JP6247241B2 - Antibody prescription - Google Patents

Description

The present invention relates to shear and temperature stable antibody formulations.

  Proteins are larger and more complex than traditional organic and inorganic drugs (ie, having a large number of functional groups in addition to complex three-dimensional structures), and the formulation of such proteins poses unique problems. For proteins that remain biologically active, the formulation must preserve at least the complete conformational integrity of the amino acid core sequence of the protein while at the same time protecting multiple functional groups of the protein from degradation. Don't be. Protein degradation pathways can be chemically unstable (ie, any process that involves modification of the protein by bond formation at the cleavage resulting in a new chemical) or physical instability (ie, a change in protein conformation). Can be involved. Chemical instability can result in amidolysis, racemization, hydrolysis, oxidation, beta elimination or disulfide exchange. Physical instability can lead to, for example, denaturation, aggregation, precipitation or intermediate wear. The three most common proteolytic pathways are protein aggregation, amidolysis and oxidation. Cleland et al. Critical Reviews in Therapeutic Drug Carrier Systems 10 (4): 307-377 (1993).

  The CD20 molecule (also called human B lymphocyte restricted differentiation antigen or Bp35) is a hydrophobic transmembrane protein with a molecular weight of approximately 35 kD found in pre-B and mature B lymphocytes (Valentine et al. (1989) J. Biol. Chem. 264 (19): 11212-11287; and Einfield et al. (1988) EMBO J. 7 (3): 711-717). CD20 is found on the surface of over 90% B cells from peripheral blood or lymphoid organs, is expressed during early pre-B cell production, and remains until plasma cell differentiation. CD20 is present on both normal B cells as well as malignant B cells. In particular, CD20 is expressed on more than 90% of B-cell non-Hodgkin lymphoma (NHL) (Anderson et al. (1984) Blood 63 (6): 142-1433), but hematopoietic stem cells, pre-B cells, normal It has not been found in plasma cells or other normal tissues (Tedder et al. (1985) J. Immunol. 135 (2): 973-979).

  The 85 amino acid carboxyl terminal region of the CD20 protein is located in the cytoplasm. The length of the region is that of other B cell specific surface structures, such as IgM, IgD, and relatively short cytoplasmic regions of 3, 3, 28, 15, and 16 amino acids, respectively. Contrast with the α chain or β chain of an IgG heavy chain or class II histocompatibility antigen (Komaromy et al. (1983) NAR 11: 6775-6785). Of the last 61 carboxyl terminal amino acids, 21 are acidic residues, while only 2 are basic, indicating that the region has a strong net negative charge. GenBank accession number is NP. sub. --- 690605.

  CD20 may be involved in the regulation of early stage (s) in B cell activation and differentiation processes (Tedder et al. (1986) Eur. J. Immunol. 16: 881-887), a calcium ion channel. (Tedder et al. (1990) J. Cell. Biochem. 14D: 195).

  Despite the uncertainty about the actual function of CD20 that promotes B cell proliferation and / or differentiation, it is an important target for antibody-mediated therapy to control or kill B cells involved in cancer and autoimmune diseases I will provide a. In particular, the expression of CD20 in tumor cells, such as NHL, makes it an important target for antibody-mediated therapy of specific targeted therapeutic agents against CD20 positive tumor cells.

  HuMax-CD20 ™ (ofatumumab), described as a 2F2 antibody in WO2004 / 035607, is a fully human IgG1, kappa high affinity antibody that targets the CD20 molecule in the cell membrane of B cells. HuMax-CD20 ™ is in clinical development for the treatment of non-Hodgkin lymphoma (NHL), chronic lymphocytic leukemia (CLL), and rheumatoid arthritis (RA). Teleing et al., Blood, 104, pp 1793 (2004); See also Immunology, 177, pp 362-371 (2007).

  There is a need to formulate shear and temperature stable pharmaceutical formulations containing antibodies that are suitable for therapeutic use. In one embodiment, the antibody can be a monoclonal antibody. In another embodiment, the antibody includes, but is not limited to, ofatumumab, rituximab, tositumomab, ocrelizumab (2H7.v16), 11B8 or 7D8 (disclosed in WO2004 / 035607), anti-CD20 antibody disclosed in WO2005 / 103081 For example, C6, anti-CD antibodies disclosed in WO2003 / 68821, such as IMMU-106 (from Immunomedics), anti-CD20 antibodies disclosed in WO2004 / 103404, such as AME-133 (from Applied Molecular Evolution / Lilly), and Anti-CD20 antibodies disclosed in US 2003/0118592, for example anti-CD20 antibodies, including TRU-015 (from Trubion Pharmaceuticals Inc) It is possible.

International Publication No. 2004/035607 Pamphlet International Publication No. 2005/103081 Pamphlet International Publication No. 2003/68821 Pamphlet International Publication No. 2004/103404 Pamphlet US Patent Application Publication No. 2003/0118592

Cleland et al. Critical Reviews in Therapeutic Drug Carrier Systems 10 (4): 307-377 (1993) Valentine et al. (1989) J. Am. Biol. Chem. H.264 (19): 11128-11287 Einfield et al. (1988) EMBO J. et al. 7 (3): 711-717 Anderson et al. (1984) Blood 63 (6): 142-1433 Tedder et al. (1985) J. Am. Immunol. 135 (2): 973-979 Komaromy et al. (1983) NAR 11: 6775-6785 Tedder et al. (1986) Eur. J. et al. Immunol. 16: 881-887 Tedder et al. (1990) J. Org. Cell. Biochem. 14D: 195 Teleing et al., Blood, 104, pp 1793 (2004). Teleing et al. Immunology, 177, pp 362-371 (2007)

  The present invention relates to shear and temperature stable aqueous antibody formulations.

