Anti-c5 antibodies and methods of use

Abstract

To provide anti-C5 antibodies and methods of use thereof.SOLUTION: The invention provides anti-C5 antibodies and methods of using the same. In some embodiments, an isolated anti-C5 antibody of the present invention binds to an epitope within the β chain of C5 with a higher affinity at neutral pH than at acidic pH. The invention also provides isolated nucleic acids encoding an anti-C5 antibody of the present invention. The invention also provides host cells comprising a nucleic acid of the present invention. The invention also provides a method of producing an antibody comprising culturing host cells of the present invention so that the antibody is produced. The invention further provides a method of producing an anti-C5 antibody comprising immunizing an animal against a polypeptide which comprises the MG1-MG2 domain of the β chain of C5. The anti-C5 antibodies of the present invention may be for use as a medicament.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to anti-C5 antibodies and methods of using same. [Background technology]

[0002] The complement system plays a central role in the clearance of immune complexes and in the immune response to infectious agents, foreign antigens, virus-infected cells, and tumor cells. There are approximately 25-30 complement proteins, which are found as a complex collection of plasma proteins and membrane cofactors. Complement components achieve their immune defense function by interacting in a series of complex enzymatic cleavage and membrane binding events. The resulting complement cascade leads to the production of products with opsonic, immunoregulatory, and cytolytic functions.

[0003] It is now widely accepted that the complement system is activated through three distinct pathways: the classical, lectin, and alternative pathways. These pathways share many elements and, although initially distinct, converge and share the same terminal complement components (C5 to C9) that are involved in the activation and destruction of target cells.

[0004] The classical pathway is usually activated by the formation of antigen-antibody complexes. Independently, the first step in the activation of the lectin pathway is the binding of specific lectins, such as mannan-binding lectin (MBL), H-ficolin, M-ficolin, L-ficolin, and the C-type lectin CL-11. In contrast, the alternative pathway spontaneously undergoes low-level turnover activation, which can be easily amplified on foreign or other abnormal surfaces (bacteria, yeast, virus-infected cells, or damaged tissues). These pathways converge at the point where complement component C3 is cleaved by active proteases to yield C3a and C3b.

[0005] C3a is an anaphylatoxin. C3b binds to bacteria and other cells, as well as certain viruses and immune complexes, tagging them for removal from the circulation (a role known as opsonization). C3b also complexes with other components to form C5 convertase, which cleaves C5 into C5a and C5b.

[0006] C5 is a 190 kDa protein found in normal serum at approximately 80 μg / ml (0.4 μM). C5 is glycosylated, with approximately 1.5-3% of its mass attributable to carbohydrate. Mature C5 is a heterodimer of a 115 kDa α-chain disulfide-linked to a 75 kDa β-chain. C5 is synthesized as a 1676 amino acid single-chain precursor protein (pro-C5 precursor) (see, for example, US Pat. No. 5,993,331 and US Pat. No. 5,993,331). The pro-C5 precursor is cleaved to yield the β-chain as an amino-terminal fragment and the α-chain as a carboxyl-terminal fragment. The α-chain and β-chain polypeptide fragments are linked to each other via disulfide bonds to constitute the mature C5 protein.

[0007] Mature C5 is cleaved into C5a and C5b fragments during activation of the complement pathway. C5a is cleaved from the α-chain of C5 by C5 convertase as an amino-terminal fragment containing the first 74 amino acids of the α-chain. The remaining part of mature C5 is the fragment C5b, which contains the remainder of the α-chain disulfide-linked to the β-chain. Approximately 20% of the 11 kDa mass of C5a is attributable to carbohydrate.

[0008] C5a is another anaphylatoxin. C5b combines with C6, C7, C8, and C9 to form the membrane attack complex (MAC, C5b-9, terminal complement complex (TCC)) on the surface of target cells. When sufficient numbers of MACs are inserted into the target cell membrane, MAC pores are formed, mediating rapid osmotic lysis of the target cell.

[0009] As mentioned above, C3a and C5a are anaphylatoxins. They can induce degranulation of mast cells, which releases histamine and other inflammatory mediators, which leads to smooth muscle contraction, increased vascular permeability, leukocyte activation, and other inflammatory phenomena, such as cell proliferation leading to cell hyperplasia. C5a also functions as a chemotactic peptide that helps attract granulocytes, such as neutrophils, eosinophils, basophils, and monocytes, to the site of complement activation.

[0010] The activity of C5a is regulated by the plasma enzyme carboxypeptidase N, which removes the carboxy-terminal arginine from C5a to form the C5a-des-Arg derivative, which exhibits only 1% of the anaphylactic and polymorphonuclear chemotactic activity of unmodified C5a.

