UA126561C2 - Anti-c5 antibodies and methods of use - Google Patents

Abstract

The invention provides anti-C5 antibodies and methods of using the same. In some embodiments, an isolated anti-C5 antibody of the present invention binds to an epitope within the beta chain of C5 with a higher affinity at neutral pH than at acidic pH. The invention also provides isolated nucleic acids encoding an anti-C5 antibody of the present invention. The invention also provides host cells comprising a nucleic acid of the present invention. The invention also provides a method of producing an antibody comprising culturing a host cell of the present invention so that the antibody is produced. The invention further provides a method of producing an anti-C5 antibody comprising immunizing an animal against a polypeptide which comprises the MG1-MG2 domain of the beta chain of C5. Anti-C5 antibodies of the present invention may be for use as a medicament.

Description

1006- ch. 3061. 015-80422. g as ZO О16-56499.

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Fig. 18

This invention relates to antibodies against C5 and methods of their use. Prior art

The complement system plays a central role in the clearance of immune complexes and in immune responses to infectious agents, foreign antigens, virus-infected cells, and tumor cells.

There are approximately 25-30 complement proteins that occur as assemblies of complexes of plasma proteins and membrane cofactors. Complement components achieve their immune protective functions by interacting in a series of extensive enzymatic cleavages and membrane binding events. As a result of complement reaction cascades, products with opsonic, immunoregulatory and lytic functions are produced.

It is now generally accepted that the complement system can be activated by three distinct pathways: the classical pathway, the lectin pathway, and the alternative pathway. These pathways have many components in common, and although they have different initial steps, they converge and share the same final complement components (C5 to C9) that are responsible for the activation and destruction of target cells.

The classical pathway is usually activated due to the formation of antigen-antibody complexes.

Regardless, the first step in the activation of the lectin pathway is the binding of specific lectins, such as mannan-binding lectin (MB), H-ficolin, M-ficolin, I-ficolin, and the C-type lectin, CI-11 . In contrast, the alternative pathway spontaneously undergoes low-level reverse activation that can be easily amplified on foreign or other abnormal surfaces (bacteria, yeast, virus-infected cells, or damaged tissues). These pathways converge at the point where the complement component CZ is cleaved by an active protease, resulting in CZa and CZr.

SZa is an anaphylatoxin. CSF binds to bacterial and other cells, as well as certain viruses and immune complexes, and marks them for removal from circulation (a role known as opsonin). SZOI also forms a complex with other components, as a result of which C5 convertase is formed, which cleaves C5 into C5a and C5p.

C5 is a 190 kDa protein found in healthy serum at a concentration of approximately 80 μg/mL (0.4 μM). C5 is glycosylated by approximately 1.5-3 95 of its mass related to carbohydrate. Mature C5 is a 115 kDa alpha chain heterodimer that has a disulfide

From the connection with the beta chain of 75 kDa. C5 is synthesized as a single-chain precursor protein (pro-C5 precursor) of 1,676 amino acids (see, for example, Patent Document 1 and Patent Document 2). The pro-C5 precursor is cleaved to form the beta chain as the amino-terminal fragment and the alpha chain as the carboxyl-terminal fragment. These polypeptide fragments, the alpha chain and the beta chain, are joined together by a disulfide bond, and they make up the mature C5 protein.

Mature C5 is cleaved into C5ba and C56p fragments during complement pathway activation.

Cb5a is cleaved from the C5 alpha chain by C5-convertase as an amino-terminal fragment that includes the first 74 amino acids of the alpha chain. The rest of the mature C5 is a fragment of C5B, which contains the rest of the alpha chain linked by a disulfide bond to the beta chain. Approximately 20 95 of the mass of 11 kDa C5a is a carbohydrate.

Sba is another anaphylatoxin. C5B combines with Cb, C7, C8 and C9, forming a membrane-attacking complex (MAS, C5p-9, terminal complement complex (TCS)) on the surface of the target cell. When sufficient MACs are inserted into target cell membranes, MAC pores are formed that mediate rapid osmotic lysis of target cells.

As mentioned above, СЗа and Сб5а are anaphylatoxins. They can initiate mast cell degranulation, resulting in the release of histamine and other inflammatory mediators, which causes smooth muscle contraction, increased vascular permeability, leukocyte activation, and other inflammatory events, including cell proliferation, resulting in increased parenchymal cell content. Cb5a also functions as a chemotactic peptide that attracts granulocytes such as neutrophils, eosinophils, basophils, and monocytes to the site of complement activation.

The activity of Sba is regulated by the plasma enzyme carboxypeptidase M, which removes the carboxy-terminal arginine from Sba, thereby forming the derivative Sba-des-Agu. Sba-des-Ag9 shows only 195 anaphylactic activity and polymorphonuclear chemotactic activity of unmodified Ca.

Although a properly functioning complement system confers a powerful defense against infectious microbes, inappropriate complement regulation or activation is thought to be responsible for the pathogenesis of a variety of disorders that include, for example, rheumatoid arthritis (RA); lupus nephritis; ischemia-reperfusion injury; paroxysmal nocturnal hemoglobinuria (PMH); atypical hemolytic uremic syndrome (aNnib); dense sediment disease (000); macular degeneration (eg, age-related macular degeneration (AMD)); syndrome of hemolysis, increased activity of liver enzymes and thrombocytopenia (NEGI R-syndrome)); thrombotic thrombocytopenic purpura (TTP); spontaneous loss of the fetus; immunodeficient vasculitis; epidermolysis bullosa; recurrent fetal loss; multiple sclerosis (M); brain injury; and damage resulting from myocardial infarction, artificial circulation, and hemodialysis (see, for example, Non-Patent Document 1). Therefore, inhibition of excessive or uncontrolled activation of the complement cascade may provide clinical benefits for patients with such disorders.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare blood disorder in which red blood cells are damaged and, as a result, break down more quickly than healthy red blood cells. PMN is the result of clonal reproduction of hematopoietic stem cells with somatic mutations in the РИС-A (phosphatidylinositol glycan class A) gene, which is located on the X chromosome. Mutations in RISH-A cause an early block in the synthesis of glycosylphosphatidylinositol (GRY), a molecule that is essential for anchoring many proteins to cell surfaces. Therefore, blood cells in PMN are deficient in CRI-fixed proteins, which include proteins that regulate complement, CO55 and CO59. Under normal circumstances, these complement-regulating proteins block the formation of MAC on cell surfaces, thereby preventing erythrocyte lysis. Absence of RiI-fixed proteins causes complement-mediated hemolysis in PMN.

PMN is characterized by hemolytic anemia (reduced number of red blood cells), hemoglobinuria (the presence of hemoglobin in the urine, especially evident after sleep), and hemoglobinemia (the presence of hemoglobin in the bloodstream). Individuals suffering from PMN are known to have bouts of the disease, defined here as cases of dark urine. Hemolytic anemia is a consequence of intravascular destruction of erythrocytes by complement components. Other known symptoms include dysphasia, fatigue, impotence, thrombosis, and intermittent abdominal pain.

Eculizumab is a humanized monoclonal antibody directed against complement protein C5 and is the first therapeutic agent approved for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome

Zo (anyv) (see, for example, non-patent document 2). Eculizumab inhibits the cleavage of C5 by C5-convertase into C5ba and C5p, which prevents the formation of the final complement complex C5p-9.

Both Sba and C55-9 induce terminal complement-mediated events that are characteristic of PMN and anizh (see also U.S. Patent 3, U.S. Patent 4, U.S. Patent 5, and U.S. Patent 6).

Antibodies against C5 have been described in several reports. For example, patent document 7 describes an anti-C5 antibody that binds to the C5 alpha chain but does not bind to C5a and blocks C5 activation, but patent document 8 describes an anti-C5 monoclonal antibody that inhibits the formation of C5a . On the other hand, patent document 9 describes an anti-C5 antibody that recognizes the proteolytic site for C5-convertase on the C5 alpha chain and inhibits the conversion of C5 to C5a and C5p. Patent Document 10 describes an anti-C5 antibody that has an affinity constant of at least 1 x 107 M".

Antibodies (IdS) bind to the neonatal Es receptor (EsVp) and have a long retention time in plasma. Binding of db: to PsAp is usually observed under acidic conditions (eg, pH 6.0) and is rarely observed under neutral conditions (eg, pH 7.4).

Usually, Is are nonspecifically incorporated into cells by endocytosis, and they return to cell surfaces as a result of binding to endosomal EsAp under acidic conditions in endosomes. After that, IDS dissociates from EsAp under neutral conditions in the plasma. dDS, which failed to connect with EsAp, are destroyed in lysosomes. When the ability of Ids to bind to EsVp under acidic conditions is canceled due to mutations in its Es region, then (DS does not return with the endosomes back to the plasma, which causes a noticeable deterioration of Ids retention in plasma. In order to improve Ids retention; in plasma, a method has been reported that increases their binding to GsAp under acidic conditions. When the binding of Id(Ci) to EsAp under acidic conditions is improved due to an amino acid substitution in its Es region, then Da is more efficiently returned from the endosome back to plasma, thereby showing improved plasma retention.Meanwhile, it was also reported that Idas with enhanced binding to EsAp under neutral conditions does not dissociate from EsAp under neutral conditions in plasma, even when it returns to the cell surface due to its binding with PsNAP under acidic conditions in endosomes, and as a consequence its retention in plasma is unchanged or sometimes impaired (see, for example, non-patent document 3; non-patent document 4; because it is a non-patent document 5).

Antibodies that bind to antigens in a pH-dependent manner have recently been reported (see, for example, Patent Document 11 and Patent Document 12). These antibodies strongly bind to antigens under neutral conditions in plasma and dissociate from antigens under acidic conditions in endosomes. After dissociation from antigens, antibodies acquire the ability to bind to antigens once again, when they return from the endosomes back to the plasma with the help of EsAp. Therefore, a single antibody molecule can repeatedly bind to numerous antigen molecules. In general, the retention of the antigen in the plasma is much shorter than the retention of the antibody, which has the aforementioned mechanism of mediated EsNAP recycling. Therefore, when an antigen binds to an antibody, then the antigen usually exhibits prolonged retention in the plasma, resulting in an increase in the concentration of the antigen in the plasma. On the other hand, it has been reported that the above-described antibodies that bind to antigens in a pH-dependent manner eliminate antigens from plasma faster than conventional antibodies because they dissociate from antigens inside endosomes during the process of EsNAP-mediated recycling. Patent Document 13 also describes a computer simulation analysis that demonstrates that an antibody with pH-dependent binding directed against C5 can prolong the knockdown effect on the antigen.

List of citations

Patent documents

Patent Document 1 - US Patent Mo 6,355,245

Patent Document 2 - US Patent Mo 7,432,356

Patent document 3-MO 2005/074607

Patent document 4-40 2007/106585

Patent document 5-40 2008/069889

Patent document 6--MO 2010/054403

Patent document 7-MO 95/29697

Patent document 8-MO 02/30985

Patent document 9-0 2004/007553

Patent document 10-М/О 2010/015608

Patent document 11-М/О 2009/125825

Patent document 12-М/О 2011/122011

Patent document 13-М/О 2011/111007

Non-patent documents

Non-patent document 1-Noiegz ei ai!., Ittipoi. German 223:300-316 (2008)

Non-patent document 2-YuOtuyiCyk ei aY., Te Opsoiodivi 13(9):993-1000 (2008)

Non-patent document 3-Ueipa ei ai!., / Ittipoi. 182(12): 7663-7671 (2009)

Non-Patent Document 4-Oana-Mappap ei a!., In Vioi. Snet 282(3): 1709-1717 (2007)

Non-patent document 5-OaiHAsdpa ei ai., u. And so on. 169(9):5171-5180 (2002)

The essence of the invention

Technical task

The task of the invention is to obtain antibodies against C5 and methods of their use.

Solving the problem

The invention provides antibodies against C5 and methods of their use.

In some embodiments, the selected anti-C5 antibody of the present invention binds to an epitope within the C5 beta chain. In some embodiments, the isolated anti-C5 antibody of the present invention binds to an epitope within the domain of MC21-M(2 beta-chain of C5. In some embodiments, the isolated anti-C5 antibody of the present invention binds to an epitope within the fragment that consists of amino acids 33-124 of the beta chain (5EO IJU MO: 40) of C5 In some embodiments, the selected anti-C5 antibody of the present invention binds to an epitope within the beta chain (ZEO IJU MO: 40) of C5, which includes at least one fragment , selected from the group consisting of amino acids 47-57, 70-76 and 107-110. In some embodiments, the isolated anti-C5 antibody of the present invention binds to an epitope within the beta-chain fragment (ZEO IU MO: 40) of C5 , which includes at least one amino acid residue selected from the group consisting of (11048, Azro1, Niv70, Niv72, I uzIOOU and Nivi 10 sequence of FEO IJOO MO: 40. In further embodiments, the antibody binds to C5 with higher affinity at at neutral pH than at acidic pH. In further embodiments, the antibody binds to C5 with higher affinity at pH 7.4 than at pH 5.8. In another embodiment, the isolated anti-C5 antibody of the present invention binds to the same epitope as the antibody listed in Table 2. In further embodiments, the antibody binds to the same epitope as the antibody listed in Table 2, with higher affinity at pH 7.4 than at pH 5.8. In a further embodiment, the anti-C5 antibody of the present invention binds to the same epitope as the antibody listed in Table 7 or 8. In further embodiments, the antibody binds to the same epitope as the antibody listed in Table 7 or 8, with higher affinity at pH 7.4 than at pH 5.8.

In certain embodiments, the antibody against C 5 of the present invention competes for binding to

C5 with an antibody that includes a MH and MI pair selected from: (a) MH 5EO IO MO: I and MI 5EO IYUO MO: 11; (B) MN 5EO IYUO MO: 5 and MI 5EO IYU MO: 15; (c) MN 5EO IO MO: 4 and MI 5EO IYUO MO: 14; (4) MN 5EO

IO MO: 6 and MI 5EO IU MO: 16; (is) MN 5EO IO MO: 2 and MI 5EO IO MO: 12; (I) MN 5EO IU MO: Z ta

MI ZEO IO MO: 13; (9) MN 5EO IO MO: 9 and MI 5EO IU MO: 19; (p) MN 5EO IO MO: 7 and MI 5EO IU

MO: 17; (Y) MN BEO IO MO: 8 and MI 5EO IO MO: 18; and () MN 5EO IO MO: 10 and MI 5EO IO MO: 20.

In further embodiments, the anti-C5 antibody binds to C5 with higher affinity at neutral pH than at acidic pH. In further embodiments, the anti-C5 antibody binds to C5 with higher affinity at pH 7.4 than at pH 5.8.

In some embodiments, the isolated antibody against C5 of the present invention has a characteristic selected from the group consisting of: (a) the antibody contacts amino acids 051 and KI09 of C5 (5EBO IO MO: 39); (p) the affinity of the antibody to C5 (5EO IU MO: 39) is higher than the affinity of the antibody to the mutant C5, which includes the E48A substitution in 5EO IU MO: 39; or (c) the antibody binds to the C5 protein, which consists of the amino acid sequence FEO IU MO: 39, at pH 7.4, but does not bind to the C5 protein, which consists of the amino acid sequence 5EO IO MO: 39 with the substitution H72U, at pH 7.4. In further embodiments, the antibody binds to C5 with higher affinity at neutral pH than at acidic pH. In further embodiments, the antibody binds to C5 with higher affinity at pH 7.4 than at pH 5.8.

In some embodiments, the selected anti-C5 antibody of the present invention inhibits activation

C5. In some embodiments, the selected anti-C5 antibody of the present invention inhibits the activation of the HB85H C5 variant. In some embodiments, the selected anti-C5 antibody of the present invention is a monoclonal antibody. In some embodiments, the isolated anti-C5 antibody of the present invention is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the selected anti-C5 antibody of the present invention is an antibody fragment that binds to C5. In some embodiments, the selected anti-C5 antibody of the present invention is a full-length yes or Id antibody.

In some embodiments, the isolated antibody against C 5 of the present invention includes (a) NUV-

NZ, which includes the amino acid sequence OXIS1UH" x with RTINAMXAa x 5, where Xi is C or A, X" is

M, O or O, Xz is T or Y, Xa is M or H, X5 is Y or mm (ZEO I MO: 128), (5) NUV-I 3, which includes the amino acid sequence ОХХХХ2Ма55USМХ». where X is 5, C, M or T, X" is E or

K, Xz is A, T or H (5EO IO MO: 131), and (c) NUV-H2, which includes the amino acid sequence

ХХХгTaBOАХзЗУХАХ5МХКа, where X is C, A or C, X» is Y or E, Xz is T, O or E, Xa is Y,

K or O, X5 is 5, O or E, Xv is A or M (5EO IO MO: 127).

In some embodiments, the isolated antibody against C 5 of the present invention includes (a) NUV-

NO, which includes the amino acid sequence 55UUCH1 x 2, where Xy is M or U, X" -tsne Sabo A (5BO 10 MO: 126) (B) NMBA-H2, which includes the amino acid sequence

ХХХгТАБОАХзЗУХАХ5МУХвКа, where X is C, A or C, Xo is Y or EC, Xz is T, O or E, Xa is Y,

K or O, X5 is 5, O or E, Xv is A or M (5EO IJU MO: 127), and (c) NUV-NZ, which includes the amino acid sequence OX ХХ»2 x zRTNAMXAa x 5, where X - is C or A, Xe is M, O or 0, Xz is T or U, Xa is Y or H, Xv is Y or mm (5EO IO MO: 128). In further embodiments, the antibody comprises (a) NMV-1 1, which comprises the amino acid sequence ХІАБОХ»ХХХХХХХА, where Xy is O or NO, Xg is M, O or C, Xz is C or 5, Xa is O, K or 5 (5EO IU MO: 129); (c) NUV-1I2, which includes the amino acid sequence SAU x 2Xz5, where Xy is K, E or T, Xo" is I or T, Xz is A, H, E or O (5EO IO MO: 130) ; and (c) NUV-I3, which includes the amino acid sequence

OKHITHx2Ua55USMH", where Xy is 5, C, M or T, X" is E or K, Xz is A, T or H (5EO IO MO: 131).

In some embodiments, the isolated antibody against C 5 of the present invention includes (a) NUV-

Y1, which includes the amino acid sequence ХІАБОХ»ХХзохХХАІ A, where Xy is O or B, Xg is M, o or C, Xz is B or 5, Xa is 0, K or 5 (5EO IO MO: 129); (B) NUV-I2, which includes the amino acid sequence SABH I x 2Xz5, where Xy is K, E or T, X»- thisY or T, Xz- thisA, H, E or OO (5EBO IO MO: 130); and (c) NMUV-I3, which includes the amino acid sequence

OKHITHx2Ua55USMH", where Xy is 5, C, M or T, X" is E or K, Xz is A, T or H (5EO IO MO: 131).

In some embodiments, the selected anti-C5 antibody of the present invention includes the framework region of the ENT variable domain of the heavy chain, which includes the amino acid sequence 60 according to any of BEO IO MO: 132-134; EN2, which includes the amino acid sequence according to any of 5EO IYUO MO: 135-136; EVZ, which includes the amino acid sequence of any of the 5EO IU

MO: 137-139; and EN4, which includes the amino acid sequence of any of BEO IU MO: 140-141. In some embodiments, the isolated anti-C5 antibody of the present invention includes the EVIT framework region of the light chain variable domain, which includes the amino acid sequence of any of 5BO I MO: 142-143; EB2, which includes the amino acid sequence according to any of 5BEO AND MO: 144-145; BEVZ, which includes the amino acid sequence according to any of 5EBEO IO MO: 146-147; and EB4, which includes the amino acid sequence 5EO IO MO: 148.

In some embodiments, the selected anti-C5 antibody of the present invention includes (a) a UN sequence that is at least 95 to 95 identical to the amino acid sequence of any of the 5EO IO MO: 10, 106-110; (5) an MI sequence that is at least 95% identical to the amino acid sequence of any of 5EO IO MO: 20, 111-113; or (c) a UN sequence as in (a) and an MI sequence as in (B). In further embodiments, the antibody includes the sequence MN according to any of 5EO IU MO: 10, 106-110. In further embodiments, the antibody comprises an MI sequence. according to any of 5EO 10 MO: 20,111-113.

The invention provides an antibody that includes the MH sequence according to any of the 5EO IU MO: 10,106-110, and the MI sequence according to any of the 5EO IU MO: 20,111-113.

The invention also provides isolated nucleic acids that encode an antibody against C5 of the present invention. The invention also provides host cells that contain the nucleic acid of the present invention. The invention also provides a method of obtaining an antibody, which includes the cultivation of the host cell of the present invention, so that the antibody is produced.

The invention further proposes a method of obtaining antibodies against C5. In some embodiments, the method comprises immunizing an animal against a polypeptide that includes a Ma1-

MOa2g (5EBEO IU MO: 43) beta-chain C5. In some embodiments, the method includes immunizing an animal against a polypeptide that includes a region that corresponds to amino acids at positions 33 to 124 of the beta chain (ZEO IU MO: 40) C5. In some embodiments, the method includes immunizing the animal against a polypeptide that includes at least one fragment selected from amino acids 47-57, 70-76, and 107-110 of the beta chain (ZEO IO MO: 40) of C5. In some embodiments, the method includes immunizing an animal against a polypeptide that includes

A fragment of the beta chain (5EO AND MO: 40) of C5, which includes at least one amino acid selected from Sis48, Azrobi1, Ni 70, Ni 72, uzIO9Tanivo.

The invention also provides a pharmaceutical composition that includes an antibody against C5 of the present invention and a pharmaceutically acceptable carrier.

Antibodies against C5 of the present invention can be prescribed for use as a medicinal product. Antibodies against C5 of the present invention may be prescribed for use in the treatment of a complement-mediated disease or condition involving excessive or uncontrolled activation of C5. Antibodies against C5 of the present invention can be prescribed for use in increasing the clearance of C5 from plasma.

Antibodies against C5 of the present invention can be used in the production of medicinal products. In some embodiments, the medicament is for the treatment of a complement-mediated disease or condition involving excessive or uncontrolled activation

C5. In some embodiments, the drug is intended to increase clearance of C5 from plasma.

The invention also provides a method of treating an individual who has a complement-mediated disease or condition involving excessive or uncontrolled activation of C5.

In some embodiments, the method includes administering to the individual an effective amount of the antibody against C5 of the present invention. The invention also offers a method of increasing clearance

C5 from the individual's plasma. In some embodiments, the method includes administering to the individual an effective amount of the antibody against C5 of the present invention to increase the clearance of C5 from the plasma.

Brief description of illustrative material

Fig. 1

Figure 1 illustrates the epitope-specific sorting of antibodies against C5, as described in Example 2.2. Antibodies grouped in one epitope group are outlined with a bold line.

Fig. 2A

Figure 2A illustrates the sensograms of VIASONE (registered trademark) antibodies against

C5 at pH 7.4 (solid line) and at pH 5.8 (dashed line) to evaluate the dependence on pH, as described in Example 3.2. SRAOZO5, SEAOZO7, SEAOZbb, SEAOBOY, SEAO538 and SEAOB599 are antibodies grouped in epitope group C, as described in Example 2.2.

Fig. 28

Figure 28 illustrates the sensograms of VIASONE (registered trademark) antibodies against

C5 at pH 7.4 (solid line) and at pH 5.8 (dashed line) to evaluate the dependence on pH, as described in Example 3.2. SEAObb6b, SEAObE72, and SEAOb675 are antibodies clustered in the C epitope group, and SEAOZ330 and SEAOZ41 are antibodies clustered in the B epitope group, as described in

Examples 2.2. 305I 05 is a humanized CEAOZO5 antibody, as described in Example 2.3.

Fig. WITH

Figure C illustrates Western blot analysis of fragments derived from the beta chain

C5 (amino acids 19-180, 161-340, 321-500 and 481-660 5EO IO MO: 40) and which are fused to the WTO-label as described in Example 4.1. SEAOZO5, SEAOZO7, SEAOZbb, SEAOBOY, SEAOB5Z38, SEAOB99,

SEAObbb, SEAOb72, and SEAObB75 are antibodies grouped in epitope group C Antibody against ST is a positive control antibody. The position of C5 fragments (46-49 kDa) fused with OT is indicated by an arrow.

Fig. 4

Figure 4 illustrates the sensograms of VIASOVE (registered trademark) antibodies against

C5 in relation to the Ma21-MO2 domain of the beta chain of C5, as described in Example 4.3. The top panel shows the results of CEAOZO5 (solid line), CEAOZO7 (dashed line), CEAOZbb (dashed line) and eculizumab (dotted line), Middle panel shows the results of CEAOBO1 (solid line), CEAO599 (dashed line), CEAO538 (dashed line) and eculizumab (dashed line). The bottom panel shows the results of SEAOb66 (solid line), SEAO672 (dashed line), SEAObE75 (dashed line) and eculizumab (dashed line). SEAOZO5, SEAOZO7, SEAOZbb, SEAOBOY, SEAOB38, SEAOB99, SEAObbb, SEAOb672 and

SEAOb75 is an antibody grouped in epitope group C. Eculizumab is a control antibody against

C5.

Fig. BA

Figure 5A illustrates Western blot analysis of peptide fragments originating from the Ma1-Ma2 domain (amino acids 33-124, 45-124, 52-124, 33-111, 33-108, and 45-111 5EO IUO MO: 40) and which are merged with label 251 as described in Example 4.4. The anti-451 antibody is used as the antibody for the reaction. The position of the fused from 51 C5 fragments (35-37 kDa) is indicated by an arrow.

Fig. 5B

Figure 5B illustrates Western blot analysis of peptide fragments originating from the Ma1-Ma2 domain (amino acids 33-124, 45-124, 52-124, 393-111, 33-108 and 45-111 5EO and MO: 40 ) and which are merged with label 25, as described in Example 4.4. SEAOZO5 is used as an antibody for the reaction.

Fig. 5C

Figure 5C summarizes the binding reactions of anti-C5 antibodies to fragments derived from the beta-chain of C5 as described in Example 4.4. Fragments to which anti-C5 antibodies bind are grouped into epitope group C (SEAOZO5, SEAOZO7, SEAOZb6b, SEAOBO1, SEAOB538, SEAOB99,

SEAObbb, SEAOb72 and SEAOb75) are shown in gray, fragments with which they do not bind are shown in white.

Fig. 6

Figure 6 illustrates Western blot analysis of C5 point mutants in which E48, 051 and

K109 of the beta chain is substituted with alanine (E484A, 051A and K1O9A, respectively), as described in Example 4.5. In the left panel, eculizumab (an anti-C5 antibody that binds to the alpha chain) is used as the reaction antibody, and the position of the C5 alpha chain (approximately 113 kDa) is indicated by an arrow. In the right panel, CEAOZO5 (clustered in epitope group C, it binds to the beta chain) is used as an antibody for the reaction, and the position of the beta chain

C5 (approximately 74 kDa) is indicated by an arrowhead.

Fig. 7

Figure 7 presents VIASONVE (registered trademark) sensograms demonstrating the interaction of eculizumab-P76004 (upper panel) or 305 05 (lower panel) with C5 mutants as described in Example 4.6. Sensograms were obtained by injecting C5-m/i (thick solid curve), C5-E48A (curve with short dashes), C5-Y51A (curve with long dashes) and C5-KIO9A (thin solid curve), respectively, along the sensor surface with immobilized eculizumab-P76004 or 3051 05. Eculizumab is a control antibody against

C5. 305I 05 is a humanized SEAOZOB5 antibody (clustered in epitope group C) as described in

Examples 2.3.

Fig. 8

Figure 8 presents VIASONVE (registered trademark) sensograms showing the interaction of 305 05 with Ni-substituted C5 mutants to assess pH dependence as described in Example 4.7. Sensograms were obtained as a result of injection of C5-m/i (thick solid curve), C5-H7OM (curve with long dashes), C5-H72U (curve with short dashes),

C5-NIT1OM (dashed curve) and C5-H7OM--NITOM (thin solid curve), respectively, along the sensor surface with immobilized 305105. To assess pH-dependent interactions, the antibody-antigen complexes were allowed to dissociate at pH 7.4, followed by additional dissociation at pH 5.8 (indicated by an arrow).

Fig. BY

Figure 9A illustrates inhibition of complement-activated liposome lysis by anti-C5 antibodies as described in Example 5.1. The results are shown for antibodies SEAOZO5, SEAOZ07,

SEAOzbb, SEAOBOY, SEAO5Z38, SEAOB99, SEAObbb, SEAOb72 and SEAOb75, grouped in epitope group C, as described in Example 2.2.

Fig. 98

Figure 9B illustrates inhibition of complement-activated liposome lysis by anti-C5 antibodies as described in Example 5.1. The results are shown for antibodies SEAOZZ3ZO and

SEAOZA1, grouped in epitope group B, as described in Example 2.2.

Fig. 10A

Figure 10A illustrates inhibition by anti-C5 antibodies of C5ba generation as described in

Examples 5.2. C5a concentrations were quantified in supernatants obtained during the liposome lysis assay shown in Figure 9A.

Fig. 108

Figure 108 illustrates inhibition by anti-C5 antibodies of C5ba generation as described in

Examples 5.2. C5a concentrations were quantified in the supernatants obtained during the liposome lysis assay shown in Figure 98.

Fig. 11

Figure 11 illustrates the inhibition by antibodies against C5 of complement-activated hemolysis, as described in Example 5.3. Complements were activated using the classical pathway.

Fig. 12

Figure 12 illustrates the inhibition by antibodies against C5 of complement-activated hemolysis, as described in Example 5.4. Complements were activated using an alternative pathway.

Fig. 13

Figure 13 illustrates the temporal dynamics of human C5 plasma concentration after intravenous administration of only human C5 or human C5 and anti-human C5 antibodies during assessment of C5 clearance in mice as described in Example 6.2. SEAOZO5, SEAOZO7, SEAOZ66,

SEAOBi, SEAOB538, SEAOB99, SEAObbb, SEAOb72, and SEAOb75 are antibodies clustered in epitope group C, and SEAOZZO and SEAOZ341 are antibodies clustered in epitope group B, as described in Example 2.2.

Fig. 14

Figure 14 illustrates the temporal dynamics of the concentration of human anti-C5 antibody in plasma after intravenous administration of human C5 and anti-human C5 antibody during the evaluation of the pharmacokinetics of the antibody in mice as described in Example 6.3. SEAOZO5, SEAOZO7, SEdDOZ66,

SEAOBi, SEAOB538, SEAOB99, SEAObbb, SEAObE7T2, and SEAOb75 are antibodies clustered in epitope group C, and SEAOZZ3O and SEAOZ41 are antibodies clustered in epitope group B, as described in Example 2.2.

Fig. 15

Figure 15 illustrates inhibition of complement-activated liposome lysis by anti-C5 antibodies as described in Example 9.1. Results for antibodies 3051 015-5I(2422, 305 016-52x422, 305I 018-5Ш0422, 305I 019-52:422, 3051 020-512422 and 3051 O20-512:1115 are shown.

Fig. 16

Figure 16 illustrates inhibition of complement-activated liposome lysis by anti-C5 antibodies as described in Example 9.1. The results for antibodies Z305I015-za115Ta305I 023-51 (3429) are shown.

Fig. 17

Figure 17 illustrates inhibition of complement-activated liposome lysis by anti-C5 antibodies as described in Example 9.1. Results for antibodies 305I O22-5(2115, 305 022-5Ц12422, 305I O23-51231115 and 305I 023-5123422 are shown.

Fig. 18

Figure 18 illustrates the inhibition by antibodies against C5 of the generation of C5a, as described in Example 9.2. Sba concentrations were quantified in supernatants obtained during the liposome lysis assay shown in Figure 15.

Fig. 19

Figure 19 illustrates the inhibition by antibodies against C5 of the generation of C5a, as described in Example 9.2. Sba concentrations were quantified in the supernatants obtained during the liposome lysis assay shown in Figure 16.

Fig. 20

Figure 20 illustrates the inhibition of complement activity in monkey plasma by antibodies against

C5, as described in Example 9.3. Antibodies against C5 were administered to Javan macaques, and complement activity in monkey plasma was measured using a hemolysis assay.

Fig. 21

Figure 21 illustrates the inhibition of the biological activity of native C5 (M/T) and C5 variants (MI451, 844920, M802I, B885H, BZ280, 0966, 51310M and E1437O0) by an anti-C5 antibody (eculizumab) as described in Example 9.4.

Fig. 22

Figure 22 illustrates the inhibition of the biological activity of natural C5 (M/T) and variants of C5 (M145I, 84490, M802I, vB885H, 9280, 0966, 51310M and E1437O0) by an antibody against C5 (variant 305), as described in Example 9.4.

Fig. 23

Figure 23 illustrates the inhibition by anti-C5 antibodies (WMO441 and variant 305) of complement-activated liposome lysis as described in Example 9.5.

Fig. 24

Figure 24 illustrates the temporal dynamics of the plasma concentration of C5 in Javan macaques after intravenous administration of an anti-human C5 antibody during the assessment of C5 clearance in Javan macaques as described in Example 10.2.

Fig. 25

Figure 25 illustrates the time dynamics of the concentration of anti-human C5 antibody in plasma after intravenous administration of an anti-human C5 antibody during the evaluation of the pharmacokinetics of the antibody in Javan macaques, as described in Example 10.3.

Fig. 26

Figures 26A and 26B illustrate the crystal structure of Rab 305 bound to the (PS5)- domain

Human Ma C5 as described in Example 11.6. Figure 26A illustrates an asymmetric unit. Ma

Zo is shown as a surface and Raf 305 is shown as ribbons (dark gray - heavy chain, light gray - light chain). Figure 268 illustrates molecules 1 and 2 superimposed on each other (dark gray - molecule 1, light gray - molecule 2).

Fig. 27A

Figure 27A illustrates the contact site epitope for Rab 305 on the MO domain as described in Example 11.6. Figure 27A illustrates the mapping of epitopes in the amino acid sequence of MO (dark gray - closer than 3.0 angstroms, light gray - closer than 4.5 angstroms).

Fig. 278

Figure 27B illustrates the contact site epitope for Rab 305 on the MO domain as described in Example 11.6. Figure 27B illustrates epithet mapping in the crystal structure (dark gray spheres closer than 3.0 angstroms, light gray branches closer than 4.5 angstroms).

Fig. 28A

Figure 28A illustrates a close-up of the interactions of E48, 051 and K109 (represented as branches) with Har 305 (represented as a surface) as described in Example 11.7.

Fig. 28831

Figure 288 illustrates the interactions between E48 and its environment (dark gray dashed line - hydrogen bond with Rab, light gray dashed line - water mediated hydrogen bond), as described in Example 11.7.

Fig. 28C

Figure 28C illustrates the interactions between 051 and its surroundings (dark gray dashed line - hydrogen bond with Raf) as described in Example 11.7.

Fig. 280

Figure 280 illustrates the interactions between KI109 and its surroundings (dark gray dotted line - hydrogen bond with Rab, light gray dotted line - salt bridge with NN SONZ095), as described in Example 11.7.

Fig. 29A

Figure 29A illustrates a close-up of the interactions of H7O, H72, and H110 (shown as branches) with bar 305 (shown as a surface) as described in Example 11.8, in the same orientation as in Figure 28A.

Fig. 298

Figure 298 illustrates the interactions between H7O and its environment, as described in Example 11.8.

The histidine residue is represented by branches and lattices). The hydrogen bond is indicated by a dashed line.

Fig. 29C

Figure 29C illustrates the interactions between H72 and its environment as described in Example 11.8.

The histidine residue is represented by branches and lattices). The hydrogen bond is indicated by a dashed line.

Fig. 290

Figure 290 illustrates the interactions between NI 10 and its environment, as described in Example 11.8.

The histidine residue is represented by branches and lattices). The distance between NI 10 and H-SOVZ NITOOS is shown by a dashed line.

Description of variants of implementation

The methods and procedures described herein or referred to herein are generally well understood and are generally practiced using conventional methodology by those skilled in the art, such as, for example, the widely used methodologies described in a!., MoiIesshciag Siopipd: A Garogaiyugu Mapiai Za eayop (2001) Soia brhipd

Narog Iarogayugu Rgev55, Soii brhipud Nagbrog, M.M.; Sctepi Rgoiosoi5 ip MoiIesshiag Vioiodu (H.M.

A!yzibeii, ei ai. Edv., (2003)); in the Meshoavs ip Epgutoiodu series (Asadetis Rgevs5, Ips.): RSV 2: A

Rgasiisa! Arrgoasi (M.U. MasRNegzop, V.O. Natez apa a.V8. Tauiog eav5. (1995)), Napoumu apa GI ape, edz5. (1988) Apiirodievs, A I arogaiyugu Mapiai, apa Apita! Sei!Yi Siyige (V.I. EgezNpeu, Yiddish. (1987));

Oiidopisiyeoiide Zupipeviv (M... Sai, ed., 1984); MeiShoodv ip Moiesshiag Vioodu, Nytapa Rgezv; Sow

Vioiodu: A I arogaiogu Moierook (UE Seii5, ed., 1998) Asadetis Rgev5; Apita! This! Siyitge (V.I.

EgezPpeu), united, 1987); Ipodisiyop about Seyi apa Tivvye SyPyge (U.R. MashSheg apa R.E. Nobetgiv, 1998)

Riepyt Rhev5; Seyi apa Tizvye SiyPyge: I arogayugu Rgosedigez (A. Oouye, U.V. Styynv5, apa O.a.

Memei, editor, 1993-8) U. MMIeu apa bopv; Napabsok oi Ekhregitepia! Ittypoiodu (O.M. M/vig apa b.b.

Vias Kuueyi, ed.); Sepe Tgapeoieg Mesyug5 g Mattaiyap SeiYiv (9U.M. MiPeg apa M.R. Sapov, ed., 1987);

RSV: Te Roiutegaze SNaip Reasiip, (Miiiiiv yei ai!., edv., 1994); Sitepi Rgoyusoi5 ip Ittypoiodu (UE Soiidap eyi ai., edv5., 1991); pop Rgoiosoyiv ip Moiesshiag Vioodu (M/Peu apa bopv, 1999);

Ititipobioiodu (S.A. dapezhau apa R. Tgameg5, 1997); Apiirodievs (R. Ripsy, 1997); Apiirodiev: A

From Rgasiisai Arrgoasp! (0. Satsu., unit, IV Rhev5, 1988-1989); Moposiope! Apiirodiev: A Rgasiisai

Arrgoasi (R. 5perpega apa S. Oeap, eadv., Ohio Opimegeyu Rgez55, 2000); Ovipd Apiirodiev: A

Garogaiogu Mapia! (E. Napyom apa 0. Gape (So briipud Nagrog Iarogaiogu Rgevzv, 1999); Te

Apiirodiez (M. 7apeyi apa 9. 0. Sarga, ed., Nagplood Asadetis RybiizNegv, 1995); and Sapseg:

Rgipsirieh apa Rgasiise oi Opsoiodu (MU.T. Oemia ei a)., edv., U.V. And Irripsoi Sotrapu, 1993).