  The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

  One embodiment is suitable for full-length monoclonal anti-CD20 antibody formulations, but can also be used to formulate other types of antibodies, eg, polyclonal antibodies, or monoclonal antibodies or fragments of polyclonal antibodies.

  In one embodiment, the invention relates to an anti-CD20 antibody formulation comprising a therapeutically effective amount of an anti-CD antibody, wherein the formulation comprises 10-100 mM sodium acetate, 25-100 mM sodium chloride, 0.5- It further contained 5% arginine free base, 0.02-0.2 mM EDTA, 0.01-0.2% polysorbate 80, and was adjusted to pH 5.0-7.0.

  In another embodiment, the invention relates to an anti-CD20 antibody formulation comprising an anti-CD20 antibody in the concentration range of 20-300 mg / mL, wherein the formulation comprises 50 mM sodium acetate, 51 mM sodium chloride, 1% arginine free base. , 0.05 mM EDTA, 0.02% polysorbate 80, and adjusted to pH 5.5. In another embodiment, the antibody is an anti-CD20 antibody fragment, eg, a monoclonal antibody fragment. A preferred anti-CD20 antibody is ofatumumab.

  In one embodiment, the invention relates to an ofatumumab formulation comprising a therapeutically effective amount of ofatumumab, wherein the formulation comprises 10-100 mM sodium acetate, 25-100 mM sodium chloride, 0.5-5% arginine release. It further contained a base, 0.02-0.2 mM EDTA, 0.01-0.2% polysorbate 80, and was adjusted to pH 5.0-7.0.

  In one embodiment, the invention relates to an ofatumumab formulation comprising ofatumumab in a concentration range of 20-300 mg / mL, wherein the formulation comprises 50 mM sodium acetate, 51 mM sodium chloride, 1% arginine free base, 0.05 mM EDTA, 0.02% polysorbate 80 was further included, and the pH was adjusted to 5.5.

  In yet another embodiment, the invention relates to an anti-CD20 antibody formulation that is stable for at least 2 years. In another embodiment, the invention relates to an anti-CD20 antibody formulation that is stable at temperatures up to at least 55 ° C. In another embodiment, the invention relates to an anti-CD20 antibody formulation that is stable for at least 2 years at a temperature of about 5 ° C. In another embodiment, the invention relates to an anti-CD20 antibody formulation that is stable for at least 3 months at a temperature of about 25 ° C. In another embodiment, the invention is an anti-CD20 antibody formulation that is stable for at least 1 month at a temperature of about 40 ° C. In another embodiment, the invention relates to an anti-CD20 antibody formulation that is stable for at least 1 day at a temperature of about 55 ° C. In another embodiment, the invention is an anti-CD0 antibody formulation that is stable for at least 1 day at a temperature range of about 5-55 ° C. with shaking. In another embodiment, the invention is about 5-25 ° C, 5-35 ° C, 5-45 ° C, 10-25 ° C, 10-35 ° C, 10-45 ° C, 10-55 ° C, 20 with shaking. It relates to an anti-CD20 antibody formulation that is stable for at least 1 day at a temperature range of -35 ° C, 20-45 ° C, or 20-55 ° C.

  In another embodiment, the invention provides that the antibody is about 20-300 mg / mL, 50-300 mg / mL, 100-300 mg / mL, 150-300 mg / mL, 200-300 mg / mL, or 250-300 mg / mL. Relates to an anti-CD20 antibody formulation present in an amount.

  In another embodiment, the invention relates to an anti-CD20 antibody formulation in which sodium acetate is present in an amount of about 50 mM, 40 mM, 45 mM, 55 mM, or 60 mM. In other embodiments, sodium acetate is present in an amount of 10-100 mM, 20-100 mM, 30-100 mM, 40-100 mM, 50-100 mM, 60-100 mM, 70-100 mM, 25-80 mM, or 30-70 mM. You may do it.

  In yet another embodiment, the present invention relates to an anti-CD20 antibody formulation in which acetic acid is present (about 100 mM acetic acid) to adjust to about pH 5.5. In other embodiments, the pH may be adjusted to pH 5.0, 5.5, 6.0, 6.5 or 7.0. In still other embodiments of the invention, NaOH or HCl is used to adjust the pH to 5.0, 5.5, 6.0, 6.5 or 7.0.

  In yet another embodiment, the invention relates to an anti-CD20 antibody formulation in which sodium chloride is present in an amount of about 51 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 52 mM, 53 mM, 54 mM, 55 mM. In other embodiments, sodium chloride may be present in an amount of 25-100 mM, 35-90 mM, 45-80 mM, 25-70 mM, or 45-70 mM.

  In another embodiment, the invention relates to an anti-CD20 antibody formulation in which arginine free base is present in an amount of about 1%, 0.7%, 1.3%, or 2.0%. In other embodiments, the arginine free base is 0.5-5.0%, 0.5-2.0%, 0.5-2.5%, 0.5-3.0%, 0.5 It may be -3.5%, 0.5-4.0%, or 0.5-4.5%.

  In another embodiment, the invention relates to an anti-CD20 antibody formulation in which EDTA is present in an amount of about 0.05 mM, 0.03 mM, 0.04 mM, or 0.06 mM. In other embodiments, the EDTA is 0.02 mM to 0.2 mM, 0.02 mM to 0.1 mM, 0.02 mM to 0.15 mM, 0.04 mM to 0.1 mM, 0.03 mM to 0.15 mM, or It may be present in an amount of 0.03 mM to 0.2 mM.