[0011] A properly functioning complement system provides a robust defense against infectious microorganisms, but inappropriate regulation or activation of complement has been implicated in the pathogenesis of a variety of disorders, including, for example, rheumatoid arthritis (RA); lupus nephritis; ischemia-reperfusion injury; paroxysmal nocturnal hemoglobinuria (PNH); atypical hemolytic uremic syndrome (aHUS); dense deposit disease (DDD); macular degeneration (e.g., age-related macular degeneration (AMD)); hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome; thrombotic thrombocytopenic purpura (TTP); spontaneous fetal loss; oligoimmune vasculitis; epidermolysis bullosa; recurrent abortion; multiple sclerosis (MS); traumatic brain injury; and injury resulting from myocardial infarction, cardiopulmonary bypass, and hemodialysis (see, for example, Non-Patent Document 1). Thus, inhibition of excessive or uncontrolled activation of the complement cascade can provide clinical benefit to patients with such diseases.

[0012] Paroxysmal nocturnal hemoglobinuria (PNH) is a rare blood disorder in which red blood cells are damaged and therefore destroyed more rapidly than normal red blood cells. PNH results from the clonal expansion of hematopoietic stem cells that carry somatic mutations in the PIG-A (phosphatidylinositol glycan class A) gene, located on the X chromosome. Mutations in PIG-A lead to an early inhibition of synthesis of glycosylphosphatidylinositol (GPI), a molecule required to anchor many proteins to the cell surface. As a result, blood cells in PNH lack GPI-anchored proteins, including the complement regulatory proteins CD55 and CD59. Under normal circumstances, these complement regulatory proteins block the formation of MAC on the cell surface, thereby preventing lysis of red blood cells. The absence of GPI-anchored proteins causes complement-mediated hemolysis in PNH.

[0013] PNH is characterized by hemolytic anemia (a reduction in red blood cell count), hemoglobinuria (the presence of hemoglobin in the urine, especially noticeable after sleep), and hemoglobinemia (the presence of hemoglobin in the bloodstream). Individuals affected by PNH have been known to have attacks, defined herein as the appearance of dark urine. Hemolytic anemia is due to the intravascular destruction of red blood cells by complement components. Other known symptoms include speech impairment, fatigue, erectile dysfunction, thrombosis, and recurrent abdominal pain.

[0014] Eculizumab is a humanized monoclonal antibody against the complement protein C5, and is the first therapeutic agent approved for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS) (see, for example, Non-Patent Document 2). Eculizumab inhibits the cleavage of C5 by C5 convertase into C5a and C5b, thereby preventing the generation of the terminal complement complex C5b-9. Both C5a and C5b-9 cause the terminal complement-mediated events characteristic of PNH and aHUS (see also Patent Documents 3, 4, 5, and 6).

[0015] Several reports have described anti-C5 antibodies. For example, Patent Document 7 described an anti-C5 antibody that binds to the α-chain of C5 but not to C5a and blocks the activation of C5, while Patent Document 8 described an anti-C5 monoclonal antibody that inhibits the formation of C5a. On the other hand, Patent Document 9 described an anti-C5 antibody that recognizes a proteolytic site for C5 convertase on the α-chain of C5 and inhibits the conversion of C5 to C5a and C5b. Patent Document 10 described an anti-C5 antibody that inhibits the conversion of C5 to C5a and C5b by recognizing a proteolytic site for C5 convertase on the α-chain of C5 at least 1×10 7 M -1 described an anti-C5 antibody with an affinity constant of

[0016] Antibodies (IgG) bind to neonatal Fc receptors (FcRn) and have a long plasma residence time. Binding of IgG to FcRn is typically observed under acidic conditions (e.g., pH 6.0) and rarely observed under neutral conditions (e.g., pH 7.4). Typically, IgG is nonspecifically taken up into cells via endocytosis and returns to the cell surface by binding to endosomal FcRn under acidic conditions in endosomes. IgG then dissociates from FcRn under neutral conditions in plasma. IgG that fails to bind to FcRn is degraded in lysosomes. If the FcRn-binding ability of IgG under acidic conditions is lost by introducing a mutation into its Fc region, IgG is not recycled from endosomes to plasma, which leads to a significant decrease in the plasma retention of IgG. In order to improve the plasma retention of IgG, a method for 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 into its Fc region, the IgG is recycled from endosomes to plasma more efficiently, and thereby shows improved plasma retention. On the other hand, it has also been reported that IgG with enhanced FcRn binding under neutral conditions does not dissociate from FcRn under neutral conditions in plasma, even when it returns to the cell surface via its binding to FcRn under acidic conditions in endosomes, and as a result, its plasma retention remains unchanged or even deteriorates (see, for example, Non-Patent Document 3; Non-Patent Document 4; Non-Patent Document 5).