I. DEFINITION

Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. 5ipdieiop ei ai., Oisiopagu oi Mistorioodu apa MoiIesshiag Vioiodu 2pa ed., u.

MuUyeu 4 ope (Mem Moi, MM 1994), and Mas, Admapsei Ogdapis SNetivzngu Veasiyopv,

Mespapiztv apa bigisiige 4 ed., dopp UMIeu 5 op (Mem Hogk, MU 1992) offer the person skilled in the art a general guide to many of the terms used in this application. All references cited in this specification, including patent applications and publications, are incorporated by reference in their entirety.

For the purpose of interpreting this description of the invention, the following definitions will apply where appropriate, terms used in the singular will also be used in the plural, and vice versa. It should be understood that the terminology used in this specification is intended to describe only specific embodiments and is not intended to be limiting. In the event that any proposed definition below conflicts with any document incorporated by reference in this specification, the proposed definition below will control. A "human acceptor framework region" for the purposes of this specification is a framework region that includes the amino acid sequence of a light chain variable domain (M) framework region or a heavy chain variable domain (MH) framework region derived from a human immunoglobulin framework region or a consensus framework region person as defined below. A human acceptor framework region "derived from" a human immunoglobulin framework region or a human consensus framework region may include the same amino acid sequence thereof, or it may have changes in the amino acid sequence. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, C or less, or 2 or less. In some embodiments, the human light chain variable region acceptor framework is identical in sequence to the human immunoglobulin light chain variable region framework sequence or the human consensus framework region sequence. "Affinity" refers to the strength of the set of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the inherent binding affinity that reflects a 1:11 interaction between the members of a binding pair (eg, antibody and antigen). The affinity of the molecule X to its partner M can generally be represented by the dissociation constant (Ka). Affinity can be measured using conventional methods known in the art, including methods described herein. Specific illustrative examples of embodiments of binding affinity measurement are described below.

An "affinity matured" antibody means an antibody with one or more changes in one or more hypervariable regions (HVRs), compared to an original antibody that does not have such changes, and such changes lead to an improvement in the affinity of the antibody to the antigen.

The terms "anti-C5 antibody" and "antibody that binds to C5" mean an antibody that is capable of binding to C5 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent when it targets C5 . In one embodiment, the degree of binding of an anti-C5 antibody to an unrelated protein that is not C5 is less than 10 95 of the binding of the antibody to C5, as measured, for example, by radioimmunoassay (NIA). In certain embodiments, the antibody that binds to C5 has a dissociation constant (Ka) that is 1 µM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0 .01 nM or less or 0.001 nm or less (e.g., 108 M or less, e.g., 103 M to 10-33 M, e.g., 109 M to 10-33 M). In certain embodiments, an anti-C5 antibody binds to a C5 epitope that is conserved among C5 from other animal species.

The term "antibody" as used herein is used in its broadest sense to encompass various antibody constructs, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigenicity. causative activity. "Antibody fragment" means a molecule other than an intact antibody that includes a portion of an intact antibody that binds to an antigen to which the intact antibody binds.

Examples of antibody fragments include, but are not limited to, Em, Rab, Bar", Bar-5n,

Kab)"; diatila; linear antibodies; single-chain antibody molecules (for example, 5cEm) and multispecific antibodies formed from antibody fragments. "Antibody that binds to the same epitope" as the reference antibody means an antibody that blocks the binding of the reference antibody to its antigen in a competitive assay, and/or conversely, the reference antibody blocks the binding of that antibody to its antigen in a competitive assay This description provides an example of a competitive binding assay.

The term "chimeric" antibody means an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from another source or species. "Class" of an antibody means the type of constant domain or constant region inherent in its heavy chain. There are five main classes of antibodies: IA, 40, I9ZE, da and IOM, and several of them can be further divided into subclasses (isotypes), for example, Ids, IDS", IdSz, a, IA: and IddA".

The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

The term "cytotoxic agent", as used herein, means a substance that inhibits or interferes with cellular function or results in cell death or destruction.

Cytotoxic agents include, but are not limited to, radioactive isotopes (eg

A", 191, 125, 90, Ve"86, Ve88 t! Bi2!2, rZg, pp and radioactive isotopes and); chemotherapeutic agents or drugs (eg, methotrexate, adriamycin, periwinkle alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibition agents; enzymes and their fragments, such as nucleotic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various antitumor and anticancer agents described below. "Effector functions" mean those biological types of activity inherent in the E-region of an antibody, as they differ depending on the isotype of the antibody. Examples of antibody effector functions include:

binding of Sid and complement-dependent cytotoxicity (SOC); binding to the Es receptor; antibody-dependent cell-mediated cytotoxicity (ACC); phagocytosis; downregulation of cell surface receptors (eg, B-cell receptor) and B-cell activation. An "effective amount" of an agent, such as a pharmaceutical composition, means an amount that is effective at the dosage and for the necessary time periods to achieve the desired therapeutic or prophylactic result.

The term "epitope" includes any determinant that has the property of being bound by an antibody. An epitope is a region of an antigen that is bound by an antibody that hits the antigen and that includes specific amino acids that directly contact the antibody. Epitope determinants can include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and they can have specific three-dimensional structural characteristics and/or specific charge characteristics.

In general, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and/or macromolecules.

The term "Pc region" in this description is used to define the C-terminal region of the immunoglobulin heavy chain, which contains at least part of the constant region. The term includes

E-regions of the native sequence and variant E-regions. In one embodiment, the Es region of the human ds heavy chain extends from Sub226 or Pg230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (I uz447) of the E-region may or may not be present. Unless otherwise indicated in this description, the numbering of amino acid residues in the E-region or constant region follows the European Nomenclature system, also called the EMO-index, as described in Karai ei ai., bedcepse5z oi Rgoivii5 oi

Ittypoiodis! Ipiegevi, Battle Ed. Rybiis Neatsy begmise, Maiyopa! Ipziyshev oi Neapy, VeiShezaa, MO, 1991. "Framework site" or "EV" means the residues of the variable domain, different from the residues of hypervariable regions (HIV). The EB of the variable domain generally consists of four EB domains: EB, ЕН2, ЕВЗ and ЕН4. Therefore, the sequences of NUA and EC are usually arranged in the following order in MN (or MI): ЕВ1-НИ(11)-ЕН2-Н2(И2)-ЕВЗ-НЗИИ 3)-ЕВА.

The terms "full-length antibody", "intact antibody" and "complete antibody" are used in

In this specification, interchangeably, they mean an antibody that has a structure substantially similar to that of a native antibody, or that has heavy chains that contain an E region as defined herein.

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 such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived from them, regardless of the number of passages. The progeny may not be completely identical to the original cell in terms of nucleic acid content, but may contain mutations. This description includes mutant progeny that have the same function or biological activity as determined by screening or selection for the original transformed cell. A "human antibody" is an antibody that has an amino acid sequence that matches the amino acid sequence of an antibody that is produced by a human or a human cell, or that originates from a non-human source and that uses human antibody spectra or other antibody coding sequences a person This definition of a human antibody specifically excludes a humanized antibody that includes antigen-binding residues that are not human. "Human consensus framework region" is a framework region that represents the most frequently occurring amino acid residues in the selection of MI or MN human immunoglobulin framework sequences. In general, the selection of MI or MN human immunoglobulin sequences is from a subset of variable domain sequences. In general, a subgroup of sequences is a subgroup, as indicated in Kabayi ei aY., Zedyepsev oi Rgoivip5 oi Ittipoiodis! Ipiegevi, Ey

Eaiiop, MIN Rybiisaiop 91-3242, Weshezaa MO (1991), my!5. 1-3. In one embodiment, for

MI, the subgroup is a subgroup of kappa I, as indicated in Kabayi Yei aYi., cf. above. In one embodiment, for MN, the subgroup is a subgroup of PI, as specified in Kabaye yi a!., cf. above. "Humanized" antibody means a chimeric antibody that includes amino acid residues

Non-human NMUs and amino acid residues from human EVs. In certain embodiments, a humanized antibody will include substantially all of at least one, or typically two, variable domains in which all or substantially all of the NMAs (e.g., SOVA) correspond to the 60 hypervariable regions of a non-human antibody, and all or substantially all of the EC correspond to the framework regions of human antibodies. A humanized antibody can optionally include at least a portion of the constant region of the antibody that is derived from a human antibody. A "humanized form" of an antibody, eg, a non-human antibody, means an antibody that has been humanized.

The term "hypervariable region" or "HMA" as used herein refers to each of the regions of the variable domain of an antibody that are hypervariable in sequence ("complementarity determining regions" or "CORs") and/or form structurally defined loops ( "hypervariable loops"), and/or contain antigen-contacting residues ("antigen contacts"). In general, antibodies include six NMEs: three in MN (NI, H2, NZ) and three in MI. (I1, 12, 13). Examples of NMEs in this specification include: (a) hypervariable loops that occur at amino acid residues 26-32 (I 1), 50-52 (I 2), 91-96 (I 3), 26-32 (NI ), 53-55 (H2) and 96-101 (NZ) (Spoipia, y. Moi. Vioi. 196:901-917 (1987)); (B) OWLs that occur at amino acid residues 24-34 (I 1 ), 50-56 (12), 89-97 (13), 31-35B6 (NI), 50-65 (H2g) and 95-102 (NZ) (Kabai ei ai,

Zedyepsez oi Rgoivin5 oi Ittipoiodis! Ipiegevi, Battle Ed. Rybiis Neatsy Zeguise, MIN, Veijpezaa, MO (1991)); (c) antigenic contacts that occur at amino acid residues 2706-36 (11), 46-55 (12), 89-96 (I 3), 30-356 (NI), 47-58 (H2) and 93-101 (NZ) (MasSaisht ei ai. 9). My. Vioi 262:732-745 (1996)); and (4) combinations (a), (B) and/or (c), which include amino acid residues of NUA 46-56 (I 2), 47-56 (I 2), 48-56 (I 2), 49- 56 (12), 26-35 (NO), 26-35B (NO), 49-65 (Н2г), 93-102 (НЗ) and 94-102 (НЗ).

Unless otherwise indicated, NME residues and other residues in the variable domain (eg, EP residues) are numbered in this specification according to Kabayi et al., see above. An "immunoconjugate" is an antibody conjugated to one or more heterologous molecule(s), including, but not limited to, a cytotoxic agent. An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human primates and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human. An "isolated" antibody is an antibody that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to greater than 95 95 or 99 95

By purity as determined by, for example, electrophoresis (eg sodium dodecyl sulfate polyacrylamide gel (505-RACE)), isoelectric focusing (IEE), capillary electrophoresis) or chromatography (eg ion-exchange or reverse-phase HPLC (HPLC) ). For an overview of methods for evaluating antibody purity, see, for example, Niaitap ei ai!., u). Spgotaiod"g. B 848:79-87 (2007). "Isolated" nucleic acid means a nucleic acid molecule that has been separated from the components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain that molecule nucleic acid, but there is a nucleic acid molecule present outside the chromosome or at a chromosomal position that is different from the natural chromosomal position. "Secluded nucleic acid that encodes an anti-C5 antibody" means one or more nucleic acid molecules that encode a heavy chain and a light chain antibodies (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors and such nucleic acid molecule(s) that are present in one or more locations in the host cell.

The term "monoclonal antibody" as used herein means an antibody derived from a population of essentially homogeneous antibodies; that is, the individual antibodies contained in the population are identical and/or bind to the same epitope, excluding possible antibody variants, such as those that have natural mutations or that arise during the preparation of a monoclonal antibody, while such variants are usually present in small amounts. Unlike polyclonal antibody preparations, which usually include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Therefore, the definition "monoclonal" indicates the nature of the antibody as obtained from a substantially homogeneous population of antibodies, and is not interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention can be obtained by a variety of methods, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals that contain all or part of loci of human immunoglobulin, while such methods and examples of other methods 60 of obtaining monoclonal antibodies are described in this description of the invention.

"Nude antibody" means an antibody that is not conjugated with a heterologous component (eg, a cytotoxic component) or a radioactive label. The naked antibody may be present in the pharmaceutical formulation.

The term "native antibodies" means natural immunoglobulin molecules with different structures.

For example, native DS antibodies; - these are heterotetrameric glycoproteins with a mass of about 150,000 daltons, which consist of two identical light chains and two identical heavy chains that have a disulfide bond. From the M- to the C-terminus, each heavy chain has a variable region (VR), also called the variable heavy domain or the heavy chain variable domain, followed by three constant domains (CDN,

SNI and SNZ). Similarly, from the M- to the C-terminus, each light chain has a variable region (MU), also called a variable light domain or light chain variable domain, followed by a constant light (Si!) domain. The light chain of an antibody can be classified into one of two types, called k (kappa) and X (lambda), based on the amino acid sequence of its constant domain.

The term "package insert" is used to refer to the instructions that are traditionally included in the commercial packaging of therapeutic products and which contain information regarding the indications, uses, dosage, methods of administration, complex therapy, contraindications and/or warnings regarding the use of such therapeutic products. "Percent amino acid sequence identity (96)" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve maximum sequence identity in percentages, with any conservative substitutions not considered part of sequence identity.

Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways within the skill in the art, for example using publicly available computer software such as VI AT, VI A5ZT-2, APOM or Medaiidp (OMA5ZTAR). Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximal alignment over the full length of the sequences being compared. However, for the purposes of the present invention, amino acid sequence identity values in percent (90) are generated using the A CM-2 sequence comparison computer program.

The computer program for comparing the sequences of AG ISM-2 was developed by Sepepiesy,

Ips., and the initial program was submitted together with user documentation to the Office

United States Copyright Office (05 Sorugpidni Oyise), UMazpipdiop O0.S., 20559, where it is registered under number 05 Sorugpidni VNeadivigayop Mo. THO510087. The AGISYAM-2 program is publicly available from Sepepiesp, Ips., boshii bap Egapsiso, Calif., or it can be downloaded from the source program. The A'SM-2 program should be downloaded for use on the MIH operating system, in particular the digital SHOMIH M4.00. All sequence comparison parameters are set by the AGISM-2 program, and they do not change.

In cases where the AGIS2M-2 program is used to compare amino acid sequences, the amino acid sequence identity in percent of a given amino acid sequence A with respect to, against, or with a given amino acid sequence B (which can alternatively be rephrased as "a given amino acid sequence A" that has overall or in its part, a certain identity of amino acid sequences in percentages with respect to, against or with a given amino acid sequence B) is calculated as follows: multiplied by 100 fraction X/U; where X is the number of amino acid residues counted as identical matches by the sequence alignment program A Y2M-2 in this program alignment of A and B, and where M is the total number of amino acid residues in B. It is clear that where the length of the amino acid sequence A is not equal to the length of amino acid sequence B, the percent identity of amino acid sequences A and B will not be equal to the percent identity of amino acid sequences B and A. Unless otherwise specifically indicated, all percentage amino acid sequence identity values used in this specification are obtained using the AGISM-2 computer program as described in the previous paragraph.

The term "pharmaceutical composition" means a preparation that has a form that allows the biological activity of the active ingredient contained therein to be effective and that does not contain additional components that are unacceptably toxic to the subject to whom the composition will be administered.

"Pharmaceutically acceptable carrier" means an ingredient in a pharmaceutical formulation, other than the active ingredient, that is non-toxic to the subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizing agent, or preservative.

The term "C5" as used herein encompasses any native C5 from any source: vertebrates, including mammals such as primates (eg, humans and monkeys) and rodents (eg, mice and rats). Unless otherwise specified, the term "C5" refers to a human C5 protein having the amino acid sequence shown in 5EO IUO MO: 39 and which contains the beta chain sequence shown in 5EO I MO: 40. The term includes "full-length" unprocessed C5, as well as any form of C5 obtained as a result of processing in the cell. The term also covers naturally occurring variants of C5, such as spliced variants or allelic variants.

The amino acid sequence of the human C5 example is given in 5EO IU MO: 39 ("natural type" or "M/UT" C5). The amino acid sequence of an example of a human C5 beta chain is given in BEO

IO MO: 40. Amino acid sequences of examples of the MC, MO2 and ME1-MO2 domains of the beta chain

Human C5 is given in 5EO IYUO MO: 41, 42 and 43, respectively. The amino acid sequences of C5 examples of Javan macaques (supotaYidy5 topKeu) and mice are given in 5EO IO MO: 44 and 105, respectively. Amino acid residues 1-19 5EO IO MO: 39, 40, 43, 44 and 105 correspond to a signal sequence that is removed during processing in the cell, as a result of which it is not contained in the corresponding example of the amino acid sequence.

As used herein, the term "treatment" (and its corresponding grammatical forms, such as "treat" or "treat") means a clinical intervention intended to attempt to alter the natural course of a disease in the individual being treated, and may be performed either for the purpose of prevention, or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, reduction of symptoms, reduction of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction of disease progression, improvement or palliation of disease state, and remission or improved prognosis. In some embodiments, the antibodies of the present invention are used to delay the development of the disease or slow down the development of the disease.

The term "variable region" or "variable domain" means a domain of heavy or light

From the chain of the antibody, which is involved in the binding of the antibody to the antigen. The heavy chain and light chain (MH and MI, respectively) variable domains of a native antibody typically have similar structures, with each domain comprising four conserved framework regions (EB) and three hypervariable regions (HVR). (See, for example, Kipai ei a!., Kibu Ittipoiodo, 6" ed.,

M.N. Egeetap apa So., Rade 91 (2007).) A single UN or Mi domain may be sufficient to provide antigen-binding specificity. In addition, antibodies that bind to a specific antigen can be isolated by using the MN or MU domain from an antibody that binds to that antigen to screen a library of complementary MN or Mi domains, respectively. See, for example, Ropoiapo ei ai., U. Ittipoi. 150:880-887 (1993); SiaiKkwop ei ai!., Mayte 352:624-628 (1991).

The term "vector" as used herein means a nucleic acid molecule capable of generating another nucleic acid to which it is linked. The term includes a vector as a self-replicating nucleic acid structure as well as a vector incorporated into the genome of the host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably attached. Such vectors are referred to herein as "expression vectors".

I. COMPOSITIONS AND METHODS

In one aspect, the invention is based, in part, on anti-C5 antibodies and their uses. In certain embodiments, antibodies that bind to C5 are provided. Antibodies of the invention are useful, for example, in the diagnosis or treatment of a complement-mediated disease or condition involving excessive or uncontrolled activation of C5.

A. Examples of antibodies against C5

In one aspect, the invention provides isolated antibodies that bind to C5. In certain embodiments, the anti-C5 antibody of the present invention binds to an epitope within the C5 beta chain. In certain embodiments, the anti-C5 antibody of the present invention binds to an epitope within the M21-MOc2 domain of the C5 beta chain. In certain embodiments, an anti-C5 antibody binds to an epitope within a fragment that consists of amino acids 19-180 of the C5 beta chain. In certain embodiments, the anti-C5 antibody binds to an epitope within the Ma domain (amino acids 20-124 5EO IU MO: 40 (5EO IO MO: 41)) of the C5 beta chain. In certain embodiments, an anti-C5 antibody binds to an epitope within a fragment consisting of amino acids 33-124 of the C5 beta chain (5EO I MO: 40). In another embodiment, the antibody does not bind to a fragment that is shorter than a fragment that consists of amino acids 33-124 of the C5 beta chain, for example, to a fragment that consists of amino acids 45-124, 52-124, 33-111 , 353-108 or 45-111 beta-chain C5 (5EO IU

MO: 40).

In another aspect, the invention provides anti-C5 antibodies that exhibit pH-dependent binding characteristics. As used herein, the term "pH-dependent binding" means that an antibody exhibits "weakened binding to C5 at acidic pH, compared to its binding at neutral pH" (for the purposes of this specification, both terms may be used as interchangeable). For example, antibodies "with characteristics of pH-dependent binding" include antibodies that bind to C5 with higher affinity at neutral pH than at acidic pH. In certain embodiments, the antibodies of the present invention bind to C5 with an affinity at neutral pH of 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 or more times exaggerates the affinity at acidic pH. In some embodiments, antibodies bind to C5 with higher affinity at pH 7.4 than at pH 5.8. In further embodiments, the antibodies of the present invention bind to C5 with an affinity at pH 7.4 that is at least 2.

Z, 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,200, 400, 1000, 10000 or more times exaggerates affinity at pH 5.8. The "affinity" of an antibody to C5 for the purposes of this specification is expressed as antibody KO. KO of an antibody means the equilibrium dissociation constant of the interaction of an antibody with an antigen. The higher the KO value for the binding of an antibody to its antigen, the weaker is its binding affinity to this particular antigen. Therefore, as used herein, the expression "higher affinity at neutral pH than at acidic pH" (or the equivalent expression "pH-dependent binding") means that the KO for binding of an antibody to C5 at acidic pH is greater than

KO for binding of antibodies to C5 at neutral pH. For example, in the context of the present invention, an antibody is considered to bind to C5 with higher affinity at neutral pH than at acidic pH, if the binding affinity of the antibody to C5 at acidic pH is at least 2-fold greater than the binding affinity of the antibody with C5 at neutral pH. Thus, the present invention includes antibodies that bind to C5 at acidic pH with a pH of 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, 10,000 or more times exaggerates KO binding of antibody to C5 at neutral pH. In another embodiment, the KO antibody value at neutral pH can be 10-77 M, 108 M, 109 M, 1070 M, 10-17 M, 10-72 M or less. In another embodiment, the KO antibody value at acidic pH can be 10 M, 108 M, 10-7 M, 105 M or more.

In further embodiments, an antibody is considered to bind C5 with higher affinity at neutral pH than at acidic pH if the C5 antibody binding coefficient at pH 5.8 is at least 2-fold greater than the C5 antibody binding coefficient at pH 7.4. In some embodiments, the proposed antibodies bind to C5 at pH 5.8 with an OR that is at least 3,5,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10,000 or more times exaggerates KO binding of antibody to C5 at pH 7.4. In another embodiment, the value of KO antibody at pH 7.4 can be 10- M, 109 M, 109 M, 1079 M, 107

M, 1072 M or less. In another embodiment, the KO value of the antibody at pH 5.8 can be 107 M, 103 M, 10 M, 105 M or more.

The binding properties of an antibody to a specific antigen can also be expressed as the Ka of the antibody, the KO of the antibody means the dissociation rate constant of the antibody to a specific antigen, and it is defined in inverse seconds (ie, a second).

Ka indicates a weaker binding of the antibody to its antigen. Therefore, this invention includes antibodies that bind to C5 with a higher Ka value at acidic pH than at neutral pH. The present invention includes antibodies that bind to C5 at acidic pH with a Ka that 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 or more times exaggerates the Ka of antibodies that bind to C5 at neutral pH. In another embodiment, the Ka value of the antibody at neutral pH may be 10-1/s, 103 1/s, 102 1/s, 105 1/s, 105 1/s or less. In another embodiment, the Ka value of the antibody at acidic pH can be 10-1/s, 1072 1/s, 107 1/s or more. The invention also includes antibodies that bind to C5 with a higher Ka value at pH 5.8 than at pH 7.4. The present invention includes antibodies that bind to C5 at pH 5.8 with a Ka that is at least 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 or more times exaggerates the Ka of antibodies that bind to C5 at pH 7.4. In another embodiment, the value

Ka antibody at pH 7.4 can be 1072 1/s, 103 1/s, 102 1/s, 105 1/s, 106 1/s or less. In another embodiment, the Ka value of the antibody at pH 5.8 can be 103 1/s, 102 1/s, 107 1/s or more.

In certain cases, "weakened binding to C5 at acidic pH compared to its binding at neutral pH" is expressed as the ratio of the KO value of the antibody that binds to C5 at acidic pH to the KO value of the antibody that binds to C5 at neutral pH (or vice versa). For example, for the purposes of the present invention, an antibody may be considered to exhibit "weakened binding to C5 at acidic pH compared to its binding at neutral pH" if the antibody exhibits a ratio of KO under acidic conditions / KO under neutral conditions of 2 or more. In certain examples, the ratio of KO at pH 5.5 / KO at pH 74 for the antibody of the present invention is 2 or more. In certain examples of variants, the ratio of KO in acidic conditions / KO in neutral conditions for the antibody of the present invention can be 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 or more. In another embodiment, the KO antibody value at neutral pH may be 10-M, 108 M, 109 M, 1079 M, 10-7 M, 10-72 M or less. In another embodiment, the KO antibody value at acidic pH can be 107 M, 108 M, 107 M, 105 M or more. In further cases, for purposes of the present invention, an antibody can be considered to exhibit "weakened binding to C5 at acidic pH compared to its binding at neutral pH" if the antibody exhibits a ratio of KO at pH 5.8 / KO at pH 7.4 which is 2 or more.

In certain examples of variants, the ratio of KO at pH 5.8 / KO at pH 7.4 for the antibody of the present invention can be 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 or more. In another embodiment, the KO antibody value at pH 7.4 can be 10-M, 10-8 M, 10-9 M, 10-79 M, 10-77 M, 10-72 M or less. In another embodiment, the KO value of the antibody at pH 5.8 can be 109 M, 108 M, 10- M, 10-6 M or more.

In certain cases, "weakened binding to C5 at acidic pH compared to its binding at neutral pH" is expressed as the ratio of the Ka value of the antibody that binds to C5 at acidic pH to the Ka value of the antibody that binds to C5 at neutral pH (or vice versa). For example, for the purposes of this invention, it can be assumed that

An antibody exhibits "weakened binding to C5 at acidic pH compared to its binding at neutral pH" if the antibody exhibits a ratio of Ka under acidic conditions / Ka under neutral conditions of 2 or greater. In certain examples, the ratio Ka at pH 5.5 / Ka at pH 7.4 for the antibody of the present invention is 2 or more. In certain examples of variants, the ratio Ka in acidic conditions / Ka in neutral conditions for the antibody of the present invention can be 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 or more.

In further examples, the ratio Ka at pH 5.8 / Ka at pH 7.4 for the antibody of the present invention can be 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 or more. In another embodiment, the value

Ka antibody at neutral pH can be 1072 1/s, 103 1/s, 1072 1/s, 105 1/s, 105 1/s or less. In a further embodiment, the Ka value of the antibody at pH 7.4 may be 102 1/s, 103 1/s, 102 1/s, 105 1/s, 105 1/s or less. In another embodiment, the Ka value of the antibody at acidic pH can be 103 1/s, 102 1/s, 107 1/s or more. In a further embodiment, the Ka value of the antibody at pH 5.8 may be 10 1/s, 1072 1/s, 107 1/s or more.

As used herein, the term "acidic pH" refers to a pH of 4.0 to 6.5. The term "acidic pH" includes a pH value of any of 4.0, 4,1,4,2,4,3,4,4,4,5,4,6,4,7,4,8,4 ,9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 61, 6 ,2, 6.3, 6.4 and 6.5. In particular aspects, the "acidic pH" is 5.8.

As used herein, the term "neutral pH" refers to a pH of 6.7 to about 10.0. The term "neutral pH" includes a pH of any of 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7 ,6, 7,7, 7,8, 7,9, 8,0, 8,1, 8,2, 8,3, 8,4, 8,5, 8,6, 8,7, 8,8 , 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 and 10.0. In particular aspects, "neutral pH" is ?7.4.

The value of KO and the value of Ka, as expressed in this description, can be determined using a biosensor based on surface plasmon resonance to characterize antibody-antigen interactions. (See, e.g., Example C in this specification). The value of KO and the value of Ka can be determined at 25 "C or at 37 "C.

In certain embodiments, the anti-C5 antibody of the present invention binds to an epitope within the C5 beta chain, which consists of the MOE1T domain (ZEO IO MO: 41). In certain embodiments, the anti-C5 antibody of the present invention binds to an epitope within the beta chain

(ZEO IO MO: 40) C5, which includes at least one fragment selected from the group consisting of amino acids 47-57, 70-76 and 107-110. In certain embodiments, the anti-C5 antibody of the present invention binds to an epitope within the beta-chain fragment (5EO IO MO: 40) of C5, which includes at least one amino acid selected from the group consisting of TNg47, C1Ish48, AIa49,

Rpeb5o, Azr5b1, AIab2, TNg53, I uz57, Nivz70, Ma171, Niz72, beg74, SiIshi76, Mai107, 5e108, I uye109 and

Niz110. In certain embodiments, the anti-C5 antibody of the present invention binds to an epitope within the beta-chain fragment (5EO I MO: 40) of C5, which includes at least one amino acid selected from the group consisting of C1/sh48, Avr5b1, Niv70, Level 72, Level 109 and Level 110.

In certain embodiments, the binding of the anti-C5 antibody of the present invention to the mutant C5 is weakened compared to its binding to native C5, where the mutant C5 has at least one amino acid substitution at a position selected from the group consisting of C1Ish48, Avro1,

Nibt72 and Gu5109. In another embodiment, the pH-dependent binding of an anti-C5 antibody of the present invention to a C5 mutant is attenuated compared to its pH-dependent binding to native C5, wherein the C5 mutant has at least one amino acid substitution at a position selected from the group consisting of consists of Niv70, Nibz72 and Nibz110. In a further embodiment, the mutant

The C5 amino acid at the position selected from Sis48, A5p51 and Gu5109 is substituted with alanine, and the amino acid at the position selected from Niz70, Niz72 and Niz110 is substituted by tyrosine.

In certain embodiments, the antibody against C5 of the present invention competes for binding to

C5 with an antibody that includes a pair of MH and Mi selected from the following: (a) MH 5EO IO MO: 1 and MI

ZEO on MO: 11; (B) MN 5EO IO MO: 22 and MI 5EO IO MO: 26; (c) MN 5EO IO MO: 21 and MI 5EO IO

MO: 25; (4) MN 5EO IO MO: 5 and MI 5EO IU MO: 15; (e) МН 5EO IYU MO: 4 and MI 5EO IYUO MO: 14; (1)

MN 5EO IO MO: 6 and MI 5EO IO MO: 16; (9) MN 5EO IU MO: 2 and MI 5EO IU MO: 12; (n) MN 5EO I

MO: Z and MI 5EO IO MO: 13; () MN 5EO IO MO: 9 and MI 5EO IO MO: 19; () MN 5EO IO MO: 7 and MI

ZEO IO MO: 17; (K) MN 5EO IO MO: 8 and MI 5EO IO MO: 18; (1) MN 5EO IO MO: 23 and MI 5EO I

MO: 27 and (t) MN 5EO IO MO: 10 and MI 5EO AND MO: 20.

In certain embodiments, the anti-C5 antibody of the present invention binds to C5 and contacts the amino acid A5rb5i (051) 5EO IO MO: 39. In additional embodiments, the anti-C5 antibody of the present invention binds to C5 and contacts the amino acid I uz109 (K 109)

ZEO IO MO: 39. In a further embodiment, the anti-C5 antibody of the present invention binds to C5 and contacts amino acid Avr5bi1 (051) and amino acid I uz109 (K109) BEO IJ MO: 39.

In certain embodiments, the binding of the anti-C5 antibody of the present invention to the C5 mutant is weakened compared to its binding to native C5, where the C5 mutant has a substitution

Siin48AIa (E48A) in 5EO IO MO: 39. In another embodiment, the pH-dependent binding of the anti-C5 antibody of the present invention to the C5 mutant is weakened, compared to its pH-dependent binding to native C5, where the C5 mutant has a substitution Ss1ish48Aia (E48A) in 5EO IYU MO: 39.

In a further embodiment, the anti-C5 antibody binds to the C5 protein, which consists of the amino acid sequence 5ESO IO MO: 39, but does not bind to the C5 protein, which consists of the amino acid sequence FEO IUO MO: 39 with the H72U substitution, where the protein

C5 and C5 protein with H72MU substitution are prepared and screened under the same conditions. In a further embodiment, the anti-C5 antibody binds to the C5 protein, which consists of the amino acid sequence FEO IU MO: 39 at pH 7.4, but does not bind to the C5 protein with the H72U substitution at pH 7.4.

Without being bound by any particular theory, it can be predicted that the binding of an anti-C5 antibody to C5 is weakened (or almost lost) when an amino acid residue in C5 is replaced by another amino acid, meaning that this amino acid residue in C5 is crucial for interactions between the anti-C5 antibody C5 and C5 and that the antibody can recognize an epitope around this amino acid residue in C5.

In this invention, it was determined that a group of antibodies against C5, which compete with each other or bind to the same epitope, can demonstrate the characteristics of pH-dependent binding. Among amino acids, histidine, whose pKa value is approximately 6.0 to 6.5, can have different proton dissociation states between neutral and acidic pH. Therefore, the histidine residue in C5 is able to contribute to pH-dependent interactions between the antibody against

C5 and C5. Without being bound by any particular theory, it is anticipated that an anti-C5 antibody may recognize a conformational structure around a histidine residue in C5 that is variably dependent on pH. This prediction may be consistent with the experimental results described below that the pH dependence of an anti-C5 antibody is reduced (or almost lost) when the histidine residue in C5 is replaced by another amino acid (ie, an anti-C5 antibody with pH-dependent binding characteristics binds to mutant C5 bo with a substituted histidine with an affinity similar to that of natural C5 at neutral pH, while the same antibody binds to a mutant C5 with a substituted histidine with a higher affinity compared to the affinity when binding to natural C5 at acidic pH) .

In certain embodiments, the anti-C5 antibody of the present invention binds to C5 from more than one species of animal. In further embodiments, the anti-C5 antibody binds to C5 from a human and a non-human animal. In further embodiments, the anti-C5 antibody binds to C5 from a human and a monkey (for example, a Javan macaque (supotaiYidv5), a rhesus macaque, a monkey, a chimpanzee, or a baboon).

In one aspect, the invention provides anti-C5 antibodies that inhibit C5 activation.

In certain embodiments, anti-C5 antibodies are provided that prevent the cleavage of C5, resulting in the formation of Cba and C5b, which in turn prevents the generation of anaphylatoxic activity associated with Cba, and also prevents the assembly of the membrane attack complex C5p-9 (MAS). , associated with S5r. In certain embodiments, anti-C5 antibodies are provided that block the conversion of C5 to C5ba and C5B by C5 convertase.

In certain embodiments, anti-C5 antibodies that block access to the convertase are provided

C5 to the cleavage site in C5. In certain embodiments, anti-C5 antibodies are provided that block the hemolytic activity that results from C5 activation. In further embodiments, anti-C5 antibodies of the present invention inhibit activation of C5 by a classical pathway and/or an alternative pathway.

In one aspect, the invention provides anti-C5 antibodies that inhibit the activation of the C5 variant. The term "variant C5" means a genetic variant of C5 that is the result of a genetic variation, such as a mutation, polymorphism, or allelic variation. Genetic variation may include deletion, substitution or insertion of one or more nucleotides. A C5 variant can have one or more genetic variations in C5. In certain embodiments, variant C5 has a biological activity similar to the biological activity of native C5. Such variant C5 may have at least one variation selected from the group consisting of M145I, 84492, M802I, ВА885Н,

B8O280, 0966, 51310M and E14370. In this description, B885H, for example, denotes a genetic variation where arginine at position 885 is replaced by histidine. In certain embodiments, the anti-C5 antibody of the present invention inhibits the activation of both native C5 and at least one C5 variant selected from the group consisting of M145I, 4492, M802I, B885H, 9280, 0966,

Z1310M Tag14370.

In one aspect, the invention provides an anti-C5 antibody that comprises at least one, two, three, four, five, or six hypervariable regions (HVRs) selected from the following: (a)

NUV-NI, which includes the amino acid sequence according to any of BEO IYUO MO: 45-54; (c) NUV-

H2, which includes the amino acid sequence according to any of 5EO IU MO: 55-64; (c) NMV-NZ, which includes the amino acid sequence according to any of the BEO IU MO: 65-74; (8) NUV-11, which includes the amino acid sequence of any of 5EO AND MO: 75-84; (is) NUV-12, which includes the amino acid sequence according to any of 5EO AND MO: 85-94; and (u) NUV-IZ, which includes the amino acid sequence according to any of 5EO IU MO: 95-104.

In one aspect, the invention provides an antibody that includes at least one, at least two, or all three sequences of HMA MH selected from the following: (a) NUV-NI, which includes an amino acid sequence of any of 5EO IYUO MO: 45-54; (p) NUMV-H2, which includes the amino acid sequence according to any of 5EO IYUO MO: 55-64; and (c) NUV-NZ, which includes the amino acid sequence of any of 5EO IYUO MO: 65-74. In one embodiment, the antibody includes a NUV-NZ that includes the amino acid sequence of any of 5EO IU MO: 65-74. In another embodiment, the antibody includes a NUN-NZ that includes an amino acid sequence of any of 5EO I MO: 65-74, and a NMV-IZ that includes an amino acid sequence of any of 5EO 10 MO: 95-104. In a further embodiment, the antibody includes a NUV-NZ that includes an amino acid sequence following any of 5EO IO MO: 65-74,

NMV-Y 3, which includes the amino acid sequence of any of the 5EO IYUO MO: 95-104, and NUV-

BO H2, which includes the amino acid sequence according to any of 5BEO IO MO: 55-64. In a further embodiment, the antibody includes (a) NUV-NI, which includes the amino acid sequence according to any of 5EO IO MO: 45-54; (p) NUV-H2, which includes the amino acid sequence according to any of 5EO IO MO: 55-64; and (c) NUV-NZ, which includes the amino acid sequence of any of ZEO IU MO: 65-74.

In one aspect, the invention provides an antibody that includes at least one, at least two, or all three NMA Mi sequences selected from the following: (a) NMV-I71, which includes the amino acid sequence of any of 5EO IJOO MO: 75-84; (p) NMUV-12, which includes the amino acid sequence according to any of 5EO and MO: 85-94; and (c) NMV-IZ, which includes the amino acid sequence of any of 5EO IO MO: 95-104. In one embodiment, the antibody 60 comprises (a) NUV-11, which comprises the amino acid sequence of any of the 5EO IU

MO: 75-84; (p) NUV-1I2, which includes the amino acid sequence according to any of BEO IU MO: 85-94; and (c) NMV-II 3, which includes the amino acid sequence of any one of BEO IO MO: 95-104.