  In another embodiment, the invention relates to an anti-CD20 antibody formulation in which polysorbate 80 is present in an amount of about 0.02%, 0.015%, or 0.025%. In other embodiments, polysorbate 80 is 0.01-0.1%, 0.01-0.15%, 0.02-0.2%, 0.02-0.15%, 0.01- It may be present in an amount of 0.25%, or 0.01-0.05%.

  In another embodiment, the invention relates to a method of treating a disease involving cells expressing CD20 by administering to a mammal an anti-CD20 antibody formulation of the invention comprising a therapeutically effective amount of an anti-CD20 antibody. Where the formulation comprises 10-100 mM sodium acetate, 25-100 mM sodium chloride, 0.5-5% arginine free base, 0.02-0.2 mM EDTA, 0.01-0.2% polysorbate 80. In addition, the pH was adjusted to 5.0 to 7.0. Examples of “diseases involving cells expressing CD20” that can be treated (eg, ameliorated) or prevented include, but are not limited to, tumorigenic diseases and immune diseases such as autoimmune diseases. Examples of tumorigenic diseases that can be treated and / or prevented include progenitor B cell lymphoblastic leukemia / lymphoma and mature B cell tumors such as B cell chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL) Follicular lymphoma (FL), cutaneous follicular central lymphoma, marginal, including B-cell prolymphocytic leukemia, lymphoid plasma cell lymphoma, mantle cell lymphoma (MCL), low-grade, intermediate-grade and high-grade FL Band B cell lymphoma (MALT, nodal and splenic), hairy cell leukemia, diffuse large B cell lymphoma, Burkitt lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disease, Waldens B-cell lymphoma, including NHL, including trauma macroglobulinemia and anaplastic large cell lymphoma (ALCL) It is. Examples of immune diseases involving B cells expressing CD20 that can be treated and / or prevented include psoriasis, psoriatic arthritis, dermatitis, systemic scleroderma and sclerosis, inflammatory bowel disease (IBD), Crohn's disease Ulcerative colitis, respiratory distress syndrome, meningitis, encephalitis, uveitis, glomerulonephritis, eczema, asthma, atherosclerosis, leukocyte adhesion deficiency, multiple sclerosis, Raynaud's syndrome, Sjogren's syndrome, young Type diabetes, Reiter's disease, Behcet's disease, immune complex nephritis, IgA nephropathy, IgM polyneuropathy, immune-mediated thrombocytopenia such as acute idiopathic thrombocytopenic purpura and chronic idiopathic thrombocytopenic purpura, Hemolytic anemia, myasthenia gravis, lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis (RA), atopic dermatitis, pemphigus, Graves' disease, bridge Thyroiditis, Wegener's granulomatosis, Omen's syndrome, chronic renal failure, acute infectious mononucleosis include HIV and herpes virus associated diseases. Further examples are severe acute respiratory distress syndrome and chorioretinitis. Still further examples are diseases and disorders caused by infection of B cells with viruses such as Epstein-Barr virus (EBV). Yet a further example is COPD.

  In yet another embodiment, the invention provides a method of treating a disease involving cells expressing CD20 by administering to a mammal an anti-CD20 antibody formulation of the invention comprising a therapeutically effective amount of an anti-CD20 antibody. Wherein the formulation is 10-100 mM sodium acetate, 25-100 mM sodium chloride, 0.5-5% arginine free base, 0.02-0.2 mM EDTA, 0.01-0.2% polysorbate 80 The stable antibody formulation is adjusted to pH 5.0 to 7.0, and is administered to mammals by oral administration, parenteral administration, intranasal administration, vaginal administration, rectal administration, tongue administration, sublingual Administration, buccal administration, transdermal administration, intravenous administration, or subcutaneous administration.

  In yet another embodiment, the invention relates to a disease involving cells expressing CD20 by administering to a mammal an anti-CD20 antibody formulation of the invention comprising an anti-CD20 antibody in a concentration range of 20-300 mg / mL. Regarding the method of treatment, the formulation now further included 50 mM sodium acetate, 51 mM sodium chloride, 1% arginine free base, 0.05 mM EDTA, 0.02% polysorbate 80, and was adjusted to pH 5.5. A preferred anti-CD20 antibody is ofatumumab.

  It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to further illustrate the claimed invention. .

  The accompanying drawings are included to provide a further understanding of the invention, and are a part of and constitute a part of this specification, several embodiments of the invention, and principles of the invention. It is explained together with explanations useful for explaining.

FIG. 1 shows two formulations of 20 mg / mL (30 mM citrate, 100 mM NaCl, pH 6.5) anti-CD20 antibody standard formulation (RefMat). FIG. 2 shows two 20 mg / mL (50 mM sodium acetate, sodium chloride (51 mM), 1% arginine free base, 0.05 mM EDTA, 0.02% polysorbate 80, pH adjusted to 5.5 with HCl). 1 shows one embodiment of the present invention (PlatForm) formulation of an anti-CD20 antibody. FIG. 3 illustrates a comparison of the thermal stability of anti-CD20 antibodies in a formulation embodiment of the present invention (PlatForm) and standard formulation buffer (RefMat) by DSC. Thermodynamically, the two formulations are similar as seen in their DSC profiles because the change in apparent Tm is less than 0.5 ° C. between formulations.

  One embodiment of the invention relates to shear and temperature stable antibody formulations.

  In another embodiment, the present invention provides the unexpected stability found in formulations under co-stress conditions of elevated temperature and shaking at 55 ° C.