[0017] Recently, antibodies that bind to antigens in a pH-dependent manner have been reported (see, for example, Patent Document 11 and Patent Document 12). These antibodies bind strongly to antigens under neutral conditions in plasma and dissociate from antigens under acidic conditions in endosomes. After dissociation from antigens, the antibodies can bind to antigens again when recycled to plasma via FcRn. Thus, a single antibody molecule can bind to multiple antigen molecules repeatedly. In general, the plasma retention of antigens is much shorter than that of antibodies with the above-mentioned FcRn-mediated recycling mechanism. Thus, when an antigen binds to an antibody, the antigen usually exhibits extended plasma retention, which leads to an increase in the plasma concentration of the antigen. Meanwhile, it has been reported that the above-mentioned antibodies that bind to antigens in a pH-dependent manner dissociate from antigens in endosomes during the FcRn-mediated recycling process, and therefore clear antigens from plasma more quickly than typical antibodies. Patent Document 13 also describes a computer modeling analysis showing that antibodies that bind to C5 in a pH-dependent manner can prolong antigen knockdown. [Prior art documents] [Patent documents]

[0018] [Patent Document 1] U.S. Patent No. 6,355,245 [Patent Document 2] U.S. Patent No. 7,432,356 [Patent Document 3] WO2005 / 074607 [Patent Document 4] WO2007 / 106585 [Patent Document 5] WO2008 / 069889 [Patent Document 6] WO2010 / 054403 [Patent Document 7] WO95 / 29697 [Patent Document 8] WO02 / 30985 [Patent Document 9] WO2004 / 007553 [Patent Document 10] WO2010 / 015608 [Patent Document 11] WO2009 / 125825 [Patent Document 12] WO2011 / 122011 [Patent Document 13] WO2011 / 111007 [Non-patent literature]

[0019] [Non-Patent Document 1] Holers et al., Immunol. Rev. 223:300-316 (2008) [Non-Patent Document 2] Dmytrijuk et al., The Oncologist 13(9):993-1000 (2008) [Non-Patent Document 3] Yeung et al., J Immunol. 182(12): 7663-7671 (2009) [Non-Patent Document 4] Datta-Mannan et al., J Biol. Chem. 282(3):1709-1717 (2007) [Non-Patent Document 5] Dall'Acqua et al., J. Immunol. 169(9):5171-5180 (2002) Summary of the Invention [Problem to be solved by the invention]

[0020] It is an object of the present invention to provide anti-C5 antibodies and methods of using the same. [Means for solving the problem]

[0021] The present invention provides anti-C5 antibodies and methods of using same.

[0022] In some embodiments, the isolated anti-C5 antibody of the present invention binds to an epitope within the beta chain of C5. In some embodiments, the isolated anti-C5 antibody of the present invention binds to an epitope within the MG1-MG2 domain of the beta chain of C5. In some embodiments, the isolated anti-C5 antibody of the present invention binds to 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 present invention binds to an epitope within a fragment consisting of at least one fragment selected from the group consisting of amino acids 47-57, 70-76, and 107-110 of the beta chain of C5 (SEQ ID NO:40). In some embodiments, the isolated anti-C5 antibody of the present invention binds to an epitope within a fragment consisting of at least one amino acid residue selected from the group consisting of Glu48, Asp51, His70, His72, Lys109, and His110 of SEQ ID NO:40. In a further embodiment, the antibody binds to C5 with a higher affinity at neutral pH than at acidic pH. In a further embodiment, the antibody binds to 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 a further embodiment, the antibody binds to the same epitope as the antibody described in Table 2 with a higher affinity at pH 7.4 than at pH 5.8. In a further embodiment, the anti-C5 antibody of the invention binds to the same epitope as the antibody described in Table 7 or 8. In a further embodiment, the antibody binds to the same epitope as the antibody described in Table 7 or 8 with a higher affinity at pH 7.4 than at pH 5.8.