In one aspect, an antibody of the invention comprises (a) an MN domain that includes at least one, at least two, or all three NMA MN sequences selected from the following: (i) a NUMB-NI that includes an amino acid sequence after any of 5EO AND MO: 45-54, (ii) NUV-H2, which includes the amino acid sequence according to any of 5EO IU MO: 55-64, and (ii) NUV-NZ, which includes the amino acid sequence according to any of 5EO I MO: 65 -74; and (p) an MI domain that includes at least one, at least two, or all three NME MI sequences selected from the following: (i) NUV-

Y1, which includes the amino acid sequence of any of 5EO IU MO: 75-84, (ii) NMV-I 2, which includes the amino acid sequence of any of 5EO I MO: 85-94, and (c) NMV- And 3, which includes the amino acid sequence according to any of BEO IU MO: 95-104.

In another aspect, the invention provides an antibody that includes (a) NMV-NI, which includes the amino acid sequence of any of 5EO IYUO MO: 45-54; (p) NUMV-H2, which includes the amino acid sequence according to any of 5EO 10 MO: 55-64; (c) NUV-NZ, which includes the amino acid sequence according to any of 5EO IYUO MO: 65-74; (a) NMV-11, which includes the amino acid sequence of any of 5EO AND MO: 75-84; (is) NUV-12, which includes the amino acid sequence according to any of 5EO AND MO: 85-94; and (u) NUV-IZ, which includes the amino acid sequence according to any of 5EO IU MO: 95-104.

In one aspect, the invention provides an anti-C5 antibody that includes at least one, two, three, four, five, or six NMAs selected from the following: (a) NUV-NI, which includes an amino acid sequence of any of 5EO IU MO : 45, 54, 117, 126; (5) NUV-H2, which includes the amino acid sequence according to any of 5EO AND MO: 55, 64, 118-120, 127; (c) NUV-

NZ, which includes the amino acid sequence according to any of the 5EO IU MO: 65, 74, 121, 128; (and)

NMVA-Y1, which includes the amino acid sequence according to any of 5EO IO MO: 75, 84, 122, 129; (is) NUV-1I2, which includes the amino acid sequence according to any of 5EO IO MO: 85, 94, 123-124, 130; and (i) NMV-II 3, which includes the amino acid sequence according to any of the BEO IJU MO: 95, 104, 125, 131.

In one aspect, the invention provides an antibody that includes at least one, at least two, or all three HMA MN sequences selected from the following: (a) NUV-NI, which includes

The amino acid sequence according to any of the 5EO IU MO: 45, 54, 117, 126; (5) NUV-H2, which includes the amino acid sequence according to any of 5EO IU MO: 55, 64, 118-120,127; and (c)

NMUV-NZ, which includes the amino acid sequence according to any of the 5EO IU MO: 65, 74121, 128.

In an aqueous embodiment, the antibody comprises a NUV-NZ, which includes an amino acid sequence following any of the 5EO IO MO: 65, 74, 121, 128. In another embodiment, the antibody includes a NUV-NZ, which includes an amino acid sequence following any of the 5EO IUO MO: 65, 74, 121, 128, and NMRV-I 3, which includes the amino acid sequence of any of the 5EO IU MO: 95, 104, 125, 131. In a further embodiment, the antibody includes NMV-NZ, which includes the amino acid sequence of any of the 5EO IUO MO: 65, 74, 121, 128, NMV-Y 3, which includes the amino acid sequence of any of the 5EO IUO MO: 95, 104, 125, 131, and NUV-H2, which includes the amino acid sequence of any of 5EO IO MO: 55, 64, 118-120, 127.

In a further embodiment, the antibody includes (a) NUV-NI, which includes the amino acid sequence according to any of 5EBEO IO MO: 45, 54, 117, 126; (B) NMV-H2, which includes the amino acid sequence according to any of BEO IO MO: 55, 64, 118-120,127; and (c) NUV-NZ, which includes the amino acid sequence of any of the 5EO IU MO: 65, 74, 121,128.

In another aspect, the invention provides an antibody that includes at least one, at least two, or all three NMA Mi sequences selected from the following: (a) HMV-I71, which includes the amino acid sequence of any of 5EO IU MO: 75, 84, 122 , 129; (5) NMUV-I2, which includes the amino acid sequence according to any of the 5EO IYUO MO: 85, 94, 123-124, 130; and (c)

NMV-Y 3, which includes the amino acid sequence according to any of the BEO IYUO MO: 95, 104, 125, 131.

In one embodiment, the antibody includes (a) NMA-I1, which includes the amino acid sequence of any of 5EO and MO: 75, 84, 122, 129; (p) NMUV-1I2 which includes the amino acid sequence according to any of 5EO IO MO: 85, 94, 123-124, 130; and (c) NMV-Y 3, which includes the amino acid sequence of any of the 5EO IU MO: 95,104,125,131.

In one aspect, an antibody of the invention comprises (a) an MH domain that includes at least one, at least two, or all three sequences of the MH NUV selected from the following: (i) a NUV-NI that includes an amino acid sequence following any of the 5EO IO MO: 45, 54, 117, 126, (her) NUV-H2, which includes the amino acid sequence according to any of the 5EO IO MO: 55, 64, 118-120, 127, and (iii)

NMUV-NZ, which includes the amino acid sequence according to any of the 5EO IO MO: 65, 74, 121, 128; and (b) an MI domain that includes at least one, at least two, or all three NMA MI sequences 60 selected from the following: (i) NMUV-11 that includes an amino acid sequence of any of the 5ESO

IO MO: 75, 84, 122, 129, (its) NUV-I2, which includes the amino acid sequence according to any of

ZEO IJU MO: 85, 94, 123-124, 130, and (c) NMV-I 3, which includes the amino acid sequence of any of the 5EO IJU MO: 95, 104, 125,131.

In another aspect, the invention provides an antibody that includes (a) NMV-NI, which includes the amino acid sequence of any of 5EO IU MO: 45, 54, 117, 126; (5) NUV-H2, which includes the amino acid sequence according to any of 5EO IU MO: 55, 64, 118-120,127; (c) NUV-

NZ, which includes the amino acid sequence according to any of 5EO 10 MO: 65, 74121128; (and)

NMVA-Y1, which includes the amino acid sequence according to any of 5EO IO MO: 75, 84, 122, 129; (e) NUV-I2, which includes the amino acid sequence according to any of 5EO and MO: 85, 94, 123-124, 130; and (i) NMV-II 3, which includes the amino acid sequence according to any of the BEO IJU MO: 95, 104, 125, 131.

In certain embodiments, any one or more amino acids of the anti-C5 antibody as proposed above are substituted at the following NME positions: (a) in NUV-NI (5EO IO MO: 45) at positions 5 and 6; (B) in NUV-N2 (ZEO IO MO: 55) in positions 1, 3, 9, 11, 13 and 15; (c) at NUV-NZ (5EO

IO MO: 65) in positions 2, 5, 6, 12 and 13; (4) in NMV-I1 (ZEO IO MO: 75) in positions 1, 5, 7 and 9; (is) in NUV-I2 (ZEO IO MO: 85) in positions 4, 5 and 6 and () in NMV-I Z (5EO IO MO: 95) in positions 2, 4 and 12.

In certain embodiments, the substitutions are conservative substitutions as suggested herein. In certain embodiments, any one or more of the following substitutions can be made in any combination: (a) in NMA-NI (ZEO IO MO: 45) IOM or SbA; (5) in NUV-N2 (ZEO IYUO MO: 55) SI A or c, ZE, T90 or E, Mi IC or O, 5130 or E, or AT5U; (c) in NUV-NZ (ZEO and MO: 65) C2A, M5O or 0, T6U, M12H, or I 13; (4) in NMA-I1 (5EO and MO: 75) OT1B, MO or c, 75, BOK or 5; (is) in NUV-I2 (ZEO IO MO: 85) CAT or E, I 571 or ABbN, AbE or AbO; (1) in NUV-IZ (ZEO IO MO: 95) Сб25, С2М or С2Т, Е«4К; or A12T or A12H.

All possible combinations of the above-mentioned substitutions are covered by the 5EO IU MO consensus sequences: 126, 127, 128, 129, 130 and 131 for NUV-NI, NUV-H2, NMUV-NZ,

НМВ-И1, НМВ-12 and НМП-1 3, respectively.

In any of the above embodiments, the anti-C5 antibody is humanized.

In one embodiment, the anti-C5 antibody comprises HMV, as in any of the above

In embodiments, it also includes a human acceptor framework, such as a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-C5 antibody comprises an HMP, as in any of the aforementioned embodiments, and also comprises an MN or MI that includes an EB sequence, where the EB sequences are as follows. For the heavy chain variable domain EV! includes the amino acid sequence for any of the 5EO IU MO: 132-134, EB2 includes the amino acid sequence for any of the 5EO IO MO: 135-136, EAZ includes the amino acid sequence for any of the 5EO

IO MO: 137-139, EN4 includes the amino acid sequence according to any of 5EO IU MO: 140-141.

For the variable domain of the light chain, ERNI includes the amino acid sequence after any of the 5EO IUO MO: 142-143, EN2 includes the amino acid sequence after any of the 5EO IU

MO: 144-145, EVZ includes the amino acid sequence of any of 5EO IU MO: 146-147, EB4 includes the amino acid sequence of 5EO IO MO: 148.

In another aspect, the anti-C5 antibody comprises a heavy chain variable domain (HCH) sequence that is at least 90 Fo, 91 95, 92 95, 93 U, 94 95, 95 Uo, 96 U, 97 Uo, 98 Fo, 99 90 or 100 95 identical amino acid sequence according to any of 5EO IYUO MO: 1-10. In certain embodiments, the UN sequence that is at least 90 95, 91 Fo, 92 Fo, 93 Fo, 94 Fo, 95 95, 9695, 9796, 9895, or 9995 identical contains substitutions (eg, conservative substitutions), insertions, or deletions relative to the reference sequence, however, an anti-C5 antibody that includes such a sequence retains the ability to bind to C5. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in any of

ZEO IU MO: 1-10. In certain embodiments, the substitutions, insertions, or deletions occur at sites outside the NMNC (e.g., in EVs). Optionally, the anti-C5 antibody includes the sequence

MH in any of the 5EO IO MO: I-10, including post-translational modifications of this sequence.

In a particular embodiment, the UN includes one, two or three NMUEs selected from the following: (a)

NMV-NI, which includes the amino acid sequence according to any of the 5EO IO MO: 45-54, (r) NUV-

H2, which includes the amino acid sequence of any of 5EO IJOO MO: 55-64, and (c) NUV-NZ, which includes the amino acid sequence of any of BEO AND MO: 65-74.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises a light chain variable domain (MI) sequence that is at least at 90 95, 91 9", 92 Uv, 93 U, 94 Or, 95 9, 96 9", 97 U, 98 9", 99 95 or 100 95 identical amino acid sequence for any 60 of 5EO IO MO: 11-20. In certain embodiments, an Mi sequence that is at least 90 95

9195, 92 95, 9395, 94 95, 9595, 96 95, 97 95, 9895, or 99 95 is identical, contains substitutions (eg, conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-C5 antibody that includes such sequence, retains the ability to communicate with

C5. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in any of the FEO IUO MO: 11-20. In certain embodiments, substitutions, insertions, or deletions occur at sites outside the NMA (eg, in the EN). Optionally, the anti-C5 antibody includes the sequence of MI in any of FEO IO MO: 11-20, including post-translational modifications of this sequence. In a particular embodiment, MI includes one, two or three

A NUV selected from the following: (a) NUV-11 comprising an amino acid sequence of any of

ZEO AND MO: 75-84, (r) NUV-12, which includes the amino acid sequence according to any of 5BEO 10

MO: 85-94, and (c) NMV-I 3, which includes the amino acid sequence of any of 5EO IU MO: 95-104.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises MH, as in any of the embodiments provided above, and Mi, as in any of the embodiments provided above. In one embodiment, the antibody comprises MH and MI sequences. in any of 5BEO 10 MO: 1-Y and in any of 5BO I MO: 11-20, respectively, including post-translational modifications of such sequences.

In another aspect, the anti-C5 antibody comprises a heavy chain variable domain (HCH) sequence that is at least 90 Fo, 91 95, 92 95, 93 U, 94 95, 95 Uo, 96 U, 97 Uo, 98 Fo, 99 90 or 100 95 identical amino acid sequence according to any of 5EO 10 MO: 10, 106-110.

In certain embodiments, an MN sequence that is at least 90 95 , 91 95 , 92 95 , 93 95 , 9495 , 9595 , 9695 , 9795 , 9895 , or 9995 identical contains substitutions (eg, conservative substitutions), insertions, or deletions relative to the reference sequence, however, an anti-C5 antibody that includes such a sequence retains the ability to bind to C5.

In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in any of the FEO IU MO: 10, 106-110. In certain embodiments, substitutions, insertions, or deletions occur at sites outside the NMA (eg, in the EN). Optionally, the antibody against C5 includes the sequence of MH in any of the FEO IJU MO: 10, 106-110, including post-translational modifications of this sequence. In a special variant, the implementation of MH includes

From one, two or three NMAs selected from the following: (a) NMV-NI, which includes the amino acid sequence of any of 5EBEO IO MO: 45, 54, 117, 126, (B) NMV-H2, which includes the amino acid sequence the sequence of any of 5EO IU MO: 55, 64, 118-120, 127, and (c) NUV-NZ, which includes the amino acid sequence of any of BEO IU MO: 65, 74, 121, 128.

In another aspect, the anti-C5 antibody comprises a MH sequence that is at least 90 95, 91 95, 92 95, 93 Uo, 94 95, 95 96, 96 9o, 97 Fo, 98 95, 99 95, or 100 95 identical in amino acid sequence to any of 5EO 10 MO: 10, 106-110. In certain embodiments, the MH sequence that is at least 90 95 , 91 9 , 92 95 , 93 о , 94 Fo , 95 U , 96 Fo , 97 U , 98 95 , or 99 95 is identical, contains a substitution (e.g., conservative substitutions ), insertions or deletions relative to the reference sequence, but an anti-C5 antibody that includes such a sequence retains the ability to bind to C5. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in any of 5EO 10 MO: 10, 106-110.

In certain embodiments, the substitutions, insertions, or deletions occur at sites outside the NMV (e.g., in the EB). Optionally, the anti-C5 antibody includes the UN sequence in any of ZEO IU MO: 10, 106-110, including post-translational modifications of this sequence.

In a particular embodiment, the UN includes one, two or three NMUEs selected from the following: (a)

NMV-NI, which includes the amino acid sequence of any of the 5EO IU MO: 45, 54, 117, 126, (b) NUV-H2, which includes the amino acid sequence of any of the 5EO IU MO: 55, 64, 118 - 120, 127, and (c) NUV-NZ, which includes the amino acid sequence according to any of the 5EO IU MO: 65, 74, 121128.

In another aspect, the anti-C5 antibody comprises a MH sequence that is at least 90 95, 91 95, 92 95, 93 Uo, 94 95, 95 96, 96 Uo, 97 Fo, 98 95, 99 95, or 100 95 identical in amino acid sequence to 5EO AND MO: 10. In certain embodiments, the MH sequence is an amino acid sequence according to ES IO MO: 10. In certain embodiments, the UN sequence is at least at 90 9, 91 9", 92 Ov, 93 U, 94 Fo, 95 U, 96 9", 97 Fo, 98 95 or 99 95 is identical, contains substitutions (eg, conservative substitutions), insertions or deletions relative to the reference sequence, but an anti-C5 antibody that includes such a sequence retains the ability to bind to C5 . In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in the 5EO IO MO: 10. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the NMV (eg, in the EB). 60 Optionally, the anti-C5 antibody includes the sequence MH in 5EO IU MO: 10, including post-translational modifications of this sequence. In a special embodiment, the MH includes one, two or three NMAs selected from the following: (a) NMV-NI, which includes the amino acid sequence according to 5EO IO MO: 54, (B) NUV-H2, which includes the amino acid sequence according to BEO

IO MO: 64, and (c) NUV-NZ, which includes the amino acid sequence at 5EO IO MO: 74.

In another aspect, the anti-C5 antibody comprises a MH sequence that is at least 90 95, 91 95, 92 95, 93 Uo, 94 95, 95 96, 96 Uo, 97 Fo, 98 95, 99 95, or 100 95 identical in amino acid sequence to 5BEO AND MO: 106. In certain embodiments, the MH sequence is an amino acid sequence according to 5EO IJ MO: 106. In certain embodiments, the sequence

MH that is at least 90 95, 9195, 9295, 9395, 94 95, 9595, 96 95, 97 95, 9895, or 99 95 identical, contains substitutions (eg, conservative substitutions), insertions, or deletions relative to the reference sequence, but the antibody against C5, which includes such a sequence, retains the ability to bind to C5. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in the 5EO IU MO: 106. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the NMV (eg, in the EB). Optionally, the antibody against C5 includes the sequence of MH in 5EO IU MO: 106, including post-translational modifications of this sequence. In a special embodiment, the MH includes one, two or three NMAs selected from the following: (a) NMV-NI, which includes the amino acid sequence according to BEO IU MO: 117, (p) NUV-H2, which includes the amino acid sequence according to BEO

IO MO: 118, and (c) NUV-NZ, which includes the amino acid sequence at 5EO IU MO: 121.

In another aspect, the anti-C5 antibody comprises a MH sequence that is at least 90 95, 91 95, 92 95, 93 Uo, 94 95, 95 96, 96 Uo, 97 Fo, 98 95, 99 95, or 100 95 identical in amino acid sequence to 5BEO I0O MO: 107. In certain embodiments, the MH sequence is an amino acid sequence according to 5EO IU MO: 107. In certain embodiments, the sequence

MH that is at least 90 95, 9195, 9295, 9395, 94 95, 9595, 96 95, 97 95, 9895, or 99 95 identical, contains substitutions (eg, conservative substitutions), insertions, or deletions relative to the reference sequence, but the antibody against C5, which includes such a sequence, retains the ability to bind to C5. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in the BEO IU MO: 107. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the HMV (e.g.,

Zo in EV). Optionally, the antibody against C5 includes the sequence MH in 5EO IU MO: 107, including post-translational modifications of this sequence. In a special embodiment, the MH includes one, two or three NMAs selected from the following: (a) NMV-NI, which includes the amino acid sequence according to BEO IU MO: 117, (p) NUV-H2, which includes the amino acid sequence according to BEO

IO MO: 119, and (c) NUV-NZ, which includes the amino acid sequence at 5EO IU MO: 121.

In another aspect, the anti-C5 antibody comprises a MH sequence that is at least 90 95, 91 95, 92 95, 93 Uo, 94 95, 95 96, 96 Uo, 97 Fo, 98 95, 99 95, or 100 95 identical in amino acid sequence to 5BEO AND MO: 108. In certain embodiments, the MH sequence is an amino acid sequence according to 5EO IJ MO: 108. In certain embodiments, the sequence

MH that is at least 90 95, 9195, 9295, 9395, 94 95, 9595, 96 95, 97 95, 9895, or 99 95 identical, contains substitutions (eg, conservative substitutions), insertions, or deletions relative to the reference sequence, but the antibody against C5, which includes such a sequence, retains the ability to bind to C5. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in the 5EO IU MO: 108. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the NMV (eg, in the EB). Optionally, the antibody against C5 includes the sequence MH in 5EO IU MO: 108, including post-translational modifications of this sequence. In a special embodiment, the MH includes one, two or three NMAs selected from the following: (a) NMV-NI, which includes the amino acid sequence according to BEO IU MO: 117, (p) NUV-H2, which includes the amino acid sequence according to BEO

IO MO: 118, and (c) NUV-NZ, which includes the amino acid sequence at 5EO IU MO: 121.

In another aspect, the anti-C5 antibody comprises a MH sequence that is at least 90 95, 91 95, 92 95, 93 Uo, 94 95, 95 96, 96 Uo, 97 Fo, 98 95, 99 95, or 100 95 identical in amino acid sequence to 5BEO AND MO: 109. In certain embodiments, the MH sequence is an amino acid sequence according to 5EO IJ MO: 109. In certain embodiments, the sequence

MH that is at least 90 95, 9195, 9295, 9395, 94 95, 9595, 96 95, 97 95, 9895, or 99 95 identical, contains substitutions (eg, conservative substitutions), insertions, or deletions relative to the reference sequence, but the antibody against C5, which includes such a sequence, retains the ability to bind to C5. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in the 5EO IU MO: 109. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the NMV (eg, 60 in the EB). Optionally, the antibody against C5 includes the sequence of MH in 5EO IU MO: 109, including post-translational modifications of this sequence. In a special embodiment, the MH includes one, two or three NMAs selected from the following: (a) NMV-NI, which includes the amino acid sequence according to BEO IU MO: 117, (p) NUV-H2, which includes the amino acid sequence according to BEO

IO MO: 118, and (c) NUV-NZ, which includes the amino acid sequence at 5EO IU MO: 121.

In another aspect, the anti-C5 antibody comprises a MH sequence that is at least 90 95, 91 95, 92 95, 93 Uo, 94 95, 95 96, 96 Uo, 97 Fo, 98 95, 99 95, or 100 95 identical in amino acid sequence to 5BEO I0O MO: 110. In certain embodiments, the MH sequence is an amino acid sequence according to 5EO IU MO: 110. In certain embodiments, the sequence

MH that is at least 90 95, 9195, 9295, 9395, 94 95, 9595, 96 95, 97 95, 9895, or 99 95 identical, contains substitutions (eg, conservative substitutions), insertions, or deletions relative to the reference sequence, but the antibody against C5, which includes such a sequence, retains the ability to bind to C5. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in the 5EO IU MO: 110. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the NMV (eg, in the EB). Optionally, the antibody against C5 includes the sequence MH in 5EO IU MO: 110, including post-translational modifications of this sequence. In a special embodiment, the MH includes one, two or three NMAs selected from the following: (a) NMV-NI, which includes the amino acid sequence according to BEO IU MO: 117, (p) NUV-H2, which includes the amino acid sequence according to BEO

IO MO: 120, and (c) NUV-NZ, which includes the amino acid sequence for 5EO IU MO: 121.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises a light chain variable domain (MI) sequence that is at least at 90 95, 91 9", 92 Uv, 93 U, 94 Or, 95 9, 96 9", 97 U, 98 9", 99 95 or 100 95 identical amino acid sequence according to any of 5EO IO MO: 20, 111-113. In certain embodiments, an MI sequence that is at least 90 Fo, 91 95, 92 Fo, 93 Y, 94 95, 95 90, 96 95, 97 Y, 98 95, or 99 95 identical contains a substitution (eg, conservative substitutions) , insertions or deletions relative to the reference sequence, but an anti-C5 antibody that includes such a sequence retains the ability to bind to

C5. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in any of the FEO IU MO: 20, 111-113. In certain embodiments, substitutions, insertions, or deletions occur at sites outside the NMA (eg, in the EN). arbitrarily

The antibody against C5 includes the MI sequence in any of the FEO IJU MO: 20, 111-113, including post-translational modifications of this sequence. In a particular embodiment, the MI includes one, two, or three NMAs selected from the following: (a) NMUV-I1, which includes the amino acid sequence of any of 5EO I MO: 75, 84122, 129, (B) NMUV-I2, which includes the amino acid sequence of any of 5EO IO MO: 85, 94, 123-124, 130, and (c) NMV-I 3, which includes the amino acid sequence of any of BEO IU MO: 95,104, 125,131.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises an Mi that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9995%, or 100% amino acid identical to sequences according to 5EO IO MO: 20. In certain variants of the implementation of the sequence of MI. is the amino acid sequence according to 5EO IO MO: 20. In certain embodiments, the MI sequence is at least at 90 95, 91 9", 92 95, 93 U", 94 U, 95 9", 96 9, 97 U", 98 95 or 99 95 is identical, contains substitutions (eg, conservative substitutions), insertions or deletions relative to the reference sequence, but an anti-C5 antibody that includes such a sequence retains the ability to bind to C5. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in the 5EO IU MO: 20. In certain embodiments, the substitutions, insertions, or deletions occur at sites outside the NMV (eg, in EN). Optionally, the antibody against C5 includes the MI sequence in 5EO IJOO MO: 20, including post-translational modifications of this sequence. In a special embodiment, the MI includes one, two or three NMAs selected from the following: (a) NMUV-I1, which includes the amino acid sequence according to FEO IU MO: 84, (5) NUV-12, which includes the amino acid sequence according to ZEO I

BO MO: 94, and (c) NMV-I 3, which includes the amino acid sequence according to 5EO 10 MO: 104.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises an Mi that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9995%, or 100% amino acid identical to sequences according to EC IJU MO: 111. In certain variants of the implementation of the sequence

MI is the amino acid sequence of 5EO AND MO: 111. In certain embodiments, an MI sequence that is at least at 90 95, 91 9", 92 95, 93 U", 94 U, 95 9", 96 9, 97 U" , 98 95 or 99 95 is identical, contains substitutions (for example, conservative substitutions), insertions or deletions relative to the reference sequence, but an anti-C5 antibody that includes such a sequence retains the ability to bind to C5. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in the 5EO IU MO: 111. In certain embodiments, 60 the substitutions, insertions, or deletions occur in regions outside the NMV (e.g.,

in EV). Optionally, the anti-C5 antibody includes the MI sequence in 5EO IO MO: 111, including post-translational modifications of this sequence. In a special embodiment, the MI includes one, two or three NMAs selected from the following: (a) NMUV-I1, which includes the amino acid sequence according to BEO IJOO MO: 122, (p) NUV-1I2, which includes the amino acid sequence according to FEO

IO MO: 123, and (c) NUV-I 3, which includes the amino acid sequence for 5EO I MO: 125.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises an Mi that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9995%, or 100% amino acid identical to sequences according to 5EO IYUO MO: 112. In certain variants of the implementation of the sequence

MI is an amino acid sequence according to 5EO AND MO: 112. In certain embodiments, an MI sequence that is at least at 90 95, 91 9", 92 95, 93 U", 94 U, 95 9", 96 9, 97 U" , 98 95 or 99 95 is identical, contains substitutions (for example, conservative substitutions), insertions or deletions relative to the reference sequence, but an anti-C5 antibody that includes such a sequence retains the ability to bind to C5. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in the 5EO IU MO: 112. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the NMV (eg, in the EB). Optionally, the anti-C5 antibody includes the MI sequence in 5EO IO MO: 112, including post-translational modifications of this sequence. In a special embodiment, the MI includes one, two or three NMAs selected from the following: (a) NMUV-I1, which includes the amino acid sequence according to BEO IJOO MO: 122, (p) NUV-1I2, which includes the amino acid sequence according to FEO

IO MO: 123, and (c) NUV-I 3, which includes the amino acid sequence for 5EO I MO: 125.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises an Mi that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9995%, or 100% amino acid identical to sequences according to EC IJU MO: 113. In certain variants of the implementation of the sequence

MI is an amino acid sequence according to 5EO AND MO: 113. In certain embodiments, an MI sequence that is at least at 90 95, 91 9", 92 95, 93 U", 94 U, 95 9", 96 9, 97 U" , 98 95 or 99 95 is identical, contains substitutions (for example, conservative substitutions), insertions or deletions relative to the reference sequence, but an anti-C5 antibody that includes such a sequence retains the ability to bind to C5. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in the 5EO IU MO: 113. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the NMV (eg, in the EB). Optionally, the anti-C5 antibody includes the MI sequence in 5EO IYUO MO: 113, including post-translational modifications of this sequence. In a special embodiment, the MI includes one, two or three NMAs selected from the following: (a) NMUV-I1, which includes the amino acid sequence according to BEO IJOO MO: 122, (p) NUV-1I2, which includes the amino acid sequence according to BEO

IO MO: 124, and (c) NUV-I 3, which includes the amino acid sequence of 5EO I MO: 125.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises MH as described in any of the embodiments provided above and Mi as in any of the embodiments provided above. In one embodiment, the antibody comprises sequences

MN and MI in any of 5EO IO MO: 10, 106-110 and in any of 5EO IU MO: 20, 111-113, respectively, including post-translational modifications of these sequences. In one embodiment, the antibody comprises the MN sequence of 5EO IU MO:10 and the MI sequence of 5EO IU

MO: 20. In one embodiment, the antibody comprises the MH sequence for 5EO IO MO: 106 and the MI sequence for 5BO IO MO: 111. In one embodiment, the antibody comprises the MH sequence for 5EO IU MO: 107 and the MI sequence for 5EO IUO MO: 111. In one embodiment, the antibody includes the MH sequence for 5EO IO MO: 108 and the MI sequence for 5EO I

MO: 111. In one embodiment, the antibody comprises the MN sequence of 5EO IU MO: 109 and the MI sequence of 5BO IU MO: 111. In one embodiment, the antibody comprises the MN sequence of 5EO IU MO: 109 and the MI sequence of 5EO IUO MO: 112. In one embodiment, the antibody includes the MH sequence of 5EO IO MO: 109 and the MI sequence of 5EO I

BO MO: 113. In one embodiment, the antibody comprises the MN sequence for 5EO IJU MO: 110 and the MI sequence for 5EO IJU MO: 113.

In one aspect, an anti-C5 antibody is provided, wherein the antibody comprises a UN sequence that comprises (a) NUV-NI, which includes the amino acid sequence of BEO IU MO: 54, (p) NUV-H2, which includes the amino acid sequence of 5EO I MO: 64, and (c) NUV-NZ, which includes the amino acid sequence of 5EO IUO MO: 74, and the MI sequence, which contains (a) NUV-I1, which includes the amino acid sequence of BEO IO MO: 84; (5) NUV-12, which includes the amino acid sequence for 5EO IU MO: 94; and (c) NMV-II 3, which includes the amino acid sequence according to ES

IO MO: 104.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises a sequence of UN, 60 which comprises (a) NUV-NI, which comprises the amino acid sequence according to BEO IYUO MO: 117, (p) NUV-

H2, which includes the amino acid sequence of 5EO I MO: 118, and (c) NUV-NZ, which includes the amino acid sequence of 5EO IO MO: 121, and the MI sequence, which contains (a) NUV-11, which includes the amino acid sequence of 5EO 10 MO: 122; (p) NUV-I2, which includes the amino acid sequence for 5EO IO MO: 123; and (c) NUV-IZ, which includes the amino acid sequence according to BEO IYUO MO: 125.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises a UN sequence that comprises (a) NUV-NI, which includes the amino acid sequence according to BEO IYUO MO: 117, (p) NUV-

H2, which includes the amino acid sequence of 5EO IUO MO: 119, and (c) NUV-NZ, which includes the amino acid sequence of 5EO IO MO: 121, and the sequence of MI, which contains (a) NUV-11, which includes the amino acid sequence of 5EO 10 MO: 122; (p) NUV-I2, which includes the amino acid sequence for 5EO IO MO: 123; and (c) NUV-IZ, which includes the amino acid sequence according to BEO IYUO MO: 125.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises a UN sequence comprising (a) NUV-NI, which comprises the amino acid sequence of BEO AND MO: 117, (p) NUV-

H2, which includes the amino acid sequence of 5EO I MO: 118, and (c) NUV-NZ, which includes the amino acid sequence of 5EO IO MO: 121, and the MI sequence, which contains (a) NUV-11, which includes the amino acid sequence of 5EO 10 MO: 122; (p) NUV-I2, which includes the amino acid sequence for 5EO IO MO: 124; and (c) NUV-IZ, which includes the amino acid sequence according to BEO IYUO MO: 125.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises a UN sequence that comprises (a) NUV-NI, which includes the amino acid sequence according to BEO IYUO MO: 117, (p) NUV-

H2, which includes the amino acid sequence of 5EO IO MO: 120, and (c) NMA-NZ, which includes the amino acid sequence of 5EO IO MO: 121, and the sequence of MI, which contains (a) NUV-I1, which includes the amino acid sequence of 5EO I

MO: 122; (c) NUV-12, which includes the amino acid sequence for 5EO IO MO: 124; and (c) NUV-

And 3, which includes the amino acid sequence according to 5BEO IU MO: 125.

In certain embodiments, the anti-C5 antibody of the present invention includes MH, as in any of the embodiments proposed above, and a heavy chain constant region that includes an amino acid sequence following any of 5EO IO MO: 33, 34, 35, 114115 and 116.

In certain embodiments, the anti-C5 antibody of the present invention includes an MI, as in any of the embodiments proposed above, and a light chain constant region that includes the amino acid sequence of any of BEO IU MO: 36, 37 and 38.

In another aspect, the invention provides an antibody that binds to the same epitope as the anti-C5 antibody provided herein. For example, in certain embodiments, an antibody that binds to the same epitope as an antibody provided in

Tables 2. As demonstrated by the working examples below, all of the anti-C5 antibodies shown in Table 2 are clustered in the same epitope group for C5, and they exhibit pH-dependent binding characteristics.

In an additional aspect, the invention provides an antibody that binds to the same epitope as the antibody provided herein. In a further aspect, the invention provides an antibody that binds to the same epitope as the antibody described in

Tables 7 or 8. In certain embodiments, an antibody is provided that binds to an epitope within a fragment that consists of amino acids 33-124 of the C5 beta chain (5EO IU

MO: 40). In certain embodiments, an antibody is provided that binds to an epitope within the C5 beta chain (5EO IO MO: 40) that includes at least one fragment selected from the group consisting of amino acids 47-57, 70-76, and 107 -110. In certain embodiments, an antibody is provided that binds to an epitope within the C5 beta chain fragment (5EO I

MO: 40), which includes at least one amino acid selected from the group consisting of THg47,

SiIsh48, AIa49, Rneb5O, Azr5b1, AIab2, Tyg53, Iuv57, Niv70, Ma!71, Nibz72, bZeg74, Sishub, Ma1IO7,

Zepov, I UziO" and Nivi 10. In another embodiment, the epitope of the antibody against C5 of the present invention is a conformational epitope.

In a further aspect of the invention, the anti-C 5 antibody according to any of the aforementioned embodiments is a monoclonal antibody, including a chimeric antibody, a humanized antibody, and a human antibody. In one embodiment, the anti-C5 antibody is an antibody fragment, for example, Em, Rab, Raf 5cEm, diabody, or E(ab)2 fragment. In another embodiment, the antibody is a full-length antibody, for example, an intact Da or Ids4 antibody, or another antibody class or isotype as defined herein.

In a further aspect, an anti-C5 antibody according to any of the aforementioned embodiments may include any of the features, alone or in combination, as described in Sections 1-7 below.

1. Antibody affinity

In certain embodiments, an antibody provided herein has a dissociation constant (Ka) that is 1 µM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less than or 0.001 nM or less (for example, 10"M or less, for example, from 10"M to 102M, for example, from 10"M to 10" 13 M).

In one embodiment, Ka is measured using an isotope-labeled antigen (IAB) binding assay. In one embodiment, the NIA is performed with the Rabr version of the antibody of interest and its antigen. For example, the binding affinity of an EPar solution to an antigen is measured by equilibrating Bar with the minimum concentration of labeled (I) antigen in the presence of titration series of unlabeled antigen, followed by capture of the bound antigen with a plate coated with an anti-Rab antibody (see, for example, Spec. ai., u). My. Vioi 293:865-881 (1999)). In order to set the conditions of analysis for tablets with many cells MISVOTITEV (registered trademark) (TPepto

Zsiepiyis) were coated overnight with 5 μg/ml anti-Rab capture antibody (Surrey!abz) in 50 mM sodium carbonate (pH 9.6) and then blocked with 2 95 (w/v) bovine serum albumin in PB5 for 2 to 5 hours at room temperature (approximately 23°C). In a non-adsorbing plate (Mips 2269620), 100 pM or 26 pM H25C antigen was mixed with serial dilutions of the Rar of interest (e.g., which is consistent with an anti-MESE antibody score, Rar-12, in Rheevzia eli a!., Sapseg Rev. 57:4593-4599 (1997)).The slave of interest is then incubated overnight; however, the incubation may be continued for a longer period (e.g., about 65 hours). , to ensure that equilibration has been achieved. The mixtures are then transferred to a capture plate for incubation at room temperature (eg, for one hour). The solution is then removed and the plate is washed eight times with 0.195 polysorbate 20 (PM/EEM-20 (registered trademark) in RV 5. When the tablets are dried, then add 150 μl/cell to the scintillator (MISVO5SIMT-20; RasKaga) and tablets are counted on a TORSOSHMT "m (RasKaga) gamma counter for 10 minutes. Concentrations of each Rab that give less than 20 95 of maximal binding or exactly 2095 of it are selected for use in the competitive binding assay .

According to another embodiment, Ka is measured using surface plasmon resonance analysis VIASOVE (registered trademark). For example, analysis using VIASONVE (registered trademark)-2000 or VIASOVE (registered trademark)-3000 (VIASORVE (registered trademark), Ips.,

Rizsaiaulau, MU) is performed at 25 "C with immobilized CM5 antigen chips at approximately 10 response units (RU) In one embodiment, carboxymethylated dextran biosensor chips (CM5, VIASOVE (registered trademark), Ips.) are activated with M-ethyl -M'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EC) and M-hydroxysuccinimide (MH) according to the supplier's instructions. The antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (approximately 0.2 μM ) prior to injection at a flow rate of 5 μl/min to achieve approximately 10 response units (RI) of bound protein. After injection of antigen, 1 M ethanolamine is injected to block unreacted groups. To measure kinetics, two-fold serial dilutions of Rab (from 0.78 to 500 nm) are injected into РВ5 with a surfactant (РВ5Т) of 0.05 95 polysorbate 20 (ТМ/ЕЕМ-207м) at 25"C with a flow rate of approximately 25 μl/minute. Association constants (Cop) and dissociation constants (Which are calculated using the one-to-one binding model

Iapdtiig (VIASONE (registered trademark) EmaYinayop boiimage version 3.2) by simultaneous comparison of association and dissociation sensograms. The equilibrium constant of dissociation (Ka) is calculated as the ratio koi/kop. See, for example, Spep ei ai., u. My. Axes 293:865-881 (1999). If the association constant exceeds 105 M" s" in the above-mentioned surface plasmon resonance analysis, then the association constant can be determined using the fluorescence quenching method in which the increase or decrease in the intensity of fluorescent radiation is measured (excitation - 295 nm; emission - 340 nm, 16 nm bandwidth ) at 25 "С 20 nM antibodies against the antigen (Rab form) in RVE, pH 7.2, in the presence of increasing concentrations of the antigen, which is measured in a spectrometer, such as a spectrophotometer capable of measuring by the stopped-flow method (Amim Ipvigitepov), or a spectrophotometer 5IM-AMIMSO "m series 8000 (Tneptobresigopis) with a cuvette where mixing is carried out. 2. Fragments of antibodies

In certain embodiments, an antibody provided herein is a fragment of an antibody. Antibody fragments include, but are not limited to, fragments of Rab, Rab",

Ear-5H, K(ar)2, Em and 5cEm and other fragments described below. For a review of certain fragments of antibodies, see Niazop et al., May. May 9:129-134 (2003). For an overview of the 5sem fragments, see, for example, RiisKiip, in Te RIPaptasiodu oi Moposiopa! Apiirroadie5, my. 113, Nosepriga apa

Mooge eeav., (Zrgipdeg-Mepad, Mem Moi), pp. 269-315 (1994); see also U/O 93/16185 and US Patent Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a discussion of Bar and E(ar)2 fragments that include reutilization receptor binding epitope residues and that have an extended half-life of ip mimo.