  A further embodiment of the invention is a more stable formulation compared to a standard formulation (eg, 30 mM citrate, 100 mM NaCl, pH 6.5). The formulation of the present invention showed a decrease in precipitation (transparent state) when exposed to stress conditions, while the standard formulation was agglomerated. As seen in its DSC (Differential Scanning Calorimeter) profile, this result was unpredictable because the two formulations were thermodynamically similar.

  In describing the present invention, certain terms are used as defined below.

  The term “protein formulation” or “antibody formulation” is an additional component that is toxic to the subject to which the formulation will be administered, such that the biological activity of the active ingredient is clearly effective. Refers to a preparation that does not contain

  “Pharmaceutically acceptable” excipients (vehicles, additives) are those that can be suitably administered to a subject mammal to provide an effective amount of the active ingredient used. For example, excipient concentration is also related to injection suitability.

  A “stable” formulation is one in which the protein therein essentially retains its physical and / or chemical stability and / or biological activity upon storage. Various analytical methods for measuring protein stability are available in the art, such as Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured for a selected time at a selected temperature. Preferably, the formulation is stable at ambient temperature or 40 ° C for at least 1 month and / or stable at 2-8 ° C for at least 1-2 years. Furthermore, it is desirable that the formulation can be stable after freezing (eg, up to -70 ° C.) and thawing of the product.

  Little change in aggregation, precipitation and / or denaturation as observed by color and / or transparency appearance tests or as measured by UV light scattering (measuring visualized aggregates) or size exclusion chromatography (SEC) Or, if not indicated at all, a protein “retains its physical stability” in a biopharmaceutical formulation. SEC measures soluble aggregates that are not necessarily precursors of visualized aggregates.

  A protein "retains its chemical stability" in a biopharmaceutical formulation if the chemical stability at a given time is such that the protein is believed to retain its biological activity as described below. Chemically degraded species can be biologically active and chemically unstable. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Chemical changes may involve size correction (eg, clipping) that can be assessed using SEC, eg, SDS-PAGE and / or matrix-related laser desorption ionization / time-of-flight mass spectrometry (MALDI / TOF MS). Other types of chemical changes include, for example, charge changes that can be evaluated by ion exchange chromatography (eg, those resulting from denaturation).

  If the change in the biological activity of the antibody at a given time is within about 10% (within assay error) of the biological activity shown at the time the pharmaceutical formulation is determined as determined in the antigen binding assay, the antibody is “Retain its biological activity” in the formulation. Other “biological activity” assays for antibodies are described in detail below.

  The term “isotonic” means that the intended formulation has essentially the same osmotic pressure as human blood. In one embodiment, isotonic formulations of the present invention will generally have an osmotic pressure in the range of 250-350 mOsm. In other embodiments, isotonic formulations of the present invention will have an osmotic pressure of about 350-450 mOsm. In yet another embodiment, an isotonic formulation of the present invention will have an osmotic pressure of greater than 450 mOsm. Isotonicity can be measured, for example, using a vapor pressure or freezing osmometer.

  As used herein, “buffer” refers to a buffered solution that resists changes in pH by the action of its acid-base conjugate components. In one embodiment, the buffer of the present invention has a pH in the range of about 4.5 to about 6.0; in another embodiment, about 4.8 to about 5.8; in a further embodiment, about 5. Having a pH of 5. Examples of buffers that would adjust the pH in the range include acetate (eg, sodium acetate), succinate (eg, sodium succinate), gluconate, histidine, citrate and other organic acid buffers Is mentioned. If a freeze-thaw stable formulation is desired, the buffer is preferably not phosphate.

  In the context of the present invention, in the context of the present invention, a “therapeutically effective amount” of an antibody refers to an amount effective for the prevention or treatment of a disorder for which the antibody is effective in therapy. A “disorder” is any condition that would benefit from treatment with an antibody. This includes chronic and acute disorders or diseases, including pathologies that predispose the mammal to the disorder in question. In a preferred embodiment, the “disorder” is a disease involving cells that express CD20.

  A “preservative” is a compound that can be included in a formulation to inherently reduce bacterial action, and thus, for example, facilitates the manufacture of a versatile formulation. Examples of potential preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl group is a long chain compound), and benzethonium chloride. Other types of preservatives include aromatic alcohol analogs such as phenol, butyl alcohol and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. Is mentioned. The most preferred preservative herein is benzyl alcohol.

  The term “antibody” is used in the broadest sense and specifically indicates monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and the desired biological activity. As long as it includes antibody fragments.

  “Antibody fragments” comprise a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab ′, F (ab ′) 2, and Fv fragments; diabodies; linear antibodies; single chain antibody molecules; and multiple antibodies formed from antibody fragments. .

  As used herein, the term “monoclonal antibody” refers to a group of individual antibodies, including substantially homologous antibodies, ie, groups that are identical except for naturally occurring variants that may be present in small amounts. An antibody obtained from Monoclonal antibodies are highly specific and are raised against a single antigenic site. Furthermore, in contrast to normal (polyclonal) antibody preparations that typically contain different antibodies made against different determinants (epitopes), each monoclonal antibody is made against a single determinant of the antigen. . The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous group of antibodies, and is not to be construed as a required group of antibodies by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be made by the hybridoma method first described in Kohler et al., Nature 256: 495 (1975) or can be made by recombinant DNA methods (e.g., U.S. Patent No. No. 4,816,567). “Monoclonal antibodies” can also be obtained using techniques described, for example, in Clackson et al., Nature 352: 624-626 (1991) and Marks et al., J. Mol. Biol. 222: 581-597 (1991). It can be isolated from a phage antibody library.

