KR102226975B1 - Anti-c5 antibodies and methods of use - Google Patents

Abstract

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

Description

Anti-C5 antibody and method of use {ANTI-C5 ANTIBODIES AND METHODS OF USE}

The present invention relates to anti-C5 antibodies and methods of using the same.

The complement system plays a central role in the elimination of immune complexes and in the immune response to infectious agents, foreign antigens, virus-infected cells and tumor cells. There are about 25 to 30 complement proteins, found as a complex collection of plasma proteins and membrane cofactors. The complement components achieve their immune defense function by interacting in a series of complex enzymatic cleavage and membrane binding reactions. The resulting complement chain reaction leads to the production of products with opsonic, immunomodulatory and cytolytic functions.

Currently, it is widely accepted that the complement system can be activated through three distinct pathways: the classical pathway, the lectin pathway, and the alternative pathway. These pathways share many components, which differ in their early stages, but converge and share the same terminal complement components (C5 to C9) responsible for activation and destruction of target cells.

Classical pathways are normally activated by the formation of antigen-antibody complexes. Independently, the first step in activation of the lectin pathway is the binding of specific lectins such as mannan-binding lectin (MBL), H-ficoline, M-ficoline, L-ficoline and C-type lectin CL-11. In contrast, alternative pathways spontaneously generate low levels of metabolic activation, which activation can easily be augmented on foreign or other abnormal surfaces (bacteria, yeast, viral infected cells or damaged tissue). These pathways converge at the point where complement component C3 is cleaved by the active protease to produce C3a and C3b.

C3a is anaphylatoxin. C3b binds to bacteria and other cells, as well as certain viruses and immune complexes, and labels them for elimination in circulation (a role known as opsonins). C3b also forms a complex with other components, producing a C5 convertase that cleaves C5 into C5a and C5b.

C5 is a 190 kDa protein found at about 80 μg/ml (0.4 μM) in normal serum. C5 is glycosylated, with about 1.5 to 3% of its mass due to carbohydrates. Mature C5 is a 115 kDa alpha chain heterodimer disulfide bonded to a 75 kDa beta chain. C5 is synthesized as a single-chain precursor protein (pro-C5 precursor) of 1676 amino acids (see, for example, Patent Document 1 and Patent Document 2). The pro-C5 precursor is cleaved to produce a beta chain as an amino terminal fragment and an alpha chain as a carboxyl terminal fragment. The alpha-chain and beta-chain polypeptide fragments are linked to each other by disulfide bonds to constitute a mature C5 protein.

Mature C5 is cleaved into C5a and C5b fragments upon activation of complement pathways. C5a is cleaved from the alpha chain of C5 as an amino terminal fragment containing the first 74 amino acids of the alpha chain by C5 convertase. The remainder of mature C5 is a fragment C5b containing the remainder of the alpha chain disulfide bonded to the beta chain. About 20% of the 11 kDa mass of C5a comes from carbohydrates.

C5a is another anaphylatoxin. C5b binds with C6, C7, C8 and C9 to form a membrane attack complex (MAC, C5b-9, terminal complement complex (TCC)) on the surface of the target cell. When a sufficient number of MACs are inserted into the target cell membrane, MAC pores are formed to mediate rapid osmotic lysis of the target cells.

As mentioned above, C3a and C5a are anaphylatoxins. They can cause mast cell degranulation, which releases histamine and other inflammatory mediators, leading to other inflammatory phenomena, including smooth muscle contraction, increased vascular permeability, leukocyte activation, and cell proliferation leading to apoptosis. C5a also acts as a chemotactic peptide that serves to attract granulocytes such as neutrophils, eosinophils, basophils and monocytes to the complement activation site.

The activity of C5a is regulated by the plasma enzyme carboxypeptidase N, which removes the carboxy-terminal arginine from C5a to produce a C5a-des-Arg derivative. C5a-des-Arg represents only 1% of the anaphylactic activity and polymorphonuclear chemotaxis activity of unmodified C5a.

A properly functioning complement system provides strong defense against infectious microorganisms, but inadequate regulation or activation of complement has been implicated in the development of a variety of diseases, including, for example: rheumatoid arthritis (RA); Lupus nephritis; Ischemia-reperfusion injury; Paroxysmal nocturnal hemoglobinuria (PNH); Atypical hemolytic uremic syndrome (aHUS); High density deposition disease (DDD); Macular degeneration (eg, age-related macular degeneration (AMD)); Hemolytic, increased liver enzymes and low platelet (HELLP) syndrome; Thrombotic thrombocytoptic purpura (TTP); Natural heritage; Pauci-immune vasculitis; Bullous epidermolysis; Recurrent miscarriage; Multiple sclerosis (MS); Traumatic brain injury; And injuries resulting from myocardial infarction, cardiopulmonary bypass, and hemodialysis (see, for example, Non-Patent Document 1). Therefore, inhibition of excessive or uncontrolled activation of the complement chain reaction can provide clinical benefit to patients with these diseases.

Paroxysmal nocturnal hemoglobinuria (PNH) is an uncommon blood disorder in which red blood cells are weakened and destroyed more rapidly than normal red blood cells. PNH results from clonal expansion of hematopoietic stem cells with somatic mutations in the PIG-A (phosphatidylinositol glycan class A) gene located on the X chromosome. Mutations in PIG-A cause early blockade in the synthesis of glycosylphosphatidylinositol (GPI), a molecule necessary for immobilization of many proteins on the cell surface. Consequently, PNH blood cells are deficient in GPI-fixed proteins, including complement-regulatory proteins CD55 and CD59. Under normal circumstances, the complement-regulatory proteins prevent red blood cell lysis by blocking the formation of MACs on the cell surface. The absence of GPI-fixed protein results in complement-mediated hemolysis in PNH.

PNH is characterized by hemolytic anemia (reduced number of red blood cells), hemoglobinuria (the presence of hemoglobin in the urine, particularly pronounced after sleep), and hemoglobinemia (the presence of hemoglobin in the bloodstream). Individuals with PNH are known to have seizures defined here as the development of dark urine. Hemolytic anemia is due to intravascular destruction of red blood cells by complement components. Other known symptoms include aphasia, fatigue, impotence, thrombosis and recurrent abdominal pain.

Eculizumab is a humanized monoclonal antibody directed against complement protein C5 and is the first treatment approved for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS) (e.g., Non-Patent Document 2 Reference). Eculizumab inhibits cleavage of C5 to C5a and C5b by C5 convertase, thereby preventing the production of terminal complement complex C5b-9. Both C5a and C5b-9 cause terminal complement-mediated reactions characteristic of PHN and aHUS (see also Patent Document 3, Patent Document 4, Patent Document 5, and Patent Document 6).

Several reports have described anti-C5 antibodies. For example, Patent Document 7 describes an anti-C5 antibody that binds to the alpha chain of C5 but does not bind to C5a and blocks the activation of C5, whereas Patent Document 8 describes an anti-C5 monoclonal antibody that inhibits C5a production. Is described. On the other hand, Patent Document 9 describes an anti-C5 antibody that recognizes a proteolytic site for C5 converting enzyme on the alpha chain of C5 and inhibits the conversion of C5 to C5a and C5b. Patent Document 10 describes an anti-C5 antibody having an affinity constant of at least 1 x 10 ⁷ M ^-1.

The antibody (IgG) binds to the neonatal Fc receptor (FcRn) and has a long plasma retention time. Binding of IgG to FcRn is typically observed under acidic conditions (e.g., pH 6.0), and the binding is rarely observed under neutral conditions (e.g., pH 7.4). Typically, IgG is non-specifically incorporated into cells via endocytosis and returned to the cell surface by binding to the endosome FcRn under acidic conditions in the endosome. The IgG is then dissociated from FcRn under neutral conditions in plasma. IgG that cannot bind to FcRn is degraded in lysosomes. When the FcRn binding ability of IgG under acidic conditions is excluded by introducing a mutation in its Fc region, IgG is not recycled from endosomes into plasma, resulting in a significant reduction in plasma retention of IgG. In order to improve the plasma retention of IgG, a method of enhancing its FcRn binding under acidic conditions has been reported. When the FcRn binding of IgG under acidic conditions is improved by introducing amino acid substitutions in its Fc region, the IgG is more effectively recycled from the endosomes to the plasma, resulting in improved plasma retention. On the other hand, IgG having increased FcRn binding under neutral conditions does not dissociate from FcRn under neutral conditions in plasma even when the IgG returns to the cell surface by its binding to FcRn under acidic conditions in endosomes, and consequently It has also been reported that its plasma retention remains unchanged or rather worsens (see, for example, Non-Patent Document 3; Non-Patent Document 4; Non-Patent Document 5).

Recently, antibodies that bind to antigens in a pH-dependent manner have been reported (see, for example, Patent Document 11 and Patent Document 12). The antibodies bind strongly to the antigen under neutral conditions in plasma and dissociate from the antigen under acidic conditions in endosomes. After dissociation from the antigen, the antibodies are able to bind to the antigen again when recycled to the plasma through FcRn. Thus, a single antibody molecule can bind repeatedly to several antigenic molecules. In general, the plasma retention of antigens is much shorter than antibodies with the aforementioned FcRn-mediated recycling mechanism. Therefore, when the antigen is bound to the antibody, the antigen normally exhibits prolonged plasma retention, resulting in an increase in the plasma concentration of the antigen. On the other hand, the aforementioned antibodies that bind to the antigen in a pH-dependent manner remove the antigen from the plasma more rapidly than typical antibodies because they dissociate from the antigen in the endosomes during the FcRn-mediated recycling process. Patent Document 13 also describes a computer modeling analysis showing that antibodies with pH-dependent binding induced to C5 can prolong antigen knockdown.

U.S. Patent No. 6,355,245 U.S. Patent No. 7,432,356 International Patent Publication No. 2005/074607 International Patent Publication No. 2007/106585 International Patent Publication No. 2008/069889 International Patent Publication No. 2010/054403 International Patent Publication No. 95/29697 International Patent Publication No. 02/30985 International Patent Publication No. 2004/007553 International Patent Publication No. 2010/015608 International Patent Publication No. 2009/125825 International Patent Publication No. 2011/122011 International Patent Publication No. 2011/111007

Holers et al., Immunol. Rev. 223:300-316 (2008) Dmytrijuk et al., The Oncologist 13(9):993-1000 (2008) Yeung et al., J Immunol. 182(12): 7663-7671 (2009) Datta-Mannan et al., J Biol. Chem. 282(3):1709-1717 (2007) Dall'Acqua et al., J. Immunol. 169(9):5171-5180 (2002)

It is an object of the present invention to provide an anti-C5 antibody and a method of using the same.

The present invention provides anti-C5 antibodies and methods of using the same.

In some embodiments, an isolated anti-C5 antibody of the invention binds to an epitope within the beta chain of C5. In some embodiments, an isolated anti-C5 antibody of the invention binds an epitope within the MG1-MG2 domain of the beta chain of C5. In some embodiments, an isolated anti-C5 antibody of the invention binds an epitope within a fragment consisting of amino acids 33-124 of the beta chain of C5 (SEQ ID NO: 40). In some embodiments, the isolated anti-C5 antibody of the invention is within the beta chain of C5 (SEQ ID NO: 40) comprising one or more fragments selected from the group consisting of amino acids 47-57, 70-76, and 107-110. Binds to the epitope. In some embodiments, the isolated anti-C5 antibody of the present invention is a beta chain of C5 comprising one or more amino acid residues selected from the group consisting of Glu48, Asp51, His70, His72, Lys109 and His110 of SEQ ID NO: 40 (SEQ ID NO: 40). In another embodiment, the antibody binds C5 with a higher affinity at neutral pH than at acidic pH. In another embodiment, the antibody binds C5 with a higher affinity at pH 7.4 than at pH 5.8. In another embodiment, the isolated anti-C5 antibody of the invention binds to the same epitope as the antibody described in Table 2. In another embodiment, the antibody binds the same epitope as the antibody described in Table 2 with a higher affinity at pH 7.4 than at pH 5.8. In another embodiment, the anti-C5 antibodies of the invention bind to the same epitope as the antibodies described in Tables 7 or 8. In another embodiment, the antibody binds the same epitope as the antibody described in Tables 7 or 8 with a higher affinity at pH 7.4 than at pH 5.8.

In certain embodiments, an anti-C5 antibody of the invention competes for binding to C5 with an antibody comprising a VH and VL pair selected from: (a) a VH of SEQ ID NO: 1 and a VL of SEQ ID NO: 11; (b) the VH of SEQ ID NO: 5 and the VL of SEQ ID NO: 15; (c) the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 14; (d) the VH of SEQ ID NO: 6 and the VL of SEQ ID NO: 16; (e) the VH of SEQ ID NO: 2 and the VL of SEQ ID NO: 12; (f) the VH of SEQ ID NO: 3 and the VL of SEQ ID NO: 13; (g) the VH of SEQ ID NO: 9 and the VL of SEQ ID NO: 19; (h) the VH of SEQ ID NO: 7 and the VL of SEQ ID NO: 17; (i) the VH of SEQ ID NO: 8 and the VL of SEQ ID NO: 18; And (j) a VH of SEQ ID NO: 10 and a VL of SEQ ID NO: 20. In another embodiment, the anti-C5 antibody binds C5 with a higher affinity at neutral pH than at acidic pH. In another embodiment, the anti-C5 antibody binds C5 with a higher affinity at pH 7.4 than at pH 5.8.

In some embodiments, an isolated anti-C5 antibody of the invention has the characteristics selected from the group consisting of: (a) the antibody is in contact with amino acids D51 and K109 of C5 (SEQ ID NO: 39); (b) the affinity of the antibody for C5 (SEQ ID NO: 39) is greater than the affinity of the antibody for the C5 mutant consisting of the E48A substitution of SEQ ID NO: 39; (c) The antibody binds to the C5 protein consisting of the amino acid sequence of SEQ ID NO: 39 at pH 7.4, but does not bind to the C5 protein consisting of the amino acid sequence of SEQ ID NO: 39 having the H72Y substitution at pH 7.4. In another embodiment, the antibody binds C5 with a higher affinity at neutral pH than at acidic pH. In another embodiment, the antibody binds C5 with a higher affinity at pH 7.4 than at pH 5.8.

In some embodiments, an isolated anti-C5 antibody of the invention inhibits the activation of C5. In some embodiments, an isolated anti-C5 antibody of the invention inhibits activation of C5 variant R885H. In some embodiments, an isolated anti-C5 antibody of the invention is a monoclonal antibody. In some embodiments, an isolated anti-C5 antibody of the invention is a human antibody, humanized antibody or chimeric antibody. In some embodiments, an isolated anti-C5 antibody of the invention is an antibody fragment that binds C5. In some embodiments, the isolated anti-C5 antibody of the invention is a full length IgG1 antibody or a full length IgG4 antibody.

In some embodiments, an isolated anti-C5 antibody of the invention comprises (a) the amino acid sequence DX ₁ GYX ₂ X ₃ PTHAMX ₄ X ₅ , wherein X ₁ is G or A and X ₂ is V, Q or D. , X ₃ is T or Y, X ₄ is Y or H, X ₅ is L or Y) HVR-H3 comprising (SEQ ID NO: 128), (b) amino acid sequence QX ₁ TX ₂ VGSSYGNX ₃ (wherein , X ₁ is S, C, N or T, X ₂ is F or K, X ₃ is A, T or H) (SEQ ID NO: 131) HVR-L3, and (c) amino acid sequence X ₁ IX ₂ TGSGAX ₃ YX ₄ AX ₅ WX ₆ KG (where X ₁ is C, A or G, X ₂ is Y or F, X ₃ is T, D or E, and X ₄ is Y, K or Q, X ₅ is S, D or E, and X ₆ is A or V) (SEQ ID NO: 127).

In some embodiments, an isolated anti-C5 antibody of the invention comprises (a) the amino acid sequence SSYYX ₁ X ₂ , wherein X ₁ is M or V and X ₂ is C or A (SEQ ID NO: 126). HVR-H1, (b) amino acid sequence X ₁ IX ₂ TGSGAX ₃ YX ₄ AX ₅ WX ₆ KG (where X ₁ is C, A or G, X ₂ is Y or F, and X ₃ is T, D Or HVR-H2 comprising E, X ₄ is Y, K or Q, X ₅ is S, D or E, and X ₆ is A or V) (SEQ ID NO: 127), and (c) amino acid sequence DX ₁ GYX ₂ X ₃ PTHAMX ₄ X ₅ (where X ₁ is G or A, X ₂ is V, Q or D, X ₃ is T or Y, X ₄ is Y or H, and X ₅ is L or Y) (SEQ ID NO: 128). In another embodiment, the antibody comprises (a) the amino acid sequence X ₁ ASQX ₂ IX ₃ SX ₄ LA, wherein X ₁ is Q or R, X ₂ is N, Q or G, and X ₃ is G or S And, X ₄ is D, K or S) (SEQ ID NO: 129), including HVR-L1; (b) amino acid sequence GASX ₁ X ₂ X ₃ S (wherein X ₁ is K, E or T, X ₂ is L or T, and X ₃ is A, H, E or Q) (SEQ ID NO: 130) HVR-L2 comprising a; And (c) amino acid sequence QX ₁ TX ₂ VGSSYGNX ₃ (where X ₁ is S, C, N or T, X ₂ is F or K, and X ₃ is A, T or H) (SEQ ID NO: 131) It includes HVR-L3 comprising a.

In some embodiments, an isolated anti-C5 antibody of the invention comprises (a) the amino acid sequence X ₁ ASQX ₂ IX ₃ SX ₄ LA, wherein X ₁ is Q or R and X ₂ is N, Q or G, HVR-L1 comprising X ₃ is G or S, and X ₄ is D, K or S) (SEQ ID NO: 129); (b) amino acid sequence GASX ₁ X ₂ X ₃ S (wherein X ₁ is K, E or T, X ₂ is L or T, and X ₃ is A, H, E or Q) (SEQ ID NO: 130) HVR-L2 comprising a; And (c) amino acid sequence QX ₁ TX ₂ VGSSYGNX ₃ (where X ₁ is S, C, N or T, X ₂ is F or K, and X ₃ is A, T or H) (SEQ ID NO: 131) It includes HVR-L3 comprising a.

In some embodiments, an isolated anti-C5 antibody of the invention comprises a heavy chain variable domain framework FR1 comprising the amino acid sequence of any one of SEQ ID NOs: 132-134; FR2 comprising the amino acid sequence of any one of SEQ ID NOs: 135 and 136; FR3 comprising the amino acid sequence of any one of SEQ ID NOs: 137 to 139; And FR4 comprising the amino acid sequence of any one of SEQ ID NOs: 140 and 141. In some embodiments, an isolated anti-C5 antibody of the invention comprises a light chain variable domain framework FR1 comprising the amino acid sequence of any one of SEQ ID NOs: 142 and 143; FR2 comprising the amino acid sequence of any one of SEQ ID NOs: 144 and 145; FR3 comprising the amino acid sequence of any one of SEQ ID NOs: 146 and 147; And FR4 comprising the amino acid sequence of SEQ ID NO: 148.

In some embodiments, an isolated anti-C5 antibody of the invention comprises (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 10 and 106-110; (b) a VL sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 20 and 111 to 113; Or (c) the VH sequence in (a) and the VL sequence in (b). In another embodiment, the antibody comprises the VH sequence of any one of SEQ ID NOs: 10 and 106-110. In another embodiment, the antibody comprises the VL sequence of any one of SEQ ID NOs: 20 and 111-113.

The present invention includes the VH sequence of any one of SEQ ID NOs: 10 and 106 to 110 and the VL sequence of any one of SEQ ID NOs: 20 and 111 to 113.

The invention also provides an isolated nucleic acid encoding an anti-C5 antibody of the invention. The invention also provides a host cell comprising the nucleic acid of the invention. The invention also provides a method for producing an antibody, comprising culturing the host cell of the invention so that the antibody is produced.

The invention also provides a method of making an anti-C5 antibody. In some embodiments, the method comprises immunizing the animal against a polypeptide comprising the MG1-MG2 domain of the beta chain of C5 (SEQ ID NO: 43). In some embodiments, the method comprises immunizing the animal against a polypeptide comprising a region corresponding to amino acids at positions 33 to 124 of the beta chain of C5 (SEQ ID NO: 40). In some embodiments, the method comprises immunizing the animal against a polypeptide comprising one or more fragments selected from amino acids 47-57, 70-76, and 107-110 of the beta chain of C5 (SEQ ID NO: 40). . In some embodiments, the method comprises immunizing an animal against a polypeptide comprising a fragment of the beta chain of C5 (SEQ ID NO: 40) comprising one or more amino acids selected from Glu48, Asp51, His70, His72, Lys109 and His110. Includes steps.

The invention also provides a pharmaceutical formulation comprising an anti-C5 antibody of the invention and a pharmaceutically acceptable carrier.

The anti-C5 antibody of the present invention may be for use as a medicament. Anti-C5 antibodies of the invention may be for use in treating complement-mediated diseases or conditions involving excessive or uncontrolled activation of C5. The anti-C5 antibody of the present invention may be for use in enhancing the removal of C5 from plasma.

The anti-C5 antibody of the present invention can be used in the manufacture of a medicament. In some embodiments, the medicament is for treatment of a complement-mediated disease or condition involving excessive or uncontrolled activation of C5. In some embodiments, the medicament is for enhancing the removal of C5 from plasma.

The invention also provides a method of treating a subject having a complement-mediated disease or condition that involves excessive or uncontrolled activation of C5. In some embodiments, the method comprises administering to the individual an effective amount of an anti-C5 antibody of the invention. The invention also provides a method of enhancing the removal of C5 from plasma in a subject. In some embodiments, the method comprises administering to the individual an effective amount of an anti-C5 antibody of the invention to enhance removal of C5 from plasma.

1 illustrates epitope binning of anti-C5 antibodies, as described in Example 2.2. Antibodies classified in the same epitope bin are shown in the box with a bold line.
Figure 2a illustrates the BIACORE (registered trademark) sensorgram of anti-C5 antibodies at pH 7.4 (solid line) and pH 5.8 (dashed line) to assess pH-dependence, as described in Example 3.2. will be. CFA0305, CFA0307, CFA0366, CFA0501, CFA0538 and CFA0599 are antibodies classified as epitope C, as described in Example 2.2.
2B illustrates Biacore® sensorgrams of anti-C5 antibodies at pH 7.4 (solid line) and pH 5.8 (dashed line) to assess pH-dependence, as described in Example 3.2. As described in Example 2.2, CFA0666, CFA0672 and CFA0675 are antibodies classified as epitope A, and CFA0330 and CFA0341 are antibodies classified as epitope B. 305LO5 is a humanized antibody of CFA0305, as described in Example 2.3.
3 is a Western for C5 beta chain-derived fragments fused to a GST-tag (amino acids 19 to 180, 161 to 340, 321 to 500 and 481 to 660 of SEQ ID NO: 40), as described in Example 4.1. Illustrative of Western Blot analysis. CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672 and CFA0675 are antibodies classified as epitope C. Anti-GST antibody is a positive control. The location of the GST-fused C5 fragments (46-49 kDa) is indicated by arrows.
4 is a diagram illustrating a Biacore (registered trademark) sensorgram of an anti-C5 antibody against the MG1-MG2 domain of the C5 beta chain, as described in Example 4.3. The upper panel shows the results of CFA0305 (solid line), CFA0307 (dashed line), CFA0366 (dashed line) and eculizumab (dotted line). The middle panel shows the results of CFA0501 (solid line), CFA0599 (dashed line), CFA0538 (dashed line) and eculizumab (dotted line). The lower panel shows the results of CFA0666 (solid line), CFA0672 (dashed line), CFA0675 (dashed line) and eculizumab (dotted line). CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672 and CFA0675 are antibodies classified as epitope C. Eculizumab is a control anti-C5 antibody.
5A shows MG1-MG2 domain-derived peptide fragments fused to a GST-tag (amino acids 33 to 124, 45 to 124, 52 to 124, 33 to 111, 33 of SEQ ID NO: 40, as described in Example 4.4. To 108, and 45 to 111). Anti-GST antibodies are used as reaction antibodies. The location of the GST-fused C5 fragment (35-37 kDa) is indicated by arrows.
5B shows MG1-MG2 domain-derived peptide fragments fused to a GST-tag (amino acids 33 to 124, 45 to 124, 52 to 124, 33 to 111, 33 of SEQ ID NO: 40, as described in Example 4.4. To 108, and 45 to 111). CFA0305 is used as the reaction antibody.
5C summarizes the binding reaction of anti-C5 antibodies to C5 beta chain-derived fragments, as described in Example 4.4. Fragments to which anti-C5 antibodies classified as epitope C (CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675) bind are shown in gray, and fragments that do not bind to the antibodies are shown in white. Indicated.
6 illustrates Western blot analysis for C5 point mutants (E48A, D51A and K109A, respectively) in which E48, D51 and K109 of the beta chain were substituted with alanine, as described in Example 4.5. In the left panel, eculizumab (anti-C5 antibody, alpha chain binding agent) is used as a reaction antibody, and the position of the C5 alpha chain (about 113 kDa) is indicated by an arrow. In the right panel, CFA0305 (classified as epitope C, beta chain binding agent) is used as a reaction antibody, and the position of the beta chain of C5 (about 74 kDa) is indicated by an arrow.
7 shows a Biacore® sensorgram showing the interaction of eculizumab-F760G4 (top panel) or 305LO5 (bottom panel) with C5 mutants, as described in Example 4.6. Sensorgrams are shown above the sensor surface immobilized with eculizumab-F760G4 or 305LO5, C5-wt (solid bold curve), C5-E48A (short-dashed curve), C5-D51A (long-dashed curve), and C5-K109A. (Thin solid curve) was obtained by each injection. Eculizumab is a control anti-C5 antibody. 305LO5 is a humanized antibody of CFA0305 (classified as epitope C), as described in Example 2.3.
Figure 8 shows a Biacore (registered trademark) sensorgram showing the interaction of 305LO5 and C5 His mutants to evaluate the pH-dependence, as described in Example 4.7. Sensorgrams are shown above the sensor surface immobilized with 305LO5, C5-wt (solid thick curve), C5-H70Y (long-dashed curve), C5-H72Y (short-dashed curve), C5-H110Y (dotted curve), and C5. -H70Y+H110Y (thin solid curve) was obtained by each injection. Antibody/antigen complexes were dissociated at pH 7.4 to assess pH-dependent interactions, followed by further dissociation at pH 5.8 (indicated by arrows).
9A illustrates inhibition of complement-activated liposome lysis by anti-C5 antibodies, as described in Example 5.1. As described in Example 2.2, the results of CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675 classified as epitope C are shown.
9B illustrates inhibition of complement-activated liposome lysis by anti-C5 antibodies, as described in Example 5.1. As described in Example 2.2, the results of antibodies CFA0330 and CFA0341 classified as epitope B are shown.
10A illustrates inhibition of C5a production by anti-C5 antibodies, as described in Example 5.2. The C5a concentration was quantified in the supernatant obtained during the liposome dissolution analysis described in FIG. 9A.
10B illustrates inhibition of C5a production by anti-C5 antibodies, as described in Example 5.2. The C5a concentration was quantified in the supernatant obtained during the liposome dissolution analysis described in FIG. 9B.
11 illustrates inhibition of complement-activated hemolysis by anti-C5 antibodies, as described in Example 5.3. Complement was activated through classical pathways.
12 illustrates inhibition of complement-activated hemolysis by anti-C5 antibodies, as described in Example 5.4. Complement was activated through an alternative pathway.
13 illustrates the time course of human C5 concentration in plasma after intravenous administration of human C5 alone or human C5 and anti-human C5 antibodies in mice evaluating C5 clearance, as described in Example 6.2. As described in Example 2.2, CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675 are antibodies classified as epitope C, and CFA0330 and CFA0341 are antibodies classified as epitope B.
14 illustrates the time course of plasma anti-human C5 antibody concentration after intravenous administration of human C5 and anti-human C5 antibodies in mice evaluating antibody pharmacokinetics, as described in Example 6.3. As described in Example 2.2, CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675 are antibodies classified as epitope C, and CFA0330 and CFA0341 are antibodies classified as epitope B.
15 illustrates inhibition of complement-activated liposome lysis by anti-C5 antibodies, as described in Example 9.1. The results of antibodies 305LO15-SG422, 305LO16-SG422, 305LO18-SG422, 305LO19-SG422, 305LO20-SG422, and 305LO20-SG115 are shown.
16 illustrates inhibition of complement-activated liposome lysis by anti-C5 antibodies, as described in Example 9.1. The results of antibodies 305LO15-SG115 and 305LO23-SG429 are shown.
Figure 17 illustrates the inhibition of complement-activated liposome lysis by anti-C5 antibodies, as described in Example 9.1. The results of antibodies 305LO22-SG115, 305LO22-SG422, 305LO23-SG115, and 305LO23-SG422 are shown.
18 illustrates inhibition of C5a production by anti-C5 antibodies, as described in Example 9.2. The C5a concentration was quantified in the supernatant obtained during the liposome dissolution assay described in FIG. 15.
Figure 19 illustrates the inhibition of C5a production by anti-C5 antibodies, as described in Example 9.2. The C5a concentration was quantified in the supernatant obtained during the liposome dissolution assay described in FIG. 16.
Figure 20 illustrates the inhibition of complement activity in monkey plasma by anti-C5 antibodies, as described in Example 9.3. Anti-C5 antibody was administered to cynomolgus monkeys, and complement activity in the monkey's plasma was measured in a hemolysis assay.
Figure 21 shows the wild type C5 (WT) and C5 variants (V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N, and E1437D) by anti-C5 antibody (eculizumab) as described in Example 9.4. To illustrate the inhibition of biological activity.
Figure 22 is the organism of wild type C5 (WT) and C5 variants (V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N, and E1437D) by anti-C5 antibody (305 variant) as described in Example 9.4. To illustrate the inhibition of activity.
23 illustrates inhibition of complement-activated liposome hemolysis by anti-C5 antibodies (BNJ441 and 305 variants), as described in Example 9.5.
Figure 24 illustrates the time course of plasma cynomolgus C5 concentration after intravenous administration of anti-human C5 antibody in cynomolgus monkeys evaluating C5 clearance, as described in Example 10.2.
Figure 25 illustrates the time course of plasma anti-human C5 concentration after intravenous administration of anti-human C5 antibody in cynomolgus monkeys to evaluate antibody pharmacokinetics, as described in Example 10.3.
26A and 26B illustrate the crystal structure of 305 Fab bound to the human C5(hC5)-MG1 domain, as described in Example 11.6. 26A illustrates an asymmetric unit. MG1 is represented as a surface and 305 Fab is shown as a ribbon (dark gray: heavy chain, light gray: light chain). 26B illustrates overlapping molecules 1 and 2 (dark gray: molecule 1, light gray: molecule 2).
Figure 27A illustrates the epitope of the 305 Fab contact region on the MG1 domain, as described in Example 11.6. Figure 27A illustrates epitope mapping in the MG1 amino acid sequence (dark gray: closer than 3.0 Å, light gray: closer than 4.5 Å).
Figure 27B illustrates the epitope of the 305 Fab contact region on the MG1 domain, as described in Example 11.6. Figure 27B illustrates epitope mapping within the crystal structure (dark gray spheres: closer than 3.0 Å, light gray bars: closer than 4.5 Å).
28A illustrates an enlarged view of the interaction of E48, D51 and K109 (shown as a bar) and 305 Fab (shown as a surface), as described in Example 11.7.
28B illustrates the interaction between E48 and its environment (dark gray dotted line: hydrogen bonding with Fab, light gray dotted line: water-mediated hydrogen bonding), as described in Example 11.7.
28C illustrates the interaction between D51 and its environment (dark gray dotted line: hydrogen bonding with Fab), as described in Example 11.7.
28D illustrates the interaction between K109 and its environment (dark gray dotted line: hydrogen bonding with Fab, light gray dotted line: salt crosslinking with H-CDR3_D95), as described in Example 11.7.
29A illustrates an enlarged view of the interaction of H70, H72, and H110 (shown as a bar) and 305 Fab (shown as a surface), as described in Example 11.8, in the same orientation as FIG. 28A.
29B illustrates the interaction between H70 and its environment, as described in Example 11.8. The histidine residues are shown as rods and meshes. Hydrogen bonds are indicated by dotted lines.
29C illustrates the interaction between H72 and its environment, as described in Example 11.8. The histidine residues are represented by rods and meshes. Hydrogen bonds are indicated by dotted lines.
29D illustrates the interaction between H110 and its environment, as described in Example 11.8. The histidine residues are represented by rods and meshes. The distance between H110 and H-CDR3_H100c is indicated by a dotted line.

The techniques and procedures described or referenced herein are generally well-recognized, and are typically used by those skilled in the art using methods conventionally, such as the widely used methods described in the following documents. Used: Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney), ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993)].

I. Definition

Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons ( New York, NY 1992)] provides general guidance to many of the terms used herein to those skilled in the art. All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.

In order to understand this specification, the following definitions will apply and, where appropriate, terms used in the singular will also include the plural, and vice versa. It is to be understood that the terminology used herein is only intended to describe specific embodiments and is not intended to be limiting. If any of the definitions set forth below contradict any document incorporated herein by reference, the definitions set forth below are to be adjusted.

In the case of the present application, the "acceptor human framework" is a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human common framework as defined below. It is a framework comprising the amino acid sequence of. Receptor human frameworks "derived from" the human immunoglobulin framework or the human consensus framework may comprise the same amino acid sequence thereof, or the framework may contain amino acid sequence changes. 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, 3 or less, or 2 or less. In some embodiments, the VL receptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

"Affinity" refers to the strength of the sum 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 an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can generally be expressed as the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Certain exemplary and representative embodiments for measuring affinity are described below.

An “affinity enhanced” antibody refers to an antibody that has one or more mutations in one or more hypervariable regions (HVRs) as compared to a parent antibody that does not have a mutation that causes an improvement in the affinity of the antibody for the antigen.

The terms “anti-C5 antibody” and “an antibody that binds to C5” refer to an antibody capable of binding C5 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting C5. In one embodiment, the degree of binding of the anti-C5 antibody to an irrelevant non-C5 protein is less than about 10% of the antibody that binds to C5 as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to C5 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., 10 ^-8 M or less, For example, it has a dissociation constant (Kd) of ^{10 -8} to 10 ^-13 M, for example, 10 ^-9 to 10 ^{-13 M).} In certain embodiments, the anti-C5 antibody binds an epitope of C5 that is conserved among C5 from different species.

The term "antibody" herein is used in the broadest sense and includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments when exhibiting the desired antigen-binding activity. (But not limited to), includes a variety of antibody constructs.

"Antibody fragment" refers to a molecule other than an intact antibody comprising a portion of an intact antibody that binds to an antigen to which an intact antibody binds. Examples of antibody fragments include Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; Diabody; Linear antibodies; Single chain antibody molecules (eg, scFv); And multispecific antibodies generated from antibody fragments.

The reference antibody and "antibody that binds to the same epitope" refer to an antibody that blocks the binding of the reference antibody to the antigen in a competition assay, and the reference antibody blocks the binding of the antibody to the antigen in a competition assay. Representative competition assays are provided herein.

The term “chimeric” antibody refers to 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 a different source or species.

The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG and IgM, several of which are subclasses (isotypes), e.g. IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , It can be further classified as IgA ₁ and IgA _2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma and mu, respectively.

The term “cytotoxic agent” as used herein refers to a substance that inhibits or interferes with cellular function and/or causes cell death or destruction. Cytotoxic agents include radioactive isotopes (e.g., At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² , and radioactive isotopes of Lu) ; Chemotherapy or chemotherapy drugs (e.g. methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other inter A calating agent); Growth inhibitors; Enzymes and fragments thereof such as nucleolytic enzymes; Antibiotics; Toxins, including small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; And various anti-tumor agents or anti-cancer agents disclosed below.

“Effector function” refers to a biological activity due to the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; Antibody-dependent cell-mediated cytotoxicity (ADCC); Phagocytosis; Down regulation of cell surface receptors (eg, B cell receptors); And B cell activation.

"Effective amount" of a medicament, eg, a pharmaceutical formulation, refers to an amount effective at the dosage and for the time required to achieve the desired therapeutic or prophylactic result.

The term “epitope” includes any determinant capable of being bound by an antibody. An epitope is a region of an antigen that is bound by an antibody that targets the antigen, and contains specific amino acids that are in direct contact with the antibody. Epitope determinants may include chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural characteristics and/or specific charge characteristics. In general, antibodies specific for a particular target antigen will preferentially recognize the epitope on the target antigen in a complex mixture of proteins and/or macromolecules.

The term “Fc region” is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region runs from Cys226, or from Pro230 to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the number of amino acid residues in the Fc region or constant region is described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991], it follows the EU numbering system, also called the EU Index.

“Framework” or “FR” refers to variable domain residues other than hypervariable region (HVR) residues. The FR of the variable domain generally consists of four FR domains: FR1, FR2, FR3 and FR4. Thus, HVR and FR sequences generally appear in the following order in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms "full-length antibody", "intact antibody" and "whole antibody" refer to an antibody having a structure substantially similar to that of a native antibody herein or having a heavy chain containing an Fc region as defined herein. Are used interchangeably to

The terms “host cell”, “host cell line” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of the cells. Host cells include “transformants” and “transformed cells”, which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The progeny may not be completely identical to the parent cell in terms of nucleic acid content and may contain mutations. Mutant progeny that have the same function or biological activity as selected or selected in the originally transformed cell are included in the present invention.

A “human antibody” is an antibody that has an amino acid sequence corresponding to the amino acid sequence of an antibody produced by human or human cells or derived from a human antibody repertoire or from a non-human source using other human antibody-encoding sequences. The above definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen-binding moieties.

The “human consensus framework” is a framework representing the amino acid residues most commonly present in the selection of human immunoglobulin VL or VH framework sequences. In general, the selection of human immunoglobulin VL or VH sequences is made from a subgroup of variable domain sequences. In general, this subgroup of sequences is described in Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3] . In one embodiment, for VL, the subgroup is subgroup Kappa I as in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al., supra.

