi) arginine at position 8 in

sequences SEQ ID NO: 72; and (bq) threonine at position 9 in

SEQ ID NO: 72.

In one embodiment, the anti-IL-8 antibody specified in Section B of the specification contains one or more amino acid substitution (s) at position (s) selected from the group consisting of the following positions: ( a) alanine at position 6 in

sequences SEQ ID NO: 68; (b) glycine at position 8 in

sequences SEQ ID NO: 68; (c) tyrosine at position 9 in

sequences SEQ ID NO: 68; (d) arginine at position 11 in

sequences SEQ ID NO: 68; and (e) tyrosine at position 3 in

SEQ ID NO: 69.

In one embodiment, the anti-IL-8 antibody specified in Section B of the specification contains a combination (s) of amino acid substitutions at positions selected from the group consisting of the following: (a) alanine at position 6 in SEQ ID NO: 68; (b) glycine at position 8 in SEQ ID NO: 68; (c) tyrosine at position 9 in SEQ ID NO: 68; (d) arginine at position 11 in SEQ ID NO: 68; and (e) tyrosine at position 3 in SEQ ID NO: 69.

In one embodiment, the anti-IL-8 antibody specified in Section B of the specification contains amino acid substitutions at the following positions: (a) tyrosine at position 9 in SEQ ID NO: 68; (b) arginine at position 11 in SEQ ID NO: 68; and (c) tyrosine at position 3 in SEQ ID NO: 69.

In one embodiment, the anti-IL-8 antibody specified in Section B of the specification contains amino acid substitutions at the following positions: (a) alanine at position 6 in SEQ ID NO: 68; (b) glycine at position 8 in SEQ ID NO: 68; (c) tyrosine at position 9 in SEQ ID NO: 68; (d) arginine at position 11 in SEQ ID NO: 68; and (e) tyrosine at position 3 in SEQ ID NO: 69.

In one embodiment, the anti-IL-8 antibody referred to in Section B of the description comprises: (a) an alanine to aspartic acid substitution at position 6 in SEQ ID NO: 68; (b) substitution of arginine for proline at position 11 in SEQ ID NO: 68; and (c) substitution of tyrosine for histidine at position 3 in SEQ ID NO: 69.

In one embodiment, the anti-IL-8 antibody specified in Section B of the description comprises: (a) replacing glycine with tyrosine at position 8 in SEQ ID NO: 68; and (b) substitution of tyrosine for histidine at position 9 in SEQ ID NO: 68.

In one embodiment, the anti-IL-8 antibody referred to in Section B of the description comprises: (a) an alanine to aspartic acid substitution at position 6 in SEQ ID NO: 68; (b) substitution of glycine for tyrosine at position 8 in SEQ ID NO: 68; (c) substitution of tyrosine for histidine at position 9 in SEQ ID NO: 68; (d) substitution of arginine for proline at position 11 in SEQ ID NO: 68; and (e) substitution of tyrosine for histidine at position 3 in SEQ ID NO: 69.

In one embodiment, the anti-IL-8 antibody referred to in Section B of the specification comprises an HVR-H2 that comprises the amino acid sequence of SEQ ID NO: 73.

In one embodiment, the anti-IL-8 antibody referred to in Section B of the description contains HVR-H3, which contains the amino acid sequence of SEQ ID NO: 74.

In one embodiment, the anti-IL-8 antibody referred to in Section B of the description contains HVR-H1, which contains the amino acid sequence of SEQ ID NO: 67, HVR-H2, which contains the amino acid sequence of SEQ ID NO: 73, and HVR- H3, which contains the amino acid sequence of SEQ ID NO: 74.

In one embodiment, the anti-IL-8 antibody specified in Section B of the specification contains one or more amino acid substitution (s) at position (s) selected from the group consisting of the following positions: ( a) serine at position 8 in SEQ ID NO: 70; (b) asparagine at position 1 in SEQ ID NO: 71; (c) leucine at position 5 in SEQ ID NO: 71; and (d) glutamine at position 1 in SEQ ID NO: 72. In another embodiment, the anti-IL-8 antibody comprises a combination of any 2, 3, or all 4 of these substitutions.

In one embodiment of the invention, the anti-IL-8 antibody specified in Section B of the description contains a combination (s) of amino acid substitutions at position (s) selected from the group consisting of the following positions: (a) serine at position 8 in SEQ ID NO: 70; (b) asparagine at position 1 in SEQ ID NO: 71; (c) leucine at position 5 in SEQ ID NO: 71; and (d) glutamine at position 1 in SEQ ID NO: 72. In another embodiment, the anti-IL-8 antibody comprises a combination of any 2, 3, or all 4 of these substitutions.

In one embodiment, the anti-IL-8 antibody specified in Section B of the description contains amino acid substitutions at positions: (a) asparagine at position 1 in SEQ ID NO: 71; (b) leucine at position 5 in SEQ ID NO: 71; and (c) glutamine at position 1 in SEQ ID NO: 72.

In one embodiment, the anti-IL-8 antibody specified in Section B of the specification contains amino acid substitutions at positions: (a) serine at position 8 in SEQ ID NO: 70; (b) asparagine at position 1 in SEQ ID NO: 71; (c) leucine at position 5 in SEQ ID NO: 71; and (d) glutamine at position 1 in SEQ ID NO: 72.

In one embodiment, the anti-IL-8 antibody specified in Section B of the specification comprises: (a) replacing asparagine with lysine at position 1 in SEQ ID NO: 71; (b) substitution of leucine for histidine at position 5 in SEQ ID NO: 71; and (c) replacing glutamine with lysine at position 1 in SEQ ID NO: 72.

In one embodiment, the anti-IL-8 antibody specified in Section B of the specification comprises: (a) replacing serine with glutamic acid at position 8 in SEQ ID NO: 70; (b) substitution of asparagine for lysine at position 1 in SEQ ID NO: 71; (c) substitution of leucine for histidine at position 5 in SEQ ID NO: 71; and (c) replacing glutamine with lysine at position 1 in SEQ ID NO: 72.

In one embodiment, the anti-IL-8 antibody referred to in Section B of the specification contains an HVR-L2 that contains the amino acid sequence of SEQ ID NO: 75.

In one embodiment, the anti-IL-8 antibody specified in Section B of the description contains HVR-L3, which contains the amino acid sequence of SEQ ID NO: 76.

In one embodiment, the anti-IL-8 antibody specified in Section B of the description contains HVR-L1, which contains the amino acid sequence of SEQ ID NO: 70, HVR-L2, which contains the amino acid sequence of SEQ ID NO: 75, and HVR- L3, which contains the amino acid sequence of SEQ ID NO: 76.

In one embodiment, the anti-IL-8 antibody specified in Section B of the specification contains amino acid substitutions at the following positions: (a) alanine at position 55 in SEQ ID NO: 77; (b) glycine at position 57 in SEQ ID NO: 77; (c) tyrosine at position 58 in SEQ ID NO: 77; (d) arginine at position 60 in SEQ ID NO: 77; (e) glutamine at position 84 in SEQ ID NO: 77; (f) a serine at position 87 in SEQ ID NO: 77; and (g) tyrosine at position 103 in SEQ ID NO: 77.

In one embodiment, the anti-IL-8 antibody referred to in Section B of the description comprises: (a) an alanine to aspartic acid substitution at position 55 in SEQ ID NO: 77; (b) substitution of glycine for tyrosine at position 57 in SEQ ID NO: 77; (c) substitution of tyrosine for histidine at position 58 in SEQ ID NO: 77; (d) substitution of arginine for proline at position 60 in SEQ ID NO: 77; (e) substitution of glutamine for threonine at position 84 in SEQ ID NO: 77; (f) substitution of serine for aspartic acid at position 87 in SEQ ID NO: 77; and (g) substitution of tyrosine for histidine at position 103 in SEQ ID NO: 77.

In one embodiment, the anti-IL-8 antibody referred to in Section B of the description comprises a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO: 78.

In one embodiment, the anti-IL-8 antibody referred to in Section B of the specification comprises a light chain variable region that comprises the amino acid sequence of SEQ ID NO: 79.

In one embodiment, the anti-IL-8 antibody referred to in Section B of the description comprises a heavy chain variable region that contains the amino acid sequence of SEQ ID NO: 78 and a light chain variable region that contains the amino acid sequence of SEQ ID NO: 79. An anti-IL-8 antibody that contains a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 78 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 79 may be an anti-IL-8 antibody that binds IL- 8 in a pH-dependent manner. An anti-IL-8 antibody that contains a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 78 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 79 may be an anti-IL-8 antibody that maintains IL stability -8-neutralizing activity in vivo (for example, in plasma). An anti-IL-8 antibody that contains a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 78 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 79 may be an antibody of low immunogenicity.

In an alternative aspect of the invention, the anti-IL-8 antibodies referred to in Section B of the specification also include antibodies having a pH-dependent affinity for IL-8 and containing at least one amino acid substitution in at least any of the following amino acid sequences: (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 102; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 103; (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 104; (d) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 105; (e) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 106; and (e) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 107.

In an alternative aspect of the invention, the anti-IL-8 antibodies referred to in Section B of the specification also include antibodies having a pH-dependent affinity for IL-8 and containing at least one amino acid substitution in at least any of the following amino acid sequences: (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 108; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 109; (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 110; (d) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 111; (e) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 112; and (f) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 113.

In an alternative aspect of the invention, the anti-IL-8 antibodies referred to in Section B of the specification also include antibodies having a pH-dependent affinity for IL-8 and containing at least one amino acid substitution in at least any of the following amino acid sequences: (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 114; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 115; (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 116; (d) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 117; (e) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 118; and (e) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 119.

In an alternative aspect of the invention, the anti-IL-8 antibodies referred to in Section B of the specification also include antibodies having a pH-dependent affinity for IL-8 and containing at least one amino acid substitution in at least any of the following amino acid sequences: (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 120; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 121; (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 122; (d) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 123; (e) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 124; and (f) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 125.

In an alternative aspect of the invention, the anti-IL-8 antibodies referred to in Section B of the specification also include antibodies having a pH-dependent affinity for IL-8 and containing at least one amino acid substitution in at least any of the following amino acid sequences: (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 126; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 127; (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 128; (d) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 129; (e) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 130; and (e) HVR-L3, which contains the amino acid sequence of SEQ IDNO: 131.

In an alternative aspect of the invention, the anti-IL-8 antibodies referred to in Section B of the specification also include antibodies having a pH-dependent affinity for IL-8 and containing at least one amino acid substitution in at least any of the following amino acid sequences: (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 132; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 133; (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 134; (d) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 135; (e) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 136; and (e) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 137.

In an alternative aspect of the invention, the anti-IL-8 antibodies referred to in Section B of the specification also include antibodies having a pH-dependent affinity for IL-8 and containing at least one amino acid substitution in at least any of the following amino acid sequences: (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 138; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 139; (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 140; (d) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 141; (e) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 142; and (f) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 143.

In one embodiment, the anti-IL-8 antibody specified in Section B of the specification has an IL-8 neutralizing activity. IL-8 neutralizing activity means the ability to inhibit the biological activity of IL-8, or may mean the ability to inhibit binding to the IL-8 receptor.

In an alternative aspect of the invention, the anti-IL-8 antibody referred to in Section B of the specification is an anti-IL-8 antibody that binds to IL-8 in a pH-dependent manner. In the context of section B of the description, an antibody to IL-8 that binds IL-8 in a pH-dependent manner means an antibody whose binding affinity for IL-8 at acidic pH is lower than that for IL-8 at neutral pH. For example, pH-dependent antibodies to IL-8 include antibodies that have higher affinity for IL-8 at neutral pH than at acidic pH. In one embodiment, the anti-IL-8 antibody specified in section B of the description has an affinity for IL-8 at neutral pH, which is at least 2, 3, 5, 10, 15, 20, 25, 30, 35 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 times or more higher than the affinity at acidic pH. Binding affinity can be measured by surface plasma resonance methods (eg, BIACORE®), but not limited to them. The association reaction rate constant (k _on ) and the dissociation reaction rate constant (k _off ) can be calculated using the BIACORE® T200 Evaluation software (GE Healthcare) using a simple 1: 1 Langmuir binding model by simultaneously approximating the association and dissociation sensorgrams. The equilibrium constant of the dissociation reaction ((KD) is calculated as the ratio k _off / k _on . For screening for antibodies whose binding affinity depends on pH, ELISA, Kinetic Exclusion Assay (KinExA ™) and other assays as well as surface plasmon resonance methods (such as BIACORE®) pH-dependent IL-8 binding capacity refers to the ability to bind to IL-8 in a pH-dependent manner In addition, whether the antibody has the ability to repeatedly bind IL-8 can be evaluated by the methods described in WO 2009/125825.

In one embodiment, it is preferred that the anti-IL-8 antibody specified in Section B of the description has a low dissociation constant (KD) for IL-8 at neutral pH. In one embodiment of the invention, the dissociation constant for the antibody specified in section B, which characterizes binding to IL-8 at neutral pH, is, for example, 0.3 nM or less (but not limited to only the specified value). In one embodiment, the dissociation constant for the antibody of Section B, which binds to IL-8 at neutral pH, is, for example, 0.1 nM or less (but not limited to only the specified value) In one embodiment the dissociation constant for the antibody specified in section B, characterizing binding to IL-8 at neutral pH, is, for example, 0.03 nM or less (but not limited to only the specified value).

In one embodiment, it is preferred that the anti-IL-8 antibody described in Section B of the description has a low dissociation constant (KD) for IL-8 at pH 7.4. In one embodiment of the invention, the dissociation constant for the antibody specified in section B, characterizing binding to IL-8 at pH 7.4, is, for example, 0.3 nM or less (but not limited to only the specified value). In one embodiment of the invention, the dissociation constant for the antibody specified in section B, which characterizes binding to IL-8 at pH 7.4, is, for example, 0.1 nM or less (but not limited to only the specified value). In one embodiment of the invention, the dissociation constant for the antibody specified in section B, which characterizes binding to IL-8 at pH 7.4, is, for example, 0.03 nM or less (but not limited to only the specified value)

In one embodiment, it is preferred that the anti-IL-8 antibody specified in Section B of the description has a high dissociation constant (KD) for IL-8 at acidic pH. In one embodiment of the invention, the dissociation constant for the antibody specified in section B, which characterizes binding to IL-8 at acidic pH, is, for example, 3 nM or more (but not limited to only the specified value). In one embodiment of the invention, the dissociation constant for the antibody specified in section B, which characterizes binding to IL-8 at acidic pH, is, for example, 10 nM or more (but not limited to only the specified value). In one embodiment of the invention, the dissociation constant for the antibody specified in section B, which characterizes binding to IL-8 at acidic pH, is, for example, 30 nM or more (but not limited to only the specified value).

In one embodiment, it is preferred that the anti-IL-8 antibody specified in Section B of the description has a high dissociation constant (KD) for IL-8 at pH 5.8. In one embodiment of the invention, the dissociation constant for the antibody specified in section B, which characterizes binding to IL-8 at pH 5.8, is, for example, 3 nM or more (but not limited to only the specified value). In one embodiment of the invention, the dissociation constant for the antibody specified in section B, which characterizes binding to IL-8 at pH 5.8, is, for example, 10 nM or more (but not limited to only the specified value). In one embodiment of the invention, the dissociation constant for the antibody specified in section B, characterizing binding to IL-8 at pH 5.8, is, for example, 30 nM or more (but not limited to only the specified value).

In one embodiment of the invention, it is preferred that the binding affinity of the anti-IL-8 antibody specified in Section B of the description to IL-8 is higher at neutral pH than at acidic pH.

In one embodiment, the ratio of the dissociation constant at acidic pH and neutral pH [KD (acidic pH) / KD (neutral pH)] of the anti-IL-8 antibody specified in section B is, for example, 30 or more (but not limited to specified value only). In one embodiment, the ratio of the dissociation constants at acidic pH to neutral pH [KD (acidic pH) / KD (neutral pH)] of the anti-IL-8 antibody specified in section B is, for example, 100 or more, for example, 200 , 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000 or 9500 (but not limited to only them).

In one embodiment of the invention, the ratio of the dissociation constant at pH 5.8 and pH 7.4 [KD (pH 5.8) / KD (pH 7.4)] of the anti-IL-8 antibody specified in section B is, for example, 30 or more (but not limited to only the specified value). In one embodiment, the ratio of the dissociation constants at pH 5.8 and pH 7.4 [KD (pH 5.8) / KD (pH 7.4)] of the anti-IL-8 antibody specified in Section B is, for example , 100, for example, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000 , 8500, 9000, or 9500 (but not limited to).

In one embodiment, it is preferred that the anti-IL-8 antibody specified in Section B of the specification has a high dissociation rate constant (k _off ) at acidic pH. In one embodiment, the reaction rate constant of the dissociation reaction of the antibody specified in section B of the description at an acidic pH is, for example, 0.003 (1 / s) or more (but not limited only to the specified value). In one embodiment of the invention, the reaction rate constant of the dissociation reaction of the antibody specified in section B of the description at acidic pH is, for example, 0.005 (1 / s) or more (but not limited only to the specified value). In one embodiment, the reaction rate constant of the dissociation reaction of the antibody specified in section B of the description at acidic pH is, for example, 0.01 (1 / s) or more (but not limited to only the specified value).

In one embodiment of the invention, it is preferred that the anti-IL-8 antibody specified in section B of the description has a high dissociation rate constant (k _off ) at pH 5.8. In one embodiment, the reaction rate constant for the dissociation of the antibody specified in Section B at pH 5.8 is, for example, 0.003 (1 / s) or more (but not limited to this value). In one embodiment, the reaction rate constant of the dissociation reaction of the antibody specified in Section B of the description at pH 5.8 is, for example, 0.005 (1 / s) or more (but not limited to only the specified value). In one embodiment, the reaction rate constant of the dissociation reaction of the antibody specified in Section B of the description at pH 5.8 is, for example, 0.01 (1 / s) or more (but not limited to only the specified value).

In one embodiment, it is preferred that the anti-IL-8 antibody specified in Section B of the specification retains stable IL-8 neutralizing activity in solution (eg, in PBS). Whether the activity remains stable in solution can be assessed by determining whether the IL-8 neutralizing activity of the antibody specified in section B of the description added to the solution changes after storage for a period of time at a specific temperature relative to the activity prior to addition to the solution. In one embodiment, the storage period is, for example, 1, 2, 3, or 4 weeks (but not limited to). In one embodiment, the storage temperature is, for example, but not limited to 25 ° C, 30 ° C, 35 ° C, 40 ° C, or 50 ° C. In one embodiment of the invention, the storage temperature is, for example, 40 ° C (but not limited to only the specified); and the storage period is, for example, 2 weeks (but not limited to only specified). In one embodiment of the invention, the storage temperature is, for example, 50 ° C (but not limited only to the specified); and the storage period is, for example, 1 week (but not limited to only specified).

In one embodiment, it is preferred that the anti-IL-8 antibody described in Section B of the specification retains stable IL-8 neutralizing activity in vivo (eg, in plasma). Whether the activity remains stable in vivo can be assessed by determining whether the IL-8 neutralizing activity of the antibody specified in section B of the description added to the plasma of an animal (e.g., mouse) or human changes after storage for a specified period of time at a specified temperature relative to activity prior to addition to plasma. In one embodiment, the storage period is, for example, 1, 2, 3, or 4 weeks (but not limited to). In one embodiment, the storage temperature is, for example, but not limited to 25 ° C, 30 ° C, 35 ° C, or 40 ° C. In one embodiment of the invention, the storage temperature is, for example, 40 ° C (but not limited to only the specified); and the storage period is, for example, 2 weeks (but not limited to only specified).

In one embodiment, the rate of cellular uptake of the anti-IL-8 antibody described in Section B is higher when the antibody is complexed with IL-8 than the uncomplexed antibody. The anti-IL-8 antibody referred to in section B of the specification is more readily absorbed by cells when it forms a complex with IL-8 outside the cells (eg, in plasma) than when it does not form a complex with IL-8.

In one embodiment of the invention, it is preferred that the predicted immunogenicity of the anti-IL-8 antibody specified in Section B of the description, as expected in a human host, is reduced. "Low immunogenicity" can mean (but is not limited to only indicated), for example, that after administration, the antibody to IL-8 does not induce an immune response in a living organism, in at least half or more of the individuals to whom the antibody is administered in a sufficient amount during the period time sufficient to achieve therapeutic efficacy. Induction of an immune response can involve the production of antibodies against a drug. The term “low production of anti-drug antibody” is used interchangeably with the term “low immunogenicity”. The level of immunogenicity in humans can be determined using a T-cell epitope prediction program. These programs for predicting T cell epitopes include Epibase (Lonza), iTope / TCED (Antitope), EpiMatrix (EpiVax), etc. EpiMatrix is a system for predicting the immunogenicity of a protein of interest based on the automatic construction of sequences of peptide fragments by sectioning the amino acid sequence of the protein for which it is required to predict its immunogenicity into nine amino acids and then calculating their ability to bind to the eight major alleles of GCS class II (DRB1 * 0101, DRB1 * 0301, DRB1 * 0401, DRB1 * 0701, DRB1 * 0801, DRB1 * 1101, DRB1 * 1301 and DRB1 * 1501) (Clin. Immunol. 131 (2), 2009, pp. 189-201). Sequences in which the amino acid amino acid sequence of the anti-IL-8 antibody has been modified can be analyzed using the T-cell epitope prediction programs described above for the reduced immunogenicity sequences generated. Preferred amino acid modification sites for reducing the immunogenicity of an anti-IL-8 antibody specified in Section B of the specification include, but are not limited to, the amino acids at position 81 and / or position 82b according to Cabot numbering in the anti-IL-8 antibody heavy chain sequence indicated in SEQ ID NO: 78.

One of the embodiments of the invention, referred to in Section B of the description, are methods of enhancing the elimination of IL-8 from the body of an individual as compared to using a reference antibody, comprising administering the anti-IL-8 antibody specified in Section B of the description to the individual. One of the embodiments of the invention referred to in section B of the description is the use of the antibody to IL-8, specified in section B of the description, to enhance the elimination of IL-8 from the body of the individual, compared with using a reference antibody. One of the embodiments of the invention referred to in Section B of the specification is an anti-IL-8 antibody specified in Section B of the specification, intended for use to enhance the elimination of IL-8 from an individual as compared to using a reference antibody. One of the embodiments of the invention referred to in Section B of the specification is the use of the anti-IL-8 antibody specified in Section B of the specification for the preparation of pharmaceutical compositions designed to enhance the elimination of IL-8 in vivo as compared to using a reference antibody. One of the embodiments of the invention specified in section B of the description, are pharmaceutical compositions containing the antibody to IL-8 specified in section B of the description, designed to enhance the elimination of IL-8 compared to using a reference antibody. One of the embodiments of the invention referred to in Section B of the description are methods of enhancing the elimination of IL-8 over the use of a reference antibody, comprising administering the anti-IL-8 antibody specified in Section B of the description to an individual. In one of the embodiments of the invention specified in section B of the description, the reference antibody means an antibody to IL-8 before modification to obtain the antibody specified in section B of the description, or an antibody, the binding affinity for IL-8 in which is strong as in acidic and at neutral pH. The reference antibody can be an antibody comprising the amino acid sequence of SEQ ID NOs: 83 and 84 or SEQ ID NOs: 89 and 87.

One of the embodiments of the invention specified in Section B of the description are pharmaceutical compositions containing the antibody to IL-8 specified in Section B of the description, characterized in that the antibody to IL-8 specified in Section B of the description binds to IL-8 and then with the extracellular matrix. One of the embodiments of the invention referred to in Section B of the specification is the use of the anti-IL-8 antibody specified in Section B of the specification for preparing pharmaceutical compositions characterized in that the antibody to IL-8 specified in Section B of the specification binds to IL -8 and then with the extracellular matrix.

In any of the above embodiments, the anti-IL-8 antibody may be a humanized antibody.

In one aspect of the invention, the antibody referred to in Section B of the description comprises a heavy chain variable region according to any of the above-described embodiments and a light chain variable region according to any of the above-described embodiments. In one embodiment, the antibody referred to in Section B of the specification contains the heavy chain variable region of SEQ ID NO: 78 and the light chain variable region of SEQ ID NO: 79, and may also contain post-translational modifications in said sequences.

In a further aspect of the invention, an anti-IL-8 antibody as defined in any of the above embodiments may have any of the features, individually or in combination, described in sections 1-7 below.

1. Chimeric antibody and humanized antibody

In some embodiments, the antibody identified in Section B of the specification is a chimeric antibody. Certain chimeric antibodies are described, for example, in US Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81, 1984, cc. 6851-6855. In one example, a chimeric antibody comprises a non-human variable region (eg, a variable region from an antibody from mice, rats, hamsters, rabbits, or non-human primates such as monkeys) and a human constant region.

In some embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity in humans while maintaining the specificity and affinity of the parent non-human antibody. Typically, a humanized antibody contains one or more variable domains in which HVRs, for example CDRs (or portions thereof) are derived from non-human antibodies, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody may optionally contain at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (eg, the antibody from which the HVR residues are derived), for example, to maintain or improve the specificity or affinity of the antibody.

и др., Methods 36, 2005, сс. 43-60 (описание «перестановки FR»); и Osbourn и др., Methods 36, 2005, сс. 61-68 и Klimka и др., Br. J. Cancer 83, 2000, сс. 252-260 (описание подхода «направленной селекции» для перестановки FR).Information on humanized antibodies and methods for their creation is summarized, for example, in Almagro, Front. Biosci. 13, 2008, pp. 1619-1633, and are also described in Riechmann et al., Nature 332, 1988, pp. 323-329; Queen et al., Proc. Natl. Acad. Sci. USA 86, 1989, pp. 10029-10033; US Nos. 5821337, 7527791, 6982321 and 7087409; Kashmiri et al., Methods 36, 2005, pp. 25-34 (description of SDR (a-CDR) transplantation); Padlan, Mol. Immunol. 28, 1991, pp. 489-498 (description of "applying a new coating");

et al., Methods 36, 2005, pp. 43-60 (description of "FR permutation"); and Osbourn et al., Methods 36, 2005, pp. 61-68 and Klimka et al., Br. J. Cancer 83, 2000, pp. 252-260 (describing a "directed selection" approach for FR permutation).

Human frameworks that can be used for humanization include, but are not limited to: frameworks selected using "best fit" techniques (see, for example, Sims et al., J. Immunol. 151, 1993, pp. 2296-2308; framework regions derived from the consensus sequence of a particular subset of human light and heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA 89, 1992, pp. 4285- 4289 and Presta et al., J. Immunol. 151, 1993, pp. 2623-2632); human mature (with somatic mutations) framework regions or human germline framework regions (see, for example, Almagro and Fransson, Front. Biosci 13, 2008, pp. 1619-1633); and framework regions obtained from screening FR libraries (see, for example, Vasa et al., J. Biol. Chem. 272, 1997, pp. 10678-10684 and Rosok et al., J. Biol. Chem. 271, 1996, pp. 22611-22618).

2. Antibody fragments

In some embodiments, the antibody described in Section B of the specification is an antibody fragment. Antibody fragments include, but are not limited to, such fragments as Fab, Fab ', Fab'-SH, F (ab') ₂ , Fv and scFv, and other fragments described below. For a review of some antibody fragments, see Hudson et al. Nat. Med. 9, 2003, pp. 129-134. For a review of scFv fragments, see, for example, Plueckthun in: The Pharmacology of Monoclonal Antibodies, ed. Rosenburg and Moore, Springer, New York, vol. 113, 1994, pp. 269-315; see also WO 93/16185; and US Nos. 5571894 and 5587458.

Dimeric antibodies are antibody fragments with two antigen-binding sites, which can be bivalent or bispecific (see, for example, EP 0404097; WO 1993/01161; Hudson et al. Nat. Med. 9, 2003, pp. 129-134; and Holliger et al., Proc. Natl. Acad. Sci. USA 90, 1993, pp. 6444-6448). Trimeric and tetrameric antibodies are also described in Hudson et al., Nat. Med. 9, 2003, pp. 129-134).

Single domain antibodies are antibody fragments containing all or part of the variable domain of the heavy chain, or all or part of the variable domain of the light chain of an antibody. In some embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, Massachusetts; see, for example, US Pat. No. 6,248,516 B1). Antibody fragments can be generated by a variety of techniques, including, but not limited to, proteolytic cleavage of an intact antibody, as well as using recombinant host cells (eg, E. coli or phage) as described in section B herein.

3. Human antibody

In some embodiments, the antibody specified in Section B of the specification may be a human antibody. Human antibodies can be produced using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5, 2001, cc. 368-374 and Lonberg, Curr. Opin. Immunol. 20, 2008, cc. 450-459. Human antibodies can be produced by administering an immunogen to a transgenic animal modified to produce intact human antibodies or intact antibodies with human variable regions in response to challenge with the antigen. These animals typically contain all or part of the human immunoglobulin loci instead of the endogenous immunoglobulin loci that are either present outside the chromosomes or integrated arbitrarily into the animal's chromosomes. In such transgenic mice, endogenous immunoglobulin loci are usually inactivated. For a review of methods for producing human antibodies in transgenic animals see Lonberg, Nat. Biotech. 23, 2005, pp. 1117-1125 (see, for example, also in US No. 6075181 and US No. 6150584 description of XENOMOUSE ™ technology; in US No. 5770429 description of HUMAB ™ technology; in US No. 7041870 description of K-M MOUSE ™ technology and in the publication of US patent application 2007/0061900 description of VELOCIMOUSE ™ technology).

Human variable regions from intact antibodies obtained with these animals can be further modified, for example, by combining with another human constant region.

Human antibodies can be generated using hybridoma-based techniques. Human myeloma and murine-human heteromyeloma cell lines have been described for the production of human monoclonal antibodies (see, for example, Kozbor, J. Immunol. 133, 1984, pp. 3001-3005; Brodeur et al., Monoclonal Antibody Production Techniques and Applications , Marcel Dekker, Inc., New York, 1987, pp. 51-63 and Boerner et al., J. Immunol. 147, 1991, pp. 86-95). Human antibodies generated using human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA 103, 2006, cc. 3557-3562. Additional methods include those described, for example, in US No. 7189826 (description of obtaining monoclonal human antibodies of the IgM type from hybridoma cell lines), and Ni, Xiandai Mianyixue 26, 2006, cc. 265-268 (description of human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology 20, 2005, cc. 927-937 and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology 27, 2005, cc. 185-191. Human antibodies can also be generated by isolating the variable domain sequences of Fv clones selected from phage display libraries that were generated using human antibodies. These variable domain sequences can then be combined with the desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

4. Antibodies obtained from libraries

The antibodies mentioned in section B of the description can be isolated by screening combinatorial libraries of antibodies with the desired activity or activities. For example, a variety of techniques are known in the art for generating phage display libraries and screening these libraries for antibodies having the desired binding characteristics. These methods are summarized, for example, in Hoogenboom et al., Methods in Molecular Biology 178, 2001, cc. 1-37, and are also described, for example, in McCafferty et al., Nature 348, 1990, pp. 552-554; Clackson et al., Nature 352, 1991, pp. 624-628; Marks et al., J. Mol. Biol. 222, 1992, pp. 581-597; Marks and Bradbury, Methods in Molecular Biology 248, 2003, cc. 161-175; Sidhu et al., J. Mol. Biol. 338, 2004, cc. 299-310; Lee et al., J. Mol. Biol. 340, 2004, cc. 1073-1093; Fellouse, Proc. Natl. Acad. Sci. USA 101, 2004, cc. 12467-12472 and Lee et al. J. Immunol. Methods 284, 2004, cc. 119-132.

In some phage display techniques, populations of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in phage libraries, which are then screened for antigen-binding phage according to the method described in Winter et al., Ann ... Rev. Immunol. 12, 1994, pp. 433-455. Phage typically exposes antibody fragments either as single chain Fv fragments (scFv) or as Fab fragments.

Alternatively, it is possible to clone an untreated ("naive") population (eg, from a human body), obtaining a single source of antibodies to a wide range of foreign as well as its own antigens without any immunization, as described in Griffiths et al., EMBO J. 12, 1993, pp. 725-734.

And finally, "naive" libraries can also be obtained by synthesis methods by cloning non-rearranged V-gene segments from stem cells and using PCR primers containing a random sequence to encode hypervariable CDR3 regions and to carry out rearrangement in vitro according to the method, described by Hoogenboom and Winter, J. Mol. Biol. 227, 1992, cc. 381-388. Phage libraries of human antibodies are described, for example, in the following patent publications: US No. 5750373 and US Patent Application Publications 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936 and 2009/0002360. As used herein, antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments.

5. Multispecific antibody

In some embodiments, the antibody referred to in Section B of the specification may be, for example, a multispecific antibody, eg, a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have specificities that bind to at least two different sites.

In some embodiments, one of the binding specificities provides binding to IL-8 and the other to any other antigen.

In some embodiments, the bispecific antibodies can bind to two different epitopes on IL-8. Bispecific antibodies can also be used to localize cytotoxic agents to cells that express IL-8. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.