  “Humanized” forms of non-human (eg, murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Most humanized antibodies have non-human species (donor antibodies) such as mice, rats, rabbits or non-human primates whose residues from the recipient's hypervariable region have the desired specificity, affinity and ability. Is a human immunoglobulin (recipient antibody) that is replaced by residues from the hypervariable region. In some cases, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further purify the antibody. In general, a humanized antibody has all or substantially all of the hypervariable regions equivalent to those of a non-human immunoglobulin, and all or substantially all of the FR regions are of a human immunoglobulin sequence. Substantially all of at least one, typically two variable regions will be included. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details, see Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593- See 596 (1992).

  “Single-chain Fv” or “sFv” antibody fragments comprise the VH and VL regions of an antibody, wherein the regions are present in a single polypeptide chain. In general, the Fv polypeptide further comprises a polypeptide linker between the VH and VL regions that allows the sFv to form the desired structure of antibody binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, N.Y., pp. 269-315 (1994).

  The term “diabody” is a small molecule having two antigen binding sites, the fragment comprising a heavy chain variable region (VH) linked to a light chain variable region (VL) in the same polypeptide chain (VH and VL). Refers to an antibody fragment. By using a linker that is too short to combine between two regions on the same chain, the region is combined with the complementary region of another chain to create two antigen binding sites. Diabodies are described in further detail, for example, in EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).

  The expression “linear antibody” as used herein refers to the antibody described in Zapata et al. Protein Eng. 8 (10): 1057-1062 (1995). That is, these antibodies comprise a set of tandem Fd segments (VH--CH--VH1--CH1) that form a set of antigen binding regions. Linear antibodies can be bispecific or monospecific.

  The formulated antibody is preferably essentially pure and desirably essentially homogeneous (ie, free of contaminating proteins, etc.). By “essentially pure” antibody is meant a composition comprising at least about 90%, preferably at least about 95% by weight of antibody, based on the total weight of the composition. By “essentially homogeneous” antibody is meant a composition comprising at least 99% by weight of antibody relative to the total weight of the composition.

  “Treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those that should be prevented.

  A “mammal” to be treated includes, but is not limited to, mammals, including humans, livestock and farm animals, and zoo sports or pet animals such as dogs, horses, cats, and cows. Any animal classified as an animal.

  “Stress conditions” refer to an environment that is chemically and physically unsuitable for proteins and can impart unacceptable protein stability (eg, heat, shear, chemical stress).

  Size exclusion chromatography is a chromatographic method in which particles are separated based on their size or hydrodynamic volume.

  Dynamic light scattering is a method for measuring the time dependence of protein scattering. Traditionally, the time dependence is handled to obtain the hydrodynamic radius of the molecule.

  “DSC” refers to differential scanning calorimetry: DSC collection parameters include but are not limited to 1 mg / ml protein, 5-80 ° C. scan with 70 ° C./hr scan rate and 15 min wait It can be prewait. A buffer-buffer scan is first obtained and can be subtracted from the raw data. Data can be buffer corrected, protein concentration normalized, and then plotted. Aggregation can prevent reference correction.

  The following examples further illustrate the present invention. The examples are not intended to limit the scope of the invention, but provide a further understanding of the invention.

  The invention is further illustrated by the following examples which are intended to illustrate the invention. These examples are not intended to be construed as limiting the scope of the invention. Many modifications and variations of the present invention are possible in light of the disclosure herein and are therefore within the scope of the invention. The following examples are conducted using standard methods, which are well known and familiar to those skilled in the art, except where otherwise described in detail.

Example 1.1: Platform Formulation Buffer Preparation In one embodiment of the present invention, 4 liters of acetate buffer was prepared. In this embodiment, the final buffer consisted of 50 mM sodium acetate, 0.05 mM EDTA, 51 mM NaCl, 1.0% arginine, 0.02% polysorbate 80, pH 5.5. A buffer was prepared by dissolving sodium acetate trihydrate, disodium edetate (EDTA), polysorbate 80 and L-arginine free base in 3.5 L of deionized water. As soon as the pH was adjusted to 5.5 using 3N HCl, the volume was increased to 4.0 L and the buffer was filtered using a 0.45 μm filter unit. The buffer can then be stored at 2-8 ° C. until use. The phrase “%” in the present application indicates “volume%”.

Example 2.1: Preparation of ofatumumab in platform formulation buffer In one embodiment of the present invention, ofatumumab was formulated into platform formulation (50 mM sodium acetate, 51 mM NaCl, 0.05 mM EDTA, 0.02% polysorbate 80, and 1 (Diafiltrated to 0.0% arginine (free base)) and concentrated to stabilize. Ofatumumab was diafiltered into a platform formulation using a lab-scale tangential flow system with three membranes. After diafiltration into platform buffer, ofatumumab was concentrated to a maximum concentration of 179 mg / mL. It took about 3 days to complete all the process and the yield was 96.1%. A portion of 179 mg / mL was diluted with platform formulation buffer so that a concentration range of approximately 20-179 mg / mL could be studied.