[0023] In certain embodiments, the anti-C5 antibodies of the invention compete for binding to C5 with an antibody comprising a VH and VL pair selected from the following: (a) a VH of SEQ ID NO:1 and a VL of SEQ ID NO:11; (b) a VH of SEQ ID NO:5 and a VL of SEQ ID NO:15; (c) a VH of SEQ ID NO:4 and a VL of SEQ ID NO:14; (d) a VH of SEQ ID NO:6 and a VL of SEQ ID NO:16; (e) a VH of SEQ ID NO:2 and a VL of SEQ ID NO:12; (f) a VH of SEQ ID NO:3 and a VL of SEQ ID NO:13; (g) a VH of SEQ ID NO:9 and a VL of SEQ ID NO:19; (h) a VH of SEQ ID NO:7 and a VL of SEQ ID NO:17; (i) a VH of SEQ ID NO:8 and a VL of SEQ ID NO:18; and (j) a VH of SEQ ID NO:10 and a VL of SEQ ID NO:20. In further embodiments, the anti-C5 antibody binds to C5 with greater affinity at neutral pH than at acidic pH, hi further embodiments, the anti-C5 antibody binds to C5 with greater affinity at pH 7.4 than at pH 5.8.

[0024] In some embodiments, the isolated anti-C5 antibody of the present invention has a property selected from the group consisting of: (a) the antibody contacts 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 a C5 variant consisting of an E48A substitution of SEQ ID NO:39; or (c) the antibody binds to a C5 protein consisting of the amino acid sequence of SEQ ID NO:39 at pH 7.4, but does not bind to a C5 protein consisting of the amino acid sequence of SEQ ID NO:39 with an H72Y substitution at pH 7.4. In further embodiments, the antibody binds to C5 with a higher affinity at neutral pH than at acidic pH. In further embodiments, the antibody binds to C5 with a higher affinity at pH 7.4 than at pH 5.8.

[0025] In some embodiments, the isolated anti-C5 antibody of the present invention inhibits the activation of C5. In some embodiments, the isolated anti-C5 antibody of the present invention inhibits the activation of C5 mutant R885H. In some embodiments, the isolated anti-C5 antibody of the present invention is a monoclonal antibody. In some embodiments, the isolated anti-C5 antibody of the present invention is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the isolated anti-C5 antibody of the present invention is an antibody fragment that binds to C5. In some embodiments, the isolated anti-C5 antibody of the present invention is a full-length IgG1 antibody or a full-length IgG4 antibody.

[0026] In some embodiments, the isolated anti-C5 antibody of the present invention comprises: (a) the amino acid sequence DX 1 GYX 2 X 3 PTHAMX 4 X 5 (where X 1 is G, A, X 2 is V, Q or D, X 3 is T, Y, X 4 is Y, H, or X 5 is L or Y) (SEQ ID NO: 128); and (b) HVR-H3 comprising the amino acid sequence QX 1 TX 2 VGSSYGNX 3 (where X 1 is S, C, N or T, X 2 is F, K, X 3 (c) an HVR-L3 comprising the 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, F, X 3 is T, D or E, X 4 is Y, K or Q, X 5 is S, D or E, X 6 is A or V) (SEQ ID NO: 127).

[0027] In some embodiments, the isolated anti-C5 antibody of the invention comprises: (a) the amino acid sequence SSYYX 1 X 2 (where X 1 is M, V, X 2 is C or A) (SEQ ID NO: 126); and (b) HVR-H1 comprising the 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, F, X 3 is T, D or E, X 4 is Y, K or Q, X 5 is S, D or E, X 6 is A or V) (SEQ ID NO: 127), and (c) an HVR-H2 having the amino acid sequence DX 1 GYX 2 X 3 PTHAMX 4 X 5 (where X 1 is G, A, X 2 is V, Q or D, X 3 is T, Y, X 4 is Y, H, or X 5 and HVR-H3 comprising the amino acid sequence X 1 ASQX 2 IX 3 SX 4 LA (where X 1 is Q, R, X 2 is N, Q or G, X 3 is G, S, or X 4 is D, K or S) (SEQ ID NO: 129); (b) an HVR-L1 comprising the amino acid sequence GASX 1 X 2 X 3 S (where X 1 is K, E or T, X 2 is L, T, X 3is A, H, E or Q) (SEQ ID NO: 130); and (c) HVR-L2 comprising the amino acid sequence QX 1 TX 2 VGSSYGNX 3 (where X 1 is S, C, N or T, X 2 is F, K, X 3 is A, T or H) (SEQ ID NO: 131).

[0028] In some embodiments, the isolated anti-C5 antibody of the present invention comprises: (a) the amino acid sequence X 1 ASQX 2 IX 3 SX 4 LA (where X 1 is Q, R, X 2 is N, Q or G, X 3 is G, S, or X 4 is D, K or S) (SEQ ID NO: 129); (b) an HVR-L1 comprising the amino acid sequence GASX 1 X 2 X 3 S (where X 1 is K, E or T, X 2 is L, T, X 3 is A, H, E or Q) (SEQ ID NO: 130); and (c) HVR-L2 comprising the amino acid sequence QX 1 TX 2 VGSSYGNX 3 (where X 1 is S, C, N or T, X 2 is F, K, X 3 is A, T or H) (SEQ ID NO: 131).