Techniques for creating multispecific antibodies include, but are not limited to, the recombinant co-expression of two pairs of heavy chain-light chains of immunoglobulins with different specificities (see Milstein and Cuello, Nature 305, 1983, pp. 537-540, WO 93/08829 and Traunecker et al., EMBO J. 10.1991, pp. 3655-3659), and knob-in-hole technology (providing a protrusion-in-hole interaction, see, for example, US No. 5731168). Multispecific antibodies can also be produced by generating controlled electrostatic stimuli to produce antibody molecules with heterodimeric Fc (WO 2009/089004); cross-linking two or more antibodies or fragments (see, for example, US No. 4676980 and Brennan et al., Science 229, 1985, pp. 81-83); the use of leucine zippers to produce bispecific antibodies (see, for example, Kostelny et al., J. Immunol. 148, 1992, pp. 1547-1553); the use of "dimeric antibody (diabodi)" technology to create bispecific antibody fragments (see, eg, Hollinger et al., Proc. Natl. Acad. Sci. USA 90, 1993, pp. 6444-6448); and the use of single chain Fv (scFv) dimers (see, for example, Gruber et al., J. Immunol. 152, 1994, pp. 5368-5374); and obtaining trispecific antibodies according to the method described, for example, in Tutt et al., J. Immunol. 147, 1991, pp. 60-69).

Also included within the scope of the present invention are engineered antibodies with three or more functional antigen binding sites, including "octopus antibodies" (see, for example, US 2006/0025576).

An antibody or fragment thereof referred to in section B of the present description refers to a "dual-acting Fab" or "DAF" containing an antigen-binding site that binds to IL-8 as well as to another antigen other than it (see, for example, US 2008/0069820).

6. Antibody variants

The amino acid sequence of antibody variants can be obtained by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by synthesizing peptides. These modifications include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequences of the antibody. Any combination of deletions, insertions and substitutions can be used to obtain the final construct, provided that the final construct is an antibody that has the desired characteristics as set forth in the context of Section B of the description.

One of the embodiments of the invention mentioned in section B of the description are antibody variants having one or more amino acid substitutions. These substitution sites can be at any position in the antibody. Amino acids suggested as conservative substitutions are shown in Table 10 under the heading “conservative substitutions”. Amino acids suggested as typical substitutions that result in more significant changes are shown in Table 10 under the heading "typical substitutions" and are described below with reference to amino acid side chain classes.

: His, Lys, Arg; (5) остатки, которые влияют на ориентацию цепи: Gly, Pro; (6) ароматические: Trp, Tyr, Phe.Amino acids can be grouped based on common side chain properties as follows: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4)

: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.

By non-conservative substitutions is meant the replacement of a representative of one of the specified classes with a representative from another class.

Insertions into amino acid sequences include amino and / or carboxy terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as insertions of one or more amino acid residues into the sequence. An example of the result of carrying out terminal insertions is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion of the N- or C-terminus of an antibody with an enzyme (eg, for ADEPT (antibody directed prodrug therapy enzyme) or a polypeptide that prolongs the serum half-life of an antibody.

7. Variants of glycosylation

In one embodiment, the antibodies referred to in Section B of the specification may be glycosylated antibodies. The addition or deletion of glycosylation sites in an antibody can be easily accomplished by altering the amino acid sequence to create or remove glycosylation sites.

If the antibody contains an Fc region, then the sugar chain attached to it can be changed. Native antibodies that are produced by mammalian cells typically contain a branched biantenal oligosaccharide, which is typically N-linked to the Asn297 CH2 domain of the Fc region (see, for example, Wright et al., TIBTECH 15, 1997 , pp. 26-32). The oligosaccharide may include, for example, mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and fucose attached to GlcNAc in the stem of the biantennial oligosaccharide structure. In one embodiment, the oligosaccharide in the antibody specified in Section B of the description is modified to create antibody variants with certain improved properties.

8. Fc-region variants

In one embodiment, one or more amino acid modifications are introduced into the Fc region of the antibody described in Section B, thereby creating a variant Fc region. Fc region variants include variants in which one, two, three, or more amino acids have been modified (e.g., by substitution) in the native sequence of a human Fc region (e.g., human IgG1, IgG2, IgG3 or IgG4 Fc regions ).

The anti-IL-8 antibody referred to in Section B of the specification may contain an Fc region that has at least one of, but is not limited to, the following five properties: (a) increased binding affinity to FcRn of the Fc region relative to affinity binding to FcRn of the native Fc region at acidic pH; (b) reduced binding affinity of the Fc region to pre-present ADA relative to the binding affinity of the native Fc region to pre-present ADA; (c) an extended plasma half-life of the Fc region relative to the plasma half-life of the native Fc region; (d) decreased clearance from plasma of the Fc region relative to clearance from plasma of the native Fc region; and (e) reduced binding affinity of the Fc region to the effector receptor relative to the binding affinity of the native Fc region to the effector receptor. In some embodiments, the Fc region has 2, 3, or 4 of the above properties. In one embodiment, the Fc region variants include variants with increased FcRn binding affinity at acidic pH. Fc region variants with increased FcRn binding affinity include (but are limited to) Fc region variants in which the FcRn binding affinity is increased up to 2, 3, 4, 5, 10, 15, 20, 30, 50, or 100 times compared to the FcRn binding affinity of an antibody containing the Fc region of native IgG.

In one embodiment, the Fc region variant comprises a safe and advantageous Fc region variant that does not bind to preexisting ADA while simultaneously having an improved plasma half-life. In the context of Section B of the specification, the term "ADA" refers to an endogenous antibody having binding affinity for an epitope on a therapeutic antibody. In the context of Section B of the specification, the term "preexisting ADA" refers to a detectable antibody to a drug that is in the patient's blood prior to administration of the therapeutic antibody to the patient. Preexisting ADA includes rheumatoid factor. Fc region variants with low binding affinity for preexisting ADA include, but are not limited to, Fc region variants whose ADA binding affinity is reduced to 1/10 or less, 1/50 or less, or 1/100 or less compared to the ADA binding affinity of an antibody that contains the Fc region of native IgG.

In one embodiment, a variant Fc region comprises Fc region variants that have low binding affinity for complement proteins or that do not bind to complement proteins. Complement proteins include C1q. Fc region variants with low binding affinity for complement proteins include (but are not limited to) Fc region variants whose binding affinity for complement proteins is reduced to 1/10 or less, 1/50 or less, or 1/100 or less. compared to the binding affinity of complement proteins of an antibody that contains the Fc region of native IgG.

In one embodiment, an Fc region variant comprises an Fc region variant that has low binding affinity for effector receptors or that does not have binding affinity for an effector receptor. Effector receptors include, but are not limited to, FcγRI, FcγRII, and FcγRIII. FcγRI includes, but is not limited to, FcγRIa, FcγRIb, and FcγRIc, as well as their subtypes. FcγRII includes (but is not limited to) FcγRIIa (which has two allotypes: R131 and H131) and FcγRIIb. FcγRIII includes (but is not limited to) FcγRIIIa (which has two allotypes: V158 and F158) and FcγRIIIb (which has two allotypes: FcγRIIIb-NA1 and FcγRIIIb-NA2). Fc region variants with low binding affinity to effector receptors include, but are not limited to, Fc region variants whose binding affinity to effector receptors is reduced to at least 1/10 or less, 1/50 or less, or 1/100 or less than the binding affinity of an antibody that contains the Fc region of native IgG.

In one embodiment, a variant Fc region comprises an Fc region containing one or more amino acid substitutions at any of the positions selected from the group consisting of positions 235, 236, 239, 327, 330, 331, 428, 434, 436, 438 and 440 according to EU numbering compared to native Fc region.

In one embodiment, a variant Fc region comprises an Fc region that contains amino acid substitutions at positions 235, 236, 239, 428, 434, 436, 438 and 440 according to EU numbering, compared to the native Fc region.

In one embodiment, a variant Fc region comprises an Fc region that contains amino acid substitutions at positions 235, 236, 327, 330, 331, 428, 434, 436, 438 and 440 according to EU numbering, compared to native Fc -area.

In one embodiment, a variant Fc region comprises an Fc region that contains one or more amino acid substitutions selected from the group consisting of L235R, G236R, S239K, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R, and S440E.

In one embodiment, a variant Fc region comprises an Fc region that contains the amino acid substitutions M428L, N434A, Y436T, Q438R, and S440E.

In one embodiment, a variant Fc region comprises an Fc region that contains amino acid substitutions L235R, G236R, S239K, M428L, N434A, Y436T, Q438R, and S440E.

In one embodiment, a variant Fc region comprises an Fc region that contains amino acid substitutions L235R, G236R, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R, and S440E.

In one embodiment, the anti-IL-8 antibody specified in Section B of the description comprises the amino acid sequence of SEQ ID NO: 80 and / or the amino acid sequence of SEQ ID NO: 82. An anti-IL-8 antibody that comprises the amino acid sequence of SEQ ID NO : 80 and / or the amino acid sequence of SEQ ID NO: 82 can be an anti-IL-8 antibody that binds IL-8 in a pH-dependent manner. An anti-IL-8 antibody that contains the amino acid sequence of SEQ ID NO: 80 and / or the amino acid sequence of SEQ ID NO: 82 can maintain stable IL-8 neutralizing activity in vivo (eg, in plasma). An anti-IL-8 antibody that contains the amino acid sequence of SEQ ID NO: 80 and / or the amino acid sequence of SEQ ID NO: 82 may be an antibody of low immunogenicity. An anti-IL-8 antibody that contains the amino acid sequence of SEQ ID NO: 80 and / or the amino acid sequence of SEQ ID NO: 82 may contain an Fc region whose FcRn binding affinity is increased at acidic pH (e.g., pH 5.8) by compared to the affinity of FcRn binding of the native Fc region at acidic pH. An anti-IL-8 antibody that contains the amino acid sequence of SEQ ID NO: 80 and / or the amino acid sequence of SEQ ID NO: 82 may contain an Fc region whose binding affinity to a pre-existing ADA is reduced compared to the binding affinity of the native Fc region to previously present ADA. An anti-IL-8 antibody that contains the amino acid sequence of SEQ ID NO: 80 and / or the amino acid sequence of SEQ ID NO: 82 may contain an Fc region with a plasma half-life that is prolonged compared to the native Fc region. An anti-IL-8 antibody that contains the amino acid sequence of SEQ ID NO: 80 and / or the amino acid sequence of SEQ ID NO: 82 may contain an Fc region whose binding affinity for effector receptors is reduced compared to the native Fc region. In a further embodiment, the anti-IL-8 antibody comprises a combination of any of 2, 3, 4, 5, 6, or all 7 of the properties described above.

In one embodiment, the anti-IL-8 antibody specified in Section B of the description contains the amino acid sequence of SEQ ID NO: 81 and / or the amino acid sequence of SEQ ID NO: 82. Anti-IL-8 antibody that contains the amino acid sequence of SEQ ID NO : 81 and / or the amino acid sequence of SEQ ID NO: 82 can be an anti-IL-8 antibody that binds IL-8 in a pH-dependent manner. An anti-IL-8 antibody that contains the amino acid sequence of SEQ ID NO: 81 and / or the amino acid sequence of SEQ ID NO: 82 can maintain stable IL-8 neutralizing activity in vivo (eg, in plasma). An anti-IL-8 antibody that contains the amino acid sequence of SEQ ID NO: 81 and / or the amino acid sequence of SEQ ID NO: 82 may be an antibody of low immunogenicity. An anti-IL-8 antibody that contains the amino acid sequence of SEQ ID NO: 81 and / or the amino acid sequence of SEQ ID NO: 82 may contain an Fc region whose FcRn binding affinity is increased at acidic pH (e.g., pH 5.8) by compared to the affinity of FcRn binding of the native Fc region at acidic pH. An anti-IL-8 antibody that contains the amino acid sequence of SEQ ID NO: 81 and / or the amino acid sequence of SEQ ID NO: 82 may contain an Fc region whose binding affinity to a pre-existing ADA is reduced compared to the binding affinity of the native Fc region to previously present ADA. An anti-IL-8 antibody that contains the amino acid sequence of SEQ ID NO: 81 and / or the amino acid sequence of SEQ ID NO: 82 may contain an Fc region, the plasma half-life of which is prolonged compared to the native Fc region. An anti-IL-8 antibody that contains the amino acid sequence of SEQ ID NO: 81 and / or the amino acid sequence of SEQ ID NO: 82 may contain an Fc region whose binding affinity to effector receptors is reduced compared to the native Fc region. In a further embodiment, the anti-IL-8 antibody comprises a combination of any of 2, 3, 4, 5, 6, or all 7 of the properties described above.

Some embodiments of the invention relate to an antibody variant as defined in Section B of the specification that has some, but not all, effector functions. An antibody variant can be a promising candidate in cases where some effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks the ability to bind to FcγR (and therefore likely lacks ADCC activity), but retains the ability to bind to FcRn.

Primary cultured cells mediating ADCC, i.e. NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII and FcγRIII. Data on FcR expression on hematopoietic cells are summarized in Table 3, p. 464 in Ravetch and Kinet, Annu. Rev. Immunol. 9, 1991, cc. 457-492. Examples of in vitro assays for the ADCC activity of a molecule of interest include, but are not limited to, the assays described in US Pat. No. 5,500,362 (see, for example, Hellstrom et al., Proc. Natl. Acad. Sci. USA 83, 1986, cc. 7059-7063 and Hellstrom et al., Proc. Nat'l Acad. Sci. USA 82, 1985, 1499-1502); US No. 5821337 (see Bruggemann et al., J. Exp. Med. 166, 1987, cc. 1351-1361). In an alternate embodiment may be used non-radioactive methods of analysis (see, for example, non-radioactive cytotoxicity ACTI ™ analysis based on flow cytometry (firm CellTechnology, Inc. of Mountain View, California);.. And nonradioactive cytotoxicity analysis CytoTox 96 ^® (company Promega, Madison Peripheral blood mononuclear cells (PBMCs) and natural killer cells (NKs) are useful effector cells for these assays.

Alternatively or additionally, the ADCC activity of the antibody variant of interest can be assessed in vivo, eg, in an animal model, eg, as described in Clynes et al., Proc. Natl. Acad. Sci. USA 95, 1998, cc. 652-656. C1q binding assays can also be performed to confirm that the antibody cannot bind C1q and therefore lacks CDC activity (see, for example, the description of the ELISA for evaluating C1q and C3c binding in WO 2006/029879 and WO 2005/100402). To assess complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202, 1996, pp. 163-171; Cragg et al., Blood 101, 2003, pp. 1045- 1052; and Cragg, Blood 103, 2004, 2738-2743). Determination of FcRn binding and clearance / half-life in vivo can also be performed using methods known in the art (see, for example, Petkova et al., Int'l. Immunol. 18 (12), 2006, pp. 1759- 1769).

Antibodies with reduced effector function include antibodies with substitution of one or more of the following Fc region residues, such as 238, 265, 269, 270, 297, 327 and 329 (see, for example, US No. 6,737,056). These Fc mutants include Fc mutants with substitutions at two or more of the following amino acid positions: 265, 269, 270, 297 and 327, including the so-called "DANA" Fc region mutant carrying an alanine substitution of residues 265 and 297 (US No. 7332581).

Several variants of antibodies with increased or decreased ability to bind to FcR have been described (see, for example, US No. 6,737,056; WO 2004/056312 and Shields et al. J. Biol. Chem. 276, 2001, cc. 6591-6604).

Antibodies with extended blood half-life and improved FcRn binding activity at acidic pH are described in US 2005/0014934. The disclosed antibodies contain an Fc region with one or more substitutions that improve the binding of the Fc region to FcRn. These Fc region variants include variants with substitutions at one or more positions selected from 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378 , 380, 382, 413, 424 or 434 in the Fc region, for example, replacing the position 434 in the Fc region (US No. 7371826).

Additional examples concerning other Fc region variants are also described in Duncan, Nature 322, 1988, pp. 738-740; in US Nos. 5648260 and 5624821 and in WO 1994/29351.

9. Antibody derivatives

In some embodiments, the antibody described in Section B of the description can be further modified to contain additional non-protein fragments known in the art and readily available. Fragments suitable for antibody derivatization include, but are not limited to, water-soluble polymers. Examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane copolymers, copolymers / maleic anhydride, polyamino acids (either homopolymers or random copolymers) and dextran- or poly (n-vinylpyrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (for example, polyvinyl alcohol mixtures), Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers attached to an antibody can vary and, if more than one polymer is attached, they can be the same or different molecules. In general, the amount and / or type of polymers used for derivatization can be determined based on (but not limited to) such features as the specific properties or functions of the antibody to be improved, whether the antibody derivative is intended to be used in therapy under certain conditions, and etc.

Another embodiment of the invention are conjugates of the anti-IL-8 antibody specified in Section B of the description and a non-proteinaceous moiety that can be selectively heated by irradiation. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102, 2005, cc. 11600-11605). The radiation can be of any wavelength and includes (but is not limited to) wavelengths that do not damage normal cells, but that heat the non-protein moiety to a temperature that kills the cells closest to the antibody-non-protein moiety conjugate.

B. Recombination and composition techniques

The anti-IL-8 antibodies specified in section B of the description can be prepared using recombination techniques and compositions such as those described in US Pat. No. 4,816,567. One embodiment of the invention is (are) isolated nucleic acid (s) encoding the anti-IL-8 antibody specified in Section B of the description. Said nucleic acid (s) may encode an amino acid sequence containing an antibody VL and / or an amino acid sequence containing an antibody VH (eg, an antibody light and / or heavy chain). A further embodiment of the invention is (are) one or more vectors (eg, expression vectors), which (s) contains (s) the specified nucleic acid (s). One of the embodiments of the invention is a host cell containing the specified (s) nucleic acid (s). In one of these embodiments of the invention, the host cell contains (for example, as a result of transformation with the indicated vectors): (1) a vector containing a nucleic acid that encodes an antibody VL and an antibody VH, or (2) a first vector containing a nucleic acid, which encodes the VL of the antibody; and a second vector containing the nucleic acid that encodes the VH of the antibody. In one embodiment, the host cell is a eukaryotic cell (eg, Chinese hamster ovary (CHO) cell or lymphoid cell (eg, Y0-, NS0-, Sp20-cell).

One embodiment of the invention is a method for producing an anti-IL-8 antibody as defined in Section B of the specification, wherein the method comprises culturing a host cell containing a nucleic acid that encodes the aforementioned anti-IL-8 antibody under conditions suitable for expression of the antibody, and optionally isolating the antibody (eg, from a host cell or host cell culture medium).

For recombinant production of anti-IL-8 antibody, nucleic acid (s) encoding the antibody, for example, as described above, are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell ... The specified nucleic acid (s) can be easily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that have the ability to specifically bind to nucleic acids encoding the heavy and light chains of an antibody).

Suitable host cells for cloning or expressing antibody-encoding vectors include the prokaryotic or eukaryotic cells described in Section B of this disclosure. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc-associated effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, US Nos. 5648237, 5789199, and 5840523 (see also Charlton in: Methods in Molecular Biology, ed. Lo B.K.C., Humana Press, Totowa, NJ, Vol. 248, 2003, pp. 245-254, describing the expression of antibody fragments in E. coli.) After expression, the antibody can be recovered from the bacterial cell paste as a soluble fraction and can be further purified.

In addition to prokaryotic organisms, eukaryotic microorganisms, such as filamentous fungi or yeast, including strains of fungi and yeast, whose glycosylation pathways have been "humanized", can be used as hosts suitable for cloning or expressing vectors that encode antibodies or a fully human glycosylation scheme (see Gerngross, Nat. Biotech. 22, 2004, pp. 1409-1414 and Li et al., Nat. Biotech. 24, 2006, pp. 210-215).

Host cells that can be used to express the glycosylated antibody are also obtained from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells are insect cells, and plant cells can also be used. Were identified numerous strains of baculoviruses and corresponding insect cells suitable for them as hosts, especially for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts (see, eg, US Nos. 5959177, 6040498, 6420548, 7125978 and 6417429 (description of PLANTIBODIES ™ technology for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, you can use mammalian cell lines that are adapted to growth in suspension. Other examples of suitable mammalian host cell lines are monkey kidney cell line CV1 transformed with OB40 (COS-7); human embryonic kidney cell line (HEK 293 cells or 293 cells, described, for example, in Graham et al., J. Gen. Virol., 36, 1977, pp. 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, described, for example, in Mather, Biol. Reprod., 23, 1980, pp. 243-251); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76,); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); bovine rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor cells (MMT 060562); TRI cells, described, for example, in Mather et al., Annals N.Y. Acad. Sci., 383, 1982, pp. 44-68); MRC 5 cells and FS4 cells.

Other valuable mammalian host cell lines are Chinese hamster ovary (CHO) cells, including DHFR ^- -CHO cells (Urlaub et al. Proc. Natl. Acad. Sci. USA, 77, 1980, 4216-4220); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines that can be used to produce antibodies, see, for example, Yazaki and Wu, in: Methods in Molecular Biology, ed. V.K.S. Lo, Humana Press, Totowa, NJ, vol. 248, 2003, cc. 255-268.

The antibody referred to in Section B of the description obtained by culture, in which the host cells described above carry nucleic acids that encode the antibody under conditions suitable for antibody expression, can be isolated from the inside or outside of the host cell (media, milk, etc.) ) and purified as an almost homogeneous antibody. Isolation / purification techniques that are generally used to purify polypeptides can be suitably used to isolate and purify antibodies; however, the methods are not limited to the above examples. The antibody can be appropriately separated and purified, for example, by appropriate selection and combination of column chromatography, filtration, ultrafiltration, desalting, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, and other methods. Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reversed phase chromatography, and adsorption chromatography. This chromatography can be performed, for example, using liquid chromatography such as HPLC and HPLC. Columns for use in affinity chromatography can be a protein A column or a protein G column. A protein A column includes, for example, Hyper D, POROS, Sepharose F.F. (Pharmacia), etc.

Taking into account the characteristics of antibodies to IL-8, such as increased activity of binding to the extracellular matrix and increased uptake by cells of the complexes described above, in the description section presents methods for selecting antibodies with increased activity of binding extracellular matrix and antibodies with increased uptake by cells. One of the embodiments of the invention, referred to in section B of the description, are methods for producing an antibody to IL-8, which contains a variable region, the binding activity of which with IL-8 depends on pH, including the steps of: (a) assessing the binding between the antibody to IL- 8 and extracellular matrix; (b) selection of antibodies to IL-8, characterized by strong binding to the extracellular matrix; (c) culturing a host that contains a vector carrying a nucleic acid that encodes an antibody; and (d) isolating the antibody from the cellular environment.

Binding to the extracellular matrix can be assessed by any method known to those of ordinary skill in the art, but not specifically limited to them. For example, assays can be performed using an ELISA system to detect binding between an antibody and an extracellular matrix, for which the antibody is added to the extracellular matrix immobilized on the plate and a labeled anti-antibody antibody is added thereto. Alternatively, these assays can be performed, for example, using an electrochemiluminescence (ECL) method in which a mixture of an antibody and a ruthenium-labeled antibody is added to an extracellular matrix immobilized on a plate, and the binding between the antibody and the extracellular matrix is assessed based on the electrochemiluminescence of ruthenium.

The anti-IL-8 antibody, the binding of which to the extracellular matrix was assessed in step (a) above, can be an individual antibody or contacted IL-8. "Selection of an anti-IL-8 antibody characterized by strong binding to the extracellular matrix" in step (b) means that the anti-IL-8 antibody is selected based on a criterion according to which the value characterizing the binding between the extracellular matrix and the anti-IL-8 antibody is higher than the value characterizing the binding between the extracellular matrix and the control antibody when assessing the binding to the extracellular matrix, and may exceed it, for example, in 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 times or more; however, the ratio is not specifically limited to the above examples. Except for the presence of IL-8, preferably the conditions are the same in the step of assessing the binding between the anti-IL-8 antibody and the extracellular matrix. The control anti-IL-8 antibody used to compare anti-IL-8 antibodies with multiple modifications may be an unmodified anti-IL-8 antibody. In this case, the conditions are preferably the same except for the presence of IL-8. In particular, in one of the embodiments of the invention indicated in section B of the description, an antibody with a higher value characterizing the binding between the extracellular matrix is selected from several and anti-IL-8 antibodies that do not contact IL-8. In another embodiment of the invention, referred to in section B of the description, an antibody with a higher value characterizing binding between the extracellular matrix is selected from several anti-IL-8 antibodies that are in contact with IL-8. In an alternative embodiment, “selection of an anti-IL-8 antibody characterized by strong binding to the extracellular matrix” in step (b) means that the antibody can be selected based on a criterion according to which the binding between the antibody and the extracellular matrix varies depending on the presence of IL- 8 when evaluating extracellular matrix binding. The ratio of the value characterizing the binding to the extracellular matrix of the anti-IL-8 antibody in contact with IL-8 and the value characterizing the binding to the extracellular matrix of the anti-IL-8 antibody without contact with IL-8 can be, for example, 2-1000. In addition, the ratio between the values can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000.

B. Analyzes

The anti-IL-8 antibodies provided in Section B of this specification can be identified, screened, or characterized by their physical / chemical properties and / or biological activities using various assays known in the art.

1. Assays of binding and other assays

According to one aspect of the invention, the antibodies described in Section B of the description can be evaluated for their antigen binding activity using known methods such as ELISA, Western blotting, kinetic exclusion assay (KinExA ™) and surface plasma resonance using such devices like BIACORE (GE Healthcare).

In one embodiment, the binding affinity can be measured using BIACORE T200 (GE Healthcare) as follows. An appropriately selected capture protein (eg protein A / G (PIERCE)) is immobilized on a CM4 sensor chip (GE Healthcare) by amine coupling and the antibody of interest is allowed to capture. Then a diluted antigen solution and a rolling buffer (as a reference solution: for example, 0.05% Tween-20, 20 mM ACES, 150 mM NaCl, pH 7.4) are injected for interaction of antigen molecules with the antibody "captured" on the sensor chip. The sensor chip is regenerated using 10 mM glycine x HCl solution (pH 1.5). The measurements are carried out at a predetermined temperature (eg 37 ° C, 25 ° C or 20 ° C). The association reaction rate constant k _on (1 / Ms) and the dissociation reaction rate constant k _off (1 / s), which are both kinetic parameters, are calculated from the sensograms obtained from the measurement. The KD (M) of each antibody for antigen is calculated based on these constants. Each parameter is calculated using BIACORE T200 Evaluation software (GE Healthcare).

In one embodiment, IL-8 can be quantified according to the method described below. Anti-human IL-8 antibody, which contains the constant region of mouse IgG, is immobilized on the plate. A solution containing IL-8 linked to a humanized anti-IL-8 antibody that does not compete with the aforementioned anti-human IL-8 antibody is added in aliquots to the immobilized plate. After mixing, the biotinylated anti-human Igκ light chain antibody is added and allowed to react for a period of time. Then sulfo-labeled streptavidin is also added and allowed to react for a period of time. Assay buffer is then added and immediately measured with a SECTOR Imager 2400 (Meso Scale Discovery).

2. Analysis of activity

One of the objects of the invention are assays for the identification of anti-IL-8 antibodies with biological activity. Biological activity includes, for example, IL-8 neutralizing activity and activity against blocking IL-8 signals. Section B of the description presents antibodies having the indicated biological activity in vivo and / or ex vivo.

One of the embodiments of the invention is a method for determining the level of neutralization of IL-8, which does not limit the scope of the invention, and the specified activity can also be determined using the methods described below. The PathHunter ™ CHO-K1 CXCR2 β-arrestin cell line (DiscoveRx cat # 93-0202C2) is an artificial cell line designed to express human CXCR2, known as the human IL-8 receptor, and emitting chemiluminescence upon receipt of signals from the human IL-8. After adding human IL-8 to the cell culture medium, the cells emit chemiluminescence depending on the concentration of added human IL-8. When human IL-8 is added in combination with an anti-human IL-8 antibody to the culture medium, the chemiluminescence of cells is reduced or undetectable compared to the option without the addition of an antibody, since the anti-human IL-8 antibody can block the transduction of IL-8 signals. In particular, a more potent neutralizing activity of an antibody against human IL-8 corresponds to a lower level of chemiluminescence; and the weaker neutralizing activity of the antibody against human IL-8 corresponds to a higher level of chemiluminescence. Thus, the neutralizing activity against human IL-8 of an anti-human IL-8 antibody can be determined by evaluating the above difference.

D. Pharmaceutical compositions

Pharmaceutical compositions containing the anti-IL-8 antibody referred to in the context of Section B herein can be prepared by mixing the anti-IL-8 antibody of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th Ed. . ed. Osol A., 1980) in the form of lyophilized compositions or aqueous solutions.

Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used and include, but are not limited to: buffers such as phosphate, citrate buffers, and other organic acid buffers; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkylparabens such as methyl- or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol and meta); low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as poly (vinylpyrrolidone); amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal-containing complexes (eg, Zn-protein complexes); and / or non-ionic surfactants such as TWEEN ™, PLURONICS ™ or polyethylene glycol (PEG).

As used herein, examples of pharmaceutically acceptable carriers also include interstitial drug dispersing agents such as soluble neutral active glycoproteins of hyaluronidases (sHASEGP), for example human soluble hyaluronidase glycoproteins PH-20, such as rHuPH20 (BYLENEX International, company Inc.). Several examples of sHASEGP and methods of their use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect of the invention, sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.

Examples of lyophilized antibody compositions are described in US No. 6267958. Aqueous antibody compositions include those described in US No. 6171586 and WO 2006/044908 and the compositions described in WO 2006/044908 include a histidine acetate buffer.

In the context of Section B of the present description, the composition may also contain more than one active substance, if necessary for the particular indication to be treated, preferably substances with additional activities that do not adversely affect each other. Such active substances must be present in combination in amounts effective to achieve the intended purpose.

The active substances can also be incorporated into microcapsules, for example, obtained by means of coacervation or interfacial polymerization methods, for example, in hydroxymethylcellulose or gelatin microcapsules and poly (methyl methacrylate) microcapsules, respectively, in colloidal drug administration systems (for example, liposomes, albumin microspheres, nanoparticles and nanocapsules) or in a macroemulsion. Such methods are described in Remington's Pharmaceutical Sciences, 16th ed., Ed. Osol A., 1980.

Sustained release formulations can be prepared. Suitable examples of sustained-release formulations are semi-permeable matrices of solid hydrophobic polymers, including the anti-IL8 antibody specified in Section B of the specification, such matrices are shaped articles such as films or microcapsules.

Compositions to be used in vivo generally must be sterile. Sterility can be easily achieved, for example, by filtration through sterile filtration membranes.

E. Therapeutic Methods and Compositions

In some embodiments, the anti-IL-8 antibodies described in Section B of the description are used in therapeutic methods.

One aspect of the invention is an anti-IL-8 antibody for use as a pharmaceutical composition. An alternative aspect of the invention is an anti-IL-8 antibody for the treatment of a disease in which IL-8 is present in excess. One embodiment of the invention is an anti-IL-8 antibody for use in methods of treating a disease in which IL-8 is present in an excessive amount. One of the embodiments of the invention referred to in Section B of the description are methods of treating an individual having a disease in which IL-8 is present in an excessive amount (for example, a disease associated with the presence of IL-8 in an excessive amount), which include administering to the individual an effective amount of anti-IL-8 antibody. Another embodiment of the invention referred to in Section B of the specification are anti-IL-8 antibodies for use in these methods. One of the embodiments of the invention specified in section B of the description is a pharmaceutical composition containing in an effective amount an antibody to IL-8, which is used to treat a disease in which IL-8 is present in an excessive amount. One of the embodiments of the invention mentioned in section B of the description is the use of an antibody to IL-8 for the preparation of a pharmaceutical composition for the treatment of a disease in which IL-8 is present in an excessive amount. One of the embodiments of the invention referred to in section B of the description is the use in an effective amount of antibodies to IL-8 for the treatment of a disease in which IL-8 is present in an excessive amount. Diseases in which IL-8 is present in excess include, but are not limited to, inflammatory skin diseases such as inflammatory keratosis (eg, psoriasis), atopic dermatitis, and contact dermatitis; autoimmune diseases such as chronic inflammatory diseases including rheumatoid arthritis, systemic lupus erythematosus (SLE) and Behcet's disease; inflammatory bowel diseases such as Crohn's disease and ulcerative colitis; inflammatory liver diseases such as hepatitis B, hepatitis C, alcoholic hepatitis, and drug-induced allergic hepatitis; inflammatory kidney disease such as glomerulonephritis; inflammatory respiratory diseases such as bronchitis and asthma; chronic inflammatory vascular diseases such as atherosclerosis; multiple sclerosis, oral ulcer, chorditis and inflammation associated with the use of artificial organs and / or artificial blood vessels; malignant tumors such as ovarian cancer, lung cancer, prostate cancer, stomach cancer, breast cancer, melanoma, head and neck cancer, and kidney cancer; infection-related sepsis; cystic fibrosis; fibrosis of the lung; and acute lung damage.