Example 3.1: Preparation of Ofatumumab in Standard and Platform Formula for Direct Appearance (GA) Comparison For direct comparison of appearance over 12 weeks and shaking experiments, anti-CD20 antibody (ofatumumab) was administered at 20 mg / mL. Prepared in standard formulation and platform (one embodiment of the invention) formulation at concentrations.
Standard and platform formulated anti-CD20 antibodies were filtered using a low protein binding 0.2 μm membrane filter. After filtration, each formulation was filled in 3 mL into a 5 cc vial using aseptic operation in a clean draft, capped and crimped. Two vials of each formulation were temperature controlled and placed on a shaker. The vial was shaken at 325 rpm at a temperature of 55 ° C. While heating and shaking, the appearance was observed as described in Example 3.2 at regular intervals over a period of 42 hours. Figures 1 and 2 show the standard and platform formulations, respectively, 18.5 hours after heat shaking. The overall appearance results of the shaking study showed that the standard formulation would produce particles over time when shaken at a temperature of 55 ° C faster than the platform.

Example 3.2: GA, 18.5 hours shaking study-Appearance ofatumumab, 20 and 100 mg / mL
The appearance (GA) of anti-CD20 monoclonal antibody shaking study samples is shown in the table below. GA was achieved using general methods that can be used for IgG antibody solutions that describe color, clarity and visible particulate matter.

Example 4: Measurement of thermal stability of ofatumumab solution in standard and platform buffers by differential scanning calorimetry (DSC) In order to properly finish the test by DSC, scans of buffer alone and protein were obtained. The protein in the standard and platform formulations was diluted to 1 mg / mL as shown in Example 4.1. Data was obtained by setting the DSC to equilibrate for 15 minutes before each scan and scan at 5-80 ° C. at a scan rate of 70 ° C./hour. The volume of the DSC sample cells was approximately 0.5 mL. After obtaining a buffer and protein scan, the buffer scan could then be subtracted from the protein scan. The protein concentration in the sample was obtained to correct the concentration in each scan (see Example 4.2). Tun value, ° C, onset of denaturation, Tm, ° C, at peak transition (denaturation temperature and peak width at T1 / 2, ° C, half height (indicating changes in tertiary structure and cooperativity of transition) ) Was obtained for each formulation of offatumumab (see Example 4.3). The actual DSC scan can be seen in FIG. Based on DSC results, either ofatumumab in standard or platform formulations would be expected to have similar thermal stability because it has a similar DSC profile.

Example 4.1: Sample preparation for biophysical characterization of ofatumumab pH study:

Example 4.2: A280 measurement

Example 4.3: DSC results

  In a more detailed embodiment, an anti-CD20 antibody formulation of the invention treats a subject with a carcinogenic disorder, eg, a B cell lymphoma, eg, a disorder characterized by the presence of tumor cells that express CD20, including NHL. Can be used for Examples of carcinogenic diseases that can be treated and / or prevented include progenitor B cell lymphoblastic leukemia / lymphoma and mature B cell tumors such as B cell chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B cell prolymphocytic leukemia, lymphoid plasma cell lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), cutaneous follicular central lymphoma, marginal zone, including low grade, intermediate grade and high grade FL B cell lymphoma (MALT type, nodal and splenic), hairy cell leukemia, diffuse large B cell lymphoma, Burkitt lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenstrae B cell lymphomas, including mumacroglobulinemia, and anaplastic large cell lymphoma (ALCL), including NHL It is.

  Further examples of non-Hodgkin lymphoma include lymphoma-like granulomatosis, primary exudative lymphoma, intravascular large B-cell lymphoma, mediastinal large B-cell lymphoma, heavy chain disease (γ, μ, and α disease) Lymphomas induced by therapy with immunosuppressive agents, such as cyclosporine-induced lymphoma and methotrexate-induced lymphoma.

  In further embodiments, the anti-CD20 antibody formulations of the invention can be used to treat Hodgkin lymphoma.

  Examples of immune disorders (diseases) involving cells expressing CD20 that can be treated and / or prevented by the anti-CD20 antibody formulations of the invention include autoimmune diseases such as psoriasis, psoriatic arthritis, dermatitis, systemic strong Dermatosis and sclerosis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, respiratory distress syndrome, meningitis, encephalitis, uveitis, glomerulonephritis, eczema, asthma, atherosclerosis, leukocytes Adhesion failure, multiple sclerosis, Raynaud's syndrome, Sjogren's syndrome, juvenile diabetes, Reiter's disease, Behcet's disease, immune complex nephritis, IGA nephropathy, IGM polyneuropathy, immune-mediated thrombocytopenia, eg acute idiopathic Thrombocytopenic purpura and chronic idiopathic thrombocytopenic purpura, hemolytic anemia, myasthenia gravis, lupus nephritis, systemic lupus erythematosus, Rheumatism (RA), atopic dermatitis, pemphigus, Graves' disease, Hashimoto's thyroiditis, Wegner's granulomatosis, Omen's syndrome, chronic renal failure, acute infectious mononucleosis, HIV, and herpes virus-related diseases . Further examples are acute respiratory distress syndrome and chorioretinitis. In addition, other diseases and disorders include those caused or mediated by infection of B cells with viruses such as Epstein-Barr virus (EBV).