[0029] In some embodiments, the isolated anti-C5 antibody of the present invention comprises a heavy chain variable domain framework FR1 comprising any one of the amino acid sequences of SEQ ID NOs: 132 to 134; a FR2 comprising any one of the amino acid sequences of SEQ ID NOs: 135 to 136; a FR3 comprising any one of the amino acid sequences of SEQ ID NOs: 137 to 139; and a FR4 comprising any one of the amino acid sequences of SEQ ID NOs: 140 to 141. In some embodiments, the isolated anti-C5 antibody of the present invention comprises a light chain variable domain framework FR1 comprising any one of the amino acid sequences of SEQ ID NOs: 142 to 143; a FR2 comprising any one of the amino acid sequences of SEQ ID NOs: 144 to 145; a FR3 comprising any one of the amino acid sequences of SEQ ID NOs: 146 to 147; and a FR4 comprising the amino acid sequence of SEQ ID NO: 148.

[0030] In some embodiments, the isolated anti-C5 antibody of the present 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, 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, 111-113; or (c) the VH sequence of (a) and the VL sequence of (b). In further embodiments, the antibody comprises a VH sequence of any one of SEQ ID NOs: 10, 106-110. In further embodiments, the antibody comprises a VL sequence of any one of SEQ ID NOs: 20, 111-113.

[0031] The present invention provides antibodies comprising any one of the VH sequences of SEQ ID NOs: 10, 106 to 110, and any one of the VL sequences of SEQ ID NOs: 20, 111 to 113.

[0032] The invention also provides isolated nucleic acids encoding anti-C5 antibodies of the invention. The invention also provides host cells comprising nucleic acids of the invention. The invention also provides methods of producing antibodies, comprising culturing host cells of the invention such that the antibody is produced.

[0033] The present invention further provides a method for producing an anti-C5 antibody. In some embodiments, the method comprises immunizing an animal against a polypeptide comprising the MG1-MG2 domain of the β chain of C5 (SEQ ID NO: 43). In some embodiments, the method comprises immunizing an animal against a polypeptide comprising a region corresponding to amino acids at positions 33-124 of the β chain of C5 (SEQ ID NO: 40). In some embodiments, the method comprises immunizing an animal against a polypeptide comprising at least one fragment selected from amino acids 47-57, 70-76, and 107-110 of the β chain of C5 (SEQ ID NO: 40). In some embodiments, the method comprises immunizing an animal against a polypeptide comprising a fragment of the β chain of C5 (SEQ ID NO: 40) comprising at least one amino acid selected from Glu48, Asp51, His70, His72, Lys109, and His110.

[0034] The present invention also provides a pharmaceutical formulation comprising an anti-C5 antibody of the present invention and a pharma- ceutically acceptable carrier.

[0035] The anti-C5 antibody of the present invention can be used as a pharmaceutical.The anti-C5 antibody of the present invention can be used in the treatment of complement-mediated diseases or conditions involving excessive or uncontrolled activation of C5.The anti-C5 antibody of the present invention can be used in enhancing the clearance of C5 from plasma.

[0036] The anti-C5 antibody of the present invention can be used in the manufacture of medicine.In some embodiments, the medicine is for treating complement-mediated disease or condition that involves excessive or uncontrolled activation of C5.In some embodiments, the medicine is for enhancing the clearance of C5 from plasma.

[0037] The present invention also provides a method for treating an individual with a complement-mediated disease or condition involving 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 present invention. The present invention also provides a method for enhancing the clearance of C5 from plasma in an individual. In some embodiments, the method comprises administering to the individual an effective amount of an anti-C5 antibody of the present invention to enhance the clearance of C5 from plasma. [Brief description of the drawings]