"Humanized" antibody refers to a chimeric antibody comprising amino acid residues from a non-human HVR and amino acid residues from a human FR. In certain embodiments, the humanized antibody will comprise substantially all of one or more, and typically two, variable domains, wherein all or almost all of the HVRs (e.g., CDRs) correspond to non-human antibodies and , All or almost all of the FRs correspond to human antibodies. The humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. The "humanized form" of an antibody, eg, a non-human antibody, refers to an antibody that has undergone humanization.

The term “hypervariable region” or “HVR”, as used herein, refers to a loop that is hypervariable in sequence (“complementary determining region” or “CDR”) and/or structurally defined (“hypervariable loop”). It refers to each of the regions of an antibody variable domain that forms and/or contains an antigen-contacting moiety (“antigen contact”). In general, antibodies comprise 6 HVRs: 3 in VH (H1, H2, H3) and 3 in VL (L1, L2, L3). Representative HVRs herein include: (a) amino acid residues 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1), 53-55(H2), And a hypervariable loop present in 96-101 (H3) (Chothia, J. Mol. Biol. 196:901-917 (1987)); (b) present at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3). CDR (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NIH, Bethesda, MD (1991)); (c) present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3). Antigen contacting moieties (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)); And (d) HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49 Combinations of (a), (b) and/or (c), including -65 (H2), 93-102 (H3), and 94-102 (H3).

Unless otherwise indicated, HVR residues, and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

An “immunoconjugate” is an antibody conjugated to one or more heterologous molecule(s), including, but not limited to, cytotoxic agents.

An “individual” or “subject” is a mammal. Mammals include livestock (e.g. cattle, sheep, cats, dogs and horses), primates (e.g. human and non-human primates, e.g. monkeys), rabbits and rodents (e.g. mice and rats) , But is not limited to this. In certain embodiments, the individual or subject is a human.

An “isolated” antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focus (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC) method. When measured, it is purified to a purity greater than 95% or 99%. For review of methods for evaluating antibody purity, see, for example, Flatman et al, J. Chromatogr. B 848:79-87 (2007).

"Isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid typically includes a nucleic acid molecule contained in a cell containing the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

An “isolated nucleic acid encoding an anti-C5 antibody” includes the nucleic acid molecule(s) in a single vector or separate vectors and the nucleic acid molecule(s) present at one or more positions in a host cell, including antibody heavy and light chains. It refers to one or more nucleic acid molecules encoding (or fragments thereof).

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homologous antibodies. That is, the individual antibodies constituting the population are the same, except for possible variant antibodies (the variants are generally present in trace amounts), for example, containing natural mutations or occurring in the manufacture of monoclonal antibody preparations, and/or , Bind to the same epitope. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of the monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier “monoclonal” denotes the properties of the antibody as obtained from a substantially homogeneous population of antibodies and should not be understood as requiring the preparation of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention include hybridoma methods, recombinant DNA methods, phage-display methods, and methods using transgenic animals containing all or part of a human immunoglobulin locus. But not limited) can be prepared by a variety of techniques, the above and other representative methods for producing monoclonal antibodies are described herein.

“Naked antibody” refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or a radiolabel. Naked antibodies can be present in pharmaceutical formulations.

“Native antibody” refers to a natural immunoglobulin molecule having a variety of structures. For example, a native IgG antibody is a heterotetrameric glycoprotein of about 150,000 Daltons, consisting of two identical light chains and two identical heavy chains disulfide-linked. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

The term “package insert” refers to instructions typically included in a commercial package of a therapeutic product, including information on the indications, usage, dosage, dosage regimen, combination therapy, contraindications, and/or precautions for use of the therapeutic product. It is used to say.

"Percent (%) amino acid sequence identity" with respect to the reference polypeptide sequence means that, if necessary, after aligning the sequences and introducing gaps, any conservative substitutions are made to achieve maximum percent sequence identity. It is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, not considered to be part of Alignment to determine percent amino acid sequence identity can be accomplished in the art using publicly available computer software such as, for example, BLAST, BLAST-2, ALIGN, or Megalin (DNASTAR) software. It can be achieved in a variety of ways that belong. Those skilled in the art can determine the appropriate parameters for aligning the sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. However, the value of% amino acid sequence identity in the sense of the present specification is prepared using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was produced by Genentech, Inc., and the source code is in Washington, D.C., USA. It was submitted with user documentation to the U.S. Copyright Office of 20559, and is registered here under the U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. of South San Francisco, CA, or may be compiled from source code. The ALIGN-2 program must be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and are unchanged.

In situations where ALIGN-2 is used for amino acid sequence comparison, the% amino acid sequence identity of a given amino acid sequence A for, relative to or against a given amino acid sequence B (both alternatively, for a given amino acid sequence B, its Can be expressed as a given amino acid sequence A that has or contains a specific% amino acid sequence identity compared to or against) is calculated as follows: 100 x fraction X/Y. Here, X is the number of amino acid residues evaluated as the same match by the sequence alignment program ALIGN-2 in the alignment of the above program of A and B, and Y is the total number of amino acid residues in B. It will be appreciated that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the% amino acid sequence identity of A to B will not be equal to the% amino acid sequence identity of B to A. Unless specifically stated otherwise, all% amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

The term "pharmaceutical formulation" refers to a formulation that is in a form for making the biological activity of the active ingredient contained therein effective, but does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is to be administered.

"Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation other than the active ingredient, which is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

The term “C5”, as used herein, is any natural from any vertebrate source, including mammals such as primates (eg, humans and monkeys) and rodents (eg, mice and rats). Includes C5. Unless otherwise indicated, the term "C5" refers to a human C5 protein having the amino acid sequence shown in SEQ ID NO: 39 and containing the beta chain sequence shown in SEQ ID NO: 40. The term includes "full-length," untreated C5, as well as any form of C5 resulting from treatment in the cell. The term also includes natural variants of C5, such as splice variants or allelic variants. A representative human C5 amino acid sequence is shown in SEQ ID NO: 39 (“wild type” or “WT” C5). The amino acid sequence of a representative beta chain of human C5 is shown in SEQ ID NO: 40. Representative amino acid sequences of MG1, MG2 and MG1-MG2 domains of the beta chain of human C5 are shown in SEQ ID NOs: 41, 42 and 43, respectively. Amino acid sequences of representative cynomolgus monkey and murine C5 are shown in SEQ ID NOs: 44 and 105, respectively. Amino acid residues 1 to 19 of SEQ ID NOs: 39, 40, 43, 44 and 105 correspond to signal sequences that are removed upon treatment in the cell and are missing from the corresponding representative amino acid sequence.

As used herein, “treatment” (and its grammatical variations such as “treat” or “treating”) refers to a clinical intervention in an attempt to alter the natural course of the subject being treated, for prophylaxis or It can be performed during the course of clinical pathology. Preferred effects of treatment include prevention of the occurrence or recurrence of the disease, alleviation of symptoms, alleviation of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, improvement or alleviation of disease state, and remission or improved Including, but not limited to, prognosis. In some embodiments, the antibodies of the invention are used to delay the progression of a disease or to slow the progression of a disease.

The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain involved in binding an antibody to an antigen. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies generally have a similar structure, with each domain comprising 4 conserved framework regions (FR) and 3 hypervariable regions (HVR). (See, eg, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to provide antigen-binding specificity. In addition, antibodies that bind to a specific antigen can be isolated using VH or VL domains from antibodies that bind to the antigen in order to search for libraries of each of the complementary VL or VH domains (for example, literature [ Portolano et al., J. Immunol. 150:880-887 (1993); Clackson et al., Nature 352:624-628 (1991)).

The term “vector”, as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid construct as well as the vector incorporated into the genome of the host cell into which it has been introduced. Certain vectors are capable of directing the expression of the nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors”.

II. Composition and method

In one aspect, the invention is based, in part, on anti-C5 antibodies and uses thereof. In certain embodiments, an antibody that binds to C5 is provided. The antibodies of the present invention are useful, for example, in the diagnosis or treatment of complement-mediated diseases or disorders involving excessive or uncontrolled activation of C5.

A. Representative anti-C5 antibodies

In one aspect, the invention provides an isolated antibody that binds to C5. In certain embodiments, an anti-C5 antibody of the invention binds to an epitope within the beta chain of C5. In certain embodiments, the anti-C5 antibody binds to an epitope within the MG1-MG2 domain of the beta chain of C5. In certain embodiments, the anti-C5 antibody binds to an epitope within a fragment consisting of amino acids 19 to 180 of the beta chain of C5. In certain embodiments, the anti-C5 antibody binds to an epitope within the MG1 domain of the beta chain of C5 (amino acids 20 to 124 of SEQ ID NO: 40 (SEQ ID NO: 41)). In certain embodiments, the anti-C5 antibody binds to an epitope within a fragment consisting of amino acids 33 to 124 of the beta chain of C5 (SEQ ID NO: 40). In another embodiment, the antibody is a shorter fragment than a fragment consisting of amino acids 33 to 124 of the beta chain of C5, e.g. amino acids 45 to 124, 52 to 124, and 33 of the beta chain of C5 (SEQ ID NO: 40). To 111, 33 to 108, or 45 to 111.

In another aspect, the present invention provides an anti-C5 antibody that exhibits pH-dependent binding properties. As used herein, the expression “pH-dependent binding” means that the antibody exhibits “reduced binding to C5 at acidic pH compared to its binding at neutral pH” (for this disclosure, two expressions All can be used interchangeably). For example, antibodies "having pH-dependent binding properties" include antibodies that bind C5 with a higher affinity at neutral pH than at acidic pH. In certain embodiments, the antibodies of the invention are at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, at neutral pH than at acidic pH. It binds to C5 with at least 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 times higher affinity. In some embodiments, the antibody binds C5 with a higher affinity at pH 7.4 than at pH 5.8. In another embodiment, the antibody of the invention is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 at pH 7.4 than at pH 5.8. , 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 times higher affinity to C5.

In the present disclosure, the "affinity" of an antibody for C5 is expressed in terms of the KD of the antibody. The KD of an antibody refers to the equilibrium dissociation constant of the antibody-antigen interaction. The higher the KD value for antibody binding to an antigen, the weaker its binding affinity for that particular antigen. Thus, as used herein, the expression “higher affinity at neutral pH than at acidic pH” (or the equivalent expression “pH-dependent binding”) means that the KD for antibody binding to C5 at acidic pH is neutral. It means greater than the KD for antibody binding to C5 at pH. For example, in the context of the present invention, an antibody is better at neutral pH than at acidic pH if the KD of antibody binding to C5 at acidic pH is at least twice the KD of antibody binding to C5 at neutral pH. It is considered to bind C5 with high affinity. Thus, the present invention is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 than the KD of antibody binding to C5 at neutral pH. , 80, 85, 90, 95, 100, 200, 400, 1000, 10000 times or more. In another embodiment, the KD value of the antibody at neutral pH may be 10 ^-7 M, 10 ^-8 M, 10 ^-9 M, 10 ^-10 M, 10 ^-11 M, 10 ^-12 M or less. In another embodiment, the KD value of the antibody at an acidic pH may be 10 ^-9 M, 10 ^-8 M, 10 ^-7 M, 10 ^-6 M or higher.

In another embodiment, the antibody is C5 with a higher affinity at neutral pH than at acidic pH if the KD of antibody binding to C5 at pH 5.8 is at least twice the KD of antibody binding to C5 at pH 7.4. Is considered to bind to. In some embodiments, provided antibodies are at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, greater than the KD of antibody binding to C5 at pH 7.4. It binds to C5 at pH 5.8 under a KD that is at least 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 times greater. In another embodiment, the KD value of the antibody at pH 7.4 may be 10 ^-7 M, 10 ^-8 M, 10 ^-9 M, 10 ^-10 M, 10 ^-11 M, 10 ^-12 M or less. In another embodiment, the KD value of the antibody at pH 5.8 may be 10 ^-9 M, 10 ^-8 M, 10 ^-7 M, 10 ^-6 M or higher.

The binding properties of an antibody to a particular antigen can also be expressed in terms of the kd of the antibody. The kd of an antibody refers to the dissociation rate constant of the antibody related to a specific antigen and is expressed in terms of the ^{reciprocal of seconds (ie, seconds- 1 ).} Increasing the kd value indicates weaker binding of the antibody to the antigen. Therefore, the present invention includes antibodies that bind C5 under higher kd values at acidic pH than at neutral pH. The present invention is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 than the kd of antibody binding to C5 at neutral pH. , 85, 90, 95, 100, 200, 400, 1000, 10000 times greater kd at acidic pH under an antibody that binds to C5. In another embodiment, the kd value of the antibody at neutral pH is 10 ^-2 1 / s, 10 ^-3 1 / s, 10 ^-4 1 / s, 10 ^-5 1 / s, 10 ^-6 1 / s or less. I can. In another embodiment, the kd value of the antibody at acidic pH may be 10 ^-3 1/s, 10 ^-2 1/s, 10 ^-1 1/s or more. The invention also includes antibodies that bind C5 under a higher kd value at pH 5.8 than at pH 7.4. The present invention is at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 than the kd of antibody binding to C5 at pH 7.4. , 90, 95, 100, 200, 400, 1000, 10000 times greater kd at pH 5.8 at pH 5.8. In another embodiment, the kd value of the antibody at pH 7.4 is 10 ^-2 1/s, 10 ^-3 1/s, 10 ^-4 1/s, 10 ^-5 1/s, 10 ^-6 1/s or less. I can. In another embodiment, the kd value of the antibody at pH 5.8 may be 10 ^-3 1/s, 10 ^-2 1/s, 10 ^-1 1/s or more.

In certain cases, "reduced binding to C5 at acidic pH compared to its binding at neutral pH" is the ratio of the KD value of antibody binding to C5 at acidic pH to the KD value of antibody binding to C5 at neutral pH. (Or vice versa). For example, in the present invention, when an antibody exhibits an acid/neutral KD ratio of 2 or more, the antibody can be considered to exhibit "reduced binding to C5 at acidic pH compared to its binding at neutral pH". have. In certain exemplary embodiments, the pH 5.8/pH 7.4 KD ratio for the antibodies of the invention is at least 2. In certain exemplary embodiments, the acidic/neutral KD ratio for the antibodies of the invention is 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 KD value of the antibody at neutral pH can be 10 ^-7 M, 10 ^-8 M, 10 ^-9 M, 10 ^-10 M, 10 ^-11 M, 10 ^-12 M or less. In another embodiment, the KD value of the antibody at an acidic pH may be 10 ^-9 M, 10 ^-8 M, 10 ^-7 M, 10 ^-6 M or higher. In another case, in the present invention, when the antibody exhibits a pH 5.8/pH 7.4 KD ratio of 2 or more, the antibody is said to exhibit "reduced binding to C5 at acidic pH compared to its binding at neutral pH". Can be considered. In certain exemplary embodiments, the pH 5.8/pH 7.4 KD ratio for the antibodies of the invention is 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 KD value of the antibody at pH 7.4 may be 10 ^-7 M, 10 ^-8 M, 10 ^-9 M, 10 ^-10 M, 10 ^-11 M, 10 ^-12 M or less. In another embodiment, the KD value of the antibody at pH 5.8 may be 10 ^-9 M, 10 ^-8 M, 10 ^-7 M, 10 ^-6 M or higher.

In certain cases, “reduced binding to C5 at acidic pH compared to its binding at neutral pH” is the ratio of the kd value of antibody binding to C5 at acidic pH to the kd value of antibody binding to C5 at neutral pH ( Or vice versa). For example, in the present invention, when an antibody exhibits an acidic/neutral kd ratio of 2 or more, the antibody can be considered to exhibit "reduced binding to C5 at acidic pH compared to its binding at neutral pH". have. In certain exemplary embodiments, the pH 5.8/pH 7.4 kd ratio for the antibodies of the invention is at least 2. In certain exemplary embodiments, the acidic/neutral kd ratio for the antibodies of the invention is 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 exemplary embodiment, the pH 5.8/pH 7.4 kd ratio for the antibodies of the invention is 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, It may be 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more. In another embodiment, the kd value of the antibody at neutral pH can be 10 ^-2 1/s, 10 ^-3 1/s, 10 ^-4 1/s, 10 ^-5 1/s, 10 ^-6 1/s or less. have. In another embodiment, the kd value of the antibody at pH 7.4 may be 10 ^-2 1/s, 10 ^-3 1/s, 10 ^-4 1/s, 10 ^-5 1/s, 10 ^-6 1/s or less. have. In another embodiment, the kd value of the antibody at an acidic pH may be 10 ^-3 1/s, 10 ^-2 1/s, 10 ^-1 1/s or more. In another embodiment, the kd value of the antibody at pH 5.8 may be 10 ^-3 1/s, 10 ^-2 1/s, 10 ^-1 1/s or more.

As used herein, the expression “acidic pH” means a pH of 4.0 to 6.5. The expression "acidic pH" is 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, 6.1 , 6.2, 6.3, 6.4, and 6.5. In certain embodiments, the “acidic pH” is 5.8.

As used herein, the expression “neutral pH” means a pH of 6.7 to about 10.0. The expression "neutral pH" means 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 certain embodiments, the “neutral pH” is 7.4.

As shown herein, KD values and kd values can be measured using a surface plasmon resonance-based biosensor to characterize antibody-antigen interactions (see, for example, Example 3 herein). The KD value and the kd value can be measured at 25°C or 37°C.

In certain embodiments, an anti-C5 antibody of the invention binds to an epitope within the beta chain of C5 consisting of the MG1 domain (SEQ ID NO: 41). In certain embodiments, the anti-C5 antibody of the invention comprises an epitope in the beta chain of C5 (SEQ ID NO: 40) comprising one or more fragments selected from the group consisting of amino acids 47-57, 70-76, and 107-110. To be combined. In certain embodiments, the anti-C5 antibody of the invention consists of Thr47, Glu48, Ala49, Phe50, Asp51, Ala52, Thr53, Lys57, His70, Val71, His72, Ser74, Glu76, Val107, Ser108, Lys109, and His110. It binds to an epitope in a fragment of the beta chain of C5 (SEQ ID NO: 40) containing one or more amino acids selected from the group. In certain embodiments, the anti-C5 antibody of the present invention is within a fragment of the beta chain of C5 (SEQ ID NO: 40) comprising one or more amino acids selected from the group consisting of Glu48, Asp51, His70, His72, Lys109, and His110. Binds to the epitope. In certain embodiments, the binding of an anti-C5 antibody of the invention to the C5 mutant is reduced compared to its binding to wild-type C5, wherein the C5 mutant is selected from the group consisting of Glu48, Asp51, His72, and Lys109. Has one or more amino acid substitutions at the position. In another embodiment, the pH-dependent binding of the anti-C5 antibody of the invention to the C5 mutant is reduced compared to its pH-dependent binding to wild-type C5, wherein the C5 mutant is His70, His72, and His110 It has one or more amino acid substitutions at a position selected from the group consisting of. In another embodiment, in the C5 mutant, the amino acid at the position selected from Glu48, Asp51, and Lys109 is substituted with alanine, and the amino acid at the position selected from His70, His72, and His110 is substituted with tyrosine.

In certain embodiments, an anti-C5 antibody of the invention competes for binding to C5 with an antibody comprising VH and VL selected from: (a) a VH of SEQ ID NO: 1 and a VL of SEQ ID NO: 11; (b) a VH of SEQ ID NO: 22 and a VL of SEQ ID NO: 26; (c) a VH of SEQ ID NO: 21 and a VL of SEQ ID NO: 25; (d) the VH of SEQ ID NO: 5 and the VL of SEQ ID NO: 15; (e) the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 14; (f) the VH of SEQ ID NO: 6 and the VL of SEQ ID NO: 16; (g) the VH of SEQ ID NO: 2 and the VL of SEQ ID NO: 12; (h) the VH of SEQ ID NO: 3 and the VL of SEQ ID NO: 13; (i) the VH of SEQ ID NO: 9 and the VL of SEQ ID NO: 19; (j) the VH of SEQ ID NO: 7 and the VL of SEQ ID NO: 17; (k) the VH of SEQ ID NO: 8 and the VL of SEQ ID NO: 18; (l) a VH of SEQ ID NO: 23 and a VL of SEQ ID NO: 27; And (m) a VH of SEQ ID NO: 10 and a VL of SEQ ID NO: 20.

In certain embodiments, an anti-C5 antibody of the invention binds to C5 and contacts amino acid Asp51 (D51) of SEQ ID NO: 39. In another embodiment, the anti-C5 antibody of the invention binds to C5 and contacts amino acid Lys109 (K109) of SEQ ID NO: 39. In another embodiment, the anti-C5 antibody of the invention binds to C5 and contacts amino acids Asp51 (D51) and amino acids Lys109 (K109) of SEQ ID NO: 39.

In certain embodiments, the binding of an anti-C5 antibody of the invention to the C5 mutant is reduced relative to its binding to wild-type C5, wherein the C5 mutant has a Glu48Ala(E48A) substitution of SEQ ID NO: 39. In another embodiment, the pH-dependent binding of the anti-C5 antibody of the invention to the C5 mutant is reduced compared to its pH-dependent binding to wild-type C5, wherein the C5 mutant is Glu48Ala of SEQ ID NO: 39 ( E48A) has a substitution.

In another embodiment, the anti-C5 antibody binds to the C5 protein consisting of the amino acid sequence of SEQ ID NO: 39, but not the C5 protein consisting of the amino acid sequence of SEQ ID NO: 39 with an H72Y substitution, wherein the C5 protein and H72Y The substituted C5 protein is prepared and selected under the same conditions. In another embodiment, the anti-C5 antibody binds to the C5 protein consisting of the amino acid sequence of SEQ ID NO: 39 at pH 7.4, but not the H72Y substituted C5 protein at pH 7.4.

Without wishing to be bound by any particular theory, the binding of the anti-C5 antibody to C5 is reduced (or almost lost) when an amino acid residue on C5 is replaced with another amino acid residue, which means that the amino acid residue on C5 is reduced to the anti-C5 antibody and It is crucial for the interaction between and can be assumed to mean that the antibody is capable of recognizing the epitope around the amino acid residue on C5.

In the present invention, it was found that a group of anti-C5 antibodies that compete with each other or bind to the same epitope may exhibit pH-dependent binding properties. Among amino acids, histidine with a pKa value of about 6.0 to 6.5 may have different proton dissociation states between neutral and acidic pH. Therefore, the histidine residue on C5 can contribute to the pH-dependent interaction between the anti-C5 antibody and C5. Without being bound by any particular theory, it can be assumed that the anti-C5 antibody is capable of recognizing the conformational structure around the histidine residue on C5, which is variable with pH. This speculation may be consistent with the experimental results described below: The pH-dependence of the anti-C5 antibody is reduced (or almost lost) when the histidine residue on C5 is replaced with another amino acid (i.e., pH-dependent binding. Anti-C5 antibodies with properties bind to the histidine mutants of C5 with a similar affinity for wild-type C5 at neutral pH, whereas the same antibodies bind to the histidine mutants of C5 with a higher affinity than wild-type C5 at acidic pH) .

In certain embodiments, anti-C5 antibodies of the invention bind C5 from more than one species. In another embodiment, the anti-C5 antibody binds to C5 from human and non-human animals. In another embodiment, the anti-C5 antibody binds C5 from humans and monkeys (eg, cynomolgus, rhesus macaque, marmoset, chimpanzee or baboon).

In one aspect, the invention provides an anti-C5 antibody that inhibits the activation of C5. In certain embodiments, an anti-C5 that prevents cleavage of C5 that produces C5a and C5b, thereby preventing the occurrence of anaphylactic activity associated with C5a, as well as the construction of the C5b-9 membrane attack complex (MAC) associated with C5b. Antibodies are provided. In certain embodiments, anti-C5 antibodies are provided that block the conversion of C5 to C5a and C5b by C5 convertase. In certain embodiments, anti-C5 antibodies are provided that block access of the C5 convertase to the cleavage site on C5. In certain embodiments, anti-C5 antibodies are provided that block hemolytic activity caused by activation of C5. In another embodiment, the anti-C5 antibodies of the invention inhibit the activation of C5 by the classical and/or alternative pathways.

In one aspect, the present invention provides an anti-C5 antibody that inhibits the activation of the C5 variant. C5 variant refers to a genetic variant of C5 resulting from genetic variation such as mutation, polymorphism or allelic variation. Genetic variations may include deletions, substitutions or insertions of one or more nucleotides. The C5 variant may contain one or more genetic variations in C5. In certain embodiments, the C5 variant has similar biological activity to wild type C5. Such C5 variants may include one or more variants selected from the group consisting of V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N, and E1437D. Here, for example, R885H refers to a genetic mutation in which arginine at position 885 is substituted with histidine. In certain embodiments, the anti-C5 antibodies of the invention inhibit the activation of both wild-type C5 and one or more C5 variants selected from the group consisting of V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N and E1437D.

In one aspect, the present invention provides a combination of (a) HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 45 to 54; (b) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 55 to 64; (c) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 65 to 74; (d) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 75 to 84; (e) HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 85 to 94; And (f) at least one, two, three, four, five or six hypervariable regions (HVRs) selected from HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 95 to 104. Anti-C5 antibodies are provided.

In one aspect, the present invention provides a combination of (a) HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 45 to 54; (b) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 55 to 64; And (c) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 65 to 74. It provides an antibody comprising one or more, two or more, or all three VH HVR sequences. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 65-74. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 65-74, and HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 95-104. In another embodiment, the antibody is HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 65-74, HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 95-104, and SEQ ID NO: 55- It includes HVR-H2 comprising the amino acid sequence of any one of 64. In another embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 45-54; (b) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 55 to 64; And (c) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 65 to 74.

In another aspect, the present invention provides (a) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 75 to 84; (b) HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 85 to 94; And (c) one or more, two or more, or all three VL HVR sequences selected from HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 95 to 104 is provided. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 75-84; (b) HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 85 to 94; And (c) HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 95 to 104.

In another aspect, the antibody of the present invention comprises (a) (i) HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 45 to 54; (ii) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 55 to 64; And (iii) a VH domain comprising one or more, two or more, or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 65 to 74; And (b) (i) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 75 to 84; (ii) HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 85 to 94; And (iii) a VL domain comprising one or more, two or more, or all three VL HVR sequences selected from HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 95 to 104.

In another aspect, the present invention provides (a) HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 45 to 54; (b) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 55 to 64; (c) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 65 to 74; (d) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 75 to 84; (e) HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 85 to 94; And (f) HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 95 to 104 is provided.

In one aspect, the present invention provides a combination of (a) HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 45, 54, 117 and 126; (b) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 55, 64, 118 to 120 and 127; (c) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 65, 74, 121 and 128; (d) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 75, 84, 122 and 129; (e) HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 85, 94, 123, 124 and 130; And (f) at least one, two, three, four, five or six HVRs selected from HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 95, 104, 125 and 131. -C5 antibody is provided.

In one aspect, the present invention provides a combination of (a) HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 45, 54, 117 and 126; (b) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 55, 64, 118 to 120 and 127; And (c) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 65, 74, 121 and 128. It provides an antibody comprising one or more, two or more, or all three VH HVR sequences. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 65, 74, 121 and 128. In another embodiment, the antibody is HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 65, 74, 121 and 128 and HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 95, 104, 125 and 131. -L3 included. In another embodiment, the antibody is HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 65, 74, 121 and 128, HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 95, 104, 125 and 131 -L3, and HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 55, 64, 118 to 120 and 127. In another embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 45, 54, 117 and 126; (b) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 55, 64, 118 to 120 and 127; And (c) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 65, 74, 121 and 128.

In another aspect, the present invention provides a combination of (a) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 75, 84, 122 and 129; (b) HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 85, 94, 123, 124 and 130; And (c) HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 95, 104, 125, and 131. It provides an antibody comprising one or more, two or more, or all three VL HVR sequences. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 75, 84, 122 and 129; (b) HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 85, 94, 123, 124 and 130; And (c) HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 95, 104, 125 and 131.

In another aspect, the antibody of the invention comprises (a) (i) HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 45, 54, 117 and 126; (ii) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 55, 64, 118 to 120 and 127; And (iii) a VH domain comprising one or more, two or more, or all three VH HVR sequences selected from HVR-H3 comprising an amino acid sequence of any one of SEQ ID NOs: 65, 74, 121 and 128; And (b) (i) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 75, 84, 122 and 129; (ii) HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 85, 94, 123, 124 and 130; And (iii) a VL domain comprising one or more, two or more, or all three VL HVR sequences selected from HVR-L3 comprising an amino acid sequence of any one of SEQ ID NOs: 95, 104, 125 and 131.

In another aspect, the present invention provides a combination of (a) HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 45, 54, 117 and 126; (b) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 55, 64, 118 to 120 and 127; (c) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 65, 74, 121 and 128; (d) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 75, 84, 122 and 129; (e) HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 85, 94, 123, 124 and 130; And (f) HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 95, 104, 125 and 131 is provided.

In certain embodiments, any one or more amino acids of the anti-C5 antibody as provided above are substituted at the following HVR positions: (a) at HVR-H1 (SEQ ID NO: 45), at positions 5 and 6; (b) at HVR-H2 (SEQ ID NO: 55), at positions 1, 3, 9, 11, 13 and 15; (c) at HVR-H3 (SEQ ID NO: 65), at positions 2, 5, 6, 12 and 13; (d) at HVR-L1 (SEQ ID NO: 75), at positions 1, 5, 7 and 9; (e) at HVR-L2 (SEQ ID NO: 85), at positions 4, 5 and 6; And (f) at HVR-L3 (SEQ ID NO: 95), at positions 2, 4 and 12.

In certain embodiments, the substitution is a conservative substitution as provided herein. In certain embodiments, any one or more of the following substitutions may be made in any combination: (a) in HVR-H1 (SEQ ID NO: 45), M5V or C6A; (b) in HVR-H2 (SEQ ID NO: 55), C1A or G, Y3F, T9D or E, Y11K or Q, S13D or E, or A15V; (c) in HVR-H3 (SEQ ID NO: 65), G2A, V5Q or D, T6Y, Y12H, or L13Y; (d) in HVR-L1 (SEQ ID NO: 75), Q1R, N5Q or G, G7S, D9K or S; (e) in HVR-L2 (SEQ ID NO: 85), K4T or E, L5T, or A6H, A6 E, or A6Q; (f) In HVR-L3 (SEQ ID NO: 95), C2S, C2N, or C2T, F4K or A12T or A12H.

All possible combinations of the above substitutions are in the consensus sequence of SEQ ID NOs: 126, 127, 128, 129, 130 and 131 for HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3, respectively. Is covered by

In any of the above embodiments, the anti-C5 antibody is humanized. In one embodiment, the anti-C5 antibody comprises an HVR as in any of the above embodiments, and also comprises a receptor human framework, such as a human immunoglobulin framework or a human common framework. In another embodiment, the anti-C5 antibody comprises an HVR as in any of the above embodiments, and also comprises a VH or VL comprising a FR sequence, wherein the FR sequence is as follows. For the heavy chain variable domain, FR1 comprises the amino acid sequence of any one of SEQ ID NOs: 132 to 134, FR2 comprises the amino acid sequence of any one of SEQ ID NOs: 135 and 136, and FR3 is any one of SEQ ID NOs: 137 to 139 It includes the amino acid sequence of, and FR4 includes the amino acid sequence of any one of SEQ ID NOs: 140 and 141. For the light chain variable domain, FR1 comprises the amino acid sequence of any one of SEQ ID NOs: 142 and 143, FR2 comprises the amino acid sequence of any one of SEQ ID NOs: 144 and 145, and FR3 is any one of SEQ ID NOs: 146 and 147 It includes the amino acid sequence of, and FR4 includes the amino acid sequence of SEQ ID NO: 148.

In another embodiment, the anti-C5 antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% relative to the amino acid sequence of any one of SEQ ID NOs: 1-10. , A heavy chain variable domain (VH) sequence with 99% or 100% sequence identity. In certain embodiments, VH sequences with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are substituted with respect to the reference sequence (e.g. , Conservative substitutions), insertions or deletions, but retaining the ability to bind to C5. In certain embodiments, a total of 1-10 amino acids have been substituted, inserted and/or deleted in any one of SEQ ID NOs: 1-10. In certain embodiments, substitutions, insertions or deletions occur in regions other than the HVR (ie, in the FR). Optionally, the anti-C5 antibody comprises a VH sequence in any one of SEQ ID NOs: 1 to 10, including post-translational modifications of the sequence. In certain embodiments, the VH is (a) HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 45-54, (b) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 55-64, and (c) one, two or three HVRs selected from HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 65 to 74.

In another embodiment, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100 for the amino acid sequence of any one of SEQ ID NOs: 11-20. Anti-C5 antibodies are provided comprising a light chain variable domain (VL) with% sequence identity. In certain embodiments, VL sequences with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are substituted with respect to the reference sequence (e.g. , Conservative substitutions), insertions or deletions, but retaining the ability to bind to C5. In certain embodiments, a total of 1-10 amino acids have been substituted, inserted and/or deleted in any one of SEQ ID NOs: 11-20. In certain embodiments, substitutions, insertions or deletions occur in regions other than the HVR (ie, in the FR). Optionally, the anti-C5 antibody comprises a VL sequence in any one of SEQ ID NOs: 11 to 20, including post-translational modifications of the sequence. In certain embodiments, the VL is (a) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 75-84, (b) HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 85-94, and (c) 1, 2 or 3 HVRs selected from HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 95 to 104.

In another aspect, an anti-C5 antibody is provided comprising a VH in any of the embodiments provided above and a VL in any of the embodiments provided above. In one embodiment, the antibody comprises a VH and VL sequence in each of SEQ ID NOs: 1 to 10 and SEQ ID NOs: 11 to 20, respectively, including post-translational modifications of the sequences.

In another embodiment, the anti-C5 antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, relative to the amino acid sequence of any one of SEQ ID NOs: 10 and 106 to 110, It includes a heavy chain variable domain (VH) sequence with 98%, 99% or 100% sequence identity. In certain embodiments, VH sequences with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are substituted with respect to the reference sequence (e.g. , Conservative substitutions), insertions or deletions, but retaining the ability to bind to C5. In certain embodiments, a total of 1-10 amino acids have been substituted, inserted and/or deleted in any one of SEQ ID NOs: 10 and 106-110. In certain embodiments, substitutions, insertions or deletions occur in regions other than the HVR (ie, in the FR). Optionally, the anti-C5 antibody comprises a VH sequence in any one of SEQ ID NOs: 10 and 106 to 110, including post-translational modifications of the sequence. In certain embodiments, the VH is (a) HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 45, 54, 117 and 126, (b) any one of SEQ ID NOs: 55, 64, 118-120 and 127 HVR-H2 comprising an amino acid sequence, and (c) HVR-H3 comprising an amino acid sequence of any one of SEQ ID NOs: 65, 74, 121 and 128.

In another embodiment, the anti-C5 antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, relative to the amino acid sequence of any one of SEQ ID NOs: 10 and 106 to 110, Includes VH sequences with 98%, 99% or 100% sequence identity. In certain embodiments, VH sequences with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are substituted with respect to the reference sequence (e.g. , Conservative substitutions), insertions or deletions, but retaining the ability to bind to C5. In certain embodiments, a total of 1-10 amino acids have been substituted, inserted and/or deleted in any one of SEQ ID NOs: 10 and 106-110. In certain embodiments, substitutions, insertions or deletions occur in regions other than the HVR (ie, in the FR). Optionally, the anti-C5 antibody comprises a VH sequence in any one of SEQ ID NOs: 10 and 106 to 110, including post-translational modifications of the sequence. In certain embodiments, the VH is (a) HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 45, 54, 117 and 126, (b) any one of SEQ ID NOs: 55, 64, 118-120 and 127 HVR-H2 comprising an amino acid sequence, and (c) HVR-H3 comprising an amino acid sequence of any one of SEQ ID NOs: 65, 74, 121 and 128.

In another embodiment, the anti-C5 antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100 relative to the amino acid sequence of SEQ ID NO: 10. Includes VH sequences with% sequence identity. In certain embodiments, the VH sequence is the amino acid sequence of SEQ ID NO: 10. In certain embodiments, VH sequences with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are substituted with respect to the reference sequence (e.g. , Conservative substitutions), insertions or deletions, but retaining 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 SEQ ID NO: 10. In certain embodiments, substitutions, insertions or deletions occur in regions other than the HVR (ie, in the FR). Optionally, the anti-C5 antibody comprises a VH sequence in SEQ ID NO: 10, including post-translational modifications of the sequence. In certain embodiments, the VH comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 54, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 64, and (c) the amino acid sequence of SEQ ID NO: 74. 1, 2 or 3 HVRs selected from HVR-H3 comprising.

In another embodiment, the anti-C5 antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100 relative to the amino acid sequence of SEQ ID NO: 106. Includes VH sequences with% sequence identity. In certain embodiments, the VH sequence is the amino acid sequence of SEQ ID NO: 106. In certain embodiments, VH sequences with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are substituted with respect to the reference sequence (e.g. , Conservative substitutions), insertions or deletions, but retaining 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 SEQ ID NO: 106. In certain embodiments, substitutions, insertions or deletions occur in regions other than the HVR (ie, in the FR). Optionally, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 106, including post-translational modifications of the sequence. In certain embodiments, the VH comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118, and (c) the amino acid sequence of SEQ ID NO: 121. 1, 2 or 3 HVRs selected from HVR-H3 comprising.

In another embodiment, the anti-C5 antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100 relative to the amino acid sequence of SEQ ID NO: 107. It is a VH sequence with% sequence identity. In certain embodiments, the VH sequence comprises the amino acid sequence of SEQ ID NO: 107. In certain embodiments, VH sequences with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are substituted with respect to the reference sequence (e.g. , Conservative substitutions), insertions or deletions, but retaining 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 SEQ ID NO: 107. In certain embodiments, substitutions, insertions or deletions occur in regions other than the HVR (ie, in the FR). Optionally, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 107, including post-translational modifications of the sequence. In certain embodiments, the VH comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 119, and (c) the amino acid sequence of SEQ ID NO: 121. 1, 2 or 3 HVRs selected from HVR-H3 comprising.