Diatyles are antibody fragments with two antigen-binding sites, which can be bivalent or bispecific. See, for example, EP 404,097; M/O 1993/01161; Niazop eyi a!I., Mai. Honey. 9:129-134 (2003); and Noiipodeg eyi a!., Rgos. Mai). Asad 5 BOTH 90:6444-6448 (1993).

Triatila and tetratila are also described in Nidzop eiai., Mai. Honey. 9:129-134 (2003).

Single-domain antibodies are antibody fragments that include all or part of the variable domain of the heavy chain or all or part of the variable domain of the light chain of the antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (Juotapii5, Ips., MMaynat, MA; see, for example, US Pat. Mob, 248,516).

Antibody fragments can be obtained in a variety of ways, including, but not limited to, proteolytic digestion of an intact antibody, as well as production by recombinant host cells (eg, E. soii or phage), as described herein. 3. Chimeric and humanized antibodies

In certain embodiments, an antibody provided herein is a chimeric antibody.

Certain chimeric antibodies are described, for example, in US Pat. No. 4,816,567; and Mogtizop ei ai!., Rgos.

May Asad Axes BOTH 81:6851-6855 (1984)). In one example, the chimeric antibody comprises a non-human variable region (eg, a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region. In a further example, a chimeric antibody is a "switched class" antibody in which the class or subclass of the original antibody has been changed. Chimeric antibodies include their antigen-binding fragments.

In certain embodiments, the chimeric antibody is a humanized antibody. Usually, the antibody, which is not human, is humanized in order to reduce the immunogenicity for humans, at the same time

The specificity and affinity of the original antibody, which is not human, is preserved. Generally, a humanized antibody comprises one or more variable domains in which the NUN, e.g.

EBs (or parts thereof) are derived from a non-human antibody, and EBs (or parts thereof) are derived from human antibody sequences. A humanized antibody will optionally also include at least a portion of a human constant region. In some embodiments, some residues

ECs in a humanized antibody are replaced with appropriate residues from a non-human antibody (eg, an antibody from which NUR residues are derived), for example to restore or improve the specificity or affinity of the antibody.

An overview of humanized antibodies and methods of obtaining them is given, for example, in AItaadgo apa

Ekapv5zop, Egopi. Anyway. 13:1619-1633 (2008), and they are further described, for example, in Wiesptapp et al., Maite 332:323-329 (1988); Oyeep ei ai., Rgos. Mai! Asad 5 BOTH 86:10029-10033 (1989); US Patent Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kavptitgi eyi a!., Meityoa" 36:25-34 (2005) (describing the transplantation of a specificity-determining region (50NA)); Radiap,

MY. And so on. 28:489-498 (1991) (describing "transposition" ("esipasipad")); Ba! Asdpa ei ai.,

Meipodz 36:43-60 (2005) (describing the "EV permutation" ("EV zp!Shiiipa")); and O5roshpt her ai.,

Meinpodz 36:61-68 (2005) and KiitkKa ei ai., Vg. U. Sapseg. 83:252-260 (2000) (describing a "guided selection" ("Ziidea zeiIesiop") approach to EB mixing).

Human framework regions that can be used for humanization include, but are not limited to: framework regions that are selected using the "best fit" ("bevi-yiyi") method (see, for example, 5bitv5 ei ai.,.). And so on! 151:2296 (1993)); framework regions that are derived from the consensus human antibody sequence of a certain subset of the variable regions of the light and heavy chains (see, for example, Sapeg yi ai., Rgos. Mai).

Asad bsi BOTH 89:4285 (1992); and Rgezia evy ai., U. Ittipoi. 151:2623 (1993)); mature (somatically mutated) human scaffolds or human germline scaffolds (see, e.g., AItadgto apa Egapozop, Egopi. Viovsi. 13:1619-1633 (2008)); and framework sites obtained as a result of screening of EC libraries (see, for example, Vasa ei ai., y. Vioi. Spet. 272:10678-10684 (1997) and Rozok yi a!., 9. Vioi. Snet. 271:22611 -22618(1996)). 4. Human antibodies

In certain embodiments, the antibody provided herein is a human antibody.

Human antibodies can be obtained using various methods known in the art. Antibodies of a person are described in general in maps BiIK apa maps de Whipkey, Si. Orip. Rnaptasoi 5:368-374 (2001) and

Goprego, Sit. Orip. And so on. 20:450-459 (2008).

Human antibodies can be obtained by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to inoculation with the antigen. Such animals usually contain all or part of human immunoglobulin loci that replace endogenous immunoglobulin loci, or that are extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are usually inactivated. For an overview of methods of obtaining human antibodies from transgenic animals, see I oprego, Maia. Vioyesp. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe the CHEMOMOYBE "M technology: U.S. Patent No. 5,770,429, which describes the NOMAV (registered trademark) technology; U.S. Patent No. 7,041,870, which describes the K-M technology

MOBE (registered trademark) and US patent application publication

MO 2007/0061900, which describes the MEGOSIMOO5BE technology (registered trademark).

Human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with a distinct human constant region.

Human antibodies can also be obtained by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines have been described for the production of human monoclonal antibodies. (See, for example, Ko?rog U. Ittipoi., 133:3001 (1984); Vgodeig ei ai.,

Moposiope! Apiirodu Rhodisiup Tesppidnez apa Arriisaiop5, pp. 51-63 (OekKKeg, Ips., Mem/mok, 1987); and Voyeteg evy ai., ). And so on. 147:86 (1991)). Human antibodies obtained by technology using a human B-cell hybridoma are also described in i. Mai! Asad

All together. BOTH 103:3557-3562 (2006). Additional methods include methods described, for example, in US Pat. No. 7,189,826 (which describes the production of monoclonal antibodies (human BM from hybridoma cell lines)) and Mo, Hiapdaii Miapuihie 26(4) 265-268 (2006) (which describes hybridomas of the type "human- human"). Technology using a human hybridoma (technology

Triomi) (Tiota iesppoYodu) is also described in MoiIteg5, Nizioiodu apa NiziorainoYodu 20(3): 927-93 7 (2005) and MoPtegv, Meinodz apa Ripaipd5 in Ehregitepiai apa Siipisa! Rnapthasoiodu 27(3): 185-191 (2005).

Human antibodies can also be generated by isolating sequences of the variable domain of the Em clone selected from human-derived phage display libraries. Such variable domain sequences can then be combined with the desired human constant domain. Methods of selecting human antibodies from antibody libraries are described below. 5. Antibodies originating from libraries

Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies with the desired activity or types of activity. For example, various methods of generating phage display libraries and screening such libraries for antibodies with desired binding characteristics are known in the art. An overview of such methods is given, for example, in Noodeproot essay ai.

Toioula, MU, 2001), and they are also described, for example, in MsSanepu ei ai., Maishge 348:552-554;

Siaskzop ei ai., Maite 352:624-628 (1991); Maikz ei ai., U. Moi. Axes 222:581-597 (1992); MaiQkw,

My.MoOI. Vioi 248:161-175 (Go, ed., Nytap Rhev55, Toiomzha, Moscow State University, 2003); Poor women, U. Moi. Vioi 338(2):299-310 (2004); And ee ei ai., 9. My. Vioi 340(5):1073-1093 (2004); RePoive, Rhos. Mai! Asad from the U.S. BOTH 101(34)12467-12472 (2004); And ee ev! ai., 9U. And so on. Meinodz" 284(1-2):119-132 (2004).

In certain phage display methods, the MH and MI gene repertoires are separately cloned by polymerase chain reaction (PCR) and arbitrarily recombined into phage libraries that can then be screened for antigen binding of the phage as described in

Muipieg eyi a. App. Weum. And so on. 12:433-455 (1994). Phage usually displays antibody fragments either as single-chain Em fragments (c5cEm) or as Rar fragments. Libraries from immunized sources offer highly immunogen-affinity antibodies without the need for hybridoma construction.

Alternatively, the native repertoire can be cloned (e.g., from a human) to obtain a single source of antibodies for a wide range of both autoantigens and non-autoantigens without any immunization, as described in Siiiiine et al., EMBO 3. 12:725- 734 (1993). Finally, native libraries can also be obtained by synthesis by cloning segments of the unrearranged U gene from stem cells using ROS primers that contain an arbitrary sequence to encode the hypervariable regions of SOBZ3 and performing ip myo rearrangement as described in Noodeproot, .). My. Vioi 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: Pat

US Patent No. 5,750,373 and US Patent Publications MoMeo 2005/0079574, 2005/0119455, 2005/0266000, 60 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936 and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody libraries are believed to be the human antibodies or human antibody fragments described herein. 6. Multispecific antibodies

In certain embodiments, the antibody provided herein is a multispecific antibody, for example, a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificity to at least two different sites. In certain embodiments, one of the binding specificities is specific to C5, and the other is specific to any other antigen. In certain embodiments, bispecific antibodies can bind to two different C5 epitopes. Bispecific antibodies can also be used to localize cytotoxic agents to cells that express C5.

Bispecific antibodies can be obtained as full-length antibodies or as antibody fragments.

Methods for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two heavy chain and light chain immunoglobulin pairs that have excellent specificity (see Miivivip apa Sceio, Maishgte 305:537 (1983)),

MO 93/08829 and TgaipesKeg et al., EMVO .. 10:3655 (1991)), and a "protrusion-into-recess" type design (see, for example, US Patent Mo 5,731,168). Multispecific antibodies can also be obtained by engineering the effects of electrostatic regulation to obtain heterodimeric Es antibody molecules (M/02009/089004А1); by cross-linking two or more antibodies or fragments (see, for example, US Pat. No. 4,676,980 and

Vgeppap vy a!., Zsiepse, 229:81 (1985)); by using a leucine fastener to obtain bispecific antibodies (see, for example, Kovzie!pu ei ai., y. Ittipoi. 148(5):1547-1553 (1992)); by using the "diatyl" technology to obtain fragments of bispecific antibodies (see, for example, NoiYipadeg yei ai!., Rgos. Mai!I. Asad. 5si. OBA 90:6444-6448 (1993)); and by using single-chain Em dimers (5CsEm) (see, for example, Cshybreg eyi ai., u). And so on. 152:5368 (1994)); and by preparing trispecific antibodies as described, for example, in

That ey ai., y. And so on. 147:60 (1991).

Also included in this description are engineered antibodies with three or more functional antigen-binding sites, including "octopus antibodies" ("osiorib apiirodiev") (see, for example, O5 2006/0025576).

zo In this specification, an antibody or fragment also includes a "dual-acting EAB" (Yua! Asiipu EAB) or "AR" that includes an antigen-binding site that binds to C5 as well as another, distinct, antigen (see for example, О52008/0069820). 7. Variants of antibodies

In certain embodiments, variant amino acid sequences of the antibodies proposed herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. Variants of the amino acid sequences of the antibody can be obtained by making appropriate modifications of the nucleotide sequence that encodes the antibody, or by synthesizing peptides. Such modifications include, for example, deletions from, and/or insertions into, and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, provided that the final construct inherent desirable characteristics, such as antigen binding a. Variants of substitutions, insertions and deletions

In certain embodiments, antibody variants that have one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include NMA and EB. Conservative substitutions are listed in Table 1 under the heading "preferred substitutions". The most significant changes are suggested in Table 1 under the heading "typical substitutions" and are described below with reference to classes of amino acid side chains. Amino acid substitutions can be made in the antibody of interest, and the products can be screened for desired activity, eg, retention/improvement of antigen binding, reduced immunogenicity, or improved AOSS or SOC.

Table 1

Amino acids can be grouped according to the general properties of the side chains: (1) hydrophobic: norleucine, Mei, Aa, Mai, I ei, Ne; (2) neutral hydrophilic: Suv, Zeg, TNg, Azp, Sip; (3) acidic: Avr, Sy; (4) alkaline: Niv, I uv, Agu; (5) residues that affect chain orientation:

STU, Rgo; and (6) aromatic: Tgr, Tug, Rne.

Non-conservative substitutions will involve exchanging a member of one of these classes for another class.

One type of substitution-derived variant involves the substitution of one or more residues of the hypervariable region of the original antibody (eg, humanized antibody or human antibody). Generally, the resulting variant(s) selected for further investigation will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, decreased immunogenicity) compared to the original antibody and/or will have in fact, certain biological properties of the original antibody are preserved. An example of a substitution-derived variant is an affinity-mature antibody that can be obtained in a convenient manner, for example, using phage display-based affinity maturation methods, such as those described herein. Briefly, one or more NMA residues are mutated, and antibody variants are displayed on the phage envelope and screened for specific biological activity (eg, binding affinity).

Changes (e.g., substitutions) can be made to the NMV, for example, to improve the affinity of the antibody. Such changes can be carried out at variable points ("poivzroiv") of NMEA, that is, at residues encoded by codons that undergo mutations with a high frequency during the process of somatic maturation (see, for example, Spomzhanigu, Meipoaz» Moi. Visi. 207:179- 196 (2008)), and/or on residues that contact the antigen, while the resulting MH or MI variant is tested for binding affinity. Affinity maturation due to construction and re-selection from secondary libraries is described, for example, in Noodeproot's essay ai.

Viodou 178:1-37 (O'"Viiep ei aiY., ed. Nytap Rgev5, Toioula, MU, (2001). In some embodiments, affinity maturation makes various changes in variable genes selected for

From maturation, by any of a number of methods (eg, error-prone PCR, strand transfer, or oligonucleotide-directed mutagenesis). A secondary library is then created. This library is then screened to identify antibody variants with the desired affinity. In another way of achieving diversity, they are used

HMV-directed approaches, in which several NMA residues are randomized (for example, 4-6 residues simultaneously). Residues of NMA involved in antigen binding can

To be specifically identified, for example, using alanine-scanning mutagenesis or modeling. In particular, SOV-NZ and SOV-Y Z are often chosen as targets.

In certain embodiments, substitutions, insertions or deletions can be made within one or more HMPs as long as such changes do not significantly reduce the ability of the antibody to bind to the antigen. For example, conservative changes (eg, conservative substitutions as suggested herein) that do not significantly reduce binding affinity can be made in the NMA. Such changes can be, for example, in NMA outside the residues in contact with the antigen. In certain embodiments of the variant sequences of UN and MI proposed above, each of the NMCs is either unchanged or contains no more than one, two, or three amino acid substitutions.

A useful way to identify residues or regions of an antibody that can be targeted for mutagenesis is called "alanine-scanning mutagenesis," as described in Science, 5th Science 244:1081-1085 (1989). In this method, a target residue or group of residues (e.g., charged residues such as agu, azr, Piv, Iuz, and dish) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of antibody with antigen. Further substitutions can be made at amino acid positions, demonstrating functional sensitivity to initial substitutions. Alternatively or additionally, the crystal structure of the antigen-antibody complex identifies the points of contact between the antibody and the antigen. Such contact residues or neighboring residues can be targets, or they can be deleted as replacement candidates. Options can be screened to determine if they contain the desired properties.

Insertions of amino acid sequences include amino- and/or carboxyl-terminal fusions, the length of which varies in the range from one residue to polypeptides that contain a hundred or more residues, as well as insertions in the sequence of one or multiple amino acid residues. Examples of terminal insertions include an antibody with an M-terminal methionyl residue.

Other options for inserting an antibody molecule include fusing the M- or C-terminus of the antibody with an enzyme (eg, AYUERT) or a polypeptide that extends the half-life of the antibody in serum.

Z. Variants of glycosylation

In certain embodiments, an antibody of the present invention is modified to increase or decrease the degree to which the antibody is glycosylated. Addition of glycosylation sites to an antibody or their deletion can be traditionally performed by changing the amino acid sequence to create or delete one or more glycosylation sites.

Where the antibody includes an E region, the carbohydrate attached to it can be changed. Native antibodies produced by mammalian cells usually include a branched 2-antennary oligosaccharide, which is usually attached by M-crosslinking to the A5zp297 domain of the CH2 E region. See, for example, MUgidni eyi ai., TIVTESN 15:26-32 (1997). This oligosaccharide may include various carbohydrates, such as mannose, M-acetylglucosamine (CysMAc), galactose and sialic acid, as well as fucose, attached to the CisCyma in the "stem" of the 2-antennary oligosaccharide structure. In some embodiments, modification of the oligosaccharide in the antibodies of the invention can be performed to create antibody variants with certain improved properties.

In one embodiment, antibody variants are provided that have a hydrocarbon structure that lacks fucose attached (directly or indirectly) to the E region.

For example, the amount of fucose in such an antibody can be from 1,905 to 80,9b, from 1,95 to 65,95, from 595 to 65,965, or from 2,095 to 40,95. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain on Avzp297 relative to the sum of all glycostructures (eg, complex, hybrid, and high-mannose structures) attached to

Avp297, measured by MA mass spectroscopy! 0I-TOE, as described, for example, in

MO2008/077546. A5zp297 denotes an asparagine residue located at approximately position 297 in the E-site (European nomenclature of residues of E-sites); however, Azp297 can also be located approximately -7- C amino acids to the right or left of position 297, i.e., between positions 294 and 300, resulting from minor sequence variations in antibodies. Such fucosylation variants may have an improved function of AUCOS. See, for example, US MoMe patent publications 05 2003/0157108 (Rgezia, I.); 5 2004/0093621 (Kuozhsha Nakko Koauo So., 4). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: 5 2003/0157108; UMO 2000/61739; UMO 2001/29246; 60 O52003/0115614; 005 2002/0164328; 005 2004/0093621; 005 2004/0132140; 05 2004/0110704;

O52g004/0110282; 005 2004/0109865; UMO 2003/085119; MO 2003/084570; MO 2005/035586;

MO 2005/035778; MO 2005/053742; MO 2002/031140; OkKa?laki ei ai., U. Moi. Vioi 33671 239-1249 (2004); Matape-ONpiki ei ai., Vioiesn. Vioepud. 87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Hei 13 CHO cells deficient in protein fucosylation (VirKai et al., Ag. Biospet. Viorpuz. 249:533-545 (1986); published US Patent Application 2003/0157108, Rzezia, I ; and M/O2004/056312, Adatvs ei ai., especially in

Example 11), and knockout cell lines, such as CHO cells, which are knockout for the alpha-1,6-fucosyltransferase gene, ROT8, (see, e.g., Uatape-Oppiki et al., Bioesn. Bioepd. 87:614 (2004 );

Further, variants of antibodies with halved oligosaccharides are proposed, for example, oligosaccharides in which the 2-antennary oligosaccharide attached to the Ps region of the antibody is halved with M-acetylglucosamine (C1isMAc). Such antibody variants may have reduced fucosylation and/or improved AOSS function. Examples of such variants of antibodies are described, for example, in UUO 2003/011878 (Ueap-Maigei and I.); US patent Mo 6,602,684 (Shtapa ei a!) and 05 2005/0123546 (Otapa ei aI.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the E-region are also offered. Such antibody variants may have improved SOC function. Such variants of antibodies are described, for example, in

MO 1997/30087 (Raiye! eyi aI.); UUO 1998/58964 (Raii, 5.) and MO 1999/22764 (Vas, 5.). c Variants of E-plots

In certain embodiments, one or more amino acid modifications can be made in the Es region of the antibody proposed herein, thereby generating a variant of the Es region.

A variant ERs region may include a sequence of a human Es region (e.g., Es regions

human IS, Ids2, DOZ or Ids4) that has an amino acid modification (for example, a substitution) at one or more amino acid positions.

In certain embodiments, the invention provides an antibody variant that possesses some, but not all, of the effector functions that make it a desirable candidate for an application in which the half-life of the ip mimo antibody is important, while certain effector functions (such as SOC and

AOSS) are optional or harmful. Cytotoxicity assays of ip miigo and/or ip mimo can be performed to confirm reduction/depletion of SOC and/or AOSS activity. For example,

Assays of binding to the Es receptor (Es!) can be performed to verify that the antibody lacks binding to Es-haMMa-E (as a result of which insufficient activity of AOSS is likely), but retains the ability to bind to EsAp. Primary cells for mediation

ARSCs, MK cells, express only Es- and MmMa-VIII, while monocytes express Es-gamma-

VI, Rs-tamMa-VII and Es-gtamMa-VI. EsA expression on hematopoietic cells is summarized in

Table C on page 464 in the article Nameisn apa Kipei, Appy. German And so on. 9:457-492 (1991).

Non-limiting examples of ip myo assays for assessing the AUCS activity of a molecule of interest are described in US Pat.

Rgos. Magician! Asad si. OBA 83:7059-7063 (1986)) and NeiYibtot ei ai., Rhos. Magician! Asad axis BOTH 82:1499-1502 (1985); in US Patent No. 5,821,337 (see Vgiddetapp et al., U. Ehr. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be used (see, for example, ASTI"M non-radioactive cytotoxicity assay for flow cytometry (SePTesNpo|odu, Ips. Moshpiaip Miemu, SA); and SuiyuTox 96 (registered trademark) non-radioactive cytotoxicity assay (Rgoteda, Madizhop, Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer cells (NKs). Alternatively or additionally, the AUC activity of a molecule of interest can be assessed by IP mimo, for example, in an animal model, such as , as described in Siupez ei ai., Rgos.

Magician! Asad bsi BOTH 95:652-656 (1998). Seed binding assays can also be performed to confirm that the antibody is unable to bind to Seed and, as a result, lacks SOC activity. See, for example, EGI5ZA with connection to Sid and SZs in M/O2006/029879 and

MO2005/100402. To assess complement activation, it is possible to perform an analysis of SOS (see, for example, Saapo-Zapiogo ei aiYi., )U. And so on. MeiShosdv 202:163 (1996); Studd eyi ai., Viosa 101:1045-1052 (2003); and Stado ei ai., Viosa 103:2738-2743 (2004)). Definition of linking with

EsNAp and clearance/half-life of ip mimo can also be performed using methods known in the art (see, e.g., Reikoma vii., IP. Ittipoi. 18(12):1759-1769 (2006)).

Antibodies with reduced effector function include antibodies with replacement of one or more residues of the ES region: 238, 265, 269, 270, 297, 327 and 329 (US patent Mo 6,737,056). Such Es mutants include Es mutants with substitutions at two or more of the following amino acid positions: 265,269, 270, 297, and 327, including the so-called "SAMA" Es mutant with substitution of residues 265 and 297 for alanine (US patent Mo 7,332,581).

Certain variants of antibodies with improved or weakened binding to EsVp have been described. (See, e.g., US Pat. No. 6,737,056; MO 2004/056312 and Publ., 9. Biol. Spec. 9(2): 6591-6604 (2001)).

In certain embodiments, the variant of the antibody includes an Es region with one or more amino acid substitutions that improve AJOSS, for example, substitutions at positions 298, 333 and/or 334 of the Es region (European Nomenclature of Residues).

In some embodiments, the Es region is modified to alter (ie, enhance or attenuate) Cd binding and/or complement-dependent cytotoxicity (CTO), for example, as described in US Pat. No. 6,194,551, M/O1999/51642 and Idysodye of her ai.,

U. Ittipoi. 164:4178-4184 (2000).

Antibodies with a prolonged half-life and improved binding to the neonatal Es receptor (ERsVp), which is responsible for the transfer of maternal ideas to the fetus (Syshueg ei ai., y).

And so on. 117:587 (1976) and Kit et al., U. Ittipoi. 24:249 (1994)), described in 052005/0014934 (Nipup et al.). These antibodies include an Es region with one or more substitutions in it that improve binding of the Es region to EsNp. Such ES variants include variants with substitutions at one or more residues of the ES site: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, for example, with a substitution of residue 434

ES plots (US patent Mo 7,371,826).

See also Juipsap, Mayte 322:738-40 (1988); US patent Mo 5,648,260; US patent

Mo. 5,624,821 and Mo. 1994/29351, which consider other examples of E-site variants. and. Antibody variants constructed using cysteine

In certain embodiments, it may be desirable to create cysteine-engineered antibodies, such as ShiomMABr, in which one or more antibody residues are replaced by cysteine residues. In special variants, the replacement of residues is carried out on the available sites of the antibody. As a result of the substitution of these residues with cysteine, reactive thiol groups are located in accessible sites of the antibody and can be used to link the antibody with other components, such as drug components or "linker drug" components, to form an immunoconjugate, as described below in this description. In certain embodiments, any one or more of the following residues may be substituted

With cysteine: M205 (Cabot's nomenclature) E-sections of the light chain; A118 (European Nomenclature) E-sections of heavy chain and 5400 (European Nomenclature) E-sections of heavy chain. Antibodies constructed using cysteine can be prepared as described, for example, in US Patent No. 7,521,541. e. Derived antibodies

In certain embodiments, an antibody provided herein can be further modified to contain additional non-proteinaceous components that are known in the art and readily available. Components suitable for obtaining antibody derivatives include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene copolymer and maleic anhydride, polyamino acids (or homopolymers, or random copolymers), and dextran or poly(p-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (eg, glycerins), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde can have advantages during production due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Typically, the amount and/or type of polymers used for derivatization can be determined based on property considerations that include, but are not limited to, specific properties or functions of the antibody intended to be improved so that the derived antibody is used in therapy under defined conditions. conditions, etc.

In another embodiment, conjugates of an antibody and a non-proteinaceous component are provided, which can be selectively heated by exposure to radiation. In one embodiment, the non-bilious-like component is a carbon nanotube (Kath Yei Ai!., Rgos. Mai!. Asad. zZsi. OBA 102:11600-11605 (2005)). The radiation can be of any wavelength, including, but not limited to, a wavelength that is not harmful to normal cells, but heats the non-proteinaceous component to a temperature at which cells close to 60 non-proteinaceous component of the antibody die.

B. Recombinant methods and compositions

Antibodies can be obtained using recombinant methods and compositions, for example, as described in US Pat. No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-C5 antibody described herein is provided. Such a nucleic acid can encode an amino acid sequence that includes a UM antibody and/or an amino acid sequence that includes a MH antibody (eg, the light and/or heavy chains of the antibody). In a further embodiment, one or more vectors (eg, expression vectors) are provided that include such nucleic acid. In a further embodiment, a host cell is provided that contains such a nucleic acid. In one such embodiment, the host cell contains (eg, has been transformed by): (1) a vector that includes a nucleic acid that encodes an amino acid sequence that includes Mi. antibodies, and an amino acid sequence that includes MH antibodies, or (2) a first vector that includes a nucleic acid that encodes an amino acid sequence that includes Mi. antibodies, and a second vector that includes a nucleic acid that encodes an amino acid sequence that includes MH antibodies. In one embodiment, the host cell is a eukaryotic cell, for example, a Chinese Hamster Ovary (CHO) cell, or a lymphoid cell (for example, a MO cell, M50, 5p20). In one embodiment, a method of producing an anti-C5 antibody is provided, wherein the method comprises culturing a host cell that contains a nucleic acid encoding an antibody as provided above under conditions suitable for expression of the antibody, and optionally obtaining the antibody from the host cell (or the culture medium of the host cell).

To recombinantly obtain an antibody against C5, the nucleic acid that encodes this antibody, for example, as described above, is isolated and inserted into one or more vectors for subsequent cloning and/or expression in a host cell. Such nucleic acid can be easily isolated and sequenced using conventional methods (for example, using oligonucleotide probes that are able to specifically bind to the genes that encode the heavy and light chains of the antibody).

Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, especially when glycosylation and effector functions of Es are not

With the necessary For expression of antibody fragments and polypeptides in bacteria, see, for example, US Patent Nos. 5,648,237, 5,789,199 and 5,840,523. (See also Spapop,

Method ip Moiesshiag Vioiodu, Moi. 248 (V.K.S. I o, ed., Nytapa Rgev5, Toioma, MU, 2003), pp. 245-254, where the expression of antibody fragments in E. soy is described). After expression, the antibody can be isolated from the bacterial cell paste in the form of a soluble fraction and further purified.

In addition to prokaryotic, eukaryotic microbes such as mycelial fungi or yeast are suitable hosts for cloning or expression of antibody-encoding vectors, and they include fungal and yeast strains whose glycosylation cascades have been "humanized", resulting in an antibody with a partial or full profile of human glycosylation. Look

Segpdgov5, Mai. Vioyesn. 22:1409-1414 (2004), and | and her a!., Mai. Vioyesn. 24:210-215 (2006).

Suitable host cells for expression of the glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified that can be used in association with insect cells, especially for transfection of 5-rodorieg cells.

Tgidiregaa.

Plant cell cultures can also be used as hosts. See, for example, US Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe the RI AMTIVOOIE5" technology for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to growth in suspension can be used. Other examples of useful mammalian host cell lines are the monkey kidney CUT line transformed with 5U40 (СО5-7); human embryonic kidney line (293 or 293 cells, as described, for example, in Stapat 6th ai.,

U. ep Migoi. 36:59 (1977)); newborn hamster kidney cells (HNK); mouse cells

Sertoli (TMA4 cells, as described, for example, in Maineg, Vioi. Nergoid. 23:243-251 (1980)); monkey kidney cells (CM1); African green monkey kidney cells (MENVO-76); human cervical carcinoma cells (HCC); dog kidney cells (MSC); gray rat liver cells (VA ZA); human lung cells (M/138); human liver cells (Ner a2); mouse mammary gland tumor cells (MMT 060562); TVI cells, as described, for example, in Maineg and I,, Appai5 M.M. Asai. si. 383:44-68 (1982); MAC 5 cells and E54 cells. Other useful mammalian host cell lines include Chinese Hamster Ovary (HNO) cells, including ONEV" HNO cells (pa ei ai., Rhos. Mai!. Asad. 5si. OBA 77:4216 (1980)); and myeloma cell lines such as MO, M5O, and 5r2/0.For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Magaki apa My, Meinodz ip Moiessciag Wioiodu, Moi. 248 (V.K.S. Io, ed., Nytapa Rgevs5, Toioula, MU), pp. 255-268 (2003).

Polyclonal antibodies are mainly formed in animals as a result of numerous subcutaneous (5c) or intraperitoneal (ip) injections of the corresponding antigen and adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic for the species to be immunized, for example snail lymph hemocyanin, serum albumin, bovine thyroglobulin or soy trypsin inhibitor, using a bifunctional or derivatizing agent, e.g. maleimidobenzoyl sulfosuccinimide ester (conjugation via cysteine residues), M-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, 5OCI" or B'M-C-ME, where B and

IN! are different alkyl groups.

Animals (usually non-human mammals) are immunized against the antigen, immunogenic conjugates, or derivatives by combining 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with C volumes of complete Freund's adjuvant and administering this solution by transdermal injection at multiple sites. One month thereafter, animals are re-stimulated with 1/5 to 1/10 of the original amount of peptide or conjugate in complete adjuvant

Freund by subcutaneous injection in numerous places. 7-14 days after that, blood is taken from the animals, and the serum is analyzed for the antibody titer. Animals are stimulated until the titer stabilizes. Preferably, the animal is stimulated with a conjugate of the same antigen, but conjugated with a different protein and/or through a different cross-linking reagent. Conjugates can also be obtained in recombinant cell culture as fusion proteins. In addition, agents that promote aggregation, such as alum, are suitably used to enhance the immune response.

Monoclonal antibodies are derived from a population of essentially homogeneous antibodies, that is, the individual antibodies that make up the population are identical except for possible natural mutations and/or post-translational modifications (eg, isomerization, amidation) that may be present in small amounts. Therefore, the term "monoclonal" indicates the nature of the antibody, when it is not a mixture of different antibodies.

For example, monoclonal antibodies can be obtained using the hybridoma method, which was first described in Copieg et al., Mashte 256(5517):495-497 (1975). In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described above in this specification to generate lymphocytes that either produce or are capable of producing antibodies that will specifically bind to the protein being used for immunization. Alternatively, lymphocytes can be immunized ip myigo.

The immunizing agent will usually include an antigenic protein or a fusion variant thereof.

In general, any of the peripheral blood lymphocytes (PBV) are used if human-derived cells are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with immortal cell lines using a suitable fusion agent such as polyethylene glycol to form a hybridoma cell (Schdipud, Moposiopa! Apiirodiev:

Rgipsiriez apa Rgasiise, Asadetis Rgez5 (1986), pp. 59-103).

Immortal cell lines are usually transformed mammalian cells, particularly myeloma cells, which are derived from rodents, cattle, and humans. Usually, rat or mouse myeloma cell lines are used. Hybridoma cells obtained in this way are seeded and grown in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of unfused primary myeloma cells. For example, if the primary myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (NORAT or NRAT), the hybridoma culture medium will usually include hypoxanthine, aminopterin, and thymidine (HAT medium), which are substances that prevent the growth of NORET-deficient cells.

Preferred immortal myeloma cells are cells that fuse efficiently, support sustained high-level antibody production by selected antibody-producing cells, and are sensitive to an environment such as a NAT environment. Among them, myeloma lines of mice are preferred, for example, which are derived from MORS-21 and MRO-11 mouse tumors, available from ZaiYIK Ipziyshe Sei! Oiztibshiop Sepieg, Zap Oiedo, Sa pogpia O5A, and 5P-2 cells (and their derivatives, for example, X63-Aa8-653), available from Ategisap Ture Siyste SoPPesiyup, Mapazzav, Migaipia

BA Human myeloma cell lines and murine-human heteromyeloma cell lines for the production of human monoclonal antibodies have also been described (Kogbog vai. U. Ittipoi. 133(6):3001-3005

(1984); Wgodeig her a)., Moposiopa! Apiibodu Rgodisip Thesppidnez apa Arriisayop5, Magse! Oekkeg",

Ips., Mem HOIK, pp. 51-63 (1987)).

The culture medium in which the hybridoma cells grow is analyzed for the production of monoclonal antibodies against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by an immunoprecipitation assay or by an antibody binding assay such as a radioimmunoassay (RIA) or a solid-phase enzyme-linked immunosorbent assay (ELISA). Such methods and assays are known in the art. For example, binding affinity can be determined using a Scatchard assay, see Myp5op, Apai. Viospet. 107(1):220-239(1980).

After identification of hybridoma cells that produce antibodies of the desired specificity, affinity, and/or activity, these clones can be subcloned using limiting dilution methods and grown according to standard methods (Ciadipd, supra).

Suitable culture media for this purpose include, for example, O-MEM or VRMI-1640 media. In addition, hybridoma cells can be grown i.p. as tumors in a mammal.

Monoclonal antibodies that are secreted by subclones are conveniently isolated from culture medium, ascites fluid, or serum by conventional immunoglobulin purification methods, such as, for example, protein A sepharose chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

Antibodies can be obtained by immunizing a suitable host animal against the antigen.

In one embodiment, the antigen is a polypeptide that includes full-length C5.

In one embodiment, the antigen is a polypeptide that includes the beta chain (5EO IU MO: 40) of C5. In one embodiment, the antigen is a polypeptide that includes the Ma1-mMa2 domain (ZEO IU MO: 43) of the C5 beta chain. In one embodiment, the antigen is a polypeptide that includes the Ma domain (ZEO IU MO: 41) of the C5 beta chain. In one embodiment, the antigen is a polypeptide that includes a region that corresponds to amino acids at positions 19 to 180 of the C5 beta chain. In one embodiment, the antigen is a polypeptide that includes a region that corresponds to amino acids at positions 33 to 124 of the C5 beta chain. In one embodiment, the antigen is a polypeptide that includes at least one fragment selected from amino acids 47-57, 70-76 and 107-110 of the beta chain (5EO IU MO: 40) of C5. In one embodiment, the antigen is a polypeptide that includes a C5 beta chain fragment that includes at least one amino acid selected from the group consisting of TNg47, c1shi48, AIa49, Rpebo,

A5r5b1, AiIab2, TAg53, I uz57, Niz70, Ma171, Niz72, Zeg74, Sishi76, MaI107, Zet08, I uz109 and Niv110.

In one embodiment, the antigen is a polypeptide that includes a C5 beta chain fragment that includes at least one amino acid selected from the group consisting of C1Ish48, Avzro1,

Niz70, Niv72, Guz109 and Nib110. Also included in this invention are antibodies obtained as a result of immunization of an animal against an antigen. These antibodies may include any of the features, alone or in combination, as described above under "Examples of Anti-C5 Antibodies".

S. Analyzes

The anti-C5 antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or various types of biological activity using a variety of assays known in the art. 1. Binding assays and other analyses

In one aspect, the antibody of the invention is tested for its antigen-binding activity, for example, using known methods, such as EIGI5BA, Western blotting, VIASOVE (registered trademark), and the like.

In another aspect, competitive assays can be used to identify an antibody that competes for binding to C5 with an anti-C5 antibody described herein.

In certain embodiments, when such a competing antibody is present in excess, it blocks (eg, attenuates) the binding of the reference antibody to C5 by at least 10 95 , 15 96 , 20 toe, 25 toe, 20 toe, 35 toe, 40 toe , 45 to, 50 Zo, 25 o, 6o0 to, 65 to, 70 to, 75595 or more.

In some cases, binding is inhibited by at least 80 95, 85 95, 90 95, 9595 or more.

In certain embodiments, such a competing antibody binds to the same epitope (eg, a linear or conformational epitope) that is bound by an anti-C5 antibody described herein (eg, an anti-C5 antibody listed in Table 2). Detailed examples of methods for mapping the epitope to which an antibody binds are provided in Moiits, "Eriore

Marripa Rgoyusoi!v5, " in Meitoavz ip Moiesshag Vioiodu moyo. 66 (Nytapa Rgezz, Toioua, MU) (1996).

In an example of a competitive assay, immobilized C5 is incubated in a solution containing a first labeled (reference) antibody that binds to C5 and a second unlabeled antibody that is tested for its ability to compete with the first antibody for binding to C5. The second antibody may be present in the supernatant of the hybridoma. As a control, immobilized

C5 is incubated in a solution that includes the first labeled antibody, but does not include the second unlabeled antibody. After incubation under conditions that promote binding of the first antibody to C5, the remaining unbound antibody is removed and the number of labels associated with immobilized C5 is measured. If the number of labels associated with immobilized C5 is significantly reduced in the test sample compared to the control sample, this indicates that the second antibody competes with the first antibody for binding to C5. See Napom apa IGape,

Apiirodiev: And Aroghaiogu Mapiai! sp. 14 (Soyd Zrgipud Nagbog I aroghaigu, Soiy 5rgipd Nabog, MU) (1988).