An alternative embodiment of the invention referred to in Section B of the specification is an anti-IL-8 antibody for use in inhibiting the accumulation of IL-8 with biological activity. "Suppression of IL-8 accumulation" can be achieved by preventing an increase in the amount of previously present IL-8 in vivo or by reducing the amount of previously present IL-8 in vivo. One of the embodiments of the invention mentioned in section B of the description is an antibody to IL-8, designed to suppress the accumulation of biologically active IL-8 in an individual. As used herein, “IL-8 present in vivo” can refer to IL-8 in combination with an antibody to IL-8 or to free IL-8; alternatively, may refer to total IL-8 as a sum. As used herein, "present in vivo" can mean "secreted from outside cells in vivo". One of the embodiments of the invention referred to in section B of the description is a method for suppressing the accumulation of IL-8 having biological activity, which includes the step of administering an effective amount of anti-IL-8 antibody. One of the embodiments of the invention specified in section B of the description is a pharmaceutical composition designed to suppress the accumulation of IL-8 with biological activity, which contains an effective amount of an antibody to IL-8. One of the embodiments of the invention referred to in section B of the description is the use of an antibody to IL-8 for the preparation of a pharmaceutical composition designed to suppress the accumulation of IL-8 with biological activity. One of the embodiments of the invention referred to in section B of the description is the use of an effective amount of antibodies to IL-8 to suppress the accumulation of IL-8 with biological activity. In one embodiment, the anti-IL-8 antibody described in section B of the description suppresses the accumulation of IL-8 compared to the anti-IL-8 antibody, which does not have a pH-dependent binding activity. An "individual" in the context of any of the above embodiments is preferably a human.

An alternative embodiment of the invention referred to in Section B is an anti-IL-8 antibody for use in inhibiting angiogenesis (eg, neoangiogenesis). One of the embodiments of the invention described in section B of the description is a method of inhibiting neoangiogenesis in an individual, which includes the introduction in an effective amount of antibodies to IL-8 to the individual, as well as an antibody to IL-8, intended for use in the method. One of the embodiments of the invention, specified in section B of the description, is a pharmaceutical composition designed to inhibit neoangiogenesis, which contains an effective amount of anti-IL-8 antibody. One of the embodiments of the invention, referred to in section B of the description, is the use of an antibody to IL-8 for the preparation of a pharmaceutical composition intended to inhibit neoangiogenesis. One of the embodiments of the invention referred to in section B of the description is the use of an effective amount of antibodies to IL-8 to inhibit neoangiogenesis. An "individual" in the context of any of the above embodiments is preferably a human.

An alternative embodiment of the invention referred to in Section B of the specification is an anti-IL-8 antibody for use in inhibiting the facilitation of neutrophil migration. One of the embodiments of the invention referred to in section B of the description is a method of inhibiting the facilitation of migration of neutrophils in an individual, which include the introduction in an effective amount of antibodies to IL-8 to the individual; and an anti-IL-8 antibody for use in the method. One of the embodiments of the invention referred to in section B of the description, are pharmaceutical compositions designed to inhibit the facilitation of migration of neutrophils in an individual, which contain an effective amount of antibody to IL-8. One of the embodiments of the invention referred to in section B of the description is the use of an antibody to IL-8 to obtain a pharmaceutical composition designed to inhibit the facilitation of neutrophil migration in an individual. One of the embodiments of the invention, referred to in section B of the description, is the use in an effective amount of antibodies to IL-8 to inhibit the facilitation of migration of neutrophils in an individual. An "individual" in the context of any of the above embodiments is preferably a human.

An alternative embodiment of the invention, as described in Section B, is a pharmaceutical composition comprising an anti-IL-8 antibody as described herein, for example, for use in any of the above therapeutic methods. In one embodiment, the pharmaceutical composition comprises the anti-IL-8 antibodies specified in Section B of the specification and a pharmaceutically acceptable carrier.

The antibody referred to in section B of the description can be used either individually or in combination with other agents intended for therapy. For example, the antibody described in Section B of the description can be co-administered with at least one additional therapeutic agent.

The antibody referred to in Section B of the description (and optionally any additional therapeutic agent) can be administered by any suitable route, including parenteral, intrapulmonary and intranasal, and, if necessary, used for topical treatment, administration to a lesion. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be done by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending on, inter alia, whether the treatment is short-term or chronic. You can use a variety of dosing regimens, including, but not limited to, single administration or multiple administrations at different times, bolus administration, and pulsed infusion.

Preferably, the antibody referred to in Section B of the specification is formulated, dosed, and administered in accordance with good clinical practice. Considered in this context, factors include the specific disorder to be treated, the specific mammal to be treated, the clinical condition of the individual patient, the cause of the disorder, the region of administration of the agent, the route of administration, the schedule of administration, and other factors known to the medical practitioner. The antibody can, but is not required, be included in the composition in combination with one or more other agents currently used to prevent and treat the disorder in question.

The effective amount of these other agents depends on the amount of antibody present in the composition, the type of disorder or treatment, and other factors noted above. They are usually used in the same doses and using the same routes of administration as described in this description, or in an amount ranging from 1 to 99% of the dose indicated in section B of this description, or in any dose and with using any route that empirical / clinical judgment is appropriate.

For the prevention or treatment of a disease, the acceptable dose of the antibody specified in Section B of the description (when used alone or in combination with one or more other additional therapeutic agents) depends on the type of disease to be treated, the type of antibody, the severity and course of the disease, and whether an antibody for preventive or therapeutic purposes, prior therapy, patient history, and antibody response. The antibody can be administered to a patient once or in a series of treatments. Depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (e.g., 0.1-10 mg / kg) of the antibody may represent the initial possible dose to be administered to a patient, for example, via one or more separate treatments or by continuous infusion. One example of typical daily doses would be from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. When repeated administrations are used over several days or longer, depending on the condition, treatment should generally be continued until symptoms of the disease are suppressed. One example of a dose of antibody is from about 0.05 to about 10 mg / kg. Thus, one or more of the following doses can be administered to a patient: about 0.5 mg / kg, for example 2.0 mg / kg, for example 4.0 mg / kg, or, for example, 10 mg / kg (or any combination thereof). These doses can be administered in divided doses, for example, every week or every three weeks (for example, so that the patient receives from about two to about twenty, for example, about six doses of the antibody). An initial higher loading dose may be used, followed by the administration of one or more lower doses. Using conventional methods and analyzes, the effect of said therapy can be easily monitored.

E. Products

Another object of the invention, referred to in section B of the description, are products that contain products used for the treatment, prevention and / or diagnosis of the above disorders. The specified product is a container and a label or package insert that is placed on or enclosed in the container. Acceptable containers are, for example, cans, vials, syringes, IV bags, etc. The containers can be made from various materials such as glass or plastic. The container contains a composition that, alone or in combination with another composition, is effective in treating, preventing and / or diagnosing a condition, and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial fitted with a plug that can be pierced with a hypodermic needle). At least one active ingredient in the composition is an antibody specified in section B of the description. The label or package insert indicates that the composition is used to treat the selected condition.

In addition, the article of manufacture may include (a) a first container containing a composition, wherein the composition comprises an antibody as defined in Section B of the description; and (b) a second container containing a composition, wherein the composition contains an additional cytotoxic or other therapeutic agent. In accordance with these embodiments of the invention, described in Section B, the article of manufacture may include a package insert that contains information that the compositions may be used to treat a particular condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container with a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. In addition, it can include other products that are commercially and consumer-oriented, such as other buffers, diluents, filters, needles and syringes.

Those of ordinary skill in the art will appreciate, based on well-known technological knowledge in the art, that section B of the description may include all combinations of the full embodiments of the invention or portions thereof disclosed herein, unless it is technologically unacceptable.

Sections A, B or C descriptions

All documents cited in the present description in the section "Background of the invention" are included in this description by reference.

It should be borne in mind that in the context of the present description, the expression "and / or" includes the meaning of all concepts specified before and after the phrase "and / or", which includes all combinations of concepts associated with the phrase as appropriate.

While various elements are described herein with terms such as first, second, third, fourth, etc., it should be understood that such concepts do not impose restrictions on the elements. These concepts are used only to distinguish one element from other elements, and it should be borne in mind that, for example, the first element can be designated as the second element, and similarly, the second element can be designated as the first element without deviating from the scope of the sections. A, B and C descriptions.

Unless specifically indicated otherwise, or unless it is inappropriate in the context of the description, all terms in the singular are meant to include the plural, and any terms used in the present description in the plural also include the singular.

The terminology used in this description is used only for the purpose of describing specific embodiments of the invention and is not intended to limit the invention. Unless otherwise indicated, all concepts (including technical and scientific concepts) used in this description are to be interpreted as having the same meaning generally understood by those of ordinary skill in the art to which sections A, B and In descriptions, and should not be interpreted in an idealized or overly formal sense.

In the context of the present description, it is understood that the term "contains" defines the presence of the described elements (representatives, stages, elements, numbers, etc.), unless the context clearly indicates otherwise; and the concept does not exclude the presence of other elements (representatives, stages, elements, numbers, etc.).

The embodiments in sections A, B and C of the description are presented with reference to schematic illustrations, which may be enlarged for clarity.

Unless the context of the present description indicates otherwise, the numerical values used in it should be understood as values in a certain range, which is within the technical competence of one of ordinary skill in the art. For example, the expression "1 mg" should be understood as "about 1 mg" with certain variations. For example, the expression "1-5 elements" should be understood as if it were specifically written "1 element, 2 elements, 3 elements, 4 elements, 5 elements", unless this is contrary to the context.

Examples of

Sections A, B and C below are illustrated in more detail in examples 1-4 and 21-23, examples 5-7, 19 and 20 and examples 8-19, respectively, but these examples should not be construed as limiting the scope of the invention. It should be understood that various other embodiments of the invention may be practiced based on the general description provided above.

Example 1

Production of pH-Dependent Human IL-6 Receptor Binding Antibodies with Increased pI

Fv4-IgG1, described in WO 2009/125825, is an antibody that binds to the human IL-6 receptor in a pH-dependent manner and contains VH3-IgG1 (SEQ ID NO: 24) as the heavy chain and VL3-CK (SEQ ID NO: 32) as a light chain. To increase the pI of Fv4-IgG1, amino acid substitutions were introduced into the Fv4-IgG1 variable region that decreased the amount of negatively charged amino acids (such as aspartic acid and glutamic acid) while increasing the amount of positively charged amino acids (such as arginine and lysine). More specifically, VH3 (High_pI) -IgG1 (SEQ ID NO: 25) was obtained as a heavy chain with an increased pI by replacing glutamic acid at position 16 with glutamine in the heavy chain of VH3-IgG1, glutamic acid at position 43 with arginine, glutamine at position 64 to lysine and glutamic acid at position 105 to glutamine, where positions are given according to Cabot's numbering. Similarly, VL3 (High_pI) -CK (SEQ ID NO: 33) was obtained as a light chain with an increased pI by replacing serine at position 18 with arginine in the light chain VL3-CK, arginine at position 24 for glutamine, glutamic acid at position 45 to lysine, glutamic acid at position 79 to glutamine and glutamic acid at position 107 to lysine, where positions are given according to Cabot's numbering. When a substitution at position 79 of VL3-CK was introduced, modifications were simultaneously introduced that resulted in the substitution of alanine at position 80 for proline and alanine at position 83 for isoleucine, although they were not carried out with the aim of increasing the pI.

Using the method described in Reference Example 2, the following antibodies were obtained: (a) Low_pI-IgG1 containing VH3-IgG1 as the heavy chain and VL3-CK as the light chain; (b) Middle_pI-IgG1 containing VH3-IgG1 as heavy chain and VL3 (High_pI) -CK as light chain; and (c) High_pI-IgG1 containing VH3 (High_pI) -IgG1 as heavy chain and VL3 (High_pI) -CK as light chain.

The theoretical pI value was then calculated for each of the resulting antibodies using the GENETYX-SV / RC version 9.1.0 (GENETYX CORPORATION) using methods known in the art (see, for example, Skoog et al., Trends Analyt. Chem. 5 (4), 1986, pp. 82-83). It was assumed that the side chains of all cysteine residues in the antibody molecule form disulfide bonds, and therefore, the contribution from the side chains of cysteine to the pKa value was excluded in the calculation.

The calculated theoretical pI values are presented in Table 3. While the theoretical pI value for Low_pI-IgG1 was 6.39, the values for Middle_pI-IgG1 and High_pI-IgG1 were 8.70 and 9.30, respectively, indicating that the theoretical pI values increased in a stepwise manner.

WO 2011/122011 describes Fv4-IgG1-F11 (hereinafter referred to as Low_pI-F11) and Fv4-IgG1-F939 (hereinafter referred to as Low_pI-F939), whose FcRn-mediated uptake by cells increased after the introduction of amino acid substitutions into the Fc region of Fv4-IgG1 and imparting FcRn-binding ability under neutral pH conditions. In addition, WO 2013/125667 describes Fv4-IgG1-F1180 (hereinafter referred to as Low_pI-F1180), the FcγR-mediated uptake of which by cells increased after the introduction of amino acid substitutions in the Fv4-IgG1 Fc region in order to increase its FcγR-binding ability in conditions of neutral pH. At the same time, an amino acid modification was introduced into Fv4-IgG1-F1180 to increase antibody retention in plasma by increasing its binding to FcRn under acidic pH conditions in endosomes. The antibodies presented below were obtained by increasing the pI of antibodies containing these new Fc region variants.

More specifically, in each of the constructs VH3-IgG1-F11 (SEQ ID NO: 30) and VH3-IgG1-F939 (SEQ ID NO: 26) described in WO 2011/122011 and VH3-IgG1-F1180 (SEQ ID NO : 28) described in WO 2013/125667 substituted glutamic acid at position 16 for glutamine, glutamic acid at position 43 for arginine, glutamine at position 64 for lysine and glutamic acid at position 105 for glutamine, where positions are given according to Cabot numbering , to obtain VH3 (High_pI) -F11 (SEQ ID NO: 31), VH3 (High_pI) -F939 (SEQ ID NO: 27) and VH3 (High_pI) -F1180 (SEQ ID NO: 29), respectively, as heavy chains with increased pI.

Using these heavy chains, the following antibodies were prepared using the method described in Reference Example 2: (1) Low_pI-F939 containing VH3-IgG1-F939 as the heavy chain and VL3-CK as the light chain; (2) Middle_pI-F939 containing VH3 (High_pI) -F939 as heavy chain and VL3-CK as light chain; (3) High_pI-F939 containing VH3 (High_pI) -F939 as the heavy chain and VL3 (High_pI) -CK as the light chain; (4) Low_pI-F1180 containing VH3-IgG1-F1180 as heavy chain and VL3-CK as light chain; (5) Middle_pI-F1180 containing VH3-IgG1-F1180 as heavy chain and VL3 (High_pI) -CK as light chain; (6) High_pI-F1180 containing VH3 (High_pI) -F1180 as the heavy chain and VL3 (High_pI) -CK as the light chain; (7) Low_pI-F11 containing VH3-IgG1-F11 as heavy chain and VL3-CK as light chain; and (8) High_pI-F11 containing VH3 (High_pI) -F11 as the heavy chain and VL3 (High_pI) -CK as the light chain.

Then, the theoretical pI value for each of the obtained antibodies was calculated using the GENETYX-SV / RC software, version 9.1.0 (GENETYX CORPORATION) using a method similar to that described earlier. The calculated theoretical pI values are presented in Table 3. For all antibodies containing the novel Fc region variants, the theoretical pI values increased stepwise in the following order: Low_pI, Middle_pI, and High_pI.

Example 2

Effects of antibodies with increased pI, characterized by pH-dependent binding, leading to antigen elimination

(2-1) In vivo assay of antibodies with pI-regulated pH-dependent binding to human IL-6 receptor

As demonstrated below, in vivo assays were performed using various pH-dependent binding to human IL-6 receptor antibodies obtained in Example 1: Low_pI-IgG1, High_pI-IgG1, Low_pI-F939, Middle_pI-F939, High_pI-F939, Low_pI -F1180, Middle_pI-F1180 and High_pI-F1180.

Soluble human IL-6 receptor (also referred to as "hsIL-6R") prepared by the method described in Reference Example 3, anti-human IL-6 receptor antibody and Sanglopor human immunoglobulin preparation were administered simultaneously to human FcRn transgenic mice ( B6.mFcRn - / -. HFcRn Tg mouse line 32 + / +, Jackson Laboratories; see Methods Mol. Biol. 602, 2010, pp. 93-104) and then evaluated the in vivo kinetics of soluble human IL-6 receptor ... A solution containing a mixture of soluble human IL-6 receptor, antibody to human IL-6 receptor and Sanglopor (at concentrations of 5 μg / ml, 0.1 mg / ml and 100 mg / ml, respectively), was injected once at a dose of 10 ml / kg through the tail vein. Since the anti-human IL-6 receptor antibody was present in sufficient excess relative to the soluble human IL-6 receptor, it was assumed that almost all of the soluble human IL-6 receptor would bind to the antibody. Blood was collected 15 minutes, 7 hours, one day, two days, three days, and seven days after administration. The collected blood was immediately centrifuged at 4 ° C and 15,000 rpm for 15 min to obtain plasma. The separated plasma was stored in a freezer at -20 ° C or below until measurement.

(2-2) Measurement of plasma concentration of soluble human IL-6 receptor by electrochemiluminescence method

Plasma concentration of soluble human IL-6 receptor in mice was measured by electrochemiluminescence method. Samples of soluble human IL-6 receptor were prepared, the concentration of which was adjusted to 250, 125, 62.5, 31.25, 15.61, 7.81 or 3.90 pg / ml to construct a calibration curve, and mouse plasma samples diluted 50 times, respectively. The samples were mixed with anti-human IL-6R monoclonal antibody (R&D) labeled with ruthenium using sulfo-labeled NHS ester (Meso Scale Discovery), biotinylated anti-human IL-6R antibody (R&D) and tocilizumab (no. CAS: 375823-41-9), which was an antibody that binds human IL-6 receptor, and then allowed to react overnight at 37 ° C. The final concentration of tocilizumab was adjusted to 333 μg / ml. The reaction solutions were then added to a streptavidin-coated Gold Multi-ARRAY plate (Meso Scale Discovery). After standing for another hour at room temperature, the reaction solution was washed. Then, immediately after the Read Buffer T (× 4) (Meso Scale Discovery) was added to the plate, measurements were made using a SECTOR Imager 2400 (Meso Scale Discovery). Soluble human IL-6 receptor concentration was calculated based on the response from a calibration curve using the analytical software, SOFTmax PRO (Molecular Devices).

The observed changes in plasma concentration of soluble human IL-6 receptor in human FcRn transgenic mice after intravenous administration are shown in FIG. 1, 2 and 3. FIG. 1 shows the effect of increased antigen elimination when the pI of the variable region is increased in the case of the native constant region of IgG1. FIG. 2 demonstrates the effect of increased antigen elimination when the pI of the variable region was increased in an antibody that was confirmed to bind to FcRn under neutral pH conditions (F939). FIG. 3 demonstrates the effect of increased antigen elimination when the pI of the variable region was increased in an antibody whose FcγR-binding capacity was increased under neutral pH conditions (F1180).

In all cases, it has been demonstrated that by increasing the pI of antibodies it is possible to increase the rate of antigen elimination in the case of antibodies with pH-dependent binding. It was also demonstrated that by further increasing the binding capacity for FcRn or FcγR under neutral pH conditions, it is possible to further increase the rate of antigen elimination compared to the case when only the pI is increased in antibodies with pH-dependent binding (comparing Fig. 1 to Fig. 2 and 3).

(2-3) In vivo fluid analysis of antibodies with pI-regulated pH-dependent binding to human IL-6 receptor

The in vivo assay described below was performed using various pH-dependent binding to human IL-6 receptor antibodies prepared in Example 1: Low_pI-IgG1, High_pI-IgG1, Low_pI-F11 and High_pI-F11.

An infusion osmotic minipump (model 2004; Alzet) containing soluble human IL-6 receptor was implanted subcutaneously in the back of human FcRn transgenic mice (B6.mFcRn - / -. HFcRn Tg mice line 32 + / +, Jackson Laboratories; Methods Mol. Biol. 602, 2010, pp. 93-104), creating model animals in which the concentration of soluble human IL-6 receptor in plasma is maintained at a constant level. Model animals were injected with antibodies to the human IL-6 receptor and, after administration, the kinetics of the antibodies in vivo was assessed.

More specifically, an anti-mouse CD4 monoclonal antibody prepared by a method known in the art was injected once at a dose of 20 mg / kg into the tail vein to suppress the production of neutralizing antibodies to soluble human IL-6 receptor that can be produced by the mouse itself. Then, an infusion pump containing soluble human IL-6 receptor at a concentration of 92.8 μg / ml was implanted subcutaneously into the back of the mouse. Three days after implantation of the infusion pump, antibodies to the human IL-6 receptor were injected into the tail vein once at a dose of 1 mg / kg. Blood was taken from mice 15 minutes, 7 hours, 1 day, 2 days, 3 or 4 days, 6 or 7 days, 13 or 14 days, 20 or 21 days, and 27 or 28 days after administration of antibodies to the human IL-6 receptor ... The collected blood was immediately centrifuged at 15000 rpm and 4 ° C for 15 minutes to obtain plasma. The separated plasma was stored in a freezer at -20 ° C or below until measurement.

(2-4) Measurement of hsIL-6R plasma concentration by electrochemiluminescence method

Plasma hsIL-6R concentration in mice was measured by electrochemiluminescence method. Samples of hsIL-6R, the concentration of which was adjusted to 250, 125, 62.5, 31.25, 15.61, 7.81 or 3.90 pg / ml to construct a calibration curve, and analyzed plasma samples of mice, diluted 50 times or more, mixed with an anti-human IL-6R monoclonal antibody (R&D) labeled with ruthenium using a sulfo-labeled NHS ester (Meso Scale Discovery), a biotinylated anti-human IL-6R antibody (R&D) and tocilizumab, and allowed to react overnight at 37 ° C. The final concentration of tocilizumab was adjusted to 333 μg / ml. The reaction solutions were then added to a streptavidin-coated Gold Multi-ARRAY plate (Meso Scale Discovery). After standing for another hour at room temperature, the reaction solution was washed. Then, immediately after adding to the plate the reading buffer (Read Buffer T (× 4)) (Meso Scale Discovery), measurements were carried out using a SECTOR Imager 2400 (Meso Scale Discovery). The concentration of soluble human IL-6R receptor was calculated based on the response from a calibration curve using the analytical software, SOFTmax PRO (Molecular Devices).

Changes in measured human IL-6 receptor concentration are shown in FIG. 4. As in the case of an antibody, the Fc region of which was the Fc region of native IgG1 (High_pI-IgG1), in the case of an antibody that contained a new variant of the Fc region with an increased ability to bind to FcRn under neutral pH conditions (High_pI-F11 ), the concentration of soluble human IL-6 receptor in plasma decreased in the case of administration of the high pI antibody (which is also referred to as "High_pI") as compared to the case of administration of the low pI antibody (which is also referred to as "Low_pI").

Without going into any particular theory, the results obtained in these experiments can also be explained as follows: when the Fc region of the administered antibody is the same as that of the native IgG-type antibody, then it is believed that the uptake by cells occurs in mainly through nonspecific absorption (pinocytosis). Therefore, since the cell membrane is negatively charged, the higher the pI of the administered antibody-antigen complex (i.e., when the charge of the molecule as a whole becomes more positive), the faster the complex can reach the cell membrane and the easier non-specific uptake can take place. When an antibody with an increased pI forms a complex with an antigen, then this complex as a whole is also characterized by an increased pI in comparison with the complex formed by the parent antibody and antigen; therefore, cellular uptake may increase. Thus, by increasing the pI of an antibody characterized by pH-dependent binding to an antigen, it is possible to further increase the rate or level of antigen elimination from plasma and to maintain the concentration of antigen in plasma at a lower level.

In these examples, an increase in the pI of an antibody is accomplished by introducing amino acid substitutions that decrease the number of negatively charged amino acids and / or increase the number of positively charged amino acids that can be displayed on the surface of the antibody molecule in the variable region of the antibody. It will be apparent to those of skill in the art that the effects achieved with such an increase in pI are largely (or practically) independent of the type of target antigen or amino acid sequence that the antibody has, but would be expected to be dependent on the pI. For example, WO 2007/114319 and WO 2009/041643 outline the following provisions.

Since the molecular weight of the IgG-type antibody is large enough, its main metabolic pathway does not include renal excretion. IgG-type antibodies that have Fc are known to have a long half-life because they are recycled through the rescue pathway of FcRn expressed in cells, including blood vessel endothelial cells, and IgG is believed to be metabolized primarily in endothelial cells. More specifically, it is believed that IgGs that are nonspecifically taken up by endothelial cells are recycled by binding to FcRn, and molecules that cannot bind to FcRn are metabolized. IgGs whose FcRn binding capacity is reduced have a shorter blood half-life, and conversely, the blood half-life can be lengthened by increasing their ability to bind to FcRn. Thus, in the previously known methods of controlling the kinetic characteristics of IgG in blood, a modification of Fc was used to change its ability to bind to FcRn; however, in the working examples in WO 2007/114319 (mainly concerning methods for replacing amino acids in the FR region) and WO 2009/041643 (mainly concerning methods for replacing amino acids in the CDR region), it is demonstrated that regardless of the type of target antigen by modifying the pI variable region of an antibody can control its blood half-life without modifying the Fc. It is believed that the rate of nonspecific uptake of an IgG-type antibody by endothelial cells depends on the physicochemical Coulomb interaction between a negatively charged cell surface and an IgG-type antibody. Therefore, it is believed that a decrease (increase) in pI of an IgG-type antibody and thereby a decrease (increase) in Coulomb interactions decreases (increases) its nonspecific uptake by endothelial cells and, therefore, decreases (increases) its metabolism in endothelial cells, thereby allowing to control pharmacokinetic characteristics in plasma. Since the Coulomb interaction between endothelial cells and the negative charge of the cell surface is a physicochemical interaction, it is believed that this interaction is largely independent of the antibody-forming amino acid sequence per se. Therefore, the methods for monitoring plasma pharmacokinetic characteristics described herein are applicable not only to specific antibodies, but can be widely applied to any polypeptide containing an antibody variable region. As used herein, a decrease (increase) in Coulomb interactions means a decrease (increase) in the Coulomb force, which is an attractive force and / or an increase (decrease) in the Coulomb force, which is a repulsive force.

Amino acid substitutions for the above purposes can be a single amino acid substitution or a combination of several amino acid substitutions. Some embodiments of the invention relate to a method of introducing a single amino acid substitution, or a combination of several amino acid substitutions, at a position (s) exposed on the surface of an antibody molecule. Alternatively, multiple introduced amino acid substitutions can be conformationally close to each other. When creating the invention, the idea was put forward that, for example, when replacing amino acids that can be displayed on the surface of an antibody molecule with positively charged amino acids (preferably arginine or lysine) or using already existing positively charged amino acids (preferably arginine or lysine) , it may be preferable to additionally replace one or more amino acids that are conformationally close to the indicated amino acids (in some cases even one or more amino acids "immersed" inside the antibody molecule) with positively charged amino acids to result in a state with locally grouped positive charges, located in conformationally close positions. In the context of the present description, the definition of "conformationally close (s) position (s)" does not impose specific restrictions, but it may, for example, mean a state in which one amino acid substitution or several amino acid substitutions are introduced within 20 Angstroms, preferably within 15 Angstroms or more preferably within 10 Angstroms of each other. Whether amino acid substitutions of interest are at a position exposed on the surface of an antibody molecule or whether amino acid substitutions are at proximal positions can be determined using known techniques such as X-ray crystallography.

Thus, given that pI is one indicator characterizing the total charge of the molecule, and that the charges embedded inside the antibody molecule and the charges located on the surface of the antibody molecule are considered the same, when creating the invention it was concluded that by creating antibody molecules with a broad and comprehensive consideration of the effects of all charges, including not only pI, but also surface charges and local clustering of charges of antibody molecules, it is possible to further increase the rate of antigen elimination from plasma and to maintain the concentration of antigen in plasma at even lower levels.

Receptors such as FcRn or FcγR are expressed on the cell membrane, and it is believed that antibodies with increased affinity for FcRn or FcγR under neutral pH conditions are absorbed by cells mainly through these Fc receptors. Since the cell membrane is negatively charged, the administered antibody-antigen complex reaches the cell membrane faster when its pI is high (when the charge of the molecule as a whole is shifted towards more positive values), and absorption by the Fc receptor can occur more easily. Therefore, antibodies that have increased affinity for FcRn or FcγR under neutral pH conditions, as well as increased pI, are characterized by increased uptake by cells through Fc receptors when they form a complex with antigens. Therefore, the rate of elimination of antigens from plasma by antibodies that bind to antigens in a pH-dependent manner and have increased affinity for FcRn or FcγR under neutral pH conditions can be increased by increasing their pI and the plasma antigen concentration can be maintained at lower levels.

Example 3

Evaluation of the ability of antibodies with pH-dependent binding, having increased pI, to bind to the extracellular matrix

(3-1) Assessment of the ability to bind to the extracellular matrix

The following experiment was carried out to evaluate the effects of conferring pH-dependent antigen-binding properties to antibodies and further modification of pI on their extracellular matrix binding capacity.

Using a method similar to that described in example 1, three types of antibodies with different pIs were obtained as antibodies characterized by pH-dependent binding to the IL-6 receptor: Low_pI-IgG1, Middle_pI-IgG1 and High_pI-IgG1. As canonical antibodies not having a pH-dependent binding to the IL-6 receptor, using the method described in Example 2, Low_pI (NPH) -IgG1 was obtained containing H54 (SEQ ID NO: 34) and L28 (SEQ ID NO: 35), and High_pI (NPH) -IgG1 containing H (WT) (SEQ ID NO: 36) and L (WT) (SEQ ID NO: 37), respectively, described in WO 2009/125825 ,.

For these antibodies, a theoretical pI value was calculated by a method similar to that described in Example 1, the results are shown in Table 4. It was also demonstrated that antibodies that do not have a pH-dependent binding to the IL-6 receptor were characterized by an increased similarity of pI values with antibodies which possessed pH-dependent binding.

(3-2) Evaluation of the binding of antibodies to the extracellular matrix by electrochemiluminescence (ECL) method

The extracellular matrix (cell basement membrane matrix, BD Matrigel Basement Membrane Matrix; manufactured by BD) was diluted to a concentration of 2 mg / ml using TBS (Takara). The diluted extracellular matrix was added to a 96-well plate MULTI-ARRAY, High bind, Bare (manufactured by Meso Scale Discovery: MSD) at 5 μl per well and immobilized overnight at 4 ° C. Then, 20 mM ACES buffer, pH 7.4, containing 150 mM NaCl, 0.05% Tween 20, 0.5% BSA and 0.01% NaN _{3 was} added to the plate for blocking. Antibodies to be evaluated were diluted to a concentration of 30, 10, and 3 μg / ml using 20 mM ACES buffer, pH 7.4 (ACES-T buffer), containing 150 mM NaCl, 0.05% Tween 20 and 0.01% NaN ₃ , and then further diluted using 20 mM buffer buffer, pH 7.4, containing 150 mM NaCl, 0.01% Tween 20, 0.1% BSA and 0.01% NaN ₃ (dilution buffer) to reach the final concentration 10, 3.3 and 1 μg / ml, respectively. The diluted antibody solutions were added to the plate, from which the blocking solution was removed, and the plate was shaken at room temperature for 1 h. The antibody solutions were removed, ACES-T buffer containing 0.25% glutaraldehyde was added and then allowed to stand for 10 min, the plate was washed with D-PBS (manufactured by Wako Pure Chemical Industries) containing 0.05% Tween 20. Antibodies for ECL detection were prepared by conjugation with sulfo-tagged goat anti-human IgG (gamma) (manufactured by Zymed Laboratories) with using an NHS ester with a sulfo-tag (manufactured by MSD). Antibodies for detection were diluted in dilution buffer to a concentration of 1 μg / ml, added to the plate and then shaken in the dark at room temperature for 1 hour. Antibodies for detection were removed and a 2-fold diluted solution prepared by diluting MSD reading buffer was added Read Buffer T (4 ×) (manufactured by MSD) with ultrapure water; and then the luminescence level was measured with a SECTOR Imager 2400 (manufactured by MSD).

The results are shown in FIG. 5. It was found that for antibodies characterized by pH-dependent binding, and for antibodies that were not characterized by pH-dependent binding, binding to the extracellular matrix increased with an increase in their pI. In addition, it was unexpectedly found that the effect of increasing binding to the extracellular matrix with increasing pI was significant for antibodies having pH-dependent binding to antigen. In other words, it was found that an antibody whose binding to an antigen is pH dependent and which has a high pI (High_pI-IgG1) has a higher affinity for the extracellular matrix.

Without going into any particular theory, the results obtained in these experiments can also be explained as follows. It is known that the introduction of histidine modifications into the variable region of an antibody is one of the methods for imparting a pH-dependent antigen-binding property to an antibody (see, for example, WO 2009/125825). Histidine carries an imidazoyl group on its side chain and is uncharged under conditions from neutral pH to basic pH, but is known to be positively charged under conditions of acidic pH. Taking this property of histidine into account, it is possible, by introducing histidine into the variable region of an antibody, primarily in the CDR located near the site of interaction with the antigen, to change the charge and conformation in the region of interaction with the antigen when passing from neutral to acidic pH conditions. Such antibodies can be expected to have an affinity for antigen that varies in a pH-dependent manner. It should be obvious to those skilled in the art that the effects obtained from such introduction (s) of histidine is largely (or practically) independent of the type of target antigen or amino acid sequence that the antibody has, but depends on the site of introduction of histidine. or the amount of introduced histidine residues.