In another embodiment, the anti-C5 antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100 relative to the amino acid sequence of SEQ ID NO: 108. It is a VH sequence with% sequence identity. In certain embodiments, the VH sequence comprises the amino acid sequence of SEQ ID NO: 108. In certain embodiments, VH sequences with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are substituted with respect to the reference sequence (e.g. , Conservative substitutions), insertions or deletions, but retaining 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 SEQ ID NO: 108. In certain embodiments, substitutions, insertions or deletions occur in regions other than the HVR (ie, in the FR). Optionally, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 108, including post-translational modifications of the sequence. In certain embodiments, the VH comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118, and (c) the amino acid sequence of SEQ ID NO: 121. 1, 2 or 3 HVRs selected from HVR-H3 comprising.

In another embodiment, the anti-C5 antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100 relative to the amino acid sequence of SEQ ID NO: 109. It is a VH sequence with% sequence identity. In certain embodiments, the VH sequence comprises the amino acid sequence of SEQ ID NO: 109. In certain embodiments, VH sequences with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are substituted with respect to the reference sequence (e.g. , Conservative substitutions), insertions or deletions, but retaining 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 SEQ ID NO: 109. In certain embodiments, substitutions, insertions or deletions occur in regions other than the HVR (ie, in the FR). Optionally, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 109, including post-translational modifications of the sequence. In certain embodiments, the VH comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118, and (c) the amino acid sequence of SEQ ID NO: 121. 1, 2 or 3 HVRs selected from HVR-H3 comprising.

In another embodiment, the anti-C5 antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100 relative to the amino acid sequence of SEQ ID NO: 110. It is a VH sequence with% sequence identity. In certain embodiments, the VH sequence comprises the amino acid sequence of SEQ ID NO: 110. In certain embodiments, VH sequences with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are substituted with respect to the reference sequence (e.g. , Conservative substitutions), insertions or deletions, but retaining 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 SEQ ID NO: 110. In certain embodiments, substitutions, insertions or deletions occur in regions other than the HVR (ie, in the FR). Optionally, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 110, including post-translational modifications of the sequence. In certain embodiments, the VH comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 120, and (c) the amino acid sequence of SEQ ID NO: 121. 1, 2 or 3 HVRs selected from HVR-H3 comprising.

In another embodiment, the anti-C5 antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, relative to the amino acid sequence of any one of SEQ ID NOs: 20 and 111-113, It includes a light chain variable domain (VL) with 98%, 99% or 100% sequence identity. In certain embodiments, VL sequences with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are substituted with respect to the reference sequence (e.g. , Conservative substitutions), insertions or deletions, but retaining the ability to bind to C5. In certain embodiments, a total of 1-10 amino acids have been substituted, inserted and/or deleted in any one of SEQ ID NOs: 20 and 111-113. In certain embodiments, substitutions, insertions or deletions occur in regions other than the HVR (ie, in the FR). Optionally, the anti-C5 antibody comprises a VL sequence in any one of SEQ ID NOs: 20 and 111 to 113, including post-translational modifications of the sequence. In certain embodiments, the VL comprises (a) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 75, 84, 122 and 129; (b) HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 85, 94, 123, 124 and 130; And (c) HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 95, 104, 125 and 131.

In another embodiment, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 20. Anti-C5 antibodies comprising VL are provided. In certain embodiments, the VL sequence is the amino acid sequence of SEQ ID NO: 20. In certain embodiments, VL sequences with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are substituted with respect to the reference sequence (e.g. , Conservative substitutions), insertions or deletions, but retaining 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 SEQ ID NO: 20. In certain embodiments, substitutions, insertions or deletions occur in regions other than the HVR (ie, in the FR). Optionally, the anti-C5 antibody comprises a VL sequence in SEQ ID NO: 20, including post-translational modifications of the sequence. In certain embodiments, the VL comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 84; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 94; And (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 104.

In another embodiment, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 111. Anti-C5 antibodies comprising VL are provided. In certain embodiments, the VL sequence is the amino acid sequence of SEQ ID NO: 111. In certain embodiments, VL sequences with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are substituted with respect to the reference sequence (e.g. , Conservative substitutions), insertions or deletions, but retaining 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 SEQ ID NO: 111. In certain embodiments, substitutions, insertions or deletions occur in regions other than the HVR (ie, in the FR). Optionally, the anti-C5 antibody comprises the VL sequence in SEQ ID NO: 111, including post-translational modifications of the sequence. In certain embodiments, the VL comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123; And (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125.

In another embodiment, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 112 Anti-C5 antibodies comprising VL are provided. In certain embodiments, said VL sequence is the amino acid sequence of SEQ ID NO: 112. In certain embodiments, VL sequences with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are substituted with respect to the reference sequence (e.g. , Conservative substitutions), insertions or deletions, but retaining 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 SEQ ID NO: 112. In certain embodiments, substitutions, insertions or deletions occur in regions other than the HVR (ie, in the FR). Optionally, the anti-C5 antibody comprises the VL sequence in SEQ ID NO: 112, including post-translational modifications of the sequence. In certain embodiments, the VL comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123; And (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125.

In another embodiment, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 113. Anti-C5 antibodies comprising VL are provided. In certain embodiments, said VL sequence is the amino acid sequence of SEQ ID NO: 113. In certain embodiments, VL sequences with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are substituted with respect to the reference sequence (e.g. , Conservative substitutions), insertions or deletions, but retaining 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 SEQ ID NO: 113. In certain embodiments, substitutions, insertions or deletions occur in regions other than the HVR (ie, in the FR). Optionally, the anti-C5 antibody comprises the VL sequence in SEQ ID NO: 113, including post-translational modifications of the sequence. In certain embodiments, the VL comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 124; And (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125.

In another aspect, an anti-C5 antibody is provided comprising a VH in any of the embodiments provided above and a VL in any of the embodiments provided above. In one embodiment, the antibody comprises a VH and VL sequence in any one of SEQ ID NOs: 10 and 106 to 110 and any one of SEQ ID NOs: 20 and 111 to 113, respectively, including post-translational modifications of the sequences. In one embodiment, the antibody comprises the VH sequence of SEQ ID NO: 10 and the VL sequence of SEQ ID NO: 20. In one embodiment, the antibody comprises the VH sequence of SEQ ID NO: 106 and the VL sequence of SEQ ID NO: 111. In another embodiment, the antibody comprises the VH sequence of SEQ ID NO: 107 and the VL sequence of SEQ ID NO: 111. In another embodiment, the antibody comprises the VH sequence of SEQ ID NO: 108 and the VL sequence of SEQ ID NO: 111. In another embodiment, the antibody comprises the VH sequence of SEQ ID NO: 109 and the VL sequence of SEQ ID NO: 111. In another embodiment, the antibody comprises the VH sequence of SEQ ID NO: 109 and the VL sequence of SEQ ID NO: 112. In another embodiment, the antibody comprises the VH sequence of SEQ ID NO: 109 and the VL sequence of SEQ ID NO: 113. In another embodiment, the antibody comprises the VH sequence of SEQ ID NO: 110 and the VL sequence of SEQ ID NO: 113.

In one embodiment, (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 54, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 64, and (c) HVR comprising the amino acid sequence of SEQ ID NO: 74 VH sequence containing -H3; And (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 84, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 94, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 104. Anti-C5 antibodies comprising the containing VL sequence are provided.

In another embodiment, (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118, and (c) comprising the amino acid sequence of SEQ ID NO: 121 VH sequence containing HVR-H3; And (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125. Anti-C5 antibodies comprising the containing VL sequence are provided.

In another embodiment, (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 119, and (c) comprising the amino acid sequence of SEQ ID NO: 121 VH sequence containing HVR-H3; And (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125. Anti-C5 antibodies comprising the containing VL sequence are provided.

In another embodiment, (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118, and (c) comprising the amino acid sequence of SEQ ID NO: 121 VH sequence containing HVR-H3; And (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 124, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125. Anti-C5 antibodies comprising the containing VL sequence are provided.

In another embodiment, (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 120, and (c) comprising the amino acid sequence of SEQ ID NO: 121 VH sequence containing HVR-H3; And (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 124, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125. Anti-C5 antibodies comprising the containing VL sequence are provided.

In certain embodiments, the anti-C5 antibody of the invention comprises a heavy chain constant region comprising the VH in any of the embodiments provided above and the amino acid sequence of any one of SEQ ID NOs: 33, 34, 35, 114, 115 and 116. Includes. In certain embodiments, an anti-C5 antibody of the invention comprises a light chain constant region comprising the VL in any of the embodiments provided above and an amino acid sequence of any one of SEQ ID NOs: 36, 37 and 38.

In another aspect, the invention provides an antibody that binds to the same epitope as an anti-C5 antibody provided herein. For example, in certain embodiments, antibodies are provided that bind to the same epitope as the antibodies described in Table 2. As demonstrated by the examples below, all anti-C5 antibodies described in Table 2 were classified as the same epitope bin of C5 and exhibited pH-dependent binding properties.

In another aspect, the invention provides antibodies that bind to the same epitope as the antibodies provided herein. In another aspect, the invention provides antibodies that bind to the same epitope as the antibodies described in Tables 7 or 8. In certain embodiments, antibodies are provided that bind to an epitope within a fragment consisting of amino acids 33 to 124 of the beta chain of C5 (SEQ ID NO: 40). In certain embodiments, antibodies are provided that bind to an epitope in the beta chain of C5 (SEQ ID NO: 40) comprising one or more fragments selected from the group consisting of amino acids 47-57, 70-76, and 107-110. In certain embodiments, comprises one or more amino acids selected from the group consisting of Thr47, Glu48, Ala49, Phe50, Asp51, Ala52, Thr53, Lys57, His70, Val71, His72, Ser74, Glu76, Val107, Ser108, Lys109, and His110. An antibody that binds to an epitope in a fragment of the C5 beta chain (SEQ ID NO: 40) is provided. In another embodiment, the epitope of the anti-C5 antibody of the invention is a conformational epitope.

In another aspect of the invention, the anti-C5 antibodies according to any of the above embodiments are monoclonal antibodies, including chimeric, humanized or human antibodies. In one embodiment, the anti-C5 antibody is an antibody fragment, eg, Fv, Fab, Fab', scFv, diabody or F(ab') ₂ fragment. In another embodiment, the antibody is a full length antibody, e.g., an intact IgG1 or IgG4 antibody or another antibody class or isotype as defined herein.

In another aspect, an anti-C5 antibody according to any of the above embodiments may comprise any of the features, alone or in combination, as described in Sections 1 to 7 below.

1. Antibody affinity

In certain embodiments, the antibodies provided herein are 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., 10 ^-8 M or less, e.g. For example, it has a dissociation constant (Kd) of 10 ^-8 to 10 ^-13 M, for example, 10 ^-9 to 10 ^{-13 M).}

In one embodiment, Kd is measured by radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using the Fab version of the antibody in question and its antigen. ^{For example, the solution binding affinity of Fab for the antigen is determined by equilibrating the Fab with a minimum concentration of (125} I)-labeled antigen in the presence of a serial titration of an unlabeled antigen, and then converting the bound antigen to an anti-Fab antibody. -Measured by capture with coated plates (see, eg, Chen et al., J. Mol. Biol . 293:865-881 (1999)). To set the conditions for the assay, a microtiter (MICROTITER®) multi-well plate (Thermo Scientific) was placed in 50 mM sodium carbonate (pH 9.6) at 5 μg/ml of the capture anti-Fab antibody. It was coated overnight with (Cappel Labs) and then blocked for 2 to 5 hours with 2% (w/v) bovine serum albumin in PBS at room temperature (about 23° C.). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM of [ ¹²⁵ I]-antigen is mixed with serial dilutions of the Fab in question (eg, Presta et al., Cancer Res). . 57: 4593-4599 (1997)] anti -VEGF antibody, to match the evaluation of Fab-12) in the. The Fab is then incubated overnight, but the incubation may last longer (eg, about 65 hours) to ensure equilibrium is achieved. Thereafter, the mixture is transferred to a capture plate for incubation at room temperature (eg, for about 1 hour). The solution is then removed and the plate washed 8 times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. When the plate is dry, 150 μl/well of scintillant (MICROSCINT-20™; Packard) is added, and the plate is placed in a TOPCOUNT™ gamma counter (Packard) for 10 minutes. Count on phase. The concentration of each Fab that provides up to 20% of maximal binding is selected for use in competitive binding assays.

According to another embodiment, Kd is measured using Biacore® surface plasmon resonance assay. For example, analysis using Biacore (registered trademark)-2000 or Biacore (registered trademark)-3000 (Biacore (registered trademark), Inc., Piscataway, NJ, USA) was performed at 25° C. for about 10 reactions. It is carried out using the immobilized antigen CM5 chip in the response unit (RU). In one embodiment, a carboxymethylated dextran biosensor chip (CM5, Biacore®, Inc.) is N-ethyl-N'-(3-dimethylaminopropyl)-carbodiyi according to the supplier's instructions. Activated with mid hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The antigen is diluted with 10 mM sodium acetate (pH 4.8) to 5 μg/ml (about 0.2 μM) and then injected at a flow rate of 5 μl/min to achieve about 10 response units (RU) of coupled protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) were added in PBS containing 0.05% polysorbate 20 (Tween-20™) surfactant at 25° C. at a flow rate of about 25 μl/min ( PBST). The binding rate (k _on ) and dissociation rate (k _off ) are a simple one-to-one Langmuir binding model (Biacore® Evaluation Software) by simultaneously fitting the binding and dissociation sensorgrams. It is calculated using version 3.2). The equilibrium dissociation constant (Kd) is _{calculated as the ratio k off} /k _on (see, eg, Chen et al., J. Mol. Biol. 293:865-881 (1999)). ^{When the on-rate exceeds 10 6} M ^-1 s ^{- 1} by the surface plasmon resonance analysis method described above, the on-rate is a spectrophotometer, for example, a spectrophotometer equipped with a stop-flow rectifier. 20 nM in PBS (pH 7.2) in the presence of increasing concentrations of antigen as measured on (Aviv Instruments) or 8000-series SLM-AMINCO™ spectrophotometer with stirring cuvette (ThermoSpectronic) The anti-antigen antibody (Fab form) can be measured using a fluorescence quenching technique that measures the increase or decrease in the fluorescence emission intensity at 25°C (excitation = 295 nm; emission = 340 nm, 16 nm passband). have.

2. Antibody fragment

In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') ₂ , Fv and scFv fragments, and other fragments described below. For review of specific antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For review of scFv fragments, see, eg, Pleckthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994)]; See also International Patent Publication No. 93/16185; And U.S. Patent Nos. 5,571,894 and 5,587,458. _{For a review of Fab and F(ab') 2} fragments that contain regenerative receptor binding epitope residues and have an increased half-life in vivo, see US Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites, which may be bivalent or bispecific (eg, European Patent No. 0 404 097; International Patent Publication No. 1993/01161; Hudson et al. , Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or part of the heavy chain variable domain, or all or part of the light chain variable domain of the antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, Mass.); See, for example, U.S. Patent No. 6,248,516).

Antibody fragments are, as described herein, proteolytic cleavage of intact antibodies, and recombinant host cells (e.g., Escherichia coli (Escherichia coli ) or phage).

3. Chimeric and humanized antibodies

In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, eg, in US Pat. Nos. 4,816,567; And Morrison et al. , Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984). In one example, chimeric antibodies include non-human variable regions (eg, variable regions derived from non-human primates such as mice, rats, hamsters, rabbits, or monkeys) and human constant regions. In another example, a chimeric antibody is a “class converted” antibody in which the class or subclass has been changed from the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while maintaining the specificity and affinity of the non-human parent antibody. In general, a humanized antibody comprises one or more variable domains in which the HVR, eg, the CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. The humanized antibody will optionally also comprise at least a portion of a human constant region. In some embodiments, some of the FR residues in a humanized antibody are corresponding to from a non-human antibody (e.g., an antibody from which HVR residues are derived), e.g., to restore or improve antibody specificity or affinity. Is substituted with a residue.

Humanized antibodies and methods for their preparation are described, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), for example, Riechmann et al. , Nature 332:323-329 (1988); Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989)]; US Patents 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describes specificity determining region (SDR) grafting); See Padlan, Mol. Immunol. 28:489-498 (1991)] (describes "resurfacing");Dall'Acqua et al., Methods 36:43-60 (2005) (describes “FR shuffling”); And Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer 83:252-260 (2000)] (which describes a "directed selection" approach to FR shuffling).

Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using the "best-fit" method (see, for example, Sims et al. , J. Immunol. 151:2296-2308 (1993)); Framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (eg, Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-4289 (1992); and Presta et al., J. Immunol. 151:2623-2632 (1993)); Human mature (somatic mutated) framework regions or human germline framework regions (see, eg, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); And framework regions derived from FR library searches (eg Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be generated using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharma. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

Human antibodies can be prepared 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 antigenic stimulation. Such animals typically contain all or part of a human immunoglobulin locus, which replaces the endogenous immunoglobulin locus, is extrachromosomally, or is randomly integrated into the animal's chromosome. In these transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, US Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™ technology; U.S. Patent No. 5,770,429 describing HUMAB (registered trademark) technology; See U.S. Patent No. 7,041,870, describing K-M mouse (registered trademark) technology, and U.S. Patent Publication No. 2007/0061900, describing VELOCIMOUSE technology. Human variable regions from intact antibodies produced by these animals can be further modified, for example by binding with different human constant regions.

Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described (see, eg, Kozbor, J. Immunol. 133:3001 (1984); Brodeur et al., Monoclonal Antibody. Production Techniques and Applications, pp. 51-63 (Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol. 147:86 (1991)). Human antibodies produced by human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006). Other methods include, for example, US Pat. No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue 26(4):265-268 (2006). ] (Which describes a human-human hybridoma). Human hybridoma technology (Trioma technology) is also described in Vollmers, Histology and Histopathology 20(3):927-937 (2005) and Vollmers, Methods and Findings in Experimental and Clinical Pharmacology 27(3):185. -191 (2005)].

Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. The variable domain sequence can then be combined with the human constant domain of interest. Techniques for selecting human antibodies from antibody libraries are described below.

5. Library-derived antibodies

Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies having the desired activity or activities. For example, various methods are known in the art for constructing a phage display library and screening the library for antibodies having the desired binding properties. Such methods are outlined in, for example, Hoogenboom et al., Methods in Molecular Biology 178:1-37 (2001), and are described, for example, in McCafferty et al., Nature 348:552-554 ; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks, Meth. Mol. Biol. 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and randomly recombined in a phage library, followed by Winter et al., Ann. Rev. Immunol. 12:433-455 (1994)]. Phage typically displays antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, as described in Grifiths et al., EMBO J. 12:725-734 (1993), a naive repertoire was cloned (eg, from humans) to provide extensive non- It is possible to provide a single source of antibodies against autologous and also autologous antigens. Finally, naive libraries are also described in Hoogenboom and Winter, J. Mol. Biol. 227:381-388 (1992), cloned non-rearranged V-gene segments from stem cells, and used PCR primers containing random sequences to encode and test highly variable CDR3 regions. It can be made synthetically by achieving rearrangement in the tube. Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373 and U.S. Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936 and 2009/0002360 are included.

Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecific antibody, eg, a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for two or more different sites. In certain embodiments, one of the binding specificities is for C5 and the other is for any other antigen. In certain embodiments, bispecific antibodies are capable of binding two different epitopes of C5. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing C5. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.

As a technique for producing a multispecific antibody, the recombinant co-expression of two immunoglobulin heavy-light chain pairs having different specificities (Milstein and Cuello, Nature 305:537-540 (1983)); International Patent Publication 93 /08829; and Traunecker et al., EMBO J. 10:3655-3659 (1991)), and "knob-in-hole" manipulation (e.g., U.S. Patent 5,731,168). Multispecific antibodies can also be used by manipulating the electrostatic steering effect for producing antibody Fc-heterodimer molecules (International Patent Publication No. 2009/089004A1); By crosslinking two or more antibodies or fragments (see, eg, US Pat. No. 4,676,980; and Brennan et al., Science 229:81-83 (1985)); By generating bispecific antibodies using leucine zippers (see, eg, Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)); By using “diabody” technology to prepare bispecific antibody fragments (see, eg, Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); And by using single-chain Fv (sFv) dimers (see, eg, Gruber et al., J. Immunol. 152:5368-5374 (1994)); And, eg, Tutt et al., J. Immunol. 147:60-69 (1991)].

Engineered antibodies having three or more functional antigen binding sites, including “Octopus antibodies”, are also included herein (see, eg, US 2006/0025576).

The antibody or fragment herein also includes a “dual acting Fab” or “DAF” comprising an antigen binding site that binds to C5 as well as other different antigens (see, eg, US Patent Publication No. 2008/0069820). .

7. Antibody variants

In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletion of residues from the amino acid sequence of the antibody, and/or insertion of residues into the sequence and/or substitution of residues within the sequence. Any combination of deletion, insertion and substitution can be performed to obtain the final construct, but the final construct must possess the desired properties, such as antigen-binding properties.

a. Substitution, insertion and deletion variants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Corresponding sites for substitutional mutagenesis include HVR and FR. Conservative substitutions are shown in Table 1 under the heading "Preferred substitutions". More substantial changes are provided in Table 1 under the heading "exemplary substitutions" and as further described below with respect to the amino acid side chain class. Amino acid substitutions can be introduced into the antibody of interest, and products can be selected for a desired activity, such as maintenance/improved antigen binding activity, reduced immunogenicity, or improved ADCC or CDC.

Amino acids can be classified according to their usual side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilicity: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Gin; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; And (6) aromatic: Trp, Tyr, Phe.

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

One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (eg, a humanized or human antibody). In general, the resulting variant(s) selected for further study will have a change (e.g., improvement) in certain biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody. / Or will substantially retain the specific biological properties of the parent antibody. Exemplary substitutional variants are affinity enhanced antibodies that can conveniently be generated, for example, using phage display-based affinity enhancing techniques as described herein. Briefly, one or more HVR residues are mutated and variant antibodies are displayed on phage and screened for specific biological activity (eg, binding affinity).

For example, to improve antibody affinity, mutations (eg, substitutions) can be made in the HVR. Such mutations are HVR “hotspots,” ie residues encoded by codons that mutate at high frequencies during the somatic maturation process (see, eg, Chowdhury, Methods Mol. Biol. 207:179-196 (2008) )], and/or at a residue in contact with the antigen, wherein the resulting variant VH or VL is tested for binding affinity. Affinity enhancement by constructing a secondary library and re-selecting therefrom is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (2002). In some embodiments of affinity enhancement, changes in a variable gene selected for enhancement by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). Is introduced. Then, a secondary library is built. The library is then screened to identify any antibody variants with the desired affinity. Other methods of introducing changes include an HVR-oriented approach in which several HVR residues (eg, 4 to 6 residues at a time) are randomly selected. HVR residues involved in antigen binding can be specifically identified using, for example, alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are particularly often targeted.

In certain embodiments, substitutions, insertions or deletions may occur within one or more HVRs so long as the variation does not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative variations that do not substantially reduce binding affinity (eg, conservative substitutions as provided herein) can be made in the HVR. The mutation can occur outside of the antigen-contacting residue in the HVR. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unmutated or contains no more than 1, 2 or 3 amino acid substitutions.

A useful method for the identification of residues or regions of an antibody that can be targeted for mutagenesis is called “alanine scanning mutagenesis” as described in Cunningham, Science 244:1081-1085 (1989). In this method, in order to determine whether the interaction of the antibody with the antigen is affected, the residue or group of the target residues (e.g., charged residues such as arg, asp, his, lys and glu) is identified and neutralized. Or a negatively charged amino acid (eg, alanine or polyalanine). Additional substitutions may be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex to identify the point of contact between the antibody and the antigen. The contacting moieties and adjacent moieties can be targeted or removed as candidates for substitution. Variants can be screened to determine if they possess the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to a polypeptide containing more than a hundred residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include fusion to the N- or C-terminus of the antibody to an enzyme (eg, ADEPT) or a polypeptide that increases the serum half-life of the antibody.

b. Glycosylation variants

In certain embodiments, antibodies provided herein are mutated to increase or decrease the degree to which the antibody is glycosylated. The addition or deletion of a glycosylation site to an antibody can conveniently be accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

When the antibody comprises an Fc region, the carbohydrate bound thereto may be mutated. Native antibodies produced by mammalian cells typically comprise branched biantennary oligosaccharides, generally N-linked to Asn297 of the CH2 domain of the Fc region (e.g., See Wright, TIBTECH 15:26-32 (1997)). Oligosaccharides can include a variety of carbohydrates, such as mannose, N-acetyl glucosamine (GlcNAc), galactose and sialic acid, as well as fucose bound to GlcNAc in the "stem" of the biantennary oligosaccharide structure. have. In some embodiments, modifications of oligosaccharides can be made in the antibodies of the invention to generate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydrate structure that lacks fucose bound (directly or indirectly) to the Fc region. For example, the amount of fucose in the antibody may be 1 to 80%, 1 to 65%, 5 to 65%, or 20 to 40%. The amount of fucose is, for example, as described in International Patent Publication No. 2008/077546, as measured by MALDI-TOF mass spectrometry, all glycostructures bound to Asn297 (e.g., complexes, hybrids And the sum of the mannose high content structure), as measured by calculating the average amount of fucose in the sugar chain at Asn297. Asn297 refers to an asparagine residue located approximately at position 297 in the Fc region (EU numbering system for Fc region residues); Asn297 may also be located upstream or downstream of about +/-3 amino acids of position 297, ie, between positions 294 and 300, due to minor sequence changes in the antibody. The fucosylated variant may have an improved ADCC function (e.g., U.S. Patent Publication No. 2003/0157108 (Presta, L.)); U.S. Patent Publication No. 2004/0093621 (Kyo Wa Hakko Kogyo Co., Ltd.). Examples of publications relating to “defucosylated” or “fucose-deficient” antibody variants include: US Patent Publication No. 2003/0157108; International Patent Publication No. 2000/61739; International Patent Publication No. 2001/29246; US Patent Publication No. 2003/0115614; US Patent Publication No. 2002/0164328; US Patent Publication No. 2004/0093621; US Patent Publication No. 2004/0132140; US Patent Publication No. 2004/0110704; US Patent Publication No. 2004/0110282; US Patent Publication No. 2004/0109865; International Patent Publication No. 2003/085119; International Patent Publication No. 2003/084570; International Patent Publication No. 2005/035586; International Patent Publication No. 2005/035778; International Patent Publication No. 2005/053742; International Patent Publication No. 2002/031140; Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004)]. Examples of cell lines capable of producing afucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986)]; US Patent Publication No. 2003/0157108, Presta L.; And International Patent Publication No. 2004/056312, Adams et al., in particular Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells. (See, for example, Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda et al. , Biotechnol. Bioeng. 94:680-688 (2006)); and International Patent Publication No. 2003 /085107) are included.

For example, antibody variants are also provided having biantennary oligosaccharides bound to the Fc region of the antibody and bisecting oligosaccharides bisected by GlcNAc. The antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants include, for example, International Patent Publication No. 2003/011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); And US Patent Publication No. 2005/0123546 (Umana et al.). Antibody variants having one or more galactose residues in the oligosaccharide bound to the Fc region are also provided. The antibody variants may have improved CDC function. The antibody variants include, for example, International Patent Publication No. 1997/30087 (Patel et al.); International Patent Publication No. 1998/58964 (Raju, S.); And International Patent Publication No. 1999/22764 (Raju S).

c. Fc region variant

In certain embodiments, Fc region variants can be generated by introducing one or more amino acid modifications into the Fc region of an antibody provided herein. Fc region variants may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising amino acid modifications (e.g., substitutions) at one or more amino acid positions.

In certain embodiments, the present invention provides that the half-life of the antibody in vivo is important, but has some, but not all, effector functions that make certain effector functions (e.g., complement and ADCC) a preferred candidate for unnecessary or detrimental applications. Consider antibody variants. In vitro and/or in vivo cytotoxicity assays can be performed to confirm reduction/depletion of CDC and/or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks Fc gamma R binding (and thus tends to lack ADCC activity), but retains the ability to bind FcRn. NK cells, the primary cells for mediating ADCC, express only Fc gamma RIII, whereas monocytes express Fc gamma RI, Fc gamma RII and Fc gamma RIII. FcR expression on hematopoietic cells is described in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991)]. Non-limiting examples of in vitro assays for assessing ADCC activity of a given molecule are described in US Pat. No. 5,500,362 (see, for example, Hellstrom et al., Proc. Natl. Acad. Sci. USA 83:7059-7063 ( 1986); and Hellstrom et al., Porc. Natl. Acad. Sci. USA 82:1499-1502 (1985)); US Patent No. 5,821,337 (see Brueggemann et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be used (e.g., ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, CA, USA); And CytoTox 96 (registered trademark) non-radioactive cytotoxicity assay (see Promega, Madison, Wis.). Useful effector cells for these assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule in question can be determined in vivo, eg, in Clynes et al., Proc. Nat'l. Acad. Sci. USA 95:652-656 (1998)]. C1q binding assays can also be performed to confirm that the antibody is unable to bind C1q and thus lacks CDC activity (e.g., in International Patent Publication No. 2006/029879 and International Patent Publication No. 2005/100402. C1q and C3c binding ELISA). To assess complement activation, CDC analysis can be performed (eg, Gazzano-Santoro et al., J. Immunol. Methods 202:163-171 (1996); Cragg et al., Blood 101: 1045-1052 (2003); and Cragg et al., Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half-life measurements can also be performed using methods known in the art (eg Petkova et al., Int'l. Immunol. 18(12):1759-1769). (2006)].

Antibodies with reduced effector function include antibodies with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (US Pat. No. 6,737,056). The Fc mutant includes a so-called "DANA" Fc mutant having a substitution of residues 265 and 297 to alanine, and an Fc mutant having a substitution at two or more of amino acid positions 265, 269, 270, 297 and 327 Included (US Pat. No. 7,332,581).

Certain antibody variants with improved or reduced binding to FcR have been described (eg, US Pat. No. 6,737,056; International Patent Publication No. 2004/056312; and Shields et al., J. Biol. Chem. 276:6591-6604 (2001)).

In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333 and/or 334 (the EU numbering system of residues) of the Fc region. .

In some embodiments, for example, US Pat. No. 6,194,551, International Patent Publication No. 99/51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000)], mutations that result in mutated (ie, improved or reduced) Clq binding and/or complement dependent cytotoxicity (CDC) are made in the Fc region.

The neonatal Fc receptor (FcRn) responsible for increased half-life and delivery of complement IgG to the fetus (Guyer et al., J. Immunol. 117:587-593 (1976); and Kim et al., J. Immunol 24:2429-2434 (1994)]) have been described in US Patent Publication No. 2005/0014934 (Hinton et al.). The antibodies include an Fc region having one or more substitutions that improve the binding of the Fc region to FcRn among them. The Fc variant includes Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 And variants having a substitution at one or more of 434, eg, substitution of Fc region residue 434 (US Pat. No. 7,371,826).

For other examples of Fc region variants, see also Duncan, Nature 332:738-740 (1988); US Patent No. 5,648,260; U.S. Patent No. 5,624,821; And International Patent Publication No. 94/29351.

d. Cysteine engineered antibody variants

In certain embodiments, it may be desirable to generate cysteine engineered antibodies, eg, “thioMAbs,” in which one or more residues of the antibody have been substituted with cysteine residues. In certain embodiments, the substituted moiety is present at an accessible site of the antibody. By substituting these residues with cysteine, a reactive thiol group is located at an accessible site of the antibody, and the antibody is conjugated to another residue, such as a drug residue or a linker-drug residue, to create an immunoconjugate as further described herein. Can be used to In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering system) of the light chain; Heavy chain A118 (EU numbering system); And S400 (EU numbering system) of the heavy chain Fc region. Cysteine engineered antibodies can be prepared, for example, as described in US Pat. No. 7,521,541.

e. Antibody derivative

In certain embodiments, antibodies provided herein may be further modified to contain additional non-proteinaceous moieties known in the art and readily available. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly -1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propropylene glycol homopolymer , Polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol (eg glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. Polymers may have any molecular weight and may or may not be branched. The number of polymers bound to the antibody may vary, and if more than one polymer is bound, they may be the same or different molecules. In general, the number and/or type of polymers used for derivatization include, but are not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used in therapy under defined conditions, etc. It can be determined on the basis of.

In another embodiment, a conjugate of an antibody and a non-proteinaceous moiety that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005).) The radiation can be at any wavelength. It may have, but is not harmful to normal cells, but includes, but is not limited to, the wavelength of heating the non-proteinaceous moiety to a temperature at which cells close to the antibody-non-proteinaceous moiety die.

B. Recombinant method and composition

Antibodies can be prepared using recombinant methods and compositions as described, for example, in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-C5 antibody described herein is provided. The nucleic acid may encode an amino acid sequence including VL and/or an amino acid sequence including VH of an antibody (eg, light chain and/or heavy chain of an antibody). In another embodiment, one or more vectors (eg, expression vectors) comprising said nucleic acid are provided. In another embodiment, a host cell comprising said nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., transformed with): (1) a nucleic acid encoding the amino acid sequence comprising the VL of the antibody and the amino acid sequence comprising the VH of the antibody. A vector comprising, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, eg, a Chinese Hamster Ovary (CHO) cell or a lymphatic cell (eg, Y0, NS0, Sp20 cell). In one embodiment, comprising culturing a host cell comprising a nucleic acid encoding an antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium), Methods of making anti-C5 antibodies are provided.

For recombinant production of anti-C5 antibodies, for example, nucleic acids encoding antibodies as described above are isolated and inserted into one or more vectors for further cloning and/or expression in host cells. The nucleic acid can be easily isolated and sequenced using conventional procedures (eg, by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of an 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 Fc effector functions are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, US Pat. Nos. 5,648,237, 5,789,199 and 5,840,523 (see also Charlton, which describes the expression of antibody fragments in Escherichia coli. , Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254). After expression, the antibody can be isolated from the bacterial cell paste in the soluble fraction and can be further purified.

In addition to prokaryotic cells, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable for antibody-encoding vectors, including fungal and yeast strains where the glycosylation pathway is "humanized" resulting in the production of antibodies with partially or completely human glycosylation patterns It is a cloning or expression host (see Gerngross, Nat. Biotech. 22:1409-1414 (2004); and Li et al., Nat. Biotech. 24:210-215 (2006)).

Host cells suitable for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. In particular, Spodoptera frigiferda (Spodoptera frugiperda ) For transfection of cells, many baculovirus strains have been identified that can be used with insect cells.

Plant cell cultures can also be used as hosts (e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 (Plantibody for producing antibodies in transgenic plants). PLANTIBODIES™ technology description).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growing in a suspended state may be useful. Other examples of useful mammalian host cell lines include the monkey kidney CV1 cell line transformed by SV40 (COS-7); Human fetal kidney cell lines (eg, 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59-74 (1977)); Baby hamster kidney cells (BHK); Mouse sertoli cells (eg, TM4 cells as described in Mother, Biol. Reprod. 23:243-252 (1980)); Monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); Human cervical cancer cells (HELA); Canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); Human lung cells (W138); Human hepatocytes (Hep G2); Mouse breast cancer cells (MMT 060562); See, for example, Mother et al., Annals NY Acad. Sci. TRI cells as described in 383:44-68 (1982); MRC5 cells; And FS4 cells. Other useful mammalian host cell is DHFR mainly ^_ CHO cells (as described [Urlaub et al, Proc Natl Acad Sci USA 77:..... 4216-4220 (1980)]) Chinese hamster ovary (CHO) cells, including; And myeloma cell lines such as Y0, NS0 and Sp2/0. For review of specific mammalian host cell lines suitable for antibody production, see, eg, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003)].

Polyclonal antibodies are preferably increased in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of relevant antigens and adjuvants. Bifunctional agents or derivatizing agents, such as maleimidobenzoyl sulfosuccinimide ester (conjugated via cysteine residue), N-hydroxysuccinimide (via lysine residue), glutaraldehyde, succinic anhydride, SOCl ₂ Or a protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin or soybean, using ^{R 1} N=C=NR (where R and R ^{1 are different alkyl groups).} It may be useful to conjugate the relevant antigen to a trypsin inhibitor.

Animals (typically non-human mammals), for example, 100 μg or 5 μg of protein or conjugate (for rabbit or mouse, respectively) are mixed with 3 volumes of Freund's complete adjuvant and the solution is Immunization against antigens, immunogenic conjugates or derivatives by intradermal injection at several sites. After 1 month, animals are boosted with 1/5 to 1/10 of the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at several sites. After 7-14 days, the animals are bled and the serum is analyzed for antibody titer. Animals are boosted until titer plateaus are reached. Preferably, animals are boosted with conjugates of the same antigen, but conjugated to different proteins and/or conjugated via different crosslinking reagents. Conjugates can also be prepared as protein fusions in recombinant cell culture. In addition, a coagulant such as alum is suitably used to enhance the immune response.

Monoclonal antibodies are obtained from a population of substantially homologous antibodies. That is, the individual antibodies constituting the population are identical except for possible natural mutations and/or post-translational modifications (eg, isomerization, amidation) that may be present in trace amounts. Thus, the modifier “monoclonal” denotes the properties of an antibody as not being a mixture of separate antibodies.

For example, monoclonal antibodies are described in Kohler et al. Nature 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 to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. . Alternatively, lymphocytes can be immunized in vitro.

The immunizing agent will typically comprise an antigenic protein or a fusion variant thereof. In general, peripheral blood lymphocytes (PBL) are used when cells of human origin are desired, or spleen cells or lymph node cells are used when a non-human mammalian source is desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, for example polyethylene glycol, to produce hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59 -103]).

Immortalized cell lines are typically transformed mammalian cells, especially myeloma cells of rodent, bovine and human origin. Typically, rat or mouse myeloma cell lines are used. The hybridoma cells prepared as above are preferably inoculated and grown in an appropriate culture medium containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for hybridomas is typically hypoxanthine, substances that prevent the growth of HGPRT-deficient cells, Will include aminopterin and thymidine ((HAT medium).