In another example of a competitive assay, the VIASOVE (registered trademark) assay is used to determine the ability of a test anti-C5 antibody to compete for binding to C5 with a second (reference) anti-C5 antibody. In a further aspect, in which the VIASOVE (registered trademark) instrument (eg, the VIASOVE (registered trademark) 3000) is used according to the manufacturer's recommendations, C5 protein is captured on the VIASOVE (registered trademark) CM5 chip using a standard method known in the art to to obtain a surface with applied C5.

Typically 200-800 resonance units of C5 bind to the chip (this is the amount that allows easy measurement of binding levels, but which can be easily saturated at the concentrations of test antibody used). Two antibodies (ie, test and reference antibody) to be evaluated for their ability to compete with each other are mixed at a 1:11 molar ratio of binding sites in a suitable buffer to form the test mixture. When concentrations are calculated based on the binding site, then the molecular weight of the test or reference antibody is considered to be the total molecular weight of the corresponding antibody divided by the number of C5 binding sites on the antibody. The concentration of each antibody (ie, test and reference antibodies) in the test mixture must be high enough to readily saturate the binding sites for that antibody on the C5 molecules captured on the VIASORNE (registered trademark) chip. The test and reference antibodies in the mixture have the same molar concentration (based on binding), which is usually between 1.00 and 1.5 micromoles (based on the binding site). Separate solutions are also prepared, which contain only the tested antibody and only

A reference antibody. The test antibody and the reference antibody in these solutions must be in the same buffer and have the same concentration and conditions as those of the mixture being tested. The mixture to be tested, which contains the test antibody and the reference antibody, is run over a C5-coated VIASONE (registered trademark) chip, and the total number of bindings is recorded. The chip is then processed like this

C5 in such a way as to remove the bound test antibody or reference antibody without damaging the bound C5 chip. Typically, this is done by processing the MM NS chip for 60 seconds. The solution of only the tested antibody is then passed over the surface with applied C5 and the number of bindings is recorded. The chip is processed again to remove all bound antibody without damaging the C5 bound to the chip. The solution of only the reference antibody is then passed over the surface with applied C 5 and the number of bindings is recorded. The maximum theoretical binding of the mixture of test antibody and reference antibody is then calculated, and this is the sum of the binding of each antibody (ie, test and reference) when separately passed over the C5 surface. If the actual recorded binding of the mixture is less than this theoretical maximum, then the test antibody and the reference antibody compete with each other for binding to

C5. Therefore, in general, the competitive anti-C5 antibody tested is an antibody that will bind to C5 in the VIASOVE (registered trademark) blocking assay described above, such that during the assay and in the presence of the anti-C5 reference antibody, the reported binding is between 80 95 to 0.1 95 (eg between 80 95 » to 4 95) of the maximum theoretical binding, specifically 75595 to 0.195 (eg 75595 - 4905) of the maximum theoretical binding and more specifically 70 95 to 0 .1 95 (eg, 7095 - 45905) of the maximum theoretical binding (as defined above) of the test antibody and the reference antibody in combination.

In certain embodiments, the antibody against C5 of the present invention competes for binding to

C5 with an antibody that includes a pair of MN and MI selected from the antibody SEAOZ341 and SEAOZZO. In some embodiments, the anti-C5 antibody competes for binding to C5 with an antibody selected from:

SEAOB538, SEAOB1, SEAOB99, SEAOZO7, SEAOZbb, SEAOb75 and SEAObE72. In some embodiments, the anti-C5 antibody competes for binding to C5 with the SEAOZ29 antibody. In some embodiments, the anti-C5 antibody competes for binding to C5 with the SEAOb66 antibody.

In certain embodiments, the antibody against C5 of the present invention competes for binding to

C5 with an antibody that includes a pair of MH and MI antibodies SEAOZO5 or 3051 05.

In further embodiments, the anti-C5 antibody binds to C5 with higher affinity at neutral pH than at acidic pH. In certain embodiments, an anti-C5 antibody of the present invention competes for binding to C5 with an antibody that includes a UN and MI pair selected from:

SEAOB538, SEAOB1, SEAOB99, SEAOZO7, SEAOZb6b, SEAOBE75 and SEAOB672. In certain embodiments, the anti-C5 antibody competes for binding to C5 with the SEPAOb66b6 antibody. In further embodiments, the anti-C5 antibody binds to C5 with higher affinity at pH 7.4 than at pH 5.8.

In further embodiments, the anti-C5 antibody binds to C5 with higher affinity at neutral pH than at acidic pH. In certain embodiments, the anti-C5 antibody of the present invention competes for binding to C5 with an antibody that includes a pair of MH and Mi antibodies

SEAOZO5 or 3051 05. In further embodiments, the anti-C5 antibody binds to C5 with higher affinity at pH 7.4 than at pH 5.8.

In certain embodiments, the antibody against C5 of the present invention competes for binding to

C5 with an antibody that includes a pair of MH and MI selected from MH for 5EO IO MO: 22 and MI for 5EO IO MO: 26, or MH for 5EO IO MO: 21 and MI for 5EO IO MO: 25. In some embodiments, an anti-C5 antibody competes for binding to C5 with an antibody that includes a pair of MH and MI selected from: (a) MH for 5EO IO MO: 5 and MI for 5EO IU MO: 15; (5) MN for 5EO IO MO: 4 and MI for 5EO IYUO MO: 14; (c) MN for the Ministry of Education and Culture of the Ministry of Education: 6 and MI for the Ministry of Education and Culture of the Ministry of Education and Culture of the Ministry of Education: 16; (8) MN for 5EO IO MO: 2 and MI for 5EO IU MO: 12; (is) MN for 5EO IO MO: Z and MI for 5EO IO MO: 13; (I) MN for 5EO IO MO: 1 and MI for 5EO and MO: 11; (9) MN for 5EOIU MO: 9 and MI for 5EO IO MO: 19; (n) MN for 5EO IO MO: 7 and MI for 5EO IO MO: 17; and

MN for 5EO IO MO: 8 and MI for 5EO IO MO: 18. In some embodiments, the anti-C5 antibody competes for binding to C5 with an antibody that includes MN for 5EO IO MO: 23 and MI for 5EO IUO MO: 27 In some embodiments, an anti-C5 antibody competes for binding to C5 with an antibody that includes a MN for 5EO IO MO: 7 and an MI for BEO IO MO: 17.

In certain embodiments, the antibody against C5 of the present invention competes for binding to

C5 with an antibody that includes a pair of MH and MI selected from: (a) MH for 5EO IO MO: 1 and MI for 5EO I

MO: 11; (5) MN for 5EO IO MO: 22 and MI for 5EO IO MO: 26; (c) MN for 5EO IO MO: 21 and MI for 5EO

IO MO: 25; (4) MN for 5EO IO MO: 5 and MI for 5EO IO MO: 15; (is) MN for 5EO IO MO: 4 and MI for 5EO

IO MO: 14; () MN for 5EO IO MO: 6 and MI for 5EO IO MO: 16; (9) MN for 5EO IO MO: 2 and MI for 5EO

IO MO: 12; (p) MN for 5EO IO MO: Z and MI for 5EO IO MO: 13; (Y) MN for 5EO IO MO: 9 and MI for 5ZEO

IO MO: 19; Y) MN for 5EO IO MO: 7 and MI for 5EO IO MO: 17; (K) MN for 5EO IO MO: 8 and MI for 5EO

IO MO: 18; (1) MN for 5EO IO MO: 23 and MI for 5EO IO MO: 27; and (t) MN for 5EO IO MO: 10 and MI for

ZEO and MO: 20.

In certain embodiments, the antibody against C5 of the present invention competes for binding to

C5 with an antibody that includes a pair of MH and MI selected from (a) MH for 5EO IO MO: 22 and MI for 5EO IU

MO: 26; (5) MN for 5EO IO MO: 21 and MI for 5EO IU MO: 25; (c) MN for 5EO IO MO: 5 and MI for 5EO I

MO: 15; (8) MN for 5EO IO MO: 4 and MI for 5EO IU MO: 14; (is) MN for 5EO IO MO: 6 and MI for 5EO Ir

MO: 16; ()) MN for 5EO IO MO: 2 and MI for 5EO IU MO: 12; (9) MN for 5EO IO MO: Z and MI for 5EO Ir

MO: 13; (n) MN for 5EO IO MO: 9 and MI for 5EO IO MO: 19; (i) MN for 5EO IO MO: 7 and MI for 5EO Ir

MO: 17; (Y) MN for 5EO IO MO: 8 and MI for 5EO IO MO: 18; (K) MN for 5EO IO MO: 23 and MI for 5EO Ir

MO: 27.

In certain embodiments, the antibody against C5 of the present invention competes for binding to

C5 with an antibody that includes a pair of MH and Mi selected from MH for 5EO IO MO: 1 and MI for 5EO AND MO: 11, or MH for 5EO IO MO: 10 and MI for 5EO IU MO: 20.

In further embodiments, the anti-C5 antibody binds to C5 with higher affinity at neutral pH than at acidic pH. In some embodiments, the anti-C5 antibody binds to C5 with higher affinity at neutral pH than at acidic pH and competes for binding to C5 with an antibody that includes an MH and MI pair selected from: (a) MH by BEO and MO: 1 and Mi for 5EO IYU MO: 11; (5) MN for 5EO IO MO: 5 and MI for 5EO IO MO: 15; (c) MN for 5EO IYUO MO: 4 and MI for 5EO IU MO: 14; (8) MN for 5EO IO MO: 6 and MI for 5EO IU MO: 16; (is) MN for 5EO IU MO: 2 and MI for BEO IO MO: 12; (U) MN for 5EO IO MO: Z and MI for 5EO IO MO: 13; (9) MN for 5EO IU MO: 9 and MI for 5EO IO MO: 19; (n) MN for 5EO IO MO: 7 and MI for 5EO IU MO: 17; (y) MN for 5EO and MO: 8 and MI for 5EO IO MO: 18; and () MN for 5EO IUO MO: 10 and MI for 5EO IO MO: 20. In further embodiments, the anti-C5 antibody binds to C5 with higher affinity at pH 7.4 than at pH 5.8.

In some embodiments, the anti-C5 antibody binds to C5 with higher affinity at neutral pH than at acidic pH and competes for binding to C5 with an antibody that 60 comprises an MH and MI pair selected from: (a) MH for 5EO IO MO: 5 and MI for 5EO IO MO: 15; (B) UN for

ZEO IO MO: 4 and MI for 5EO IYUO MO: 14; (c) MN for 5EO IO MO: 6 and MI for 5EO IO MO: 16; (a) MN for

ZEO IYUO MO: 2 and MI for 5EO IO MO: 12; (is) MN for 5EO IO MO: Z and MI for 5EO IO MO: 13; (In MN for

ZEO IO MO: 1 and MI for 5EO IO MO: 11; (4) MN for 5ZEO IO MO: 9 and MI for 5EO IO MO: 19; (n) MN for

ZEO IU MO: 7 and MI for 5EO IO MO: 17; and () MN for 5EO IO MO: 8 and MI for 5EO IU MO: 18. In further embodiments, the anti-C5 antibody binds to C5 with higher affinity at pH 7.4 than at pH 5.8.

In some embodiments, the anti-C5 antibody binds to C5 with higher affinity at neutral pH than at acidic pH and competes for binding to C5 with an antibody that includes an MH and MI pair selected from MH for 5EO IO MO: 1 and MI for 5EO IO MO: 11 or MN for 5EO Ir

MO: 10 and MI for 5EO IU MO: 20. In further embodiments, the anti-C5 antibody binds to C5 with higher affinity at pH 7.4 than at pH 5.8.

In certain embodiments, whether the anti-C5 antibody of the present invention binds to a certain epitope can be determined as follows: point mutants of C5, in which the amino acid (except for alanine) in C5 is replaced by alanine, are expressed in 293 cells, and binding of the antibody against C5 with C5 mutants is tested using EHI5A, Western blotting or

VIASOVE (registered trademark); wherein a significant attenuation or cessation of binding of the anti-C5 antibody to the C5 mutant relative to its binding to native C5 indicates that the anti-C5 antibody binds to an epitope that includes such amino acid in C5. In certain embodiments, the amino acid in C5 that should be replaced with alanine is selected from the group consisting of C1Ish48, Avrob1, Niv70, Niv72, I in'109 and Niz110 of the C5 beta chain (5EO IU

MO: 40). In further embodiments, the amino acid in C5 that should be replaced with alanine is

Azr51 or I ugIO9 beta-chain C5 (ZEO IU MO: 40).

In another embodiment, whether an anti-C5 antibody with pH-dependent binding characteristics binds to a particular epitope can be determined as follows: C5 point mutants in which the histidine residue in C5 is replaced by another amino acid (eg, tyrosine) are expressed in cells 293, and the binding of anti-C5 antibodies to C5 mutants is tested using EGISA, Western blotting or VIASOVE (registered trademark); where a significant weakening of anti-C5 antibody binding to native C5 at acidic pH relative to its binding to mutant C5 at acidic pH indicates that the anti-C5 antibody

C5 binds to an epitope that includes such a histidine residue in C5. In further embodiments, the binding of the anti-C5 antibody to native C5 at neutral pH is not significantly weakened relative to its binding to mutant C5 at neutral pH. In certain embodiments, the histidine residue in C5 to be replaced with another amino acid is selected from the group consisting of Nib70, Niz72, and Nib110 of the C5 beta chain (5EO IO MO: 40).

In a further embodiment, the Nib70 histidine residue is replaced by tyrosine. 2. Analyzes of activity

In one aspect, assays are provided to identify anti-C5 antibodies that have biological activity. Biological activity may include, for example, inhibition of activity

C5, preventing the cleavage of C5, resulting in the formation of Cba and C5p, blocking the access of C5 convertase to the cleavage site in C5, blocking the hemolytic activity caused by the activation of C5, etc. Antibodies having such biological activity as ip mimo and/or ip miiko are also offered.

In certain embodiments, an antibody of the invention is tested for such biological activity.

In certain embodiments, whether a test antibody inhibits the cleavage of C5 into C5ba and C5p is determined by methods described in, e.g., Izeptap et al., U Ittipo). 124(1):326-331 (1980). In another embodiment, this is determined by methods of specific detection of Cba and/or C5b cleaved proteins, for example, using EGISA or Western blotting. Where the C5 cleavage product (i.e. C5ba and/or

C5b) is detected in the presence of the test antibody (or as a result of contact with it), the test antibody is identified as an antibody capable of inhibiting the cleavage of C5.

In certain embodiments, the concentration and/or physiological activity of Sba can be measured using methods such as chemotactic assays, VIA, or EHI5A (See, e.g.,

Maga apa 7chuashek 4. Siip. Ipmevi 50(3):606-616 (1971)).

In certain embodiments, whether the test antibody blocks the access of C5 convertase to

C5, determined by methods of detecting protein interactions between C5 and C5 convertase, for example, EGIZA or VIASOVE (registered trademark). Where interactions are weakened in the presence of (or by contact with) the test antibody, the test antibody is identified as an antibody capable of blocking C5-to-C5 convertase 60 access.

In certain embodiments, C5 activity can be measured as a function of its ability to lyse cells in a subject's body fluids. The ability of C 5 to lyse or deplete cells can be measured by methods well known in the art, for example, by conventional hemolytic assays such as the hemolysis assay described by Kabai apa

Mauyeg (yedz), Ehregitepia! Ittipospetivigh, 2pa Eyop, 135-240, 5rgiprdtyeYia, 1, Sat Tnpotav (1961), pages 135-139, using a traditional variant of such an analysis, such as the method of hemolysis of chicken erythrocytes, as described in, for example, Niitep ei ai., M Epai. U. Mea. 350(6): 552-559 (2004). In certain embodiments, C5 activity or its inhibition is quantified using the CH5bed assay. Analysis of CH5Oed is a method of measuring the total classical activity of complement in serum. This test is a lysis assay that uses antibody-sensitized red blood cells as an activator of the classical complement pathway and various dilutions of test serum to determine the amount required for 50 95 lysis (CH5O) to occur. The percentage ratio of hemolysis can be determined, for example, using a spectrophotometer. The analysis of СН5ООед allows you to perform an indirect measurement of the formation of the terminal complement complex (TCC), because by themselves

TSS are directly responsible for the hemolysis that is measured. Inhibition of C5 activation can also be detected and/or measured using the methods described below and exemplified in the working examples. Using assays of these or other suitable types, it is possible to screen for candidate antibodies capable of inhibiting C5 activation. In certain embodiments, inhibiting C5 activation includes reducing C5 activation in the assay by at least 5 95, 10 96, 1595, 20 95, 2595, 30 90, 35 95, or 40 95 or more compared to the effect of a negative control antibody under similar conditions. In some embodiments, this refers to inhibition of C5 activation by at least 45 95, 50 95, 55 95, 60 Zo, 65 Us, 70 90, 75 o, 80 95, 85 9, 90 95, or 95 95 or more. p. Immunoconjugates

In some embodiments, the invention provides immunoconjugates comprising an anti-C5 antibody disclosed herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal,

Of plant or animal origin, or their fragments), or radioactive isotopes.

In one embodiment, the immunoconjugate is an antibody-drug conjugate (AOD) in which the antibody is conjugated to one or more drugs, including, but not limited to, maytansinoid (see US Pat. Nos. 5,208,020, 5,416,064 and E.P. ER 0 425 235 VI); auristatin, such as the OE and OPE components of the drug monomethylauristatin (MMAE and MMABE) (see US Patent Nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin; calichemycin or a derivative thereof (see US Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Niptap et al., Sapseg Nez. 53:3336-3342 (1993); and I ode eyi a)I. , Sapseg Vev. 58:2925-2928 (1998)); an anthracycline, such as daunomycin or doxorubicin (see Kgai? eyi a!., Sitepi Med. Spet. 13:477-523 (2006); Uinteu ewi a!., Viogdapis a Med. Spet. I eige 16:358-362 ( 2006); Togdom evi ai., Viosopi. Snet. 16:717-721 (2005); Madu ei a!I., Ros. Mai). Asad 5 BOTH 97:829-834 (2000); Viromsnik ei ai!., Viooga. 2 Med. Snet I eyetv 12:1529-1532 (2002); Kipd eyi aiYi.,, 9. May. Spent 45:4336-4343 (2002); and U.S. Pat

Mo 6,630,579); methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tezetaxel and ortataxel; trichothecene; and SS1065.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A-chain, non-binding active diphtheria toxin fragments, A-chain exotoxin (from Rzepdotopah aegidipose), A-chain of ricin, A-chain of abrin, A-chain of modecin, alpha-sarcin, AiIespiez ogai proteins, diantin proteins, proteins of Rnuylias Ategisapa (RARI,

RARP and PAP-5), an inhibitor obtained from bitter cucumber (Totogaisa sNagapia), curcin, crotin, an inhibitor obtained from soapwort (Zaraopagla oNicipa|i5), gelonin, mitogelin, restrictocin, phenomycin, enomycin and trichothecene.

In another embodiment, the immunoconjugate includes an antibody described herein conjugated to a radioactive atom to form a radioactive conjugate. A variety of radioactive isotopes are available for the production of radioactive conjugates. Examples include A, 91, 25, mo De!86, Be88, pz, Vig12, rzg, Prg!2 and radioactive isotopes I and. When the radioactive conjugate is used for detection, then it can include a radioactive atom for scintigraphic studies , such as ІС99т or 1123, or a spin tag for nuclear magnetic resonance (MMA) imaging (also known as magnetic resonance imaging, TII),

such as iodine-123 ("iodipe-123 adaipe"), iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, magnesium, and iron.

Antibody-cytotoxic agent conjugates can be obtained using a variety of bifunctional protein cross-linking agents, such as M-succinimidyl-3-(2-pyridyldithio)propionate (5POP), succinimidyl-4-(M-maleimidomethyl)cyclohexane-1-carboxylate ( ZMSS), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate NS), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene) . For example, ricin immunotoxin can be obtained as described in MiieNa 6vi ai.,

Zsiepse 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene-triaminopentaacetic acid (MX-OTRA) is an applied chelating agent for conjugation of a radioactive nucleotide with an antibody. See UU094/11026. The linker may be a "cleavable linker" that promotes the release of cytotoxic drugs into the cell. For example, you can use an acid-resistant linker, a peptidase-sensitive linker, a light-resistant linker, a dimethyl linker, or a disulfide-containing linker (SpNaigi et al., Sapseg Vezv.

B2:127-131 (1992); US patent Mo 5,208,020).

Immunoconjugates or antibody-drug conjugates (AODs) described herein, which are particularly contemplated, but not limited to, are such conjugates that are prepared by means of cross-linking reagents that include, but are not limited to , VMR5, EMSO5,

CMV5, NVUB5, I 0-5MSS, MV5, MRVN, 5VAR, 5IA, 5IAV, MOSS, 5MRV, ZMRN, sulfo-EMO5, sulfo-C2MV5, sulfo-KMY5, sulfo-MV5, sulfo-5IAV, sulfo-5MOS, and sulfo-5MRV, and

ZUZV (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g. from

Riegse Viotesnpoiodu, Ips., Vosktoga, IS., 0.5. AND).

E. Methods and compositions for diagnosis and detection

In certain embodiments, any anti-C5 antibody provided herein is useful for detecting the presence of C5 in a biological sample. The term "detection" as used herein includes quantitative and qualitative detection. In certain embodiments, the biological sample includes a cell or tissue, such as serum, whole blood, plasma, biopsy sample, tissue sample, cell suspension, saliva, sputum, oral fluid, cerebrospinal fluid, amniotic fluid, ascitic fluid, milk, colostrum, secretion mammary gland, lymph, urine, sweat, lacrimal fluid, gastric juice, synovial fluid, abdominal fluid, intraocular fluid and mucus.

In one embodiment, an anti-C5 antibody is provided for use in a diagnostic or detection method. In a further aspect, a method of presence detection is proposed

C5 in a biological sample. In certain embodiments, the method includes contacting the biological sample with an anti-C5 antibody as described herein under conditions that allow the anti-C5 antibody to bind to C5, and detecting whether a complex has formed between the anti-C5 antibody and C5. This method can be ip myto or ip mimo. In one embodiment, an antibody against C5 is used to select subjects suitable for treatment with an antibody against C5, such as when C5 is a biological marker for patient selection.

In another embodiment, a method is provided for selecting an individual who has a complement-mediated disease or condition involving excessive or uncontrolled C5 activation and who is suitable for therapy that includes an anti-C5 antibody of the present invention. In certain embodiments, the method includes (a) detecting a genetic variation in C5 derived from an individual, and (B) selecting an individual suitable for therapy that includes an anti-C5 antibody of the present invention when the genetic variation is found in C5 that comes from the individual. In another embodiment, a method of selecting a therapy for an individual having a complement-mediated disease or condition involving excessive or uncontrolled activation of C5 is provided. In certain embodiments, the method comprises (a) detecting a genetic variation in the C5 derived from the individual, and (B) selecting a therapy that includes an anti-C5 antibody of the present invention for the individual when the genetic variation is detected in the C5 derived from the individual .

In another embodiment, a method of treating an individual having a complement-mediated disease or condition involving excessive or uncontrolled activation of C5 is provided. In certain embodiments, the method includes (a) detecting a genetic variation in C5 derived from an individual, (B) selecting an individual that is suitable for therapy that includes an anti-C5 antibody of the present invention when the genetic variation is found in C5 derived from the individual, and (c) administration of an antibody against C5 of the present invention to the individual.

In another embodiment, an anti-C5 antibody of the present invention is provided for use in the treatment of an individual who has a complement-mediated disease or condition involving excessive or uncontrolled activation of C5. In certain embodiments, an individual is treated with an anti-C5 antibody of the present invention when a genetic variation is detected in C5 derived from the individual.

In another embodiment, it is proposed to use ip miigo genetic variation in C5 to select an individual who has a complement-mediated disease or condition involving excessive or uncontrolled activation of C5, and who is suitable for therapy that includes an anti-C5 antibody of the present invention. In certain embodiments, an individual is selected as being eligible for therapy when this genetic variation is found in

C5, originating from an individual. Another embodiment provides the use of ip myo genetic variation in C5 to select a therapy for an individual who has a complement-mediated disease or condition involving excessive or uncontrolled activation of C5.

In certain embodiments, a therapy that includes an anti-C5 antibody of the present invention is selected for an individual when a genetic variation is detected in C5 derived from the individual.

Some patients who have a genetic variation in C5 have been reported to show a poor response to therapy that includes an existing anti-C5 antibody (Mibpitiga et al., M. Epdi. U. Med. 370:632-639 (2014)). It is recommended that such a patient be treated with a therapy that includes an anti-C5 antibody of the present invention, as such an antibody has inhibitory activity on the activation of C5 variants as well as natural C5, as shown in the working examples below.

Detection of genetic variation in C5 can be carried out using a method known from the prior art. Such a method may include, but is not limited to, sequencing, PCR (PCR), reverse transcriptase PCR (RT-PCR), and a hybridization-based method such as Southern blotting or Northern blotting. C5 variants may include at least one genetic variation. The genetic variation can be selected from the group consisting of MI451, 84492, U802I, B885H, 9280, 096bM, 51310M and E14370. In this description, A885H, for example, denotes a genetic variation where arginine at position 885 is replaced by histidine.

In certain embodiments, variant C5 has biological activity similar to biological activity

From the activity of natural C5.

Examples of disorders that can be diagnosed using the antibodies of the invention include rheumatoid arthritis (RA); systemic lupus erythematosus (5IE); lupus nephritis; ischemic reperfusion injury (II); asthma; paroxysmal nocturnal hemoglobinuria (PMH); hemolytic-uremic syndrome (HO) (for example, atypical hemolytic-uremic syndrome (anib5)) dense sediment disease (000); opticomyelitis (MMO); multifocal motor neuropathy (MMM); multiple sclerosis (M); systemic sclerosis; macular degeneration (eg, age-related macular degeneration (AMD)); syndrome of hemolysis, increased activity of liver enzymes and thrombocytopenia (NEG I R); thrombotic thrombocytopenic purpura (TTP); spontaneous loss of the fetus; epidermolysis bullosa; recurrent fetal loss; pre-eclampsia; brain injury; severe myasthenia gravis; cold agglutination disease; syndrome

Segrena; dermatomyositis; bullous pemphigoid; phototoxic reactions; hemolytic uremic syndrome associated with Shiga E. soy toxin; typical or infectious hemolytic uremic syndrome (HO); glomerulonephritis C3Z3; vasculitis associated with antineutrophil cytoplasmic antibodies (AMSA); humoral and vascular graft rejection; antibody-mediated acute rejection (AMEA); dysfunction of transplantation; myocardial infarction; allogeneic transplant; sepsis; ischemic heart disease; congenital angioedema; dermatomyositis; Graves' disease; atherosclerosis; the disease

Alzheimer's (Ab); Huntington's disease; Creutzfeldt-Jakob disease; Parkinson's disease; various types of cancer; wound; septic shock; damage to the spinal cord; uveitis; diabetes-related eye diseases; retrolental fibroplasia; glomerulonephritis; membranous nephritis; IdA - nephropathy; acute respiratory failure syndrome in adults (ANVOB); chronic obstructive pulmonary disease (COPD); cystic fibrosis; hemolytic anemia; paroxysmal cold hemoglobinuria; anaphylactic shock; allergy; osteoporosis; osteoarthritis; autoimmune thyroiditis; type I diabetes; psoriasis; ordinary blister; autoimmune hemolytic anemia (AINA); idiopathic thrombocytopenic purpura (IHR); Goodpasture's syndrome; Degos disease; antiphospholipid syndrome (ARB); catastrophic AR5 (CARB); cardiovascular disorder; myocarditis; cerebrovascular disorder; disorder of peripheral vessels; vasorenal disorder; mesenteric/enteric vascular disorder; vasculitis; Schenlein-Henoch's purpura nephritis; Takayasu's disease; dilated cardiomyopathy; diabetic angiopathy; Kawasaki disease (arthritis); for venous gas embolus (MY); restenosis after stent placement; rotational atherectomy;

membranous nephropathy; Guillain-Barre syndrome; Fisher's syndrome; antigen-induced arthritis; synovial inflammation; viral infections; bacterial infections; fungal infections and damage resulting from myocardial infarction, artificial blood circulation and hemodialysis.

In certain embodiments, labeled anti-C5 antibodies are provided. Labels include, but are not limited to, labels or components that are directly detected (such as fluorescent, chromophoric, electron-impermeable, chemiluminescent, and radioactive labels), and components such as enzymes or ligands; which are revealed indirectly, for example, through an enzymatic reaction or molecular interaction.

Examples of labels include, but are not limited to, radioisotopes 32P, 140, 1251 ZN and "1, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly and bacterial luciferase luciferase (US patent Mo 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HAP), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, saccharide oxidases such as glucose oxidase, galactose oxidase, and glucose-β6-phosphate dehydrogenase , heterocyclic oxidases such as uricase and xanthine oxidase coupled to an enzyme that uses hydrogen peroxide to oxidize the initial dye such as HAP, lactoperoxidase or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, persistent free radicals, and the like.

E. Pharmaceutical compositions

Anti-C5 antibody pharmaceutical compositions, as described herein, are obtained by mixing such an antibody, which has a desired degree of purity, with one or more arbitrary pharmaceutically acceptable carriers (Ketipdiopye Rnaptaseshisa! Zsiepsez 161y eaiiop, Ozvoi, A.

EYA9. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the doses and concentrations used and include, but are not limited to: buffers such as phosphate, citrate and other organic acids; antioxidants, which include 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 t-cresol); low molecular weight (less than

From approximately 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; 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 EOTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (for example, 2p-protein complexes) and/or nonionic surfactants such as polyethylene glycol (PEG). In this description, examples of pharmaceutically acceptable carriers also include agents for dispersing drugs in the intercellular space, such as soluble neutrally active hyaluronidase glycoproteins (5zNABESR), for example, soluble human hyaluronidase glycoproteins pH-20, such as "NiPH2gO (NMI EMEH (registered trademark) ,

Vahieg Ipiegaiiopai, Ips.). Certain examples of 56NABESR and methods of their application, including

INiRN2O, described in US MoMo publications 2005/0260186 and 2006/0104968. In one aspect, 5NABESR is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

Examples of lyophilized pharmaceutical formulations of antibodies are described in US Pat

Mo 6,267,958. Aqueous pharmaceutical formulations of antibodies include formulations described in US Pat

Mo 6,171,586 and M/O 2006/044908, while the pharmaceutical formulations of the latter reference include a histidine-acetate buffer.

In this description, the pharmaceutical composition may also contain more than one active ingredient if necessary for a particular therapeutic indication, preferably those ingredients having compatible modes of activity that do not adversely affect each other. Such active ingredients are suitably present in the combination in an amount effective for the intended purpose.

Active ingredients can be placed in microcapsules obtained, for example, by coacervation methods or by interfacial polymerization, for example, hydroxymethyl cellulose microcapsules or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g. liposomes , albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in a macroemulsion. Such methods are described in Rnappaseshisa's Vetipdyopye! bsiepsez 161 eaiiop, Oz5oi, A. Ea. (1980).

Sustained-release drugs are available. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers,

containing an antibody, while matrices are formed products, for example, films or microcapsules.

Pharmaceutical formulations that are intended for ip administration are usually sterile.

Sterility can easily be achieved, for example, by filtering through sterile filter membranes. and. Therapeutic methods and compositions

The anti-C5 antibody provided herein can be used in therapeutic methods.

In one aspect, an anti-C5 antibody for use as a medicament is provided. In further aspects, an anti-C5 antibody is provided for use in the treatment of a complement-mediated disease or condition involving excessive or uncontrolled activation of C5. In certain embodiments, an anti-C5 antibody is provided for use in a method of treatment. In certain embodiments, the invention provides an anti-C5 antibody for use in a method of treating an individual having a complement-mediated disease or condition involving excessive or uncontrolled activation of C5, the method comprising administering to the individual an effective amount of an anti-C5 antibody. In one such embodiment, the method also includes administering to the individual an effective amount of at least one additional therapeutic agent. An "individual" according to any of the aforementioned embodiments is preferably a human.

When the antigen is a soluble protein, then as a result of binding of the antibody to its antigen, the half-life of the antigen in the plasma can be prolonged (ie, the clearance of the antigen from the plasma can decrease), since the antibody itself has a longer half-life in the plasma and it serves as a carrier for the antigen. This occurs due to the recirculation of the antigen-antibody complex with the help of

EsAp through the endosomal pathway in the cell (Noorepiap, Mai. Neum. Ittipoi. 7(9):715-725 (2007)).

However, an antibody with pH-dependent binding characteristics, which binds to its antigen in a neutral extracellular environment while releasing it into acidic endosomal compartments after entry into cells, is expected to have unmatched properties in terms of antigen neutralization and clearance compared to with its analogue, which binds in a pH-independent way (Iamzha eyii ai., Mai. Vioiesp... 28(11):1203-1207 (2010);

Zo Remapabouipa ei ai!., tAbBz 5(6):851-859 (2013); MO 2009/125825).

In further embodiments, the invention provides an antibody against C 5 for use in increasing the clearance of C 5 from plasma. In certain embodiments, the invention provides an anti-C5 antibody for use in a method of increasing plasma clearance of C5 in an individual, the method comprising administering to the individual an effective amount of an anti-C5 antibody to increase plasma clearance of C5. In one embodiment, an anti-C5 antibody increases the clearance of C5 from plasma compared to a traditional anti-C5 antibody that does not have pH-dependent binding characteristics. An "individual" according to any of the aforementioned embodiments is preferably a human.

In further embodiments, the invention provides an anti-C5 antibody for use in inhibiting the accumulation of C5 in plasma. In certain embodiments, the invention provides an anti-C5 antibody for use in a method of inhibiting the accumulation of C5 in plasma in an individual, the method comprising administering to the individual an effective amount of an anti-C5 antibody to inhibit the accumulation of C5 in plasma. In one embodiment, the accumulation of C5 in the plasma is the result of the formation of an antigen-antibody complex. In another embodiment, an anti-C5 antibody inhibits the accumulation of C5 in plasma compared to a traditional anti-C5 antibody that does not have pH-dependent binding characteristics. An "individual" according to any of the aforementioned embodiments is preferably a human.

The anti-C5 antibody of the present invention can inhibit the activation of C5. In further embodiments, the invention provides an anti-C5 antibody for use in inhibiting C5 activation. In certain embodiments, the invention provides an anti-C5 antibody for use in a method of inhibiting C5 activation in an individual, the method comprising administering to the individual an effective amount of an anti-C5 antibody to inhibit C5 activation. In one embodiment, C5-mediated cytotoxicity is inhibited by inhibiting C5 activation. An "individual" according to any of the aforementioned embodiments is preferably a human.

In a further aspect, the invention proposes the use of an antibody against C5 in the production or preparation of a medicinal product. In one embodiment, the medicament is for the treatment of a complement-mediated disease or condition involving excessive or uncontrolled activation of C5. In a further embodiment, the medicament is for use in a method of treating a complement-mediated disease or condition involving excessive or uncontrolled activation

C5, wherein the method comprises administering to an individual having a complement-mediated disease or condition involving excessive or uncontrolled activation of C5, an effective amount of the drug. In one such embodiment, the method also includes administering to the individual an effective amount of at least one additional therapeutic agent. An "individual" according to any of the aforementioned embodiments is preferably a human.

In a further embodiment, the drug is intended to increase clearance

C5 from plasma. In a further embodiment, the drug is intended for use in a method of increasing plasma clearance of C5 in an individual, the method comprising administering to the individual an effective amount of the drug to increase plasma clearance of C5.

In one embodiment, an anti-C5 antibody increases the clearance of C5 from plasma compared to a traditional anti-C5 antibody that does not have pH-dependent binding characteristics. An "individual" according to any of the aforementioned embodiments is preferably a human.

In a further embodiment, the drug is intended to inhibit the accumulation of C5 in plasma. In a further embodiment, the drug is intended for use in a method of inhibiting the accumulation of C5 in plasma in an individual, the method including administering to the individual an effective amount of the drug to inhibit the accumulation of C5 in plasma. In one embodiment, the accumulation of C5 in the plasma is the result of the formation of an antigen-antibody complex. In another embodiment, the antibody against

C5 inhibits the accumulation of C5 in plasma compared to a traditional anti-C5 antibody, which does not have pH-dependent binding characteristics. An "individual" according to any of the aforementioned embodiments is preferably a human.

The anti-C5 antibody of the present invention can inhibit the activation of C5. In a further embodiment, the drug is intended to inhibit C5 activation. In a further embodiment, the drug is intended for use in a method of inhibiting C5 activation in an individual, the method comprising administering to the individual an effective amount of a drug to inhibit C5 activation. In one embodiment, cytotoxicity,

Zo is mediated by C5, suppressed due to inhibition of C5 activation. An "individual" according to any of the aforementioned embodiments is preferably a human.

In a further aspect, the invention provides a method of treating a complement-mediated disease or condition involving excessive or uncontrolled activation

C5. In one embodiment, the method comprises administering to an individual having a complement-mediated disease or condition involving excessive or uncontrolled activation of C5, an effective amount of an anti-C5 antibody. In one such embodiment, the method also includes administering to the individual an effective amount of at least one additional therapeutic agent. An "individual" according to any of the aforementioned embodiments is preferably a human.

In a further aspect, the invention proposes a method of increasing the clearance of C 5 from plasma in an individual. In one embodiment, the method includes administering to the individual an effective amount of an anti-C5 antibody to increase the clearance of C5 from the plasma. In one embodiment, an anti-C5 antibody increases the clearance of C5 from plasma compared to a traditional anti-C5 antibody that does not have pH-dependent binding characteristics. In one embodiment, "Individual" is a human.

In a further aspect, the invention proposes a method of inhibiting the accumulation of C5 in the plasma of an individual. In one embodiment, the method includes administering to the individual an effective amount of an anti-C5 antibody to inhibit the accumulation of C5 in the plasma. In one embodiment, the accumulation of C5 in the plasma is the result of the formation of an antigen-antibody complex. In another embodiment, an anti-C5 antibody inhibits the accumulation of C5 in plasma compared to a traditional anti-C5 antibody that does not have pH-dependent binding characteristics. In one embodiment, an "individual" is a human.

The anti-C5 antibody of the present invention can inhibit the activation of C5. In a further aspect, the invention provides a method of inhibiting C5 activation in an individual. In one embodiment, the method includes administering to the individual an effective amount of an anti-C5 antibody to inhibit C5 activation. In one embodiment, C5-mediated cytotoxicity is inhibited by inhibiting C5 activation. In one embodiment, an "individual" is a human.