[0038] [Figure 1] Figure 1 shows epitope binning of anti-C5 antibodies as described in Example 2.2. Antibodies classified into the same epitope bin are boxed in bold. [Figure 2A] Figure 2A shows BIACORE® sensorgrams of anti-C5 antibodies at pH 7.4 (solid line) and pH 5.8 (dashed line) to assess pH dependency, as described in Example 3.2. As described in Example 2.2, CFA0305, CFA0307, ​​CFA0366, CFA0501, CFA0538, and CFA0599 are antibodies classified as epitope C. [Figure 2B] Figure 2B shows BIACORE® sensorgrams of anti-C5 antibodies at pH 7.4 (solid line) and pH 5.8 (dashed line) to assess pH dependency, as described in Example 3.2. As described in Example 2.2, CFA0666, CFA0672, and CFA0675 are antibodies classified as epitope C, and CFA0330 and CFA0341 are antibodies classified as epitope B. As described in Example 2.3, 305LO5 is a humanized antibody of CFA0305. [Diagram 3]Figure 3 shows Western blot analysis for fragments derived from the C5 β chain fused to a GST tag (amino acids 19-180, 161-340, 321-500, and 481-660 of SEQ ID NO:40) as described in Example 4.1. CFA0305, CFA0307, ​​CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675 are antibodies classified as epitope C. Anti-GST antibody serves as positive control. The position of the GST-fused C5 fragment (46-49 kDa) is marked with an arrow. [Figure 4] Figure 4 shows BIACORE® sensorgrams of anti-C5 antibodies against the MG1-MG2 domain of the C5 β-chain, as described in Example 4.3. The upper panel shows the results of CFA0305 (solid line), CFA0307 (dashed line), CFA0366 (dash-dotted line), and eculizumab (dotted line). The middle panel shows the results of CFA0501 (solid line), CFA0599 (dashed line), CFA0538 (dash-dotted line), and eculizumab (dotted line). The lower panel shows the results of CFA0666 (solid line), CFA0672 (dashed line), CFA0675 (dash-dotted 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. [Figure 5A] FIG. 5A shows Western blot analysis for peptide fragments derived from the MG1-MG2 domains fused to a GST tag (amino acids 33-124, 45-124, 52-124, 33-111, 33-108, and 45-111 of SEQ ID NO:40) as described in Example 4.4. Anti-GST antibody is used as the antibody for the reaction. The position of the GST-fused C5 fragment (35-37 kDa) is marked with an arrow. [Figure 5B]Figure 5B shows a Western blot analysis of peptide fragments derived from the MG1-MG2 domains fused to a GST tag (amino acids 33-124, 45-124, 52-124, 33-111, 33-108, and 45-111 of SEQ ID NO:40) as described in Example 4.4. CFA0305 is used as the antibody for the reaction. [Figure 5C] Figure 5C is a summary of the binding reactions of anti-C5 antibodies to fragments derived from the C5 β chain as described in Example 4.4. Fragments bound by the anti-C5 antibodies classified as epitope C (CFA0305, CFA0307, ​​CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675) are shown in grey, and fragments to which they do not bind are shown in white. [Figure 6] Figure 6 shows a Western blot analysis of C5 point mutants in which E48, D51, and K109 of the β chain are replaced with alanine (E48A, D51A, and K109A, respectively) as described in Example 4.5. In the left panel, eculizumab (anti-C5 antibody, α chain binder) is used as the antibody for the reaction, and the position of the α chain of C5 (about 113 kDa) is marked with an arrow. In the right panel, CFA0305 (classified as epitope C, β chain binder) is used as the antibody for the reaction, and the position of the β chain of C5 (about 74 kDa) is marked with an arrowhead. [Figure 7] Figure 7 presents BIACORE® sensorgrams showing the interaction of eculizumab-F760G4 (upper panel) or 305LO5 (lower panel) with C5 variants as described in Example 4.6. The sensorgrams were obtained by injection of C5-wt (thick solid line), C5-E48A (short dashed line), C5-D51A (long dashed line), and C5-K109A (thin solid line) over the sensor surface immobilized with eculizumab-F760G4 or 305LO5, respectively. 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]Figure 8 presents BIACORE® sensorgrams showing the interaction of 305LO5 with C5 His mutants to assess pH dependency as described in Example 4.7. The sensorgrams were obtained by injecting C5-wt (thick solid line), C5-H70Y (long dashed line), C5-H72Y (short dashed line), C5-H110Y (dotted line), and C5-H70Y+H110Y (thin solid line) onto the sensor surface immobilized with 305LO5, respectively. To assess pH-dependent interactions, the antibody / antigen complex was dissociated at pH 7.4, followed by further dissociation at pH 5.8 (indicated by arrows). [Figure 9A] Figure 9A shows inhibition of complement-activated liposome lysis by anti-C5 antibodies, as described in Example 5.1. Results are shown for CFA0305, CFA0307, ​​CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675, which were classified as epitope C, as described in Example 2.2. [Figure 9B] Figure 9B shows inhibition of complement-activated liposome lysis by anti-C5 antibodies, as described in Example 5.1. Shown are results for antibodies CFA0330 and CFA0341, classified in epitope B, as described in Example 2.2. [Figure 10A] Figure 10A shows the inhibition of C5a generation by anti-C5 antibodies as described in Example 5.2. The concentration of C5a was quantified in the supernatant obtained during the liposome lysis assay as described in Figure 9A. [Figure 10B] Figure 10B shows inhibition of C5a generation by anti-C5 antibodies as described in Example 5.2. The concentration of C5a was quantified in the supernatant obtained during the liposome lysis assay as described in Figure 9B. [Figure 11] Figure 11 shows inhibition of complement-activated hemolysis by anti-C5 antibodies, as described in Example 5.3. Complement was activated via the classical pathway. [Figure 12] Figure 12 shows inhibition of complement-activated hemolysis by anti-C5 antibodies, as described in Example 5.4. Complement was activated via the alternative pathway. [Figure 13] Figure 13 shows the time course of plasma human C5 concentrations following intravenous administration of human C5 alone or human C5 with anti-human C5 antibodies in mice to assess 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. [Figure 14] Figure 14 shows the