Preferred immortalized myeloma cells are cells that are effectively fused and promote stable high level production of antibodies by selected antibody-producing cells and are sensitive to media such as HAT media. Among these, murine myeloma cell lines, e.g., cell lines derived from MOPC-21 and MPC-11 mouse tumors available from Salk Institute Cell Distribution Center, San Diego, CA, and Manor, Virginia, USA SP-2 cells (and derivatives thereof, such as X63-Ag8-653) available from Sus' American Type Culture Collection are preferred. Human myeloma and mouse-human xenomyeloma cell lines have also been described for the generation of human monoclonal antibodies (Kozbor et al., J Immunol. 133(6):3001-3005 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987), pp. 51-63]).

The culture medium in which hybridoma cells are grown is analyzed for the production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibody produced by hybridoma cells is determined by immunoprecipitation, or by an in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). do. Such techniques and assays are known in the art. For example, binding affinity is described in Munson, Anal Biochem. 107(1):220-239 (1980)].

After hybridoma cells producing antibodies with the desired specificity, affinity and/or activity have been identified, the clones are subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). I can. Culture medium suitable for the above purpose includes, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as tumors in mammals.

Monoclonal antibodies secreted by subclones are, for example, culture medium, ascites, by conventional immunoglobulin purification procedures such as protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. Or is appropriately separated from serum.

Antibodies can be generated by immunizing an appropriate host animal against an antigen. In one embodiment, the antigen is a polypeptide comprising full length C5. In one embodiment, the antigen is a polypeptide comprising the beta chain of C5 (SEQ ID NO: 40). In one embodiment, the antigen is a polypeptide comprising the MG1-MG2 domain of the beta chain of C5 (SEQ ID NO: 43). In one embodiment, the antigen is a polypeptide comprising the MG1 domain of the beta chain of C5 (SEQ ID NO: 41). In one embodiment, the antigen is a polypeptide comprising a region corresponding to an amino acid at positions 19 to 180 of the beta chain of C5. In one embodiment, the antigen is a polypeptide comprising a region corresponding to an amino acid at positions 33 to 124 of the beta chain of C5. In one embodiment, the antigen is a polypeptide comprising one or more fragments selected from amino acids 47-57, 70-76, and 107-110 of the beta chain of C5 (SEQ ID NO: 40). In one embodiment, the antigen is at least one amino acid selected from the group consisting of Thr47, Glu48, Ala49, Phe50, Asp51, Ala52, Thr53, Lys57, His70, Val71, His72, Ser74, Glu76, Val107, Ser108, Lys109, and His110. It is a polypeptide containing a fragment of the beta chain of C5 containing. In one embodiment, the antigen is a polypeptide comprising a fragment of the beta chain of C5 comprising one or more amino acids selected from the group consisting of Glu48, Asp51, His70, His72, Lys109, and His110. Antibodies produced by immunizing animals against an antigen are also included in the present invention. The antibodies may contain any of the features, alone or in combination, as described in the “Tritorial Anti-C5 Antibodies” above.

C. Analysis method

Anti-C5 antibodies provided herein can be identified, screened or characterized for their physical/chemical properties and/or biological activity by various assays known in the art.

1. Binding method and other method

In one embodiment, the antibody of the present invention is tested for its antigen binding activity by known methods such as, for example, ELISA, Western Blot, Biacore® and the like.

In another embodiment, competition assays can be used to identify antibodies that compete with the anti-C5 antibodies described herein for binding to C5. In certain embodiments, when the competing antibody is present in excess, the antibody inhibits binding of the reference antibody to C5 by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%. , 50%, 55%, 60%, 65%, 70%, 75% or more block (e.g., reduce). In some cases, binding is inhibited by at least 80%, 85%, 90%, 95% or more. In certain embodiments, the competing antibody binds to the same epitope (eg, linear or conformational epitope) bound by the anti-C5 antibody described herein. Detailed exemplary methods for mapping epitopes to which antibodies bind are described in Morris, “Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ) (1996).

In an exemplary competition assay, in a solution comprising 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, Incubate the immobilized C5. The second antibody may be present in the hybridoma supernatant. As a control, immobilized C5 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to C5, excess unbound antibody is removed, and the amount of the label bound to the immobilized C5 is measured. If the amount of label bound to immobilized C5 is substantially reduced in the test sample compared to the control sample, it indicates that the second antibody competes with the first antibody for binding to C5 (Harlow and Lane, Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) (1988)).

In another exemplary competition assay, a Biacore® assay is used to determine the ability of a test anti-C5 antibody to compete with binding to C5 by a second (reference) anti-C5 antibody. In another aspect of operating a Biacore® device (e.g. Biacore® 3000) according to the manufacturer's recommendations, the C5 protein is known in the art to create a C5-coated surface. Captured on a CM5 Biacore® chip using standard techniques. Typically 200 to 800 resonance units of C5 are coupled to the chip (an amount that provides an easily measurable level of binding but can be easily saturated by the concentration of the test antibody used). The two antibodies to be evaluated for their ability to compete with each other (i.e., the test antibody and the reference antibody) were mixed at the binding site in a 1:1 molar ratio in a suitable buffer to create a test mixture. When calculating the concentration based on the binding site, the molecular weight of the test antibody or reference antibody is estimated as the total molecular weight of the corresponding antibody divided by the number of C-binding sites on the antibody. The concentration of each antibody (i.e., test antibody and reference antibody) in the test mixture should be high enough to readily saturate the binding site for the antibody on the C5 molecule captured on the Biacore® chip. The test antibody and reference antibody in the mixture are present at the same molar concentration (by binding basis), typically 1.00 to 1.5 μM (by binding site). Separate solutions containing only the test antibody and only the reference antibody are also prepared. The test antibody and reference antibody in the above solutions should be present in the same buffer and at the same concentration and conditions as in the test mixture. The test mixture containing the test antibody and the reference antibody is transferred onto a C5-coated Biacore® chip and the total amount of binding is recorded. The chip is then treated in such a way that the bound test antibody or reference antibody is removed without damaging the chip-bound C5. Typically, the treatment is carried out by treating the chip with 30 mM HCl for 60 seconds. Then, a solution of only the test antibody is transferred onto the C5-coated surface and the amount of binding is recorded. The chip is again treated to remove all bound antibodies without damaging the chip-bound C5. Then, a solution of only the reference antibody is transferred onto the C5-coated surface and the amount of binding is recorded. Next, the maximum theoretical binding of the mixture of test antibody and reference antibody is calculated, the amount being the sum of the binding of each antibody (i.e., test and reference antibody) as it migrates only over the C5 surface. If the actual recorded binding of the mixture is below this theoretical maximum, the test antibody and reference antibody compete with each other for binding to C5. Thus, in general, the competing test anti-C5 antibody is determined in the Biacore® blocking assay, during the assay and in the presence of the reference anti-C5 antibody, the recorded binding is the maximum theoretical binding of the test antibody and the reference antibody together. 80% to 0.1% (e.g., 80% to 4%) of, especially 75% to 0.1% (e.g., 75% to 4%) of the maximum theoretical binding, more particularly the maximum theoretical binding (as defined above) ) To be 70% to 0.1% (e.g., 70% to 4%) of C5.

In certain embodiments, an anti-C5 antibody of the invention competes for binding to C5 with an antibody comprising a VH and VL pair selected from antibodies CFA0341 and CFA0330. In some embodiments, the anti-C5 antibody competes with an antibody selected from CFA0538, CFA0501, CFA0599, CFA0307, CFA0366, CFA0675, and CFA0672 for binding to C5. In some embodiments, the anti-C5 antibody competes with antibody CFA0329 for binding to C5. In some embodiments, the anti-C5 antibody competes with antibody CFA0666 for binding to C5.

In certain embodiments, an anti-C5 antibody of the invention competes for binding to C5 with an antibody comprising a VH and VL pair of antibody CFA0305 or 305LO5.

In another embodiment, the anti-C5 antibody binds C5 with a higher affinity at neutral pH than at acidic pH. In certain embodiments, an anti-C5 antibody of the invention competes for binding to C5 with an antibody comprising a VH and VL pair selected from CFA0538, CFA0501, CFA0599, CFA0307, CFA0366, CFA0675, and CFA0672. In some embodiments, the anti-C5 antibody competes with antibody CFA0666 for binding to C5. In another embodiment, the anti-C5 antibody binds C5 with a higher affinity at pH 7.4 than at pH 5.8.

In another embodiment, the anti-C5 antibody binds C5 with a higher affinity at neutral pH than at acidic pH. In certain embodiments, an anti-C5 antibody of the invention competes for binding to C5 with an antibody comprising a VH and VL pair of antibody CFA0503 or 305LO5. In another embodiment, the anti-C5 antibody binds C5 with a higher affinity at pH 7.4 than at pH 5.8.

In certain embodiments, the anti-C5 antibody of the invention comprises a VH and VL pair selected from the VH of SEQ ID NO: 22 and the VL of SEQ ID NO: 26, or the VH of SEQ ID NO: 21 and the VL of SEQ ID NO: 25 for binding to C5. Competes with the containing antibody. In some embodiments, the anti-C5 antibody competes for binding to C5 with an antibody comprising a VH and VL pair selected from: (a) a VH of SEQ ID NO: 5 and a VL of SEQ ID NO: 15; (b) the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 14; (c) the VH of SEQ ID NO: 6 and the VL of SEQ ID NO: 16; (d) the VH of SEQ ID NO: 2 and the VL of SEQ ID NO: 12; (e) the VH of SEQ ID NO: 3 and the VL of SEQ ID NO: 13; (f) the VH of SEQ ID NO: 1 and the VL of SEQ ID NO: 11; (g) the VH of SEQ ID NO: 9 and the VL of SEQ ID NO: 19; (h) the VH of SEQ ID NO: 7 and the VL of SEQ ID NO: 17; And (i) the VH of SEQ ID NO: 8 and the VL of SEQ ID NO: 18. In some embodiments, the anti-C5 antibody competes with an antibody comprising the VH of SEQ ID NO: 23 and the VL of SEQ ID NO: 27 for binding to C5. In some embodiments, the anti-C5 antibody competes with an antibody comprising the VH of SEQ ID NO: 7 and the VL of SEQ ID NO: 17 for binding to C5.

In certain embodiments, an anti-C5 antibody of the invention competes for binding to C5 with an antibody comprising a VH and VL pair selected from: (a) a VH of SEQ ID NO: 1 and a VL of SEQ ID NO: 11; (b) a VH of SEQ ID NO: 22 and a VL of SEQ ID NO: 26; (c) a VH of SEQ ID NO: 21 and a VL of SEQ ID NO: 25; (d) the VH of SEQ ID NO: 5 and the VL of SEQ ID NO: 15; (e) the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 14; (f) the VH of SEQ ID NO: 6 and the VL of SEQ ID NO: 16; (g) the VH of SEQ ID NO: 2 and the VL of SEQ ID NO: 12; (h) the VH of SEQ ID NO: 3 and the VL of SEQ ID NO: 13; (i) the VH of SEQ ID NO: 9 and the VL of SEQ ID NO: 19; (j) the VH of SEQ ID NO: 7 and the VL of SEQ ID NO: 17; (k) the VH of SEQ ID NO: 8 and the VL of SEQ ID NO: 18; (l) a VH of SEQ ID NO: 23 and a VL of SEQ ID NO: 27; And (m) a VH of SEQ ID NO: 10 and a VL of SEQ ID NO: 20.

In certain embodiments, an anti-C5 antibody of the invention competes for binding to C5 with an antibody comprising a VH and VL pair selected from: (a) a VH of SEQ ID NO: 22 and a VL of SEQ ID NO: 26; (b) a VH of SEQ ID NO: 21 and a VL of SEQ ID NO: 25; (c) the VH of SEQ ID NO: 5 and the VL of SEQ ID NO: 15; (d) the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 14; (e) the VH of SEQ ID NO: 6 and the VL of SEQ ID NO: 16; (f) the VH of SEQ ID NO: 2 and the VL of SEQ ID NO: 12; (g) the VH of SEQ ID NO: 3 and the VL of SEQ ID NO: 13; (h) a VH of SEQ ID NO: 9 and a VL of SEQ ID NO: 19; (i) the VH of SEQ ID NO: 7 and the VL of SEQ ID NO: 17; (j) the VH of SEQ ID NO: 8 and the VL of SEQ ID NO: 18; And (k) a VH of SEQ ID NO: 23 and a VL of SEQ ID NO: 27.

In certain embodiments, the anti-C5 antibody of the invention in binding to C5 comprises a VH and VL pair selected from the VH of SEQ ID NO: 1 and the VL of SEQ ID NO: 11, or the VH of SEQ ID NO: 10 and the VL of SEQ ID NO: 20. Competes with the containing antibody.

In another embodiment, the anti-C5 antibody binds C5 with a higher affinity at neutral pH than at acidic pH. In certain embodiments, the anti-C5 antibody binds C5 with higher affinity at neutral pH than at acidic pH and competes with an antibody comprising a VH and VL pair selected from the following for binding to C5: (a) The VH of SEQ ID NO: 1 and the VL of SEQ ID NO: 11; (b) the VH of SEQ ID NO: 5 and the VL of SEQ ID NO: 15; (c) the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 14; (d) the VH of SEQ ID NO: 6 and the VL of SEQ ID NO: 16; (e) the VH of SEQ ID NO: 2 and the VL of SEQ ID NO: 12; (f) the VH of SEQ ID NO: 3 and the VL of SEQ ID NO: 13; (g) the VH of SEQ ID NO: 9 and the VL of SEQ ID NO: 19; (h) the VH of SEQ ID NO: 7 and the VL of SEQ ID NO: 17; (i) the VH of SEQ ID NO: 8 and the VL of SEQ ID NO: 18; And (j) a VH of SEQ ID NO: 10 and a VL of SEQ ID NO: 20. In another embodiment, the anti-C5 antibody binds C5 with a higher affinity at pH 7.4 than at pH 5.8.

In some embodiments, the anti-C5 antibody binds C5 with higher affinity at neutral pH than at acidic pH and competes with an antibody comprising a VH and VL pair selected from the following for binding to C5: (a) A VH of SEQ ID NO: 5 and a VL of SEQ ID NO: 15; (b) the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 14; (c) the VH of SEQ ID NO: 6 and the VL of SEQ ID NO: 16; (d) the VH of SEQ ID NO: 2 and the VL of SEQ ID NO: 12; (e) the VH of SEQ ID NO: 3 and the VL of SEQ ID NO: 13; (f) the VH of SEQ ID NO: 1 and the VL of SEQ ID NO: 11; (g) the VH of SEQ ID NO: 9 and the VL of SEQ ID NO: 19; (h) the VH of SEQ ID NO: 7 and the VL of SEQ ID NO: 17; And (i) the VH of SEQ ID NO: 8 and the VL of SEQ ID NO: 18. In another embodiment, the anti-C5 antibody binds C5 with a higher affinity at pH 7.4 than at pH 5.8.

In some embodiments, the anti-C5 antibody binds C5 with higher affinity at neutral pH than at acidic pH, and the VH of SEQ ID NO: 1 and the VL of SEQ ID NO: 11, or the VH of SEQ ID NO: 10 for binding to C5. And a VH and VL pair selected from the VL of SEQ ID NO: 20. In another embodiment, the anti-C5 antibody binds C5 with a higher affinity at pH 7.4 than at pH 5.8.

In certain embodiments, whether an anti-C5 antibody of the invention binds to a particular epitope can be determined as follows: Expression in 293 cells of a C5 point mutant in which an amino acid of the phase of C5 (except alanine) is substituted for alanine. And, the binding of the anti-C5 antibody to the C5 mutant was tested by ELISA, Western blot or Biacore (registered trademark); Substantial reduction or exclusion of the binding of the anti-C5 antibody to the C5 mutant compared to its binding to wild-type C5 indicates that the anti-C5 antibody binds to an epitope comprising that amino acid on C5. In certain embodiments, the amino acid on C5 to be substituted with alanine is selected from the group consisting of Glu48, Asp51, His70, His72, Lys109, and His110 of the beta chain of C5 (SEQ ID NO: 40). In another embodiment, the amino acid on C5 to be substituted for alanine is Asp51 or Lys109 of the beta chain of C5 (SEQ ID NO: 40).

In another embodiment, whether an anti-C5 antibody with pH-dependent binding properties binds to a particular epitope can be determined as follows: a histidine residue on C5 is substituted with another amino acid (e.g., tyrosine). The resulting C5 point mutant was expressed in 293 cells, and the binding of the anti-C5 antibody to the C5 mutant was examined by ELISA, Western Blot or Biacore®; A substantial reduction in binding of the anti-C5 antibody to wild-type C5 at acidic pH compared to its binding to the C5 mutant at acidic pH indicates that the anti-C5 antibody binds to an epitope comprising the histidine residue on C5. In another embodiment, the binding of the anti-C5 antibody to wild-type C5 at neutral pH is not substantially reduced compared to its binding to the C5 mutant at neutral pH. In certain embodiments, the histidine residue on C5 to be substituted with another amino acid is selected from the group consisting of His70, His72 and His110 in the beta chain of C5 (SEQ ID NO: 40). In another embodiment, the histidine residue His70 is substituted with tyrosine.

2. Activity Assay

In one aspect, an assay is provided to identify its anti-C5 antibody having biological activity. Biological activities include, for example, inhibition of C5 activation, prevention of cleavage of C5 to produce C5a and C5b, blocking access of C5 converting enzymes to the cleavage site on C5, blocking hemolytic activity caused by activation of C5. And the like. Antibodies with such biological activity in vivo and/or in vitro are also provided.

In certain embodiments, the antibodies of the invention are tested for said biological activity.

In certain embodiments, whether the test antibody inhibits cleavage of C5 to C5a and C5b is determined by, for example, Isenman et al., J Immunol. 124(1):326-331 (1980). In another embodiment, this is determined by a method of specifically detecting truncated C5a and/or C5b proteins, such as ELISA or Western blot. When a reduced amount of cleavage product (i.e., C5a and/or C5b) is detected in the presence of the test antibody (or after contact with the test antibody), the test antibody is identified as an antibody capable of inhibiting cleavage of C5. In certain embodiments, the concentration and/or physiological activity of C5a can be determined, for example, by chemotactic assay, RIA or ELISA (see, eg, Ward and Zvaifler J. Clin. Invest. 50 ( 3):606-616 (1971)).

In certain embodiments, whether the test antibody blocks access of the C5 convertase to C5 is by a method for detection of a protein interaction between C5 convertase and C5, e.g., by ELISA or Biacore®. Is measured. When the interaction is reduced in the presence of (or after contact with) the test antibody, the test antibody is identified as an antibody capable of blocking access of the C5 convertase to C5.

In certain embodiments, C5 activity can be measured as a function of its cell-lytic ability in a subject's body fluids. The cell-lytic ability of C5 or its decrease can be determined by methods known in the art, for example, Kabat and Mayer (eds), Experimental Immunochemistry, 2nd Edition, 135-240, Springfield, IL, CC Thomas (1961. ), pages 135-139], or conventional hemolysis assays such as those described in, for example, Hillmen et al., N. Engl. J. Med. 350(6): 552-559 (2004)]. In certain embodiments, C5 activity or inhibition thereof is quantified using a CH50eq assay. The CH50eq assay is a method for measuring the total classical complement activity in serum. The assay is a lysis assay using antibody-sensitized red blood cells as activators of the classical complement pathway, and various dilutions of the test serum to determine the amount required to provide 50% lysis (CH50). The hemolysis percentage can be measured, for example, using a spectrophotometer. The CH50eq assay provides a non-indirect measurement of the TCC production, since the terminal complement complex (TCC) itself is directly responsible for the measured hemolysis. Inhibition of C5 activation can also be detected and/or measured using the methods shown and illustrated in the Examples. Using these or other suitable types of assays, candidate antibodies capable of inhibiting the activation of C5 can be screened. In certain embodiments, inhibition of C5 activation is at least 5%, 10%, 15%, 20%, 25%, 30%, 35% or 40% or more of C5 activation in the assay compared to the effect of a negative control under similar conditions. Includes reduction. In some embodiments, the inhibition refers to inhibition of C5 activation of at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more.

D. Immunoconjugate

The invention also provides for one or more cytotoxic agents, e.g. chemotherapeutic or chemotherapeutic agents, growth inhibitors, toxins (e.g., protein toxins, bacterial, fungal, enzymatically active toxins of plant or animal origin, Or fragments thereof), or an anti-C5 antibody of the present invention conjugated to a radioactive isotope.

In one embodiment, the immunoconjugate comprises maytansinoids (see, eg, U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent No. 0 425 235 B1); Auristatins such as monomethylolistatin drug residues DE and DF (MMAE and MMAF) (see US Pat. Nos. 5,635,483, 5,780,588 and 7,498,298); Dolastatin; Calichiamicin or a derivative thereof (U.S. Patent 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; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); Anthracyclines, for example daunomycin or doxorubicin (Kratz et al., urrent Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358- 362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al. , Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002)]; and US Pat. No. 6,630,579); Methotrexate; Vindesine; Taxanes such as docetaxel, paclitaxel, larotaxel and ortataxel; Tricotecin; And CC1065, including, but not limited to, an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more drugs.

In another embodiment, the immunoconjugate is a diphtheria A chain, an unbound active fragment of diphtheria toxin, an exotoxin A chain ( Pseudomonas aeruginosa ), lysine A chain, abrin A chain, modecin A chain, alpha- sacrine , Aleurites fordii protein, diantin protein, Phytolaca americana protein (PAPI, PAPII, and PAP -S), Yeoju ( momordica charantia ) inhibitors, cursin, crotin , sapaonaria officinalis inhibitors, gelonine, mitozelin, restrictocin, phenomycin, enomycin and tricotesin, including, but not limited to, enzymatically active It includes an antibody as described herein conjugated to a phosphorus toxin or fragment thereof.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to produce a radioconjugate. A variety of radioactive isotopes are available for the generation of radioconjugates. Examples include At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and radioactive isotopes of Lu. When a radioconjugate is used for detection, the radioconjugate is a radioactive element for scintillation studies, e.g., tc99m or I123, or for nuclear magnetic resonance (NMR) imaging (magnetic resonance imaging, also known as mri). Spin labels, such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Conjugates of antibodies and cytotoxic agents include various bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N -Maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), difunctional derivatives of imidoesters (e.g. dimethyl adipimidate HCl), active esters (e.g. di Succinimidyl suberate) aldehyde (e.g. glutaraldehyde), bis-azido compound (e.g. bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., Bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g. toluene 2,6-diisocyanate) and bis-active fluorine compounds (e.g. 1,5-difluoro-2 ,4-dinitrobenzene). For example, lysine immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is a representative chelating agent for conjugating radionucleotides to antibodies (International Patent Publication No. 94/11026). See issue). The linker may be a "cleavable linker" that promotes the release of cytotoxic drugs from the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers or disulfide-containing linkers (Chari et al., Cancer Res. 52:127-131 (1992)); U.S. Patent No. 5,208,020) can be used.

Immunoconjugates or ADCs are specifically, commercially available (e.g., from Pierce Biotechnology, Inc. of Rockford, IL) BMPS, EMCS, GMBS, HBVS, LC- SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, Sulfo-EMCS, Sulfo-GMBS, Sulfo-KMUS, Sulfo-MBS, Sulfo-SIAB, Sulfo-SMCC, and Sulfo-SMPB, and SVSB ( Succinimidyl-(4-vinylsulfone)benzoate), including, but not limited to, the conjugates prepared using crosslinking reagents including, but not limited to.

E. Methods and compositions for diagnosis and detection

In certain embodiments, any of the anti-C5 antibodies provided herein are useful for detecting the presence of C5 in a biological sample. The term “detection” as used herein includes quantitative or qualitative detection. In certain embodiments, the biological sample is a cell or tissue, e.g., serum, whole blood, plasma, biopsy sample, tissue sample, cell suspension, saliva, sputum, oral fluid, cerebrospinal fluid, amniotic fluid, ascites, breast milk, colostrum, mammary secretion , Lymph, urine, sweat, tear fluid, gastric fluid, synovial fluid, peritoneal fluid, lens fluid and mucus.

In one embodiment, an anti-C5 antibody is provided for use in a method of diagnosis or detection. In another aspect, a method of detecting the presence of C5 in a biological sample is provided. In certain embodiments, the method comprises contacting a biological sample with an anti-C5 antibody as described herein under conditions that allow binding of an anti-C5 antibody to C5, and a complex is formed between the anti-C5 antibody and C5. It includes detecting whether or not. The method may be an in vitro or in vivo method. In one embodiment, for example, when C5 is a biomarker for patient selection, an anti-C5 antibody is used to select a subject for treatment with an anti-C5 antibody.

In another embodiment, a method of selecting an individual with a complement-mediated disease or condition involving excessive or uncontrolled activation of C5 as suitable for treatment comprising an anti-C5 antibody of the invention is provided. In certain embodiments, the method comprises (a) detecting a genetic variation in C5 obtained from an individual, and (b) when the genetic variation is detected in C5 obtained from an individual, the individual using an anti-C5 antibody of the invention. It includes screening as suitable for the treatment to be included. In another aspect, a method of screening a treatment for an individual with 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 C5 obtained from an individual, and (b) an anti-C5 antibody of the invention for said individual when said genetic variation is detected in C5 obtained from an individual. It includes selecting a treatment comprising a.

In another embodiment, a method of treating an individual with 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 C5 obtained from the individual, and (b) when a genetic variation in C5 obtained from the individual is detected, the individual comprising an anti-C5 antibody of the invention. And (c) administering an anti-C5 antibody of the present invention to the individual.

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

In another embodiment, genetic variation in C5 to select for individuals with complement-mediated diseases or disorders involving excessive or uncontrolled activation of C5 as suitable for treatment comprising an anti-C5 antibody of the invention. The in vitro use of is provided. In certain embodiments, when a genetic variation in C5 obtained from an individual is detected, the individual is selected as suitable for the treatment. In another embodiment, an in vitro use of a genetic variation in C5 for screening for treatment for an individual with a complement-mediated disease or condition involving excessive or uncontrolled activation of C5 is provided. In certain embodiments, a treatment comprising an anti-C5 antibody of the invention is selected for the individual when a genetic variation is detected in C5 obtained from the individual.

It has been reported that some patients with genetic mutations in C5 exhibit poor responses to treatments involving existing anti-C5 antibodies (Nishimura et al., N. Engl. J. Med. 370:632-639 (2014). )]). The patient is recommended to be treated with a treatment comprising an anti-C5 antibody of the present invention, as the antibody has inhibitory activity against the activation of wild-type C5 as well as the C5 variant, as demonstrated in the examples below.

Detection of the genetic variation in C5 can be performed using methods known in the prior art. Such methods may include, but are not limited to, sequencing, PCR, RT-PCR, and hybridization-based methods, such as Southern blot or Northern blot. The C5 variant may contain one or more genetic variations. The genetic mutation may be selected from the group consisting of V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N, and E1437D. Here, R885H means, for example, a genetic mutation in which arginine at position 885 is substituted with histidine. In certain embodiments, the C5 variant has similar biological activity to wild type C5.

Representative diseases that can be diagnosed using the antibodies of the present invention include: rheumatoid arthritis (RA); Systemic lupus erythematosus (SLE); Lupus nephritis; Ischemic reperfusion injury (IRI); asthma; Paroxysmal nocturnal hemoglobinuria (PNH); Hemolytic uremic syndrome (HUS) (eg, atypical hemolytic uremic syndrome (aHUS)); High density deposition disease (DDD); Optic neuromyelitis (NMO); Multifocal motor neuropathy (MMN): multiple sclerosis (MS): systemic sclerosis; Macular degeneration (eg, age-related macular degeneration (AMD)); Hemolysis, increased liver enzymes and low platelet (HELLP) syndrome; Thrombotic thrombocytoptic purpura (TTP); Natural heritage; Bullous epidermolysis; Recurrent miscarriage; Preeclampsia; Traumatic brain injury; Myasthenia gravis; Cold agglutination disease; Sjogren syndrome; Dermatomyositis; Bullous euphorbia; Phototoxic reaction; Shiga toxin Escherichia coli-related hemolytic uremic syndrome; Typical or infectious hemolytic uremic syndrome (tHUS); C3 glomerulonephritis; Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis; Bodily fluid and vascular graft rejection; Acute antibody mediated rejection (AMR); Graft dysfunction; Myocardial infarction; Allogeneic transplant; blood poisoning; Coronary artery disease; Hereditary angioedema; Dermatomyositis; Grave's disease; Atherosclerosis; Alzheimer's disease (AD); Huntington's disease; Creutzfeld-Jacob disease; Parkinson's disease; cancer; Wound; Septic shock; Spinal cord injury; Uveitis; Diabetic eye disease; Retinopathy of prematurity; Glomerulonephritis; Membranous nephritis; Immunoglobulin A nephropathy; Adult respiratory distress syndrome (ARDS); Chronic obstructive pulmonary disease (COPD); Cystic fibrosis; Hemolytic anemia; Paroxysmal cold hemoglobinuria; Anaphylactic shock; allergy; osteoporosis; Osteoarthritis; Hashimoto's thyroiditis; Type 1 diabetes; psoriasis; pemphigus; Autoimmune hemolytic anemia (AIHA); Idiopathic thrombocytoptic purpura (ITP); Goodpasture syndrome; Degos disease; Antiphospholipid syndrome (APS); Catastrophic APS (CAPS); Cardiovascular disorders; myocarditis; Cerebrovascular disorders; Peripheral vascular disorders; Renal vascular disorders; Mesenteric/intestinal vascular disorders; Vasculitis; Henoch-Schonlein purpura nephritis; Takayasu disease; Dilated cardiomyopathy; Diabetic angiopathy; Kawasaki disease (arteritis); Venous air embolism (VGE), restenosis after stent placement Rotational atherosclerosis; Membrane nephropathy; Guillain-Barre syndrome (GBS); Fisher syndrome; Antigen-induced arthritis; Synovial inflammation; Viral infection; Bacterial infection; Fungal infection; And injuries resulting from myocardial infarction, cardiopulmonary bypass, and hemodialysis.

In certain embodiments, labeled anti-C5 antibodies are provided. Labels include labels or residues that are directly detected (e.g., fluorescence, color development, electron-high density, chemiluminescence and radioactive labels), as well as residues that are indirectly detected through, for example, enzyme reactions or molecular interactions, For example, enzymes or ligands are included, but are not limited thereto. Representative labels include radioisotopes ³² P, ¹⁴ C, ¹²⁵ I. ³ H and ¹³¹ I, fluorophores such as rare earth chelates or fluorescein and derivatives thereof, rhodamine and derivatives thereof, Dansil, Umbelli Feron, luciferase, such as firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase , Beta-galactosidase, glucoamylase, lysozyme, saccharide oxidase, such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase, HRP, lactoperoxidase or microperoxy Heterocyclic oxidase coupled with enzymes that use hydrogen peroxide to oxidize dye precursors such as polysaccharides, such as uricase and xanthine oxidase, biotin/avidin, spin labeling, bacteriophage labeling, stable free radicals, etc. Included, but not limited to.

F. Pharmaceutical formulation

Pharmaceutical formulations of anti-C5 antibodies as described herein contain one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. 1980)]), it is prepared in the form of a lyophilized formulation or aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used and include, but are not limited to: buffering agents such as phosphate, citrate and other organic acids; Antioxidants including ascorbic acid and methionine; Preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol ; Resorcinol; cyclohexanol; 3-pentanol; and m-cresol); Low molecular weight (less than about 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 EDTA; Sugars such as sucrose, mannitol, trehalose or sorbitol; Salt-generating counter-ions such as sodium; Metal complexes (eg, Zn-protein complexes); And/or non-ionic surfactants such as polyethylene glycol (PEG). Representative pharmaceutically acceptable carriers herein are also intersitial drug dispersions, e.g., soluble neutral-active hyaluronidase glycoprotein (sHASEGP), e.g. human soluble PH-20 hyaluronid. First glycoproteins, such as rHuPH20 (HYLENEX, registered trademark, Baxter International, Inc.). Certain representative sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one embodiment, the sHASEGP is admixed with one or more additional glycosaminoglycanases, such as chondroitinase.

Representative lyophilized antibody formulations are described in US Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and International Patent Publication No. 2006/044908, the latter formulation comprising a histidine-acetate buffer.

The formulations of the invention may also contain more than one active ingredient as required for the particular indication being treated, preferably ingredients with complementary activities that do not adversely affect each other. The active ingredients are suitably present together in an amount effective for the intended purpose.

The active ingredients are, for example, in microcapsules prepared by coacervation technology or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly(methylmethacrylate) microcapsules. Each capsule may be enclosed in a colloidal drug delivery system (eg, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in a macroemulsion. This technique is described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)].

Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, which matrices are in the form of shaped articles, for example films or microcapsules.

Formulations to be used for in vivo administration are generally sterile. Sterilization can be easily achieved, for example, by filtration through a sterile filtration membrane.

G. Treatment methods and compositions

Any of the anti-C5 antibodies provided herein can be used in a method of treatment.

In one aspect, an anti-C5 antibody for use as a medicament is provided. In another aspect, an anti-C5 antibody for use in treating a complement-mediated disease or condition involving excessive or uncontrolled activation of C5 is provided. In certain embodiments, anti-C5 antibodies are provided for use in a method of treatment. In certain embodiments, the invention is used in a method of treating an individual with a complement-mediated disease or condition involving excessive or uncontrolled activation of C5, comprising administering to the individual an effective amount of an anti-C5 antibody. For the following, an anti-C5 antibody is provided. In one such embodiment, the method further comprises administering to the individual an effective amount of one or more additional therapeutic agents. An “individual” according to any of the above embodiments is preferably a human.

When the antigen is a soluble protein, since the antibody itself has a longer half-life in plasma and acts as a carrier for the antigen, the binding of the antibody to that antigen results in the prolonged half-life of the antigen in plasma (i.e., a reduced antigen from plasma). Removal). This is due to the recycling of the antigen-antibody complex by FcRn through the intracellular endosome pathway (Roopenian, Nat. Rev. Immunol. 7(9):715-725 (2007)). However, an antibody with pH-dependent binding properties, which binds to its antigen in a neutral extracellular environment while releasing the antibody into the acidic endosome compartment after entry into the cell, binds to its counterpart, which binds in a pH-independent manner. It is expected to have superior properties with respect to antigen neutralization and elimination compared to (Igawa et al., Nat. Biotech.. 28(11):1203-1207 (2010); Devanaboyina et al., mAbs 5(6)). :851-859 (2013)]; International Patent Publication No. 2009/125825).

In another embodiment, the invention provides anti-C5 antibodies for use in enhancing the clearance of C5 from plasma. In certain embodiments, the invention provides an anti-C5 for use in a method of enhancing the removal of C5 from plasma in an individual comprising administering to the individual an effective amount of an anti-C5 antibody to enhance the removal of C5 from plasma. Antibodies are provided. In one embodiment, the anti-C5 antibody enhances the removal of C5 from plasma compared to a conventional anti-C5 antibody that does not have pH-dependent binding properties. An “individual” according to any of the above embodiments is preferably a human.

In another embodiment, 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 for use in a method of inhibiting the accumulation of C5 in plasma in an individual comprising administering to the individual an effective amount of an anti-C5 antibody to inhibit the accumulation of C5 in plasma. Antibodies are provided. In one embodiment, the accumulation of C5 in plasma is the result of antigen-antibody complex production. In another embodiment, the anti-C5 antibody inhibits the accumulation of C5 in plasma compared to a conventional anti-C5 antibody that does not have pH-dependent binding properties. An “individual” according to any of the above embodiments is preferably a human.

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

In another aspect, the invention provides the use of an anti-C5 antibody in the production or manufacture of a medicament. In one embodiment, the medicament is for treatment of a complement-mediated disease or condition involving excessive or uncontrolled activation of C5. In another embodiment, the medicament involves excessive or uncontrolled activation of C5, comprising administering an effective amount of the medicament to an individual with a complement-mediated disease or condition involving excessive or uncontrolled activation of C5. It is for use in a method of treating a complement-mediated disease or disease. In one such embodiment, the method further comprises administering to the individual an effective amount of one or more additional therapeutic agents. An “individual” according to any of the above embodiments is preferably a human.

In another embodiment, the medicament is for enhancing the removal of C5 from plasma. In another embodiment, the medicament is for use in a method of enhancing the removal of C5 from plasma in an individual comprising administering to the individual an effective amount of the medicament to enhance the removal of C5 from plasma. In one embodiment, the anti-C5 antibody enhances the removal of C5 from plasma compared to a conventional anti-C5 antibody that does not have pH-dependent binding properties. An “individual” according to any of the above embodiments may be a human.

In another embodiment, the medicament is for use in inhibiting the accumulation of C5 in plasma. In another embodiment, the medicament is for use in a method of inhibiting the accumulation of C5 in plasma in an individual comprising administering to the individual an effective amount of the medicament to inhibit the accumulation of C5 in plasma. In one embodiment, the accumulation of C5 in plasma is the result of antigen-antibody complex production. In another embodiment, the anti-C5 antibody inhibits the accumulation of C5 in plasma compared to a conventional anti-C5 antibody that does not have pH-dependent binding properties. An “individual” according to any of the above embodiments may be a human.

The anti-C5 antibody of the present invention can inhibit the activation of C5. In another embodiment, the medicament is for inhibiting the activation of C5. In another embodiment, the medicament is for use in a method of inhibiting the activation of C5 in an individual comprising administering to the individual an effective amount of the medicament to inhibit the activation of C5. In one embodiment, the cytotoxicity mediated by C5 is inhibited by inhibiting the activation of C5. An “individual” according to any of the above embodiments may be a human.

In another aspect, the invention provides a method of treating a complement-mediated disease or condition involving excessive or uncontrolled activation of C5. In one embodiment, the method comprises administering an effective amount of an anti-C5 antibody to an individual having a complement-mediated disease or condition involving excessive or uncontrolled activation of C5. In one such embodiment, the method further comprises administering to the individual an effective amount of one or more additional therapeutic agents. An “individual” according to any of the above embodiments may be a human.

In another aspect, the invention provides a method for enhancing the removal of C5 from plasma in a subject. In one embodiment, the method comprises administering to the individual an effective amount of an anti-C5 antibody to enhance removal of C5 from plasma. In one embodiment, the anti-C5 antibody enhances the removal of C5 from plasma compared to a conventional anti-C5 antibody that does not have pH-dependent binding properties. In one embodiment, the “individual” is a human.