WO 2009/041643 generally describes the following provisions: protein-protein interactions include hydrophobic interactions, electrostatic interactions and hydrogen bonds, and the strength of such binding can generally be characterized using a binding constant (affinity) or an apparent binding constant (avidity). pH-dependent binding, where the strength of binding changes from neutral pH conditions (eg pH 7.4) to acidic pH conditions (eg pH 5.5-6.0), depends on naturally occurring protein-protein interactions ... For example, the above binding between an IgG molecule and FcRn, which is known to be a salvage receptor for an IgG molecule, is strong under acidic pH conditions and very weak under neutral pH conditions. Histidine residues are involved in many protein-protein interactions that change in a pH-dependent manner. Since the pKa value of the histidine residue is close to 6.0-6.5, the state of proton dissociation in the histidine residue changes when passing between conditions with neutral and acidic pH. More specifically, the histidine residue is uncharged and neutral under neutral pH conditions and functions as a hydrogen scavenger; while under acidic pH conditions it is positively charged and functions as a hydrogen atom donor. It has been reported that in the case of the above interaction of the IgG-FcRn molecule, the histidine residues present on the IgG molecule are involved in pH-dependent binding (Martin et al., Mol. Cell. 7 (4), 2001, pp. 867 -877).

Consequently, the replacement of the amino acid residue involved in protein-protein interactions with a histidine residue, or the introduction of histidine into the interaction site, can impart a pH-dependent protein-protein interactions. Similar measures were taken in the case of protein-protein interactions between antibody and antigen; and by introducing histidine into the CDR sequence of an anti-chicken egg protein lysozyme antibody, a mutant antibody with reduced antigen affinity was successfully produced under acidic pH conditions (Ito et al., FEBS Lett. 309 (1), 1992, pp. 85-88). In addition, published data on antibodies that specifically bind to antigens at low pH in cancer tissues and weakly bind to the corresponding antigen under neutral pH conditions due to the introduction of histidine into the CDR sequence (WO 2003/105757).

Thus, the amino acid residue that is introduced to increase the pI is preferably lysine, arginine or histidine, which have positively charged side chains. The standard pKa values for the side chains of these amino acids are: 10.5 for lysine, 12.5 for arginine and 6.0 for histidine (Skoog et al., Trends Anal. Chem. 5 (4), 1986, pp. 82-83 ). As follows from the theory of acid-base balance, these pKa values mean that in a solution with pH 10.5, 50% of the lysine side chains are positively charged, and the remaining 50% are uncharged. As the pH of the solution increases, the proportion of positively charged side chains of lysine decreases, and in a solution with a pH of 11.5, which is 1 pH unit higher than the pKa value of lysine, the proportion of positively charged side chains becomes approximately 9%. On the other hand, as the pH of the solution decreases, the proportion of positively charged side chains increases, and in a solution with pH 9.5, which is 1 pH unit lower than the pKa value of lysine, the proportion of positively charged side chains becomes approximately 91%. This theory works in a similar way for arginine and histidine. More specifically, in a solution at neutral pH (eg, pH 7.0), almost 100% of the lysine or arginine is positively charged, while for histidine, the fraction of positively charged residues is about 9%. Therefore, although histidine is positively charged under neutral pH conditions, because this level is low compared to the level of lysine or arginine, lysine and arginine are considered to be the preferred amino acids for introduction in order to increase the pI. In addition, according to Holash et al. (Proc. Natl. Acad. Sci. 99 (17), 2002, pp. 11393-11398), although the introduction of modifications that increase the pI is considered effective in terms of modifications that enhance the binding of the extracellular matrix substitutions introducing lysine or arginine are considered for the above reasons as preferred amino acid modifications over substitutions introducing histidine.

As first established by the invention, it was possible to provide an unexpected synergistic increase in the affinity of an antibody for the extracellular matrix by combining the introduction of histidine to confer a pH-dependent antigen binding property with a pI-raising modification of the antibody. In fact, while the pI of High_pI-IgG1 was 9.30, the pI of High_pI (NPH) -IgG1 was 9.35, and therefore the indicated value for High_pI-IgG1 was slightly less. However, the actual affinity of High_pI-IgG1 for the extracellular matrix was markedly higher. This indicates that the affinity for the extracellular matrix cannot necessarily be explained by the pI level alone, and it can be argued that this fact indicates a synergistic effect due to the introduction of a combination of pI-increasing modifications and histidine modifications. This result is a phenomenon that is surprising and unexpected.

However, it should be noted that since the pKa of side chains of amino acids in a protein is greatly influenced by environmental conditions, they do not always coincide with the above theoretical pKa values. More specifically, the above description is based on general scientific theory; however, it can be easily demonstrated that numerous exceptions can occur for real proteins. For example, Hayes et al. J. Biol. Chem. 250 (18), 1975, pp. 7461-7472 indicate that when the pKa of histidine contained in myoglobin was determined experimentally, although the values obtained were around the mean (were centered) 6.0, they ranged from 5.37 to 8.05. Naturally, histidine, which has a high pKa value, should be substantially positively charged under neutral pH conditions. Thus, the aforementioned theory does not negate the pI-increasing effect of histidine introduction also in the case of a real three-dimensional protein structure. There is sufficient evidence to suggest that the amino acid modification involving the introduction of histidine, as demonstrated in the case of lysine or arginine, can have an increasing pI effect.

Example 4

Increasing pI by a single amino acid substitution in the constant region

Methods are known for increasing the pI of an antibody that binds to an antigen in a pH-dependent manner by introducing amino acid substitutions into the variable region of an antibody. In addition, it is possible to increase the pI of an antibody also by making only one amino acid substitution in the constant region of the antibody.

The method of adding a single amino acid substitution to the constant region of an antibody to increase the pI is not particularly limited, for example, it can be performed according to the method described in WO 2014/145159. As with the variable region, amino acid substitutions introduced into the constant region are preferably substitutions that decrease the amount of negatively charged amino acids (such as aspartic acid or glutamic acid) while increasing the amount of positively charged amino acids (such as arginine or lysine).

Positions for the introduction of amino acid substitutions in the constant regions are preferably, but are not limited to, positions at which amino acid side chains can be exposed on the surface of the antibody molecule. Preferred examples include a method of introducing a combination of several amino acid substitutions at positions where they can be exposed on the surface of the antibody molecule. Alternatively, the multiple introduced amino acid substitutions are preferably arranged so that they are in conformational proximity to each other. In addition, several introduced amino acid substitutions are preferably (but not limited to) substitutions for positively charged amino acids, which in certain cases results in a state in which several positive charges are in conformationally close positions. In the context of the present description, the definition of "conformationally close position" does not impose particular restrictions, but it can, for example, mean a state in which one amino acid substitution or several amino acid substitutions are introduced within 20 Angstroms, preferably within 15 Angstroms or more preferably within 10 Angstrom apart. Whether amino acid substitutions of interest are at a position exposed on the surface of an antibody molecule, or whether multiple amino acid substitutions are at proximal positions, can be determined using known techniques such as X-ray crystallography.

In addition, the method of obtaining multiple positive charges at conformationally close positions includes, in addition to the above methods, the method of using amino acids that initially have positive charges in the constant region of IgG. Examples of such positively charged amino acid positions include (a) arginine at position 255, 292, 301, 344, 355, or 416 according to EU numbering; and (b) lysine at position 121, 133, 147, 205, 210, 213, 214, 218, 222, 246, 248, 274, 288, 290, 317, 320, 322, 326, 334, 338, 340, 360 , 370, 392, 409, 414 or 439 according to EU numbering. By making substitutions for a positively charged amino acid in a position conformationally close to the indicated positively charged amino acids, it is possible to obtain several positive charges in conformationally close positions.

(4-1) Preparation of pH-dependent IgE-binding antibodies

Using the method described in Reference Example 2, the following three antibodies were prepared as pH dependent antibodies to human IgE: (1) Ab1, which is a canonical antibody containing Ab1H (SEQ ID NO: 38) as a heavy chain and Ab1L (SEQ ID NO: 39) as a light chain; (2) Ab2, which is a canonical antibody containing Ab2H (SEQ ID NO: 40) as the heavy chain and Ab2L (SEQ ID NO: 41) as the light chain; and (3) Ab3, which is a canonical antibody containing Ab3H (SEQ ID NO: 42) as the heavy chain and Ab3L (SEQ ID NO: 43) as the light chain.

(4-2) Assessment of pH dependence of binding to human IgE

The affinity of Ab1 for human IgE at pH 7.4 and pH 5.8 was evaluated as follows. Kinetic analyzes for human IgE and Ab1 were performed using a BIACORE T100 device (GE Healthcare). Measurements were carried out using the following two buffers as a rolling buffer: (1) 1.2 mM CaCl ₂ / 0.05% Tween 20, 20 mM ACES, 150 mM NaCl, pH 7.4; and (2) 1.2 mM CaCl ₂ / 0.05% Tween 20, 20 mM ACES, 150 mM NaCl, pH 5.8.

Protein A / G (ACTIGEN) in an appropriate amount was fixed on a CM4 sensor chip (GE Healthcare) using the amine coupling method to capture antibodies of interest. Then, human IgE was reacted with antibodies captured on the sensor chip by injection of a diluted IgE solution and a rolling buffer (which was used as a reference solution). One of the above buffers (1) and (2) was used as a rolling buffer, and human IgE was diluted with the appropriate buffer. To regenerate the sensor chip, 10 mM glycine-HCl was used at pH 1.5. All measurements were performed at 25 ° C. The KD (M) for human IgE was calculated for each antibody based on the association reaction rate constant ka (1 / Ms) and the dissociation rate constant kd (1 / s), which are kinetic parameters calculated from the measured sensograms. The BIACORE T100 Evaluation software (GE Healthcare) was used to calculate each parameter.

The affinity of Ab2 and Ab3 for human IgE at pH 7.4 and pH 5.8 was evaluated as follows. The binding activity (dissociation constant KD (M)) of anti-hIgE antibodies against hIgE was assessed using a BIACORE T200 device (GE Healthcare). Measurements were performed using the following two buffers as rolling buffers: (1) 1.2 mM CaCl ₂ / 0.05% Tween 20, 20 mM ACES, 150 mM NaCl, pH 7.4; and (2) 1.2 mM CaCl ₂ / 0.05% Tween 20, 20 mM ACES, 150 mM NaCl, pH 5.8.

The corresponding amount of peptide obtained by adding biotin to Lys present at the C-terminus of chemically synthesized human glypican 3 (aka, GPC3), derived from a peptide protein (having the amino acid sequence (VDDAPGNSQQATPKDNEISTFHNLGNVHSPLK (SEQ ID NO: 44)) ("biotinylated peptide GPC3 ") was added to the SA sensor chip (GE Healthcare) and immobilized on the chip using the affinity between streptavidin and biotin. Injected at an appropriate concentration of hIgE and immobilized on the chip by capture with biotinylated peptide GPC3. Antibody was injected as an analyte at an appropriate concentration hIgE and allowed to interact with hIgE on the sensor chip Then 10 mM glycine-HCl pH 1.5 was injected to regenerate the sensor chip All measurements were performed at 37 ° C. Association reaction rate constants (1 / Ms) and dissociation rate constants kd (1 / s) was calculated based on the analysis of the measurement results by approximating The curve was imaged using the BIACORE T200 Evaluation software (GE Healthcare), and the dissociation constants KD (M) were calculated from these values.

The results are shown in Table 5. For all antibodies, i. E. Ab1, Ab2 and Ab3 showed pH-dependent binding to human IgE and demonstrated that their affinity at acidic pH (pH 5.8) was sharply reduced compared to their affinity at neutral pH (pH 7.4). Therefore, administration of these antibodies to a live animal can be expected to have an accelerating effect on the elimination of human IgE antigen.

The theoretical pI (pI) values for Ab1-Ab3, calculated by a method similar to that described in example 1, are presented in table 6.

(4-3) Obtaining antibodies with increased pI by one amino acid modification in the constant region

The Ab1 obtained in Example (4-1) is an antibody containing the constant region of native human IgG1 as a constant region. Ab1H-P600 was obtained by modifying the Fc region of Ab1H, which is the heavy chain of Ab1, which consisted of replacing proline at position 238 according to EU numbering with aspartic acid and replacing serine at position 298 according to EU numbering with alanine. In addition, various Fc variants were obtained using the method described in Reference Example 2 by introducing into the Fc region of Ab1H-P600 various single amino acid substitutions indicated in Tables 7-1 and 7-2, respectively. For all Fc variants, Ab1L (SEQ ID NO: 39) was used as the light chain. The affinity of these antibodies for hFcγRII2b was comparable to the affinity of the P600 variant (data not shown).

(4-4) BIACORE Human FcγRIIb Binding Assay Using New Antibodies Containing Fc Region Variants

Binding assays of the variant Fc region of the antibody to soluble human FcγRIIb (also referred to as "hFcγRIIb") and antigen-antibody complexes were performed using a BIACORE® T200 device (GE Healthcare). Soluble hFcγRIIb was obtained in the form of a His-tagged molecule using methods known in the art. The anti-His antibody in an appropriate amount was fixed on a CM5 sensor chip (GE Healthcare) using the amine coupling method using a His capture kit (GE Healthcare) to capture hFcγRIIb. Then, the antibody-antigen complex and rolling buffer (as a reference solution) were injected and allowed to react with hFcγRIIb captured on the sensor chip. As a rolling buffer, 20 mM N- (2-acetamido) -2-aminoethanesulfonic acid, 150 mM NaCl, 1.2 mM CaCl ₂ and 0.05% (w / v) Tween 20 with pH 7.4 were used, and an appropriate buffer was also used to dilute soluble hFcγRIIb. For the regeneration of the sensor chip, 10 mM glycine-HCl with pH 1.5 was used. All measurements were performed at 25 ° C. The assay was performed on the basis of binding (RU) calculated using the sensograms obtained from the measurements, and relative values were obtained taking the P600 binding level as 1.00. The parameters were calculated using the BIACORE® T100 Evaluation software (GE Healthcare). The results are shown in Tables 7-1 and 7-2 (see the column entitled "BIACORE" in the tables) and FIG. 6. Several Fc variants have been demonstrated to have increased affinity for hFcγRIIb fixed to a BIACORE® sensor chip.

Without going into any particular theory, this result can be explained as follows. The BIACORE® sensor chip is known to be negatively charged, and this charged state can be considered to resemble the surface of a cell membrane. More specifically, it can be assumed that the binding of the antigen-antibody complex to hFcγRIIb fixed on the negatively charged BIACORE sensor chip resembles that which occurs when the antigen-antibody complex binds to hFcγRIIb present on the negatively charged surface of the cell membrane.

Antibodies obtained by introducing a pI-increasing modification into the Fc region are antibodies in which the charge of the Fc region (constant region) is a greater positive charge than the charge before the modification was introduced. Therefore, it can be assumed that the Coulomb interaction between the Fc region (positive charge) and the surface of the sensor chip (negative charge) should be enhanced as a result of the pI-increasing amino acid modification. In addition, it is to be expected that such effects should also occur on the negatively charged surface of the cell membrane; therefore, they can also be expected to have an accelerating effect on the rate or level of uptake by cells in vivo.

The above results show that the ratio of the binding level of the variant to hFcγRIIb to the binding level of Ab1H-P600 to hFcγRIIb of about 1.2 or more indicates a strong effect of charge on the binding of the antibody to hFcγRIIb on the sensor chip. Thus, the modification that is expected to provide a charge effect is, for example, the modification at position 196, 282, 285, 309, 311, 315, 345, 356, 358, 359, 361, 362, 382, 384. 385, 386, 387, 389, 399, 415, 418, 419, 421, 424 or 443 according to EU numbering. A modification at position 282, 309, 311, 315, 345, 356, 359, 361, 362, 385, 386, 387, 389, 399, 418, 419 or 443 is preferred. Amino acid substitution introduced at this position is preferably a substitution to arginine or lysine. Another example of an amino acid mutation position at which such a charge-related effect can be expected is glutamic acid at position 430 according to the EU numbering. The preferred amino acid substitution to be introduced at position 430 is a substitution for arginine or lysine, which is positively charged, or, among the uncharged residues, preferably a substitution for glycine or threonine.

(4-5) Uptake of Fc Region Variant Antibodies by hFcγRIIb Expressing Cells

To assess the level of intracellular uptake of the hFcγRIIb expressing cell line, the following assay was performed using antibodies containing the novel Fc region variant.

The MDCK cell line (Madin-Darby canine kidney cell line) constitutively expressing hFcγRIIb was obtained using known methods. Using these cells, the intracellular uptake of antigen-antibody complexes was assessed. More specifically, pHrodoRed (Life Technlogies) was used to label human IgE (antigen) according to an approved protocol and antigen-antibody complexes were prepared in culture solution with an antibody concentration of 10.8 mg / ml and an antigen concentration of 12.5 mg / ml. A culture solution containing antigen-antibody complexes was added to culture plates to the above MDCK cells that constitutively express hFcγRIIb, and incubated for 1 h, and then the fluorescence intensity from the antigen absorbed by the cells was quantified using an InCell 6000 analyzer (firm GE healthcare). The amount of antigen uptake was presented as relative values compared to the value for P600, which was taken as 1.00.

The results are shown in Tables 7-1 and 7-2 (see the column entitled “visualization” in the tables) and FIG. 7. For several Fc variants, a high level of fluorescence from the antigen in cells was detected.

Without going into any particular theory, this result can be explained as follows: antigen and antibody added to the cell culture solution form antigen-antibody complexes in the culture solution. The antigen-antibody complexes bind to hFcγRIIb expressed on the cell membrane via the Fc region of the antibody and are taken up by the cells in a receptor-dependent manner. Ab1 used in this experiment was an antibody that binds to an antigen in a pH-dependent manner; hence, the antibody can be separated by dissociation from the antigen. Since the dissociated antigen is labeled with pHrodoRed as described above, it fluoresces in endosomes. Thus, it should be considered that the higher intensity of fluorescence inside the cell compared to the control indicates that the absorption of antigen-antibody complexes by cells occurs more rapidly and more frequently.

In this case, the above ratio of the fluorescence intensity from the antigen absorbed by the cells in combination with the variants to the fluorescence intensity from the Ab1H-P600 of about 1.05 or more was considered as evidence of the presence of a charge effect on the antigen uptake by the cells. The ratio of fluorescence intensity from antigen uptake by cells in combination with variants to fluorescence intensity from Ab1H-P600 of about 1.5 or more was considered as evidence of a strong effect of charge on antigen uptake by cells. Thus, the results presented above demonstrate that by introducing a pI-raising modification at the appropriate position in the Fc region, uptake by cells can be accelerated as compared to uptake before the introduction of the modification.

An amino acid modification that provides such an effect is, for example, a modification at position 253, 254, 256, 258, 281, 282, 285, 286, 307, 309, 311, 315, 327, 330, 358, 384, 385, 387, 399, 400, 421, 433 or 434, according to EU numbering. Preferably, the modification takes place at position 254, 258, 281, 282, 285, 309, 311, 315, 327, 330, 358, 384, 399, 400, 421, 433 or 434, according to the EU numbering. The amino acid substitution introduced at this position is preferably a substitution for arginine or lysine. The position at which an amino acid substitution in the constant region is introduced to increase the pI of an antibody can be, but is not limited to, for example, the amino acid residue at position 285 according to EU numbering. An alternative example would be the amino acid substitution of the amino acid residue at position 399 according to EU numbering.

Example 5

Obtaining Fc Variants with Increased Binding to FcRn Under Acidic pH Conditions to Increase the Retention Time in Plasma

It is known that under conditions of acidic pH in endosomes, IgG-type antibodies taken up by cells are returned to plasma by binding to FcRn. Thus, IgG-type antibodies generally have a long plasma half-life compared to proteins that do not bind to FcRn. Methods are known in which this property of increasing the retention time of antibodies in plasma by increasing their affinity for FcRn under conditions of acidic pH is realized by introducing amino acid modifications into the Fc region of the antibody. More specifically, methods are known for increasing the plasma retention time of antibodies by increasing their affinity for FcRn under acidic pH conditions through amino acid modifications such as the M252Y / S254T / T256E (YTE) modification (Dall'Acqua et al. J. Biol. Chem. 281, 2006, pp. 23514-23524), modification M428L / N434S (LS) (Zalevsky et al., Nat. Biotechnol. 28, 2010, pp. 157-159) and modification N434H (Zheng et al., Clinical Pharmacology & Therapeutics 89 (2), 2011, pp. 283-290).

On the other hand, as indicated above, it is also known that Fc variants with increased affinity for FcRn under acidic pH conditions have undesirable affinity for rheumatoid factor (RF) (WO 2013/046704).

Therefore, the studies presented below were carried out in order to obtain Fc variants that allow increasing the retention time in plasma, which have a reduced ability to bind or do not have the ability to bind to rheumatoid factor.

(5-1) Obtaining antibodies containing a new variant of the Fc region

According to the method described below, Fc variants with increased affinity for FcRn were obtained under acidic pH conditions, such as variants containing the known modifications: YTE, LS or N434H, and several new Fc variants (F1847m, F1848m, F1886m, F1889m, F1927m and F1168m ).

Heavy chain coding sequences into which amino acid modifications were introduced in the heavy chain Fc region (VH3-IgG1m) of Fv4-IgG1, an antibody to human IL-6 receptor, were obtained using the method described in Reference Example 1. These heavy the chains were used to obtain the following antibodies using the method described in Reference Example 2: (a) Fv4-IgG1 containing VH3-IgG1m (SEQ ID NO: 46) as the heavy chain and VL3-CK as the light chain; (b) Fv4-YTE containing VH3-YTE (SEQ ID NO: 47) as heavy chain and VL3-CK as light chain; (c) Fv4-LS containing VH3-LS (SEQ ID NO: 48) as the heavy chain and VL3-CK as the light chain; (d) Fv4-N434H containing VH3-N434H (SEQ ID NO: 49) as the heavy chain and VL3-CK as the light chain; (e) Fv4-F1847m containing VH3-F1847m (SEQ ID NO: 50) as the heavy chain and VL3-CK as the light chain; (e) Fv4-F1848m containing VH3-F1848m (SEQ ID NO: 51) as heavy chain and VL3-CK as light chain; (g) Fv4-F1886m containing VH3-F1886m (SEQ ID NO: 52) as the heavy chain and VL3-CK as the light chain; (h) Fv4-F1889m containing VH3-F1889m (SEQ ID NO: 53) as the heavy chain and VL3-CK as the light chain; (i) Fv4-F1927m containing VH3-F1927m (SEQ ID NO: 54) as the heavy chain and VL3-CK as the light chain; and (j) Fv4-F1168m containing VH3-F1168m (SEQ ID NO: 55) as the heavy chain and VL3-CK as the light chain.

(5-2) Kinetic analysis of binding to human FcRn

Antibodies containing VH3-IgG1m or the above variant as the heavy chain and L (WT) (SEQ ID NO: 37) as the light chain were prepared using the method described in Reference Example 2, and the binding activity against human FcRn as follows.

Kinetic analyzes for human FcRn and each of the antibodies were performed using a BIACORE T100 device (GE Healthcare). Protein L (ACTIGEN) in an appropriate amount was fixed on a CM4 sensor chip (GE Healthcare) using the amine coupling method to capture antibodies of interest. The human FcRn was then reacted with antibodies captured on the sensor chip by injection of a diluted FcRn solution and a rolling buffer (used as a reference solution). 50 mM sodium phosphate, 150 mM NaCl and 0.05% (w / v) Tween 20 pH 6.0 were used as a rolling buffer, and the corresponding buffer was also used to dilute the FcRn. To regenerate the sensor chip, 10 mM glycine-HCl with pH 1.5 was used. All measurements were performed at 25 ° C. The KD (M) for human IgE was calculated for each antibody based on the association reaction rate constant ka (1 / Ms) and the dissociation rate constant kd (1 / s), which are kinetic parameters calculated from the measured sensograms. The BIACORE T100 Evaluation software (GE Healthcare) was used to calculate each parameter.

The results are shown in Table 8.

Example 6

Evaluation of the affinity of antibodies containing a variant Fc region, characterized by increased binding to FcRn under conditions of acidic pH, for rheumatoid factor

Antibodies to drugs (ADA) interfere with the efficacy and pharmacokinetic characteristics of therapeutic antibodies and sometimes cause serious side effects; thus, the clinical utility and efficacy of therapeutic antibodies may be limited by the production of ADA. Many factors influence the immunogenicity of therapeutic antibodies, and the presence of effector T cell epitopes is one such factor. In addition, the presence of ADA in a patient prior to administration of a therapeutic antibody (also referred to as a “pre-existing antibody”) can also lead to similar problems. Specifically, in the case of therapeutic antibodies for patients with autoimmune diseases such as rheumatoid arthritis (RA), rheumatoid factor (RF), which is an autoantibody to human IgG, can cause a problem inherent in "preexisting ADA". A humanized anti-CD4 IgG1 antibody with the N434H mutation has been described to provide significant binding to rheumatoid factor (Zheng et al., Clin. Pharmacol. Ther. 89 (2), 2011, pp. 283-290). Detailed study confirmed that the Asn434His mutation in human IgG1 increases the binding of rheumatoid factor to the Fc region of the antibody compared to the parental human IgG1.

Rheumatoid factor is a polyclonal autoantibody against human IgG and its epitopes in human IgG vary by clone and appear to be located in the CH2 / CH3 interface as well as in the CH3 domain, which may overlap with the FcRn binding epitope. Thus, mutations leading to an increase in the binding activity (binding affinity) for FcRn can also increase the binding activity (binding affinity) for specific clones of rheumatoid factor.

In fact, for Fc with increased affinity for FcRn at acidic pH or neutral pH, it is also known that not only the N434H modification, but also many other amino acid modifications, similarly increase the binding of Fc to rheumatoid factor (WO 2013/046704).

On the other hand, as examples, WO 2013/046704 provides several amino acid modifications that selectively suppress the affinity for rheumatoid factor without affecting the affinity for FcRn, and among them are combinations of two amino acid mutations, namely Q438R / S440E, Q438R / S440D, Q438K / S440E and Q438K / S440D. Therefore, Q438R / S440E was introduced into an Fc having the novel increased affinity under acidic pH conditions first presented herein to investigate whether binding to rheumatoid factors could be reduced.

(6-1) Rheumatoid Factor Binding Assay with Fc Region Variant Antibodies

The rheumatoid factor binding assay was performed by electrochemiluminescence (ECL) at pH 7.4 using individual serum samples (Proteogenex) obtained from 30 RA patients. A 50-fold diluted serum sample, biotinylated test antibody (1 μg / ml) and labeled with an NHS ester containing a sulfo-tag (Meso Scale Discovery), test antibody (1 μg / ml) were mixed and incubated at room temperature for 3 hours. The mixture was then added to a streptavidin-coated 96-well MULTI-ARRAY plate (Meso Scale Discovery) and the plate was incubated at room temperature for 2 hours and then washed. After reading buffer T (x4) (Meso Scale Discovery) was added to each well, the plate was immediately placed on the SECTOR imager 2400 (Meso Scale Discovery) and chemiluminescence was measured.

The analysis results are shown in FIG. 8-17. Fv4-IgG1 (Fig. 8), which is native human IgG1, showed only weak binding to rheumatoid factor, while the existing Fc variants with increased FcRn binding, Fv4-YTE (Fig. 9), Fv4-LS ( Fig. 10) and Fv4-N434H (Fig. 11), all showed significantly increased binding to rheumatoid factor of many donors. On the other hand, all new variants of the Fc region with increased binding to FcRn, Fv4-F1847m (Fig. 12), Fv4-F1848m (Fig. 13), Fv4-F1886m (Fig. 14), Fv4-F1889m (Fig. 15 ), Fv4-F1927m (Fig. 16) and Fv4-F1168m (Fig. 17), showed only weak binding to rheumatoid factor, indicating that the binding to rheumatoid factor was significantly inhibited as a result of modifications that increase binding to FcRn.

FIG. 18 shows, for each of the variants, the mean values of the affinity characterizing the binding to rheumatoid factor in the serum of 30 RA patients. All six new variants had lower affinity than the three previously known variants (YTE, LS, and N434H), and they also had lower affinity for rheumatoid factor compared to native human IgG1. Thus, from the point of view of creating for clinical purposes therapeutic antibodies with a higher affinity for FcRn for the treatment of autoimmune diseases such as rheumatoid arthritis and the like, the risk associated with rheumatoid factor, which is a problem when using existing Fc variants, was reduced for the Fc variants first presented in the present description, and, therefore, they are safer than existing known Fc variants.

Example 7

Evaluation of PK of Fc variants with increased binding to FcRn under conditions of acidic pH in cynomolgus monkeys

In example 7, the effect of increasing the retention time in plasma of cynomolgus monkeys was evaluated using the method described below using antibodies containing a new variant of the Fc region, presented in the present description, for which suppression of binding to rheumatoid factor was confirmed.

(7-1) Preparation of antibodies containing a new variant Fc region

Received the following antibodies to human IgE: (a) OHB-IgG1 containing OHBH-IgG1 (SEQ ID NO: 56) as the heavy chain and OHBL-CK (SEQ ID NO: 57) as the light chain; (b) OHB-LS containing OHBH-LS (SEQ ID NO: 58) as the heavy chain and OHBL-CK as the light chain; (c) OHB-N434A containing OHBH-N434A (SEQ ID NO: 59) as the heavy chain and OHBL-CK as the light chain; (d) OHB-F 1847m containing OHBH-F1847m (SEQ ID NO: 60) as the heavy chain and OHBL-CK as the light chain; (e) OHB-F1848m containing OHBH-F1848m (SEQ ID NO: 61) as the heavy chain and OHBL-CK as the light chain; (e) OHB-F1886m containing OHBH-F1886m (SEQ ID NO: 62) as the heavy chain and OHBL-CK as the light chain; (g) OHB-F1889m containing OHBH-F1889m (SEQ ID NO: 63) as the heavy chain and OHBL-CK as the light chain; and (h) OHB-F1927m containing OHBH-F1927m (SEQ ID NO: 64) as the heavy chain and OHBL-CK as the light chain.

(7-2) Analysis of PK of antibodies containing a new variant of the Fc region in monkeys

The kinetics of in vivo antibodies to human IgE in plasma after administration of antibodies to human IgE to cynomolgus monkeys was evaluated. A solution of antibodies to human IgE was injected once intravenously at a dose of 2 mg / kg. Blood collection was performed after 5 minutes, (two hours), seven hours, one day, two days, three days, (four days), seven days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, and 56 days after administration. The collected blood was immediately centrifuged at 4 ° C and 15,000 rpm for 5 minutes to obtain plasma. The separated plasma was stored in a freezer at -80 ° C or below until measurement. Eight types of anti-human IgE antibodies were used, namely OHB-IgG1, OHB-LS, OHB-N434A, OHB-F1847m, OHB-F1848m, OHB-F1886m, OHB-F1889m and OHB-F1927m.

(7-3) Measurement of Plasma Anti-Human IgE Antibody Concentration by ELISA

The concentration of antibodies to human IgE in the plasma of cynomolgus monkeys was measured by ELISA. First, the anti-human IgG kappa chain antibody (Antibody Solution) was added to a Nunc-Immuno Plate, MaxiSorp (Nalge Nunc International) and allowed to stand overnight at 4 ° C to obtain a plate with an immobilized anti-human IgG kappa chain antibody ... Samples were prepared for the calibration curve with a plasma concentration of 640, 320, 160, 80, 40, 20 or 10 ng / ml and samples for measurements in plasma of cynomolgus monkeys diluted 100 times or more. These samples for the calibration curve and samples for plasma measurements were prepared by addition of cynomolgus monkey IgE (a product obtained by the applicant's company) at a concentration of 1 μg / ml. After that, the samples were added to a plate with an immobilized antibody to the kappa chain of human IgG and allowed to stand at room temperature for 2 hours. Then the HRP-conjugated antibody to the human IgG gamma chain (Southern Biotech) was added and allowed to stand at room temperature for for 1 hour. After that, a chromogenic reaction was carried out using a chromogenic solution of TMB (Life Technologies) as a substrate and after termination of the reaction by adding 1H. sulfuric acid (Wako) measured the absorbance at 450 nm using a microplate reader. The concentration of anti-human IgE antibody in monkey plasma was calculated from the absorbance using a calibration curve using SOFTmax PRO analytical software (Molecular Devices). The results of measurements of changes in the concentration of antibodies to human IgE in the plasma of monkeys are shown in Fig.19. Based on the measured change in the concentration of anti-human IgE antibody in the plasma of the monkeys, clearance by elimination was calculated by time-point analysis using Phoenix WinNonlin software version 6.2 (Pharsight Corporation). The calculated pharmacokinetic parameters are presented in Table 9. Samples obtained from individuals who were positive for antibodies against the administered sample in plasma were excluded when calculating the change in antibody concentration to human IgE and plasma clearance of monkeys.

(7-4) Evaluation of antibodies to injected samples in plasma by electrochemiluminescence method

Antibodies in the plasma of monkeys to the injected samples were measured by the electrochemiluminescence method. Injection sample that has been labeled with ruthenium using a sulfo-tagged NHS ester (Meso Scale Discovery), injection sample that has been biotinylated using the EZ-Link Micro Sulfo-NHS-Biotinylation kit (Pierce), and measurement sample in the plasma of cynomolgus monkeys, mixed in equal amounts and allowed to stand overnight at 4 ° C. Samples were added to a 96-well streptavidin-coated Gold MULTI-ARRAY plate (Meso Scale Discovery), then allowed to react at room temperature for 2 hours and washed. Then, immediately after the readout buffer T (x4) (Meso Scale Discovery) was added to the plate, measurements were made using a SECTOR Imager 2400 (Meso Scale Discovery).