time course of plasma anti-human C5 antibody concentrations following intravenous administration of human C5 and anti-human C5 antibodies in mice to evaluate the pharmacokinetics of the antibodies, 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. [Figure 15] Figure 15 shows inhibition of complement-activated liposome lysis by anti-C5 antibodies, as described in Example 9.1. Results are shown for antibodies 305LO15-SG422, 305LO16-SG422, 305LO18-SG422, 305LO19-SG422, 305LO20-SG422, and 305LO20-SG115. [Figure 16] Figure 16 shows inhibition of complement-activated liposome lysis by anti-C5 antibodies, as described in Example 9.1. Results for antibodies 305LO15-SG115 and 305LO23-SG429 are shown. [Figure 17] 17 shows inhibition of complement-activated liposome lysis by anti-C5 antibodies, as described in Example 9.1. Results are shown for antibodies 305LO22-SG115, 305LO22-SG422, 305LO23-SG115, and 305LO23-SG422. [Figure 18]Figure 18 shows the inhibition of C5a generation by anti-C5 antibodies as described in Example 9.2. The concentration of C5a was quantified in the supernatant obtained during the liposome lysis assay as described in Figure 15. [Figure 19] Figure 19 shows the inhibition of C5a generation by anti-C5 antibodies as described in Example 9.2. The concentration of C5a was quantified in the supernatant obtained during the liposome lysis assay as described in Figure 16. [Figure 20] 20 shows inhibition of complement activity in monkey plasma by anti-C5 antibody, as described in Example 9.3. Anti-C5 antibody was administered to cynomolgus monkeys and complement activity in monkey plasma was measured by hemolysis assay. [Figure 21] FIG. 21 shows inhibition of biological activity of wild-type C5 (WT) and C5 mutants (V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N, and E1437D) by an anti-C5 antibody (eculizumab), as described in Example 9.4. [Figure 22] FIG. 22 shows inhibition of biological activity of wild-type C5 (WT) and C5 mutants (V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N, and E1437D) by anti-C5 antibody (305 mutant), as described in Example 9.4. [Diagram 23] FIG. 23 shows inhibition of complement-activated liposome lysis by anti-C5 antibodies (BNJ441 and the 305 mutant), as described in Example 9.5. [Figure 24] FIG. 24 shows the time course of plasma cynomolgus monkey C5 concentrations following intravenous administration of anti-human C5 antibodies in cynomolgus monkeys to assess C5 clearance, as described in Example 10.2. [Diagram 25] FIG. 25 shows the time course of plasma anti-human C5 antibody concentrations following intravenous administration of anti-human C5 antibody in cynomolgus monkeys evaluating the pharmacokinetics of the antibody, as described in Example 10.3. [Figure 26]Figures 26A and 26B show the crystal structure of 305 Fab bound to human C5 (hC5)-MG1 domain, as described in Example 11.6. Figure 26A shows the asymmetric unit. MG1 is shown in surface representation and 305 Fab is shown as ribbon (dark grey: heavy chain, light grey: light chain). Figure 26B shows superimposed molecule 1 and molecule 2 (dark grey: molecule 1, light grey: molecule 2). [Figure 27A] Figure 27A shows the epitope of the 305 Fab contact region on the MG1 domain, as described in Example 11.6. Figure 27A shows the epitope mapping on the MG1 amino acid sequence (dark grey: closer than 3.0 Å, light grey: closer than 4.5 Å). [Figure 27B] Figure 27B shows the epitope of the 305 Fab contact region on the MG1 domain, as described in Example 11.6. Figure 27B shows the epitope mapping on the crystal structure (dark grey spheres: closer than 3.0 Å, light grey sticks: closer than 4.5 Å). [Figure 28A] FIG. 28A shows a close-up view of the interaction of E48, D51, and K109 (stick representation) with 305 Fab (surface representation), as described in Example 11.7. [Figure 28B] FIG. 28B shows the interactions of E48 with its surroundings (dark grey dotted lines: hydrogen bonds with Fab, light grey dotted lines: hydrogen bonds via water) as described in Example 11.7. [Figure 28C] FIG. 28C shows the interactions of D51 with its surroundings (dark grey dotted line: hydrogen bonds with Fab) as described in Example 11.7. [Figure 28D] FIG. 28D shows the interactions of K109 with its surroundings (dark grey dotted line: hydrogen bond with Fab, light grey dotted line: salt bridge with H-CDR3_D95) as described in Example 11.7. [Figure 29A] FIG. 29A shows a close-up view of the interactions of H70, H72, and H110 (stick representation) with 305 Fab (surface representation) in the same orientation as FIG. 28A, as described in Example 11.8. [Figure 29B]Figure 29B shows the interactions of H70 with its surroundings, as described in Example 11.8. This histidine residue is shown in stick and mesh representation. Hydrogen bonds are shown as dotted lines. [Figure 29C] Figure 29C shows the interactions of H72 with its surroundings, as described in Example 11.8. This histidine residue is shown in stick and mesh representation. Hydrogen bonds are shown as dotted lines. [Figure 29D] Figure 29D shows the interactions of H110 with its surroundings as described in Example 11.8. The histidine residue is shown in stick and mesh representation. The distance between H110 and H-CDR3_H100c is shown as a dotted line. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] Description of the Aspects The techniques and procedures described or cited herein are generally well understood and may be found in, for example, 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 (FM Ausubel, et al. eds., (2003));the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987));Oligonucleotide Synthesis (MJ Gait, ed., 1984);Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney), ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (DM Weir and C.C.Blackwell, eds.);Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987);PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994);Current Protocols in Immunology (JE Coligan et al., eds., 1991);Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (CA 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), using conventional techniques commonly employed by those of skill in the art.