In a further aspect, the invention provides pharmaceutical compositions comprising any of the anti-C5 antibodies provided herein, for example, for use in any of the above-mentioned therapeutic methods. In one embodiment, the pharmaceutical composition comprises any of the anti-C5 antibodies proposed herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition includes any of the anti-C 5 antibodies proposed herein and at least one additional therapeutic agent.

In a further aspect, the pharmaceutical composition is for the treatment of a complement-mediated disease or condition involving excessive or uncontrolled activation

C5. In a further embodiment, the pharmaceutical composition is designed to increase the clearance of C5 from the plasma. In one embodiment, an anti-C5 antibody increases the clearance of C5 from plasma compared to a traditional anti-C5 antibody that does not have pH-dependent binding characteristics. In a further embodiment, the pharmaceutical composition is designed to inhibit the accumulation of C5 in plasma. In one embodiment, the accumulation of C5 in the plasma is the result of the formation of an antigen-antibody complex. In another embodiment, an anti-C5 antibody inhibits the accumulation of C5 in plasma compared to a traditional anti-C5 antibody that does not have pH-dependent binding characteristics. The anti-C5 antibody of the present invention can inhibit the activation of C5. In a further embodiment, the pharmaceutical composition is designed to inhibit C5 activation. In one embodiment, C5-mediated cytotoxicity is inhibited by inhibiting C5 activation. In one embodiment, the pharmaceutical composition is administered to an individual who has a complement-mediated disease or condition involving excessive or uncontrolled activation of C5. An "individual"' in any of the above-mentioned embodiments is preferably a human.

In one aspect, the individual has natural C5. In another aspect, the individual has the C5 variant.

In certain embodiments, variant C5 has biological activity similar to that of natural C5. Such variant C5 may include at least one variation selected from the group consisting of M145I, 84492, M802I, B885H, 29280, 0966, 51310M and E14370. In this description

B885H, for example, denotes a genetic variation where arginine at position 885 is replaced by histidine.

In a further aspect, the invention provides methods for producing a medicinal product or pharmaceutical composition, wherein the methods include mixing any of the anti-C5 antibodies proposed herein with a pharmaceutically acceptable carrier,

For example, for use in any of the above-mentioned therapeutic methods. In one embodiment, the methods of preparing the drug or pharmaceutical composition also include adding at least one additional therapeutic agent to the drug or pharmaceutical composition.

In certain embodiments, the complement-mediated disease or condition involving excessive or uncontrolled activation of C5 is selected from the group consisting of rheumatoid arthritis (RA); systemic lupus erythematosus (5IE); lupus nephritis; ischemic reperfusion injury (IVI); asthma; paroxysmal nocturnal hemoglobinuria (PMH); hemolytic-uremic syndrome (HE) (for example, atypical hemolytic-uremic syndrome (anNib5)); dense sediment diseases (000); opticomyelitis (MMO); multifocal motor neuropathy (MMM); multiple sclerosis (MS); systemic sclerosis; macular degeneration (for example, age-related macular degeneration (AMD)); syndrome of hemolysis, increased activity of liver enzymes and thrombocytopenia (NEI R); thrombotic thrombocytopenic purpura (TTP); spontaneous fetal loss; epidermolysis bullosa; recurrent fetal loss; pre-eclampsia; brain injury; severe myasthenia gravis; cold agglutination diseases of Segren's syndrome; dermatomyositis; bullous pemphigoid; phototoxic reactions; hemolytic uremic syndrome associated with Shiga E. soy toxin; typical or infectious hemolytic uremic syndrome of NI); glomerulonephritis

C3; vasculitis associated with antineutrophil cytoplasmic antibodies (AMSA); humoral and vascular graft rejection; antibody-mediated acute rejection (AMA); dysfunctions of transplantation; myocardial infarction; allogeneic transplant; sepsis; ischemic heart disease; congenital angioedema; dermatomyositis; Graves' disease; atherosclerosis; Alzheimer's disease (AD); diseases

Huntington; Creutzfeldt-Jakob disease; Parkinson's disease; various types of cancer; wound; septic shock; damage to the spinal cord; uveitis; diabetes-related eye diseases; retrolental fibroplasia; glomerulonephritis; membranous nephritis; IdA-nephropathy; acute respiratory failure syndrome in adults (ANOB); chronic obstructive pulmonary disease (COPD); cystic fibrosis; hemolytic anemia; paroxysmal cold hemoglobinuria; anaphylactic shock; allergies; osteoporosis; osteoarthritis; autoimmune thyroiditis; type I diabetes; psoriasis; ordinary blister; autoimmune hemolytic anemia (AINA); idiopathic thrombocytopenic purpura (ITP); Goodpasture's syndrome; Degos disease;

antiphospholipid syndrome (ARB); catastrophic ARB (CARB); cardiovascular disorder; myocarditis; cerebrovascular disorder; disorder of peripheral vessels; vasorenal disorder; mesenteric loenteric vascular disorder; vasculitis; Schenlein-Henoch's purpura of nephritis; Takayasu's disease; dilated cardiomyopathy; diabetic angiopathy; Kawasaki disease (arthritis); venous gas embolus (VGE); restenosis after stent placement; rotational atherectomy; membranous nephropathy; Guillain-Barre syndrome; Fisher's syndrome; antigen-induced arthritis; synovial inflammation; viral infections; bacterial infections; fungal infections and damage resulting from myocardial infarction, artificial blood circulation and hemodialysis.

In certain embodiments, the complement-mediated disease or condition is an ophthalmic disease condition. In further embodiments, the ophthalmic condition is macular degeneration. In further embodiments, macular degeneration is AMO. In further embodiments, AMO is a dry form of AMO.

In certain embodiments, the complement-mediated disease or condition is paroxysmal nocturnal hemoglobinuria (PNH).

In certain embodiments, the complement-mediated disease or condition is myocardial infarction.

In certain embodiments, the complement-mediated disease or condition is rheumatoid arthritis (RA).

In certain embodiments, the complement-mediated disease or condition is osteoporosis or osteoarthritis.

In certain embodiments, the complement-mediated disease or condition is inflammation.

In certain embodiments, the complement-mediated disease or condition is cancer.

Antibodies of the present invention can be used either alone or in combination with other agents during treatment. For example, an antibody of the present invention can be administered together with at least one additional therapeutic agent.

Such combination therapies as mentioned above include combination administration (where two or more therapeutic agents are included in the same or separate pharmaceutical formulation) and

A separate administration, in which case the administration of the antibody of the present invention may occur before, simultaneously and/or after the administration of an additional therapeutic agent or agents. In one embodiment, the administration of the anti-C5 antibody and the administration of the additional therapeutic agent are performed relative to each other within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days

An antibody of the invention (and any additional therapeutic agent) may be administered by any suitable route, including parenteral, intrapulmonary, and intranasal administration and, if desired for topical treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. The administration of the dose can be carried out by any suitable method, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or continuous. Various drug administration regimens are contemplated herein, including, but not limited to, single or multiple timed administration, bolus administration, or pulsed infusion.

Antibodies of the present invention will be included in the pharmaceutical composition, dosed and administered according to a method consistent with good medical practice. Factors to be considered in this context 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 site of delivery of the agent, the route of administration, the mode of administration, and other factors known to those skilled in the art. The antibody is not required, but is optionally included in a pharmaceutical formulation with one or more agents currently being used to prevent or treat a given disorder. The effective amount of such other agents depends on the amount of antibody present in the pharmaceutical formulation, the type of disorder or treatment, and other factors discussed above. they are generally administered at the same doses and with the routes of administration described herein, or from about 1 95 to 99 95 of the doses described herein, or at any dose or by any route of administration that empirically/ clinically determined to be appropriate.

For the prevention or treatment of a disease, the appropriate dosage of the antibody of the present invention (when used alone or in combination with one or more additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody,

the severity and course of the disease, whether the antibody is being administered prophylactically or curatively, prior therapy, the patient's medical history and reaction to the antibody, and the discretion of the treating physician. The antibody is conveniently administered to the patient once or during treatment sessions. Depending on the type and severity of the disease, approximately 1 μg/kg to 15 mg/kg (e.g., 0.1 mg/kg to 10 mg/kg) of the antibody may be a candidate starting dose for administration to the patient, either by, for example, a single or more separate administrations, either by continuous infusion. A single usual daily dose can range from about 1 mcg/kg to 100 mg/kg or more, depending on the factors mentioned above. For multiple administrations over several days or longer, depending on the condition, treatment will usually be continued until the desired suppression of disease symptoms occurs. One exemplary antibody dosage will range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or any combination thereof) can be administered to a patient. Such doses can be administered at intervals, for example, every week or every three weeks (eg, so that the patient receives from about two to about twenty or, for example, six doses of the antibody). An initial higher loading dose followed by one or more lower doses may be administered. The progress of this treatment is easily monitored using traditional methods and tests.

It will be understood that any of the above formulations and therapeutic methods can be carried out using an immunoconjugate of the invention instead of or in addition to an antibody against

C5.

N. Industrial products

In one aspect, the invention provides an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above. An article of manufacture includes a container and a label or insert that either accompanies the container or is placed on the container. Suitable containers include, for example, bottles, ampoules, syringes, IV bags, and the like. Containers can be made of different materials, such as glass or plastic. The container contains a composition that alone or in combination with another composition is effective for the treatment, prevention or

For the diagnosis of the condition, it may have a means for sterile access (for example, the container may be a bag with a solution for intravenous injections or an ampoule that has a stopper pierced by a hypodermic needle). At least one active agent in the composition is an antibody of the invention. The label or package insert states that the composition is used to treat the condition of choice. In addition, the industrial product may contain (a) a first container with a composition contained therein, where the composition contains an antibody of the invention; and (B) a second container with a composition contained therein, wherein the composition contains an additional cytotoxic agent or a therapeutic agent of another action. An article of manufacture in this embodiment of the invention may also include an insert indicating that the compositions may be used to treat a particular condition. Alternatively or additionally, the industrial product may also include a second (or third) container that contains a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may also include other materials desirable from a commercial and consumer standpoint, including other buffers, diluents, filters, needles, and syringes.

It is clear that any of the above-mentioned industrial products can include the immunoconjugate of the invention instead of or in addition to the antibody against C5.

Examples

Next, examples of methods and compositions of the invention are described. It is clear that other various implementations may be used in practice, taking into account the general description provided above.

Example 1

Obtaining C5 1.1. Expression and purification of recombinant human and cynomolgus macaque C5 Recombinant

Human C5 (MSVI SepVapkK accession number: МРО0О1726.2, 5ЕО IХО МО: 39) was transiently expressed using the cell line Egeesziuile293-E (Tnepto RivesNeg, Sapizrad, CA, USA).

Conditioned mediums expressing human C5 were diluted with an equal volume of water (pi), then applied to an anion-exchange column of O-zerpagose EE or O-zerpagose HP (SE Neainsageye,

Orrzaia, Sweden) followed by mass gradient elution. Fractions containing human C5 were collected, and then the concentration of MacSi salt and pH were adjusted to 80 mM and pH 6.4, respectively. 60 The obtained sample was applied to a cation exchange column 5Р-zerpagose HP (SE Neainsage, Orrzaia,

Sweden) and eluted in a Mass gradient. Fractions containing human C5 were collected and loaded onto a CNT ceramic hydroxyapatite column (Vio-RNaai I Abogaitiev, Negsshez, CA, USA). The human C5 eluate was then loaded onto a 5iregaeh 200 gel filtration column (CE Peainsage, Orrzaia,

Sweden). Fractions containing human C5 were combined and stored at -150 °C.

Expression and purification of recombinant C5 from the Javan macaque (MSVI SepVapkK accession number: ХРО0О5580972, 5EO IЮО MO: 44) was performed in the same way as the human counterpart. 1.2. Purification of Javanese macaque C5 (supoC5) from plasma

A sample of cynomolgus macaque plasma was applied to agarose 551 7 (Iplimodep, West Oviedo, CA, USA) followed by elution with 100 mM sodium acetate, pH 3.5. Fractions containing supoC5 were immediately neutralized and run on an HP column with Protein A (SE Neainsage, Orrzaia, Sweden) followed by agarose with Peptide M (Iplimodep, West Oviedo, CA, USA). The flow through fraction was then directed to a Ziregdeh 200 gel filtration column (SE Neainsage, Orrzaia, Sweden).

Fractions containing supoC5 were combined and stored at -80 °C.

Example 2

Obtaining antibodies against C5 2.1. Antibody screening

Antibodies against C5 were obtained, selected and analyzed as follows.

MAMU rabbits (New Zealand White rabbits) aged 12 to 16 weeks were immunized with human C5 and/or monkey C5 (50-100 μg/dose/rabbit) by intradermal injection. This dose was repeated 4-5 times within 2 months. One week after the final immunization, the spleen and blood were collected from the immunized rabbits. Antigen-specific B cells were stained with labeled antigen, sorted using an ECM cell sorter (BASB5 apya Sh, VO) and placed in 96-well plates at a density of one cell/cell together with 25,000 cells/well of EI/4 cells (Enteroreap SoiPesiip oi Seyi Syitgev) and activated conditioned medium of rabbit T-cells, diluted 20 times, and then cultured for 7-12 days. EI 4 cells were treated with mitomycin C (BZidta, Sai Mo. M4287) for 2 hours and washed three times beforehand.

Activated conditioned medium of rabbit T cells was obtained by culturing rabbit thymocytes in VRMI-1640 containing phytohemagglutinin-M (Vospe, Sai Mo. 1 1082132-

Zo 001), phorbol 12-myristate 13-acetate (bidta, Sai Mo. RI585) and 2 95 ЕВ5. After culturing, B-cell culture supernatants were collected for further analysis, and the debris was stored at low temperatures.

The EP IBA assay was used to test the specificity of antibodies in the culture supernatant of B cells. A layer of streptavidin (Sepebgiri, Sai Mo. 7202043) was applied to

MAKHIZOgr with 384 cells (Mips, Sai Mo. 164688) at 50 nM in РВ5 for 1 hour at room temperature. The tablets were then blocked with ViosKipyaa Ope (Masaiai Tezdie, Sai Mo. 03953-95) diluted 5-fold. Human or monkey C5 was labeled with MNO-RES4-Viosiip (RIEVSE, Sai Mo. 21329) and added to blocked EIGI5BA plates, incubated for 1 hour and washed.

Culture supernatants of B cells were added to EGI5A plates, incubated for 1 hour and washed. Binding was detected using a horseradish peroxidase conjugate with a goat anti-rabbit IS antibody (VETNMUI, Sai Mo. AT20-11 IR), and then AVT5 (KRI, Sai Mo. 50-66-06) was added.

The EGISA assay was used to assess the pH-dependent binding of antibodies against C5.

Es goat against DS rabbit (VETNMI, Sai Mo. A120-111 A), diluted to 1 μg/ml RVB(-) was added to MAHI5ogr with 384 cells (Mips, Sai Mo. 164688), incubated for 1 hour at room temperature and blocked ViosKipiaa Ope (Masaiai Tezdie, Sai Mo. 03953-95), diluted 5 times. After incubation, the plates were washed and B cell culture supernatants were added. Plates were incubated for 1 hour, washed, and 500 pM biotinylated human or monkey C5 was added and incubated for 1 hour. After incubation, the plates were washed and incubated with ME5 buffer pH 5.8 (20 mM ME5, 150 mM Masi and 1.2 mM

Sas12) or ME5 buffer pH 5.8 (20 mM MEZ5, 150 mM Masi and 1 mM EOTA) for 1 hour at room temperature. After incubation, the binding of biotinylated C5 was detected using a horseradish peroxidase-streptavidin conjugate (TPepto 5siepiis, Sai Mo. 21132), and then AVT5 (KRI., Sai Mo. 50-66-06) was added.

The Axis VEYUOZ384 system (RaiYi and Zsiepse5) was used to evaluate the affinity and pH-dependent binding of antibodies against C5. Antibodies secreted into the culture supernatant of B cells were loaded onto the tip of a Protein A biosensor (Rai!I ie bsiepsev5) and immersed in 50 nM human or monkey C5 in ME5 buffer pH 7.4 to analyze binding kinetics. Dissociation kinetics was analyzed both in ME5 buffer pH 7.4 and in ME5 buffer pH 5.6.

A total of 41,439 B-cell lines were screened for affinity and pH-dependent binding to human or monkey C5 and 677 lines were selected and labeled with CEAO0O0O1-0677. RNA of selected lines was purified from debris of cryopreserved cells using sets 28-96

Oiisk-AMA (2UMO VEZEAVSN, Sai Mo. A1053). The DNA encoding the variable regions of the heavy chain of the antibody was amplified in the selected lines using PCR with reverse transcription and recombined with the DNA encoding the constant region of the heavy chain E760c214 (5EO I MO: 33) or 939234 (5EO IO MO: 34). The DNA encoding the variable regions of the light chain of the antibody was amplified using ROSA with reverse transcription and recombined with the DNA encoding the constant region of the COMTOS light chain (5EO IO MO: 36). The genes of the heavy and light chains of the existing humanized antibody against C5, eculizumab (EsyHn-c2c4, 5EO IO MO: 29 and Esii -

KO, 5EO IO MO: 30). DNA encoding MH (EsyH, 5EO IO MO: 31) was fused in frame with DNA encoding a modified CH IdDa4 of human (E760C214, 5EO IO MO: 33), and DNA encoding MI (Esii, 5EO IO MO: 32), were fused in frame with DNA encoding the constant region of the KO light chain (ZEO IO MO: 37).

Each of the fused coding sequences was also cloned into an expression vector. Antibodies were expressed in РЕгебиюе"M 293-E cells (Ipigtodep) and purified from the culture supernatant to assess functional activity. The neutralizing activity of the antibodies was assessed by testing the inhibition of complement activity using a liposome lysis assay as described in Example 5.1. 2.2. Analysis of binding of the epitope using IBA sandwich EI

Antibodies against C5 with high affinity, pH dependence and neutralizing activity were selected for further analysis. Sandwich EG IZA analysis was used to group selected antibodies into different epitope groups binding to the same C5 protein epitopes or overlapping epitopes. Unlabeled captured antibodies were diluted PB5 (-) to 1 μg/ml and added to MAHIBOogr plates with 384 cells (Mips, Sai Mo. 164688). Plates were incubated for 1 hour at room temperature and blocked with 5-fold diluted ViosKipd Ope (Masaiai

Tezdie, Sai Mo. 03953-95). Plates were incubated for 1 hour, washed, 2 nM human C5 added and incubated for 1 hour. After incubation, the plates were washed and labeled detection antibodies were added (1 μg/ml, biotinylated with MNe-RESA-VioIP). After

After incubation for 1 hour, binding of the biotinylated antibody was detected with the help of horseradish peroxidase-streptavidin conjugate (TPepto 5bsiepiis, Sai Mo. 21132), after which added

AVTZ5 (KRI, Sai Mo. 50-66-06).

All anti-C5 antibodies were used as a capture antibody and a detection antibody and paired in full. As shown in Figure 1, mutually competing antibodies were grouped into 7 epitope groups: SEAOb668, SEAOZ334 and SEAOZ319 were grouped into epitope group A,

SEAOb47, SEPAOB589, SEAOZ41, SEPAOb6Z39, SEAOb6Z35, SRAOZZO and SEAOZ18 were grouped into epitope group B, SEAO538, SEAOBOY, SERAOB99, SEAOZO7, SEAOZbb, SEAOZO5, SEAOb75, SEAObbb and

SEAOb72 was grouped into epitope group C, eculizumab and SEAOZ322 were grouped into epitope group 0,

SEAOZ329 was grouped into epitope group E, SEAOZ359 and SEAO217 were grouped into epitope group E, and

SEAOB579, SERAOZ328 and SEAO272 were grouped into epitope group C. Figure 1 shows epitope binding of some chimeric antibodies against C5. The sequences of MH and Mi antibodies against C5, grouped in epitope group C, are shown in Table 2.

Table 2

Antibodies against C5 grouped in epitope C 01 8BOYUMOoi 77777711

SEAOZO? | 2 | 12 | 46 | 56 | 66 | 76 | 86 | YaVg96

SEAoOBOi | 4 | 14 | 48 | 58 | 68 | 78 | 88 | Yu G(98

SEAO"38 | 5 | 15 | 49 | 59 | 69 | 79 | 89 | 99

SEdoOB99 | 6 | 16 | 50 | 60 | 70 | 80 | 90 00

SEAOb7a | 8 | 18 | 52 | 62 | 72 | 82 | 92 | 102 сСЕАоОб75 | 9 | 19 | 53 | 6 with | 73 | 83 | 93 | 03 2.3. Humanization and optimization

Humanization of the variable region of some antibodies against C5 was performed in order to reduce the possible immunogenicity of antibodies. The hypervariable regions (HVR) of the rabbit anti-C5 antibody were transplanted onto the homologous framework regions (HR) of the human antibody, using the traditional approach for transplantation of OWL (Maishge 321:522-525 (1986)). Genes encoding humanized MH and MI were synthesized and combined with modified human CH (52402, 5EO IU

MO: 35) and human C Idb (5KI, 5EBO 10 MO: 38), respectively, and each of the combined sequences was cloned into an expression vector.

A number of mutations and combinations of mutations were investigated to identify mutations and combinations of mutations that improved the binding properties of certain leader antibodies. Numerous mutations were then made in the humanized variable regions to increase the affinity of binding to C5 at neutral pH or to decrease the affinity of binding to C5 at acidic pH. As a result, from SEAOZO5 we received one of the optimized variants, 305 05 (MN, 5EO IO MO: 10; MI,

ZEO IU MO: 20; NUMV-NI, 5EO AND MO: 54; NUV-N2, 5EO IO MO: 64; NUV-NZ, 5EO and MO: 74; NUV-

IT, BEO IU MO: 84; NMV-12, 5EO IO MO: 94; and NMV-I 3, 5EO IO MO: 104).

Antibodies were expressed in HEK293 cells co-transfected with a mixture of heavy and light chain expression vectors and purified with Protein A.

Example C

Characteristics of binding of antibodies against C5 3.1. Expression and purification of recombinant antibodies

Recombinant antibodies were transiently expressed using the cell line Egeeziuye293-

E (TNepto Rivspeg, Sagpizrady, SA, USA). Purification of antibody-expressing conditioned media was performed using the traditional method using Protein A.

If necessary, further gel filtration was performed. 3.2. Assessment of dependence on pH

Kinetic parameters of anti-C5 antibodies against recombinant human C5 were evaluated at pH 7.4 and pH 5.8 at 37 °C using a 1200 VIASONVE (registered trademark) device (CE Neainsagje). Rgod/a (Riegsje) was immobilized on a CMA4 sensor chip , using an amine coupling kit (ZhbE Neainsage) according to the recommended CE settings

Neainsage. Antibodies and analytes were diluted in the respective mobile buffers, ACE5 pH 7.4 and pH 5.8

Zo (20 mm ACE5, 150 mm Masi, 1.2 mm Sasi», 0.05 95 Tzhsheep 20, 0.005 95 Mam»). Each antibody was captured on the sensor surface using PgA/(u. Antibody capture levels were typically 60-90 resonance units (RU). Recombinant human C5 was then injected at concentrations of 10 nM and 20 nM or 20 nM and 40 nM, followed by dissociation.

The surface was regenerated with 20 mM Mahon. Kinetic parameters at both pH conditions were determined by fitting the sensograms to a 1:1 binding model using evaluation software, 1200 VIASOVE (registered trademark), version 2.0 (CE Neainsage). The sensograms of all antibodies are shown in Figures 2A and 28. The rate of association (ka), the rate of dissociation (Ka) and the binding affinity (KO) of the antibodies are shown in Table 3. All antibodies, with the exception of SEAOZZO (MN, 5EO IO MO: 21 and MI, 5EO IU MO: 25) and SEAOZA (MN, ZEO IO MO: 22 and MI, 5EO IO MO: 26), showed a relatively higher dissociation rate at pH 5.8 than at pH 7.4.

Table C

Kinetic parameters of antibodies against C5 at pH 7.4 and pH 5.8 of the antibody | ka | ka | KO | ka | ka | co (

3.3. Cross-reactivity testing

In order to observe the cross-reactivity of anti-C5 antibodies with respect to human C5 (pC5) and Javan macaque C5 (supoC5), a VIASONVE (registered trademark) kinetic analysis was performed. The assay settings were the same as described in Example 3.2.

Recombinant supoC5 was injected at concentrations of 2, 10, and 50 nM. Kinetic parameters were determined using the same data fitting as described in Example 3.2. Kinetic parameters and binding affinity at pH 7.4 are shown in Table 4. Kinetic parameters for PS5 shown in Table 4 are results from Example 3.2. All anti-C5 antibodies, with the exception of SEAOb672, showed comparable KO against Co and supoC5. KO antibodies

SEAOb672 in relation to supoC5 was 8 times weaker than in relation to PS.

Table 4

Kinetic parameters and binding affinity of antibodies against C5 in relation to pC5 and supo Сb at pH 74 of the antibody | ka | ka | KO | ka | ka | co

Example 4

Mapping of epitopes in relation to antibodies against C5 4.1. Binding of anti-C5 mAbs to peptides derived from the beta-chain of C5

Monoclonal antibodies against C5 (MAB) were tested for their binding to peptides derived from the C5 beta chain using Western blot analysis. Peptides C5: 19-180, 161-340, 321-500 and 481-660, - fused with a 5T tag (raEH-41-I, aE Neainsage I Me Zsiepse5, 28-9545-49), were expressed in E. soybean ( ONbairna, TOMOVO, OMA-903). E. soya samples were collected after incubation with 1 mM isopropyl-beta-O-i-thiogalactopyranoside (IRTS) for 5 hours at 37 "C and centrifuged at 20,000 x 9 for 1 minute, and a pellet was obtained. The pellet was suspended in a buffer solution for samples (2IU) (U/ako, 191-13272) and used for Western blot analysis. The expression of each peptide was confirmed with an antibody against 51 (Abeat, ar9085) (Figure 3). The arrow points to C5 peptides fused to 451 ( 46-49 kDa). MAB against C5: SEAOZO5, SEAOZO7,

SEAOBbb, SEAOB1, SEAOB538, SEAOB99, SEAOBbb, SEAOB672 and SEAOB75 are bound to the 19-180 C5 peptide (Figure 3). 4.2. Expression and purification of the Ma1-MOa2 domain (1-2253 human C5 Recombinant domain

Ma1-ma2 (5EO IOI MO: 43) human C5 beta-chain was transiently expressed using the Egebiuie293-E cell line (TNepto ReevesNeg, Sapzrad, CA, USA). conditioned environments,

Those expressing the M(1-M(2) domain were diluted with 72 volumes of thiolO water and then loaded onto an O-Zerpagose EE anion-exchange column (SE Neainsage, Orrzaia, Sweden). The flow-through fraction from the anion-exchange column was adjusted to pH 5, 0 and loaded onto a 5P-zerpagose NR cation exchange column (SE Peaysageje, Orrzaia, Sweden) and eluted in a Mass gradient. Fractions containing the MO1-M(2) domain were obtained from the eluent and then loaded onto a Ziregdeh 75 gel filtration column (SE Peinsagje, Orrzaia, Sweden), balanced with 1 x PB5. Fractions containing the Ma1-Magg domain were then pooled and stored at -80 °C. 4.3. Binding capacity to the MC1-Ma2 domain

The binding capacity of anti-C5 antibodies to the MaI1-Ma2 domain was measured using the same assay settings as described in Example 3.2, except that the measurements were performed only at pH 7.4. The Ma1-Ma2 domain was injected at concentrations of 20 nM and 40 nM. As shown in Figure 4, all antibodies, with the exception of eculizumab-76004, showed enhanced binding, indicating that these antibodies bind to

Ma1-Ma2. Eculizumab-P76004, known as an antibody that binds to the alpha chain, did not show binding to the MC1-Ma2 domain.

4.4. Binding of mAb against C5 to peptides originating from the ME1-MOS2 domain of C5 MAB against

C5 was tested for their binding to peptides originating from the domain

Ma1-meg, using western blot analysis. Peptides C5: 33-124, 45-124, 52-124, 33-111, 33-108 and 45-111 (5EO IU MO: 40) - fused with the C5T tag were expressed in E. soya. E. soybean samples were collected after incubation with IM IRT for 5 hours at 37 "C, and centrifuged at 20,000 x 9 for 1 minute, and a pellet was obtained. The pellet was suspended in sample buffer solution (2ME xx and used for Western blot analysis. The expression of each peptide derived from

C5, confirmed with antibody against 51 (Figure 5A). SEAOZO5 bound only to peptide 33-124 (Figure 58). SEAOZO5 bound to the beta chain of recombinant human C5 (C5) (approximately 70 kDa), which was used as a control. Figure 5C summarizes the reaction of mAbs against C5 with C5-derived peptides. 4.5. Anti-C5 mAb binding to C5 mutants

Since the analysis of the crystal structure predicted that three amino acid residues in the beta chain of C5 - E48, 051 and K109 - are involved in the binding between C5 and MAB against

C5, then mAbs against C5 were tested for their binding to point mutants of human C5 using Western blot analysis. C5 point mutants in which any of E48, 051, and CT109 were replaced by alanine were expressed in E5293 cells by lipofection. Culture media were collected 5 days after lipofection and then used for Western blotting. 505-RACE was performed under reducing conditions. The results are shown in Figure 6. Eculizumab bound to the alpha chain of native (M/T) C5 and three point mutants of C5, while SEAOZ3O5 bound strongly to the beta chain of M/T C5, weakly bound to mutant C5 E48A did not bind to the beta-chain of C5 05O1A and KI1TO9A mutants, indicating that these three amino acid residues are involved in the antibody/antigen interaction. Table 5 shows the summarized results of the Western blot analysis of MAB against C5 (SEAOZO5, SEAOZO07,

SEAOZbb, SEAOBOY, SEAOBZ38, SEAOBbB99, SEAObbb, SEAOb2 and SEAOb75). MAbs against C5 are grouped into one epitope group C, but the binding profiles differ slightly between antibodies, suggesting that the binding sites of C5 with MAbs against C5 are located close to each other, but they are not identical.

Table 5

Generalization of the MAB reaction against C5 with C5 mutants 1111111111ММТ11 11111 БВА | OA | KovA

SEAOZOYE SI1117яю1Я11111ю11-11

SEAOZO? СЯ71117zhyu1 11111711

SEAOZVE SI11171yayu 11111111

SvAOBOT SG! 71117zhyu1 11117111

SEAOBZV S Y711717yayu 11111711

SEAOBYAYA /Y7771717yayu 11111711

SEAObbE S Y717117yayu 11111711

SEAOBUa S Y117yayu 11111711 -1111x (SEAObYa» Yizh Y71717171yuk1 1111-11

From 4.6. Analysis of the binding of VIASOVE (registered trademark) anti-C5 antibodies to C5 mutants

In order to test whether the E48, (551 and K1I09 residues are really involved in the antibody-antigen interaction, a VIASOVE (registered trademark) binding assay was performed. Three C5 mutants - E48A, (251A and KITO9A) were prepared as described in

Examples 4.5. Samples of culture supernatants containing mutant C5 overexpressed in E5293 cells were prepared at a concentration of 40 μg/ml mutant C5.

For VIASOVE (registered trademark) binding analysis, the sample was diluted 10x with VIASONE (registered trademark) mobile buffer (ACE5 pH 7,4,10 mg/ml B5A, 1 mg/ml carboxymethyldextran) to a final concentration of 4 μg/ml sample mutant C5.

The interactions of the three C5 mutants with anti-C5 antibodies were evaluated at 37 °C using a 1200 VIASOVE (registered trademark) (cE Neainsagje) using the assay conditions described in Example 3.2. ACE5 buffer pH 7.4 containing 10 mg/ml B5A, 1 mg/ml carboxymethyldextran, was used as a mobile buffer. Eculizumab-?P76004 and 305I 05 were captured on different flow cells using a mouse monoclonal anti-human des antibody that is specific for the Es fragment (CE NeinNsage). Flow cell 1 was used as reference surface. Native and mutant C5 proteins were injected onto the sensor surface at a concentration of 4 μg/ml to interact with the captured antibodies. At the end of each assay cycle, the sensor surface was regenerated with 3M MosSb. The results were analyzed using the Via Emaicai software, version 2.0 ( SE

Neainsage). The curves of the reference flow cell (flow cell 1) and blank injections of the moving buffer were subtracted from the curves of the flow cell with captured antibodies.

As shown in Figure 7, all three C5 mutants were able to bind to eculizumab with a binding profile similar to native C5. For 305 05, all three mutants showed a lower binding response to 305 05 compared to natural C5. Mutants Ю5ТА and КИ09А reduced the binding of С5 with 305И 05 to the initial level. 4.7. Identification of Nib5 residues in C5 that contribute to the pH-dependent interaction between anti-C5 antibody and C5

Analysis of the crystal structure revealed that three histidine residues in human C5 are located in the antibody/antigen interaction zone. A histidine residue with a normal pKa of approximately 6.0 is known to promote pH-dependent protein-protein interaction (Idama et al., Viospit

Journal Asia 1844(11):1943-1950 (2014)). In order to investigate which of the Nib residues in the antibody/antigen interaction zone contribute to the pH-dependent interactions between the anti-C5 antibody and

C5, VIASOVE (registered trademark) binding analysis was performed. Three mutants

Human C5 with a single mutation of Niv (H7OU, H72U and NI 10U) and a mutant with a double mutation of Nivb (H7OM and NI 10U) were obtained as follows: mutants with a single Niv in which any of H7O, H72 and NI 10 is replaced by tyrosine, and a double Nizh mutant in which both H7O and NI10 are replaced by tyrosine were expressed in E5293 cells by lipofection. Antigen binding properties of Nih-substituted C5 mutants with 30505, a pH-dependent anti-C5 antibody, were determined using a modified VIASONVE (registered trademark) assay as described in Example 4.6. Briefly, an additional dissociation phase at pH 5.8 was performed in the VIASOVE (registered trademark) assay immediately after the dissociation phase at pH 7.4 in order to assess the pH-dependent dissociation of antibody-antigen complexes formed at pH 7.4. The rate of dissociation at pH 5.8 was determined by processing and

From data matching, using software curve matching bsgtirreg 2.0 (Vioi odis 5oimage).

As shown in Figure 8, a single mutation of Nis in C5 to H7O or NO 10 and a double mutation of Nib (H7O and NO 10) did not affect the binding of C5 to 305I05 at neutral pH. Meanwhile, a single Niz mutation on H72 showed a significant disruption of C5 binding to 305 05. The dissociation constants at pH 5.8 of Ni-substituted C5 mutants and native C5 protein (C5-mi) are given in

Tables 6. As shown in Table 6, native C5 exhibited the fastest dissociation from 30505 at pH 5.8 among the C5 antigens tested. A single Niz mutation on Н7О0 showed an almost two-fold lower dissociation rate at pH 5.8, and a single Ni mutation on

NO 10 caused a slightly lower dissociation rate at pH 5.8 compared to natural C5.

A double mutation on both H7O and NI 10 resulted in a greater effect on pH-dependent binding, with the rate of dissociation at pH 5.8 almost three times lower than that of native C5.

Table 6

Values of dissociation constants at pH 5.8 for Niz C5 mutants that bind to 305I 05

Example 5

Inhibitory activity of antibodies against C 5 in relation to activation of C 5 5.1. Inhibition of complement-activated liposome lysis by MAB antibodies against C5

Anti-C5 mAbs were tested for inhibition of complement activity using a liposome lysis assay. 30 μl of healthy human serum (6.7 9c) (Viorgedis, ZENO18) was mixed with 20 μl of diluted MAB in a 96-well plate and incubated on a shaker for 30 minutes at 25 °C. Liposomes sensitized with anti-dinitrophenyl antibodies (Ashokii CH5BO,

Mmako, 995-40801), was transferred to each cell and the plate was placed on a shaker for 2 minutes at 25 "C. 50 μl of substrate solution (Ashiokkii CH5O) was added to each cell and mixed by shaking for 2 minutes at 25 "C. The final mixture was incubated at 37 "C for 40 minutes, after which the optical density (00) of the mixture was measured at 340 nm.

The percentage of liposome lysis was determined as 100 x OO ml-OO background of serum and liposomes. 0599, 0666, 0672 and 0675 inhibited the lysis of liposomes. Two pH-independent antibodies SEDOZ30 and 0341 also inhibited lysis (Figure 9B). 5.2. Inhibition of C5a formation by MAb antibodies against C5

Anti-C5 mAbs were tested for Cb5a formation during liposome lysis to confirm that anti-C5 mAbs inhibit cleavage of C5 into C5a and C5B. The level of C5a in supernatants from the analysis of liposome lysis was quantified using the Cb5a EGIBA kit (Nazuvietv, Rugoz7). All MAbs inhibited Sba formation in the supernatants in a dose-dependent manner (Figures 10A and 108B). 5.3. Inhibition of complement-activated hemolysis by antibodies MAB against C5 MAB against

C5 was tested for inhibition of classical complement activity in a hemolytic assay.

Chicken erythrocytes (CsBVS5) (Hippomaimme geseagsp, IS05-0810) were washed with gelatin/veronal buffered saline containing 0.5 mM MaOsi»g and 0.15 mM Sasig (CMV---) (Voviop

Viorgodisov, IVV-Z00X), and then sensitized with an antibody against chicken ABC (KosKiapa 103-4139) at a concentration of 1 μg/ml for 15 minutes at 4 "C. The cells were then washed with SUVik and suspended in the same buffer with a density of 5 x 107 cells/ml. In a separate plate with 96 round-bottom cells for the microtest, 50 μl of healthy human serum (20 9bo) (Viorgedisje, FENO19) was mixed with 50 μl of diluted MAB and incubated on a shaker at 37 "C for 30 minutes. 60 μl of a suspension of sensitized SVVS was then added to cells containing serum, and the antibody mixture was incubated at 37 °C for 30 minutes. After incubation, the tablet was centrifuged at 1000 x 4 for 2 minutes at 4 °C. Supernatants (100 μL) were transferred to cells on a 96-well flat-bottom microassay plate to measure OO at 415 nm with a reference wavelength of 630 nm.

The percentage of hemolysis was determined as 100 x OO md-OO serum and svvs))/ KOJ without mal-JUOJ background serum and svvs)|.

Example 6

Study of the pharmacokinetics of monoclonal antibodies against C5 from human C5 in mice 6.1. IP mimo test using S57VI /6 mice

The kinetics of IP by human C5 (Sairiospet) and anti-human C5 antibodies were evaluated after administration of only human C5 or human C5 and anti-human C5 antibodies to C57VI/6 mice (IP

By the way of Vioiodis! Kezotsgse Sepige, Zipdaroge). Human C5 solution (0.01 mg/ml) or a mixture solution containing human C5 and anti-human C5 antibody (0.01 mg/ml and 2 mg/ml (SEAOZ305-

E760c24, SEAOZO7-E760024, SEAOZb66-E76004, SEAAOBO1-E760c24, SEAO538-E76004, SEAOB599-

E760c24, SEAObbb-Е760024, SEAOb72-Е760054 and SEAOb75-Е76004) or 0.2 mg/ml (SEAOZ330-

E76004 and SEAOZ341-E76004, respectively), were injected once in a dose of 10 ml/kg into the tail vein.