As a result, it was confirmed that all of the new Fc variants were characterized by a significantly increased retention time in plasma compared to the Fc region of native IgG1.

(7-5) Analysis of PK Fc variants in mice

The experiment described below was carried out to compare F1718, which is an Fc variant described in WO 2013/046704, and F1848m, which is an Fc variant first obtained by the present invention, as Fc variants designed to increase FcRn binding when acidic pH.

Heavy chain coding sequences into which amino acid modifications were introduced in the Fc region of the heavy chain (VH3-IgG1) Fv4-IgG1 (anti-human IL-6 receptor antibody) were obtained using the method described in reference example 1. Using these heavy chains were obtained the following antibodies using the method described in reference example 2: (a) Fv4-IgG1 containing VH3-IgG1 as heavy chain and VL3-CK as light chain; and (b) Fv4-F1718 containing VH3-F1718 (SEQ ID NO: 65) as the heavy chain and VL3-CK as the light chain.

The above anti-human IL-6 receptor antibodies were injected once at a dose of 1 mg / kg into the tail vein of human FcRn transgenic mice (B6.mFcRn - / -. HFcRn Tg mouse line 32 + / +; Jackson Laboratories, Methods Mol. Biol. 602, 2010, pp. 93-104). Blood was collected 15 minutes, 7 hours, 1 day, 2 days, 3 days, 7 days, 14 days, 21 days, and 28 days after the administration of antibodies to the human IL-6 receptor. The collected blood was immediately centrifuged at 15000 rpm and 4 ° C for 15 minutes to obtain plasma. The separated plasma was stored in a freezer at -20 ° C or below until measurement.

(7-6) Measurement of Anti-Human IL-6 Receptor Antibody Plasma Concentration by ELISA

The concentration of antibodies to the human IL-6 receptor in mouse plasma was measured by ELISA. First, an antibody to the F (ab ') ₂ fragment of human IgG (gamma chain specific) (SIGMA) was added to a Nunc-Immuno Plate, MaxiSorp (Nalge nunc International) and allowed to stand overnight at 4 ° C. to obtain a plate with immobilized anti-human IgG antibody. Samples for the calibration curve were prepared containing anti-human IL-6 receptor antibody at plasma concentrations of 0.8, 0.4, 0.2, 0.1, 0.05, 0.025 or 0.0125 μg / ml and samples for measurements in mouse plasma, diluted 100 times or more. 200 μl of soluble human IL-6 receptor at a concentration of 20 ng / ml was added to 100 μl of calibration curve samples or samples for plasma measurements, and then the mixture containing solutions were allowed to stand for 1 hour at room temperature. Thereafter, the mixed solutions were added to each well of the plate with immobilized anti-human IgG antibody and the plate was allowed to stand for 1 hour at room temperature. Subsequently, a biotinylated anti-human IL-6R antibody (R&D) was added and the reaction was allowed to proceed for 1 hour at room temperature. Thereafter, a streptavidin-PolyHRP80 conjugate (Stereospecific Detection Technologies) was added and the reaction was allowed to proceed for 1 h at room temperature and the chromogenic reaction of this reaction solution was carried out using TMB One Component HRP Microwell Substrate (BioFX Laboratories) as a substrate. After stopping the reaction by adding 1H. sulfuric acid (Showa Chemical) was measured by the absorbance in the reaction solution in each well at 450 nm using a microplate reader. The concentration of antibody in mouse plasma was calculated from the absorbance using a calibration curve using SOFTmax PRO analysis software (Molecular Devices).

The results are shown in FIG. 20. Construct F1718, which was an Fc variant for increasing FcRn binding at acidic pH, described in WO 2013/046704, did not have any prolonged effect on antibody PK, but had a plasma retention time equivalent to that of native IgG1.

F1718 has four mutations, namely N434Y / Y436V / Q438R / S440E, introduced into the Fc region. In contrast, F1848m, first described herein, introduced four mutations, namely N434A / Y436V / Q438R / S440E. The only difference between the amino acid mutations introduced in these two types of Fc regions was that the amino acid mutation introduced at position 434 according to EU numbering was Y (tyrosine) in F1718, and A (alanine) in F1848m. Example (7-2) demonstrates that the F1848m variant had an increased plasma retention time compared to native IgG1, while the F1718 construct did not show any increase in plasma retention time. Therefore, it can be considered that A (alanine) is preferred (but this does not limit the scope of the invention) for the implementation of an amino acid mutation that should be introduced at position 434 to increase the retention time in plasma.

(7-7) Predicted score of immunogenicity of Fc variants

The formation of antibodies to drugs (ADA) affects the efficacy and pharmacokinetic characteristics of therapeutic antibodies and leads in some cases to serious side effects; and therefore, the clinical utility and drug efficacy of therapeutic antibodies can be limited by the formation of ADA. It is known that many factors affect the immunogenicity of therapeutic antibodies and, in particular, the importance of epitopes of effector T cells that carry therapeutic antibodies has been noted many times.

In silico "tools" have been developed for predicting T-cell epitopes, such as Epibase (Lonza), iTope / TCED (Antitope), and EpiMatrix (EpiVax). Using these "tools" in silico, one can predict T-cell epitopes in any amino acid sequence (Walle et al., Expert Opin. Biol. Ther. 7 (3), 2007, pp. 405-418) and the potential immunogenicity of therapeutic antibodies.

EpiMatrix was used to calculate the immunogenicity score of the evaluated Fc variants. EpiMatrix is a system for predicting the immunogenicity of a protein of interest based on the automatic construction of sequences of peptide fragments by sectioning the amino acid sequence of the protein for which it is required to predict its immunogenicity into nine amino acids and then calculating their ability to bind to the eight main alleles of GCS class II (DRB 1 * 0101 , DRB1 * 0301, DRB1 * 0401, DRB1 * 0701, DRB1 * 0801, DRB1 * 1101, DRB1 * 1301 and DRB1 * 1501) (Clin. Immunol. 131 (2), 2009, pp. 189-201).

F1718 and F1756 (N434Y / Y436T / Q438R / S440E) described in WO 2013/046704 contain the N434Y mutation. In contrast, the newly created F1848m and F1847m contain the N434A mutation.

The immunogenicity scores for these four variants, namely F1718, F1848m, F1756 and F1847m, calculated according to the method described above, are presented in Table 31 in the column entitled "EpiMatrix Score". In addition, the column entitled "tReg Adjusted Eph Score" presents the immunogenicity scores reported in the column entitled "EpiMatrix Scores" that have been corrected for Tregitope content. Tregitope is a peptide fragment sequence present in large amounts mainly in naturally occurring antibody sequences and is a sequence that is believed to inhibit immunogenicity by activating regulatory T cells (Treg).

According to these results, data presented in both the EpiMatrix Score and the tReg Adjusted Epx Score indicate that the immunogenicity scores of the N434A variants F 1848m and F 1847m were lower than those for the N434Y carriers. This suggests that A (alanine) is preferred as the amino acid mutation that needs to be introduced at position 434 to reduce the immunogenicity score.

Example 8

Preparation of humanized antibodies to human IL-8

(8-1) Preparation of humanized anti-human IL-8 hWS-4 antibody

The humanized anti-IL-8 antibody described in US Pat. No. 6,245,894 binds to human IL-8 (hIL-8) and blocks its physiological function. Modified humanized anti-IL-8 antibodies can be prepared by combining the heavy and light chain variable region sequences described in US Pat. No. 6,245,894 with virtually any of the various known human constant region sequences. Thus, the sequences of the constant region of the human antibody of these modified antibodies are not specifically limited, but as the sequences of the constant regions of the heavy chain, sequences of native human IgG1 or sequences of native human IgG4 can be used, and as sequences of constant regions of the light chain, sequences of native human kappa can be used -chains.

From the humanized anti-IL-8 antibodies described in US No. 6245894, the coding sequence for hWS4H-IgG1 (SEQ ID NO: 83), which was combined with the RVHg heavy variable region sequence and the native human anti-IgG1 heavy chain constant region sequence, was obtained by the method described in the reference example 1. In addition, the coding sequence hWS4L-k0MT (SEQ ID NO: 84), which was the combined variable region of the light chain RVLa and the sequence of the native constant region of the human kappa light chain, was obtained according to the method described in Reference Example 1. An antibody was prepared in which the above heavy chain and light chain were combined and designated as humanized WS-4 antibody (hereinafter referred to as hWS-4).

(8-2) Preparation of humanized anti-human IL-8 Hr9 antibody

A new humanized antibody was generated using non-FR human framework consensus sequences as used in hWS-4.

More specifically, the hybrid sequence of VH3-23 and VH3-64 was used as the FR1 of the heavy chain, the sequence shown in VH3-15 and VH3-49 was used as FR2, the sequence shown in VH3-72 was used as FR3 (when provided that excluded 82a according to Cabot's numbering), and the sequence presented in JH1 was used as FR4. These sequences were linked to the hWS-4 heavy chain CDR sequences to generate Hr9-IgG1 (SEQ ID NO: 85), the heavy chain of a novel humanized antibody.

Then, two types of antibodies were prepared, namely hWS-4 having hWS4H-IgG1 as the heavy chain and hWS4L-k0MT as the light chain, and Hr9 having Hr9-IgG1 as the heavy chain and hWS4L-k0MT as the light chain. In the context of section B of the present description, when referring to a light chain, in particular to Hr9, it is referred to as Hr9 / hWS4L. Antibody expression was carried out using FreeStyle 293F cells (Invitrogen) according to the protocol attached to the product. Antibodies were purified from the culture supernatant using the method described in Reference Example 2. As a result, antibodies were obtained in the amounts indicated in Table 11.

Surprisingly, it was found that the expression level of Hr9 was about 8 times higher than that of hWS-4.

(8-3) Human IL-8 Binding Activities of hWS-4 and Hr9 The affinities characterizing the binding of hWS-4 and Hr9 to human IL-8 were determined according to the method described below using a BIACORE T200 device (GE Healthcare).

A rolling buffer was used having the following composition: 0.05% Tween 20, 20 mM ACES and 150 mM NaCl (pH 7.4). Protein A / G (PIERCE) in an appropriate amount was immobilized on a CM4 sensor chip (GE Healthcare) using the amine coupling method and the antibody of interest was captured. Then, human IL-8 was reacted with the antibody captured on the sensor chip by injection of a diluted human IL-8 solution and a rolling buffer (which was used as a reference solution). A solution of the composition described above was used as a rolling buffer, and this buffer was also used to dilute human IL-8. To regenerate the sensor chip, 10 mM glycine-HCl pH 1.5 was used. All measurements were performed at 37 ° C. The KD (M) for human IgE was calculated for each antibody based on the association reaction rate constant k _on (1 / Mc) and the dissociation reaction rate constant k _off (1 / s), which are kinetic parameters calculated from sensograms obtained with measurements. The BIACORE T100 Evaluation software (GE Healthcare) was used to calculate each parameter.

The results are shown in Table 12. It was confirmed that hWS-4 and Hr9 had equivalent binding affinities for human IL-8.

For the development of antibody-containing pharmaceuticals, the level of production of antibody molecules is an important factor, and it is generally desirable that the level of production be high. Of particular note, based on the studies described above, a more suitable human consensus framework derived sequence was selected to combine with the HVR sequence of hWS-4 and Hr9 was obtained having a higher production rate while maintaining binding affinity for human IL-8.

Example 9

Generation of antibodies with pH-dependent affinity for IL-8

(9-1) Obtaining antibodies based on the modification of Hr9 to make pH dependent

Studies were conducted to impart a pH-dependent affinity for IL-8 to the Hr9 antibody obtained in example 8.

Without wishing to be bound by any particular theory, it is believed that antibodies with pH-dependent affinity for IL-8 may have the following characteristics in vivo. Antibodies administered to a living organism can bind more strongly to IL-8 under conditions that maintain a neutral pH (eg, plasma) and block its function. Some of these IL-8 / antibody complexes are taken up by cells as a result of non-specific interaction with the cell membrane (pinocytosis) (hereinafter referred to as non-specific uptake). Under conditions of acidic pH in endosomes, the binding affinities of the above antibodies to IL-8 become weak and therefore the antibody is separated from IL-8 as a result of dissociation. The antibodies that are dissociated from IL-8 can then be returned to the outside of the cell via the FcRn. The antibodies mentioned above, which have returned to the outside of the cell (into the plasma) in this way, can bind again to another IL-8 and block its function. It is believed that antibodies having pH-dependent affinity for IL-8 should be able to bind IL-8 multiple times through the above mechanism.

In contrast, in the case of an antibody that does not possess the property of the above antibody, the antibody molecule has the ability to neutralize the antigen only once, but cannot neutralize the antigen several times. In general, since an IgG-type antibody has two Fabs, one antibody molecule can neutralize two IL-8 molecules. On the other hand, antibodies that can bind IL-8 multiple times can bind IL-8 any number of times during the period of time they remain in the living body. For example, a single molecule having a pH-dependent binding to IL-8 antibody, which is absorbed by the cells ten times from the time of its administration to elimination, can neutralize as many as 20 IL-8 molecules. Therefore, an antibody that can bind several times to IL-8 has the advantage of being able to neutralize several IL-8 molecules even in the case of a small amount of antibody. On the other hand, an antibody that can bind multiple times to IL-8 has the advantage of being able to maintain a state in which it can neutralize IL-8 for a longer period of time than when administered the same amount of an antibody that does not have this property. From another point of view, an antibody that can bind several times to IL-8 has the advantage of being able to block the biological activity of IL-8 more strongly than when the antibody is administered in the same amount. which does not have this property.

To achieve these advantages, amino acid modifications, mainly histidine, were introduced into the variable regions of Hr9-IgG1 and WS4L-k0MT in order to generate antibodies that can bind IL-8 multiple times. More specifically, the variants shown in Table 13 were obtained using the methods described in Reference Examples 1 and 2.

In designations such as “Y97H” shown in Table 13, the position at which the mutation is introduced according to Cabot's numbering, the amino acid before the introduction of the mutation, and the amino acid after the introduction of the mutation are indicated. Specifically, the designation "Y97H" means that the amino acid residue at position 97 according to Cabot's numbering has been changed from Y (tyrosine) to H (histidine). In addition, when a combination of several amino acid substitutions is introduced, it is designated as “N50H / L54H” as follows.

(9-2) pH-dependent affinity for IL-8

The binding affinity for human IL-8 of the antibodies obtained in Example 9-1 was determined according to the method described below using a BIACORE T200 device (GE Healthcare). The two following rolling buffers were used: (1) 0.05% Tween 20, 20 mM ACES, 150 mM NaCl, pH 7.4; and (2) 0.05% Tween 20, 20 mM ACES, 150 mM NaCl, pH 5.8.

Protein A / G (PIERCE) in an appropriate amount was immobilized on a CM4 sensor chip (GE Healthcare) using the amine coupling method and the antibody of interest was captured. Then, human IL-8 was reacted with the antibody captured on the sensor chip by injection of a diluted human IL-8 solution and a rolling buffer (which was used as a reference solution). Any of the solutions of the composition described above was used as the rolling buffer, and this buffer was also used to dilute human IL-8. To regenerate the sensor chip, 10 mM glycine-HCl pH 1.5 was used. All measurements were performed at 37 ° C. KD (M) for each of the anti-human IgE antibodies was calculated based on the association reaction rate constant k _on (1 / Mc) and the dissociation reaction rate constant k _off (1 / s), which are kinetic parameters calculated from sensograms obtained when measuring. The BIACORE T 100 Evaluation software (GE Healthcare) was used to calculate each parameter.

The results are shown in Table 14-1. First, compared to Hr9, Hr9 / L16, which contains the L54H modification in the light chain, had slightly higher binding affinity for human IL-8 at neutral pH (pH 7.4), but lower binding affinity for human IL -8 at acidic pH (pH 5.8). On the other hand, antibodies to IL-8 (H89 / WS4L, H89 / L12 and H89 / L16), obtained by combining different light chains with H89 containing the Y97H modification in the heavy chain, all showed reduced binding affinity for human IL-8 when acidic pH; and reduced binding affinity for human IL-8 at neutral pH.

(9-3) Preparation and evaluation of antibodies modified to give pH dependence

Combinations of promising modifications identified in Example 9-2 and new amino acid mutations were evaluated, and as a result, the following combinations were identified.

Variants were prepared using the methods described in Reference Examples 1 and 2, and the binding affinity for human IL-8 was evaluated using a method similar to that described in Example 9-2.

The results are also shown in Table 14. H89 / L63, which had H89-IgG1 (SEQ ID NO: 86) as the heavy chain and L63-k0MT (SEQ ID NO: 87) as the light chain, had a binding affinity for human IL- 8 at neutral pH (pH 7.4), equivalent affinity of Hr9, and reduced at acidic pH (pH 5.8). Specifically, both k _off (dissociation reaction rate constant) and KD (dissociation constant) H89 / L63 at pH 5.8 were higher than the corresponding values for Hr9. This means that under conditions of acidic pH in endosomes, H89 / L63 had the ability to rapidly release human IL-8.

Surprisingly, it was found that H89 / L118, which has H89-IgG1 as the heavy chain and L118-k0MT (SEQ ID NO: 88) as the light chain, had a higher binding affinity (KD) for human IL-8 under neutral conditions. pH compared to Hr9, but weaker binding affinity (KD) for human IL-8 under acidic pH conditions compared to Hr9. Typically (but not limited to), when antibodies that can bind antigens multiple times are used as a pharmaceutical product, the pH-dependent antigen binding antibodies preferably have a strong binding affinity (low KD value) so that they can effectively neutralize antigens under neutral pH conditions (eg, plasma). On the other hand, antibodies preferably have a high dissociation rate constant (k _off ) and / or a low binding affinity (high KD value) so that they can release antigens more quickly under acidic pH conditions (eg, in endosomes). Compared to Hr9, H89 / L118 has acquired better properties under both neutral pH and acidic pH conditions.

Thus, suitable amino acid modifications have been identified for Hr9, such as Y97H for its heavy chain and N50H / L54H / Q89K for its light chain. It was found that it is possible to create antibodies characterized by pH-dependent binding to IL-8, having advantages as pharmaceuticals, by introducing one or a combination of several amino acid modifications selected from these modifications (but not limited to them).

Without wishing to be bound by any particular theory, it can be considered that when an antibody having pH-dependent binding to an antigen is used as a pharmaceutical, an important factor is whether or not the administered antibody can release the antigen in the endosome. In this regard, it is apparently important that there is a sufficiently weak binding (large value of the dissociation constant (KD)) under conditions of acidic pH or a sufficiently fast rate of the dissociation reaction (large value of the dissociation rate constant (k _off )). Therefore, in the experiment presented below, it was investigated whether the KD or k _{off of} H89 / L118, measured by the BIACORE method, is sufficient for antigen separation by endosomal dissociation in vivo.

Example 10

Preparation of High Affinity Antibodies for PK Analysis in Mice

There is no particular limitation on the methods for confirming the effect of antibodies on the rate of elimination of human IL-8 from mice. In one embodiment, the method comprises administering the antibody as a mixture with human IL-8 to mice and then comparing the rate of elimination of human IL-8 from the mouse plasma.

In this case, it is desirable that the reference antibody intended for use in the analysis of PK in the body of mice has a sufficiently strong binding affinity both under conditions of neutral pH and under conditions of acidic pH. Modifications were then looked for that confer high affinity to Hr9, and as a result, an antibody H998 / L63 was generated having H998-IgG1 (SEQ ID NO: 89) as the heavy chain and L63-k0MT as the light chain.

H998 / L63 was used to assess the binding affinity for human IL-8 using a method similar to that described in example 9-2. The resulting sensorgrams are shown in FIG. 21.

H998 / L63 had an unexpectedly low rate of dissociation reaction under both neutral pH and acidic pH conditions and was found to have a stronger binding affinity for IL-8 than Hr9. However, it is known that due to the mechanical limitations of the BIACORE method, analytical values such as the rate constant of the dissociation reaction (k _off ) and the dissociation constant (KD) cannot be accurately calculated in cases where protein-protein interactions are characterized by a low rate of the dissociation reaction. Since exact analytical values could not be obtained for H998 / L63, analytical values for it are not presented here. However, the results obtained in the experiment indicated that H998 / L63 had a very strong binding affinity at both neutral pH and acidic pH, and it can be used as an antibody used for comparison in the analysis of PK in mice.

Example 11

PK assay in mice using pH-dependent binding to IL-8 antibody H89 / L118

(11-1) Analysis of PK in mice using H89 / L118

The in vivo elimination rate of human IL-8 was assessed using H89 / L118 prepared in Example 9 and H998 / L63 prepared in Example 10.

The pharmacokinetic characteristics of human IL-8 were evaluated after the simultaneous administration of human IL-8 and antibodies to human IL-8 mice (line C57B L / 6J, Charles river). A solution containing a mixture of human IL-8 and anti-human IL-8 antibody (10 μg / ml and 200 μg / ml, respectively) was injected as a single dose of 10 ml / kg into the tail vein. Since the anti-human IL-8 antibody was present in sufficient excess, it was believed that at this point in time, almost all of the human IL-8 should be bound to the antibody. Blood was collected at five minutes, 2 hours, 4 hours, 7 hours, 1 day, 2 days, 3 days, 7 days, 14 days, 21 days and 28 days after administration. The collected blood was immediately centrifuged at 15000 rpm and 4 ° C for 15 minutes to obtain plasma. The separated plasma was stored in a freezer at -20 ° C or below until measurement.

(11-2) Measurement of human IL-8 plasma concentration

The concentration of human IL-8 in mouse plasma was determined by the electrochemiluminescence method. First, an anti-human IL-8 antibody (obtained in our laboratory) containing a constant region of mouse IgG was added to a 96-well MULTI-ARRAY plate (Meso Scale Discovery) and allowed to stand at room temperature for 1 hour. Then, to obtain a plate with an immobilized antibody to human IL-8, a PBS-Tween solution containing 5% BSA (w / v) was used to block at room temperature for 2 hours. Samples were prepared for constructing a calibration curve containing human IL-8 with plasma concentration 275, 91.7, 30.6, 10.2, 3.40, 1.13 or 0.377 ng / ml and samples for measurements in mouse plasma, diluted 25 times or more. The samples were mixed with hWS-4 and allowed to react overnight at 37 ° C. Thereafter, 50 μl of solutions containing the mixture was added to each well of the plate with immobilized antibody to human IL-8, and the solution was stirred at room temperature for 1 hour. The final concentration of hWS-4 was adjusted to 25 μg / ml. Then, after interaction for 1 h with a biotinylated mouse antibody to the kappa light chain of human Ig (BD Pharmingen) at room temperature and then interaction for 1 h with sulfa-labeled streptavidin (Meso Scale Discovery) at room temperature, buffer T for reading (1x) (Meso Scale Discovery) and immediately measured with a SECTOR Imager 2400 (Meso Scale Discovery). The concentration of human IL-8 was calculated based on the response using a calibration curve using SOFT Max PRO analytical software (Molecular Devices).

Plasma concentrations of human IL-8 obtained are shown in FIG. 22, and the clearance (CL) values of human IL-8 from mouse plasma are shown in Table 15.

As shown in FIG. 22, it appears that, compared to human IL-8 administered concurrently with H998 / L63, human IL-8 administered concurrently with H89 / L118 is unexpectedly rapidly eliminated from mouse plasma. In addition, CL values, which qualitatively reflect the rate of elimination of human IL-8 from mouse plasma, suggest that the rate of elimination of human IL-8 was approximately 19 times higher for H89 / L118 compared to H998 / L63.

Without going into any specific theory, based on the data obtained, the following conclusion can be drawn. Most of the human IL-8 administered simultaneously with the antibody binds to the antibody in plasma and is present in the complexes. Human IL-8 bound to H998 / L63 can exist in an antibody bound state even under acidic endosomal pH due to the antibody's inherent strong affinity. Therefore, H998 / L63 can be returned to plasma via FcRn while still complexed with human IL-8; therefore, when this happens, human IL-8 also returns to plasma at the same time. Consequently, most of the human IL-8 taken up by cells can be returned to plasma. This means that the level of elimination of human IL-8 from plasma is significantly reduced if H998 / L63 is simultaneously administered. On the other hand, as previously indicated, human IL-8 taken up by cells complexed with H89 / L118, a pH-dependent IL-8 binding antibody, can be separated by dissociation from the antibody under acidic pH conditions in the endosome. Human IL-8, separated from the antibody by dissociation, will be cleaved after transfer to the lysosome. Therefore, pH-dependent IL-8-binding antibodies can significantly accelerate the elimination of human IL-8 compared to IL-8-binding antibody such as H998 / L63, which has strong binding affinity at both acidic and neutral pH. ...

(11-3) Analysis of PK in mice at an increased dose of H89 / L118

An experiment was then carried out to verify the effect of varying the dose of H89 / L118, which was as follows. After simultaneous administration of human IL-8 and H89 / L118 (2 mg / kg or 8 mg / kg) to mice (line C57B L / 6J, Charles river), the pharmacokinetic characteristics of human IL-8 were evaluated. A solution containing a mixture of human IL-8 (2.5 μg / ml) and antibodies to human IL-8 (200 μg / ml or 800 μg / ml) was injected once into the tail vein at a dose of 10 ml / kg. It was believed that, since the antibody to human IL-8 is present in sufficient excess compared to human IL-8, then at this time almost all of the human IL-8 should be bound to the antibody. Blood was collected at 5 minutes, 7 hours, one day, two days, three days, seven days, 14 days, 21 days, and 28 days after administration. The collected blood was immediately centrifuged at 15000 rpm and 4 ° C for 15 minutes to obtain plasma. The separated plasma was stored in a freezer at -20 ° C or below until measurement.

Measurement of the concentration of human IL-8 in mouse plasma was performed by a method similar to that described in example 11-2. Plasma concentrations of human IL-8 obtained are shown in FIG. 23, and the clearance (CL) values of human IL-8 from mouse plasma are shown in Table 16.

As a result, it was confirmed that, compared to the group administered with H89 / L118 at a dose of 2 mg / kg, in the group administered with an antibody at a dose of 8 mg / kg, the elimination rate of human IL-8 was about 2 times slower.

Below in the present description, the inventors describe on a scientific basis the intended nature of one of the possible factors causing the above results, but the essence of section B is not limited to the essence of the discussion below.

Preferably, among the antibodies that are returned from the endosome to the plasma via FcRn, the proportion of antibodies bound to human IL-8 is low. With respect to human IL-8 present in the endosome, it is desirable that a large proportion of it be in free form, not bound to the antibody. When human IL-8 is co-administered with an antibody that does not have a pH-dependent affinity for IL-8, it is believed that most (almost 100%) of human IL-8 in the endosome is present in complexes with the antibody, and in free form there is a small amount (close to 0%). On the other hand, when administered in conjunction with a pH-dependent IL-8 binding antibody (eg, H89 / L118), a certain proportion of human IL-8 must be present in the endosome in free form. Hypothetically, the fraction of free form in this case can be determined as follows: [fraction of free human IL-8 in the endosome (%)] = [concentration of free human IL-8 in the endosome] / [total concentration of human IL-8 in the endosome] × 100.

Desirably, the proportion of free human IL-8 in the endosome, which is calculated according to the above equation, is higher and, for example, 20% is more preferred than 0%, 40% is more preferred than 20%, 60% more preferably than 40%, 80% more preferably than 60% and 100% more preferably than 80%.

Thus, there is a correlation between the proportion of free human IL-8 in the endosome indicated above and the binding affinity (KD) and / or the dissociation reaction rate constant (k _off ) for human IL-8 at acidic pH. It consists in the following: the weaker the binding affinity and / or the higher the rate of the dissociation reaction for human IL-8 at acidic pH, the higher the proportion of free human IL-8 in the endosome. However, in the case of antibodies having pH-dependent binding to IL-8, which lead to the fact that the proportion of free human IL-8 in the endosome is close to 100%, additional weakening of binding affinity and / or an increase in the rate of the dissociation reaction at acidic pH is not necessary. leads to an effective increase in the proportion of free human IL-8. It is easy to understand, for example, that if the proportion of free human IL-8 is increased from 99.9% even to 99.99%, then this degree of increase may not be significant.

In addition, according to the general theory of chemical equilibrium, when anti-IL-8 antibody and human IL-8 are present together and their binding reaction and dissociation reaction have reached equilibrium, the proportion of free human IL-8 is uniquely determined by three parameters: antibody concentration, antigen concentration, and dissociation constant (KD). Therefore, when the antibody concentration is high, when the antigen concentration is high, or when the dissociation constant (KD) is low, complexes are easily formed and the proportion of free human IL-8 decreases. On the other hand, when the antibody concentration is low, when the antigen concentration is low, or when the dissociation constant (KD) is high, complexing is difficult and the proportion of free human IL-8 increases.

Meanwhile, in this experiment, the rate of elimination of human IL-8 with the introduction of H89 / L118 at a dose of 8 mg / kg was lower than in the case when the antibody was administered at a dose of 2 mg / kg. Therefore, this suggests that in the endosome, the proportion of free human IL-8 decreased when the antibody was administered at a dose of 8 mg / kg, as compared to the case when the antibody was administered at a dose of 2 mg / kg. The reason for this decrease could be that increasing the antibody dose fourfold increased the concentration of the antibody in the endosome and thereby facilitated the formation of the IL-8-antibody complex in the endosome. This means that in the group that received an increased dose of antibody, the proportion of free human IL-8 in the endosome decreased and, therefore, the rate of elimination of human IL-8 decreased. This also suggests that when the antibody was administered at a dose of 8 mg / kg, the value of the dissociation constant (KD) of H89 / L118 under acidic pH conditions was insufficient to bring the content of free human IL-8 to almost 100%. More specifically, if an antibody that has a larger dissociation constant (KD) (weaker binding) under acidic pH conditions, then it can reach a state in which the proportion of free IL-8 is almost 100%, even when the antibody is introduced into dose of 8 mg / kg and the rate of elimination of human IL-8 is equivalent to that which occurs with the introduction of the antibody at a dose of 2 mg / kg.

Based on the data presented above, in order to test whether an antibody of interest with pH-dependent binding to IL-8 can provide a fraction of free human IL-8 in the endosome of almost 100%, can be tested (but not limited to) whether it is possible to increase the degree of antigen elimination action in vivo or not. For example, one method compares the rate of elimination of human IL-8 using a new pH-dependent binding to IL-8 antibody with the rate using H89 / L118, where the new antibody has weaker binding affinity at acidic pH and / or an increased rate. dissociation reactions at acidic pH versus H89 / L118. When the above novel pH-dependent IL-8 binding antibody results in a rate of elimination of human IL-8 equivalent to that of H89 / L118, this suggests that the binding affinity and / or dissociation rate of H89 / L118 at acidic pH is already at a level sufficient to achieve a fraction of free human IL-8 in the endosome of almost 100%. On the other hand, in cases where the above novel pH-dependent binding to IL-8 antibody results in a higher rate of elimination of human IL-8, this suggests that there is a possibility of an increase in the binding affinity and / or the rate of the H89 / L118 dissociation reaction at acidic pH.

Example 12

Preparation and Evaluation of H553 / L118 Antibody with pH Dependent Binding to IL-8

(12-1) Preparation of antibody H553 / L118. with pH-dependent IL-8 binding capacity

In this case, the aim of the inventors was to create antibodies having an even weaker binding affinity for human IL-8 under conditions of acidic pH and / or a higher rate of the dissociation reaction than H89 / L118.

To obtain the modified antibodies shown in Table 17, amino acid modifications were introduced, mainly using histidine, using a method similar to that described in Example 9, using H89 / L118 as a backbone. In addition, for these antibodies, the binding affinity for human IL-8 was determined using a method similar to that described in example 9-2.

A portion of these results are shown in Table 17. It has been demonstrated that the H553 / L118 antibody containing H553-IgG1 (SEQ ID NO: 90) as the heavy chain and L118-k0MT as the light chain and the H496 / L118 antibody containing H496-IgG1 (SEQ ID NO: 101) as the heavy chain and L118-k0MT as the light chain had a stronger pH dependence than H89 / L118.

The resulting H553 / L118 contained two amino acid modifications, namely Y55H and R57P, introduced into the H89 / L118 heavy chain. On the other hand, H496 / L118, obtained by introducing only R57P into the heavy chain H89 / L118, had an increased binding affinity for human IL-8 at neutral pH, but slightly altered binding affinity for human IL-8 at acidic pH, compared to H89 / L118. More specifically, the R57P modification introduced in H89 / L118 is a modification that increases the binding affinity for human IL-8 only at neutral pH, without altering the binding affinity at acidic pH. In addition, H553 / L118, obtained by introducing the Y55H modification into the heavy chain of H496 / L118, had the same or slightly increased binding affinity at neutral pH, but, on the other hand, decreased binding affinity at acidic pH compared to H89 / L118. Thus, the introduction of a combination of two amino acid modifications, Y55H and R57P, into H89 / L118 made it possible to further increase the ability to reduce binding affinity at acidic pH while maintaining or slightly increasing the binding affinity at neutral pH.