As further examples of inflammation, immunity and / or autoimmune diseases in which autoantibodies and / or excessive B lymphocyte activity are prominent and can be treated and / or prevented by the anti-CD20 antibody formulations of the present invention, the following:
Vasculitis and other vascular disorders such as microscopic polyangiitis, Churg-Strauss syndrome, and other ANCA-related vasculitis, nodular polyarteritis, essential cryoglobulinamic vasculitis , Cutaneous leukocyte vasculitis, Kawasaki disease, Takayasu arteritis, giant cell arteritis, Henoch-Schönlein purpura, primary or isolated cerebral vasculitis, erythema nodosum, obstructive thromboangitis, thrombotic platelets Secondary vasculitis (eg, hepatitis B, hepatitis C, Waldenstrom macroglobulinemia, B cells, including reduced purpura (including hemolytic uremic syndrome), and cutaneous leukocyte vasculitis Secondary to tumor, rheumatoid arthritis, Sjogren's syndrome, or systemic lupus erythematosus) Further examples are erythema nodosum, allergic vasculitis, panniculitis, Weber Christian disease, hyperglobulinemia purpura, and Buerger disease; skin disorders such as contact dermatitis, linear IgA dermatosis, Vitiligo, pyoderma gangrenosum, acquired epidermolysis bullosa, pemphigus vulgaris (including scar pemphigoid and bullous pemphigoid), alopecia areata (including systemic alopecia and complete alopecia), Herpetic dermatitis, erythema multiforme, and chronic autoimmune urticaria (including angioedema and urticaria-like vasculitis); immune-mediated cytopenias such as autoimmune neutropenia and red Connective tissue diseases such as CNS lupus, lupus erythematosus lupus, CREST syndrome, mixed connective tissue disease, polymyositis / dermatomyositis, inclusion body myositis, secondary amyloidosis, type I and I Type I cryoglobulinemia, fibromyalgia, phospholipid antibody syndrome, secondary hemophilia, relapsing polychondritis, sarcoidosis, stiff man syndrome, and rheumatic fever; further examples are eosinophilic fascia Arthritis; for example, ankylosing spondylitis, juvenile chronic arthritis, adult Still's disease, and SAPHO syndrome; further examples are sacroiliac, reactive arthritis, Still's disease, and gout; blood diseases such as Aplastic anemia, primary hemolytic anemia (including cold agglutinin syndrome), hemolytic anemia secondary to CLL or systemic lupus erythematosus; POEMS syndrome, pernicious anemia, and Waldenstrom's hyperglobulinic purpura There are further examples: agranulocytosis, autoimmune neutropenia, Franklin disease, Seligmann disease, μ chain disease, Paraneoplastic syndromes secondary to adenomas and lymphomas, and factor VIII inhibitor formation; endocrine disorders such as multi-glandular endocrine disorders, and Addison's disease; further examples are autoimmune hypoglycemia, autoimmune hypothyroidism , Autoimmune insulin syndrome, de Kervan thyroiditis, and insulin receptor antibody-mediated insulin resistance; liver-digestive diseases such as celiac disease, Whipple disease, primary biliary cirrhosis, chronic active hepatitis, And primary sclerosing cholangitis; further examples are autoimmune gastritis; nephropathy such as rapidly progressive glomerulonephritis, streptococcal post-nephritis, Goodpasture syndrome, membranous glomerulonephritis, and cryoglobulin Necrotizing; further examples are microvariable diseases; neuropathy, eg autoimmune nephropathy, polyneuropathy Lambert-Eaton myasthenia syndrome, Sydenham chorea, spinal fold, and Guillain-Barre syndrome; further examples are myelopathy / tropical spastic paraparesis, myasthenia gravis, acute inflammatory prolapse Myelogenous polyneuropathy, and chronic inflammatory demyelinating polyneuropathy; heart and lung diseases such as chronic obstructive pulmonary disease (COPD), fibrotic alveolitis, obstructive bronchiolitis, allergic Aspergillus Cystic fibrosis, Lefler syndrome, myocarditis, and pericarditis; further examples are hypersensitivity pneumonia and paraneoplastic syndromes secondary to lung cancer; allergic diseases such as bronchial asthma and high IgE syndrome Further examples are transient cataracts; ophthalmic diseases such as idiopathic chorioretinitis; infections such as parvovirus type B infection (han -And-socks syndrome); and gynecological-obstetric diseases such as recurrent miscarriage, habitual miscarriage, and intrauterine growth retardation; further examples are secondary to gynecological tumors Paraneoplastic syndromes; male reproductive diseases such as paraneoplastic syndromes secondary to testicular malignancy; and transplantation diseases such as allograft and xenograft rejection, and graft-versus-host disease.

  In one embodiment, the disease involving cells expressing CD20 is ulcerative colitis, Crohn's disease, juvenile diabetes, multiple sclerosis, immune-mediated thrombocytopenia, such as idiopathic thrombocytopenic purpura and Inflammatory, immune and / or self selected from chronic idiopathic thrombocytopenic purpura, hemolytic anemia (including autoimmune hemolytic anemia), myasthenia gravis, systemic sclerosis, or pemphigus vulgaris It is an immune disease.

  The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foreign language specification. Such modifications are intended to fall within the scope of the appended claims.

Claims (14)