In another aspect, the invention provides a method for inhibiting the accumulation of C5 in plasma in a subject. In one embodiment, the method comprises 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 plasma is the result of antigen-antibody complex production. In another embodiment, the anti-C5 antibody inhibits the accumulation of C5 in plasma compared to a conventional anti-C5 antibody that does not have pH-dependent binding properties. In one embodiment, the “individual” is a human.

The anti-C5 antibody of the present invention can inhibit the activation of C5. In another aspect, the invention provides a method for inhibiting the activation of C5 in a subject. In one embodiment, the method comprises administering to the individual an effective amount of an anti-C5 antibody to inhibit activation of C5. In one embodiment, the cytotoxicity mediated by C5 is inhibited by inhibiting the activation of C5. In one embodiment, the “individual” is a human.

In another aspect, the invention provides pharmaceutical formulations comprising any of the anti-C5 antibodies provided herein, for example, for use in any of the above methods of treatment. In one embodiment, the pharmaceutical formulation comprises any of the anti-C5 antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the anti-C5 antibodies provided herein and one or more additional therapeutic agents.

In another aspect, the pharmaceutical formulation is for treatment of a complement-mediated disease or condition involving excessive or uncontrolled activation of C5. In another embodiment, the pharmaceutical formulation is for enhancing the removal of C5 from plasma. In one embodiment, the anti-C5 antibody enhances the removal of C5 from plasma compared to a conventional anti-C5 antibody that does not have pH-dependent binding properties. In another embodiment, the pharmaceutical formulation is for use in inhibiting the accumulation of C5 in plasma. In one embodiment, the accumulation of C5 in plasma is the result of antigen-antibody complex production. In another embodiment, the anti-C5 antibody inhibits the accumulation of C5 in plasma compared to a conventional anti-C5 antibody that does not have pH-dependent binding properties. The anti-C5 antibody of the present invention can inhibit the activation of C5. In another embodiment, the pharmaceutical formulation is for inhibiting the activation of C5. In one embodiment, the cytotoxicity mediated by C5 is inhibited by inhibiting the activation of C5. In one embodiment, the pharmaceutical formulation is administered to an individual with a complement-mediated disease or condition involving excessive or uncontrolled activation of C5. An “individual” according to any of the above embodiments is preferably a human.

In one embodiment, the subject has wild type C5. In another aspect, the subject has the C5 variant. In certain embodiments, the C5 variant has similar biological activity to wild type C5. The C5 variant may include one or more mutations selected from the group consisting of V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N, and E1437D. Here, for example, R885H refers to a genetic mutation in which arginine at position 885 is substituted with histidine.

In another aspect, the present invention provides a medicament or pharmaceutical composition comprising admixing any anti-C5 antibody provided herein with a pharmaceutically acceptable carrier, e.g., for use in any of the above methods of treatment. Provides a method of preparing a formulation. In one embodiment, the method of preparing a medicament or pharmaceutical formulation further comprises adding one or more additional therapeutic agents to the medicament or pharmaceutical formulation.

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 (SLE); Lupus nephritis; Ischemic reperfusion injury (IRI); asthma; Paroxysmal nocturnal hemoglobinuria (PNH); Hemolytic uremic syndrome (HUS) (eg, atypical hemolytic uremic syndrome (aHUS)); High density deposition disease (DDD); Optic neuromyelitis (NMO); Multifocal motor neuropathy (MMN): multiple sclerosis (MS): systemic sclerosis; Macular degeneration (eg, age-related macular degeneration (AMD)); Hemolysis, increased liver enzymes and low platelet (HELLP) syndrome; Thrombotic thrombocytoptic purpura (TTP); Natural heritage; Bullous epidermolysis; Recurrent miscarriage; Preeclampsia; Traumatic brain injury; Myasthenia gravis; Cold agglutination disease; Sjogren syndrome; Dermatomyositis; Bullous euphorbia; Phototoxic reaction; Shiga toxin Escherichia coli-related hemolytic uremic syndrome; Typical or infectious hemolytic uremic syndrome (tHUS); C3 glomerulonephritis; Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis; Bodily fluid and vascular graft rejection; Acute antibody mediated rejection (AMR); Graft dysfunction; Myocardial infarction; Allogeneic transplant; blood poisoning; Coronary artery disease; Hereditary angioedema; Dermatomyositis; Graves' disease; Atherosclerosis; Alzheimer's disease (AD); Huntington's disease; Creitzfeld-Jakob disease; Parkinson's disease; cancer; Wound; Septic shock; Spinal cord injury; Uveitis; Diabetic eye disease; Retinopathy of prematurity; Glomerulonephritis; Membranous nephritis; Immunoglobulin A nephropathy; Adult respiratory distress syndrome (ARDS); Chronic obstructive pulmonary disease (COPD); Cystic fibrosis; Hemolytic anemia; Paroxysmal cold hemoglobinuria; Anaphylactic shock; allergy; osteoporosis; Osteoarthritis; Hashimoto's thyroiditis; Type 1 diabetes; psoriasis; pemphigus; Autoimmune hemolytic anemia (AIHA); Idiopathic thrombocytoptic purpura (ITP); Goodpasture syndrome; Degos disease; Antiphospholipid syndrome (APS); Catastrophic APS (CAPS); Cardiovascular disorders; myocarditis; Cerebrovascular disorders; Peripheral vascular disorders; Renal vascular disorders; Mesenteric/intestinal vascular disorders; Vasculitis; Henoch-Schenlein purpura nephritis; Takayasu disease; Dilated cardiomyopathy; Diabetic angiopathy; Kawasaki disease (arteritis); Venous air embolism (VGE), restenosis after stent placement Rotational atherosclerosis; Membrane nephropathy; Guillain-Barre syndrome (GBS); Fisher syndrome; Antigen-induced arthritis; Synovial inflammation; Viral infection; Bacterial infection; Fungal infection; And injuries resulting from myocardial infarction, cardiopulmonary bypass, and hemodialysis.

In certain embodiments, the complement-mediated disease or condition is an eye disease. In another embodiment, the eye disease is macular degeneration. In another embodiment, the macular degeneration is AMD. In another embodiment, the AMD is dry AMD.

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

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

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

The antibodies of the present invention can be used alone or in combination with other agents for treatment. For example, the antibodies of the invention can be co-administered with one or more additional therapeutic agents.

The combination treatment mentioned above includes combined administration (when two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case the administration of the antibody of the present invention is prior to administration of the additional therapeutic agent or therapeutic agents, It may occur concurrently with and/or after administration. In one embodiment, the administration of the anti-C5 antibody and the administration of the additional therapeutic agent are within about 1 month of each other, or within about 1 week, 2 weeks or 3 weeks, or about 1 day, 2 days, 3 days, 4 days, 5 days of each other. Occurs within a day or six days.

The antibodies of the invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and, if necessary for topical treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Dosing can be by any suitable route, in part, depending on whether the administration is short-term or long-term, by injection, such as, for example, intravenous or subcutaneous injection. Various dosing schedules are contemplated herein including, but not limited to, single or multiple administrations over various time points, bolus administration, and pulse infusion.

Antibodies of the invention are formulated, dosed and administered in a manner consistent with correct medical practice. Factors to consider in this regard include the particular disease being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disease, the site of delivery of the drug, the method of administration, the schedule of administration, and other factors known to the physician. Antibodies need not be formulated with one or more agents currently used to prevent or treat the disease in question, but are optionally formulated together. The effective amount of these other agents will depend on the amount of antibody present in the formulation, the type of disease or treatment, and other factors discussed above. The other agents are generally at the same dosage and under the same route of administration as described herein, or in an amount of about 1 to 99% of the dosage described herein, or any administration determined to be empirically/clinically appropriate. Used in amounts and by any route.

For the prophylaxis or treatment of a disease, the appropriate dosage of the antibody of the invention (when used alone or in combination with one or more other additional therapeutic agents) is the type of disease to be treated, the type of antibody, the severity and course of the disease, and the antibody titer for prophylactic purposes. It will depend on whether or not to be administered for therapeutic purposes, prior treatment, the patient's clinical history and response to the antibody, and the discretion of the attending physician. Antibodies are appropriately administered to the patient at one time or during a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g., 0.1 to 10 mg/kg) of antibody is, for example, by one or more separate administrations or by continuous infusion. Optionally, it may be an initial candidate dosage for administration to a patient. One typical daily dosage may range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations for several days or longer, depending on the condition, treatment is generally continued until the desired suppression of disease symptoms occurs. One exemplary dosage of the antibody is in the range of about 0.05 to about 10 mg/kg. Thus, a dose of one or more 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 the patient. The dose may be administered intermittently, eg, weekly or every 3 weeks (eg, such that the patient receives about 2 to about 20, or eg, about 6 doses of the antibody). Following the initial higher loading dose, one or more lower doses may be administered. The progress of the treatment is easily monitored by conventional techniques and assays.

It is understood that any of the above formulations or methods of treatment may be performed using the immunoconjugates of the invention in place of or in addition to an anti-C5 antibody.

H. Products

In another aspect of the present invention, a product containing substances useful for the treatment, prevention and/or diagnosis of the aforementioned diseases is provided. The product includes a container and a label or package insert on or affixed to the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. Containers can be made from a variety of materials such as glass or plastic. The container contains the composition alone, or with other compositions effective to treat, prevent and/or diagnose a disease, and may have a sterile entry port (e.g., the container may have a stopper that may be pierced by a hypodermic injection needle. It may be a bag or vial of intravenous solution with). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is to be used to treat the disease of choice. In addition, the product comprises (a) a first container in which a composition comprising an antibody of the present invention is contained; And (b) a second container contained therein with a composition comprising an additional cytotoxic agent or otherwise a therapeutic agent. The product in this embodiment of the present invention may also include a package insert indicating that the composition can be used to treat a particular disease. Alternatively or additionally, the product may be a second (or third) containing pharmaceutically acceptable buffer, e.g., bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may also include a container of. The product may also contain other materials desirable from a commercial and user standpoint, including other buffers, diluents, fillers, needles and syringes.

It is understood that any of the above products may contain the immunoconjugates of the invention instead of or in addition to an anti-C5 antibody.

Example

The following are examples of the methods and compositions of the present invention. In view of the general description provided above, it is understood that various other embodiments may be practiced.

Example One

Preparation of C5

1.1. Expression and purification of recombinant human and cynomolgus monkey C5

Recombinant human C5 (NCBI GenBank accession number NP_001726.2, SEQ ID NO: 39) was expressed using the FreeStyle 293-F cell line (Thermo Fisher, Carlsbad, CA, USA). . The conditioned medium expressing human C5 was diluted with an equal volume of milliQ water, and then placed on a Q-Sepharose FF or Q-Sepharose HP anion exchange column (GE healthcare, Uppsala, Sweden). After application, it was eluted with a NaCl gradient. Fractions containing human C5 were collected, then the salt concentration and pH were adjusted to 80 mM NaCl and pH 6.4, respectively. The resulting sample was applied on an SP-Sepharose HP cation exchange column (GE Healthcare, Uppsala, Sweden) and eluted with a NaCl gradient. Fractions containing human C5 were collected and applied to a CHT ceramic hydroxyapatite column (Bio-Rad Laboratories, Hercules, CA, USA). The human C5 eluate was then applied to a Superdex 200 gel filtration column (GE Healthcare, Uppsala, Sweden). Fractions containing human C5 were collected and stored at -150 °C.

Expression and purification of recombinant cynomolgus monkey C5 (NCBI Genbank Accession No. XP_005580972, SEQ ID No. 44) was performed in the same manner as the human counterpart.

1.2. Purification of cynomolgus monkey C5 (cynoC5) from plasma

Plasma samples from cynomolgus monkeys were applied to SSL7-agarose (Invivogen, San Diego, CA, USA) and then diluted with 100 mM sodium acetate (pH 3.5). Fractions containing cynoC5 were immediately neutralized and applied to a Protein A HP column (GE Healthcare, Uppsala, Sweden) simultaneously with peptide M agarose (Invivogen, San Diego, CA, USA). The perfusion fraction was then applied to a Superdex 200 gel filtration column (GE Healthcare, Uppsala, Sweden). Fractions containing cynoC5 were collected and stored at -80 °C.

Example 2

Generation of anti-C5 antibodies

2.1. Antibody screening

Anti-C5 antibodies were prepared, screened and analyzed as follows:

12-16 week old NZW rabbits were immunized intradermally with human C5 and/or monkey C5 (50-100 μg/dose/rabbit). The dose was repeated 4 or 5 times over a period of 2 months. One week after the last immunization, spleens and blood were collected from immunized rabbits. Antigen-specific B-cells were stained with the labeled antigen, sorted with an FCM cell sorter (FACS Aria III, BD), and 25,000 cells/well of EL4 cells (European Collection of Cell Cultures) ) And 20-fold diluted activated rabbit T-cell conditioned medium, plated on a 96-well plate at a density of 1 cell/well, and cultured for 7 to 12 days. EL4 cells were treated with mitomycin C (Sigma, Cat No. M4287) for 2 hours and washed three times in advance. Activated rabbit T-cell conditioned medium was phytohemagglutinin-M (Roche, Cat No. 1 1082132-001), phorbol 12-myristate 13-acetate (Sigma, Cat No. P1585) and 2 It was prepared by culturing rabbit thymocytes in RPMI-1640 medium containing% FBS. After incubation, the B-cell culture supernatant was collected for further analysis and the pellet was cryopreserved.

The specificity of the antibody in the B-cell culture supernatant was examined using ELISA analysis. Streptavidin (GeneScript, Cat No. Z02043) was coated with 50 nM in PBS on 384-well MAXISorp (Nunc, Cat No. 164688) at room temperature. The plate was then blocked with a 5-fold diluted Blocking One (Nacalai Tesque, Cat No. 03953-95). Human or monkey C5 was labeled with NHS-PEG4-biotin (PIERCE, Cat No. 21329), added to a blocked ELISA plate, incubated for 1 hour and washed. The B-cell culture supernatant was added to the ELISA plate, incubated for 1 hour, and washed. After detection of binding with goat anti-rabbit IgG-horseradish peroxidase (BETHYL, Cat No. A120-111P), ABTS (KPL, Cat No. 50-66-06) was added.

An ELISA assay was used to evaluate the pH-dependent binding of the antibody to C5. Goat anti-rabbit IgG-Fc (Betyl, Cat No. A120-111A) diluted to 1 μg/ml in PBS(-) was added to 384-well Maxisorp (Nunc, Cat No. 164688), and 1 at room temperature. Incubated for a period of time and blocked with a 5-fold diluted blocking source (Nakalai Tesque, Cat No. 03953-95). After incubation, the plate was washed and the B-cell culture supernatant was added. The plate was incubated for 1 hour, washed, 500 pM biotinylated human or monkey C5 was added and incubated for 1 hour. After incubation, the plate was washed and at room temperature with pH 7.4 MES buffer (20 mM MES, 150 mM NaCl and 1.2 mM CaCl ₂ ) or pH 5.8 MES buffer (20 mM MES, 150 mM NaCl and 1 mM EDTA) for 1 hour. Cultured. After incubation, binding of biotinylated C5 was detected with streptavidin-horseradish peroxidase conjugate (Thermo Scientific, Cat No. 21132), and then ABTS (KPL, Cat No. 50-66-06) was added. .

The affinity and pH-dependent binding of antibodies to C5 were evaluated using the Octet RED384 system (Pall Life Sciences). The antibody secreted in the B-cell culture supernatant was loaded onto a Protein A biosensor tip (Paul Life Sciences) and immersed in 50 nM human or monkey C5 in pH 7.4 MES buffer to analyze the binding kinetics. Dissociation kinetics were analyzed in both pH 7.4 MES buffer and pH 5.8 MES buffer.

A total of 41,439 B-cell lines were screened for affinity and pH-dependent binding to human or monkey C5, and 677 cell lines were selected and designated CFA0001-0677. RNA of the selected cell line was purified from cryopreserved cell pellets using a ZR-96 Quick-RNA kit (ZYMO RESEARCH, Cat No. R1053). In the selected cell lines, the DNA encoding the antibody heavy chain variable region was amplified by reverse transcription PCR, and recombined with the DNA encoding the heavy chain constant region F760G4 (SEQ ID NO: 33) or F939G4 (SEQ ID NO: 34). The DNA encoding the antibody light chain variable region was amplified by reverse transcription PCR and recombined with the DNA encoding the k0MTC light chain constant region (SEQ ID NO: 36). Separately, heavy and light chain genes of the existing humanized anti-C5 antibody, eculizumab (EcuH-G2G4, SEQ ID NO: 29 and EcuL-k0, SEQ ID NO: 30), were synthesized. DNA encoding VH (EcuH, SEQ ID NO: 31) was fused in frame with DNA encoding modified human IgG4 CH (F760G4, SEQ ID NO: 33), and DNA encoding VL (EcuL, SEQ ID NO: 32) was incorporated into the k0 light chain. It was fused in frame to DNA encoding the constant region (SEQ ID NO: 37). Each of the fused coding sequences was also cloned into an expression vector. Antibodies were expressed in FreeStyle™ 293-F cells (Invitrogen) and purified from culture supernatant to evaluate functional activity. Neutralizing the activity of the antibody was evaluated by examining the inhibition of complement activity using a liposome lysis assay as described in Example 5.1.

2.2 Epitope binning by sandwich ELISA

Anti-C5 antibodies with high affinity, pH dependent or neutralizing activity were selected for further analysis. Selected antibodies were sorted into different epitope bins that bind to the same or overlapping epitopes of the C5 protein using a sandwich ELISA assay. Unlabeled capture antibody was diluted to 1 μg/ml with PBS(-) and added to a 384-well Maxisorp plate (Nunc, Cat No. 164688). The plate was incubated for 1 hour at room temperature and blocked with a 5-fold diluted blocking circle (Nakalai Tesque, Cat No. 03953-95). The plate was incubated for 1 hour, washed, 2 nM human C5 was added and incubated for 1 hour. After incubation, the plate was washed and labeled detection antibody (1 μg/ml, biotinylated with NHS-PEG4-biotin) was added. After incubation for 1 hour, binding of the biotinylated antibody was detected with streptavidin-horseradish peroxidase conjugate (Thermo Scientific, Cat No. 21132), and then ABTS (KPL, Cat No. 50-66-06). Was added.

All anti-C5 antibodies were used as both capture and detection antibodies and were fully paired. As shown in Figure 1, the mutually competitive antibodies were classified into 7 epitope bins: CFA0668, CFA0334 and CFA0319 were classified as epitope A, and CFA0647, CFA0589, CFA0341, CFA0639, CFA0635, CFA0330 and CFA0318 were classified as epitope B. CFA0538, CFA0501, CFA0599, CFA0307, CFA0366, CFA0305, CFA0675, CFA0666 and CFA0672 were classified as epitope C, eculizumab and CFA0322 were classified as epitope D, and CFA0329 were classified as epitope E, and CFA0359 and CFA0359 were classified as epitope C. It was classified as epitope F, and CFA0579, CFA0328 and CFA0272 were classified as epitope G. 1 shows the epitope binning of some of the anti-C5 chimeric antibodies. The sequence of VH and VL anti-C5 antibodies classified as epitope C is listed in Table 2.

2.3. Humanization and optimization

Humanization of the variable regions of some of the anti-C5 antibodies was performed to reduce the potential immunogenicity of the antibody. The complementarity-determining region (CDR) of the anti-C5 rabbit antibody is grafted onto the homologous human antibody framework (FR) using a conventional CDR grafting approach (Nature 321:522-525 (1986)). Made it. Genes encoding humanized VH and VL were synthesized and associated with each of the modified human IgG4 CH (SG402, SEQ ID NO: 35) and human CL (SK1, SEQ ID NO: 38), and each of the bound sequences was cloned into an expression vector.

Many mutations and mutation combinations were examined to identify mutations and mutation combinations that improved the binding properties of some of the lead antibodies. Subsequently, a number of mutations were introduced into the humanized variable region to increase the binding affinity for C5 at neutral pH or to decrease the binding affinity for C5 at acidic pH. Thus, one of the optimized variants, 305LO5 (VH, SEQ ID NO: 10; VL, SEQ ID NO: 20; HVR-H1, SEQ ID NO: 54; HVR-H2, SEQ ID NO: 64; HVR-H3, SEQ ID NO: 74; HVR-L1 , SEQ ID NO: 84; HVR-L2, SEQ ID NO: 94; and HVR-L3, SEQ ID NO: 104) were generated from CFA0305.

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

Example 3

Binding characterization of anti-C5 antibodies

3.1. Expression and purification of recombinant antibodies

Recombinant antibodies were transiently expressed using the Plastyle293-F cell line (Thermo Fisher, Carlsbad, CA). Purification from the conditioned medium expressing the antibody was performed using a conventional method using protein A. Gel filtration was also performed if necessary.

3.2. pH dependence assessment

Kinetic parameters of anti-C5 antibody against recombinant human C5 were evaluated at 37° C. at pH 7.4 and pH 5.8 using a Biacore® T200 instrument (GE Healthcare). ProA/G (Pierce) was immobilized on a CM4 sensor chip using an amine coupling kit (GE Healthcare) according to the settings recommended by GE Healthcare. Antibodies and analytes were diluted with respective running buffers, ACES pH 7.4 and pH 5.8 (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl ₂ , 0.05% Tween 20, 0.005% NaN ₃ ). Each antibody was captured on the sensor surface by ProA/G. The level of antibody capture was typically between 60 and 90 resonance units (RU). Subsequently, recombinant human C5 was injected at concentrations of 10 and 20 nM or 20 and 40 nM, followed by dissociation. The surface was regenerated using 25 mM NaOH. The sensorgram was fitted to a 1:1 binding model using Biacore® T200 evaluation software, version 2.0 (GE Healthcare) to measure the kinetic parameters under both pH conditions. Sensorgrams of all antibodies are shown in Figures 2a and 2b. The binding constant (ka), dissociation constant (kd), and binding affinity (KD) of the antibodies are listed in Table 3. All antibodies except CFA0330 (VH, SEQ ID NOs: 21 and VL, SEQ ID NO: 25) and CFA0341 (VH, SEQ ID NOs: 22 and VL, SEQ ID NO: 26) showed relatively faster dissociation constants at pH 5.8 than at pH 7.4.

3.3 Cross-reactivity test

To observe the cross-reactivity of anti-C5 antibodies against human C5 (hC5) and cynomolgus monkey C5 (cynoC5), a Biacore® kinetic analysis was performed. The assay settings were the same as described in Example 3.2. Recombinant cynoC5 was injected at concentrations of 2, 10 and 50 nM. Kinetic parameters were determined by the same data fitting as described in Example 3.2. The binding kinetics and affinity at pH 7.4 are listed in Table 4. The kinetic parameters for hC5 shown in Table 4 are the results of Example 3.2. Except for CFA0672, all anti-C5 antibodies showed comparable KD for hC5 and cynoC5. The KD of CFA0672 against cynoC5 was 8 times weaker than that against hC5.

Example 4

Epitope mapping of anti-C5 antibodies

4.1. Binding of anti-C5 MAb to C5 beta chain-derived peptide

The anti-C5 monoclonal antibody (MAb) was tested y for binding to the C5 beta chain-derived peptide in Western blot analysis. C5 peptide fused to GST-tag (pGEX-4T-1, GE Healthcare Life Sciences, 28-9545-49): 19-180, 161-340, 321-500, and 481-660 Escherichia coli (DH5 alpha, TOYOBO, DNA-903). After incubation with 1 mM isopropyl beta-D-1-thiogalactopyranoside (IPTG) at 37° C. for 5 hours, an Escherichia coli sample was collected and centrifuged at 20000 xg for 1 minute to obtain a pellet. Obtained. The pellet was suspended in sample buffer (2ME+) (Wako, 191-13272) and used for Western blot analysis. Expression of each peptide was confirmed using an anti-GST antibody (Abcam, ab9085) (Fig. 3). Arrows indicate GST-fused C5 peptide (46-49 kDa). Anti-C5 MAb: CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675 bound to 19-180 of C5 (Figure 3).

4.2. Expression and purification of MG1-MG2 domain (1-225) of human C5

The recombinant MG1-MG2 domain (SEQ ID NO: 43) of the human C5 beta chain was transiently expressed using the Plastyle293-F cell line (Thermo Fisher, Carlsbad, CA). The conditioned medium expressing the MG1-MG2 domain was diluted with 1/2 volume of MilliQ water, and then applied to a Q-Sepharose FF anion exchange column (GE Healthcare, Uppsala, Sweden). The perfusion fraction from the anion exchange column was adjusted to pH 5.0, applied to an SP-Sepharose HP cation exchange column (GE Healthcare, Uppsala, Sweden) and eluted with a NaCl gradient. Fractions containing the MG1-MG2 domain were collected from the eluate. It was then applied to a Superdex 75 gel filtration column (GE Health Care, Uppsala, Sweden) equilibrated with 1 x PBS. Then, the fractions containing the MG1-MG2 domain were collected and stored at -80°C.

4.3. Binding ability to MG1-MG2 domain

The binding ability of the anti-C5 antibody to the MG1-MG2 domain was measured using the same assay settings as described in Example 3.2, except that the measurement was performed only under pH 7.4 conditions. The MG1-MG2 domain was injected at concentrations of 20 nM and 40 nM. As shown in FIG. 4, all antibodies except eculizumab-F760G4 showed an increase in binding reaction, suggesting that the antibodies were MG1-MG2 binding agents. Eculizumab-F760G4, a known alpha chain binding agent, did not show binding to the MG1-MG2 domain.

4.4. Binding of anti-C5 MAb to C5 MG1-MG2 domain-derived peptide

Anti-C5 MAb was tested for binding to the MG1-MG2 domain-derived peptide in Western blot analysis. C5 peptides fused to GST-tag: 33-124, 45-124, 52-124, 33-111, 33-108, and 45-111 (SEQ ID NO: 40) were expressed in Escherichia coli. After incubation with 1 mM IPTG at 37° C. for 5 hours, an Escherichia coli sample was collected and centrifuged at 20000 x g for 1 minute to obtain a pellet. The pellet was suspended in sample buffer (2ME+) and used for Western blot analysis. Expression of the C5-derived peptide was confirmed using an anti-GST antibody (Fig. 5A). CFA0305 only bound to peptides 33-124 (Fig. 5B). CFA0305 bound to the beta chain (about 70 kDa) of recombinant human C5 (rhC5), which was used as a control. 5C summarizes the response of anti-C5 MAb to C5-derived peptides.

4.5. Binding of anti-C5 MAb to C5 mutant

Since three amino acid residues in the C5 beta chain: E48, D51 and K109 were predicted to be involved in the binding between C5 and anti-C5-MAb by crystal structure analysis, anti-C5 MAb was used as a human C5 point in Western blot analysis. It was tested for binding to the mutant. A C5 point mutant in which any one of E48, D51 and K109 was substituted with alanine was expressed in FS293 cells by lipofection. After 5 days of lipofection, the culture medium was collected and used for Western blot. SDS-PAGE was performed under reducing conditions. The results are shown in FIG. 6. Eculizumab binds to the alpha chain of wild-type (WT) C5 and three C5 point mutants, whereas CFA0305 binds strongly to the beta chain of WT C5 and weakly to the E48A C5 mutant, and the beta of the D51A and K109A C5 mutants. By not binding to the chain, it was suggested that the three amino acid residues are involved in the antibody/antigen interaction. Table 5 shows a summary of Western blot analysis of anti-C5 MAbs (CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675). The anti-C5 MAb is classified as the same epitope C, but the binding pattern is slightly different between the antibodies, suggesting that the binding regions of C5 to the anti-C5 MAb are close to each other, but not identical.

4.6. Biacore® binding assay of anti-C5 antibodies using C5 mutants

To examine whether residues E48, G51, and K109 are actually involved in the antibody/antigen interaction, a Biacore® binding assay was performed. As described in Example 4.5, three C5 mutants were prepared: E48A, G51A, and K109A. Culture supernatant samples containing mutant C5 overexpressed in FS293 cells were prepared at 40 μg/ml mutant C5. For Biacore® binding assay, samples were mutated at 4 μg/ml using Biacore® running buffer (ACES pH 7.4, 10 mg/ml BSA, 1 mg/ml carboxymethyl dextran). It was diluted 10-fold to the final sample concentration of C5.

The interaction of the three C5 mutants with the anti-C5 antibody was evaluated using the assay conditions described in Example 3.2 using a Biacore® T200 instrument (GE Healthcare) at 37°C. ACES pH 7.4 buffer containing 10 mg/ml BSA, 1 mg/ml carboxymethyl dextran was used as a running buffer. Eculizumab-F760G4 and 305LO5 were captured on different flow cells by monoclonal mouse anti-human IgG, Fc fragment specific antibody (GE Healthcare). Flow cell 1 was used as the reference surface. Wild-type and mutant C5 proteins were injected onto the sensor surface at a concentration of 4 μg/ml to interact with the captured antibody. At the end of each analysis cycle, the sensor surface was regenerated with _{3M MgCl 2.} Results were analyzed using Bia Evaluation software, version 2.0 (GE Healthcare). The curves of the reference flow cells (flow cells 1) and the blank injections of running buffer were subtracted from the curves of flow cells with capture antibodies.

As shown in Figure 7, all three C5 mutants are capable of binding eculizumab under a similar binding profile compared to wild-type C5. In the case of 305LO5, all three mutants showed a lower binding response to 305LO5 compared to wild-type C5. The D51A and K109A mutants reduced the binding of C5 by 305LO5 to baseline levels.

4.7. Identification of His residues on C5 that contribute to the pH-dependent interaction between anti-C5 antibody and C5

As a result of crystal structure analysis, it was confirmed that three histidine residues on human C5 are located at the antibody/antigen interface. Histidine residues with a typical pKa of about 6.0 are known to contribute to pH dependent protein-protein interactions (Igawa et al., Biochim Biophys Acta 1844(11):1943-1950 (2014)). To investigate which of the His residues on the antibody/antigen interface contribute to the pH dependent interaction between the anti-C5 antibody and C5, a Biacore® binding assay was performed. Three human C5 mutants with a single His mutation (H70Y, H72Y, and H110Y), and a mutant with a double-His mutation (H70Y + H110Y) were prepared as follows: any one of H70, H72, and H110 A single His mutant substituted with tyrosine, and a double His mutant where both H70 and H110 were substituted with tyrosine were expressed in FS293 cells by lipofection. The antigen binding properties of the C5 His mutant to 305LO5, a pH-dependent anti-C5 antibody, were determined by a modified Biacore® assay as described in Example 4.6. Briefly, another dissociation step at pH 5.8 was incorporated into a Biacore® assay immediately after the dissociation step at pH 7.4 to assess the pH-dependent dissociation between antibody and antigen from complexes produced at pH 7.4. I did. The rate of dissociation at pH 5.8 was measured by processing and fitting the data using Scrubber 2.0 (BioLogic Software) curve fitting software.

As shown in Figure 8, the C5 single His mutation and double His mutation (H70 + H110) in H70 or H110 did not affect the binding of C5 to 305LO5 at neutral pH. On the other hand, a single His mutation in H72 showed a significant decrease in the binding of C5 to 305LO5. The dissociation rates at pH 5.8 for the C5 His mutant and C5-wt protein are shown in Table 6. As shown in Table 6, C5-wt showed the fastest dissociation from 305LO5 at pH 5.8 among the C5 antigens tested. The single His mutation at H70 showed an almost 2-fold slower dissociation rate at pH 5.8 compared to C5-wt, and the single His mutation at H110 showed a slightly slower dissociation rate at pH 5.8. The double His mutation in both H70 and H110 caused a greater effect on pH-dependent binding under a dissociation rate that was nearly 3 times slower than C5-wt at pH 5.8.

Example 5

Inhibitory activity of anti-C5 antibodies on C5 activation

5.1. Inhibition of complement-activated liposome lysis by anti-C5 MAb

Anti-C5 MAb was tested for inhibition of complement activity by liposome lysis assay. 30 μl of normal human serum (6.7%) (Biopredic, SER018) was mixed with 20 μl of diluted MAb in a 96-well plate and incubated on a shaker at 25° C. for 30 minutes. Liposomes sensitized with antibodies against dinitrophenyl (Autokit CH50, Wako, 995-40801) were transferred to each well and the plate was placed on a shaker at 25° C. for 2 minutes. 50 [mu]l of the substrate solution (autokit CH50) was added to each well and mixed by shaking at 25[deg.] C. for 2 minutes. After incubating the final mixture at 37° C. for 40 minutes, the mixture was measured for OD at 340 nm. The percent liposome lysis was defined as 100 x [(OD _MAb -OD _{serum and liposome background} )]/[(OD _{MAb free} -OD _{serum and liposome background} )]. 9A shows that anti-C5 MAb: CFA0305, 0307, 0366, 0501, 0538, 0599, 0666, 0672, and 0675 inhibited liposome lysis. Two non-pH-dependent antibodies: CFA0330 and 0341 also inhibited lysis (FIG. 9B ).

5.2. Inhibition of C5a production by anti-C5 MAb

Anti-C5 MAb was tested for C5a production upon liposome lysis to confirm that the anti-C5 MAb inhibited cleavage of C5 to C5a and C5b. C5a levels in the supernatant from liposome lysis were quantified using the C5a ELISA kit (R&D Systems, DY2037). All MAb dose-dependently inhibited C5a production in the supernatant (FIGS. 10A and 10B ).

5.3. Inhibition of complement-activated hemolysis by anti-C5 MAb

Anti-C5 MAb was tested for inhibition of classical complement activity in the hemolysis assay. Chicken erythrocytes (cRBC) (Innovative research, IC05-0810) were treated with gelatin/veronal-buffered saline (GVB++) containing _{0.5 mM MgCl 2} and 0.15 mM CaCl _{2 (Boston BioProducts (Boston} BioProducts), IBB-300X), and then sensitized for 15 minutes with an anti-chicken RBC antibody (Rockland 103-4139) at 1 μg/ml at 4°C. Then, the cells were washed with GVB++ and suspended in the same buffer at 5×10 ⁷ cells/ml. In a separate round-bottom 96-well microtest plate, 50 μl of normal human serum (20%) (Biopredic, SER019) was mixed with 50 μl of diluted Mab and incubated at 37° C. on a shaker for 30 minutes. Then, 60 μl of the sensitized cRBC suspension was added to the wells containing serum, and the antibody mixture was incubated at 37° C. for 30 minutes. After incubation, the plate was centrifuged for 2 minutes at 1000 xg at 4°C. The supernatant (100 μl) was transferred to a well on a flat bottom 96-well microtest plate for OD measurements at 415 nm under a reference wavelength at 630 nm. Percent hemolysis was defined as 100 x [(OD _MAb -OD _{serum and cRBCs} )]/[(OD _{MAb free} -OD _{serum and cRBCs background} )]. 11 shows that anti-C5 Mab: CFA0305 and 305LO5 inhibited the hemolysis of cRBC.

5.4. Inhibition of the alternative complement pathway by anti-C5 MAb

Hemolysis assays for alternative pathways were performed in a manner similar to classical pathway hemolysis assays. Blood collected from New Zealand white rabbits (InVivos) was mixed with the same volume of Alserver solution (Sigma, A3551), and the mixture was used as rabbit RBC (rRBC). rRBC was washed with GVB supplemented with 2 mM MgCl ₂ and 10 mM EGTA and suspended at 7 ^{×10 8} cells/ml in the same buffer. In a round-bottom 96-well microtest plate, 40 μl of normal human serum (25%) (Biopredic, SER019) was mixed with 40 μl of diluted Mab and incubated at 37° C. on a shaker for 30 minutes. Subsequently, 20 μl of rRBC suspension was added to the wells containing serum, and the antibody mixture was incubated at 37° C. for 60 minutes. After incubation, the plate was centrifuged for 2 minutes at 1000 xg at 4°C. The supernatant (70 μl) was transferred to a well on a flat bottom 96-well microtest plate for OD measurements at 415 nm under a reference wavelength at 630 nm. 12 shows that anti-C5 Mab: CFA0305 and CFA0672 inhibit the hemolysis of rRBC, suggesting that these antibodies inhibit the alternative complement pathway.

Example 6

Pharmacokinetic study of anti-C5 monoclonal antibody using human C5 in mice

6.1. In vivo examination using C57BL/6 mice

Human C5 alone or human C5 and anti-human C5 antibodies were administered to C57BL/6 mice (Invivos or Biological Resource Center, Singapore) followed by human C5 (Calbiochem) and anti-human The in vivo kinetics of the C5 antibody was evaluated. Human C5 solution (0.01 mg/ml) or human C5 and anti-human C5 antibodies (0.01 mg/ml and 2 mg/ml, respectively (CFA0305-F760G4, CFA0307-F760G4, CFA0366-F760G4, CFA0501-F760G4, CFA0538-F760G4, respectively) , CFA0599-F760G4, CFA0666-F760G4, CFA0672-F760G4, and CFA0675-F760G4) or a solution of a mixture containing 0.2 mg/ml (CFA0330-F760G4, and CFA0341-F760G4)] at a dose of 10 ml/kg. It was administered once within. In this case, the anti-human C5 antibody is present in excess compared to human C5, so it is assumed that almost all human C5 will bind to the antibody. Blood was collected 5 minutes, 7 hours, 1 day, 2 days, 3 days, and 7 days after administration. The collected blood was immediately centrifuged at 14,000 rpm and 4° C. for 10 minutes to separate plasma. The separated plasma was stored in a refrigerator at -80 °C before analysis. Anti-human C5 antibodies used were as follows: CFA0305-F760G4, CFA0307-F760G4, CFA0330-F760G4, CFA0341-F760G4, CFA0366-F760G4, CFA0501-F760G4, CFA0538-F760G4, CFA0599-F760G4, CFA0599-F760G4, CFA0599-F760G4, CFA0599-F760G4 CFA0672-F760G4, and CFA0675-F760G4.

6.2. Measurement of total human C5 plasma concentration by electrochemiluminescence (ECL) analysis

The concentration of total human C5 in mouse plasma was measured by ECL.