(12-2) Analysis of PK in mice using H553 / L118

Evaluation of the rate of elimination of human IL-8 from mice using H553 / L118 was carried out using a method similar to that described in example 11-2. Plasma concentrations of human IL-8 obtained are shown in FIG. 24, and the clearance (CL) values of human IL-8 from mouse plasma are shown in Table 18.

As a result, no large differences were found between H553 / L118 and H89 / L118 when comparing the data obtained in mice injected with the antibody at a dose of 2 mg / kg; however, when comparing data obtained in mice injected with the antibody at a dose of 8 mg / kg, it was confirmed that H553 / L118 accelerated the elimination of human IL-8 by 2.5 times or so compared to H89 / L118. On the other hand, for H553 / L118 there was no difference in the rate of elimination of human IL-8 when administered at doses of 2 mg / kg and 8 mg / kg, and for H89 / L118 there was no decrease in the rate of antigen elimination due to an increase antibody doses.

One of the reasons (but not limited to it) for which such results were obtained may be the following. H533 / L118 provided an equivalent rate of elimination of human IL-8 when administered at doses of 2 mg / kg and 8 mg / kg. This may indicate that the fraction of free IL-8 in the endosome could reach a level close to 100%, since the binding of IL-8 H553 / L118 at acidic pH is rather weak even when administered at a dose of 8 mg / kg. In other words, this suggests that while H89 / L118 may provide the maximum depletion effect of human IL-8 when administered at a dose of 2 mg / kg, its effects may be attenuated when administered at a high dose of about 8 mg / kg. On the other hand, H553 / L118 can provide the maximum human IL-8 depletion effect even when administered at a high dose of 8 mg / kg.

(12-3) Assessment of stability using H553 / L118

It has been demonstrated that H553 / L118 is an antibody that can accelerate the elimination of human IL-8 from mice more than H89 / L118. However, in order for this antibody to retain its inhibitory effect on human IL-8 for a long period of time in vivo, it is also important that the IL-8 neutralizing activity remains stable (stability of the IL-8 neutralizing activity of this antibody) over a period of time. as long as the administered antibody is present in vivo (eg, in plasma). Therefore, the stability of these antibodies in mouse plasma was assessed using the method described below.

Mouse plasma was collected from the blood of C57BL / 6J mice (Charles River) using a method known in the art. 200 μl of 200 mM PBS (Sigma, P4417) was added to 800 μl of mouse plasma to make a volume of 1 ml. In addition, sodium azide was added to a final concentration of 0.1% as an antiseptic. Then added to the above plasma each of the antibodies (Hr9, H89 / L118 and H553 / L118) to a final concentration of 0.2 mg / ml. At this point in time, a portion of the sample was taken as an initial sample. The rest of the sample was stored at 40 ° C. After storage for one week and two weeks, a portion of each sample was taken and used as a sample stored for one week and a sample stored for two weeks. All samples were frozen at -80 ° C and stored until analysis.

The anti-IL-8 antibodies contained in mouse plasma were then analyzed for their neutralizing human IL-8 activity as follows: CXCR1 and CXCR2 are known receptors for human IL-8. PathHunter®, CHO-K1 CXCR2 β-arrestin cell line (DiscoveRx Co., catalog number 93-0202C2) expresses human CXCR2 and is a cell line engineered to emit electrochemiluminescence when mediated by human IL-8 signals. The human IL-8 neutralizing activity possessed by an anti-human IL-8 antibody can be assessed (but not limited to) using the cell. When human IL-8 is added to a cell culture solution, a certain level of chemiluminescence is emitted, which depends on the concentration of added human IL-8. When human IL-8 and anti-human IL-8 antibody are added together to a culture solution, human IL-8 signal transduction can be blocked by binding of anti-human IL-8 antibody to human IL-8. As a result, the chemiluminescence caused by the addition of human IL-8 will be inhibited by the anti-human IL-8 antibody and the chemiluminescence should be weaker than when no antibody is added, or there may be no chemiluminescence at all. Therefore, when the human IL-8 neutralizing activity possessed by the antibody becomes stronger, the degree of chemiluminescence becomes weaker; and when the neutralizing activity of an antibody to human IL-8 becomes weaker, the degree of chemiluminescence becomes stronger.

The same is the case with the antibody, which was added to mouse plasma and stored for a certain period of time. If the neutralizing activity of the antibody in the mouse plasma does not change during storage, then the degree of chemiluminescence indicated above before and after storage should not change. On the other hand, in the case of an antibody whose neutralizing activity decreases upon storage in mouse plasma, the degree of chemiluminescence when using the stored antibody should increase as compared to that before storage.

The above cell line was then used to investigate whether the neutralizing activity of the antibody is retained during storage in mouse plasma. The cell line was first suspended in Cell Plating 0 AssayComplete ™ reagent and then plated into a 384-well plate at 5000 cells / well. One day after the start of cell culture, the experiment described below was performed to determine the concentration of human IL-8 to be added. Serially diluted human IL-8 solutions, which contained final concentrations of human IL-8 from 45 nM (400 ng / ml) to 0.098 nM (0.1 ng / ml), were added to the cell culture solution. The detection reagent was then added according to the protocol attached to the product and the relative level of chemiluminescence was determined using a chemiluminescence detector. On the basis of the obtained data, the reactivity of the cells towards human IL-8 was confirmed and the concentration of human IL-8 suitable for confirming the neutralizing activity of antibodies to human IL-8 was determined. In this case, the concentration of human IL-8 was adjusted to 2 nM.

Then, the mouse plasma added with the above antibody to human IL-8 was used to evaluate the neutralizing activity of the antibodies contained therein. Human IL-8 in the concentration defined above and mouse plasma containing the above anti-human IL-8 antibody were added to the cell culture. The amount of mouse plasma to be added was determined to contain graded concentrations of anti-human IL-8 antibody ranging from 2 μg / ml (13.3 nM) to 0.016 μg / ml (0.1 nM). The detection reagents were then added according to the protocol attached to the product, and the relative levels of chemiluminescence were determined using a chemiluminescence detector.

In this case, relative values were calculated, representing the relative levels of chemiluminescence for each concentration of antibody, taking the average relative level of chemiluminescence in the wells without human IL-8 and antibody added as 0%, and taking the average relative level of chemiluminescence in the wells to which only human IL-8, but no antibody added, over 100%.

The results of the human IL-8 inhibition assay using human CXCR2 expressing cells are shown in FIG. 25A, which shows the results obtained for the original sample (without preservative treatment in mouse plasma), in FIG. 25B, which shows the results obtained for samples stored at 40 ° C for one week, and in Fig. 25B, which shows the results obtained with samples stored at 40 ° C for two weeks.

The results indicate that there was no difference between Hr9 and H89 / L118 in the neutralizing activity of human IL-8 before and after storage in mouse plasma. On the other hand, H553 / L118 showed a decrease in human IL-8 neutralizing activity after storage for two weeks. Therefore, the human IL-8 neutralizing activity of H553 / L118 is rapidly reduced in mouse plasma compared to that of Hr9 and H89 / L118, and H553 / L118 was found to be an antibody having unstable properties in terms of IL-8 neutralizing activity.

Example 13

Preparation of antibodies with a reduced score of predicted immunogenicity calculated using the in silico system

(13-1) Predicted immunogenicity score of various IL-8 binding antibodies

The formation of antibodies to drugs (ADA) affects the efficacy and pharmacokinetic characteristics of therapeutic antibodies and leads in some cases to serious side effects; and therefore, the clinical utility and drug efficacy of therapeutic antibodies can be limited by the formation of ADA. It is known that the immunogenicity of therapeutic antibodies is influenced by many factors and, in particular, the importance of effector T-cell epitopes in therapeutic antibodies has been noted many times.

In silico "tools" have been developed for predicting T-cell epitopes, such as Epibase (Lonza), iTope / TCED (Antitope), and EpiMatrix (EpiVax). Using these “tools” in silico, one can predict T-cell epitopes in any amino acid sequence (Walle et al., Expert Opin. Biol. Ther. 7 (3), 2007, ee. 405-418) and the potential immunogenicity of therapeutic antibodies.

EpiMatrix was used to calculate the immunogenicity score of the evaluated Fc variants. EpiMatrix is a system for predicting the immunogenicity of a protein of interest based on the automatic construction of sequences of peptide fragments by sectioning the amino acid sequence of the protein for which it is required to predict its immunogenicity into nine amino acids and then calculating their ability to bind to the eight main alleles of GCS class II (DRB1 * 0101, DRB1 * 0301, DRB1 * 0401, DRB1 * 0701, DRB1 * 0801, DRB1 * 1101, DRB1 * 1301 and DRB1 * 1501) (Clin. Immunol. 131 (2), 2009, e. 189-201).

The immunogenicity scores of the heavy chains and light chains of each anti-IL-8 antibody, which were calculated according to the method described above, are presented in Table 19 under the column entitled "EpiMatrix Score". In addition, the column entitled "tReg Adjusted Epx Score" presents the immunogenicity scores reported in the EpiMatrix Score, which have been corrected for the Tregitope content. Tregitope is a peptide fragment sequence present in large amounts mainly in naturally occurring antibody sequences and is a sequence that is believed to inhibit immunogenicity by activating regulatory T cells (Treg).

In addition, for these points, the column entitled "Sum" presents the sum of the points for the heavy and light chains.

According to these results, data reported in both the EpiMatrix Score and the tReg Adjusted Epx Score suggest that the immunogenicity scores for H89 / L118, H496 / L118, and H553 / L118 are lower than those for hWS. -4, which is a known humanized anti-human IL-8 antibody.

In addition, using the EpiMatrix, the predicted rate of ADA formation for the antibody molecule as a whole can be compared by comparing the heavy chain and light chain scores with the actual rate of ADA formation due to various commercially available antibodies. The results of this analysis are shown in FIG. 26. Due to the limitations of the system, FIG. 26 uses the following designations: “WS4” for hWS-4, “HR9” for Hr9, “H89L118” for H89 / L118, “H496L118” for H496 / L118, and “H553L118” for H553 / L118.

It is known that the frequency of ADA formation in the human body for various commercial antibodies is 45% for campata (alemtuzumab), 27% for rituxan (rituximab) and 14% for zenapax (daclizumab), which are shown in FIG. 26. On the other hand, while the frequency of ADA formation predicted from the amino acid sequences was 10.42% for hWS-4, which is a known humanized anti-human IL-8 antibody, the frequencies for the newly identified antibodies H89 / L118 (5.52%), H496 / L118 (4.67%) or H553 / L118 (3.45%) were significantly lower compared to the incidence for hWS-4.

(13-2) Obtaining modified antibodies with reduced scores of predicted immunogenicity

As indicated above, the immunogenicity scores for H89 / L118, H496 / L118, and H553 / L118 were lower than for hWS-4; however, as shown in Table 19, the heavy chain immunogenicity scores are greater than the light chain scores, suggesting that there is still room for improvement in the heavy chain amino acid sequences, primarily in terms of immunogenicity. Therefore, a search was performed for amino acid modifications of the variable region of the heavy chain H496, which can reduce the score of immunogenicity. A careful search yielded three variants, H496v1, in which the alanine at position 52c according to Cabot's numbering was replaced by aspartic acid, H496v2, in which glutamine at position 81 was replaced by threonine, and H496v3, in which the serine at position 82b was replaced for aspartic acid. In addition, a variant H1004 was obtained containing all three of these modifications.

The results of calculating the points of immunogenicity using a method similar to the method described in example 13-1 are presented in table 20.

The three heavy chains, H496v1, H496v2 and H496v3, each containing one modification, had lower immunogenicity scores than H496. In addition, in the case of H1004, which contained a combination of the three modifications, a significant improvement in the immunogenicity score was achieved.

In this case, it was found that in addition to L118, L395 is a light chain suitable for use in combination with H1004. Therefore, when calculating the points of immunogenicity, both a combination with L118 and a combination with L395 were used. As shown in Table 20, H1004 / L118 and H1004 / L395, which are combinations of heavy chain and light chain, also had very low immunogenicity scores.

The frequency of ADA formation was then predicted for these combinations using a method similar to that described in Example 13-1. The results are shown in FIG. 27. FIG. 27 the following designations are used: "V1" for H496v1 / L118, "V2" for H496v2 / L118, "V3" for H496v3 / L118, "H1004L118" for H1004 / L118H "H1004L395" for H1004 / L395.

Surprisingly, it was found that H1004 / L118 and H1004 / L395, which had significantly lower immunogenicity scores, also had improved ADA rates, with a predicted value of 0%.

(13-3) Measurement of IL-8 binding affinity H1004 / L395

Received H1004 / L395, which is an antibody containing H1004-IgG1m (SEQ ID NO: 91) as the heavy chain and L395-k0MT (SEQ ID NO: 82) as the light chain. The binding affinity of H1004 / L395 for human IL-8 was measured according to the method described below using a BIACORE T200 device (GE Healthcare).

Used two of the following rolling buffers, measurements were performed at the appropriate temperatures: (1) 0.05% Tween20, 40 mm ACES, 150 mm NaCl, pH 7.4, 40 ° C; and (2) 0.05% Tween20, 40 mM ACES, 150 mM NaCl, pH 5.8, 37 ° C.

Protein A / G (PIERCE) in an appropriate amount was immobilized on a CM4 sensor chip (GE Healthcare) using the amine coupling method and antibodies of interest were captured. Then, a diluted human IL-8 solution or a rolling buffer (which was used as a reference solution) was injected to effect the interaction of antibodies captured on the sensor chip with human IL-8. One of the above solutions was used as a rolling buffer, and the corresponding buffers were also used to dilute human IL-8. To regenerate the sensor chip, we used 25 mM NaOH and 10 mM glycine-HC! (pH 1.5). The KD (M) for each antibody, characterizing binding to human IL-8, was calculated based on the association reaction rate constant k _on (1 / Mc) and the dissociation rate constant k _off (1 / s), which are the kinetic parameters calculated based on sensograms obtained during measurements. The BIACORE T200 Evaluation software (GE Healthcare) was used to calculate each parameter.

The measurement results are shown in Table 21. As compared with H89 / L118, H1004 / L395, having a lower score immunogenicity was equivalent value KD for human IL-8 at neutral pH, but higher values of k _off and KD at acidic pH; and it was found that it had the ability to quickly be separated by dissociation from IL-8 in the endosome.

Example 14

Preparation and Evaluation of Antibody H1009 / L395 with pH Dependent Binding to IL-8

(14-1) Preparation of various antibodies having pH-dependent binding to IL-8

Received H1004 / L395, which has a pH-dependent IL-8-binding ability, as well as a reduced score of immunogenicity, carrying out the assessment according to the method described in example 13. After that, a special study was carried out to obtain variants that had the indicated preferred properties, and stability in mouse plasma.

Based on H1004 / L395, the following modified antibodies were obtained by introducing various modifications.

A total of 36 types of antibodies were obtained by combining 18 types of heavy chains and the two types of light chains described above. For these antibodies, various evaluations were performed according to the method described below.

Binding affinities for human IL-8 under neutral and acidic pH conditions were measured by a method similar to that described in Example 13-3. Among the results obtained, Table 22 shows KD at pH 7.4 and KD and k _off at pH 5.8.

Then, using the method described below, the stability against binding of IL-8 was evaluated during storage of antibodies in PBS.

The corresponding antibodies were dialyzed overnight against D-PBS (Sigma-Aldrich) and then the concentration of each of the antibodies was adjusted to 0.1 mg / ml. At this point in time, some antibody samples were collected as parental samples. The remaining samples were stored at 50 ° C for one week and then collected as samples for an accelerated test at an elevated temperature.

Binding affinity for IL-8 was then measured by the BIACORE method using stock samples and accelerated samples at elevated temperature.

Binding levels of human IL-8 to modified antibodies were analyzed using a BIACORE T200 device (GE Healthcare). Measurements were carried out at 40 ° C using 0.05% Tween20, 40 mM ACES and 150 mM NaCl pH 7.4 as a rolling buffer.

Protein A / G (PIERCE) in an appropriate amount was immobilized on a CM4 sensor chip (GE Healthcare) using the amine coupling method and antibodies of interest were captured. Then, a diluted human IL-8 solution or a rolling buffer (which was used as a reference solution) was injected to effect the interaction of antibodies captured on the sensor chip with human IL-8. A rolling buffer was also used to dilute human IL-8. To regenerate the sensor chip, 25 mM NaOH and 10 mM glycine-HCl (pH 1.5) were used. The measured binding level of human IL-8 and the amount of antibodies captured at this binding level were calculated using the BIACORE T200 Evaluation software (GE Healthcare).

The number of human IL-8 binding per corresponding 1000 RU amount of captured antibody was calculated for the original samples and samples for accelerated testing at elevated temperature. In addition, the ratio of the binding level of human IL-8 for the parent samples to the binding level for the samples for the accelerated test at elevated temperature was calculated.

The resulting ratios of the binding level of human IL-8 for the parent samples to the binding level for the samples for the accelerated test at elevated temperature are also presented in Table 22.

Based on the above study, the antibody H1009 / L395 was obtained, which was an antibody containing H1009-IgG1m (SEQ ID NO: 92) as the heavy chain and L395-k0MT as the light chain.

As shown in Table 22, compared to H89 / L118, H1009 / L395 had a slightly higher binding affinity for human IL-8 at neutral pH, but, on the other hand, a lower binding affinity at acidic pH, which means that its pH the dependence was even stronger. In addition, when placed under stringent conditions such as 50 ° C in PBS, H1009 / L395 had slightly higher stability in binding to IL-8 compared to that of H89 / L118.

Therefore, H1009 / L395 was chosen as an antibody whose neutralizing activity in mouse plasma can be maintained at a stable level while maintaining its pH-dependent ability to bind IL-8.

(14-2) Assessment of stability H1009 / L395

Then, by a method similar to that described in Example 12-3, it was assessed whether the IL-8 neutralizing activity of H1009 / L395 remained stable in mouse plasma. This experiment used H1009 / L395-F1886s, which will be described in more detail in Example 19. This antibody had the same variable region as H1009 / L395, and its constant region had modifications that increase FcRn binding under acidic pH conditions, and modifications reducing its binding to FcγR, compared to native human IgG1. The variable region H1009 / L395, primarily the region around the HVR, is responsible for binding to human IL-8 and neutralizing IL-8 activity of this antibody, and it is believed that modifications introduced into the constant region do not affect these properties.

Evaluation of stability in mouse plasma was carried out as follows. 150 μl of 200 mM phosphate buffer (pH 6.7) was added to 585 μl of mouse plasma. Then sodium azide was added as an antiseptic at a final concentration of 0.1%. Each antibody (Hr9, H89 / L118 or H1009 / L395-F1886s) was added to the above mouse plasma at a final concentration of 0.4 mg / ml. At this point in time, a portion of the sample was taken as the original sample. The remaining sample was stored at 40 ° C. After 1 week and two weeks after the start of storage, a portion of each sample was taken and used as a sample stored for 1 week and a sample stored for 2 weeks. All samples were frozen at -80 ° C and stored until each analysis.

Measurement of neutralizing human IL-8 activity was carried out using human CXCR2 expressing cells using a method similar to that described in Example 12-3. However, the concentration of human IL-8 that was used to confirm the neutralizing activity of the anti-human IL-8 antibody in this experiment was 1.2 nM.

The results of the human IL-8 inhibition assay using the above antibodies and human CXCR2 expressing cells are shown in FIG. 28A showing the results for the original sample (which was not stored in mouse plasma), FIG. 28B, which shows the results for samples that were stored at 40 ° C for 1 week, and in FIG. 28B, which shows the results for samples that were stored at 40 ° C for 2 weeks.

As a result, it was unexpectedly found that the neutralizing activity of H1009 / L395-F1886s against human IL-8 persisted even after storage in mouse plasma at 40 ° C for 2 weeks, and the neutralizing activity of human IL-8 was more stable than in case H553 / L118.

(14-3) Analysis of PK in mice using H1009 / L395

The rate of elimination of human IL-8 by antibody H1009 / H395 in mice was assessed using the following method. H1009 / L395, H553 / L118 and H998 / L63 were used as antibodies. Administration to mice and collection of blood, as well as measurement of plasma concentration of human IL-8 in mice were performed using the method described in example 11.

The resulting plasma concentration of human IL-8 is shown in FIG. 29, and data on the value of clearance of human IL-8 (CL) from mouse plasma are presented in table 23.

As a result, it was found that the rate of elimination of human IL-8 from the body of mice when H1009 / L395 was administered at a dose of 2 mg / kg was equivalent to the rate of elimination when H553 / L118 was administered, and it was found that when H1009 / L395 was used, almost 100% was achieved - high level of free IL-8 in the endosome. It was found that the clearance (CL), which is a quantitative indicator of the rate of elimination of human IL-8 from the plasma of mice, is approximately 30 times higher than when using H998 / L63.

The effect of increasing the rate of elimination of human IL-8 can be explained as follows (but not limited to). As a rule, in a living organism, in which the concentration of antigens is maintained at approximately constant levels, the rate of formation and the rate of elimination of antigens should also be maintained at approximately constant levels. When antibodies are administered under such conditions, even in cases where the rate of antigen formation is not affected, the rates of antigen elimination may be altered by the formation of antigen-antibody complexes. As a rule, since the rate of antigen elimination is higher than the rate of antibody elimination, the rate of elimination of antigens that form complexes with the antibody decreases in such cases. When the rate of antigen elimination decreases, the concentration of antigen in plasma increases, but the level of increase in this case can also be determined as the ratio of the rate of elimination when the antigen is present in an unbound state and the rate of elimination when the antigen forms a complex. This means that, compared to the rate of elimination when the antigen is present in an unbound state, if the rate of elimination during complex formation is reduced by 1/10, then the concentration of the antigen in the plasma of the body to which the antibody is injected can increase up to about 10 times compared to concentration before antibody administration. In the context of the present description, clearance (CL) can be used as the rate of elimination. More specifically, the increase in antigen concentration (antigen accumulation) that occurs after the introduction of the antibody into the body can be determined by the CL of the antigen in each condition, i.e. before the administration of the antibody and after the administration of the antibody.

As used herein, the presence of an approximately 30-fold difference in CL of human IL-8 when administered with H998 / L63 and H1009 / L395 suggests that there may be an approximately 30-fold difference between the elevation levels of human IL-8 plasma concentration when these antibodies are administered to humans ... In addition, the creation of a 30-fold difference in plasma concentration of human IL-8 suggests that there may also be an approximately 30-fold difference in the amount of antibodies required to completely block the biological activity of human IL-8 under the appropriate conditions. This means that compared to H998 / L63, H1009 / L395 can block the biological activity of IL-8 in plasma using about 1/30 of the amount, which is a very small amount of antibody. In addition, when H1009 / L395 and H998 / L63 are administered individually to a human at the same dose, H1009 / L395 can block the biological activity of IL-8 for a longer period of time with greater potency. In order to block the biological activity of IL-8 for a long period of time, it is necessary for the IL-8 neutralizing activity to be stably maintained. As demonstrated in Example 14, experiments using mouse plasma indicate that H1009 / L395 can maintain human IL-8 neutralizing activity for an extended period of time. It has also been found that H1009 / L395, which has such important distinguishing properties, is an antibody with preferred effects in terms of efficacy in neutralizing IL-8 in vivo.

Example 15

Evaluation of binding to the extracellular matrix using the pH-dependent IL-8 binding antibody H1009 / L395

The very high (30-fold magnified) effect of H1009 / L395 on the elimination of human IL-8, described in example 14, was unexpected. It is known that the rate of antigen elimination upon administration of a pH-dependent antigen-binding antibody depends on the rate of absorption of the antibody-antigen complex by cells. This means that if the rate of absorption of the pH-dependent antigen-binding antibody by the cells increases during the formation of the antigen-antibody complex as compared to the variant without the formation of the complex, then the elimination of the antigen by the pH-dependent antibody may increase. Known methods for increasing the rate of uptake of an antibody by cells include a method for imparting FcRn binding capacity to an antibody under neutral pH conditions (WO 2011/122011), a method for increasing the binding capacity of an antibody for FcγR (WO 2013/047752), and a method that accelerates complex formation, containing a multivalent antibody and a multivalent antigen (WO 2013/081143).

However, the above techniques were not used for the H1009 / L395 constant regions. In addition, although IL-8 is known to form a homodimer, human IL-8 bound by H1009 / L395 has been found to be present in monomer form since H1009 / L395 recognizes the homodimer-forming surface of human IL-8. Therefore, this antibody cannot form multivalent complexes.

More specifically, although the above procedures were not applied to H1009 / L395, H1009 / L395 has a 30-fold increased human IL-8 elimination activity.

As a result, when creating the invention, it was considered as a possible factor that can determine the above properties of pH-dependent IL-8-binding antibodies, of which H1009 / L395 is a representative. However, the discussion below describes only the possibility suggested by the applicants based on the prior art, and the essence of section B of the description is not limited to the essence of the discussion presented below.

Human IL-8 is a protein with a high isoelectric point (p1), and the theoretical isoelectric point calculated using a known method is about 10. This means that under neutral pH conditions, human IL-8 is a protein whose charge is shifted to side of positive values. pH-dependent IL-8-binding antibodies, of which H1009 / L395 is a representative, are also proteins whose charge is shifted towards positive values, and the theoretical isoelectric point of H1009 / L395 is about 9. This means that the isoelectric point of the complex formed by binding H1009 / L395, i.e. a protein that has a high isoelectric point and is initially rich in positive charges, with human IL-8, which has a high isoelectric point, should be higher than that of H1009 / L395 in an unbound state.

As described in Example 3, it can be assumed that an increase in the isoelectric point of an antibody, which includes an increase in the number of positive charges and / or a decrease in the number of negative charges on the antibody, should increase the nonspecific uptake of the antibody-antigen complex by the cells. The isoelectric point of the complex formed between the anti-IL-8 antibody and human IL-8, which has a high isoelectric point, is higher than the isoelectric point of the anti-IL-8 antibody in the unbound state, and the complex can be absorbed more rapidly by cells.

As previously described, the affinity for the extracellular matrix is also a factor that can influence cellular uptake. Therefore, in the creation of the invention, it was determined whether there is a difference in the ability to bind the extracellular matrix between the antibody in the unbound state and the complexed antibody to human IL-8.

Evaluation of the amount of antibody that binds to the extracellular matrix. using the ECL method (electrochemiluminescence)

Extracellular matrix (BD Matrigel Basement Membrane Matrix;

manufactured by BD) was diluted to a concentration of 2 mg / ml using TBS (Takara). The diluted extracellular matrix was added to a 96-well plate MULTI-ARRAY, High bind, Bare (manufactured by Meso Scale Discovery: MSD) at 5 μl per well and immobilized overnight at 4 ° C. Then, blocking was carried out with 20 mM ACES buffer, pH 7.4, containing 150 mM NaCl, 0.05% Tween 20, 0.5% BSA and 0.01% NaN ₃ .

Antibodies to be evaluated were prepared as follows. Antibody samples to be individually added were prepared by diluting each antibody to a concentration of 9 μg / ml using buffer 1 (20 mM ACES buffer containing 150 mM NaCl, 0.05% Tween20 and 0.01% NaN ₃ , pH 7, 4), and then additionally diluted them with buffer 2 (20 mM ACES buffer containing 150 mM NaCl, 0.05% Tween20, 0.1% BSA, and 0.01% NaN ₃ , pH 7.4) to a final concentration of 3 μg / ml.

On the other hand, antibody samples to be complexed with human IL-8 were prepared by adding human IL-8 at a 10-fold molar concentration relative to the antibody to the antibody sample, then diluting each antibody with Buffer-1 to obtain a concentration antibodies 9 μg / ml, respectively, and then additionally diluted each of them using buffer-2 to a final concentration of the antibody 3 μg / ml. The concentration of human IL-8 was approximately 0.6 μg / ml. The mixture was shaken at room temperature for 1 hour to form a complex.

Then, diluted solutions of only antibodies or antibodies in the complex were added to the plate, from which the blocking solution was removed, and the plate was shaken at room temperature for 1 hour. Then, after removing the solution containing unbound antibody or the solution containing the complex, buffer was added. 1 containing 0.25% glutaraldehyde. Then, after allowing the plate to stand for 10 minutes, it was washed with D-PBS (manufactured by Wako Pure Chemical Industries) containing 0.05% Tween 20. Antibodies for ECL detection were obtained by conjugation with sulfo-tagged goat anti-human IgG (gamma) (manufactured from Zymed Laboratories) using a sulfo-tagged NHS ester (manufactured by MSD). Antibodies for ECL detection were diluted in buffer-2 to a concentration of 1 μg / ml, added to the plate and then shaken in the dark at room temperature for 1 hour. Antibodies for ECL detection were removed and a 2-fold diluted solution obtained by dilution buffer for reading MSD Read Buffer T (4x) (manufactured by MSD) with ultrapure water; and then the luminescence level was measured with a SECTOR Imager 2400 (manufactured by MSD).

The results are shown in FIG. 30. It is important to note that all antibodies to IL-8, such as H1009 / L395, were characterized by a weak ability to bind to the extracellular matrix in the form of unbound antibody (-IL8), but bound to the extracellular matrix after complexing with human IL-8 (+ hIL8).

As described above, the ability of antibodies to IL-8 to acquire affinity for the extracellular matrix by binding to human IL-8 has not been elucidated. In addition, the combination of these properties with the pH-dependent binding of IL-8-binding antibodies (but not limited to only specified) allows you to increase the rate of IL-8 elimination more effectively.

Example 16

Analysis of PK in mice using non-FcRn-binding antibodies

The method below was used to confirm the formation of a complex between human IL-8 and a pH-dependent IL-8 binding antibody and to increase the uptake of this complex by cells in mice.

First, an antibody variant containing the H1009 / L395 variable region and an Fc region with a deficiency in binding affinity to various Fc receptors was obtained. In particular, as modifications designed to eliminate the ability to bind to human FcRn under acidic pH conditions, the heavy chain of H1009-IgG1 was replaced with alanine isoleucine at position 253 and aspartic acid of serine at position 254 according to EU numbering. In addition, as modifications to eliminate binding to murine FcγRs, the leucine at position 235 was replaced with arginine, the glycine at position 236 was replaced with arginine, and the series at position 239 was replaced with lysine. Received H1009-F1942m (SEQ ID NO: 93) as a heavy chain containing these four modifications. In addition, an antibody H1009 / L395-F 1942m was obtained containing H1009-F1942m as the heavy chain and L395-k0MT as the light chain.

Since an antibody that has this Fc region is deficient in binding affinity to FcRn under acidic pH conditions, it is not transferred from the endosome to the plasma. Therefore, the specified antibody is rapidly eliminated from plasma in a living organism relative to an antibody that contains the native Fc region. In this case, after absorption by cells of antibodies that contain the native Fc region, only part of them that is not saved by FcRn is cleaved after transfer to the lysosome, but in the case of antibodies containing an Fc region that does not have binding affinity to FcRn, all antibodies taken up by cells are cleaved in lysosomes. More specifically, in the case of antibodies that contain said modified Fc region, the rate of elimination of the administered antibody from the plasma may be equivalent to the rate of incorporation into cells. This means that the rate of intracellular uptake of an antibody whose binding affinity to FcRn is abolished can also be determined by measuring the rate of elimination of these antibodies from plasma.

Next, it was determined whether the intracellular uptake of the complex formed between H1009 / L395-F1942m and human IL-8 increased compared to the uptake of uncomplexed H1009 / L395-F1942m. In particular, it was determined whether the rate of elimination of the antibody from plasma would change when the antibody was administered alone and when the antibody was administered after complexation with human IL-8.

The corresponding biokinetics of the anti-human IL-8 antibody was assessed when the anti-human IL-8 antibody was administered to individually transgenic human FcRn mice (B6.mFcRn - / -. HFcRn Tg mouse line 32 + / +; Jackson Laboratories; Methods Mol Biol. 602, 2010, pp. 93-104) and when human IL-8 and an anti-human IL-8 antibody were administered simultaneously to transgenic human FcRn mice. A solution of antibodies to human IL-8 (200 μg / ml) and a solution containing a mixture of human IL-8 (10 μg / ml) and antibodies to human IL-8 (200 μg / ml) were injected individually once at a dose of 10 ml / kg into the tail vein. In this case, since the anti-human IL-8 antibody was present in substantial excess relative to human IL-8, almost all of the human IL-8 should bind to the antibody. Blood samples were obtained at 5 min, 2 h, 7 h, 1 day and 2 days after administration. The collected blood was immediately centrifuged at 4 ° C and 15,000 rpm for 15 min to obtain plasma. The separated plasma was stored in a freezer at -20 ° C or lower until measurements were taken.

Plasma concentrations of antibodies to human IL-8 in mice were measured by electrochemiluminescence. First, the biotinylated goat anti-human Ig kappa light chain antibody (IBL) was allowed to react with a streptavidin-coated Gold Multi-ARRAY plate (Meso Scale Discovery), which had been pre-blocked overnight at room temperature using a PBS-Tween solution containing 5 % (w / v) BSA, at room temperature for 1 hour to obtain a plate with an immobilized anti-human antibody. Samples for the calibration curve were prepared containing anti-human IL-8 antibody at concentrations of 3.20, 1.60, 0.800, 0.400, 0.200, 0.100 and 0.0500 μg / ml in plasma, and samples for measurement in mouse plasma, diluted in 100 times or more. Each sample was mixed with human IL-8 and then added to the immobilized anti-human antibody plate at 50 μl per well and then stirred at room temperature for 1 hour. The final human IL-8 concentration was adjusted to 333 ng / ml.