In this case, anti-human C5 antibody is present in excess of human C5, and, as a result, it is assumed that almost every human C5 binds to the antibody. Blood was drawn 5 minutes, seven hours, one day, two days, three days, and seven days after administration. The collected blood was then immediately centrifuged at 14,000 rpm and at 4 C for 10 minutes to separate the plasma. The separated plasma was stored in a freezer at -80 "C until analysis. Antibodies against human C5 used are those described above

SEAOZO5-E760c4, SEAOZO7-E760024, SEAOZZ30O-E760024, SEAOZ41-E760c24, SEAOZb66-E760C4,

SEAOBO1-E760c4, SEAOB38-Е76004, SEAOB599-Е76004, SEAOB6b-Е76004, SEAOB72-Е76064 and

SEAOb75-E760S4. 6.2. Measurement of total human C5 plasma concentration using electrochemiluminescence (ECI) analysis

The concentration of total human C5 in mouse plasma was measured using ESI.

The following method was used in the presence of SEAOZ330-E76004, SEAOZ41-P760054 or only human C5 in a plasma sample. The antibody against human C5 (Zapia Sgy7) was distributed. into an unsealed plate with 96 cells of MY TI-AKKAM (Meso Zsaie bissomegu) and left to stand overnight at a temperature of 4 "C to obtain plates with immobilized antibody against

C5 of a person. Samples for the calibration curve and samples of mouse plasma diluted 100-fold or more with 1 μg/ml of injected antibody were obtained and incubated for 30 minutes at 37 °C. The samples were then spread into plates with immobilized anti-human C5 antibody and allowed to stand for one hour at room temperature. Then BOI RO-TAS labeled anti-human antibody (Mezo Zsaie Oizsomegu) was added, and allowed to react for one hour at room temperature, and washed. Immediately after that, Keai Viyeg T (x4) (Meso Zsaiee Oibzsomegu) and performed measurements using Zesiog Itadeg 2400 (Meso zsaie bizsomegu).

The following method was used in the presence of SEAOZ305-E760024, SEAOZO7-E760C4,

SEAOZb66-Р760с4, SEAAOBO1-г760с24, SEAAO538-Р760с4, SEAOB599-Е76004, СРАОбб6-Е760С4,

SEAOB72-Е760054 or SEAOBЕ75-Е76004 in a plasma sample. Antibody against human C5 (SEAOZ329-

E93904; MN, 5EO IYUO MO: 23 and MI, 5EO IYUO MO: 27) were distributed in an unsealed 96-well MOG TI-AKKAM plate (Mezo Zsaie Oizsomegu) and left to stand overnight at 4 °C to obtain plates with immobilized antibody against C5 of a person.

Samples for the calibration curve and samples of mouse plasma diluted 100-fold or more with an acidic solution (pH 5.5) were obtained and incubated for 30 minutes at 37 °C. These samples were then distributed in plates with immobilized anti-human C5 antibody and left allowed to stand for one hour at room temperature, then labeled was added

ZY RO-TAS antibody against human C5 (SEAOZ300-E939054; MH, 5EO IUO MO: 24 and M, 5EO IU

MO: 28), and allowed to react for one hour at room temperature, and washed. Immediately after that, Keai Wiyeg T (x4) (Meso zZsaIe Oizsomegu) was distributed and measurements were made using z5esiog Itadeg 2400 (Meso zzaIe Rissomegu).

The concentration of human C5 was calculated based on the response to the calibration curve, using analytical software ZOETtah RKO (Moiesshag Oemise5). The temporal dynamics of human C5 plasma concentration after intravenous administration, measured by this method, is shown in Figure 13. The data are plotted as a percentage of the residue compared to human C5 plasma concentration after 5 minutes. 6.3. Measurement of the concentration of human anti-C5 antibodies in plasma using an assay

EC

The concentration of antibodies against human C 5 in mouse plasma was measured using EC.

A fragment of antibody E(ar)2 against human db (gamma chain specific) (Zidta) or antibody against kappa chain IdsC (Apibodu Zoishshchiop5) was distributed in an unsealed plate with 96 cells of MOSTI-AKKAM (Meso 5Zsaie Oibsomegu) and left stand overnight at a temperature of 4"C to obtain tablets with immobilized antibody or

With an antibody fragment against (390 human. Samples of the calibration curve and samples of mouse plasma diluted 100 times or more were prepared. After that, the samples were distributed in tablets with immobilized antibody or antibody fragment against human ED0 and left to stand for one hour at room temperature. Then added biotinylated anti-human dO antibody (BociPpegprioiesp) or ZOGRO-TAS-labeled anti-human EsIdS antibody (ZoshiPegprioiesi)), and left to react for one hour at room temperature, and washes were performed. After that, only when the biotinylated antibody against human DO was used, labeled ZSH RO-TAS streptavidin (Meso ZsaIe Oibzsomegu) was added, and left to react for one hour at room temperature, and washing was performed. Immediately after that, Keai Vypyeg T (x4) (Meso ZsaiIe Oiz5somegu) was distributed and measurements were performed using Besiog Iptadeg 2400 (Meso zZsaiIe Oizsomegu). The concentration of human anti-C5 antibody was calculated based on the response to the calibration curve using the analytical software 5OETtah RKO (Moiesshag Oemise5). The temporal dynamics of the concentration of human anti-C5 antibody in plasma after intravenous administration, measured by this method, is shown in Figure 14. The data are plotted as a percentage of the residue compared to the concentration of human anti-C5 antibody in plasma after 5 minutes. 6.4. Effect of pH-dependent binding of anti-human C5 antibody on clearance of human C5 i.p. mimo

pH-dependent antibodies against human C5 (SEAO305-E760214, SEAOZO7-E760c4, SEAOZbb-

E76004, SERAOB501-P76004, SEAO538-P760c04, SERAOB599-E760c4, SEAObb6b-P760c4, SEAOb72-

Е76004 and СЕАО675-Е7606054) and pH-independent antibodies against human C5 (СЕАО330-Е760.4 and СЕАОЗ341-Е760с4) were tested by ip mimo and compared the obtained concentration in plasma of antibodies against human C5 and the concentration in plasma of human C5. As shown in Figure 14, the antibody content in plasma was comparable. Meanwhile, the clearance of human C5 administered simultaneously with pH-dependent anti-human C5 antibodies was accelerated compared to the clearance of human C5 administered with pH-independent anti-human C5 antibodies (Figure 13).

Example 7

Optimization of monoclonal antibodies against C5 (variants 305)

Made a number of mutations in the optimized variable region of anti-C5 antibody 3051 05 to further improve its properties, and obtained optimized variable regions 3051 015, 60 305I 016, 305I 018, 3051 019, 3051 020, 3051 022 and 3051 023 Amino acid sequences of MH and

E. variants 305 are shown in Tables 7 and 8, respectively. The genes encoding the humanized UN were combined with a modified variant of human IdsSI CH - 505115 (5EBEO IUO MO: 114) and modified variants of human IdsS4 CH -565422 (5EO IU MO: 115) or 565429 (5EO IU MO: 116). Genes that encode humanized MI were combined with human SI (ZKI, 5EO IU MO: 38). Separately, the heavy and light chain genes encoding the humanized antibody against C5, WMO441 (8MO441H, 5EO IO MO: 149; WMO4411, 5EO IO MO: 150), were synthesized and each gene was cloned into an expression vector.

Antibodies were expressed in HEK 293 cells co-transfected with a combination of heavy and light chain expression vectors and purified with Protein A.

Table 7

NUV-NI NUV-Ng NUV-NZ , ZEO IO MO: 54 ZEO IO MO: 64 ZEO IU MO: 74 3051-05 ZESIOMO:10 ZZUMMA AIUtTavaATUKABMAKOI | OSOaMOMRTNAMNU , ZEO IO MO: 117 ZEOIO MO: 118 ZEO IO MO: 121 3051015 | FAILURE MO: 106 Z8UUMA // IDIETOZSAEUKAEMAKO OASUOURTNAMNU , ZEO IO MO: 117 ZEOIYU MO: 119 ZEO IO MO: 121 3051016 | MO FAILURE: 107 Z8UUMA // IDIETOZSAEUKAYEMUKO | OASUOURTNAMNU, ZEO IO MO: 117 ZEO IO MO: 118 ZEO IO MO: 121 305018 | FAILURE MO: 108 Z8UUMA // IDIETOZSAEUKAEMAKO OASUOURTNAMNU , ZEO IO MO: 117 ZEOIO MO: 118 ZEO IO MO: 121 3051019 | FAILURE MO: 109 Z8UUMA // IDIETOZSAEUKAEMAKO OASUOURTNAMNU , ZEO IO MO: 117 ZEOIO MO: 118 ZEO IO MO: 121 3051020 | FAILURE MO: 109 Z8UUMA // IDIETOZSAEUKAEMAKO OASUOURTNAMNU , ZEO IO MO: 117 ZEOIO MO: 118 ZEO IO MO: 121 3051022 | FAILURE OF MO: 109 Z5UUMA // ИДИЕТОЗСАЕУКАЕМАКСО ОАСУОУРТНАМНУ , ZEO IU MO: 117 ZEO IU MO: 120 ZEO IO MO: 121 3051023 ) FAILURE OF MO: 110 Z5UUMA // |DIETO5OATUKAEMAKO| OASUSURTNAMNU

Table 8

Amino acid sequences of MI variants 305

NUV-I NUVI2 NUV-Z , ZEO IU MO: 84 ZEO IO MO: 94 ZEOIU MO: 104 3055 ZBOIOMO20 | OAVOMISVBIA SABKTNU O5TKUS85UOMN , ZEOIYUMO 122 ZEO IYU MO: 123 ZEO IYU MO: 125 305-015 | ZEOTO MO: 111) VABOSIB55IA SABETEV OMTKUS55USMT , ZEO IU MO: 122 ZEO IYU MO: 123 ZEO IYU MO: 125 305-016 | ZEOIU MO: 111) VABOSIV 5VIA SABETEV OMTKUS55USMT , ZEO IU MO: 122 ZEO IU MO: 123 ZEO IU MO: 125 305-018 | ZEOIU MO: 111) VABOSIV 5VIA SABETEV OMTKUS55USMT , ZEO IU MO: 122 ZEO IU MO: 123 ZEO IU MO: 125 305-019 | ZEOIU MO: 111) VABOSIV5VIA SABETEV OMTKUS55USMT , ZEO IU MO: 122 ZEO IU MO: 123 ZEO IU MO: 125 3051020 | ZEOIO MO: 112 ) vVABOSI555IA SABETEV OMTKUS55USMT , ZEO IU MO: 122 ZEO IU MO: 124 ZEO IU MO: 125 3051022 ) ZEOIO MO: 113 | VABOSIZ5BIA SAZTTO5 OMtKUs55KHOMt , ZEO IU MO: 122 ZEO IYU MO: 124 ZEO IYU MO: 125 3051023 | ZBOIO MO:113 | vVABOSI555IA CA5TTO5 OMTKUS55USMT

Example 8

Binding characteristics of antibodies against C5 (variants 305) Kinetic parameters of antibodies against C5 in relation to human recombinant C5 were evaluated at 37 "C using the device

T200 VIASONE (registered trademark) (CE Neainsagje) under three different conditions: (1) association and dissociation measured at pH 7.4, (2) association and dissociation measured at pH 5.8, and (3) association measured at pH 7 ,4, and dissociation was measured at pH 5.8. Rgod/a (Riegse) was immobilized on the media sensor chip using an amine coupling kit (SE

NeyNsageye) according to CE Neeynsage recommended settings. Antibodies and analytes for conditions (1) and (3) were diluted in ACE5 buffer pH 7.4 (20 mM ACE5, 150 mM Masi, 1.2 mM Sasi", 0.05 95

Tmeep 20, 0.005 95 Mam"), and for condition (2) they were diluted in ACE5 buffer pH 5.8 (20 mM ACE5, 150

MM mass, 1.2 mM Sasi", 0.05 95 Tmeep 20, 0.005 95 MaM»z). Each antibody was captured on the sensor surface using PgoA/y. Antibody capture levels were usually 60-90 resonance units (RU). After that, recombinant human C5 was injected at 3 nm to 27 nm or from 13.3 nM to 129 nM, obtained as a result of threefold serial dilution, and then dissociation occurred. The surface was regenerated using 25 mM Mahon. The kinetic parameters under conditions (1) and (2) were determined by matching the sensograms with the 1:1 binding model, and the dissociation constant under condition (3) was determined by matching the sensograms with the 1:11 dissociation for the MOSC model, using the T200 software by Emaishyayop

VIASOVE (registered trademark), version 2.0 (CE NeayNsage). The pH dependence of all antibodies is shown as the ratio of dissociation constants for conditions (2) and (1).

Association constant (ka), dissociation constant (Ka), binding affinity (BO), and pH dependence are shown in Table 9. All antibodies showed a higher dissociation rate at pH 5.8 than at pH 7.4, and their dependence from pH was approximately 20-fold.

Table 9

Kinetic parameters of anti-C5 antibody variants under conditions of pH 7.4 and pH 5.8

The binding affinity of antibodies against C5 (VM.O441, eculizumab and variant 305) with recombinant human C5 at pH 7.4 and pH 5.8 was determined at 37 "C using the device

T200 VIASOVE (registered trademark) (CE NeeinNsage) to assess the effect of pH on antigen binding. Goat polyclonal antibody against human Esdb (CRI 201-10-20) was immobilized on a CM4 sensor chip using an amine coupling kit (CE

Neainsage) according to the settings recommended by the manufacturer. Antibodies and analytes were diluted in ACE5 buffer pH 7.4 or ACE5 buffer pH 5.8 containing 20 mM ACE5, 150 mM

Masi, 12 mm Sasi", 0.0595 Tmeep 20 and 0.00595 MamM". Antibodies were captured on the sensor surface using the anti-Es method, capture levels were usually 50-80 resonance units (2Ksh). Recombinant human C5 was obtained as a result of threefold serial dilution, starting from the assay conditions of 27 nM for pH 7.4 or from the assay conditions of 135 nm for pH 5.8. The surface was regenerated using 20 mM HC, 0.01 95 Gueep 20. The data were processed

Zo and aligned with the 1:1 binding model using ViaEma|ICihop 2.0 software (CE Neaiiisage).

The binding affinities (CO) of VMOU441, eculizumab and variant 305 to recombinant human C5 at pH 7.4 and pH 5.8 are shown in Table 10. Variant 305 exhibited a ratio of (CO at pH 5.8) / (CO at pH 7 ,4), which is almost 800, which is 8 times greater than the ratio for WMO441, which showed a ratio of (KO at pH 5.8) / (KO at pH 74) that was only 93.

Table 10. Day of the day - and life cycle at pH 5.8/pH 7.4

Example 9

Inhibitory activity of antibodies against C5 (variants 305) on C5 activation 9.1. Inhibition of complement-activated liposome lysis by MAB antibodies against C5

Anti-C5 mAbs were tested for inhibition of complement activity using a liposome lysis assay. 30 μl of healthy human serum (6.7 Fo) (Viorghedisje, BENO19) was mixed with 20 μl of diluted MAB in a 96-well plate and incubated on a shaker for 30 minutes at room temperature. A liposome solution sensitized with anti-dinitrophenyl antibodies (AshShoKii CH5BO, UUako, 995-40801) was transferred to each cell and the plate was placed on a shaker for 2 minutes at 37 °C. 50 μl of substrate solution (AshShoKiY CH5BO) was added to each cell and mixed by shaking for 2 minutes at 37 "С. The final mixture was incubated at 37 "C for 40 minutes, and then OO was measured at 340 nm. The percentage of liposome lysis was determined as 100 x MOU ma-OO background serum and liposomes) |/ MOU without mAn-OO background serum and liposomes) |.

Figure 15 demonstrates that MABs against C5: 305I 015-5I0422, 305I 016-52:422, 305I 018-5Д12422, 305 019-523422, 3051 020-52:422 and 3051 020-52:115 - inhibited lysis of liposomes. Antibodies with ES variants, 305 015-5(121115 and 305I O23-5C2:1429), also inhibited liposome lysis (Figure 16).

The mAb against C5 was tested for inhibition of recombinant human C5 (5EO IO MO: 39). 10 μl of C5-deficient human serum (5idta, CI 163) was mixed with 20 μl of diluted MAB and 20 μl of recombinant C5 (0.1 μg/ml) in a 96-well plate and the plate was incubated on a shaker for 1 hour at 37 °C. Liposomes (AshoKii CH5O) were transferred to each cell and the tablet was placed on a shaker for 2 minutes at 37 °C. 50 μl of substrate solution (Ashokiya

CH5O) was added to each cell and mixed by shaking for 2 minutes at 37°C. The final mixture was incubated at 37°C for 180 minutes, and then the OO was measured at 340 nm. The percentage of liposome lysis was determined as described above. Figure 17 demonstrates that MAB vs

C5: 305 022-502115, 3051 022-512422, 3051 023-50115 and 305 023-52:422 inhibited the lysis of liposomes. 9.2. Inhibition of C5ba formation by MAB antibodies against C5

Anti-C5 mAbs were tested for Cb5a formation during liposome lysis to confirm that

MAbs against C5 inhibit the cleavage of C5 into Sba and C5Bb. Levels of Sb5a in supernatants were quantified from liposome lysis analysis using the Sba EPIZA kit (HO v5uvietv, rugoz7). All MABs inhibited the formation of Sba in the supernatants in a dose-dependent manner (Figures 18 and 19). 9.3. Measurement of complement activity in Javan macaque plasma

MAb against C5 was tested for inhibition of complement activity in Javan macaque plasma. Monkeys were injected with MAB against C5 (20 mg/kg) and plasma samples were taken periodically up to day 56.

Chicken erythrocytes (CHCs) (Hippomycetes, ISO 5-0810) were washed with gelatin/veronal buffered saline containing 0.5 mM MaOsClg and 0.15 mM Sasig (CMV---) (Woviop

ViorRhodisikh, IVV-Z300X), and then sensitized with an antibody against chicken erythrocytes (NosKiapa 1033-4139) at a concentration of 1 μg/ml for 15 minutes at 4 °C. The cells were then washed

SUVaya and suspended in the same buffer at a density of 1 x 1089 cells/ml. In a separate microassay plate with 96 round-bottom cells, monkey plasma was incubated with sensitized cABS at 37 "C for 20 minutes. After incubation, the plate was centrifuged at 1000 x rpm for 2 minutes at 4 "C. Supernatants were transferred to cells in 96-well flat-bottom microassay plates for measuring OO at 415 nm with a reference wavelength of 630 nm. The percentage of hemolysis was determined as 100 x COO after the introduction of 7 oo background plasma and svvs)/ IU before the introduction of 7 or background plasma and svvs)|.

Figure 20 demonstrates that MAB against C5: 305I 015-5(2422, 305 015-5Х1:2115, 305I 016-512422, 305018-52:422, 3051 019-52:422, 3051 020-52:422, 3051 020 -52115 and 3051023-512115 - inhibited complement activity in plasma. 9.4. Inhibition of biological activity of C5 variants by MAB antibodies against C5

Anti-C5 mAbs were tested for inhibition of recombinant human C5 variants: M145I, 84492, U802I, B885H, 29280, 0966U, 51310M and E14370. Patients with PMN who have the B885H mutation in C5 have been reported to show a poor response to eculizumab (see, for example,

MivPpitigta ei ai., Mem/ Epadi. in. May 370:632-639 (2014)) Each of the human C5 variants was expressed in E5B293 cells and the supernatants were used for further investigation. 10 μl of C5-deficient human serum (Zidta, CI 163) was mixed with 20 μl of diluted MAB and 20 μl of cell culture medium containing a variant of recombinant C5 (2-3 μg/ml) in a 96-well plate and incubated on a shaker for 0.5 hours at 37 "C. Liposomes (Ashiokkii CH5BO) were transferred to each cell and the tablet was placed on a shaker for 2 minutes at 37 "C. 50 μl of the substrate solution (AshSHOKiII CH5BO) was added to each cell and mixed by shaking for 2 minutes at 37 °C.

The final mixture was incubated at 37 "C for 90 minutes, and then the OO was measured at 340 nm.

The percentage of liposome lysis was determined as described above. Figure 21 demonstrates that the MAB against C5 (eculizumab) did not inhibit the C5 B885H variant, but inhibited the other C5 variants tested.

Figure 22 demonstrates that the mAb against C5 (variant 305) inhibited all C5 variants tested. 9.5. Inhibition of complement-activated liposome lysis by MAB antibodies against C5

Anti-C5 mAbs were tested for inhibition of complement activity using a liposome lysis assay. 30 μl of healthy human serum (6.7 Fo) (Viorghedisje, BENO19) was mixed with 20 μl of diluted MAB in a 96-well plate and incubated on a shaker for 30 minutes at room temperature. A liposome solution sensitized with anti-dinitrophenyl antibodies (AshShoKii CH5BO, UUaco, 995-40801) was transferred to each cell and the plate was placed on a shaker for 2 minutes at 25 °C. 50 μl of substrate solution (AshShoKii CH5BO) was added to each cell and mixed by shaking for 2 minutes at 25 "C. The final mixture was incubated at 37 "C for 45 minutes, and then the OO was measured at 340 nm. The percentage of inhibition of liposome lysis was determined as 100 x IU MmAb-OYU background serum and liposomes) |/ IU without MAb-OYU background serum and liposomes) |.

Figure 23 demonstrates that MAb against C5-8MOU441 and variant 305 inhibited liposome lysis and that variant 305 has stronger binding activity than WMO441.

Example 10

Study of the pharmacokinetics of monoclonal antibodies against C5 (305 variants) in the Javan macaque 10.1. IP mimo test using Javan macaque

Anti-human C5 antibody IP mimo kinetics were assessed after administration of anti-human C5 antibody to a Javan macaque (Pip Mirrop Viotewais! I Arogyaugies, Tsa., Japan). A solution of human anti-C5 antibody (2.5 mg/ml) was administered once at a dose of approximately 8 ml/kg into the main vein of the forearm by infusion over 30 minutes. Blood was taken before injection and after 5 minutes, seven hours, one day, two days, three days, seven days, fourteen days, twenty one day, twenty eight days, thirty five days, forty two days, forty nine days and fifty-six days after administration. The collected blood was immediately centrifuged at 1700 x a and 4 °C for 10 minutes to separate plasma. Separated plasma for analysis

Zo was stored in a freezer at -70 "C or lower. Antibodies against human C5 were obtained as described in Example 7. 10.2. Measurement of the concentration of total C5 in the plasma of a Javan macaque using the EGISA assay

The concentration of total C5 of the Javan macaque in the plasma of the Javan macaque was measured by EHIBA. Antibody against human C5 (antibody obtained in laboratory conditions using the method described in Example 2) was distributed in a Myps-IttypoRiale tablet

Mahizogr (MaYide Myps Ipieegpaiopai) and left to stand overnight at 4 "C, resulting in plates with immobilized anti-C5 cynomolgus macaque. Samples for calibration curve and plasma samples of cynomolgus macaque, diluted 20,000 times 0.4 μg/ml of injected antibody , prepared and incubated for 60 minutes at 37 "С. After that, the samples were distributed in tablets with immobilized Javanese macaque anti-C5 and left to stand for one hour at room temperature. HPE-labeled anti-human antibody (5ocypegpVioiesi) was then added and allowed to react for thirty minutes at room temperature, and washed. After that, the horseradish peroxidase substrate was added

AUTO EPIZA NAR Zybzigaiye (KRI). The signal was measured using a tablet reader at a wavelength of 405 nm. Javanese macaque C5 concentration was calculated based on the response to the calibration curve using the analytical software 5OETtah RO (MoiIesshag Oemise5). The temporal dynamics of the plasma concentration of Javan macaque after intravenous administration, measured by this method, is shown in Figure 24. The data are plotted as a percentage of the residual compared to the concentration of Javan macaque C5 in plasma before administration. pH-dependent antibodies against human C5 (3051 015-5023422, 305I 015- zZa115, 305 016-5125422, 305I 018-5201422, 305I 019-5245422, 305I 020-5102:422, 305I 020-5(021115, 305 022- 513422, 3051 023-5(12422 and 3051 023-5(21115) demonstrated a lower accumulation of C5 in plasma compared to pH-independent antibodies against human C5. 10.3. Measurement of the concentration in plasma of antibodies against human C5 using the assay

EPZA

The concentration of antibodies against human C5 in the plasma of a Javan macaque was measured using EGI5A. The antibody against the kappa chain of human Id (Apiirodu boishiopv) was distributed in a Mips-IttipoRiaie Mahizogr (Made Mips Ipiegpaiopai) tablet and left to stand overnight at 4 "C, resulting in tablets with an immobilized anti-human Id antibody. Samples for the calibration curve were prepared and cynomolgus macaque plasma samples diluted 100-fold or more. The samples were then spread onto plates with immobilized anti-human Id antibody and allowed to stand for one hour at room temperature. Then, HPA-labeled anti-human db antibody (5pHV) was added and allowed to stand for one hour. to react for thirty minutes at room temperature, and washing was carried out. After that, the horseradish peroxidase substrate AUTO EGISA NAR Zybrvigaije (EXCEPT. The signal was measured using a tablet reader at a wavelength of 405 nm.

The concentration of human anti-C5 antibody was calculated based on the reaction according to the calibration curve, using analytical software BHOETtah RO (Moiesshiag YuOemisev).

The temporal dynamics of the concentration of human anti-C5 antibodies in plasma after intravenous administration, measured by this method, is shown in Figure 25. pH-dependent anti-C5 human antibodies (3051 015-52422, 305 015-512:115, 305I 016-505422, 305I 018 -52:422, 3051 019-5103422, 305i020-52:422, 3051020-52(2115, 3051022-5:2422, 3051023-512422 tazo5Ii 023-5:1115) demonstrated a longer half-life compared to pH-independent antibodies amy against human C5 .

Example 11

X-ray analysis of the crystal structure of the Raf variant 305 complex and the human MI C5 domain 11.1. Expression and purification of the human Ma (20-124) C5 domain

The Ma domain (amino acid residues 20-124 5EO and MO: 39), fused to tag 25 by means of a thrombin-cleavable linker (2451-MaI1), was expressed in strain B 21 SEZ of E. soya ryuse (Rgoteda) using the vector raeEX- 4T-I (sbE PNeainsage). Protein expression was induced by 0.1 mM isopropyl-beta-O-i-thiogalactopyranoside (IRTS) at 25 "C for 5 hours.

Bacterial cell debris was lysed using a Vidbrivieg cocktail (MegsK) with added lysonase (MegsK) and a complete cocktail of protease inhibitors ("sotryeie rgoiease ippiriug soskKiai") (Vosneye), and then 45T1-MY1 was purified from the soluble fraction using an azTgar column (CE Neaisage) according to the manufacturer's instructions. The α5T tag was cleaved with thrombin (bidta) and the resulting MO domain was further purified by gel filtration on a Ziregadeh 75 column (CE Neaisage). Fractions containing the MI domain were pooled and stored at - 80 "S.

From 11.2. Obtaining the Raf fragment of variant 305

Bar fragments of one of the optimized variants, derived from 305, were obtained by the traditional method, using limit digestion with papain (Kosne Oiadpozwiisv, Sai

Mo. 1047825) and then loaded onto a Protein A column (Marbiesi 5yye, CE Neainsage) to remove Es fragments, a cation exchange column (Niitar 5P NR, CE Neainsage) and a gel filtration column (Zyregah200 16/60, CE Neainsage). The fractions containing the Rab fragment were combined and stored at -80 °C. 11.3. Preparation of the Rab variant 305 complex and the human MET C5 domain Purified domain

Ma recombinant human C5 was mixed with the purified Rar fragment of variant 305 in a molar ratio of 1:11. The complex was purified by gel filtration chromatography (Zyregdeh200, magnification 10/300, (SE Neeinsage), using a column equilibrated with 20 mM NEREZ pH 7.5, 100 mM Mass. 11.4. Crystallization

The purified complexes were concentrated to approximately 10 mg/ml and crystallization was carried out by the vapor diffusion method in a sitting drop in combination with the seeding method at 4 "C. The reservoir solution consisted of 0.2 M magnesium formate dehydrate, 15.0 95 wt/vol, polyethylene glycol 3350. This turned into flat crystals within a few days. The crystal was immersed in a solution of 0.2 M magnesium formate dehydrate, 25.0 95 w/v, polyethylene glycol 3350, and 20 95 glycerol. 11.5 Data collection and structure determination

The X-ray diffraction data were measured at the output of the VI Z32KHIO irradiation from the source

ZRhipd-8. During irradiation, the crystal was constantly in a stream of nitrogen at -178 °C to maintain a frozen state, and a total of 180 X-ray diffraction images were collected using an МХ-225Н5 ССО detector (VAMOMIKH) connected to the irradiation terminal, while the crystal was rotated by 1, 0 degrees at a time.Determination of cell parameters, indexing of diffraction spots and processing of diffraction data obtained from diffraction images were performed using the program Xia2 (U. Arry. Stuvi. 43:186-190 (2010), XO5 RasKkade (Asta. Stuvi . О66:125-132 (2010)) and b5saia (Asia. Stuvi. О62:72-82 (2006)) and eventually obtained diffraction intensity data up to 2.11 angstroms of resolution. Statistical crystallographic data are shown in Table 11.

Table 11

A set of X-ray analysis data and matching characteristics

Elementary cells 11111111

Completeness (shell of the highest resolution) (95)

Vteyueg (the envelope of the highest resolution) (95) (The root mean square deviation from the ideald/-/:/ | (7777771

Length of bonds (A) a) Vteshe - UPKIHI | (PK) - "1 (yK)" T/xAKIx)Y I) (AK), where Ts(NKI) and "I (NKI)) are measurement intensity | and average intensity for the reflex with PCI indices, respectively.

P) B-factor - UPKI | Esais (PKI) |- | s (KI) І/HAKII s (AK)!І, де се and Esas are the amplitudes of the structure factor, which are observed and calculated, respectively. c) No. It is calculated with 5 95 deviation of a randomly given reflex.

The structure was determined by molecular replacement using the RNaseg program (9. Arri. Stguvi. 40:658-674 (2007)). The research model of the Rab domain was obtained from the published crystal structure of human Rab Ids4 (ROV code: ІВВ)), and the research model of the MS 1 domain was derived from the published crystal structure of human C5 (ROV code: ЗСЙ7, MaiIttipoi. 9:753-760 (2008) ). The model was built using the Sooi program (Asia Stuzi. 066:486-501 (2010)) and adjusted using the Neitaso program (Asia Stgubi. 0O67:355-367 (2011),

The crystallographic reliability factor (A) for different diffraction intensity data from 25 to 2.11 angstroms was 20.42 9o with the value of the free H-factor at 26.44 to. Characteristics of structure coordination are given in Table 11. 11.6. General structure of the Rab variant 305 complex and the C5-MOI domain

The Bar fragment of the optimized variant ("305 Rar") derived from 305 bound to the human C5-M(1 domain ("MO1") at a ratio of 1:11 and the asymmetric unit of the crystal structure contained two complexes, Molecules 1 and 2 , as depicted in Figure 26A. Molecules 1 and 2 can be mutually oriented appropriately at 0.717 angstroms of BM50 using the position of the C-alpha atom in all residues, as shown in Figure 268. The figures discussed below were obtained using Molecule 1.

In Figures 27A and 27B, the contact site epitope of Bar 305 is mapped in the MC1 amino acid sequence and crystal structure, respectively. The epitope includes amino acid residues

MEs that contain one or more atoms located within 4.5 angstroms of the Rab 305 moiety in the crystal structure. In addition, an epitope within 3.0 angstroms is highlighted on

Figure 27A. 11.7. Interactions of E48, 051 and KT109

As described in Examples 4.5 and 4.6, the anti-C5 mAbs, which include a series derived from antibody 305, were tested for binding to triome point mutants of human C5 - E48A,

О51А and КИ0О9А - with the help of binding analyzes using western blotting and

VIASOVE (registered trademark). While the 305 variants bound strongly to native C5, they bound only weakly to the C5 E48A mutant and did not bind to

With OB5BIA and КИ0О9А mutants. The crystal structure of the complex of Par305 and MI1 revealed that three amino acids: E48, 051, and K109 are all within 3.0 angstroms of Par305, forming a series of hydrogen bonds with Bar, as shown in Figure 28A. Upon closer examination, the KT09 MO1 residue sinks into the groove formed on the Rab heavy chain interaction surface and tightly interacts with Rab via three hydrogen bonds with H-SONZOIZ97, H-SOVNZM100 and H-SOVZ T1005 and via a salt bridge with NNSONZOU5 (Figure 280). 051 is located between MC1 and the Rabz3o5 heavy chain and forms two hydrogen bonds with H-SOBI beg32 and H-SOBI 5eg54, filling the space (Figure 28C). This indicates that both K109 and 051 of C5 are crucial residues for the binding of the 305 series antibodies. On the other hand, E48 is located closer to the surface and forms only one hydrogen bond with bab, suggesting that its the contribution to antibody binding will be smaller than the contribution of K109 and

O51 (Figure 288). These relationships correspond to the results of Western blotting and VIASOVE (registered trademark) binding analyzes of human C5 mutants (Examples 4.5 and 4.6). Additional Note: Residue numbering for bar amino acids is based on the Cabot nomenclature scheme. (KabBbai essays aY., Zedyepsez oi Rgoivip5 oi Ittipoiodisai!

Iptegevi, IP Ed. Ribiis Neatsi Z5eguise, Maiopai! Ipviyshez oi Neainy, VeShezaa, Ma., 1991). 11.8. Interactions of human H7O, H72 and NI 10 C5 and antibodies of the 305 series

Analysis of the crystal structure revealed that three histidine residues in human C5, namely H7O, H72 and NI 10, are included in the Rab epitope of variants 305, as shown in Figure 27A and Figure 29A. A VIASONVE (registered trademark) binding assay was performed to investigate the contribution of these histidine residues to the pH-dependent protein-protein interaction between human C5 and EPar variants 305 using human C5 mutants: H7OM, H72M, NO TOM and

H7OM--NITOU (Example 4.7). H72U caused a complete loss of binding of Rab variants 305 to C5. This C5 residue is located in the pocket formed by the SOB2 loop of the Rab 305 heavy chain and the Ma loop (I 73, 574, and E76) and tightly fills this space, as shown in Figure 29C. In addition, the H72 C5 residue forms a hydrogen bond with H-"SOV2U58." The H72gU mutation is not expected to be tolerated because there is not enough space to accommodate the bulkier tyrosine side chain. Also, a hydrogen bond with H-SOVN2U58 cannot be maintained. As for the contribution of H7O and NI 10 to the dependence on pH, the dissociation of Rab variants 305 from C5 at pH 5.8 was slowed down due to the mutations of H7OM and NI 10U. H7O forms an intramolecular hydrogen bond of 3 T53 MI, which is believed to break at pH 5.8 when protonation of H70O C5 causes a conformational change in the corresponding part of the MO1 interaction surface (Figure 29B).

Zo It is expected that for NI 10, protonation of this C5 residue will cause charge repulsion from Par 305, which may be enhanced protonation of the adjacent histidine residue H-

SOVZ NTOOs (Figure 290).

Although the above invention has been described in detail by way of illustration and examples to facilitate understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The contents of all patent and scientific literature cited in this description of the invention are specifically incorporated by reference in their entirety.