In the presence of CFA0330-F760G4, CFA0341-F760G4, or human C5 alone in plasma samples, the following method was used. Anti-human C5 antibody (Santa Cruz) was dispensed onto a multi-array 96-well empty plate (Meso Scale Discovery) and allowed to stand at 4° C. overnight to prevent anti-human C5-immobilized plates were prepared. A calibration curve sample and a mouse plasma sample diluted 100 times or more with an injection antibody of 1 μg/ml (CFA0330-F760G4 or CFA0341-F760G4) were prepared and incubated at 37° C. for 30 minutes. The samples were then dispensed onto anti-human C5-immobilized plates and allowed to stand at room temperature for 1 hour. Subsequently, SULFO-TAG-labeled anti-human IgG antibody (Meso Scale Discovery) was added, reacted at room temperature for 1 hour, and washed. Immediately after that, the Read Buffer T (x4) (Meso Scale Discovery) was dispensed and measurements were performed using a Sector Imager 2400 (Meso Scale Discovery).

The following methods were used in the presence of CFA0305-F760G4, CFA0307-F760G4, CFA0366-F760G4, CFA0501-F760G4, CFA0538-F760G4, CFA0599-F760G4, CFA0666-F760G4, CFA0672-F760G4, or CFA0675-F760G4 in plasma samples. Anti-human C5 antibody (CFA0329-F939G4; VH, SEQ ID NO: 23 and VL, SEQ ID NO: 27) was distributed on a multi-array 96-well empty plate (Meso Scale Discovery) and allowed to stand overnight at 4° C. to -A fixed plate was prepared. A calibration curve sample and a mouse plasma sample diluted 100 times or more with an acidic solution (pH 5.5) were prepared and incubated at 37° C. for 30 minutes. The samples were then dispensed onto anti-human C5-immobilized plates and allowed to stand at room temperature for 1 hour. Subsequently, sulfo-tag-labeled anti-human C5 antibody (CFA0300-F939G4; VH, SEQ ID NO: 24 and VL, SEQ ID NO: 28) was added to react at room temperature for 1 hour, followed by washing. Immediately after that, the read buffer T(x4) (Meso Scale Discovery) was dispensed and measurements were performed using a sector imager 2400 (Meso Scale Discovery).

Human C5 concentration was calculated based on the response of the calibration curve using analytical software SOFTmax PRO (Molecular Devices). As measured by the above method, the time course of plasma human C5 concentration after intravenous administration is shown in FIG. 13. Data were plotted as percentage remaining compared to plasma human C5 concentration at 5 minutes.

6.3. Anti-human C5 antibody plasma concentration measurement by ECL assay

The concentration of anti-human C5 in mouse plasma was measured by ECL. Anti-human IgG (gamma chain specific) F(ab')2 antibody fragment (Sigma) or anti-human IgG kappa chain antibody (Antibody Solutions) was added to a multi-array 96-well empty plate (meso scale). Discovery) and allowed to stand at 4° C. overnight to prepare anti-human IgG-immobilized plates. A calibration curve sample and a mouse plasma sample diluted 100 times or more were prepared. The samples were then dispensed onto anti-human IgG-immobilized plates and allowed to stand at room temperature for 1 hour. Subsequently, a biotinylated anti-human IgG antibody (Southernbiotech) or a sulfo-tag labeled anti-human IgG Fc antibody (Southern Biotech) was added to react at room temperature for 1 hour, followed by washing. Subsequently, only when a biotinylated anti-human IgG antibody was used, sulfo-tagged streptavidin (Meso Scale Discovery) was added to react at room temperature for 1 hour, followed by washing. Immediately after that, the read buffer T(x4) (Meso Scale Discovery) was dispensed and measurements were performed using a sector imager 2400 (Meso Scale Discovery). Anti-human C5 antibody concentration was calculated based on the response of the calibration curve using the analytical software Softmax Pro (Molecular Devices). The time course of the plasma anti-human C5 antibody concentration after intravenous administration as measured by the above method is shown in FIG. 14. Data were plotted as the remaining percentage compared to the anti-human C5 antibody concentration in plasma at 5 minutes.

6.4. Effect of pH dependent anti-human C5 antibody binding on clearance of human C5 in vivo

pH-dependent anti-human C5 antibodies (CFA0305-F760G4, CFA0307-F760G4, CFA0366-F760G4, CFA0501-F760G4, CFA0538-F760G4, CFA0599-F760G4, CFA0666-F760G4, CFA0672-F760G4, and CFA0675-pH-dependent) Anti-human C5 antibodies (CFA0330-F760G4, and CFA0341-F760G4) were tested in vivo, and the resulting plasma anti-human C5 antibody concentration and plasma human C5 concentration were compared. As shown in Figure 14, antibody exposure was comparable. On the other hand, the removal of human C5 administered simultaneously with the pH-dependent anti-human C5 antibody was promoted compared to the non-pH-dependent anti-human C5 antibody (FIG. 13).

Example 7

Optimization of anti-C5 monoclonal antibody (305 variant)

In order to further improve its properties, a number of mutations were introduced into the optimized variable region of anti-C5 antibody 305LO5, and the optimized variable regions 305LO15, 305LO16, 305LO18, 305LO19, 305LO20, 305LO22, and 305LO23 were generated. The amino acid sequences of VH and VL of the 305 variant are listed in Tables 7 and 8, respectively. Genes encoding humanized VH were combined with a modified human IgG1 CH variant SG115 (SEQ ID NO: 114), and a modified human IgG4 CH variant SG422 (SEQ ID NO: 115) or SG429 (SEQ ID NO: 116). The gene encoding humanized VL was combined with human CL (SK1, SEQ ID NO: 38). Separately, a humanized anti-C5 antibody, heavy and light chain genes encoding BNJ441 (BNJ441H, SEQ ID NO: 149; BNJ441L, SEQ ID NO: 150) were synthesized and cloned into expression vectors, respectively.

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

Example 8

Binding characterization of anti-C5 antibody (305 variant)

The kinetic parameters of anti-C5 antibodies against recombinant human C5 were evaluated using a Biacore® T200 instrument (GE Healthcare) at 37° C. under three different conditions: (1) both binding and dissociation. A condition that is at pH 7.4, (2) a condition where both binding and dissociation are at pH 5.8, and (3) a condition where binding is at pH 7.4 but dissociation is at pH 5.8. ProA/G (pierce) was immobilized on the CM1 sensor chip using an amine coupling kit (GE Healthcare) according to the settings recommended by GE Healthcare. In conditions (1) and (3), antibodies and analytes were diluted in ACES pH 7.4 buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl ₂ , 0.05% Tween 20, 0.005% NaN ₃ ), and condition (2 ), antibodies and analytes were diluted in ACES pH 5.8 buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl ₂ , 0.05% Tween 20, 0.005% NaN ₃ ). Each antibody was captured on the sensor surface by ProA/G. The level of antibody capture was typically between 60 and 90 resonance units (RU). Subsequently, recombinant human C5 was dissociated after injection at 3 to 27 nM or 13.3 to 120 nM prepared by 3-fold serial dilution. The surface was regenerated using 25 mM NaOH. The kinetic parameters in conditions (1) and (2) were measured by fitting the sensorgram to a 1:1 binding model, and the dissociation rate in condition (3) was Biacore® T200 evaluation software, version 2.0 ( GE Healthcare) was used to measure the sensorgram by fitting it to a 1:1 dissociation model for MCK. The pH dependence of all antibodies was expressed as the ratio of the dissociation rate of conditions (2) and (1).

The binding rate (ka), dissociation rate (kd), binding affinity (KD), and pH dependence are listed in Table 9. All antibodies showed faster dissociation at pH 5.8 than at pH 7.4, and their pH dependence was about 20 times.

The binding affinity of anti-C5 antibodies (BNJ441, eculizumab, and 305 variants) to recombinant human C5 at pH 7.4 and pH 5.8 was measured at 37° C. using a Biacore® T200 instrument (GE Healthcare). It was measured to evaluate the effect of pH upon antigen binding. Goat anti-human IgG (Fc) polyclonal antibody (KPL #01-10-20) was immobilized on a CM4 sensor chip using an amine coupling kit (GE Healthcare) according to the settings recommended by the manufacturer. Antibodies and analytes were diluted in ACES pH 7.4 buffer or ACES pH 5.8 buffer containing 20 mM ACES, 150 mM NaCl, 1.2 mM CaCl ₂ , 0.05% Tween 20 and 0.005% NaN _3. Antibodies were captured on the sensor surface using the anti-Fc method, and the level of capture was typically 50-80 resonance units (RU). Recombinant C5 was prepared by 3-fold serial dilution starting from 27 nM for the pH 7.4 assay condition or from 135 nM for the pH 5.8 assay condition. The surface was regenerated using 20 mM HCl, 0.01% Tween 20. The data were processed and fitted into a 1:1 binding model using Non-Ivelation 2.0 software (GE Healthcare).

The binding affinity (KD) of the BNJ441, eculizumab and 305 variants for recombinant human C5 at pH 7.4 and pH 5.8 are shown in Table 10. The 305 variant had a ratio of nearly 800 (KD at pH 5.8)/(KD at pH 7.4), 8 times higher than BNJ441, which showed a ratio of 93 (KD at pH 5.8)/(KD at pH 7.4). Indicated.

Example 9

Inhibitory activity of anti-C5 antibody (305 variant) on C5 activation

9.1 Inhibition of complement-activated liposome lysis by anti-C5 MAb

Anti-C5 MAb was tested for inhibition of complement activity by liposome lysis assay. 30 μl of normal human serum (6.7%) (Biopredic, SER018) was mixed with 20 μl of diluted MAb in a 96-well plate and incubated on a shaker for 30 minutes at room temperature. Liposomes sensitized with antibodies against dinitrophenyl (Autokit CH50, Waco, 995-40801) were transferred to each well and the plate was placed on a shaker at 37° C. for 2 minutes. 50 [mu]l of the substrate solution (autokit CH50) was added to each well and mixed by shaking at 37[deg.] C. for 2 minutes. After incubating the final mixture at 37° C. for 40 minutes, the OD at 340 nm was measured. The percent liposome lysis was defined as 100 x [(OD _MAb -OD _{serum and liposome background} )]/[(OD _{MAb free} -OD _{serum and liposome background} )]. Figure 15 shows that anti-C5 MAb: 305LO15-SG422, 305LO16-SG422, 305LO18-SG422, 305LO19-SG422, 305LO20-SG422, and 305LO20-SG115 inhibited liposome lysis. Two antibodies with Fc variants: 305LO15-SG115 and 305LO23-SG429 also inhibited liposome lysis (FIG. 16 ).

Anti-C5 MAb was tested for inhibition of recombinant human C5 (SEQ ID NO: 39). 10 µl of C5-deficient human serum (Sigma, C1163) was mixed with 20 µl of diluted MAb and 20 µl of recombinant C5 (0.1 µg/ml) in a 96-well plate, and for 1 hour on a shaker at 37°C. Cultured. Liposomes (autokit CH50) were transferred to each well and placed on a shaker at 37° C. for 2 minutes. 50 [mu]l of the substrate solution (autokit CH50) was added to each well and mixed by shaking at 37[deg.]C for 2 minutes. After incubating the final mixture at 37° C. for 180 minutes, the OD at 340 nm was measured. The percentage of liposome dissolution was defined as above. Figure 17 shows that anti-C5 Mab: 305LO22-SG115, 305LO22-SG422, 305LO23-SG115, and 305LO23-SG422 inhibited liposome lysis.

9.2. Inhibition of C5a production by anti-C5 MAb

Anti-C5 MAb was tested for C5a production upon liposome lysis to confirm that the anti-C5 MAb inhibited cleavage of C5 to C5a and C5b. C5a levels in the supernatant from liposome lysis assay were quantified using the C5a ELISA kit (R&D Systems, DY2037). All MAb dose-dependently inhibited C5a production in the supernatant (FIGS. 18 and 19 ).

9.3. Measurement of complement activity in cynomolgus monkey plasma

Anti-C5 MAb was tested for inhibition of complement activity in cynomolgus monkey plasma. Anti-C5 Mab was administered internally to monkeys (20 mg/kg) and plasma samples were collected periodically until day 56. Chicken red blood cells (cRBC) (Innovative Research, IC05-0810) with _{gelatin/veronal-buffered saline (GVB++) containing 0.5 mM MgCl 2} and 0.15 mM CaCl ₂ (Boston BioProducts, IBB-300X). After washing, it was sensitized at 4° C. with anti-chicken RBC antibody (Rockland 103-4139) at 1 μg/ml for 15 minutes. Then, the cells were washed with GVB++ and suspended at 1× ^{10 8} cells/ml in the same buffer. In a separate round-bottom 96-well microtest plate, monkey plasma was incubated with sensitized cRBC at 37° C. for 20 minutes. After incubation, the plate was centrifuged for 2 minutes at 1000 xg at 4°C. The supernatant was transferred to a well on a flat bottom 96-well microtest plate for OD measurements at 415 nm under a reference wavelength at 630 nm. Percent hemolysis was defined as 100 x [( _after OD administration-OD _{plasma and cRBC background} )]/[( _before OD administration-OD _{plasma and cRBC background} )]. Figure 20 shows that anti-C5 MAb: 305LO15-SG422, 305LO15-SG115, 305LO16-SG422, 305LO18-SG422, 305LO19-SG422, 305LO20-SG422, 305LO20-SG115, and 305LO23-SG115 inhibited complement activity in plasma. Show

9.4. Inhibition of biological activity of C5 variants by anti-C5 MAb

Anti-C5 MAb was tested for inhibition of recombinant human C5 variants: V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N, and E1437D. PNH patients with the R885H mutation in C5 have been reported to have a poor response to eculizumab (see, eg, Nishimura et al., New Engl. J. Med. 370:632-639 (2014)). . Each of the human C5 variants was expressed in FS293 cells, and the supernatant was used in the following studies. 10 μl of C5-deficient human serum (Sigma, C1163) is mixed with 20 μl of diluted MAb in a 96-well plate, and 20 μl of cell culture medium containing recombinant C5 variants (2-3 μg/ml), Incubated for 0.5 hours on a shaker at 37 ℃. Liposomes (autokit CH50) were transferred to each well and placed on a shaker at 37° C. for 2 minutes. 50 [mu]l of the substrate solution (autokit CH50) was added to each well and mixed by shaking at 37[deg.]C for 2 minutes. After incubating the final mixture at 37° C. for 90 minutes, the OD at 340 nm was measured. The percentage of liposome dissolution was defined as above. Figure 21 shows that anti-C5 MAb (eculizumab) did not inhibit the R885H C5 variant, but inhibited the other variants tested. Figure 22 shows that anti-C5 MAb (305 variant) inhibited all variants of C5 tested.

9.5. Inhibition of complement-activated liposome lysis by anti-C5 MAb

Anti-C5 MAb was tested for inhibition of complement activity by liposome lysis assay. 30 μl of normal human serum (6.7%) (Biopredic, SER018) was mixed with 20 μl of diluted MAb in a 96-well plate and incubated on a shaker for 30 minutes at room temperature. Liposomes sensitized with antibodies against dinitrophenyl (autokit CH50, Waco, 995-40801) were transferred to each well and the plate was placed on a shaker at 25° C. for 2 minutes. 50 [mu]l of the substrate solution (autokit CH50) was added to each well and mixed by shaking at 25[deg.] C. for 2 minutes. After incubating the final mixture at 37° C. for 45 minutes, the mixture was measured for OD at 340 nm. Percent inhibition of liposome lysis was defined as 100 x [(OD _MAb -OD _{serum and liposome background} )]/[(OD _{MAb free} -OD _{serum and liposome background} )]. 23 shows that anti-C5 MAb, BNJ441 and 305 variants inhibited liposome lysis, and that 305 variant has a stronger inhibitory activity than BNJ441.

Example 10

Pharmacokinetic study of anti-C5 monoclonal antibody (305 variant) in cynomolgus monkey

10.1. In vivo test using cynomolgus monkey

The in vivo kinetics of anti-human C5 antibodies were evaluated after administration of anti-human C5 antibodies from cynomolgus monkeys (Shin Nippon Biomedical Laboratories, Ltd., Japan). A solution of anti-human C5 antibody (2.5 mg/ml) was injected for 30 minutes and administered once at a dose of about 8 ml/kg in the lumbar cortical vein of the forefoot. Blood was collected before administration and after 5 minutes, 7 hours, 1 day, 2 days, 3 days, 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days and 56 days after administration. The collected blood was immediately centrifuged at 1,700 x g and 4° C. for 10 minutes to separate plasma. The separated plasma was stored in a refrigerator at -70°C or lower before analysis. Anti-human C5 antibodies were prepared as described in Example 7.

10.2. Determination of total cynomolgus monkey C5 plasma concentration by ELISA assay

The concentration of total cynomolgus monkey C5 in cynomolgus monkey plasma was measured by ELISA. Anti-human C5 antibody (an in-house antibody produced using the method described in Example 2) was dispensed onto Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and 4° C. An anti-cynomolgus monkey C5-immobilized plate was prepared by standing overnight at 37° C. for 60 minutes at 37° C. to prepare a calibration curve sample and a cynomolgus monkey plasma sample diluted 20000 times with an injection antibody of 0.4 μg/ml The samples were then distributed on anti-cynomolgus monkey C5-immobilized plates and allowed to stand for 1 hour at room temperature, followed by addition of HRP-labeled anti-human IgG antibody (Southern Biotech). The reaction was carried out at room temperature for 30 minutes, followed by washing, ABTS ELISA HRP substrate (KPL) was then added The signal was measured by a plate reader at a wavelength of 405 nm Cynomolgus monkey C5 concentration was analyzed by software for analysis. It was calculated based on the response of the calibration curve using Softmax Pro (Molecula Devices), as measured by the above method, the time course of the plasma cynomolgus monkey C5 concentration after intravenous administration is shown in Fig. 24. Data were plotted as the remaining percentage compared to the plasma cynomolgus monkey C5 concentration prior to dosing, pH dependent anti-human C5 antibodies (305LO15-SG422, 305LO15-SG115, 305LO16-SG422, 305LO18-SG422, 305LO19-SG422, 305LO20-SG422, 305LO20-SG115, 305LO22-SG422, 305LO23-SG422, and 305LO23-SG115) showed a lower accumulation of plasma C5 compared to the non-pH dependent anti-human C5 antibody.

10.3. Determination of anti-human C5 antibody plasma concentration by ELISA assay

The concentration of total anti-human C5 in cynomolgus monkey plasma was measured by ELISA. Anti-human IgG kappa chain antibody (Antibody Solutions) was dispensed onto Nunc-Imunoplate Maxisorp (Nalji Nunc International) and allowed to stand overnight at 4° C. to prepare anti-human IgG-immobilized plates. A calibration curve sample diluted 100 times or more and a cynomolgus monkey plasma sample were prepared. The samples were then dispensed onto anti-human IgG-immobilized plates and allowed to stand at room temperature for 1 hour. Subsequently, HRP-labeled anti-human IgG antibody (Southern Biotech) was added, reacted at room temperature for 30 minutes, and washed. Then, ABTS ELISA HRP substrate (KPL) was added. The signal was measured by a plate reader at a wavelength of 405 nm. Anti-human C5 antibody concentration was calculated based on the response of the calibration curve using the analytical software Softmax Pro (Molecular Devices). The time course of the plasma anti-human C5 antibody concentration after intravenous administration as measured by the above method is shown in FIG. 25. pH dependent anti-human C5 antibodies (305LO15-SG422, 305LO15-SG115, 305LO16-SG422, 305LO18-SG422, 305LO19-SG422, 305LO20-SG422, 305LO20-SG115, 305LO22-SG422, 305LO23-SG422, and 305LO23-SG422, and 305LO23-SG422) Showed a longer half-life compared to the non-pH dependent anti-human C5 antibody.

Example 11

X-ray crystal structure analysis of 305 variant Fab and human C5-MG1 domain complex

11.1. Expression and purification of MG1 domain (20-124) of human C5

The MG1 domain (amino acids 20 to 124 of SEQ ID NO: 39) (GST-MG1) fused to the GST-tag through a thrombin cleavable linker was transferred to Escherichia coli strain BL21 using the pGEX-4T-1 vector (GE Healthcare). It was expressed in DE3 pLysS (Promega). Protein expression was induced at 25° C. with 0.1 mM isopropyl beta-D-1-thiogalactopyranoside (IPTG) for 5 hours. Bacterial cell pellets were lysed with Bugbuster (Merck) supplemented with lysonase (Merck) and complete protease inhibitor cocktail (Roche), then GSTrap column (GE Healthcare) was placed on the manufacturer's instructions. GST-MG1 was purified from the soluble fraction using according to the following. The GST tag was digested with thrombin (Sigma), and the resulting MG1 domain was further purified with a Superdex 75 gel filtration column (GE Healthcare). Fractions containing the MG1 domain were collected and stored at -80 °C.

11.2. Preparation of Fab fragment of 305 variant

After preparing the Fab fragment of one of the optimized variants from 305 by a conventional method using restriction cleavage by papain (Roche Diagnostics, Cat No.1047825), Protein A for removing the Fc fragment Column (MabSlect SuRe, GE Healthcare), cation exchange column (HiTrap SP HP, GE Healthcare), and gel filtration column (Superdex200 16/60, GE Healthcare). Fab fragment Fractions containing the were collected and stored at -80 °C.

11.3. Preparation of 305 variant Fab and human C5-MG1 domain complex

The purified recombinant human C5-MG1 domain was mixed with the purified 305 variant Fab fragment in a 1:1 molar ratio. The complex was purified by gel filtration chromatography (Superdexx200 10/300 increase, GE Healthcare) using a column equilibrated with 25 mM HEPES pH 7.5, 100 mM NaCl.

11.4. crystallization

The purified complex was concentrated to about 10 mg/ml, and crystallization was performed at 4° C. by a sitting drop vapor diffusion method together with a seeding method. The receiver solution consisted of 0.2 M magnesium formate dehydration, 15.0% w/v polyethylene glycol 3350. This succeeded in obtaining plate-like crystals within a few days. The crystals were immersed in a solution of 0.2 M magnesium formate dehydrate, 25.0% w/v polyethylene glycol 3350, and 20% glycerol.

11.5. Data collection and structure measurement

X-ray diffraction data were measured with BL32XU in SPring-8. During the measurement, the crystals were continuously placed in a nitrogen stream at -178 °C to keep them frozen, while the crystals were rotated 1.0 degrees at a time, using the MX-225HS CCD detector (RAYONIX) coupled to the beam line. Thus, a total of 180 X-ray diffraction images were collected. Xia2 program (J. Appl. Cryst. 43:186-190 (2010)), XDS package (Acta. Cryst. D66:125-132 (2010)), and Scala (Acta. Cryst. D62:72-82 (2006)]) was used to measure cell parameters, index diffraction spots, and process diffraction data obtained from diffraction images, and finally to obtain diffraction intensity data of 2.11 Å resolution or less. I did. The statistical results of crystallographic data are shown in Table 11.

a; R _merge = Σ hkl Σ j │ Ij ( hkl )-〈 I ( hkl )〉│/Σ hkl Σj│ Ij ( hkl ) (here, Ij ( hkl ) and 〈 I ( hkl )> are the intensity of the measured value j and Is the average intensity for reflections under the exponential hkl).

b; R factor = Σ hkl │ F _calc ( hkl )│-│ F _obs ( hkl )│/Σ hkl │ F _obs ( hkl ) (where F _obs and F _calc are the observed and calculated structural factor sizes, respectively to be).

c; R _free is calculated under 5% of the reflections that are randomly invalidated.

Structure was determined by molecular substitution using the program Phaser (J. Appl. Cryst. 40:658-674 (2007)). The search model of the Fab domain was derived from the published human IgG4 Fab crystal structure (PDB code: 1BBJ), and the search model of the MG1 domain was the published human C5 crystal structure (PDB code: 3CU7, Nat. Immunol. 9:753). -760 (2008)]). A model was constructed using the Coot program (Acta Cryst. D66:486-501 (2010)) and the program Refmac5 (Acta Cryst. D67:355-367 (2011)). ) To improve. The crystallographic reliability factor (R) for the diffraction intensity data from 25-2.11 Å was 20.42%, with a Free R value of 26.44%. The statistical results of structural improvement are shown in Table 11.

11.6. Overall structure of 305 variant Fab and C5-MG1 domain complex

The Fab fragment of the optimized variant from 305 ("305 Fab") bound to the human C5-MG1 domain ("MG1") in a 1:1 ratio, and the asymmetric units of the crystal structure were two Contains complex, molecules 1 and 2. Molecules 1 and 2 can be well aligned with the C alpha atom positions at all residues at 0.717 Å RMSD, as shown in FIG. 26B. The figures discussed below were made using molecule 1.

In Figures 27A and 27B, the epitope of the 305 Fab contact region was mapped to the MG1 amino acid sequence and crystal structure, respectively. The epitope comprises an amino acid residue of MG1 containing one or more atoms located within a distance of 4.5 Å from any portion of 305 Fab in the crystal structure. In addition, epitopes within 3.0 Å are highlighted in Fig. 27A.

11.7. Interaction of E48, D51, and K109

As described in Examples 4.5 and 4.6, anti-C5 Mabs containing the 305 antibody series were transferred to three human C5 point mutants, E48A, D51A, and K109A by Western blot and Biacore® binding assay. Was tested for binding to. 305 variants strongly bound to WT C5, but they only weakly bound to the E48A C5 mutants and not to the D51A and K109A mutants. The crystal structure of the 305 Fab and MG1 complex showed that all three amino acids E48, D51, and K109 were located within 3.0 Å from 305 Fab to form a number of hydrogen bonds with the Fab, as shown in FIG. 28A. In a more detailed examination, the K109 residue of MG1 is buried in the groove formed at the interface of the heavy chain of the Fab, by three hydrogen bonds with H-CDR3_G97, H-CDR3_Y100, and H-CDR3_T100b, and by salt crosslinking with H-CDR3_D95. Interacts closely with Fab (FIG. 28D ). D51 is located between MG1 and 305 Fab, and fills the space by forming two hydrogen bonds with H-CDR1_Ser32 and H-CDR2_Ser54 (FIG. 28C). These suggest that both K109 and D51 of C5 are important residues for binding of the 305 antibody series. On the other hand, E48 is located closer to the surface and forms only one hydrogen bond with the Fab, suggesting that its contribution to antibody binding is lower than that of K109 and E51 (Fig. 28B). These relationships are consistent with the results of Western blot and Biacore® binding assays of human C5 mutants (Examples 4.5 and 4.6). Note: Residue numbers for Fab amino acids are based on the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). ).

11.8. Interaction of 305 antibody series with H70, H72, and H110 of human C5

Crystal structure analysis revealed that, as shown in Figs. 27A and 29A, three histidine residues on human C5, namely H70, H72, and H110, are included in the epitope of the 305 variant Fab. Via human C5 mutants H70Y, H72Y, H110Y, and H70Y+H110Y (Example 4.7) to investigate the contribution of these histidine residues to the pH-dependent protein-protein interaction between human C5 and 305 variant Fabs. Core(R) binding analysis was performed. As shown in Fig. 29C, the residue of C5 is located in the pocket formed by the CDR2 loop of the heavy chain of 305 Fab and the loop of MG1 ((L73, S74, and E76), and it closely fills the space. The H72 residue of C5 forms a hydrogen bond with H-CDR2_Y58. The H72Y mutation is not expected to be tolerated, as there is not enough space to accommodate the bulky side chain of tyrosine. Regarding the contribution of H70 and H110 to pH dependence, the H70Y and H110Y mutations resulted in a slower dissociation of the 305 variant Fab from C5 at pH 5.8. H70 is the T53 of MG1 and the intramolecular hydrogen. A bond is formed, which is thought to break at pH 5.8 when H70 of C5 causes a conformational change in the corresponding portion of the interaction interface of MG1 (Fig. 29B). In the case of H110, of the C5 residue. Protonation is expected to cause charge repulsion on the 305 Fab, which can be increased by protonation of the adjacent histidine residue, H-CDR3_H100c (FIG. 29D ).

Although the present invention has been described in some detail by way of illustration and example for clarity of understanding, the above description and examples should not be construed as limiting the scope of the present invention. The disclosures of all patent and scientific documents cited herein are expressly incorporated by reference in their entirety.