Then, an antibody to human IL-8 (obtained in our laboratory) containing a constant region of mouse IgG was added to the plate and allowed to react at room temperature for 1 hour. In addition, anti-mouse IgG (BECKMAN COULTER) labeled with ruthenium was added to the plate. , using a sulfo-tagged NHS ester (Meso Scale Discovery) and allowed to react for 1 hour. Then, immediately after the addition of the reading buffer T (x1) to the plate (Meso Scale Discovery), measurement was performed using a SECTOR Imager 2400 (Meso Scale Discovery). Anti-human IL-8 antibody concentration was calculated from the response using a calibration curve using SOFTmax PRO analytical software (Molecular Devices).

The resulting antibody concentration in mouse plasma is shown in FIG. 31, and data on antibody clearance under appropriate conditions are presented in Table 24.

It was found that the rate of intracellular absorption of the H1009 / L395-F1942m complex and human IL-8 increased by at least 2.2 times compared to the absorption rate of H1009 / L395-F1942m. It should be noted that in the context of the present description, "at least 2.2 times" is indicated, since for the following reason, the possibility that the value may actually be 5, 10, or 30 times is not excluded. Since the rate of elimination of human IL-8 from mouse plasma is very rapid compared to the rate of elimination of H1009 / L395-F1942m, the proportion of H1009 / L395-F1942m bound to human IL-8 in plasma rapidly decreases after administration. More specifically, even when administered concurrently with human IL-8, not all of the H1009 / L395-F1942m present in plasma is bound to human IL-8, and in fact, approximately 7 hours after administration, most of them are already in free form. Since the uptake rate was investigated under these conditions, even if the rate of intracellular uptake of the H1009 / L395-F1942m complex and human IL-8 was actually increased 5, 10 or 30 times compared to the uptake rate of H1009 / L395-F1942m, the results obtained with using this experimental system reflect this only partially; therefore, there is a possibility that the effect could be increased by more than 2.2 times or so on. Thus, based on the results obtained, according to which the rate of intracellular uptake of the complex H1009 / L395 and IL-8, as found, should exceed the actual rate of intracellular uptake of H1009 / L395 in vivo, this effect is not limited to the resulting increase in 2.2 times.

The data obtained can be interpreted as follows (but not limited only to the indicated). When H1009 / L395, which is a pH dependent IL-8 binding antibody, forms a complex with human IL-8, the complex has a higher isoelectric point and a greater positive charge than the unbound antibody. At the same time, the affinity of the complex for the extracellular matrix is higher than the affinity of the antibody in the unbound state. Properties such as an increase in the isoelectric point and an increase in binding to the extracellular matrix can be considered as factors that promote the uptake of the antibody by cells in vivo. In addition, in experiments carried out on mice, it was found that the intracellular uptake of the complex H1009 / L395 and human IL-8 increased 2.2 times or more compared to the uptake rate of H1009 / L395. Based on the foregoing, the theoretical explanation, as well as the in vitro properties and phenomena found in vivo, strongly support the hypothesis that H1009 / L395 and human IL-8 form a complex, which promotes the absorption of the complex by cells and leads to a significant increase in the elimination of human IL -8.

To date, several antibodies to IL-8 are known, but there are no data on an increase in the binding affinity to the extracellular matrix after complexation with IL-8 and an increase in the absorption of the complexes by cells.

In addition, based on the data obtained on the increase in intracellular uptake of antibodies to IL-8, when antibodies form complexes with IL-8, it can be assumed that antibodies to IL-8, which can form complexes with IL-8 in plasma, are rapidly absorbed by cells and antibodies in a free state that cannot complex with IL-8 tend to persist in plasma without being absorbed by cells. In the case when the antibody to IL-8 is pH-dependent, the antibody to IL-8, taken up by the cells, releases the IL-8 molecule in the cells and then returns to the environment outside the cells and can then bind to another IL-8 molecule; and hence the increase in intracellular uptake after complexation may have the additional effect of more potent elimination of IL-8. This means that the selection of antibodies to IL-8 with increased ability to bind to the extracellular matrix or antibodies to IL-8 with increased uptake by cells may also be another of the embodiments of the invention indicated in section B of the description.

Example 17

Prediction of the immunogenicity of the pH-dependent IL-8-binding antibody H1009 / L395 using the in silico system

Further, the immunogenicity score and the frequency of ADA formation were predicted for H1009 / L395 using a method similar to that described in example 13-1.

The results are shown in Table 25 and FIG. 32. FIG. 32 H1009 / L395 is designated "H1009L395".

The results presented in Table 25 demonstrate that H1009 / L395 has the same immunogenicity score as H1004 / L395. In addition, the frequency of ADA formation predicted for H1009 / L395 based on the results shown in FIG. 32 was 0%, and this is also similar to the value of this parameter indicated for H1004 / L395.

Thus, the predicted immunogenicity was significantly reduced in H1009 / L395 compared to the known anti-human IL-8 antibody hWS-4. Therefore, it can be assumed that H1009 / L395 should have very low immunogenicity in humans and should be able to maintain stable neutralizing activity against IL-8 for a long period of time.

Example 18

PK analysis in the body of cynomolgus monkeys using the H89 / L118 variant with an increased FcRn-binding capacity under acidic pH conditions

As described in the above examples, among antibodies that have the constant region of native IgG1 as their constant region, the pH dependent IL-8 binding antibody H1009 / L395 was an antibody with improved properties. However, these antibodies can also be used as antibodies containing amino acid substitutions in the constant region, for example, containing an Fc region with increased ability to bind to FcRn at acidic pH, examples of which are presented in example 5. Thus, H89 / L118 was used to confirm that that an Fc region with an increased ability to bind to FcRn at acidic pH can also function in a pH dependent IL-8 binding antibody.

(18-1) Preparation of H89 / L118 with a modified Fc region with increased ability to bind FcRn at acidic pH

Various modifications that increase FcRn binding described in Example 5-1 were introduced into the Fc region of H89 / L118. In particular, the following variants were obtained by introducing the modifications used in F1847m, F1848m, F1886m, F1889m, F1927m and F1168m into the Fc region of H89-IgG1: (a) H89 / L118-IgG1 construct containing H89-IgG1m (SEQ ID NO : 94) as the heavy chain and L118-K0MT as the light chain; (b) H89 / L118-F1168m containing H89-F1168m (SEQ ID NO: 95) as the heavy chain and L118-K0MT as the light chain; (c) H89 / L118-F1847m containing H89-F1847m (SEQ ID NO: 96) as the heavy chain and L118-K0MT as the light chain; (d) H89 / L118-F1848m containing H89-F1848m (SEQ ID NO: 97) as the heavy chain and L118-K0MT as the light chain; (e) H89 / L118-F1886m containing H89-F1886m (SEQ ID NO: 98) as the heavy chain and L118-K0MT as the light chain; (e) H89 / L118-F1889m containing H89-F1889m (SEQ ID NO: 99) as the heavy chain and L118-K0MT as the light chain; and (g) H89 / L118-F1927m containing H89-F1927m (SEQ ID NO: 100) as the heavy chain and L118-K0MT as the light chain. PK assays in cynomolgus monkeys using these antibodies were performed using the method described below.

(18-2) PK assays in cynomolgus monkeys using antibodies containing a new variant of the Fc region

After administration of antibodies to human IL-8 to cynomolgus monkeys, the biokinetics of antibodies to human IL-8 was evaluated. The anti-human IL-8 antibody solution was administered intravenously once at a dose of 2 mg / kg. Blood samples were obtained at 5 minutes, 4 hours, 1 day, 2 days, 3 days, 7 days, 10 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, and 56 days after administration. The collected blood was immediately centrifuged at 4 ° C and 15000 rpm for 10 min to obtain plasma. The separated plasma was stored in a freezer at -60 ° C or below, until measurements were taken.

Plasma concentrations of antibodies to human IL-8 in cynomolgus monkeys were measured by electrochemiluminescence. First, the capture Ab to the human kappa chain (h-kappa) (Antibody Solutions) was introduced into a 96-well MULTI-ARRAY plate (Meso Scale Discovery) and stirred at room temperature for 1 hour. Then the PBS-Tween solution containing 5% (w / v) BSA was used for blockade for 2 hours to obtain a plate with an immobilized anti-human antibody. Samples for the calibration curve were prepared containing anti-human IL-8 antibody at concentrations of 40.0, 13.3, 4.44, 1.48, 0.494, 0.165 and 0.0549 μg / ml in plasma, and samples were prepared for measurement in cynomolgus monkey plasma, diluted 500 times or more. 50 μl of solutions were added to each well of the plate with immobilized anti-human antibody and the solutions were stirred at room temperature for 1 hour. for 1 h. After additional addition of sulfo-labeled streptavidin (Meso Scale Discovery) and incubation for 1 h at room temperature, to ensure the reaction proceeded, read buffer T (× 1) (Meso Scale Discovery) was added to the plate and immediately measurement was carried out using a SECTOR Imager 2400 (Meso Scale Discovery). Anti-human IL-8 antibody concentration was calculated from the response using a calibration curve using SOFTmax PRO analytical software (Molecular Devices).

The results obtained, characterizing the half-life (t1 / 2) and clearance (CL) of each of the antibodies, are shown in Table 26, and changes in the concentration of antibodies in the plasma of cynomolgus monkeys are shown in FIG. 33.

The above results confirmed that all Fc region variants had a prolonged plasma retention time compared to an antibody having a native IgG1 Fc region. In particular, for H89 / L118-F1886m, the most preferred kinetics in blood was found

Example 19

Fc region with reduced binding capacity for FcγR

It is known that the Fc region of native human IgG1 binds to Fcγ receptor (s) (hereinafter referred to as FcγR) on various cells of the immune system and has effector functions, such as ADCC and ADCP, against target cells.

On the one hand, IL-8 is a soluble cytokine, and anti-IL-8 antibodies used as pharmaceuticals are expected to primarily have pharmacological actions by neutralizing IL-8 functions in areas where IL-8 is present in excess. quantity. These areas in which IL-8 is present in excess are not limited to specific areas, and, for example, may represent areas of inflammation. As is well known in these areas of inflammation, immune cells are collected and activated. Transmission of unwanted activation signals to these cells via Fc receptors and the induction of activity such as ADCC and ADCP in unwanted cells are not always necessary. Therefore, from a safety point of view, but not limited to this, it may be preferable that antibodies to IL-8 administered in vivo have a low affinity for FcγR.

(19-1) Production of modified antibodies with reduced ability to bind to FcγR

Amino acid modifications were additionally introduced into the Fc region of H1009 / L395-F1886m to reduce the ability to bind to various human and monkey cynomolgus FcγRs. In particular, H1009-F1886s (SEQ ID NO: 81) was prepared by performing each of the following substitutions in the heavy chain H1009-F1886m: an L to R substitution at position 235, a G to R substitution at position 236, and an S to K substitution at position 239 according to EU numbering. Similarly, H1009-F1974m (SEQ ID NO: 80) was prepared by replacing L with R at position 235 in H1009-F1886m and replacing G with R at position 236 according to EU numbering and replacing a region extending from position 327 to position 331 according to EU-numbering in the sequence of native human IgG4. H1009 / L395-F1886s and H1009 / L395-F1974m were prepared as antibodies having the indicated heavy chains and L395-k0MT as the light chain.

(19-2) Confirmation of affinity for various human FcγR

Then, the affinity of H1009 / L395-F1886s or H1009 / L395-F1974m for soluble forms of human or monkey cynomolgus FcγRIa or FcγRIIIa was confirmed by the following method.

Assays to assess the binding of H1009 / L395-F1886s or H1009 / L395-F1974m to soluble forms of human or monkey cynomolgus FcγRIa or FcγRIIIa were performed using the BIACORE T200 device (GE Healthcare). Soluble FcγRIa and FcγRIIIa from both human and monkey cynomolgus were prepared in the form of His-tagged molecules using methods known to those of ordinary skill in the art. A suitable amount of recombinant protein (r-protein) L (BioVision) was immobilized on a CM4 sensor chip (GE Healthcare) using the amine coupling method and the antibody of interest was captured. The soluble FcγRIa or FcγRIIIa was then injected using a rolling buffer (used as a reference solution) and allowed to react with antibodies captured on the sensor chip. HBS-EP + (GE Healthcare) was used as a rolling buffer and HBS-EP + was used to dilute soluble FcγRIa or FcγRIIIa. To regenerate the sensor chip, 10 mM glycine-HCl, pH 1.5 was used. All measurements were carried out at 20 ° C.

The results are shown in FIG. 34. As used herein, the following notation system has been used for human FcγRIa, human FcγRIIIa, cynomolgus monkey FcγRIa, and cynomolgus monkey FcγRIIIa: hFcγRIa, hFcγRIIIa, cynoFcγRIa and cynoFcγRIIIa, respectively. It was found that H1009 / L395-F1886m binds to all FcγRs, but, on the other hand, it is confirmed that H1009 / L395-F1886s and H1009 / L395-F1974m do not bind to any of the FcγRs.

(19-3) Analysis of the elimination of IL-8 from the body of mice using Fc variants

Further, for H1009 / L395-F1886s and H1009 / L395-F1974m, the rate of elimination of human IL-8 and the retention time of antibodies in the plasma of mice were determined using the following experiment. In this case, three doses of H1009 / L395-F1886s of 2 mg / kg, 5 mg / kg and 10 mg / kg were used for evaluation in order to evaluate also the dose-escalating effects of H1009 / L395-F1886s.

After simultaneous administration of human IL-8 and anti-human IL-8 antibody to human FcRn transgenic mice (B6.mFcRn - / -. HFcRn Tg mouse line 32 + / +; Jackson Laboratories; Methods Mol. Biol. 602, 2010, cc. 93-104), evaluated the biokinetics of human IL-8. A solution containing a mixture of human IL-8 (10 μg / ml) and antibodies to human IL-8 (200, 500, or 1000 μg / ml) was injected once at a dose of 10 ml / kg into the tail vein. In this case, since the anti-human IL-8 antibody was present in substantial excess relative to human IL-8, almost all of the human IL-8 should bind to the antibody. Blood samples were obtained at 5 minutes, 2 hours, 7 hours, 4 hours, 1 day, 2 days, 3 days, 7 days, 14 days, 21 days, and 28 days after administration. The collected blood was immediately centrifuged at 4 ° C and 15,000 rpm for 15 min to obtain plasma. The separated plasma was stored in a freezer at -20 ° C or lower until measurements were taken.

Plasma concentrations of human IL-8 in mice were measured using a method similar to that described in Example 11. The resulting data on plasma concentrations of human IL-8 in mice are shown in FIG. 35, and data on the clearance of human IL-8 (CL) from mouse plasma are presented in Table 27.

First, it was found that the antibody H1009 / L395 containing the Fc region of native IgG1 and H1009 / L395-F1886s containing the modified Fc region had an equivalent ability to eliminate human IL-8 when comparing the groups to which the antibodies were administered at a dose of 2 mg / kg.

In addition, it was found that when the dose of antibody H1009 / L395-F1886s was changed, no significant differences were found in the levels of human IL-8 clearance between doses of 2 mg / kg and 10 mg / kg, however, a small change in the concentration of IL-8 in plasma was detected through 1 day after administration. These results suggest with a high degree of confidence that antibodies containing the variable region H1009 / L395 have a sufficient ability to eliminate IL-8 when antibodies are administered at high doses.

(19-4) Analysis of PK Fc variants in cynomolgus monkeys

Next, the retention time of antibodies in the plasma of cynomolgus monkeys was determined using the following method using H1009 / L395-F1886s or H1009 / L395-F1974m.

The biokinetics of the anti-human IL-8 antibody was assessed in the case where the anti-human IL-8 antibody was administered individually, and in the case where the human IL-8 and the anti-human IL-8 antibody were administered simultaneously to cynomolgus monkeys. A solution of antibodies to human IL-8 (2 mg / ml) or a solution containing a mixture of human IL-8 (100 μg / kg) and antibodies to human IL-8 (2 mg / kg) was injected intravenously once at a dose of 1 ml / kg. Blood samples were obtained at 5 minutes, 4 hours, 1 day, 2 days, 3 days, 7 days, 10 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, and 56 days after administration. The collected blood was immediately centrifuged at 4 ° C and 15000 rpm for 10 min to obtain plasma. The separated plasma was stored in a freezer at about -60 ° C or below, until measurements were taken.

Plasma concentration of anti-human IL-8 antibody in cynomolgus monkeys was measured using the method described in Example 18. The resulting plasma anti-human IL-8 antibody data are shown in FIG. 36, and data characterizing the half-life (t1 / 2) and clearance (CL) of antibodies to human IL-8 from cynomolgus monkey plasma are presented in Table 28.

First, compared to Hr9 and H89 / L118, which have a native human IgG1 Fc region, H1009 / L395-F1886s, which has an improved Fc region, has been found to have a significantly prolonged plasma retention time.

In addition, when H1009 / L395-F1886s were administered concurrently with human IL-8, the change in plasma concentration was equivalent to the change when the antibody was administered alone. Based on the data obtained, the following can be assumed (but not limited to only those indicated). As described above, the intracellular uptake of the H1009 / L395 complex and human IL-8 was found to be higher than the uptake of the uncomplexed H1009 / L395. As a rule, it is believed that high molecular weight proteins can be incorporated into cells in a non-specific or receptor-dependent way, and then transferred to the lysosome and cleaved by various cleavage enzymes present in the lysosome. Therefore, if the rate of absorption of a protein into cells is increased, then the retention time of this protein in plasma is likely to deteriorate accordingly. However, the antibody has the ability to be conserved in plasma by FcRn in the endosome; and therefore, as long as FcRn rescue functions are maintained at a sufficient level, plasma retention time is not affected, even if the rate of intracellular uptake is accelerated. In this case, even when H1009 / L395-F1886s was administered simultaneously with human IL-8 cynomolgus monkeys, no effect on plasma retention time was found. This suggests the possibility that although the level of uptake of the H1009 / L395-F1886s antibody into cells is increased, the antibody is sufficiently rescued by the FcRn so that it can be released into the plasma.

In addition, for another Fc variant, H1009 / L395-F1974m, the plasma retention time equivalent to H1009 / L395-F1886s was also established. Although various modifications have been introduced into these Fc variants that reduce the ability to bind to different FcγRs as described above, they have not been found to affect the plasma retention time of the antibodies themselves. It follows from the above that, as established, the retention time in the plasma of cynomolgus monkeys of both antibodies H1009 / L395-F1886s and H1009 / L395-F1974m is significantly prolonged and is reasonably acceptable in comparison with antibodies that have the Fc region of native IgG1 ...

As demonstrated in the examples described above, due to its pH-dependent ability to bind to IL-8, coupled with rapid uptake by cells as a complex with IL-8, H1009 / L395 is the first described antibody with a significantly increased rate of elimination of human IL-8 in vivo. In addition, the binding affinity for IL-8 of this antibody under neutral pH conditions is also increased compared to the known antibody hWS-4, and the antibody can neutralize human IL-8 more strongly under neutral pH conditions corresponding to plasma conditions. In addition, it is an antibody with very high stability under plasma conditions, and in which IL-8 neutralizing activity is not reduced after in vivo administration. In addition, the Hr9 engineered H1009 / L395, which has a significantly improved production yield compared to hWS-4, is an antibody suitable for preparation from a production outlet. In addition, in silico prediction of immunogenicity for an antibody, a very low immunogenicity score was established, and this score is significantly lower compared to the known hWS-4 antibody and some other known commercially available antibodies. This means that, with a high degree of probability, it can be expected that H1009 / L395 will have a weak ability to form ADA in the human body and can be safely used for a long period of time. Thus, in comparison with the known antibodies to human IL-8, H1009 / L395 has improved qualities in various aspects and is very valuable as a pharmaceutical agent.

Antibody H1009 / L395, which has the Fc region of native IgG, has the essential and valuable properties described above; however, H1009 / L395 variants that contain a functionally improved Fc region can also be used appropriately as antibodies with increased utility. In particular, binding to FcRn can be increased under acidic pH conditions to prolong plasma retention time and to maintain action over a longer period of time. In addition, variants that contain an Fc region with an introduced modification (s) that reduce (s) the ability to bind to FcγR can be used as highly safe therapeutic antibodies to avoid unwanted activation of immune cells and the formation of cytotoxic activity in the body to which they are administered. As these Fc variants, the use of F1886s or F1974m described herein is most preferred, but these Fc variants are not limited thereto; and, if the Fc variant has similar functions, then therapeutic antibodies containing other modified Fc regions are used as an embodiment of the invention in section B of the description.

As a result, the antibodies referred to in Section B of the specification, including H1009 / L395-F1886s and H1009 / L395-F1974m, which were developed in the present invention using a comprehensive assay method, can maintain an environment where the biological activity of human IL-8 is highly inhibited. staying at a safe level and over a long period of time. In this case, levels were realized that cannot be achieved using known antibodies to IL-8, and it can be expected that these antibodies presented in section B of the description will be used as antibodies to IL-8, which are high-quality finished pharmaceutical facilities.

Example 20

Factor IXa / Factor X bispecific antibodies

The humanized factor IXa / factor X bispecific antibodies described in WO 2012/067176 bind to human factor IXa and factor X and induce blood coaggregation activity.

Humanized anti-factor IXa / factor X F8M bispecific antibody (Q499-z121 / J327-z119 / L404-k: H chain (SEQ ID NO: 330) / H chain (SEQ ID NO: 331) / total L chain ( SEQ ID NO: 332)) described in WO 2012/067176 was used in this example and F8M contains two different H-chains and two identical common L-chains. F8M was obtained by the method described in the examples in WO 2012/067176.

(20-1) Preparation of bispecific antibodies to factor IXa / factor X

The following three antibodies were prepared by the method described in Reference Example 2 as anti-factor IXa / factor X bispecific antibodies constructed from F8M: (a) F8M-F1847mv, which is a canonical antibody containing F8M-F1847mv1 (SEQ ID NO : 323) and F8M-F1847mv2 (SEQ ID NO: 324) as heavy chains and F8ML (SEQ ID NO: 325) as light chain; (b) F8M-F1868mv, which is a canonical antibody containing F8M-F1868mv1 (SEQ ID NO: 326) and F8M-F1868mv2 (SEQ ID NO: 327) as heavy chains and F8ML (SEQ ID NO: 325) as light chain; and (c) F8M-F1927mv, which is a canonical antibody containing F8M-F1927mv1 (SEQ ID NO: 328) and F8M-F1927mv2 (SEQ ID NO: 329) as heavy chains and F8ML (SEQ ID NO: 325) in as a light chain.

The heavy chain sequences include the following sequences of the same Fc variants in terms of increased ability to bind to FcRn and reduced ability to bind to rheumatoid factor mentioned in example 5:

(20-2) Pharmacokinetic study of monoclonal antibodies F8M-F1847mv, F8M-F1868mv and F8M-F1927mv in the body of cynomolgus monkeys

A pharmacokinetic study of each of the monoclonal antibodies F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv was performed after a single bolus intravenous administration at a dose of 0.6 mg / kg to a male cynomolgus monkey. Plasma concentrations of F8M-F1847mv, F8M-F1868mv and F8M-F1927mv were determined by sandwich ELISA. Pharmacokinetic parameters were calculated using WinNonlin software, version 6.4. As shown in Table 30, the half-lives of F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv were 29.3, 54.5, and 35.0 days, respectively. The F8M PK study using cynomolgus monkeys was performed on another day at a dose of 6 mg / kg and a half-life was found to be 19.4 days. Obviously, the F8M-F1847mv, F8M-F1868mv and F8M-F1927mv have longer half-lives than the F8M. This suggests that the half-life of the anti-factor IXa / X bispecific antibody can be extended by the same modification of the Fc region sequence as described in Example 5 above.

Example 21

Evaluation of plasma IgE clearance using Fab variants with increased pI

In this example, the pI-raising substitutions in the Fab fragment of antibodies resulting in increased clearance of human IgE were evaluated using pH-dependent antigen-binding antibodies. The method of adding amino acid substitutions to the variable region of an antibody to increase the pI is not limited to a particular method, for example, the method described in WO 2007/114319 or WO 2009/041643 can be used. Amino acid substitutions introduced into the variable region are preferably substitutions that decrease the amount of negatively charged amino acids (such as aspartic acid and glutamic acid) while increasing the amount of positively charged amino acids (such as arginine and lysine). In addition, amino acid substitutions can be introduced at any position in the variable region of an antibody. Sites for the introduction of amino acid substitutions are preferably, but not limited to, positions at which amino acid side chains can be exposed on the surface of an antibody molecule.

(21-1) Obtaining antibodies with increased pI by modifying amino acids in the variable region

The antibodies tested are summarized in Table 32 and Table 33.

The heavy chain, Ab1H003 (also referred to as H003, SEQ ID NO: 144), was obtained by introducing the pI-raising substitution H32R in Ab1H (SEQ ID NO: 38). Other heavy chain variants were also obtained by introducing the appropriate substitutions shown in Table 32 into Ab1H according to the method described in Reference Example 1. All heavy chain variants were expressed with Ab1L (SEQ ID NO: 39) as the light chain. The pH-dependent binding profile of this antibody is summarized in Table 5 (Ab1).

The pI-raising light chain substitution was also evaluated in a similar manner.

The light chain of Ab1L001T (which was also referred to as L001, SEQ ID NO: 164) was obtained by introducing the pI-upregulating G16K substitution in Ab1L. Other light chain variants were also obtained by introducing the appropriate substitutions shown in Table 33 into Ab1L according to the method described in Reference Example 1. All light chain variants were expressed with Ab1H as the heavy chain.

(21-2) BIACORE Human FcγRIIb Binding Assay Using Increased pI Variants

Regardless of the resulting antibodies containing Fc region variants, binding assays between soluble human FcγRIIb and antigen-antibody complexes were performed using BIACORE T200 (GE Healthcare). Soluble human FcγRIIb (NCBI, access code NM_004001.3) was obtained in the form of a His-tagged molecule using a method known in the art. Anti-His antibody in an appropriate amount was fixed on a CM5 sensor chip (GE Healthcare) by amine coupling using a His capture kit (GE Healthcare) to immobilize human FcγRIIb. Then, the antibody-antigen complex and rolling buffer (as a reference solution) were injected and allowed to react with human FcγRIIb immobilized on the sensor chip. 20 mM N- (2-acetamido) -2-aminoethanesulfonic acid, 150 mM NaCl, 1.2 mM CaCl ₂ and 0.05% (w / v) Tween 20, pH 7.4 was used as a rolling buffer and an appropriate buffer was also used to dilute soluble human FcγRIIb. To regenerate the sensor chip, 10 mM glycine-HCl, pH 1.5 was used. All measurements were carried out at 25 ° C. Assays were performed on the basis of binding (RU) calculated using the sensograms obtained from the measurements, and relative values were obtained by taking the Ab1H / Ab1L binding level (parent Ab1 antibody) as 1.00. The parameters were calculated using the BIACORET100 Evaluation software (GE Healthcare).

The results of the SPR analysis are summarized in Tables 32 and 33. Several variants were found to have an increased ability to bind to human FcγRIIb fixed to the BIACORE sensor chip.

Antibodies obtained by introducing an increase in pI modification (s) in the variable region are antibodies in which the variable region charge is more positive as compared to antibodies before the modification (s) were introduced into them. Thus, it can be assumed that the Coulomb interaction between the variable region (positive charge) and the surface of the sensor chip (negative charge) should increase with amino acid modifications that lead to an increase in pI. In addition, it is assumed that these effects similarly occur on the negatively charged surface of the cell membrane; hence, their effect on increasing the rate of absorption into cells in vivo can be expected.

As used herein, an approximately 1.2-fold or more increased binding to hFcγRIIb variants compared to binding to hFcγRIIb of parent Ab1 indicates a potent charge-driven effect on antibody binding to hFcγRIIb on a sensor chip.

Among the heavy chain variants with increased pI, the antibody with substitution (s) Q13K, G15R, S64K, T77R, D82aN, D82aG, D82aS, S82bR, E85G, or Q105R (according to Cabot numbering), alone or in combination, showed higher binding to hFcγRIIb. It is believed that a single amino acid substitution or a combination of these substitutions in the heavy chain should (are) have a strong charge-driven effect on binding to hFcγRIIb on the sensor chip. Thus, one or more positions that are expected to have an effect on increasing the rate or level of uptake by cells in vivo when introducing pI-raising modifications into the variable region of an antibody heavy chain may include, for example, positions 13, 15, 64, 77, 82a, 82b, 85 and 105 according to Cabot's numbering. The amino acid substitution introduced at the indicated position (s) can be asparagine, glycine, serine, arginine or lysine, and preferably arginine or lysine.

Among the variants of the light chain with increased pI, the antibody with the substitution (s) G16K, Q24R, A25R, S26R, E27R, Q37R, G41R, Q42K, S52K, S52R, S56K, S56R, S65R, T69R, T74K, S76R, S77R, Q79K (according to Cabot numbering), individually or in combination, had a higher binding to hFcγRIIb. It is believed that a single amino acid substitution or a combination of these substitutions in the light chain should (are) have a strong charge-mediated effect on binding to hFcγRIIb on the sensor chip. Thus, one or more positions that are expected to have an effect on increasing the rate or level of uptake by cells in vivo when introducing pI-raising modifications into the variable region of an antibody light chain may include, for example, positions 16, 24, 25, 26, 27, 37, 41, 42, 52, 56, 65, 69, 74, 76, 77 and 79 according to Cabot's numbering. The amino acid substitution introduced at the indicated position (s) can be arginine or lysine.

(21-3) Cellular uptake of antibodies containing a variant Fab region with an increased pI

To assess the level of intracellular uptake of a cell line expressing hFcγRIIb using the obtained antibodies containing a variant Fab region, an assay similar to that described above in Example (4-5) was performed, provided that the amount of absorbed antigen was expressed in relative values, taking the absorption level at using Ab1H / Ab1L (original Ab1 antibody) for 1.00.

The results of quantitative measurements of cellular uptake are summarized in Tables 32 and 33. Strong antigen fluorescence in cells was detected using several Fc variants. As used herein, an increase of about 1.5-fold or more in the fluorescence intensity of antigen taken up by cells using the variants compared to the fluorescence intensity of Ab1 may indicate a strong charge-mediated effect on cellular uptake.

It was found that among the antibodies used, an antibody with heavy chain variants with an increased pI with substitution (s) P41R, G44R, T77R, D82aN, D82aG, D82aS, S82bR, or E85G (according to Cabot's numbering), individually or in combination, led to a stronger uptake antigen cells. It is believed that a single amino acid substitution or a combination of these substitutions in the heavy chain should (are) have a strong charge-mediated effect on the uptake of antigen-antibody complex by cells. Thus, one or more positions that are expected to make the uptake of the antigen-antibody complex by cells more rapid and more frequent upon introduction of pI-raising modifications into the variable region of the heavy chain of an antibody may include, for example, positions 41, 44, 77, 82a, 82b or 85 according to Cabot's numbering. The amino acid substitution introduced at the indicated position (s) can be asparagine, glycine, serine, arginine or lysine, and preferably arginine or lysine.

It was found that among the antibodies used, an antibody with light chain variants with increased pI with substitution (s) G16K, Q24R, A25R, A25K, S26R, S26K, E27R, E27Q, E27K, Q37R, G41R, Q42K, S52K, S52R, S56R, S65R , T69R, T74K, S76R, S77R, or Q79K (according to Cabot's numbering), alone or in combination, resulted in stronger uptake of antigen by cells. It is believed that a single amino acid substitution or a combination of these substitutions in the light chain should (are) have a strong charge-related effect on the uptake of antigen-antibody complex by cells. Variants with 4 or more amino acid substitutions tended to have a stronger charge-related effect than variants with fewer amino acid substitutions. One or more positions that are expected to be uptake by cells of the antigen-antibody complex more rapidly and more frequently upon introduction of pI-raising modifications into the variable region of an antibody light chain may include, for example, positions 16, 24, 25, 26, 27, 37, 41, 42, 52, 56, 65, 69, 74, 76, 77 or 79, according to Cabot's numbering. The amino acid substitution introduced at the indicated position (s) can be glutamine, arginine or lysine, and preferably arginine or lysine.

Without going into any particular theory, this result can be explained as follows: antigen and antibody added to the cell culture solution form antigen-antibody complexes in the culture solution. The antigen-antibody complexes bind to human FcγRIIb expressed on the cell membrane through the Fc region of the antibody and are taken up by cells in a receptor-dependent manner. The antibodies used in this experiment bind to the antigen in a pH-dependent manner; therefore, the antibody can be cleaved by dissociation from the antigen in endosomes (acidic pH conditions) within cells. Since the antigen separated as a result of dissociation is transported to the lysosome and accumulates in it, it emits fluorescence inside the cells. Thus, the strong intensity of fluorescence inside the cell probably indicates that the absorption of antigen-antibody complexes by cells occurs faster and more frequently.