LIST OF SEQUENCES

"110" CHUGAI SEYAKU KABUSHIKI KAYSYA (SNOSAI 5EIMAKA KAVIZNIKI KAIBNA) "120" ANTIBODIES AGAINST C5 AND METHODS OF APPLICATION "130" C1-АТ16бОБР -1505 UR 2016-120325 -1515" 2016-06-17 - 1605 150 "170" Raiepiip megwiop 3.5 "21051 "2115 121 "2125" RAT 213" Artificial sequence "220" "223" Artificially synthesized sequence "4005 1

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"223" Artificially synthesized sequence "400" 46 "-2105" 47 -21155 "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence "400" 47 "-210" A8 "21156 "212" РВАТ 213 » Artificial sequence "2020" "223" Artificially synthesized sequence "400" 48 "-210" 49 -21155 "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence "400" 49

Shk BZh Zhuzh buk MEZH Avt « k «210» 50 -21155

From "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence "4005" 50 gift already ZVE Has ZVE

ЗB X ш "-2105 51 -21155 "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence "400" 51

Zhve bek Aziv Me BU "-2105 52 -21155 "212" RVAT 213" Artificial sequence "2020" "223" Artificially synthesized sequence

BO "400" 52 mish dav div Ti Av

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2105 53 -21155 "212" RVT 213" Artificial sequence "2020" "223" Artificially synthesized sequence "4005 53 in Zhukh iv Vb o shaEzh "-2105" 54 -21155 "212" RVT 213" Artificial sequence "2020" "223 » Artificially synthesized sequence "400" 54

Bek Bek Tez Fuk Uvi Az u 5 "2105 55 -211517 "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence "4005 55 szha ZA ех ужы бе ве ш B Же бух Жуж div already fev iv Buv

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"-2105 57 -211518 "212" RVT 213" Artificial sequence "2020" "223" Artificially synthesized sequence "4005" 57 mu sSchu "-2105 58

"212" RVAT 213" Artificial sequence "2020" "223" Artificially synthesized sequence "4005" 58

Me yiiz beech and beige ziv Viv TVzh Teh Zhuh Aiv bach zhk iv btv ZKU "2105 59 -211516 "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence "4005" 59

Me Hee Tk here is beige Si" with Jie Bush Beetle Weaved back Tv 5iz There were 5 more "2105" 60 -211516 "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence "400" 60

Khiv iv bve Without SU ZKU der Aiv bek Same visa Bek ho div Was 5 th V 15 135 "-2105" 61 -211516 "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence

From "4005 61

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-2105" 63 -211516 "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence "4005" 63

TVVzh beats Ve" Tezh zi be two Zezh Ve Te iv ayu yur" Aiv Muz SA 3 5 35 i5 "2105" 64 -211517

"212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence "400" 64

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"2105 65 "-2115 13 "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence "4005" 65

Ave ZMU bi Tukh Uvi Zh ho iVBZh Ni div es back bez "2105" 66 "-21159 "212" РVAT 213" Artificial sequence "2020" "223" Artificially synthesized sequence "400" 66 daiyu imma Ni viz Uie ХВх Az divo

X 5 "-2105" 67 "-2115 13 "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence "400" 67

Teh Bo Tv Tezh Chu avr Zi b iv Av Biyukh ayu He -2105" 68 "211511 "212" РVAT 213" Artificial sequence "2020" "223" Artificially synthesized sequence -400" 68 pi be Tukh yeh ZZHU dev ev Zav Avr dva ЮК ии их 10 "2105" 69 "211511 "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence "400" 69

With; bie TU Jacques Bu vrody Az zav oAvu ve "-210" 70 "-211512 "212" RVAT

213" Artificial sequence "223" Artificially synthesized sequence "400" 70

Uvi Bi Viv BU yvv іZe Bve ЖЕр tuyu rve Ber ynshcha "-2105 71 "211511 "212" RVT 213" Artificial sequence "2020" "223" Artificially synthesized sequence "4005" 71 av yAvr shchiU Beh Uvi uz dev bez Be Kam Bez и k 1 "-2105 72 "-21154 "212" RVT 213" Artificial sequence "2020" "223" Artificially synthesized sequence "4005" 72 pages Beh MU Same: и "-2105" 73 "211511 "212" RVT 213" Artificial sequence "2020" "223" Artificially synthesized sequence "400" 73 fany yo Zi Zah Uvi TtUzh bej yin Kia Dek fam ih 5 i6

From "2105" 74 "-2115 13 "212" RVT 213" Artificial sequence "2020" "223" Artificially synthesized sequence "400" 74 vav pi BU TUkh barv bukh Be TV Mi 5iv Mee biv Toj i 5 18 "-2105 75 "211511 "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence "4005" 75 iv 3iz beige Biz Zav bis BMUu yeah ayu without BAV i 5 her "-2105" 76 "211511 "212" RVT 213" Artificial sequence "2020" "223" Artificially synthesized sequence "400" 76 tsii div yek siv Ave y15 Tuye Bach dv yzh aka -2105 77 "-2115 13 "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence "4005" 77 "Ma Jak Beh bi Shah Uvi bu yeEzh Zaye Ave fuk ikh eh "-210" 78 "211511 "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence "400" 78

Shche iz eh za Bej Zhe meh dat Zhre de Va "-210" 79 "211511 "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence "400" 79 pai Aha Shchek si bak iiv Ba dvy Thev femi Ba

ХБ 18 -2105" 80 "211511 "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence "400" 80

ZM viv Beh BM ek viv Tech Bek div ii KE "-210" 81 "211511 "212" RVAT 213" Artificial sequence "2020" "223" Artificially synthesized sequence "400" 81

Av Aa bh BA dai ii Su Bek Viv without bach "-2105" 82 "211511 "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence "400" 82 iv iv bek C3o without ii buk Ves ee Mev BAV -2105" 83 "211511

"212" RBAT 213" Artificial sequence "2020" "223" Artificially synthesized sequence "400" 83 schi Zi Beh 5 zav Her Tezh Beh iv be Zae "-210" 84 "211511 "212" RBAT 213" Artificial sequence "2020" "223" Artificially synthesized sequence "400" 84 semi-beige lines 335 Bek Bezh be Dav -2105" 85 -21157 "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence "4005" 85 grids tank was without viv yve "-2105" 86 -21157 "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence

Zo "400" 86 pi Aiz bek Ashchi Yizha S Mach "-210" 87 -21157 "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence "400" 87 pam v Zak uy be Aa Bach -2105" 88 -21157 "212" RAT 213" Artificial sequence "2020" "223" Artificially synthesized sequence -400" 88 ka piz yeh TBZh vy viv bah i ya -2105" 89 -21157 "212" RAT 213" Artificial sequence "2020" "223" Artificially synthesized sequence

"400" 89th B "210" 90 -21157 "212" RVT 213" Artificial sequence "2020" "223" Artificially synthesized sequence "400" 90

Zak Aiv oh Tu iz liv da yi yi 5 "210" 91 -21157 "212" RVT 213" Artificial sequence "2020" "223" Artificially synthesized sequence "400" 91

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Beetle USSR yiz Zek "Bio TIE biz bek Si Uvi Bu5 Ves Ak BBa Yaeh di

Shock Si YavE Bi TV Aa be TBh Be TV ii bek bej im pav bha s-m vve KV iv ih ftj TUuh was bZb5 bvr) TVzhZ Buv UVK si Bek ek 85 Za 55

Zhuzhh BU yo TBzh BVaM si PTU ZU Zhvh Buv Uvi Be Je duv "2105 113 "2115110 "212" RAT 213" Artificial sequence "220" "223" Artificially synthesized sequence "4005 113 ker Ge Siv Mais TVzhh Bi Beh jo Zah Chek be : back Wiz Back UVU FU

ВВ» dk Uvi Zhve iyi6 Ty Ku Ak Az Kek o sia BiU ie Wes Hek Bek

Ku V 35 eat Vi Zhzhe tuyu shchi ziva eat Bko Z Z div Bo du im bmz Ha 35 40 ak

TUuk ShiU viv Beh TVZHH Tyh Bit eh Bu Uzi Bko Beh Ah Eva byuashje Bu yeh tsi» bek Pizh TBh Ber Bin Hv Zemni Zhe Ves Beh be shcha VKo

In Avr vve hiv TV TUKE bek Sez biv Ave TVh iv Uvi 1 ZE bej "2105 114 "2115 328

"212" RAT 213" Artificial sequence "223" Artificially synthesized sequence "4005" 114 visa Bek YV was Bi Bo Beh Uzi Be BzhZhe imni Azhiya ko Zhvzh Zah tv

M 5 ho ate

Zak Tvh Beh ie BU Zph Aiv Az be: U Su yiv Uvi Buv vyayu Tu that 5 For wa chem Jhe Ho bu that ku b: y ЖШЖжж (i1 ko vuh: ih ku

Ve be Zi BEo 3 TV» Uvi deh eat Vko Zah S3U Wiz without Zhvh Zah it Uvi Niz Tv Be Bho Aiz Uvi im Bib» I Vaj Seven without food ideh

Java Bezh bek Uvi Uvi TvVh Uvi Bu eh bek eh Tceeb Ye ZhMh ah tVE

TUuh HM Suv dvo" Uvi Avv" Viv was Bo Zach" Ave TVU BUz Uzi AvyuYu There was still ZO vozha Uvi iv Be Buz Bach Suz vve obuz TBu iz TVBh Suv bub5 Vko buh vu ХОВ MV

Bko div Bhb Pi ate Ajo Ah Z ko was Ua BVzh ih Be byub Already ate rkb yeshaya yev TV bez MB izz bes Av TBZH Bus sv oUSch TVh Suv

TVI UVA Uvi vav UBE Bek Nizh biz Ave BE Be UVE re Be Ave Zhyu 145 that i55 15

Teh Uvi Ba si Bi from UVI Nv Av div Buv TE Buv ME same m

U. UZH same and that Ii from virgins TU bev Zh BvVEzh Tk Ah UVA Zv. Bek otv ey tekh in Be

Between iv zvo» ThV ih Zav Z Buv BAZ Tuk ta Sta BUV UVBB Beh var scha po zhi food pi Meeb ho bek o bvh Si iz bu TB Hiz Zak obev Wiz was PUYE wai Ka KV piv Bko vka piz Bjo Sv Uvi fuj ЖЖ ema Zhjo Ky ek de zma sha sha KU ZV 40

Mmek tv There were two av UFH Bezh Be Hv Se yin and without SU Be TU ah ak BE

Vys Zhe Av» ii Ave UVI iz TEB ESV Bas wav OYI B Bko Bi two kazh uz TVH TVveE rhe BVkoOo and food dev Bezh dvo" U Bezh ba reme food Teh bah Ba eh Te Vi Bar ee vaj ha t ziv iv Bib ZE

In bbe bek was dec Uvi em Nav o ziv from Sevea Niz Aiv Viv zhTUh TE dka muzh ZAZ dev ke yzhmh Chek KO "2105115 "2115 325 "2125" RAT 213" Artificial sequence "220" "223" Artificially synthesized sequence "4005 115 from bah Tvh Buv PU Bko bej UVO KBBe BZhzhOo ben Viv Kko Suv eh Ak

Zak TV Beh BU LU TBE Az Viz im same was be) Uvi Buv aa» TK tva bus biv Zk Uvi VE Uvi Vviee TzhZhv azy bej SPU Aiv be JE BE

I ao 35 pis uzi Between Te be VEOo Aiv U i i" bek bek fu Ba uh Yavke x M skh I "i xx g ch dm , SYU i "Hu ХЖухх SU. -kh still zhk I BU - same - - Fk Ek zhk u u HU eh A to Ve khz bud K. U YAZ u iizhka Zak Bezh U vi Teh Uvi Bko eh beh beh t

That's M

In 2 Zh ol

NU t aa y Zach dav You were in Uvi Bayu fu

Mk U ku Che Nav bu bo Zah ab TV Buv Uzi Ayayu i5

Tuk Tvzh Su Avyu UZVZH Avyu Nav buh BKO Cheh dat U se yah ha Z Su zho bko Su he iz he sha ak mu Zhuk U Bzhe Bo Ku zho ko was Sk Aa Z

Ii Uvi BYU Be Bush shk shk Zhe ke Ku ih 115

MO 05 KA

Ua v se be BE BJ Mu Bo uv shM Azh Ak shu Bko ze Uvi BBe yin Bbe Be BE was Bo iz ma Kinz aka dha Mu Ba without Uvi Be fa 5 food h i y s yOZHU M her MV

Z kh a me zhvzh puh va a Uzh

NE Zhe V z Beh dk Tv BEO iv Uvi tazh SU UVA ZRK

Bar ZHE Ken Web Bi Beh dk tvE VO SHO yiv y Horrors y Z ih i 4 ih KZ - g dk p so ZHUM o y zh du ykuk v Dakh

U zak than siy Za BhEye Bach Uvi ZMU Ki dev Жжв buk Uzh E ee UA Bach Ni ZA Zv KE Be UZ u a v 345 Mokh z oz TK vk yirsguzhh ) M food Already Bk pub z zaz Uh! ti I KA Zi o: ze TE Uh zha ZK xx DALA OK Z

ZM Uvi Bi UMA Mi zvo da Buzh VE Mu Z styu 65 i73 175

Uvi bi Wa UBi fev tn UKH Zi Mav i Ave ag dec tTvE Uh vie AH A OH Ua i pe 53y iv ii

But in 1885, Uvi Zhe wav was viure re yah zha SK ah ak kah do chu baka hi Still. Va vyv Z y

Thv Za av SU BUV siv ZHUZH Bu Sbuzh was UVI eh zav a ;

What What | " ay "M her eV zhav : Zh ia 1 eh. Genre y. z. be Tykh kiv Bek be AK pe A KOZH BE KK bBuo Bezh Beh beie vu TVzhh biv Bek was AiV Tu vosuzh sha ke

IKK AK O ZK. Z Z peak not zi shchi Kz х я ж х ай LK he Хих хх ); : fo Жжж Bk iz Siv MeBe TE Buv s Vu S bi tTuzh ЩЖХВЖ Sa Bjo Bo yvke Already ish Zv Ye U schiv Vhe baz va uzh ЖЖ Sem; Already I va lyh 7 Z i Ka 35 zh x x z Zzhanukh Zhukukh; : ; and was HU kra tTuk Bzha ek Avr

Z a Uvi Bek be zahh SUB yin) Uzi buk she MBBae TUYU BKO ba wat oshchav Uvi Vek iyi bah Sta dey Vi vu z MU Yaeh ah what 55 : ke BM Ko zhi wav na BUZH Buv i ia UVI Ma tuyu s) Ж vv ke shchi vho yib ar ba bUZH yiv

Хіие вів UVI 1 tuyu s azh Za і TU Z a і Оі oko vy 55

A. zhk ko ze Kol shh gMikh tek Yake.

Yi Bho UVi food dar Beh zav Bi shShezh be bez iyem Hu;

Zzhe Zhe Kjo WEB UVA ia ayu Zach Aschro syu Zhe Z se

ZV vo ZE shUV M ikh t su s SU: ziv piz Bi gave Za bi I; 5 Uvi bayu Se bas BkU Tyuz «iv piz Bi ba ota Ve Ve

Eat for ЖЖ УВи бяю бек лка тюрошХ вому ух; niv bok ve vVut ev ShShShiV

Uvi iv a iv Z Ni A iv bek Sche du be

Kev Zah Uvi them. Ni siy Bi zy Niv lsh A hv Ku Z ze still yiv Zh zeO 305 am Zh ee bek ii: Zach Beo

ZV

"2115 325 "212" RAT 213" Artificial sequence "220" "223" Artificially synthesized sequence "4005 116 iv dec bTve dev si KE eh FaE VVe KO yin biz bue su VEVZHE dih y 5 her VE

Chek ZVzh bek SHU Bi TB div Az bo SAU Suz be) Uvi Muz Ar Tush o a ZY

Be Me sa be Uvi Tv Uz eh ber yav Bek siyu Av wife ZHE EE ke Ko 45 it Uvi Mi Zhe Be Bjo Zi Uvi be Siyu bek bek ziv ben tyu eh that 55 BO yiv bah Zak Zi Uzi TBJ Sv BzhZho bah bah yeh izh BBZ is available in Zhv

BE to Ya that bUKK Tvh Suk Be" ZV Avyu Me Mus Bkb bej dav LJh was TVi dav Buz in Z 5 j UVI "Kiz bah be bek shchi Bo Be Sv ho BEOo Kuv ShKO Av Be i95 MOV ke shchi she dka khz ZU BO I had to kill them, I was, I was, I was, I was 15, that their words are without; Mezh iv Beh je Tv ho biz Uvi Taj Sv UVi Uzi U

AO i38 148 yer Uzi Bek Miv BM Ar rho 3: Uzi Huk BV Az tzhr tuye UZ Avyu i45 her 155 ivo

With Uvi under Uvi Nivz Be iv was Te without BE As with BK Se tUuh 65 for 175

Bayu bah Х»h Tukh dk U Uvi Vazh Uvi eat Shchy Ж zhen Mav ayu Bru ich 155 Aa

Zhiko be: zv» bi yiv Za buh ba Su ev OVA dek ee Muz ku Mem

October 5

Bje Bej ek zhi zi Mu ZOV iie vezh was ziv Buv zhe pav Be We mich Be siv UVE buk Tv beo be Vko bek Ah iz z Mei zhvzh bukh wav siu Zi Hek Zam3 Tri Suv food Uvi be paU BKbe Zhuhyu Bko Yavvzh still hiv av Uvi bla eat ich bek vav pit Ba Already 023 Av ba» Zhuzh eat

Tvzh ZnE Bjo Beb Uvi May bav bah Zv se Vas Vne BOB without TuzhJ Wes dkch and Zah Uvi bvyue was Shah Azh byu Ak Zi U Av) MV Bba Yanzhe su eh Uvi without av chiv Azv without Na Aiz Na Tuzh TV»E Ak Buv yes

ZOV Z ZE ZO ii eh food bek BE

ZV

"2105 117 "-21156 "2125" RAT 213" Artificial sequence "220" "223" Artificially synthesized sequence "4005 117 "2105 118 "211517 "2125" RAT 213" Artificial sequence "220" "223" Artificially synthesized sequence "4005 » 118 visa ii Be TB bi bek bi iv biz tTuk muz MIV a zho Abe was ska

AH

"2105" 119 "211517 "2125" RAT 213" Artificial sequence "220" "223" Artificially synthesized sequence "400" 119

Moscow State University

"2105" 120 -211517 "212" RVT 213" Artificial sequence "2020" "223" Artificially synthesized sequence "400" 120

Shchi Zhiye Ri" Tv BU Zek (ii Aza TIE Zhe Was led by izh ber iv Was under "2105 121 "-2115 13 "212" RVT 213" Artificial sequence "2020" "223" Artificially synthesized sequence "400" 121

Headed SU Tech Ave o buzh bo TvVh Niv Biz Me Miv Tuzh "2105" 122 "211511 "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence "400" 122 huaziv bek iv Mu i3ye Zve bej bek beb div 3 5 i6 "2105" 123 -21157 "212" RVT 213" Artificial sequence "2020" "223" Artificially synthesized sequence "4005 123 3 in "210" 124 -21157 "212" RVT 213" Artificial sequence "2020" "223" Artificially synthesized sequence "400" 124 mi Viv ZezhE yTVh iBEzh Bi Zach "-2105 125 "-211512 "212" RVT

213" Artificial sequence "223" Artificially synthesized sequence "4005" 125 with yavo iBE Bevs UVU) bi VvE Bek Tek Bi without ЖЕ "2105" 126 -21155 "212" RVT 213" Artificial sequence "2020" "223" Artificially synthesized sequence "2020" "221" another sign "222" (5)...(5) "223" Haa is Mei or Mai "2020" "221" another sign "222" (6)...(6) "223 » Haa is Suz or Aa "4005 126 "-2105 127 -211517 "212" RVT 213" Artificial sequence "2020" "223" Artificially synthesized sequence "2020" "221" other feature

From "222" (1)...(1) "223" Haa is Suv, Aa or Spu "2020" "221" another sign "222" (3)... (3) "223" Haa is Tug or Rne "2020" "221" other sign "222" (9)...(9) "223" Haa is PTng, A5r or Siy "2020" "221" other sign "222" (11)...411) "223" Haa is Tug, I uz or SiIp "2020" "221" another sign "222" (13)...(13) "223" Haa is 5eg, Azr or Si "2020" "221" another sign " 222" (15)...(15) "223" Haa is Aa or Mai "4005" 127

Hva Kiz Hza TV ZMU Beh pi bizh He bok Hv5 diz Ha biv Ha be

"2105" 128 "-2115 13 "212" RVT 213" Artificial sequence "2020" "223" Artificially synthesized sequence "2020" "221" other sign "222" (2)... (2) "223" Haa is Siu or Aia "2020" "221" another sign "222" (5)...(5) "223" Haa is Mai, Sip or A5r "2020" "221" another sign "222" (6)... (6) "223" Haa is Tng or Tug "2020" "221" another sign "222" (12)... (12) "223" Haa is Tug or Nib "2020" "221" another sign "222" (13)...(13) "223" Haa is Yi Yei or Tug "4005" 128 dav Khaz Zi Tuh Hav ZHva Be ZHVB" Viv Aiv Meb Hva Hva "210" 129 "211511 "212" RVT 213" Artificial sequence "2020" "223" Artificially synthesized sequence "2020" "221" other feature "222" (1)...(1) "223" Haa is Sip or Aha "2020" "221" other feature "222" (5 )...(5) "223" Haa is Aup, Sip or Spu "2020" "221" another sign "222" (1)... (1) "223" Haa is Siu or 5eg "2020" "221 » another sign "222" (9)... (9) "223" Haa is Avr, I uz or 5eg "400" 129

ZHav Aziv Zek iv Hav He Hva Bek Haz food iv "-2105 130 -21157 "212" RVT 213" Artificial sequence "2020"

"223" Artificially synthesized sequence "221" another sign "222" (4)...(4) "223" Haa is I uz, Sim or TNg "2020" "221" another sign "222" (5).. .(5) "223" Haa is I ey or TNg "2020" "221" other sign "222" (6)...(6) "223" Haa is Aa, Niz, Siy or Sip "4005 130

Kya Z "2105 131 "-211512 "212" RVT 213" Artificial sequence "2020" "223" Artificially synthesized sequence "2020" "221" other sign "222" (2)... (2) "223" Haa is Zeg, Suz, Abvp or TNg "2020" "221" other sign "222" (4)... (4) "223" Haa is RNe or I uz "2020" "221" other sign "222" (12) ... (12) "223" Haa is Aa, TNg or Nib "4005 131 "iv Hav yTvE Ha Uvi Bit eh bek buh zZMt ee kah "2105132 "-2115 30 "212" RVT 213" Artificial sequence "2020" " 223" Artificially synthesized sequence "4005 132 ziv Uvi iv be) Vi (Zo Bek it ze sizhZ fev Uvi sv Bzheo st u

ZHE zm: without them she St Aiz div yvBzh ZEe Be Te dvE Viv -2105 133 "-2115 30 "212" RVT 213" Artificial sequence "2020" "223" Artificially synthesized sequence

BO "400" 133 neck USchi zZib Zenh Ua sha) ek ope 3 SAbZH Z Uvi piz Yezhhe si ZE bej Bev bko fam back beige Div Div ek sbizh kre Zhe Uvi Viv "2105 134 "-2115 30 "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence "4005 134 sh-in

Saved: Azh bdemo beige St viv from Beh 03Uu Re Jak va iv "2105 135 "-2115 14 "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence "4005 135 bkr o Uzi Ah biv iv Bho B3u Was BU without m Zhyu UVHU ku "2105" 136 "-2115 14 "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence "4005 136 them" Uvi Ah iya vla Bee shchik was 019 Zh Se Tv: May Ah

Х В 18 " "-2105 137 "-2115 32 "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence "400" 137 dez Be Zhe Zhe already Beh beyu Tvh Bekh ate ba piv Uvi Uvi Hei ZBE

Ve TMZH Aa Me zvo Bk UVI ev TV Aiz Tyh be Yeek Su div Va m zha Za "2105" 138 "-2115 32 "212" РVAT 213" Artificial sequence "2020" "223" Artificially synthesized sequence "4005 138 dka UZA TvkE yiiz bek Was" Asia Styzh Zak was Av Siv Uvi Uvi eat UVe

Her V ha i5

Mei tah dai Mei barv zho tva Avyu Ta Aia Zah Teyu Zhuk Seh Aa Ve "2105" 139 "-2115 32 "212" РВАТ 213" Artificial sequence "220" "223" Artificially synthesized sequence "4005" 139 aka Be Twe Hle bek Je Ber; the same Bek mue Leh St emo bukh feyu Ma

Mei zvy Beh Le aka Az shSchiv Avo TVh Aiiv Uvi Tk Teh Sez Aiz YavE 2105" 140 "211511 "212" РВАТ 213" Artificial sequence "220" "223" Artificially synthesized sequence "400" 140 zhk Si sv SKU xx iz; BE Tech UBi yo deEzhh "2105 141 "211511 "212" RVAT 213" Artificial sequence "220" "223" Artificially synthesized sequence "4005 141 tzhv pit DkU azh ЖVzh Ze Uvi Tyuk UVA Zak Zah "2105 142 "2115 23 "212" РВАТ 213" Artificial sequence "220" "223" Artificially synthesized sequence "4005 142 av UVK UVA Borders of TVh ziv Vaj ko be Bezh y Zah from towers Uzi sat vka UVA ZHBE Mi TV St 2105" 143 "2115 23 "212" RVAT 213" Artificial sequence "220" "223" Artificially synthesized sequence "400" 143 yve khiv siv Mach TB 0355 beige ro back beige Beb Veye div beige Uvi sh

XE 5 is 5

Zav aa Uvi TV also ЖЖ Suk "2105" 144 -211515 "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence "400" 144

Zhkv zhu iv Z Buv Bko (3 Z Aiv Bjo Ak ymiz va Zivz Teh h o t g xx

Ж сх TO их "210" 145 -211515 "212" РВАТ 213" Artificial sequence "220" "223" Artificially synthesized sequence "400" 145 ижхв тех 50 пив бета Bjo cm was div WITH Іні ІХ Хе тех "2105" 146 "-2115 32 "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence "4005" 146 pib ovi Bo bach he BBe bve siyu bek slu Bek slu TVzhЖ bayu Ve TE

Men TB iv yeh Zah Se) biv ho s3z Ave Uzi Afv Be TUh TUuh Suv "-2105" 147 "-2115 32 "212" РВАТ 213" Artificial sequence "2020" "223" Artificially synthesized sequence "400" 147

In stvi BEz bek Ake bob beige Bi bek Bi ek ZU Tk Avyuv be TBZH

Meni fnzhЖ biz Hek bek yin iv izho sib Vayu Be Aiv TvVE zh u buk SUB -2105" 148 -211510 "212" РВАТ 213" Artificial sequence "220" "223" Artificially synthesized sequence "400" 148 re slub schu Tv be Uvi For C without

"2105" 149 "2115 448 "212" RAT 213" Artificial sequence "220" "223" Artificially synthesized sequence "400" 149

Mic Ua S fei Uzhi Sv ek SU biz sa Uvi SU yiv Beka Biu Viv i 5 10 i15 bah UVA uz vi Bek Seuv be Ai yezh Bi» Miie Kiyuv Zak Lut TE

Fk Z o ko NO o t what am I going W o ke oh tu M dzhua ha bkev iv siv Tv UVA Ahe BEZ Aiv Bzhe B» iy pi fen ZRO Zhjue Maye pazh a iii Zm Bko ZKU bah z Viv Tvh ZA fuh Tvj smoh Maye Broch food AB je UVM Zhne Me Te Aka Bo Zhe o Yaeach ЖЖ Zek TV U TU

What about TV?

Men sa bek) eh bek im AkZ Yaeh biz der TvE Aiv Uvi Zheh Tukh SU kiv ze tTuk Be Be Bi Zak bej buye ze Te tu bej Ber UA cr yiv is05 ha pit biv SBU ZhVh bes VE ZhVh Uvi Veve Hek Aiv Bek Tv Zre MU Vo

Zak Zv Be ko bez div Bzhz Sv bej Asz beh TKU bek biz Zaye Zhe iv Az bo bik Suv zi Ua muz AvroTuh Be Buo Ziz zhe mi shvah 145 vo 155 io

U goes ate Av bek PU Aiz food TE bek Sbyu Uzi Niv Lives Wa zho

Te kN i75 from Uvi ik: SM bek Bek SU with Tek eh and bek Beh Uvi in TV

What about M?

UM Ve Vazh bek dev Be Sie Tv osiv TVh TUue TvBE Su Vvv Ua yo

Viv tsu Web Zhek baz Tezh fe Uvi Zhvr Was Te UZi bi BO tsu Sus stv Uvi piz was ko Byuz was BE Azv o bko Bho Uvi Wiz Ki Byuzh5 yvzhkh tUSchi bibe ie: Me Bo already Was Already Vzhe Buz ZE ЖЖ Without MeB iiv Bezh xx

ЖХБх Bu y: UZi Жвх Suz Uzi) U Uzi dr uv eh Bib (a bvyu bo shchi UVvE iv Be avy izh buye UVU Vaz" Bazh UaЬ shi Uvi Mivz Ave Aza

Muz TNzh Was without Vku from Shchi) 33 Ba avy bej TBE ZHUuzh dk Uvi Uvi

U su xxx : u. Horror hears g shu In tku Koh 1 food breath iZ tuya

Bezh Uzi ini) ЖЖ UZVE bevy bav zav dayu Ter o beo Vaz zu muzh sa ZheZ shcha ZO ZV Zo ate Su ide UVi ek Avy was ZALU yzma Bo bek Me iii BM was ZV za5 Z ZB

Ha yyuh was Viz Buv BU Siv bu" Ah sv Bu bav UVU feuj ZE Ha

Za Zav aa shko Kho VvE bi py ba Met ZV: Mus Azh b v Bej zey ХВ SzhV

ЗВ зво ЗВ ее ! y - su that g - duhy s ke Fr m s R uya y so u i h izhv Uvi ta Pit be TuUh Vko Vevh byu Siv Aza Uzi Ziyu Tr iz Bach

From In ZO

Ma pit bib Be 010 wav Ava buh Was Tvj Te yho Already Uvi im ber

Shchezhh Ark ku feyu Riv Se b Tuk bek Azhe ee Tve UVI dav izhv eh 430 y dEU TezhEv shi biz ZKU Av» UVA Ka Yavk Suk bek Uzi izh Mi Viv div hu dv ko za5 ZO am Ni es Ni buye Tah AB uv bek izhnh Bezh ema Jaazh Zam) Phi uv 5 "2105" 150 "2115 214 "2125" RAT 213" Artificial sequence "220" "223" Artificially synthesized sequence "4005 150

Beyu Se yo" Meb Teh biv Chek Bje bek Yaeh food bah biz eh Uva Mor devyu o aha Uvi SCHE Siv He Se si wav Yavy (32 dez 3 TU Bi" AiV che ate zo

Bach dai ihv buk Bib Siv buye Bk Sye Buv Aiz bko Buv im: Be Hv z

TbFk BU Aa Tk Av beyeb called Anya BBe Uvi ho meh Bk Be bek Su ya HE BO.

Zak ZKU Bach BKU TVU KvyUyu BVe TV Her TVe Kiz eh Vezh ma B vu" sing vayu Be viv TV Teh TUh Suvzv iz bab Uvi Bei av Zhvh byu Tema ve vo Za

Жве Брж осие з S.U жвж иж Uvi B ЖИ бул uz TB sbvi Aiv AV tkKko bek Uvi be Tib Be Be ho bej Avr o biv Za Buv bach 3

Zvh div dec bvi Uvi Sv ba iz; Ashi ba SB o bTuzh Bi AkU BMW Az eat UVA bi Prison Buz svi Bvr» ba» Az beo siyu bek emu fam Bezh a ie bak ti TVkK ZO Zi ber bach was Ave bek ozvzh buk fej em azh her WHO 175

Yaavk TVzhh without TV Z bek was Div dvo: o Tuye biv was Viv Buv UVA uk

Viz SUB BM In TV Viz sat down BU Zam Bek dah There was already a tank in LZH

BBe gave o Azhe ZU you Szhe ee

Claims (5)

FORMULA OF THE INVENTION 1. A pharmaceutical composition for the treatment of a complement-mediated disease or condition involving excessive or uncontrolled activation of C5, which contains an anti-C5 antibody as an active ingredient, characterized in that the antibody includes: (a) sequence of MN according to 5EO IYU MO: 106 and sequence of MI. for 5EO IYUO MO: 111; (p) sequence of MN according to 5EO and MO: 107 and sequence of MI. for 5EO IYUO MO: 111; (c) sequence of MN according to 5EO IYU MO: 108 and sequence of MI. for 5EO IYUO MO: 111; (4) sequence of MN according to 5EO IYU MO: 109 and sequence of MI. for 5EO IYUO MO: 111; (e) sequence of MH for 5EO IU MO: 109 and sequence of MI for 5EO IU MO: 112; () sequence of MH according to ZEO IYUO MO: 109 and sequence of MI according to ZEO IYUO MO: 113, or (9) sequence of MH according to 5EO IYU MO: 110 and sequence of MI. according to 5EO IJU MO: 113, and that the complement-mediated disease or condition involving excessive or uncontrolled activation of C5 is selected from the group consisting of: rheumatoid arthritis (RA); systemic lupus erythematosus (5IE); lupus nephritis; ischemic reperfusion injury (IR); asthma; paroxysmal nocturnal hemoglobinuria (PMH); hemolytic uremic syndrome (NI5B); atypical hemolytic-uremic syndrome (anise); dense sediment diseases (000); opticomyelitis (MMO); multifocal motor neuropathy (MMM); multiple sclerosis (M5); systemic sclerosis; macular degeneration; age-related macular degeneration (AMD); syndrome of hemolysis, increased activity of liver enzymes and thrombocytopenia (NEGI R); thrombotic thrombocytopenic purpura (TTP); spontaneous fetal loss; epidermolysis bullosa; recurrent fetal loss; preeclampsia; brain injury; severe myasthenia gravis; cold agglutination diseases; Segren's syndrome; dermatomyositis; bullous pemphigoid; phototoxic reaction; hemolytic uremic syndrome associated with Shiga E. soy toxin; typical or infectious hemolytic uremic syndrome (HUS); glomerulonephritis C3; vasculitis associated with antineutrophil cytoplasmic antibodies (AMSA); humoral and vascular graft rejection; antibody-mediated acute rejection (AKA); dysfunctions of transplantation; myocardial infarction; allogeneic transplant; sepsis; ischemic heart disease; congenital angioedema; dermatomyositis; Graves' disease; atherosclerosis; Alzheimer's disease (AD); Huntington's disease; Creutzfeldt-Jakob disease; Parkinson's disease; various types of cancer; wound; septic shock; damage to the spinal cord; uveitis; diabetes-related eye diseases; retrolental fibroplasia; glomerulonephritis; membranous nephritis; IdA-nephropathy; acute respiratory failure syndrome in adults (AKO5B); From chronic obstructive pulmonary disease (COPD); cystic fibrosis; hemolytic anemia; paroxysmal cold hemoglobinuria; anaphylactic shock; allergies; osteoporosis; osteoarthritis; autoimmune thyroiditis; type I diabetes! psoriasis; ordinary blister; autoimmune hemolytic anemia (AINA); idiopathic thrombocytopenic purpura (ITP); Goodpasture's syndrome; Degos disease; antiphospholipid syndrome (AR); catastrophic ARZ (SAR5B); cardiovascular disorder; myocarditis; cerebrovascular disorder; disorder of peripheral vessels; vasorenal disorder; mesenteric or enteric vascular disorder; vasculitis; Schenlein-Henoch's purpura of nephritis; Takayasu's disease; dilated cardiomyopathy; diabetic angiopathy; Kawasaki disease (arthritis); venous gas embolism (VGE); restenosis after stent placement; rotational atherectomy; membranous nephropathy; Guillain-Barré syndrome (585); Fisher's syndrome; antigen-induced arthritis; synovial inflammation; viral infections; bacterial infections; fungal infections and damage resulting from myocardial infarction, artificial blood circulation and hemodialysis. 2. Pharmaceutical composition according to claim 1, which is characterized by the fact that the antibody includes a constant region of the heavy chain, which includes the amino acid sequence according to 5EO IJU MO: 114, and a constant region of the light chain, which includes the amino acid sequence according to 5EO IJU MO: 38. 3. Pharmaceutical composition according to claim 1 or 2, which is characterized by the fact that the antibody is a full-length IDS1 antibody or a full-length Idsai antibody. 4. Use of an antibody against C5, which includes: BO (a) sequence of MH according to 5EO IU MO: 106 and sequence of MI. for 5EO IYUO MO: 111; (p) sequence of MH for 5EO IU MO: 107 and sequence of MI for 5EO IU MO: 111; (c) sequence of MN according to 5EO IYU MO: 108 and sequence of MI. for 5EO IYUO MO: 111; (4) sequence of MN according to 5EO IYU MO: 109 and sequence of MI. for 5EO IYUO MO: 111; (is) sequence of MN according to 5EO IYUO MO: 109 and sequence of MI. according to BEO IU MO: 112; () sequence of MH according to ZEO IYUO MO: 109 and sequence of MI according to ZEO IYUO MO: 113, or (9) sequence of MH according to 5EO IYU MO: 110 and sequence of MI. under 5EO IJU MO: 113, as an active ingredient of a pharmaceutical composition for the treatment of a complement-mediated disease or condition involving excessive or uncontrolled activation of C5 and which are selected from the group consisting of: rheumatoid arthritis (RA); systemic lupus erythematosus (SLE); lupus nephritis; ischemic reperfusion injury (IKI); asthma; paroxysmal nocturnal hemoglobinuria (PMH); hemolytic uremic syndrome (HUS); atypical hemolytic-uremic syndrome (aNI5Z); diseases of dense sediment (0OB); opticomyelitis (MMO); multifocal motor neuropathy (MMM); multiple sclerosis (M5); systemic sclerosis; macular degeneration; age-related macular degeneration (AMD); syndrome of hemolysis, increased activity of liver enzymes and thrombocytopenia (NEGI R); thrombotic thrombocytopenic purpura (TTP); spontaneous fetal loss; epidermolysis bullosa; recurrent fetal loss; preeclampsia; brain injury; severe myasthenia gravis; cold agglutination diseases; Segren's syndrome; dermatomyositis; bullous pemphigoid; phototoxic reaction; hemolytic uremic syndrome associated with Shiga E. soy toxin; typical or infectious hemolytic uremic syndrome (HUS); glomerulonephritis C3; vasculitis associated with antineutrophil cytoplasmic antibodies (AMSA); humoral and vascular graft rejection; antibody-mediated acute rejection (AKA); dysfunctions of transplantation; myocardial infarction; allogeneic transplant; sepsis; ischemic heart disease; congenital angioedema; dermatomyositis; Graves' disease; atherosclerosis; Alzheimer's disease (AD); Huntington's disease; Creutzfeldt-Jakob disease; Parkinson's disease; various types of cancer; wound; septic shock; damage to the spinal cord; uveitis; diabetes-related eye diseases; retrolental fibroplasia; glomerulonephritis; membranous nephritis; IdA-nephropathy; acute respiratory failure syndrome in adults (AKO5B); chronic obstructive pulmonary disease (COPD); cystic fibrosis; hemolytic anemia; paroxysmal cold hemoglobinuria; anaphylactic shock; allergies; osteoporosis; osteoarthritis; autoimmune thyroiditis; type I diabetes; psoriasis; ordinary blister; autoimmune hemolytic anemia (AINA); idiopathic thrombocytopenic purpura (ITP); Goodpasture's syndrome; Degos disease; antiphospholipid syndrome (ARB); catastrophic ARB (SARS); cardiovascular disorder; myocarditis; cerebrovascular disorder; disorder of peripheral vessels; vasorenal disorder; mesenteric or enteric vascular disorder; vasculitis; Schenlein-Henoch's purpura of nephritis; Takayasu's disease; dilated cardiomyopathy; diabetic angiopathy; Kawasaki disease (arthritis); venous gas embolism (VGE); restenosis after stent placement; rotational atherectomy; membranous nephropathy; From Guillain-Barré syndrome (585); Fisher's syndrome; antigen-induced arthritis; synovial inflammation; viral infections; bacterial infections; fungal infections and damage resulting from myocardial infarction, artificial blood circulation and hemodialysis. 5. Application according to claim 4, which differs in that the antibody includes a constant section of the heavy chain, which includes the amino acid sequence according to 5EO IU MO: 114, and a constant section of the light chain, which includes the amino acid sequence according to zEO IU MO: 38. 6. Application according to claim 4 or 5, which differs in that the antibody is a full-length antibody 901 or a full-length antibody Ide. it is VIM pak KAN KIM pi Iy i NyKmyK s u Ya ha POMELU KIM MIK UK KY CHA FORKS WE Sya AH KAMENEM MECCA MN A PI M KE NV MK HV PEV nt : I I 15 AVNONVI NIM ME t missed memes HTZ OTITI KE KE I Men are certain KU VO NK MIV I V yan: MAK ZA IUH OKO VA A MAK KM KIM UK MY ih fe abyss: ZO AVK VA KN Ki NI t ORANKY: MY KI WOVK BEK NEON KN V V KIV NA VI PI MOM KKD nention Ku OV VV V CI VEN EE ? mi adeststedh. 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