SEQUENCE LISTING <110> CHUGAI SEIYAKU KABUSHIKI KAISHA <120> ANTI-C5 ANTIBODIES AND METHODS OF USE <130> C1-A1606P <150> JP 2016-120325 <151> 2016-06-17 <160> 150 <170> PatentIn version 3.5 <210> 1 <211> 121 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 1 Gln Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Ala Ser 1 5 10 15 Leu Ala Val Thr Cys Thr Ala Ser Gly Phe Ser Ser Ser Ser Ser Tyr 20 25 30 Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Cys Ile Tyr Thr Gly Ser Gly Ala Thr Tyr Tyr Ala Ser Trp Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Thr Leu 65 70 75 80 Gln Met Thr Ser Leu Thr Val Ala Asp Thr Ala Thr Tyr Phe Cys Ala 85 90 95 Ser Asp Gly Gly Tyr Val Thr Pro Thr His Ala Met Tyr Leu Trp Gly 100 105 110 Pro Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 2 <211> 117 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 2 Gln Ser Leu Glu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Ala Ser 1 5 10 15 Leu Thr Leu Thr Cys Lys Ala Ser Gly Ile Asp Phe Ser Asn Phe Tyr 20 25 30 Tyr Ile Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Leu Ile 35 40 45 Ala Cys Ile Tyr Thr Val Ser Gly Tyr Thr Tyr Tyr Ala Ser Trp Ala 50 55 60 Lys Gly Arg Leu Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Thr Leu 65 70 75 80 Gln Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala 85 90 95 Arg Asp Leu His Ala Gly Ile Thr Asn Leu Trp Gly Pro Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 3 <211> 122 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 3 Gln Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Ala Ser 1 5 10 15 Leu Ser Leu Thr Cys Thr Ala Ala Gly Leu Asp Phe Ser Ser Ser Tyr 20 25 30 Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Ala Cys Ile Tyr Ala Gly Ser Ser Gly Ile Ile Tyr Tyr Ala Asn Trp 50 55 60 Ala Lys Gly Arg Phe Thr Ile Ser Arg Pro Ser Ser Thr Thr Val Thr 65 70 75 80 Leu Gln Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Thr Tyr Pro Thr Tyr Gly Asp Gly Gly His Ala Phe Asn Leu Trp 100 105 110 Gly Pro Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 4 <211> 117 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 4 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Ser Tyr Tyr 20 25 30 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Met Ile Tyr Gly Ser Gly Ala Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp Leu Lys Met 65 70 75 80 Thr Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gln 85 90 95 Ile Tyr Ser Gly Asp Asn Asn Asp Asn Phe Trp Gly Pro Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 5 <211> 117 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 5 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Ser Tyr Tyr 20 25 30 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Met Ile Tyr Gly Ser Gly Ala Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp Leu Lys Met 65 70 75 80 Thr Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gln 85 90 95 Ile Tyr Ser Gly Asp Asn Asn Asp Asn Phe Trp Gly Pro Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 6 <211> 117 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 6 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Leu Ser Leu Ser Asp Asn Thr 20 25 30 Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Ile Ile Ser Phe Gly Gly Asp Ala Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Pro Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Arg Ile Thr 65 70 75 80 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Val Gly 85 90 95 Ala Gly Asn Ile Phe Trp Tyr Phe Asp Leu Trp Gly Pro Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 7 <211> 116 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 7 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Thr Tyr Ala 20 25 30 Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Thr Ile Asp Thr Gly Asp Asn Ser Phe Tyr Ala Asn Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Thr 65 70 75 80 Ser Pro Thr Ala Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asn Asp 85 90 95 Gly Ser Val Tyr Asn Leu Phe Asn Leu Trp Gly Pro Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 8 <211> 110 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 8 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Gly Asn Ala 20 25 30 Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Cys Ile Tyr Thr Gly Ser Asp Thr Thr Tyr Tyr Ala Thr Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile 65 70 75 80 Ala Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly 85 90 95 Ser Gly Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser 100 105 110 <210> 9 <211> 116 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 9 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Thr Tyr Ala 20 25 30 Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Thr Ile Asp Thr Gly Asp Asn Ser Phe Tyr Ala Asn Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Thr Ser Thr Thr Val Asp Leu Lys Ile Thr 65 70 75 80 Ser Pro Thr Ala Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asn Asp 85 90 95 Gly Ser Val Tyr Asn Leu Phe Asn Leu Trp Gly Pro Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 10 <211> 123 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 10 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ser His Ser Ser 20 25 30 Tyr Tyr Val Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Gly Ala Ile Tyr Thr Gly Ser Gly Ala Thr Tyr Lys Ala Ser Trp 50 55 60 Ala Lys Gly Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Ser Asp Gly Gly Tyr Asp Tyr Pro Thr His Ala Met His Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 11 <211> 110 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 11 Asp Val Val Met Thr Gln Thr Pro Ala Ser Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Asn Ile Gly Ser Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Lys Leu Ala Ser Gly Val Ser Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Lys Glu Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys 65 70 75 80 Ala Asp Ala Val Thr Tyr Tyr Cys Gln Cys Thr Phe Val Gly Ser Ser 85 90 95 Tyr Gly Asn Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> 12 <211> 110 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 12 Ala Ile Glu Met Thr Gln Thr Pro Phe Ser Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Asn Ile Tyr Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Leu Glu Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Asp Leu Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Leu Gln Gly Tyr Ser Tyr Ser Asn 85 90 95 Val Asp Asp Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> 13 <211> 112 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 13 Ala Gln Val Leu Thr Gln Thr Pro Ser Ser Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Tyr Ser Ser 20 25 30 Asp Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 35 40 45 Leu Ile Tyr Glu Ala Ser Lys Leu Ala Ser Gly Val Pro Pro Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val 65 70 75 80 Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gly Thr Tyr Tyr Ser 85 90 95 Ser Gly Trp Tyr Phe Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> 14 <211> 110 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 14 Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Ser Ile Ser Asn Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys Leu Leu Ile 35 40 45 Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Glu Cys 65 70 75 80 Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Ser Asn 85 90 95 Val Asp Asn Thr Phe Gly Gly Gly Thr Lys Val Val Val Glu 100 105 110 <210> 15 <211> 110 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 15 Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Ser Ile Ser Asn Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys Leu Leu Ile 35 40 45 Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Glu Cys 65 70 75 80 Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Ser Asn 85 90 95 Val Asp Asn Thr Phe Gly Gly Gly Thr Thr Val Val Val Glu 100 105 110 <210> 16 <211> 110 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 16 Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ser Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Glu Ser Ile Tyr Ser Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Asp Leu Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Ser Thr Asn 85 90 95 Val His Asn Ser Phe Gly Gly Gly Thr Thr Val Val Val Glu 100 105 110 <210> 17 <211> 110 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 17 Asp Val Val Met Thr Gln Thr Pro Ala Ser Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Glu Asn Ile Tyr Ser Ala 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Leu Arg Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60 Ser Arg Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asp Ile Asn Ser 85 90 95 Val Asp Asn Thr Phe Gly Gly Gly Thr Lys Val Val Val Glu 100 105 110 <210> 18 <211> 111 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 18 Asp Val Val Met Thr Gln Thr Pro Ala Ser Val Ser Ala Pro Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Asn Ile Tyr Ser Leu 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Asp Leu Ala Ser Gly Val Pro Ser Arg Phe Arg Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Cys Thr Ala Tyr Gly Ser Ser 85 90 95 Asp Val Gly Gly Thr Phe Gly Gly Gly Thr Thr Val Val Val Glu 100 105 110 <210> 19 <211> 110 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 19 Asp Val Val Met Thr Gln Thr Pro Ala Ser Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Glu Asn Ile Tyr Ser Ala 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Leu Arg Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60 Ser Arg Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys 65 70 75 80 Ala Asp Ala Ala Ser Tyr Tyr Cys Gln Gln Tyr Tyr Asp Ile Asn Ser 85 90 95 Val Asp Asn Thr Phe Gly Gly Gly Thr Lys Val Val Val Glu 100 105 110 <210> 20 <211> 110 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 20 Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asn Ile Gly Ser Ser 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Lys Thr His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Ser Thr Lys Val Gly Ser Ser 85 90 95 Tyr Gly Asn His Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 21 <211> 117 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 21 Gln Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Glu Pro Gly Ala Ser 1 5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Trp Asn Ser Gly Tyr 20 25 30 Asp Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Ala Cys Ile Tyr Thr Gly Ser Ser Ala Asn Thr Ala Tyr Ala Asn Trp 50 55 60 Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Thr 65 70 75 80 Leu Gln Met Thr Gly Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg His Asp Asp Tyr Phe Phe Asp Leu Trp Gly Pro Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 22 <211> 115 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 22 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Tyr Thr 20 25 30 Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Cys Ile Gly 35 40 45 Tyr Ile His Ser Phe Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Glu Ile Thr 65 70 75 80 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asp Val 85 90 95 Gly Gly Ser Ser Gly Trp Asp Leu Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 23 <211> 116 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 23 Gln Ser Leu Glu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Ala Ser 1 5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe Ser Ser Gly Tyr 20 25 30 Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Cys Ile Gly Thr Ile Ser Asp Ser Thr Tyr Tyr Ala Ser Trp Ala 50 55 60 Lys Gly Arg Phe Pro Ile Ser Lys Ala Ser Ser Thr Thr Val Thr Leu 65 70 75 80 Gln Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala 85 90 95 Arg Asp Pro Tyr Ser Tyr Gly Asp Leu Trp Gly Pro Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 24 <211> 125 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 24 Gln Glu Gln Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro Glu Gly 1 5 10 15 Ser Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe Ser Ser Gly 20 25 30 Asn Trp Ile Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Ala Cys Ile Tyr Gly Gly Ser Val Gly Gly Thr Asp Tyr Ala Ser 50 55 60 Trp Ala Lys Gly Arg Phe Thr Val Ser Lys Thr Ser Ser Thr Thr Val 65 70 75 80 Thr Leu Gln Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe 85 90 95 Cys Ala Arg Met Glu Asp Gly Tyr Gly Tyr Gly Tyr Asp Thr Tyr Phe 100 105 110 Lys Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 25 <211> 114 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 25 Asp Val Val Met Thr Gln Thr Pro Ala Ser Val Ser Glu Ser Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Gln Ser Ile Gly Asn Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60 Ser Arg Ser Gly Thr Glu Pro Thr Leu Thr Asn Thr Leu Thr Ile Ser 65 70 75 80 Gly Val Gln Cys Ala Asp Val Ala Thr Tyr Tyr Cys Gln Ser Gly Trp 85 90 95 Tyr Gly Asn Ser Tyr Val Ala Ala Phe Gly Gly Gly Thr Glu Val Val 100 105 110 Val Lys <210> 26 <211> 112 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 26 Asp Val Val Met Thr Gln Thr Pro Ser Ser Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Glu Asn Ile Tyr Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu Ile 35 40 45 Tyr Asp Ala Ser Asp Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Gln Cys 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Ala Asn Tyr Asp Ser Thr Ser 85 90 95 Ser Ser Phe Gly Asn Val Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> 27 <211> 110 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 27 Asp Leu Val Met Thr Gln Thr Pro Ala Ser Val Ser Glu Pro Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Gly Thr Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Leu Gly Gly Tyr Tyr Gly Phe Ser 85 90 95 Tyr Gly Asn Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> 28 <211> 110 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 28 Asp Ile Val Met Thr Gln Thr Pro Ala Ser Val Glu Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Thr Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Leu Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gly Tyr Tyr Asp Ser Ser Thr 85 90 95 Ser Ser Tyr Val Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> 29 <211> 448 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 29 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Ser Asn Tyr 20 25 30 Trp Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Glu Tyr Thr Glu Asn Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Phe Phe Gly Ser Ser Pro Asn Trp Tyr Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys 210 215 220 Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 260 265 270 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 <210> 30 <211> 214 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 30 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Asn Val Leu Asn Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 31 <211> 122 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 31 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Ser Asn Tyr 20 25 30 Trp Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Glu Tyr Thr Glu Asn Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Phe Phe Gly Ser Ser Pro Asn Trp Tyr Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 32 <211> 107 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 32 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Asn Val Leu Asn Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 33 <211> 325 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 33 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Arg Gly Gly Pro Lys Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu 325 <210> 34 <211> 325 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 34 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Arg Gly Gly Pro Lys Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Tyr Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Tyr His Val Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu 325 <210> 35 <211> 325 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 35 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Arg Gly Gly Pro Lys Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Pro 325 <210> 36 <211> 107 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 36 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Cys 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 37 <211> 107 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 37 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 38 <211> 107 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 38 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 39 <211> 1676 <212> PRT <213> Homo sapiens <400> 39 Met Gly Leu Leu Gly Ile Leu Cys Phe Leu Ile Phe Leu Gly Lys Thr 1 5 10 15 Trp Gly Gln Glu Gln Thr Tyr Val Ile Ser Ala Pro Lys Ile Phe Arg 20 25 30 Val Gly Ala Ser Glu Asn Ile Val Ile Gln Val Tyr Gly Tyr Thr Glu 35 40 45 Ala Phe Asp Ala Thr Ile Ser Ile Lys Ser Tyr Pro Asp Lys Lys Phe 50 55 60 Ser Tyr Ser Ser Gly His Val His Leu Ser Ser Glu Asn Lys Phe Gln 65 70 75 80 Asn Ser Ala Ile Leu Thr Ile Gln Pro Lys Gln Leu Pro Gly Gly Gln 85 90 95 Asn Pro Val Ser Tyr Val Tyr Leu Glu Val Val Ser Lys His Phe Ser 100 105 110 Lys Ser Lys Arg Met Pro Ile Thr Tyr Asp Asn Gly Phe Leu Phe Ile 115 120 125 His Thr Asp Lys Pro Val Tyr Thr Pro Asp Gln Ser Val Lys Val Arg 130 135 140 Val Tyr Ser Leu Asn Asp Asp Leu Lys Pro Ala Lys Arg Glu Thr Val 145 150 155 160 Leu Thr Phe Ile Asp Pro Glu Gly Ser Glu Val Asp Met Val Glu Glu 165 170 175 Ile Asp His Ile Gly Ile Ile Ser Phe Pro Asp Phe Lys Ile Pro Ser 180 185 190 Asn Pro Arg Tyr Gly Met Trp Thr Ile Lys Ala Lys Tyr Lys Glu Asp 195 200 205 Phe Ser Thr Thr Gly Thr Ala Tyr Phe Glu Val Lys Glu Tyr Val Leu 210 215 220 Pro His Phe Ser Val Ser Ile Glu Pro Glu Tyr Asn Phe Ile Gly Tyr 225 230 235 240 Lys Asn Phe Lys Asn Phe Glu Ile Thr Ile Lys Ala Arg Tyr Phe Tyr 245 250 255 Asn Lys Val Val Thr Glu Ala Asp Val Tyr Ile Thr Phe Gly Ile Arg 260 265 270 Glu Asp Leu Lys Asp Asp Gln Lys Glu Met Met Gln Thr Ala Met Gln 275 280 285 Asn Thr Met Leu Ile Asn Gly Ile Ala Gln Val Thr Phe Asp Ser Glu 290 295 300 Thr Ala Val Lys Glu Leu Ser Tyr Tyr Ser Leu Glu Asp Leu Asn Asn 305 310 315 320 Lys Tyr Leu Tyr Ile Ala Val Thr Val Ile Glu Ser Thr Gly Gly Phe 325 330 335 Ser Glu Glu Ala Glu Ile Pro Gly Ile Lys Tyr Val Leu Ser Pro Tyr 340 345 350 Lys Leu Asn Leu Val Ala Thr Pro Leu Phe Leu Lys Pro Gly Ile Pro 355 360 365 Tyr Pro Ile Lys Val Gln Val Lys Asp Ser Leu Asp Gln Leu Val Gly 370 375 380 Gly Val Pro Val Thr Leu Asn Ala Gln Thr Ile Asp Val Asn Gln Glu 385 390 395 400 Thr Ser Asp Leu Asp Pro Ser Lys Ser Val Thr Arg Val Asp Asp Gly 405 410 415 Val Ala Ser Phe Val Leu Asn Leu Pro Ser Gly Val Thr Val Leu Glu 420 425 430 Phe Asn Val Lys Thr Asp Ala Pro Asp Leu Pro Glu Glu Asn Gln Ala 435 440 445 Arg Glu Gly Tyr Arg Ala Ile Ala Tyr Ser Ser Leu Ser Gln Ser Tyr 450 455 460 Leu Tyr Ile Asp Trp Thr Asp Asn His Lys Ala Leu Leu Val Gly Glu 465 470 475 480 His Leu Asn Ile Ile Val Thr Pro Lys Ser Pro Tyr Ile Asp Lys Ile 485 490 495 Thr His Tyr Asn Tyr Leu Ile Leu Ser Lys Gly Lys Ile Ile His Phe 500 505 510 Gly Thr Arg Glu Lys Phe Ser Asp Ala Ser Tyr Gln Ser Ile Asn Ile 515 520 525 Pro Val Thr Gln Asn Met Val Pro Ser Ser Arg Leu Leu Val Tyr Tyr 530 535 540 Ile Val Thr Gly Glu Gln Thr Ala Glu Leu Val Ser Asp Ser Val Trp 545 550 555 560 Leu Asn Ile Glu Glu Lys Cys Gly Asn Gln Leu Gln Val His Leu Ser 565 570 575 Pro Asp Ala Asp Ala Tyr Ser Pro Gly Gln Thr Val Ser Leu Asn Met 580 585 590 Ala Thr Gly Met Asp Ser Trp Val Ala Leu Ala Ala Val Asp Ser Ala 595 600 605 Val Tyr Gly Val Gln Arg Gly Ala Lys Lys Pro Leu Glu Arg Val Phe 610 615 620 Gln Phe Leu Glu Lys Ser Asp Leu Gly Cys Gly Ala Gly Gly Gly Leu 625 630 635 640 Asn Asn Ala Asn Val Phe His Leu Ala Gly Leu Thr Phe Leu Thr Asn 645 650 655 Ala Asn Ala Asp Asp Ser Gln Glu Asn Asp Glu Pro Cys Lys Glu Ile 660 665 670 Leu Arg Pro Arg Arg Thr Leu Gln Lys Lys Ile Glu Glu Ile Ala Ala 675 680 685 Lys Tyr Lys His Ser Val Val Lys Lys Cys Cys Tyr Asp Gly Ala Cys 690 695 700 Val Asn Asn Asp Glu Thr Cys Glu Gln Arg Ala Ala Arg Ile Ser Leu 705 710 715 720 Gly Pro Arg Cys Ile Lys Ala Phe Thr Glu Cys Cys Val Val Ala Ser 725 730 735 Gln Leu Arg Ala Asn Ile Ser His Lys Asp Met Gln Leu Gly Arg Leu 740 745 750 His Met Lys Thr Leu Leu Pro Val Ser Lys Pro Glu Ile Arg Ser Tyr 755 760 765 Phe Pro Glu Ser Trp Leu Trp Glu Val His Leu Val Pro Arg Arg Lys 770 775 780 Gln Leu Gln Phe Ala Leu Pro Asp Ser Leu Thr Thr Trp Glu Ile Gln 785 790 795 800 Gly Val Gly Ile Ser Asn Thr Gly Ile Cys Val Ala Asp Thr Val Lys 805 810 815 Ala Lys Val Phe Lys Asp Val Phe Leu Glu Met Asn Ile Pro Tyr Ser 820 825 830 Val Val Arg Gly Glu Gln Ile Gln Leu Lys Gly Thr Val Tyr Asn Tyr 835 840 845 Arg Thr Ser Gly Met Gln Phe Cys Val Lys Met Ser Ala Val Glu Gly 850 855 860 Ile Cys Thr Ser Glu Ser Pro Val Ile Asp His Gln Gly Thr Lys Ser 865 870 875 880 Ser Lys Cys Val Arg Gln Lys Val Glu Gly Ser Ser Ser His Leu Val 885 890 895 Thr Phe Thr Val Leu Pro Leu Glu Ile Gly Leu His Asn Ile Asn Phe 900 905 910 Ser Leu Glu Thr Trp Phe Gly Lys Glu Ile Leu Val Lys Thr Leu Arg 915 920 925 Val Val Pro Glu Gly Val Lys Arg Glu Ser Tyr Ser Gly Val Thr Leu 930 935 940 Asp Pro Arg Gly Ile Tyr Gly Thr Ile Ser Arg Arg Lys Glu Phe Pro 945 950 955 960 Tyr Arg Ile Pro Leu Asp Leu Val Pro Lys Thr Glu Ile Lys Arg Ile 965 970 975 Leu Ser Val Lys Gly Leu Leu Val Gly Glu Ile Leu Ser Ala Val Leu 980 985 990 Ser Gln Glu Gly Ile Asn Ile Leu Thr His Leu Pro Lys Gly Ser Ala 995 1000 1005 Glu Ala Glu Leu Met Ser Val Val Pro Val Phe Tyr Val Phe His 1010 1015 1020 Tyr Leu Glu Thr Gly Asn His Trp Asn Ile Phe His Ser Asp Pro 1025 1030 1035 Leu Ile Glu Lys Gln Lys Leu Lys Lys Lys Leu Lys Glu Gly Met 1040 1045 1050 Leu Ser Ile Met Ser Tyr Arg Asn Ala Asp Tyr Ser Tyr Ser Val 1055 1060 1065 Trp Lys Gly Gly Ser Ala Ser Thr Trp Leu Thr Ala Phe Ala Leu 1070 1075 1080 Arg Val Leu Gly Gln Val Asn Lys Tyr Val Glu Gln Asn Gln Asn 1085 1090 1095 Ser Ile Cys Asn Ser Leu Leu Trp Leu Val Glu Asn Tyr Gln Leu 1100 1105 1110 Asp Asn Gly Ser Phe Lys Glu Asn Ser Gln Tyr Gln Pro Ile Lys 1115 1120 1125 Leu Gln Gly Thr Leu Pro Val Glu Ala Arg Glu Asn Ser Leu Tyr 1130 1135 1140 Leu Thr Ala Phe Thr Val Ile Gly Ile Arg Lys Ala Phe Asp Ile 1145 1150 1155 Cys Pro Leu Val Lys Ile Asp Thr Ala Leu Ile Lys Ala Asp Asn 1160 1165 1170 Phe Leu Leu Glu Asn Thr Leu Pro Ala Gln Ser Thr Phe Thr Leu 1175 1180 1185 Ala Ile Ser Ala Tyr Ala Leu Ser Leu Gly Asp Lys Thr His Pro 1190 1195 1200 Gln Phe Arg Ser Ile Val Ser Ala Leu Lys Arg Glu Ala Leu Val 1205 1210 1215 Lys Gly Asn Pro Pro Ile Tyr Arg Phe Trp Lys Asp Asn Leu Gln 1220 1225 1230 His Lys Asp Ser Ser Val Pro Asn Thr Gly Thr Ala Arg Met Val 1235 1240 1245 Glu Thr Thr Ala Tyr Ala Leu Leu Thr Ser Leu Asn Leu Lys Asp 1250 1255 1260 Ile Asn Tyr Val Asn Pro Val Ile Lys Trp Leu Ser Glu Glu Gln 1265 1270 1275 Arg Tyr Gly Gly Gly Phe Tyr Ser Thr Gln Asp Thr Ile Asn Ala 1280 1285 1290 Ile Glu Gly Leu Thr Glu Tyr Ser Leu Leu Val Lys Gln Leu Arg 1295 1300 1305 Leu Ser Met Asp Ile Asp Val Ser Tyr Lys His Lys Gly Ala Leu 1310 1315 1320 His Asn Tyr Lys Met Thr Asp Lys Asn Phe Leu Gly Arg Pro Val 1325 1330 1335 Glu Val Leu Leu Asn Asp Asp Leu Ile Val Ser Thr Gly Phe Gly 1340 1345 1350 Ser Gly Leu Ala Thr Val His Val Thr Thr Val Val His Lys Thr 1355 1360 1365 Ser Thr Ser Glu Glu Val Cys Ser Phe Tyr Leu Lys Ile Asp Thr 1370 1375 1380 Gln Asp Ile Glu Ala Ser His Tyr Arg Gly Tyr Gly Asn Ser Asp 1385 1390 1395 Tyr Lys Arg Ile Val Ala Cys Ala Ser Tyr Lys Pro Ser Arg Glu 1400 1405 1410 Glu Ser Ser Ser Gly Ser Ser His Ala Val Met Asp Ile Ser Leu 1415 1420 1425 Pro Thr Gly Ile Ser Ala Asn Glu Glu Asp Leu Lys Ala Leu Val 1430 1435 1440 Glu Gly Val Asp Gln Leu Phe Thr Asp Tyr Gln Ile Lys Asp Gly 1445 1450 1455 His Val Ile Leu Gln Leu Asn Ser Ile Pro Ser Ser Asp Phe Leu 1460 1465 1470 Cys Val Arg Phe Arg Ile Phe Glu Leu Phe Glu Val Gly Phe Leu 1475 1480 1485 Ser Pro Ala Thr Phe Thr Val Tyr Glu Tyr His Arg Pro Asp Lys 1490 1495 1500 Gln Cys Thr Met Phe Tyr Ser Thr Ser Asn Ile Lys Ile Gln Lys 1505 1510 1515 Val Cys Glu Gly Ala Ala Cys Lys Cys Val Glu Ala Asp Cys Gly 1520 1525 1530 Gln Met Gln Glu Glu Leu Asp Leu Thr Ile Ser Ala Glu Thr Arg 1535 1540 1545 Lys Gln Thr Ala Cys Lys Pro Glu Ile Ala Tyr Ala Tyr Lys Val 1550 1555 1560 Ser Ile Thr Ser Ile Thr Val Glu Asn Val Phe Val Lys Tyr Lys 1565 1570 1575 Ala Thr Leu Leu Asp Ile Tyr Lys Thr Gly Glu Ala Val Ala Glu 1580 1585 1590 Lys Asp Ser Glu Ile Thr Phe Ile Lys Lys Val Thr Cys Thr Asn 1595 1600 1605 Ala Glu Leu Val Lys Gly Arg Gln Tyr Leu Ile Met Gly Lys Glu 1610 1615 1620 Ala Leu Gln Ile Lys Tyr Asn Phe Ser Phe Arg Tyr Ile Tyr Pro 1625 1630 1635 Leu Asp Ser Leu Thr Trp Ile Glu Tyr Trp Pro Arg Asp Thr Thr 1640 1645 1650 Cys Ser Ser Cys Gln Ala Phe Leu Ala Asn Leu Asp Glu Phe Ala 1655 1660 1665 Glu Asp Ile Phe Leu Asn Gly Cys 1670 1675 <210> 40 <211> 673 <212> PRT <213> Homo sapiens <400> 40 Met Gly Leu Leu Gly Ile Leu Cys Phe Leu Ile Phe Leu Gly Lys Thr 1 5 10 15 Trp Gly Gln Glu Gln Thr Tyr Val Ile Ser Ala Pro Lys Ile Phe Arg 20 25 30 Val Gly Ala Ser Glu Asn Ile Val Ile Gln Val Tyr Gly Tyr Thr Glu 35 40 45 Ala Phe Asp Ala Thr Ile Ser Ile Lys Ser Tyr Pro Asp Lys Lys Phe 50 55 60 Ser Tyr Ser Ser Gly His Val His Leu Ser Ser Glu Asn Lys Phe Gln 65 70 75 80 Asn Ser Ala Ile Leu Thr Ile Gln Pro Lys Gln Leu Pro Gly Gly Gln 85 90 95 Asn Pro Val Ser Tyr Val Tyr Leu Glu Val Val Ser Lys His Phe Ser 100 105 110 Lys Ser Lys Arg Met Pro Ile Thr Tyr Asp Asn Gly Phe Leu Phe Ile 115 120 125 His Thr Asp Lys Pro Val Tyr Thr Pro Asp Gln Ser Val Lys Val Arg 130 135 140 Val Tyr Ser Leu Asn Asp Asp Leu Lys Pro Ala Lys Arg Glu Thr Val 145 150 155 160 Leu Thr Phe Ile Asp Pro Glu Gly Ser Glu Val Asp Met Val Glu Glu 165 170 175 Ile Asp His Ile Gly Ile Ile Ser Phe Pro Asp Phe Lys Ile Pro Ser 180 185 190 Asn Pro Arg Tyr Gly Met Trp Thr Ile Lys Ala Lys Tyr Lys Glu Asp 195 200 205 Phe Ser Thr Thr Gly Thr Ala Tyr Phe Glu Val Lys Glu Tyr Val Leu 210 215 220 Pro His Phe Ser Val Ser Ile Glu Pro Glu Tyr Asn Phe Ile Gly Tyr 225 230 235 240 Lys Asn Phe Lys Asn Phe Glu Ile Thr Ile Lys Ala Arg Tyr Phe Tyr 245 250 255 Asn Lys Val Val Thr Glu Ala Asp Val Tyr Ile Thr Phe Gly Ile Arg 260 265 270 Glu Asp Leu Lys Asp Asp Gln Lys Glu Met Met Gln Thr Ala Met Gln 275 280 285 Asn Thr Met Leu Ile Asn Gly Ile Ala Gln Val Thr Phe Asp Ser Glu 290 295 300 Thr Ala Val Lys Glu Leu Ser Tyr Tyr Ser Leu Glu Asp Leu Asn Asn 305 310 315 320 Lys Tyr Leu Tyr Ile Ala Val Thr Val Ile Glu Ser Thr Gly Gly Phe 325 330 335 Ser Glu Glu Ala Glu Ile Pro Gly Ile Lys Tyr Val Leu Ser Pro Tyr 340 345 350 Lys Leu Asn Leu Val Ala Thr Pro Leu Phe Leu Lys Pro Gly Ile Pro 355 360 365 Tyr Pro Ile Lys Val Gln Val Lys Asp Ser Leu Asp Gln Leu Val Gly 370 375 380 Gly Val Pro Val Thr Leu Asn Ala Gln Thr Ile Asp Val Asn Gln Glu 385 390 395 400 Thr Ser Asp Leu Asp Pro Ser Lys Ser Val Thr Arg Val Asp Asp Gly 405 410 415 Val Ala Ser Phe Val Leu Asn Leu Pro Ser Gly Val Thr Val Leu Glu 420 425 430 Phe Asn Val Lys Thr Asp Ala Pro Asp Leu Pro Glu Glu Asn Gln Ala 435 440 445 Arg Glu Gly Tyr Arg Ala Ile Ala Tyr Ser Ser Leu Ser Gln Ser Tyr 450 455 460 Leu Tyr Ile Asp Trp Thr Asp Asn His Lys Ala Leu Leu Val Gly Glu 465 470 475 480 His Leu Asn Ile Ile Val Thr Pro Lys Ser Pro Tyr Ile Asp Lys Ile 485 490 495 Thr His Tyr Asn Tyr Leu Ile Leu Ser Lys Gly Lys Ile Ile His Phe 500 505 510 Gly Thr Arg Glu Lys Phe Ser Asp Ala Ser Tyr Gln Ser Ile Asn Ile 515 520 525 Pro Val Thr Gln Asn Met Val Pro Ser Ser Arg Leu Leu Val Tyr Tyr 530 535 540 Ile Val Thr Gly Glu Gln Thr Ala Glu Leu Val Ser Asp Ser Val Trp 545 550 555 560 Leu Asn Ile Glu Glu Lys Cys Gly Asn Gln Leu Gln Val His Leu Ser 565 570 575 Pro Asp Ala Asp Ala Tyr Ser Pro Gly Gln Thr Val Ser Leu Asn Met 580 585 590 Ala Thr Gly Met Asp Ser Trp Val Ala Leu Ala Ala Val Asp Ser Ala 595 600 605 Val Tyr Gly Val Gln Arg Gly Ala Lys Lys Pro Leu Glu Arg Val Phe 610 615 620 Gln Phe Leu Glu Lys Ser Asp Leu Gly Cys Gly Ala Gly Gly Gly Leu 625 630 635 640 Asn Asn Ala Asn Val Phe His Leu Ala Gly Leu Thr Phe Leu Thr Asn 645 650 655 Ala Asn Ala Asp Asp Ser Gln Glu Asn Asp Glu Pro Cys Lys Glu Ile 660 665 670 Leu <210> 41 <211> 105 <212> PRT <213> Homo sapiens <400> 41 Glu Gln Thr Tyr Val Ile Ser Ala Pro Lys Ile Phe Arg Val Gly Ala 1 5 10 15 Ser Glu Asn Ile Val Ile Gln Val Tyr Gly Tyr Thr Glu Ala Phe Asp 20 25 30 Ala Thr Ile Ser Ile Lys Ser Tyr Pro Asp Lys Lys Phe Ser Tyr Ser 35 40 45 Ser Gly His Val His Leu Ser Ser Glu Asn Lys Phe Gln Asn Ser Ala 50 55 60 Ile Leu Thr Ile Gln Pro Lys Gln Leu Pro Gly Gly Gln Asn Pro Val 65 70 75 80 Ser Tyr Val Tyr Leu Glu Val Val Ser Lys His Phe Ser Lys Ser Lys 85 90 95 Arg Met Pro Ile Thr Tyr Asp Asn Gly 100 105 <210> 42 <211> 101 <212> PRT <213> Homo sapiens <400> 42 Phe Leu Phe Ile His Thr Asp Lys Pro Val Tyr Thr Pro Asp Gln Ser 1 5 10 15 Val Lys Val Arg Val Tyr Ser Leu Asn Asp Asp Leu Lys Pro Ala Lys 20 25 30 Arg Glu Thr Val Leu Thr Phe Ile Asp Pro Glu Gly Ser Glu Val Asp 35 40 45 Met Val Glu Glu Ile Asp His Ile Gly Ile Ile Ser Phe Pro Asp Phe 50 55 60 Lys Ile Pro Ser Asn Pro Arg Tyr Gly Met Trp Thr Ile Lys Ala Lys 65 70 75 80 Tyr Lys Glu Asp Phe Ser Thr Thr Gly Thr Ala Tyr Phe Glu Val Lys 85 90 95 Glu Tyr Val Leu Pro 100 <210> 43 <211> 225 <212> PRT <213> Homo sapiens <400> 43 Met Gly Leu Leu Gly Ile Leu Cys Phe Leu Ile Phe Leu Gly Lys Thr 1 5 10 15 Trp Gly Gln Glu Gln Thr Tyr Val Ile Ser Ala Pro Lys Ile Phe Arg 20 25 30 Val Gly Ala Ser Glu Asn Ile Val Ile Gln Val Tyr Gly Tyr Thr Glu 35 40 45 Ala Phe Asp Ala Thr Ile Ser Ile Lys Ser Tyr Pro Asp Lys Lys Phe 50 55 60 Ser Tyr Ser Ser Gly His Val His Leu Ser Ser Glu Asn Lys Phe Gln 65 70 75 80 Asn Ser Ala Ile Leu Thr Ile Gln Pro Lys Gln Leu Pro Gly Gly Gln 85 90 95 Asn Pro Val Ser Tyr Val Tyr Leu Glu Val Val Ser Lys His Phe Ser 100 105 110 Lys Ser Lys Arg Met Pro Ile Thr Tyr Asp Asn Gly Phe Leu Phe Ile 115 120 125 His Thr Asp Lys Pro Val Tyr Thr Pro Asp Gln Ser Val Lys Val Arg 130 135 140 Val Tyr Ser Leu Asn Asp Asp Leu Lys Pro Ala Lys Arg Glu Thr Val 145 150 155 160 Leu Thr Phe Ile Asp Pro Glu Gly Ser Glu Val Asp Met Val Glu Glu 165 170 175 Ile Asp His Ile Gly Ile Ile Ser Phe Pro Asp Phe Lys Ile Pro Ser 180 185 190 Asn Pro Arg Tyr Gly Met Trp Thr Ile Lys Ala Lys Tyr Lys Glu Asp 195 200 205 Phe Ser Thr Thr Gly Thr Ala Tyr Phe Glu Val Lys Glu Tyr Val Leu 210 215 220 Pro 225 <210> 44 <211> 1676 <212> PRT <213> Macaca fascicularis <400> 44 Met Gly Leu Leu Gly Ile Leu Cys Phe Leu Ile Phe Leu Gly Lys Thr 1 5 10 15 Trp Gly Gln Glu Gln Thr Tyr Val Ile Ser Ala Pro Lys Ile Phe Arg 20 25 30 Val Gly Ala Ser Glu Asn Ile Val Ile Gln Val Tyr Gly Tyr Thr Glu 35 40 45 Ala Phe Asp Ala Thr Ile Ser Ile Lys Ser Tyr Pro Asp Lys Lys Phe 50 55 60 Ser Tyr Ser Ser Gly His Val His Leu Ser Ser Glu Asn Lys Phe Gln 65 70 75 80 Asn Ser Ala Val Leu Thr Ile Gln Pro Lys Gln Leu Pro Gly Gly Gln 85 90 95 Asn Gln Val Ser Tyr Val Tyr Leu Glu Val Val Ser Lys His Phe Ser 100 105 110 Lys Ser Lys Lys Ile Pro Ile Thr Tyr Asp Asn Gly Phe Leu Phe Ile 115 120 125 His Thr Asp Lys Pro Val Tyr Thr Pro Asp Gln Ser Val Lys Val Arg 130 135 140 Val Tyr Ser Leu Asn Asp Asp Leu Lys Pro Ala Lys Arg Glu Thr Val 145 150 155 160 Leu Thr Phe Ile Asp Pro Glu Gly Ser Glu Ile Asp Met Val Glu Glu 165 170 175 Ile Asp His Ile Gly Ile Ile Ser Phe Pro Asp Phe Lys Ile Pro Ser 180 185 190 Asn Pro Arg Tyr Gly Met Trp Thr Ile Gln Ala Lys Tyr Lys Glu Asp 195 200 205 Phe Ser Thr Thr Gly Thr Ala Phe Phe Glu Val Lys Glu Tyr Val Leu 210 215 220 Pro His Phe Ser Val Ser Val Glu Pro Glu Ser Asn Phe Ile Gly Tyr 225 230 235 240 Lys Asn Phe Lys Asn Phe Glu Ile Thr Ile Lys Ala Arg Tyr Phe Tyr 245 250 255 Asn Lys Val Val Thr Glu Ala Asp Val Tyr Ile Thr Phe Gly Ile Arg 260 265 270 Glu Asp Leu Lys Asp Asp Gln Lys Glu Met Met Gln Thr Ala Met Gln 275 280 285 Asn Thr Met Leu Ile Asn Gly Ile Ala Glu Val Thr Phe Asp Ser Glu 290 295 300 Thr Ala Val Lys Glu Leu Ser Tyr Tyr Ser Leu Glu Asp Leu Asn Asn 305 310 315 320 Lys Tyr Leu Tyr Ile Ala Val Thr Val Ile Glu Ser Thr Gly Gly Phe 325 330 335 Ser Glu Glu Ala Glu Ile Pro Gly Ile Lys Tyr Val Leu Ser Pro Tyr 340 345 350 Lys Leu Asn Leu Val Ala Thr Pro Leu Phe Leu Lys Pro Gly Ile Pro 355 360 365 Tyr Ser Ile Lys Val Gln Val Lys Asp Ala Leu Asp Gln Leu Val Gly 370 375 380 Gly Val Pro Val Thr Leu Asn Ala Gln Thr Ile Asp Val Asn Gln Glu 385 390 395 400 Thr Ser Asp Leu Glu Pro Arg Lys Ser Val Thr Arg Val Asp Asp Gly 405 410 415 Val Ala Ser Phe Val Val Asn Leu Pro Ser Gly Val Thr Val Leu Glu 420 425 430 Phe Asn Val Lys Thr Asp Ala Pro Asp Leu Pro Asp Glu Asn Gln Ala 435 440 445 Arg Glu Gly Tyr Arg Ala Ile Ala Tyr Ser Ser Leu Ser Gln Ser Tyr 450 455 460 Leu Tyr Ile Asp Trp Thr Asp Asn His Lys Ala Leu Leu Val Gly Glu 465 470 475 480 Tyr Leu Asn Ile Ile Val Thr Pro Lys Ser Pro Tyr Ile Asp Lys Ile 485 490 495 Thr His Tyr Asn Tyr Leu Ile Leu Ser Lys Gly Lys Ile Ile His Phe 500 505 510 Gly Thr Arg Glu Lys Leu Ser Asp Ala Ser Tyr Gln Ser Ile Asn Ile 515 520 525 Pro Val Thr Gln Asn Met Val Pro Ser Ser Arg Leu Leu Val Tyr Tyr 530 535 540 Ile Val Thr Gly Glu Gln Thr Ala Glu Leu Val Ser Asp Ser Val Trp 545 550 555 560 Leu Asn Ile Glu Glu Lys Cys Gly Asn Gln Leu Gln Val His Leu Ser 565 570 575 Pro Asp Ala Asp Thr Tyr Ser Pro Gly Gln Thr Val Ser Leu Asn Met 580 585 590 Val Thr Gly Met Asp Ser Trp Val Ala Leu Thr Ala Val Asp Ser Ala 595 600 605 Val Tyr Gly Val Gln Arg Arg Ala Lys Lys Pro Leu Glu Arg Val Phe 610 615 620 Gln Phe Leu Glu Lys Ser Asp Leu Gly Cys Gly Ala Gly Gly Gly Leu 625 630 635 640 Asn Asn Ala Asn Val Phe His Leu Ala Gly Leu Thr Phe Leu Thr Asn 645 650 655 Ala Asn Ala Asp Asp Ser Gln Glu Asn Asp Glu Pro Cys Lys Glu Ile 660 665 670 Ile Arg Pro Arg Arg Met Leu Gln Glu Lys Ile Glu Glu Ile Ala Ala 675 680 685 Lys Tyr Lys His Leu Val Val Lys Lys Cys Cys Tyr Asp Gly Val Arg 690 695 700 Ile Asn His Asp Glu Thr Cys Glu Gln Arg Ala Ala Arg Ile Ser Val 705 710 715 720 Gly Pro Arg Cys Val Lys Ala Phe Thr Glu Cys Cys Val Val Ala Ser 725 730 735 Gln Leu Arg Ala Asn Asn Ser His Lys Asp Leu Gln Leu Gly Arg Leu 740 745 750 His Met Lys Thr Leu Leu Pro Val Ser Lys Pro Glu Ile Arg Ser Tyr 755 760 765 Phe Pro Glu Ser Trp Leu Trp Glu Val His Leu Val Pro Arg Arg Lys 770 775 780 Gln Leu Gln Phe Ala Leu Pro Asp Ser Val Thr Thr Trp Glu Ile Gln 785 790 795 800 Gly Val Gly Ile Ser Asn Ser Gly Ile Cys Val Ala Asp Thr Ile Lys 805 810 815 Ala Lys Val Phe Lys Asp Val Phe Leu Glu Met Asn Ile Pro Tyr Ser 820 825 830 Val Val Arg Gly Glu Gln Val Gln Leu Lys Gly Thr Val Tyr Asn Tyr 835 840 845 Arg Thr Ser Gly Met Gln Phe Cys Val Lys Met Ser Ala Val Glu Gly 850 855 860 Ile Cys Thr Ser Glu Ser Pro Val Ile Asp His Gln Gly Thr Lys Ser 865 870 875 880 Ser Lys Cys Val Arg Gln Lys Val Glu Gly Ser Ser Asn His Leu Val 885 890 895 Thr Phe Thr Val Leu Pro Leu Glu Ile Gly Leu Gln Asn Ile Asn Phe 900 905 910 Ser Leu Glu Thr Ser Phe Gly Lys Glu Ile Leu Val Lys Ser Leu Arg 915 920 925 Val Val Pro Glu Gly Val Lys Arg Glu Ser Tyr Ser Gly Ile Thr Leu 930 935 940 Asp Pro Arg Gly Ile Tyr Gly Thr Ile Ser Arg Arg Lys Glu Phe Pro 945 950 955 960 Tyr Arg Ile Pro Leu Asp Leu Val Pro Lys Thr Glu Ile Lys Arg Ile 965 970 975 Leu Ser Val Lys Gly Leu Leu Val Gly Glu Ile Leu Ser Ala Val Leu 980 985 990 Ser Arg Glu Gly Ile Asn Ile Leu Thr His Leu Pro Lys Gly Ser Ala 995 1000 1005 Glu Ala Glu Leu Met Ser Val Val Pro Val Phe Tyr Val Phe His 1010 1015 1020 Tyr Leu Glu Thr Gly Asn His Trp Asn Ile Phe His Ser Asp Pro 1025 1030 1035 Leu Ile Glu Lys Arg Asn Leu Glu Lys Lys Leu Lys Glu Gly Met 1040 1045 1050 Val Ser Ile Met Ser Tyr Arg Asn Ala Asp Tyr Ser Tyr Ser Val 1055 1060 1065 Trp Lys Gly Gly Ser Ala Ser Thr Trp Leu Thr Ala Phe Ala Leu 1070 1075 1080 Arg Val Leu Gly Gln Val His Lys Tyr Val Glu Gln Asn Gln Asn 1085 1090 1095 Ser Ile Cys Asn Ser Leu Leu Trp Leu Val Glu Asn Tyr Gln Leu 1100 1105 1110 Asp Asn Gly Ser Phe Lys Glu Asn Ser Gln Tyr Gln Pro Ile Lys 1115 1120 1125 Leu Gln Gly Thr Leu Pro Val Glu Ala Arg Glu Asn Ser Leu Tyr 1130 1135 1140 Leu Thr Ala Phe Thr Val Ile Gly Ile Arg Lys Ala Phe Asp Ile 1145 1150 1155 Cys Pro Leu Val Lys Ile Asn Thr Ala Leu Ile Lys Ala Asp Thr 1160 1165 1170 Phe Leu Leu Glu Asn Thr Leu Pro Ala Gln Ser Thr Phe Thr Leu 1175 1180 1185 Ala Ile Ser Ala Tyr Ala Leu Ser Leu Gly Asp Lys Thr His Pro 1190 1195 1200 Gln Phe Arg Ser Ile Val Ser Ala Leu Lys Arg Glu Ala Leu Val 1205 1210 1215 Lys Gly Asn Pro Pro Ile Tyr Arg Phe Trp Lys Asp Ser Leu Gln 1220 1225 1230 His Lys Asp Ser Ser Val Pro Asn Thr Gly Thr Ala Arg Met Val 1235 1240 1245 Glu Thr Thr Ala Tyr Ala Leu Leu Thr Ser Leu Asn Leu Lys Asp 1250 1255 1260 Ile Asn Tyr Val Asn Pro Ile Ile Lys Trp Leu Ser Glu Glu Gln 1265 1270 1275 Arg Tyr Gly Gly Gly Phe Tyr Ser Thr Gln Asp Thr Ile Asn Ala 1280 1285 1290 Ile Glu Gly Leu Thr Glu Tyr Ser Leu Leu Val Lys Gln Leu Arg 1295 1300 1305 Leu Asn Met Asp Ile Asp Val Ala Tyr Lys His Lys Gly Pro Leu 1310 1315 1320 His Asn Tyr Lys Met Thr Asp Lys Asn Phe Leu Gly Arg Pro Val 1325 1330 1335 Glu Val Leu Leu Asn Asp Asp Leu Val Val Ser Thr Gly Phe Gly 1340 1345 1350 Ser Gly Leu Ala Thr Val His Val Thr Thr Val Val His Lys Thr 1355 1360 1365 Ser Thr Ser Glu Glu Val Cys Ser Phe Tyr Leu Lys Ile Asp Thr 1370 1375 1380 Gln Asp Ile Glu Ala Ser His Tyr Arg Gly Tyr Gly Asn Ser Asp 1385 1390 1395 Tyr Lys Arg Ile Val Ala Cys Ala Ser Tyr Lys Pro Ser Lys Glu 1400 1405 1410 Glu Ser Ser Ser Gly Ser Ser His Ala Val Met Asp Ile Ser Leu 1415 1420 1425 Pro Thr Gly Ile Asn Ala Asn Glu Glu Asp Leu Lys Ala Leu Val 1430 1435 1440 Glu Gly Val Asp Gln Leu Phe Thr Asp Tyr Gln Ile Lys Asp Gly 1445 1450 1455 His Val Ile Leu Gln Leu Asn Ser Ile Pro Ser Ser Asp Phe Leu 1460 1465 1470 Cys Val Arg Phe Arg Ile Phe Glu Leu Phe Glu Val Gly Phe Leu 1475 1480 1485 Ser Pro Ala Thr Phe Thr Val Tyr Glu Tyr His Arg Pro Asp Lys 1490 1495 1500 Gln Cys Thr Met Phe Tyr Ser Thr Ser Asn Ile Lys Ile Gln Lys 1505 1510 1515 Val Cys Glu Gly Ala Thr Cys Lys Cys Ile Glu Ala Asp Cys Gly 1520 1525 1530 Gln Met Gln Lys Glu Leu Asp Leu Thr Ile Ser Ala Glu Thr Arg 1535 1540 1545 Lys Gln Thr Ala Cys Asn Pro Glu Ile Ala Tyr Ala Tyr Lys Val 1550 1555 1560 Ile Ile Thr Ser Ile Thr Thr Glu Asn Val Phe Val Lys Tyr Lys 1565 1570 1575 Ala Thr Leu Leu Asp Ile Tyr Lys Thr Gly Glu Ala Val Ala Glu 1580 1585 1590 Lys Asp Ser Glu Ile Thr Phe Ile Lys Lys Val Thr Cys Thr Asn 1595 1600 1605 Ala Glu Leu Val Lys Gly Arg Gln Tyr Leu Ile Met Gly Lys Glu 1610 1615 1620 Ala Leu Gln Ile Lys Tyr Asn Phe Thr Phe Arg Tyr Ile Tyr Pro 1625 1630 1635 Leu Asp Ser Leu Thr Trp Ile Glu Tyr Trp Pro Arg Asp Thr Thr 1640 1645 1650 Cys Ser Ser Cys Gln Ala Phe Leu Ala Asn Leu Asp Glu Phe Ala 1655 1660 1665 Glu Asp Ile Phe Leu Asn Gly Cys 1670 1675 <210> 45 <211> 6 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 45 Ser Ser Tyr Tyr Met Cys 1 5 <210> 46 <211> 6 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 46 Asn Phe Tyr Tyr Ile Cys 1 5 <210> 47 <211> 6 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 47 Ser Ser Tyr Tyr Met Cys 1 5 <210> 48 <211> 6 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 48 Ser Ser Tyr Tyr Met Asn 1 5 <210> 49 <211> 6 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 49 Ser Ser Tyr Tyr Met Asn 1 5 <210> 50 <211> 5 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 50 Asp Asn Thr Met Thr 1 5 <210> 51 <211> 5 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 51 Thr Tyr Ala Met Gly 1 5 <210> 52 <211> 5 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 52 Gly Asn Ala Ile Asn 1 5 <210> 53 <211> 5 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 53 Thr Tyr Ala Met Gly 1 5 <210> 54 <211> 6 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 54 Ser Ser Tyr Tyr Val Ala 1 5 <210> 55 <211> 17 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 55 Cys Ile Tyr Thr Gly Ser Gly Ala Thr Tyr Tyr Ala Ser Trp Ala Lys 1 5 10 15 Gly <210> 56 <211> 17 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 56 Cys Ile Tyr Thr Val Ser Gly Tyr Thr Tyr Tyr Ala Ser Trp Ala Lys 1 5 10 15 Gly <210> 57 <211> 18 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 57 Cys Ile Tyr Ala Gly Ser Ser Gly Ile Ile Tyr Tyr Ala Asn Trp Ala 1 5 10 15 Lys Gly <210> 58 <211> 16 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 58 Met Ile Tyr Gly Ser Gly Ala Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 1 5 10 15 <210> 59 <211> 16 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 59 Met Ile Tyr Gly Ser Gly Ala Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 1 5 10 15 <210> 60 <211> 16 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 60 Ile Ile Ser Phe Gly Gly Asp Ala Tyr Tyr Ala Ser Trp Ala Lys Gly 1 5 10 15 <210> 61 <211> 16 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 61 Thr Ile Asp Thr Gly Asp Asn Ser Phe Tyr Ala Asn Trp Ala Lys Gly 1 5 10 15 <210> 62 <211> 17 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 62 Cys Ile Tyr Thr Gly Ser Asp Thr Thr Tyr Tyr Ala Thr Trp Ala Lys 1 5 10 15 Gly <210> 63 <211> 16 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 63 Thr Ile Asp Thr Gly Asp Asn Ser Phe Tyr Ala Asn Trp Ala Lys Gly 1 5 10 15 <210> 64 <211> 17 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 64 Ala Ile Tyr Thr Gly Ser Gly Ala Thr Tyr Lys Ala Ser Trp Ala Lys 1 5 10 15 Gly <210> 65 <211> 13 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 65 Asp Gly Gly Tyr Val Thr Pro Thr His Ala Met Tyr Leu 1 5 10 <210> 66 <211> 9 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 66 Asp Leu His Ala Gly Ile Thr Asn Leu 1 5 <210> 67 <211> 13 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 67 Tyr Pro Thr Tyr Gly Asp Gly Gly His Ala Phe Asn Leu 1 5 10 <210> 68 <211> 11 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 68 Gln Ile Tyr Ser Gly Asp Asn Asn Asp Asn Phe 1 5 10 <210> 69 <211> 11 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 69 Gln Ile Tyr Ser Gly Asp Asn Asn Asp Asn Phe 1 5 10 <210> 70 <211> 12 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 70 Val Gly Ala Gly Asn Ile Phe Trp Tyr Phe Asp Leu 1 5 10 <210> 71 <211> 11 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 71 Asn Asp Gly Ser Val Tyr Asn Leu Phe Asn Leu 1 5 10 <210> 72 <211> 4 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 72 Gly Ser Gly Leu One <210> 73 <211> 11 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 73 Asn Asp Gly Ser Val Tyr Asn Leu Phe Asn Leu 1 5 10 <210> 74 <211> 13 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 74 Asp Gly Gly Tyr Asp Tyr Pro Thr His Ala Met His Tyr 1 5 10 <210> 75 <211> 11 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 75 Gln Ala Ser Gln Asn Ile Gly Ser Asp Leu Ala 1 5 10 <210> 76 <211> 11 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 76 Gln Ala Ser Gln Asn Ile Tyr Ser Asn Leu Ala 1 5 10 <210> 77 <211> 13 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 77 Gln Ser Ser Gln Ser Val Tyr Ser Ser Asp Tyr Leu Ser 1 5 10 <210> 78 <211> 11 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 78 Gln Ala Ser Glu Ser Ile Ser Asn Trp Leu Ala 1 5 10 <210> 79 <211> 11 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 79 Gln Ala Ser Glu Ser Ile Ser Asn Trp Leu Ala 1 5 10 <210> 80 <211> 11 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 80 Gln Ala Ser Glu Ser Ile Tyr Ser Ala Leu Ala 1 5 10 <210> 81 <211> 11 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 81 Gln Ala Ser Glu Asn Ile Tyr Ser Ala Leu Ser 1 5 10 <210> 82 <211> 11 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 82 Gln Ala Ser Gln Asn Ile Tyr Ser Leu Leu Ala 1 5 10 <210> 83 <211> 11 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 83 Gln Ala Ser Glu Asn Ile Tyr Ser Ala Leu Ser 1 5 10 <210> 84 <211> 11 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 84 Gln Ala Ser Gln Asn Ile Gly Ser Ser Leu Ala 1 5 10 <210> 85 <211> 7 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 85 Gly Ala Ser Lys Leu Ala Ser 1 5 <210> 86 <211> 7 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 86 Gly Ala Ser Asn Leu Glu Ser 1 5 <210> 87 <211> 7 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 87 Glu Ala Ser Lys Leu Ala Ser 1 5 <210> 88 <211> 7 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 88 Arg Ala Ser Thr Leu Ala Ser 1 5 <210> 89 <211> 7 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 89 Arg Ala Ser Thr Leu Ala Ser 1 5 <210> 90 <211> 7 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 90 Ser Ala Ser Thr Leu Ala Ser 1 5 <210> 91 <211> 7 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 91 Tyr Ala Ser Thr Leu Ala Ser 1 5 <210> 92 <211> 7 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 92 His Thr Ser Asp Leu Ala Ser 1 5 <210> 93 <211> 7 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 93 Tyr Ala Ser Thr Leu Ala Ser 1 5 <210> 94 <211> 7 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 94 Gly Ala Ser Lys Thr His Ser 1 5 <210> 95 <211> 12 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 95 Gln Cys Thr Phe Val Gly Ser Ser Tyr Gly Asn Ala 1 5 10 <210> 96 <211> 12 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 96 Leu Gln Gly Tyr Ser Tyr Ser Asn Val Asp Asp Ala 1 5 10 <210> 97 <211> 12 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 97 Gln Gly Thr Tyr Tyr Ser Ser Gly Trp Tyr Phe Ala 1 5 10 <210> 98 <211> 12 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 98 Gln Gln Asp Tyr Ser Ser Ser Asn Val Asp Asn Thr 1 5 10 <210> 99 <211> 12 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 99 Gln Gln Asp Tyr Ser Ser Ser Asn Val Asp Asn Thr 1 5 10 <210> 100 <211> 12 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 100 Gln Gln Tyr Tyr Ser Ser Thr Asn Val His Asn Ser 1 5 10 <210> 101 <211> 12 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 101 Gln Gln Tyr Tyr Asp Ile Asn Ser Val Asp Asn Thr 1 5 10 <210> 102 <211> 13 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 102 Gln Cys Thr Ala Tyr Gly Ser Ser Asp Val Gly Gly Thr 1 5 10 <210> 103 <211> 12 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 103 Gln Gln Tyr Tyr Asp Ile Asn Ser Val Asp Asn Thr 1 5 10 <210> 104 <211> 12 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 104 Gln Ser Thr Lys Val Gly Ser Ser Tyr Gly Asn His 1 5 10 <210> 105 <211> 1680 <212> PRT <213> Mus musculus <400> 105 Met Gly Leu Trp Gly Ile Leu Cys Leu Leu Ile Phe Leu Asp Lys Thr 1 5 10 15 Trp Gly Gln Glu Gln Thr Tyr Val Ile Ser Ala Pro Lys Ile Leu Arg 20 25 30 Val Gly Ser Ser Glu Asn Val Val Ile Gln Val His Gly Tyr Thr Glu 35 40 45 Ala Phe Asp Ala Thr Leu Ser Leu Lys Ser Tyr Pro Asp Lys Lys Val 50 55 60 Thr Phe Ser Ser Gly Tyr Val Asn Leu Ser Pro Glu Asn Lys Phe Gln 65 70 75 80 Asn Ala Ala Leu Leu Thr Leu Gln Pro Asn Gln Val Pro Arg Glu Glu 85 90 95 Ser Pro Val Ser His Val Tyr Leu Glu Val Val Ser Lys His Phe Ser 100 105 110 Lys Ser Lys Lys Ile Pro Ile Thr Tyr Asn Asn Gly Ile Leu Phe Ile 115 120 125 His Thr Asp Lys Pro Val Tyr Thr Pro Asp Gln Ser Val Lys Ile Arg 130 135 140 Val Tyr Ser Leu Gly Asp Asp Leu Lys Pro Ala Lys Arg Glu Thr Val 145 150 155 160 Leu Thr Phe Ile Asp Pro Glu Gly Ser Glu Val Asp Ile Val Glu Glu 165 170 175 Asn Asp Tyr Thr Gly Ile Ile Ser Phe Pro Asp Phe Lys Ile Pro Ser 180 185 190 Asn Pro Lys Tyr Gly Val Trp Thr Ile Lys Ala Asn Tyr Lys Lys Asp 195 200 205 Phe Thr Thr Thr Gly Thr Ala Tyr Phe Glu Ile Lys Glu Tyr Val Leu 210 215 220 Pro Arg Phe Ser Val Ser Ile Glu Leu Glu Arg Thr Phe Ile Gly Tyr 225 230 235 240 Lys Asn Phe Lys Asn Phe Glu Ile Thr Val Lys Ala Arg Tyr Phe Tyr 245 250 255 Asn Lys Val Val Pro Asp Ala Glu Val Tyr Ala Phe Phe Gly Leu Arg 260 265 270 Glu Asp Ile Lys Asp Glu Glu Lys Gln Met Met His Lys Ala Thr Gln 275 280 285 Ala Ala Lys Leu Val Asp Gly Val Ala Gln Ile Ser Phe Asp Ser Glu 290 295 300 Thr Ala Val Lys Glu Leu Ser Tyr Asn Ser Leu Glu Asp Leu Asn Asn 305 310 315 320 Lys Tyr Leu Tyr Ile Ala Val Thr Val Thr Glu Ser Ser Gly Gly Phe 325 330 335 Ser Glu Glu Ala Glu Ile Pro Gly Val Lys Tyr Val Leu Ser Pro Tyr 340 345 350 Thr Leu Asn Leu Val Ala Thr Pro Leu Phe Val Lys Pro Gly Ile Pro 355 360 365 Phe Ser Ile Lys Ala Gln Val Lys Asp Ser Leu Glu Gln Ala Val Gly 370 375 380 Gly Val Pro Val Thr Leu Met Ala Gln Thr Val Asp Val Asn Gln Glu 385 390 395 400 Thr Ser Asp Leu Glu Thr Lys Arg Ser Ile Thr His Asp Thr Asp Gly 405 410 415 Val Ala Val Phe Val Leu Asn Leu Pro Ser Asn Val Thr Val Leu Lys 420 425 430 Phe Glu Ile Arg Thr Asp Asp Pro Glu Leu Pro Glu Glu Asn Gln Ala 435 440 445 Ser Lys Glu Tyr Glu Ala Val Ala Tyr Ser Ser Leu Ser Gln Ser Tyr 450 455 460 Ile Tyr Ile Ala Trp Thr Glu Asn Tyr Lys Pro Met Leu Val Gly Glu 465 470 475 480 Tyr Leu Asn Ile Met Val Thr Pro Lys Ser Pro Tyr Ile Asp Lys Ile 485 490 495 Thr His Tyr Asn Tyr Leu Ile Leu Ser Lys Gly Lys Ile Val Gln Tyr 500 505 510 Gly Thr Arg Glu Lys Leu Phe Ser Ser Thr Tyr Gln Asn Ile Asn Ile 515 520 525 Pro Val Thr Gln Asn Met Val Pro Ser Ala Arg Leu Leu Val Tyr Tyr 530 535 540 Ile Val Thr Gly Glu Gln Thr Ala Glu Leu Val Ala Asp Ala Val Trp 545 550 555 560 Ile Asn Ile Glu Glu Lys Cys Gly Asn Gln Leu Gln Val His Leu Ser 565 570 575 Pro Asp Glu Tyr Val Tyr Ser Pro Gly Gln Thr Val Ser Leu Asp Met 580 585 590 Val Thr Glu Ala Asp Ser Trp Val Ala Leu Ser Ala Val Asp Arg Ala 595 600 605 Val Tyr Lys Val Gln Gly Asn Ala Lys Arg Ala Met Gln Arg Val Phe 610 615 620 Gln Ala Leu Asp Glu Lys Ser Asp Leu Gly Cys Gly Ala Gly Gly Gly 625 630 635 640 His Asp Asn Ala Asp Val Phe His Leu Ala Gly Leu Thr Phe Leu Thr 645 650 655 Asn Ala Asn Ala Asp Asp Ser His Tyr Arg Asp Asp Ser Cys Lys Glu 660 665 670 Ile Leu Arg Ser Lys Arg Asn Leu His Leu Leu Arg Gln Lys Ile Glu 675 680 685 Glu Gln Ala Ala Lys Tyr Lys His Ser Val Pro Lys Lys Cys Cys Tyr 690 695 700 Asp Gly Ala Arg Val Asn Phe Tyr Glu Thr Cys Glu Glu Arg Val Ala 705 710 715 720 Arg Val Thr Ile Gly Pro Leu Cys Ile Arg Ala Phe Asn Glu Cys Cys 725 730 735 Thr Ile Ala Asn Lys Ile Arg Lys Glu Ser Pro His Lys Pro Val Gln 740 745 750 Leu Gly Arg Ile His Ile Lys Thr Leu Leu Pro Val Met Lys Ala Asp 755 760 765 Ile Arg Ser Tyr Phe Pro Glu Ser Trp Leu Trp Glu Ile His Arg Val 770 775 780 Pro Lys Arg Lys Gln Leu Gln Val Thr Leu Pro Asp Ser Leu Thr Thr 785 790 795 800 Trp Glu Ile Gln Gly Ile Gly Ile Ser Asp Asn Gly Ile Cys Val Ala 805 810 815 Asp Thr Leu Lys Ala Lys Val Phe Lys Glu Val Phe Leu Glu Met Asn 820 825 830 Ile Pro Tyr Ser Val Val Arg Gly Glu Gln Ile Gln Leu Lys Gly Thr 835 840 845 Val Tyr Asn Tyr Met Thr Ser Gly Thr Lys Phe Cys Val Lys Met Ser 850 855 860 Ala Val Glu Gly Ile Cys Thr Ser Gly Ser Ser Ala Ala Ser Leu His 865 870 875 880 Thr Ser Arg Pro Ser Arg Cys Val Phe Gln Arg Ile Glu Gly Ser Ser 885 890 895 Ser His Leu Val Thr Phe Thr Leu Leu Pro Leu Glu Ile Gly Leu His 900 905 910 Ser Ile Asn Phe Ser Leu Glu Thr Ser Phe Gly Lys Asp Ile Leu Val 915 920 925 Lys Thr Leu Arg Val Val Pro Glu Gly Val Lys Arg Glu Ser Tyr Ala 930 935 940 Gly Val Ile Leu Asp Pro Lys Gly Ile Arg Gly Ile Val Asn Arg Arg 945 950 955 960 Lys Glu Phe Pro Tyr Arg Ile Pro Leu Asp Leu Val Pro Lys Thr Lys 965 970 975 Val Glu Arg Ile Leu Ser Val Lys Gly Leu Leu Val Gly Glu Phe Leu 980 985 990 Ser Thr Val Leu Ser Lys Glu Gly Ile Asn Ile Leu Thr His Leu Pro 995 1000 1005 Lys Gly Ser Ala Glu Ala Glu Leu Met Ser Ile Ala Pro Val Phe 1010 1015 1020 Tyr Val Phe His Tyr Leu Glu Ala Gly Asn His Trp Asn Ile Phe 1025 1030 1035 Tyr Pro Asp Thr Leu Ser Lys Arg Gln Ser Leu Glu Lys Lys Ile 1040 1045 1050 Lys Gln Gly Val Val Ser Val Met Ser Tyr Arg Asn Ala Asp Tyr 1055 1060 1065 Ser Tyr Ser Met Trp Lys Gly Ala Ser Ala Ser Thr Trp Leu Thr 1070 1075 1080 Ala Phe Ala Leu Arg Val Leu Gly Gln Val Ala Lys Tyr Val Lys 1085 1090 1095 Gln Asp Glu Asn Ser Ile Cys Asn Ser Leu Leu Trp Leu Val Glu 1100 1105 1110 Lys Cys Gln Leu Glu Asn Gly Ser Phe Lys Glu Asn Ser Gln Tyr 1115 1120 1125 Leu Pro Ile Lys Leu Gln Gly Thr Leu Pro Ala Glu Ala Gln Glu 1130 1135 1140 Lys Thr Leu Tyr Leu Thr Ala Phe Ser Val Ile Gly Ile Arg Lys 1145 1150 1155 Ala Val Asp Ile Cys Pro Thr Met Lys Ile His Thr Ala Leu Asp 1160 1165 1170 Lys Ala Asp Ser Phe Leu Leu Glu Asn Thr Leu Pro Ser Lys Ser 1175 1180 1185 Thr Phe Thr Leu Ala Ile Val Ala Tyr Ala Leu Ser Leu Gly Asp 1190 1195 1200 Arg Thr His Pro Arg Phe Arg Leu Ile Val Ser Ala Leu Arg Lys 1205 1210 1215 Glu Ala Phe Val Lys Gly Asp Pro Pro Ile Tyr Arg Tyr Trp Arg 1220 1225 1230 Asp Thr Leu Lys Arg Pro Asp Ser Ser Val Pro Ser Ser Gly Thr 1235 1240 1245 Ala Gly Met Val Glu Thr Thr Ala Tyr Ala Leu Leu Ala Ser Leu 1250 1255 1260 Lys Leu Lys Asp Met Asn Tyr Ala Asn Pro Ile Ile Lys Trp Leu 1265 1270 1275 Ser Glu Glu Gln Arg Tyr Gly Gly Gly Phe Tyr Ser Thr Gln Asp 1280 1285 1290 Thr Ile Asn Ala Ile Glu Gly Leu Thr Glu Tyr Ser Leu Leu Leu 1295 1300 1305 Lys Gln Ile His Leu Asp Met Asp Ile Asn Val Ala Tyr Lys His 1310 1315 1320 Glu Gly Asp Phe His Lys Tyr Lys Val Thr Glu Lys His Phe Leu 1325 1330 1335 Gly Arg Pro Val Glu Val Ser Leu Asn Asp Asp Leu Val Val Ser 1340 1345 1350 Thr Gly Tyr Ser Ser Gly Leu Ala Thr Val Tyr Val Lys Thr Val 1355 1360 1365 Val His Lys Ile Ser Val Ser Glu Glu Phe Cys Ser Phe Tyr Leu 1370 1375 1380 Lys Ile Asp Thr Gln Asp Ile Glu Ala Ser Ser His Phe Arg Leu 1385 1390 1395 Ser Asp Ser Gly Phe Lys Arg Ile Ile Ala Cys Ala Ser Tyr Lys 1400 1405 1410 Pro Ser Lys Glu Glu Ser Thr Ser Gly Ser Ser His Ala Val Met 1415 1420 1425 Asp Ile Ser Leu Pro Thr Gly Ile Gly Ala Asn Glu Glu Asp Leu 1430 1435 1440 Arg Ala Leu Val Glu Gly Val Asp Gln Leu Leu Thr Asp Tyr Gln 1445 1450 1455 Ile Lys Asp Gly His Val Ile Leu Gln Leu Asn Ser Ile Pro Ser 1460 1465 1470 Arg Asp Phe Leu Cys Val Arg Phe Arg Ile Phe Glu Leu Phe Gln 1475 1480 1485 Val Gly Phe Leu Asn Pro Ala Thr Phe Thr Val Tyr Glu Tyr His 1490 1495 1500 Arg Pro Asp Lys Gln Cys Thr Met Ile Tyr Ser Ile Ser Asp Thr 1505 1510 1515 Arg Leu Gln Lys Val Cys Glu Gly Ala Ala Cys Thr Cys Val Glu 1520 1525 1530 Ala Asp Cys Ala Gln Leu Gln Ala Glu Val Asp Leu Ala Ile Ser 1535 1540 1545 Ala Asp Ser Arg Lys Glu Lys Ala Cys Lys Pro Glu Thr Ala Tyr 1550 1555 1560 Ala Tyr Lys Val Arg Ile Thr Ser Ala Thr Glu Glu Asn Val Phe 1565 1570 1575 Val Lys Tyr Thr Ala Thr Leu Leu Val Thr Tyr Lys Thr Gly Glu 1580 1585 1590 Ala Ala Asp Glu Asn Ser Glu Val Thr Phe Ile Lys Lys Met Ser 1595 1600 1605 Cys Thr Asn Ala Asn Leu Val Lys Gly Lys Gln Tyr Leu Ile Met 1610 1615 1620 Gly Lys Glu Val Leu Gln Ile Lys His Asn Phe Ser Phe Lys Tyr 1625 1630 1635 Ile Tyr Pro Leu Asp Ser Ser Thr Trp Ile Glu Tyr Trp Pro Thr 1640 1645 1650 Asp Thr Thr Cys Pro Ser Cys Gln Ala Phe Val Glu Asn Leu Asn 1655 1660 1665 Asn Phe Ala Glu Asp Leu Phe Leu Asn Ser Cys Glu 1670 1675 1680 <210> 106 <211> 123 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 106 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val His Ser Ser 20 25 30 Tyr Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Gly Ala Ile Phe Thr Gly Ser Gly Ala Glu Tyr Lys Ala Glu Trp 50 55 60 Ala Lys Gly Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Ser Asp Ala Gly Tyr Asp Tyr Pro Thr His Ala Met His Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 107 <211> 123 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 107 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val His Ser Ser 20 25 30 Tyr Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Gly Ala Ile Phe Thr Gly Ser Gly Ala Glu Tyr Lys Ala Glu Trp 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Ser Asp Ala Gly Tyr Asp Tyr Pro Thr His Ala Met His Tyr 100 105 110 Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 108 <211> 123 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 108 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val His Ser Ser 20 25 30 Tyr Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Gly Ala Ile Phe Thr Gly Ser Gly Ala Glu Tyr Lys Ala Glu Trp 50 55 60 Ala Lys Gly Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Ser Asp Ala Gly Tyr Asp Tyr Pro Thr His Ala Met His Tyr 100 105 110 Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 109 <211> 123 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 109 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val His Ser Ser 20 25 30 Tyr Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Gly Ala Ile Phe Thr Gly Ser Gly Ala Glu Tyr Lys Ala Glu Trp 50 55 60 Ala Lys Gly Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Ser Asp Ala Gly Tyr Asp Tyr Pro Thr His Ala Met His Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 110 <211> 123 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 110 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val His Ser Ser 20 25 30 Tyr Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Gly Ile Phe Thr Gly Ser Gly Ala Thr Tyr Lys Ala Glu Trp 50 55 60 Ala Lys Gly Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Ser Asp Ala Gly Tyr Asp Tyr Pro Thr His Ala Met His Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 111 <211> 110 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 111 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ser 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Glu Thr Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Asn Thr Lys Val Gly Ser Ser 85 90 95 Tyr Gly Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 112 <211> 110 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 112 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ser 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Glu Thr Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Asn Thr Lys Val Gly Ser Ser 85 90 95 Tyr Gly Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 113 <211> 110 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 113 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ser 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Thr Thr Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Asn Thr Lys Val Gly Ser Ser 85 90 95 Tyr Gly Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 114 <211> 328 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 114 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Arg Arg Gly Pro Lys Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ala His Tyr Thr 305 310 315 320 Arg Lys Glu Leu Ser Leu Ser Pro 325 <210> 115 <211> 325 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 115 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Arg Arg Gly Pro Lys Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Leu His Glu Ala Leu His Ala His Tyr Thr Arg Lys Glu 305 310 315 320 Leu Ser Leu Ser Pro 325 <210> 116 <211> 325 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 116 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Arg Arg Gly Pro Lys Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Leu His Glu Ala Leu His Ala His Tyr Thr Arg Lys Glu 305 310 315 320 Leu Ser Leu Ser Pro 325 <210> 117 <211> 6 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 117 Ser Ser Tyr Tyr Met Ala 1 5 <210> 118 <211> 17 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 118 Ala Ile Phe Thr Gly Ser Gly Ala Glu Tyr Lys Ala Glu Trp Ala Lys 1 5 10 15 Gly <210> 119 <211> 17 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 119 Ala Ile Phe Thr Gly Ser Gly Ala Glu Tyr Lys Ala Glu Trp Val Lys 1 5 10 15 Gly <210> 120 <211> 17 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 120 Gly Ile Phe Thr Gly Ser Gly Ala Thr Tyr Lys Ala Glu Trp Ala Lys 1 5 10 15 Gly <210> 121 <211> 13 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 121 Asp Ala Gly Tyr Asp Tyr Pro Thr His Ala Met His Tyr 1 5 10 <210> 122 <211> 11 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 122 Arg Ala Ser Gln Gly Ile Ser Ser Ser Leu Ala 1 5 10 <210> 123 <211> 7 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 123 Gly Ala Ser Glu Thr Glu Ser 1 5 <210> 124 <211> 7 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 124 Gly Ala Ser Thr Thr Gln Ser 1 5 <210> 125 <211> 12 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 125 Gln Asn Thr Lys Val Gly Ser Ser Tyr Gly Asn Thr 1 5 10 <210> 126 <211> 6 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <220> <221> misc_feature <222> (5)..(5) <223> Xaa is Met or Val <220> <221> misc_feature <222> (6)..(6) <223> Xaa is Cys or Ala <400> 126 Ser Ser Tyr Tyr Xaa Xaa 1 5 <210> 127 <211> 17 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <220> <221> misc_feature <222> (1)..(1) <223> Xaa is Cys, Ala or Gly <220> <221> misc_feature <222> (3)..(3) <223> Xaa is Tyr or Phe <220> <221> misc_feature <222> (9)..(9) <223> Xaa is Thr, Asp or Glu <220> <221> misc_feature <222> (11)..(11) <223> Xaa is Tyr, Lys or Gln <220> <221> misc_feature <222> (13)..(13) <223> Xaa is Ser, Asp or Glu <220> <221> misc_feature <222> (15)..(15) <223> Xaa is Ala or Val <400> 127 Xaa Ile Xaa Thr Gly Ser Gly Ala Xaa Tyr Xaa Ala Xaa Trp Xaa Lys 1 5 10 15 Gly <210> 128 <211> 13 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <220> <221> misc_feature <222> (2)..(2) <223> Xaa is Gly or Ala <220> <221> misc_feature <222> (5)..(5) <223> Xaa is Val, Gln or Asp <220> <221> misc_feature <222> (6)..(6) <223> Xaa is Thr or Tyr <220> <221> misc_feature <222> (12)..(12) <223> Xaa is Tyr or His <220> <221> misc_feature <222> (13)..(13) <223> Xaa is Leu or Tyr <400> 128 Asp Xaa Gly Tyr Xaa Xaa Pro Thr His Ala Met Xaa Xaa 1 5 10 <210> 129 <211> 11 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <220> <221> misc_feature <222> (1)..(1) <223> Xaa is Gln or Arg <220> <221> misc_feature <222> (5)..(5) <223> Xaa is Asn, Gln or Gly <220> <221> misc_feature <222> (7)..(7) <223> Xaa is Gly or Ser <220> <221> misc_feature <222> (9)..(9) <223> Xaa is Asp, Lys or Ser <400> 129 Xaa Ala Ser Gln Xaa Ile Xaa Ser Xaa Leu Ala 1 5 10 <210> 130 <211> 7 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <220> <221> misc_feature <222> (4)..(4) <223> Xaa is Lys, Glu or Thr <220> <221> misc_feature <222> (5)..(5) <223> Xaa is Leu or Thr <220> <221> misc_feature <222> (6)..(6) <223> Xaa is Ala, His, Glu or Gln <400> 130 Gly Ala Ser Xaa Xaa Xaa Ser 1 5 <210> 131 <211> 12 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <220> <221> misc_feature <222> (2)..(2) <223> Xaa is Ser, Cys, Asn or Thr <220> <221> misc_feature <222> (4)..(4) <223> Xaa is Phe or Lys <220> <221> misc_feature <222> (12)..(12) <223> Xaa is Ala, Thr or His <400> 131 Gln Xaa Thr Xaa Val Gly Ser Ser Tyr Gly Asn Xaa 1 5 10 <210> 132 <211> 30 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 132 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ser His 20 25 30 <210> 133 <211> 30 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 133 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val His 20 25 30 <210> 134 <211> 30 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 134 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val His 20 25 30 <210> 135 <211> 14 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 135 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly 1 5 10 <210> 136 <211> 14 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 136 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 <210> 137 <211> 32 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 137 Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val Leu Thr 1 5 10 15 Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Ser 20 25 30 <210> 138 <211> 32 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 138 Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val Leu Thr 1 5 10 15 Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Ser 20 25 30 <210> 139 <211> 32 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 139 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser 20 25 30 <210> 140 <211> 11 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 140 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 141 <211> 11 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 141 Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 142 <211> 23 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 142 Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20 <210> 143 <211> 23 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 143 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20 <210> 144 <211> 15 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 144 Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 1 5 10 15 <210> 145 <211> 15 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 145 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 1 5 10 15 <210> 146 <211> 32 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 146 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys 20 25 30 <210> 147 <211> 32 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 147 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 30 <210> 148 <211> 10 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 148 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 1 5 10 <210> 149 <211> 448 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 149 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly His Ile Phe Ser Asn Tyr 20 25 30 Trp Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Leu Pro Gly Ser Gly His Thr Glu Tyr Thr Glu Asn Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Phe Phe Gly Ser Ser Pro Asn Trp Tyr Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys 210 215 220 Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 260 265 270 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala 420 425 430 Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 <210> 150 <211> 214 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 150 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Asn Val Leu Asn Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210