OFATUMMAB, 10-100 mM sodium acetate, 25-100 mM sodium chloride, 0.5-5%, which is an anti-CD20 antibody preparation for the treatment of immune diseases and / or autoimmune diseases and is a therapeutically effective amount of anti-CD20 antibody The anti-CD-20 antibody preparation comprising arginine free base, 0.02-0.2 mM EDTA and 0.01-0.2% polysorbate 80, and adjusted to pH 5.0-7.0. The anti-CD20 antibody formulation of claim 1, wherein the anti-CD20 antibody is present in an amount of about 20-300 mg / ml. The anti-CD20 antibody formulation of claim 1, wherein the anti-CD20 antibody is present in an amount of about 50-300 mg / ml. The anti-CD20 antibody formulation of claim 1, wherein the anti-CD20 antibody is present in an amount of about 50 mg / ml.     The anti-CD20 antibody formulation of claim 1 wherein sodium acetate is present in an amount of about 50 mM.     The anti-CD20 antibody formulation of claim 1, wherein the pH of the formulation is about pH 5.5.     The anti-CD20 antibody formulation of claim 1 wherein sodium chloride is present in an amount of about 51 mM.     The anti-CD20 antibody formulation of claim 1 wherein arginine free base is present in an amount of about 1%.     The anti-CD20 antibody formulation of claim 1, wherein EDTA is present in an amount of about 0.05 mM.     The anti-CD20 antibody formulation of claim 1 wherein polysorbate 80 is present in an amount of about 0.02%.     The anti-CD20 antibody preparation according to claim 1, which is administered by a subcutaneous route. Immune diseases are psoriasis, psoriatic arthritis, dermatitis, systemic scleroderma and sclerosis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, respiratory distress syndrome, meningitis, encephalitis, uveitis Inflammation, glomerulonephritis, eczema, asthma, atherosclerosis, leukocyte adhesion failure, multiple sclerosis, Raynaud's syndrome, Sjogren's syndrome, juvenile diabetes, Reiter's disease, Behcet's disease, immune complex nephritis, IGA nephropathy , IGM polyneuropathy, immune-mediated thrombocytopenia, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, hemolytic anemia, myasthenia gravis, lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis (RA) ), Atopic dermatitis, pemphigus, Graves' disease, Hashimoto's thyroiditis, Wegner's granulomatosis, Omen's syndrome, chronic renal failure, acute infectious mononucleosis, acute Sucking syndrome, chorioretinitis, vasculitis, microscopic polyangiitis, Churg-Strauss syndrome, ANCA-related vasculitis, nodular polyarteritis, cryoglobulinamic vasculitis, cutaneous leukocyte fragility Cutaneous leukocytoclastic angiitis, Kawasaki disease, Takayasu arteritis, giant cell arteritis, Henoch-Schönlein purpura, primary or isolated cerebral vasculitis, erythema nodosum, obstructive thromboangiitis, thrombotic platelets Reduced purpura, hemolytic uremic syndrome, cutaneous leukocytoclastic vasculitis, Waldenstrom macroglobulinemia, erythema nodosum, allergic vasculitis, panniculitis, Weber Christian disease, Hyperglobulinemia purpura, Buerger's disease, contact dermatitis, linear IgA dermatosis, white Plaque, gangrenous pyoderma, acquired epidermolysis bullosa, pemphigus vulgaris, scarring pemphigus, bullous pemphigoid, alopecia areata, systemic alopecia, complete alopecia, herpes zoster Erythema, chronic autoimmune urticaria, angioedema, urticaria-like vasculitis, immune-mediated cytopenia, autoimmune neutropenia, erythroblastoma, CNS lupus, discoid erythematous lupus , CREST syndrome, polymyositis / dermatomyositis, inclusion body myositis, secondary amyloidosis, type I and II cryoglobulinemia, fibromyalgia, phospholipid syndrome, secondary hemophilia, relapsing polychondritis Sarcoidosis, stiff man syndrome, rheumatic fever, eosinophilic fasciitis, arthritis, ankylosing spondylitis, juvenile chronic arthritis, adult Still's disease, SAPHO syndrome, sacroiliac, reactive arthritis, Still's disease, gout, Non-forming Blood, primary hemolytic anemia, cold agglutinin syndrome, hemolytic anemia secondary to CLL, POEMS syndrome, pernicious anemia, Waldenstrom hyperglobulinosis purpura, agranulocytosis, autoimmune neutrophil Reduction, Franklin's disease, Seligmann's disease, μ-chain disease, paraneoplastic syndrome secondary to thymoma and lymphoma, factor VIII inhibitor formation, endocrine disorder, polyendocrine endocrine disorder, Addison's disease, autoimmune hypoglycemia , Autoimmune hypothyroidism, autoimmune insulin syndrome, de Kervan thyroiditis, insulin receptor antibody-mediated insulin resistance, liver-digestive disease, celiac disease, whipple disease, primary biliary cirrhosis, chronic Active hepatitis, primary sclerosing cholangitis, gastritis, autoimmune gastritis, nephropathy, rapidly progressive glomerulonephritis, post-streptococcal kidney Inflammation, Goodpascher's syndrome, membranous glomerulonephritis, cryoglobulinemia nephritis, autoimmune nephropathy, polyneuropathy, Lambert-Eaton myasthenic syndrome, Sydenham chorea, spinal cord fistula, Guillain Valley syndrome, myelopathy / tropical spastic paraparesis, myasthenia gravis, acute inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyneuropathy, fibrotic alveolitis, obstructive bronchiolitis, Allergic aspergillosis, cystic fibrosis, Lefler syndrome, myocarditis, pericarditis, hypersensitivity pneumonia, paraneoplastic syndrome secondary to lung cancer, bronchial asthma, high IgE syndrome, transient cataract, idiopathic chorioretinitis, Recurrent miscarriage, habitual miscarriage, intrauterine growth retardation, paraneoplastic syndrome secondary to gynecological tumors, paraneoplastic syndrome secondary to testicular malignancy, allograft and xenotransplantation Absolute reaction, and barrel depend from the group consisting of graft-versus-host disease, anti-CD20 antibody formulation according to any one of claims 1-11. Immune diseases are systemic scleroderma, systemic sclerosis, multiple sclerosis, Sjogren's syndrome, myasthenia gravis, lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis (RA), pemphigus, chronic renal failure, blood vessels The anti-CD20 antibody preparation according to any one of claims 1 to 12, which is selected from the group consisting of flames. The anti-CD20 antibody preparation according to any one of claims 1 to 13 , wherein the immune disease is selected from the group consisting of vasculitis including ANCA-related vasculitis and pemphigus including pemphigus vulgaris.

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