(21-4) Assessment of Human IgE Clearance in a Co-Injected Mouse Model

Certain anti-IgE antibodies with pH-dependent antigen binding (parent Ab1, Ab1H / Ab1L013, Ab1H / Ab1L014, Ab1H / Ab1L007) were tested in a co-injected mouse model to assess their ability to enhance IgE clearance from plasma. To create a co-injected model, C57BL6J mice (Jackson Laboratories) were injected with a single i.v. injection of IgE pre-mixed with an anti-IgE antibody, respectively. All groups received 0.2 mg / kg IgE and 1.0 mg / kg anti-IgE antibody. Total plasma IgE concentration was determined by anti-IgE ELISA. First, anti-human IgE antibody (clone 107, MABTESH) was added to a microwell plate (Nalge nunc International) and incubated for 2 hours at room temperature or overnight at 4 ° C to obtain a plate with immobilized anti-human IgE antibody. Samples to construct a standard curve and samples were mixed with an excess amount of anti-IgE antibody (obtained in our laboratory) to obtain a homogeneous immune complex. These samples were added to a plate immobilized with anti-human IgE antibody and incubated overnight at 4 ° C. These samples were then reacted sequentially with human core protein GPC3 (obtained in our laboratory), biotinylated anti-GPC3 antibody (obtained in our laboratory), streptavidin-Poly HRP80 conjugate (Stereospecific Detection Technologies) for 1 hour. After that, chemiluminescent substrate was added SuperSignal® ELISA Pico (Thermo Fisher Scientific). Chemiluminescence was determined using a SpectraMax M2 (Molecular Devices). The concentration of human IgE was calculated using SOFTmax PRO (Molecular Devices). FIG. 37 shows the profile of the time dependence of the concentration of IgE in the plasma of mice C57BL6J.

After the introduction of Fab variants with increased pI, characterized by pH-dependent antigen binding, the total concentration of IgE in plasma was lower than when using the original Ab1. These results indicate that the immune complex of antigen and antibody with an increased pI, characterized by pH-dependent antigen binding, can bind more strongly to the plasma membrane receptor such as FcγR, which increases the cellular uptake of the immune complex antigen-antibody. The antigen absorbed by the cells can be efficiently released from the antibody within the endosome, resulting in an accelerated elimination of IgE. The concentration of IgE in the body of mice treated with Ab1H / Ab1L007, which is characterized by weak efficiency in the in vitro experiment, was higher than when using other Fab variants containing antibodies with increased pI. These results also suggest that, given the in vivo plasma antigen clearance assessment, the sensitivity of the in vitro system described above, which was used to determine the fluorescence intensity using InCell 6000 analyzers, may be higher than the sensitivity of the in vitro BIACORE system described above.

Example 22

Evaluation of plasma C5 clearance using Fab variants with increased pI

In this example, pI-increasing substitutions in the Fab region of an antibody were evaluated to enhance clearance of human IgE using pH dependent antigen binding antibodies.

(22-P Preparation of C5 [Expression and Purification of Recombinant Human C5]

Recombinant human C5 (NCBI GenBank Accession Code: NP_001726.2, SEQ ID NO: 207) was transiently expressed using the FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA). Conditioned media expressing human C5 were diluted with an equal volume of milliQ water, then loaded onto a Q-Sepharose FF or Q-Sepharose HP anion exchange column (GE healthcare, Uppsala, Sweden) and eluted using a NaCl gradient. Fractions containing human C5 were pooled, then the salt concentration and pH were adjusted to 80 mM NaCl and pH 6.4, respectively. The resulting sample was loaded onto a cation exchange column packed with SP-Sepharose HP (GE healthcare, Uppsala, Sweden) and eluted using a NaCl gradient. Fractions containing human C5 were pooled and loaded onto a CHT (ceramic hydroxyapatite) column (Bio-Rad Laboratories, Hercules, Calif., USA). The human C5 eluate was then loaded onto a Superdex 200 packed gel filtration column (GE healthcare, Uppsala, Sweden). Fractions containing human C5 were pooled and stored at -150 ° C. Either recombinant human C5 obtained in our laboratory or plasma-derived human C5 (CALBIOCHEM, catalog # 204888) was used in the experiment.

Expression and purification of recombinant C5 monkey cynomolgus (NCBI GenBank, access code: XP_005580972, SEQ ID NO: 208) was performed in exactly the same manner as the human counterpart.

(22-2) Preparation of a synthetic calcium-binding motif library

Gene library of variable regions of heavy chains of antibodies, which were used as synthetic libraries of human heavy chains, consisted of 10 libraries of heavy chains. VH1-2, VH1-69, VH3-23, VH3-66, VH3-72, VH4-59, VH4-61, VH4-b, VH5-51, and VH6-1 germline frameworks were selected for this library based on frequency the germ line in human B cell populations; and the biophysical properties of V gene families. A synthetic library of human heavy chains was diversified for an antibody binding site that mimics human B cell antibody populations.

The gene library of variable regions of the light chains of antibodies was created so that it has a calcium-binding motif, and diversified in positions that can be involved in the recognition of antigens, taking into account the populations of human B-cell antibodies. The creation of a gene library of variable regions of light chains of antibodies, which has the characteristics of calcium-dependent binding to antigens, is described in WO 2012/073992.

The combination of a heavy chain variable region library and a light chain variable region library was inserted into a phagemid vector and a phagemid library was generated according to de Heard et al., Meth. Mol. Biol. 178, 2002, pp. 87-100. The trypsin cleavage site was introduced into the phagemid vector in the region of the linker between the Fab and the pIII protein. The modified M13KO7 helper phage, which had a trypsin cleavage site between the N2 and CT domains in gene III, was used to generate a phage displaying Fab.

(22-3) Isolation of calcium-dependent antibodies to C5

и др., J. Immunol. Methods 247 (1-2), 2001, сс. 191-203, Yeung и др., Biotechnol. Prog. 18(2), 2002, сс. 212-220, Jensen и др., Mol. Cell Proteomics 2(2), 2003, сс. 61-69. В качестве магнитных гранул применяли гранулы, покрытые нейтравидином (NeutrAvidin) (Sera-Mag SpeedBeads, покрытые нейтравидином) или гранулы, покрытые стрептавидином (Dynabeads М-280, покрытие стрептавидином). Человеческий С5 (фирма CALBIOCHEM, каталожный №204888) метили с помощью реагента EZ-Link NHS-ПЭГ4-Биотин (фирма PIERCE, каталожный №21329).The phage display library was diluted with TBS supplemented with BSA and CaCl ₂ at final concentrations of 4% and 1.2 mM, respectively. As a method of panning, conventional selection using magnetic beads was used according to generally accepted protocols (Junutula et al., J. Immunol. Methods 332 (1-2), 2008, pp. 41-52,

et al., J. Immunol. Methods 247 (1-2), 2001, pp. 191-203, Yeung et al. Biotechnol. Prog. 18 (2), 2002, pp. 212-220, Jensen et al., Mol. Cell Proteomics 2 (2), 2003, pp. 61-69. As magnetic beads, NeutrAvidin coated beads (Sera-Mag SpeedBeads, neutravidin coated) or streptavidin coated beads (Dynabeads M-280, streptavidin coated) were used. Human C5 (CALBIOCHEM, catalog # 204888) was labeled with EZ-Link NHS-PEG4-Biotin reagent (PIERCE, catalog # 21329).

In the initial round of phage selection, the phage display library was incubated with biotinylated human C5 (312.5 nM) for 60 min at room temperature. Phages displaying binding Fab variants were then captured using magnetic beads.

After incubation with the beads for 15 min at room temperature, the beads were washed three times with 1 ml TBS containing 1.2 mM CaCl ₂ and 0.1% Tween20 and the beads were washed twice with 1 ml TBS containing 1.2 mM CaCl ₂ ... Phages were eluted by resuspending the beads with TBS containing 1 mg / ml trypsin for 15 minutes. The eluted phages were challenged with ER2738 and rescued with a helper phage. The "rescued" phages were precipitated with polyethylene glycol, resuspended with TBS supplemented with BSA and CaCl ₂ at final concentrations of 4% and 1.2 mM, respectively, and used in the next round of panning.

After the 1st round of panning, phages were selected based on calcium dependence, in which the antibody binds more strongly to C5 in the presence of calcium ion. In the second and third rounds, panning was performed in the same manner as in the first round, except for the application of 50 nM (second round) or 12.5 nM (third round) biotinylated antigen, and finally eluted using 0.1 ml of buffer for elution (50 mM MES, 2 mM EDTA, 150 mM NaCl, pH 5.5) and was contacted with 1 μl trypsin (100 mg / ml) for selection for calcium dependence. After selection, the selected phage clones were converted into IgG format.

The binding capacity of antibodies to human C5 converted into IgG format was assessed under two different conditions: determination of association and dissociation at 1.2 mM CaCl ₂ -pH 7.4 (20 mM MES, 150 mM NaCl, 1.2 mM CaCl ₂ ) and association at 1 , 2 mM CaCl ₂ -pH 7.4 (20 mM MES, 150 mM NaCl, 1.2 mM CaCl ₂ ) and dissociation at 3 μM CaCl ₂ -pH 5.8 (20 mM MES, 150 mM NaCl, 3 μM CaCl ₂ ) at 30 ° C using the Octet RED384 system (Pall Life Sciences). 25 clones were isolated, the binding of which to the antigen depended on pH-calcium. The sensorgrams of these antibodies are shown in FIG. 38.

(22-4) Identification of the anti-C5 bispecific antibody

From the clones isolated in Example B-3, nine pH- or calcium-dependent anti-C5 antibody clones were selected for further analysis (CFP0008, 0011, 0015, 0016, 0017, 0018, 0019, 0020, 0021). Several amino acid substitutions have been introduced into the variable region of the heavy chain of CFP0016 using a method well known to those of ordinary skill in the art to improve antibody properties such as physicochemical properties. This variant of CFP0016, CFP0016H019, was used for additional analysis instead of CFP0016. The amino acid sequences of the VH and VL regions of these nine antibodies are shown in Table 34. In this table, designations in parentheses correspond to abbreviations.

Full-length genes having nucleotide sequences encoding the heavy chain and light chain of antibodies were synthesized and prepared using a method well known to those of ordinary skill in the art. Heavy chain and light chain expression vectors were prepared by inserting the resulting plasmid fragments into mammalian expression vectors. The resulting expression vectors were sequenced using a method well known to those of ordinary skill in the art. For expression of antibodies, the resulting plasmids were transiently transfected into FreeStyle293-F cells (Thermo Fisher Scientific). Purification from conditioned media expressing antibodies was performed using a method well known to those of ordinary skill in the art using r-protein A sepharose Fast Flow (GE Healthcare).

(22-5) Generation and characterization of a pH-dependent anti-C5 bispecific antibody

Bispecific antibodies that recognize two different C5 epitopes were generated by combining CFP0020 and CFP0018. The bispecific antibody was prepared in IgG format and contained two different Fab clones at each antibody binding site, and was prepared using a method well known to those of ordinary skill in the art. In this IgG-type bispecific antibody, the two heavy chains contain different heavy chain constant regions (G1dP1, SEQ ID NO: 227 and G1dN1, SEQ ID NO: 228), which effectively form a heterodimer of the two heavy chains. Bispecific antibody to C5 containing binding sites for MAb to C5 "X" and MAb to C5 "Y" was designated as "X // Y".

By introducing several amino acid substitutions in the heavy chain and light chain CDRs using a method well known to those of ordinary skill in the art, a 20 // 18 light chain combined variant was obtained, which was designated as "optimized 20 // 18" (consisting of two heavy chains: CFP0020H0261-G1dP1, SEQ ID NO: 229 and CFP0018H0012-G1dN1, SEQ ID NO: 230, and total light chain: CFP0020L233-k0, SEQ ID NO: 231).

The kinetic parameters of the optimized 20 // 18, characterizing the interaction with recombinant human C5, were evaluated under two different conditions (for example, (A) association and dissociation at pH 7.4 and (B) association at pH 7.4 and dissociation at pH 5, 8) at 37 ° C using a BIACORE T200 device (GE Healthcare). Protein A / G (Pierce, cat # 21186) or anti-human IgG (Fc) (included in the Humab Antidody Capture Kit; GE Healthcare, Cat # BR-1008-39) was immobilized on a SM4 series chip (GE Healthcare , catalog no. BR-1005-34) using the amine coupling method. Anti-C5 antibodies were captured on the immobilized molecule and then human C5 was injected. ACES pH 7.4 and pH 5.8 (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl ₂ , 0.05% Tween 20) were used as mobile buffers. Kinetic parameters at both pHs were determined by fitting sensograms to a 1: 1-RI binding model (no volumetric effect control) using BIACORE T200 Evaluation software, version 2.0 (GE Healthcare). Kinetic parameters, association reaction rate (ka), dissociation reaction rate (kd) and binding affinity (KD) at pH 7.4 and dissociation rate (kd), determined only by calculation at the dissociation phase at each pH, are presented in Table 35. For the optimized 20 // 18, a faster dissociation at pH 5.8 from the complex with human C5 was established as compared to the dissociation rate at pH 7.4.

(22-6) Obtaining antibodies with increased pI by modifying amino acids in the variable region

The antibodies tested are summarized in Tables 36 and 37.

The heavy chain, CFP0020H0261-001-G1dP1 (also referred to as 20H001, SEQ ID NO: 232), was obtained by introducing the pI-increasing substitution P41R / G44R in CFP0020H0261-G1dP1 (SEQ ID NO: 229). Likewise, the heavy chain, CFP0018H0012-002-G1dN1 (also referred to as 18H002, SEQ ID NO: 251), was prepared by introducing the pI-increasing substitution T77R / E85R in CFP0018H0012-G1dN1 (SEQ ID NO: 230). Other heavy chain variants were also obtained by introducing the corresponding substitutions indicated in Table 36 into CFP0020H0261-G1dP1 and CFP0018H0012-G1dN1, respectively, according to the method described in Reference Example 1. Heavy chain variants both CFP0020H0261-G1dP1 and CFP0018H112-G1dP1 variants expressed with CFP0020L233-k0 (SEQ ID NO: 231) as a light chain to obtain a bispecific antibody.

Similarly, the pI-raising light chain substitution was also evaluated. The light chain, CFP0020L233-001-k0 (also referred to as 20L233-001, SEQ ID NO: 271), was prepared by introducing the pI-up G16K substitution in CFP0020L233-k0. Other light chain variants were also obtained by introducing the corresponding substitutions shown in Table 37 into CFP0020L233-k0 according to the method described in Reference Example 1. All light chain variants were expressed with CFP0020H0261-G1dP1 and CFP0018H0012-G1dN1 as heavy chain, obtaining a bispecific antibody.

(22-7) BIACORE Human FcγRIIb Binding Assay Using Increased pI Variants

For the resulting antibodies containing Fc region variants, binding assays between soluble hFcγRIIb and antigen-antibody complexes were performed using a BIACORE T200 device (GE Healthcare). Soluble hFcγRIIb was obtained in the form of a His-tagged molecule using a method known in the art. Anti-His antibody in an appropriate amount was fixed on a CM5 sensor chip (GE Healthcare) by amine coupling using a His capture kit (GE Healthcare) to immobilize human FcγRIIb. Then, the antibody-antigen complex and rolling buffer (as a reference solution) were injected and allowed to react with human FcγRIIb immobilized on the sensor chip. 20 mM N- (2-acetamido) -2-aminoethanesulfonic acid, 150 mM NaCl, 1.2 mM CaCl ₂ and 0.05% (w / v) Tween 20, pH 7.4 was used as a rolling buffer and an appropriate buffer was also used to dilute soluble human FcγRIIb. To regenerate the sensor chip, 10 mM glycine-HCl, pH 1.5 was used. All measurements were performed at 25 ° C, Assays were performed based on binding (RU) calculated using sensograms obtained from measurements and relative values obtained by assuming binding level CFP0020H0261-G1dP1 / CFP0018H0012-G1dN1 / CFP0020L233-k0 (parent Ab2 antibody) for 1.00. The parameters were calculated using the BIACORET100 Evaluation software (GE Healthcare).

The results of the SPR analysis are summarized in Tables 36 and 37. Several variants were found to have an increased ability to bind to hFcγRIIb fixed to the BIACORE sensor chip. As used herein, about 1.2-fold or more increased binding to hFcγRIIb variants compared to binding to hFcγRIIb of parent Ab2 indicates a potent charge-mediated effect on antibody binding to hFcγRIIb on a sensor chip.

Among heavy chain variants with increased pI, the antibody with substitutions L63R, F63R, L82K, or S82bR (according to Cabot's numbering) had a higher binding to hFcγRIIb. It is believed that a single amino acid substitution or a combination of these substitutions in the heavy chain should have a strong charge-mediated effect on the binding to hFcγRIIb on the sensor chip. Thus, one or more positions that are expected to have an effect on increasing the rate or level of uptake by cells in vivo when introducing pI-raising modifications to the variable region (s) of an antibody heavy chain may include, for example , position 63, 82 or, 82b according to Cabot's numbering. The amino acid substitution introduced at the indicated position (s) can be arginine or lysine.

Among the light chain variants with increased pI, the antibody with substitution (s) G16K, Q27R, G41R, S52R, S56R, S65R, T69R, T74K, S76R, S77R, or Q79K (according to Cabot's numbering) had a higher binding to hFcγRIIb. It is believed that a single amino acid substitution or a combination of these substitutions in the light chain should have a potent charge-mediated effect on binding to hFcγRIIb on the sensor chip. Thus, one or more positions that are expected to have an effect on increasing the rate or level of uptake by cells in vivo when introducing pI-raising modifications into the variable region of an antibody light chain may include, for example, positions 16, 27, 41, 52, 56, 65, 69, 74, 76, 77 or 79 according to Cabot's numbering. The amino acid substitution introduced at the indicated position (s) can be arginine or lysine. Variants with 4 or more amino acid substitutions tended to have a stronger charge-related effect than variants with fewer amino acid substitutions.

(22-8) Cellular uptake of antibodies containing a variant Fab region with an increased pI

To assess the level of intracellular uptake of the hFcγRIIb expressing cell line using the resulting antibodies containing the variant Fab region, the following assay was performed.

The MDCK cell line (Madin-Darby canine kidney cell line) constitutively expressing hFcγRIIb was obtained using known methods. Using these cells, the intracellular uptake of antigen-antibody complexes was assessed. More specifically, Alexa555 (Life Technlogies) was used to label human C5 according to the validated protocol, and antigen-antibody complexes were prepared in culture solution with an antibody concentration of 10 mg / ml and an antigen concentration of 10 mg / ml. A culture solution containing antigen-antibody complexes was added to culture plates to the above MDCK cells that constitutively express hFcγRIIb, and incubated for 1 h, and then the fluorescence intensity from the antigen absorbed by the cells was quantified using an InCell 6000 analyzer (firm GE healthcare). The amount of antigen uptake was presented as relative values compared to the value for Ab2, which was taken as 1.00.

The results of a quantitative assessment of cellular uptake are summarized in Tables 36 and 37. Strong fluorescence emitted by antigen in cells was found for several variants of heavy chains and light chains.

As used herein, an approximately 1.5-fold or more increased fluorescence intensity of antigen taken up by cells using the variants compared to the fluorescence intensity using parent Ab1 indicates a strong charge-mediated effect on antigen uptake by cells.

Among the heavy chain variants with increased pI, the antibody with substitution (amy) G8R, L18R, Q39K, P41R, G44R, L63R, F63R, Q64K, Q77R, T77R, L82K, S82aN, S82bR, T83R, A85R, or E85G (according to Cabot numbering) gave to a stronger uptake of antigen by cells. It is believed that a single amino acid substitution or a combination of these substitutions in the heavy chain should have a strong charge-mediated effect on the uptake of the antigen-antibody complex by cells. Thus, one or more positions that are expected to have an effect on increasing the rate or level of uptake of the antigen-antibody complex by cells, when introduced, leading to an increase in pI modifications in the variable region (s) of the heavy chain of the antibody, may include for example, positions 8, 18, 39, 41, 44, 63, 64, 77, 82, 82a, 82b, 83 or 85 according to Cabot's numbering. The amino acid substitution introduced at the indicated position (s) can be asparagine, glycine, serine, arginine or lysine, and preferably arginine or lysine.

Among the light chain variants with increased pI, the antibody with substitutions G16K, Q27R, S27R, G41R, S52R, S56R, S65R, T69R, T74K, S76R, S77R, or Q79K (according to Cabot's numbering) resulted in stronger uptake of antigen by cells. It is believed that a single amino acid substitution or a combination of these substitutions in the heavy chain should have a strong charge-mediated effect on the uptake of the antigen-antibody complex by cells. Variants with 4 or more amino acid substitutions tended to have a stronger charge-related effect than variants with fewer amino acid substitutions. As demonstrated in Example 21, (21-3), the combination of 42K and 76R substitutions was effective against anti-IgE antibody. However, in the case of the anti-C5 antibody, the amino acid at position 42 according to Cabot's numbering is already lysine, so the effect of 42K / 76R charge change was achieved with one 76R substitution. The fact that the 76R substitution variant had a strong charge exchange effect also in the anti-C5 antibody suggests that the 42K / 76R combination has a strong charge-related effect regardless of antigen. Thus, one or more positions that are expected to have an effect on increasing the rate or level of uptake by cells of the antigen-antibody complex, when introduced, leading to an increase in pI modifications in the variable region (s) of the light chain of the antibody, may include for example, positions 16, 27, 41, 52, 56, 65, 69, 74, 76, 77 or 79 according to Cabot numbering. The amino acid substitution introduced at the indicated position (s) can be arginine or lysine.

(22-9) Assessment of C5 clearance in a co-injected mouse model

Several bispecific antibodies to C5 (parent Ab2, 20L233-005, 20L233-006 and 20L233-009) were tested in a co-injected mouse model to assess their ability to enhance plasma C5 clearance. To create a co-injected model, C57BL6J mice (Jackson Laboratories) were injected with a single i.v. injection of C5 pre-mixed with anti-C5 antibody, respectively. All groups received 0.1 mg / kg C5 and 1.0 mg / kg anti-C5 bispecific antibodies. Total plasma C5 concentration was determined by ECLIA for anti-C5 antibodies. First, an anti-human C5 IgG type antibody was added to an ECL plate and incubated overnight at 5 ° C. to obtain a plate with immobilized mouse anti-human C5 IgG. Standard curve samples and samples were mixed with rabbit anti-human IgG C5. These samples were added to a plate with immobilized mouse IgG to human C5 and incubated for 1 hour at room temperature. The samples were then reacted with HRP-conjugated anti-rabbit IgG (Jackson Immuno Research) for 1 hour. After incubation of the plate for 1 hour at room temperature, sulfa-tagged anti-HRP antibody was added. The ECL signal was determined using a SpectraMax M2 (Molecular Devices). The human C5 concentration was calculated from the ECL signal of the standard curve using SOFTmax PRO (Molecular Devices). FIG. 39 shows the profile of the time dependence of the concentration of C5 in the plasma of C57BL6J mice.

Compared to the parent Ab2, all bispecific antibodies with pI-upshift (s) substitution (s) tested in this experiment demonstrated rapid clearance of C5 from plasma. Therefore, it can be assumed that the amino acid substitution (s) T74K / S77R, S76R / Q79K and Q37R in the light chain accelerate (accelerate) the elimination of the C5-antibody immune complex in vivo as well. In addition, C5 elimination with 20L233-005 and 20L233-006 was found to be faster compared to 20L233-009, which is consistent with in vitro imaging data and BIACORE analysis.

These results suggest that even for position (s), the effect of which on antigen clearance from plasma in vivo is unlikely when evaluated either in an in vitro system based on an assessment of fluorescence intensity using an InCell 6000 analyzer, or in the in vitro BIACORE system described above. , it is possible to detect participation in clearance when using a more sensitive system in vivo. These results also suggest that, given the in vivo plasma antigen clearance assessment, the sensitivity of the in vitro system described above, which was used to determine the fluorescence intensity with the InCell 6000 analyzer, may be higher than the sensitivity of the in vitro BIACORE system described above.

Example 23

Evaluation of plasma IgE clearance using Fc region variants with increased pI

To enhance clearance of human IgE or human C5, pI-raising substitutions in the Fc region of antibodies were evaluated using pH-dependent antibodies. The method of adding amino acid substitutions to the constant region of an antibody to increase the pI is not limited to a specific method, but, for example, the method described in WO 2014/145159 can be used for this purpose.

(23-1) Obtaining antibodies with increased pI by one amino acid modification in the constant region

The antibodies tested are summarized in Table 38. The heavy chain, Ab1H-P1394m (SEQ ID NO: 307), was obtained by introducing the pI-increasing substitution Q311K into AMH. Other heavy chain variants were also obtained by introducing the corresponding substitutions shown in Table 38 into AMH according to the method described in Reference Example 1. All heavy chain variants were expressed with Ab1L as light chain.

(23-2) BIACORE Binding Assay of Human FcγRIIb Using Antibodies Containing an Increased pI Fc Region Variant

To evaluate the charge-related effect on FcRyRIIb binding of an antigen-antibody complex prepared using the antibodies shown in Table 38, an FcRγRIIb binding assay similar to that described in Example 21 (21-2) was performed. The results of the assay are shown in Table 38. As used herein, about 1.2-fold or more increased binding to hFcγRIIb using variants compared to binding to hFcγRIIb of parent Ab1 indicates a potent charge-driven effect on antibody binding to hFcγRIIb on a sensor chip.

Among the variants with an increased pI with one amino acid substitution relative to the original Ab1, for the antigen-antibody complex, where several variants designated as P1398m, P1466m, P1482m, P1512m, P1513m and P1514m were used as the antibody, the highest binding to hFcγRIIb was found. It is likely that a single amino acid substitution D413K, Q311R, P343R, D401R, D401K, G402R, Q311K, N384R, N384K, or G402K may have a potent charge-mediated effect on hFcγRIIb binding on the sensor chip.

Thus, an individual position that is expected to influence an increase in the rate or level of uptake by cells in vivo upon introduction of an increase in the pI modification to the constant region of an antibody may include, for example, positions 16, 27, 41, 52, 56 , 65, 69, 74, 76, 77 or 79 according to Cabot's numbering. The amino acid substitution introduced at this position can be arginine or lysine.

(23-3) Cellular uptake of antibodies containing a variant Fc region with an increased pI

To assess the level of intracellular uptake of the antigen-antibody complex formed using the antibodies indicated in Table 38, a cell imaging assay similar to that described in Example 21 (21-3) was performed. The results of the analysis are shown in Table 38. As used herein, an increase of about 1.5-fold or more in the fluorescence intensity of antigen taken up by cells using the variants compared to the fluorescence intensity using the parent Ab1 indicates a strong charge-mediated effect on antigen uptake by cells.

Among the variants with an increased pI with one amino acid substitution relative to the original Ab1, for the antigen-antibody complex, where several variants were used as the antibody, such as P1398m, P1466m, P1469m, P1470m, P1481m, P1482m, P1483m, P1512m, P1513m, and P1653m, stronger uptake of antigen by cells. It is likely that a single amino acid substitution D413K, Q311R, N315K, N384R, Q342K, P343R, P343K, D401R, D401K, or D413R may have a potent charge-mediated effect on the uptake of the antigen-antibody complex by cells. Thus, an individual position that is expected to influence an increase in the rate or level of uptake by cells upon introduction of an increase in pI modification to the antibody constant region may include, for example, positions 311, 315, 342, 343, 384, 401 or 413, according to EU numbering. The amino acid substitution introduced at this position can be arginine or lysine.

(23-4) Assessment of Human IgE Clearance in a Co-Injected Mouse Model

Several anti-IgE antibodies with pH-dependent antigen binding (parent Ab1, P1466m, P1469m, P1470m, P1480m, P1482m, P1512m, P 1653m) were tested in a co-injected mouse model to assess their ability to enhance plasma IgE clearance. Analyzes were carried out by a method similar to that described in example 21, (21-4). FIG. 40 shows the profiles versus time of plasma concentration in C57BL6J mice

After the introduction of variants with high pI (with only one amino acid substitution) with pH-dependent antigen binding, the total concentration of IgE in plasma was lower than when using the original Ab1, with the exception of P1480m. The P1480m variant, which was found to be weak in both in vitro experiments, did not accelerate IgE elimination. In addition, the total plasma IgE concentration in mice treated with the high pI variant without pH-dependent antigen binding was significantly higher than in the high pI variant with pH-dependent antigen binding (data not shown). These results indicate that the cellular uptake of the antigen-antibody immune complex increases with the introduction of a pI-increasing modification. Antigen taken up by cells in combination with a pH-dependent antigen-binding antibody can be efficiently released from the antibody inside the endosome, which leads to an accelerated elimination of IgE. These results also suggest that, even for a position change, the effect of which on antigen clearance from plasma in vivo is unlikely when evaluated using the in vitro BIACORE system described above, clearance participation can be detected using a more sensitive in vivo system. These results also suggest that, given the in vivo plasma antigen clearance assessment, the sensitivity of the in vitro system described above, based on the fluorescence intensity assessment using the InCell 6000 analyzer, may be higher than the sensitivity of the BIACORE in vitro system described above.

Reference example 1

Construction of expression vectors of IgG-type antibodies with amino acid substitution

Mutants were prepared using the QuickChange site-directed mutagenesis system (Stratagene) using the method described in the accompanying instructions for use. Plasmid fragments containing the mutants were inserted into expression vectors for animal cells to construct the desired expression vectors for the H chain and L chain. The nucleotide sequences of the resulting expression vectors were determined by methods known in the art.

Reference example 2

Expression and purification of IgG antibodies

Antibodies were expressed using the following method. Cell line HEK293H (Invitrogen) derived from human embryonic kidney cancer cells was suspended in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). The cells were seeded at a rate of 10 ml per dish in plates for attached cells (10 cm in diameter; CORNING) with a cell density of 5-6 × 10 ⁵ cells / ml and cultured in a CO ₂ incubator (37 ° C, 5% CO ₂ ) within 1 day. The medium was then removed by aspiration and 6.9 ml of CHO-S-SFM-II medium (Invitrogen) was added. The resulting plasmid was introduced into cells by lipofection. The resulting culture supernatants were harvested, centrifuged (about 2000 g, 5 min, room temperature) to remove cells and sterilized by filtration through a 0.22 μm MILLEX®-GV filter (Millipore) to obtain supernatants. Antibodies were purified from the resulting culture supernatants using methods known in the art using rProtein A Sepharose ™ Fast Flow (Amersham Biosciences). To determine the concentration of the purified antibody, the absorbance at 280 nm was measured using a spectrophotometer. Antibody concentrations were calculated on the basis of the stated values using the absorption coefficient calculated by the method described by Race et al., Protein Science 4, 1995, pp. 2411-2423.

Reference example 3

Purification of Soluble Human IL-6 Receptor

Recombinant soluble human IL-6 receptor, which is an antigen, was obtained by the method described below. A CHO cell line that constitutively expresses a soluble human IL-6 receptor consisting of an amino acid sequence ranging from amino acids 1 to 357 starting from the N-terminus, as described by Mullberg et al., J. Immunol. 152, 1994, pp. 4958-4968, constructed using a method known in the art. Soluble human IL-6 receptor was expressed by culturing this CHO line. Soluble human IL-6 receptor was purified from the CHO line culture supernatant using two steps: chromatography on a Blue Sepharose 6 FF column and gel filtration using column chromatography. The fraction that eluted as the main peak in the final step was used as the final purified product.

Claims (15)

1. An isolated antibody that specifically binds to IL-8, which contains (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 67, (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 73, (c) HVR -H3 containing the amino acid sequence of SEQ ID NO: 74; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 70, (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 75, and (f) HVR-L3 containing the amino acid sequence of SEQ ID NO: 76. 2. An isolated antibody that specifically binds to IL-8, which comprises a heavy chain variable region having SEQ ID NO: 78 and a light chain variable region having SEQ ID NO: 79. 3. An isolated antibody that specifically binds to IL-8, which contains a heavy chain containing the amino acid sequence of SEQ ID NO: 80 or SEQ ID NO: 81, and a light chain containing the amino acid sequence of SEQ ID NO: 82. 4. Isolated nucleic acid encoding an antibody to IL-8 according to one of paragraphs. 1-3. 5. An expression vector containing the nucleic acid of claim 4. 6. A host cell for obtaining antibodies according to one of paragraphs. 1-3, which contains a vector according to claim 5. 7. A method for producing antibodies according to one of claims. 1-3, which includes culturing the host cell according to claim 6. 8. The method of claim 7, further comprising isolating the antibody from the culture of the host cell. 9. A pharmaceutical composition for treating a disorder characterized by an excess of IL-8, which contains an effective amount of an antibody according to one of claims. 1-3 and a pharmaceutically acceptable carrier. 10. A method of treating a patient who has a disorder characterized by an excess of IL-8, which comprises administering to the individual an anti-IL-8 antibody according to one of claims. 1-3. 11. The use of antibodies to IL-8 according to one of paragraphs. 1-3 for the preparation of a pharmaceutical composition for the treatment of a disorder characterized by an excess of IL-8. 12. A method of inhibiting angiogenesis in an individual, where the method comprises administering to the individual an antibody according to one of claims. 1-3. 13. The use of antibodies to IL-8 according to one of paragraphs. 1-3 for preparing a pharmaceutical composition for inhibiting angiogenesis. 14. A method of inhibiting the facilitation of neutrophil migration in an individual, wherein the method comprises administering to the individual an antibody according to one of claims. 1-3. 15. The use of antibodies according to one of paragraphs. 1-3 for the preparation of a pharmaceutical composition designed to inhibit the facilitation of neutrophil migration.

Images