Claims (29)

A pharmaceutical formulation comprising an anti-C5 antibody for use in the treatment of a complement-mediated disease or disease involving excessive or uncontrolled activation of C5, wherein the antibody comprises:
(1) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118, and (c) HVR- comprising the amino acid sequence of SEQ ID NO: 121 VH sequence containing H3; And (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125. Containing VL sequence;
(2) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 119, and (c) HVR- comprising the amino acid sequence of SEQ ID NO: 121 VH sequence containing H3; And (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125. Containing VL sequence;
(3) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118, and (c) HVR- comprising the amino acid sequence of SEQ ID NO: 121 VH sequence containing H3; And (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 124, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125. Containing VL sequence; or
(4) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 120, and (c) HVR- comprising the amino acid sequence of SEQ ID NO: 121 VH sequence containing H3; And (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 124, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125. VL sequence containing
Including,
Complement-mediated diseases or diseases involving excessive or uncontrolled activation of C5 include rheumatoid arthritis (RA); Systemic lupus erythematosus (SLE); Lupus nephritis; Ischemic reperfusion injury (IRI); asthma; Paroxysmal nocturnal hemoglobinuria (PNH); Hemolytic uremic syndrome (HUS); Atypical hemolytic uremic syndrome (aHUS); High density deposition disease (DDD); Optic neuromyelitis (NMO); Multifocal motor neuropathy (MMN): multiple sclerosis (MS): systemic sclerosis; Macular degeneration; Age-related macular degeneration (AMD); Hemolysis, increased liver enzymes and low platelet (HELLP) syndrome; Thrombotic thrombocytoptic purpura (TTP); Natural heritage; Bullous epidermolysis; Recurrent miscarriage; Preeclampsia; Traumatic brain injury; Myasthenia gravis; Cold agglutination disease; Sjogren syndrome; Dermatomyositis; Bullous euphorbia; Phototoxic reaction; Shiga toxin Escherichia coli-related hemolytic uremic syndrome; Typical or infectious hemolytic uremic syndrome (tHUS); C3 glomerulonephritis; Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis; Bodily fluid and vascular graft rejection; Acute antibody mediated rejection (AMR); Graft dysfunction; Myocardial infarction; Allogeneic transplant; blood poisoning; Coronary artery disease; Hereditary angioedema; Dermatomyositis; Graves'disease;Atherosclerosis;Alzheimer's disease (AD); Huntington's disease; Creitzfeld-Jakob disease; Parkinson's disease; cancer; Wound; Septic shock; Spinal cord injury; Uveitis; Diabetic eye disease; Retinopathy of prematurity; Glomerulonephritis; Membranous nephritis; Immunoglobulin A nephropathy; Adult respiratory distress syndrome (ARDS); Chronic obstructive pulmonary disease (COPD); Cystic fibrosis; Hemolytic anemia; Paroxysmal cold hemoglobinuria; Anaphylactic shock; allergy; osteoporosis; Osteoarthritis; Hashimoto's thyroiditis; Type 1 diabetes; psoriasis; pemphigus; Autoimmune hemolytic anemia (AIHA); Idiopathic thrombocytoptic purpura (ITP); Goodpasture syndrome; Degos disease; Antiphospholipid syndrome (APS); Catastrophic APS (CAPS); Cardiovascular disorders; myocarditis; Cerebrovascular disorders; Peripheral vascular disorders; Renal vascular disorders; Mesenteric or intestinal vascular disorders; Vasculitis; Henoch-Schenlein purpura nephritis; Takayasu disease; Dilated cardiomyopathy; Diabetic angiopathy; Kawasaki disease (arteritis); Venous air embolism (VGE), restenosis after stent placement; Rotational atherosclerosis; Membrane nephropathy; Guillain-Barre syndrome (GBS); Fisher syndrome; Antigen-induced arthritis; Synovial inflammation; Viral infection; Bacterial infection; Fungal infection; And, a pharmaceutical formulation selected from the group consisting of injuries resulting from myocardial infarction, cardiopulmonary bypass, and hemodialysis. The method of claim 1,
The pharmaceutical formulation, wherein the antibody inhibits the activation of C5. The method of claim 1,
The pharmaceutical formulation, wherein the antibody inhibits the activation of the C5 variant R885H. The method of claim 1,
The pharmaceutical formulation, wherein the antibody is a monoclonal antibody. The method of claim 1,
The pharmaceutical formulation, wherein the antibody is a human antibody, humanized antibody or chimeric antibody. The method of claim 1,
Heavy chain variable domain framework FR1, wherein the antibody comprises the amino acid sequence of any one of SEQ ID NOs: 132 to 134; Heavy chain variable domain framework FR2 comprising the amino acid sequence of any one of SEQ ID NOs: 135 and 136; Heavy chain variable domain framework FR3 comprising the amino acid sequence of any one of SEQ ID NOs: 137 to 139; And a heavy chain variable domain framework FR4 comprising the amino acid sequence of any one of SEQ ID NOs: 140 and 141. The method of claim 1,
The light chain variable domain framework FR1, wherein the antibody comprises the amino acid sequence of any one of SEQ ID NOs: 142 and 143; Light chain variable domain framework FR2 comprising the amino acid sequence of any one of SEQ ID NOs: 144 and 145; Light chain variable domain framework FR3 comprising the amino acid sequence of any one of SEQ ID NOs: 146 and 147; And a light chain variable domain framework FR4 comprising the amino acid sequence of SEQ ID NO: 148. The method of claim 1,
The pharmaceutical formulation, wherein the antibody comprises the VH sequence of any one of SEQ ID NOs: 106 to 110. The method of claim 1,
The pharmaceutical formulation, wherein the antibody comprises the VL sequence of any one of SEQ ID NOs: 111 to 113. The method of claim 1,
The pharmaceutical formulation, wherein the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 114 and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 38. The method of claim 1,
The pharmaceutical formulation, wherein the antibody is a full-length IgG1 antibody or a full-length IgG4 antibody. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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