[0040] I. Definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this 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) provide those skilled in the art with general guidance for many of the terms used in this application. All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety.

[0041] For purposes of interpreting this specification, the following definitions shall apply and whenever applicable, terms used in the singular shall include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. In the event that any of the definitions below conflict with any document incorporated herein by reference, the definition below shall control.

[0042] An "acceptor human framework" for the purposes of this specification is a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework as defined below. An acceptor human framework "derived" from a human immunoglobulin framework or a human consensus framework may comprise those same amino acid sequences or 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 acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0043] "Affinity" refers to the strength of the total non-covalent interactions between one binding site of a molecule (e.g., an antibody) and the molecule's binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to the inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0044] An "affinity matured" antibody refers to an antibody with one or more modifications in one or more hypervariable regions (HVRs) that result in improved affinity of the antibody for antigen, compared to a parent antibody that does not possess the modifications.

[0045] The term "anti-C5 antibody" or "antibody that binds C5" refers to an antibody that can bind C5 with sufficient affinity such that it is useful as a diagnostic and / or therapeutic agent when targeted to C5. In one embodiment, the extent of binding of the anti-C5 antibody to unrelated, non-C5 proteins is less than about 10% of the binding of the antibody to C5, e.g., as measured by radioimmunoassay (RIA). In certain embodiments, an antibody that binds C5 has an affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 M) of C5. In certain embodiments, the anti-C5 antibody binds to an epitope of C5 that is conserved among C5 from different species.

[0046] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0047] An "antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 linear antibodies; single-chain antibody molecules (eg, scFv); and multispecific antibodies formed from antibody fragments.

[0048] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its own antigen in a competitive assay and / or conversely, the reference antibody blocks the binding of said antibody to its own antigen in a competitive assay. Exemplary competitive assays are provided herein.

[0049] 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.

[0050] The "class" of an antibody refers to the type of constant domain or region present in the antibody's heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into subclasses (isotypes). For example, IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2 The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0051] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes the death or destruction of cells. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitors; enzymes and fragments thereof, such as nucleases; antibiotics; toxins, such as, for example, small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin (including fragments and / or variants thereof); and various anti-tumor or anti-cancer agents, as disclosed below.

[0052] "Effector function" refers to a biological activity attributable to the Fc region of an antibody, which varies with 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; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0053] An "effective amount" of an agent (eg, a pharmaceutical formulation) refers to the amount, at dosages and for periods of time necessary, effective to achieve the desired therapeutic or prophylactic result.

[0054] The term "epitope" includes any determinant that can be bound by an antibody. An epitope is a region of an antigen that is bound by an antibody that targets the antigen and includes specific amino acids that directly contact the antibody. Epitopes can include chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural characteristics and / or specific charge characteristics. In general, an antibody specific for a particular target antigen preferentially recognizes an epitope on that target antigen in a complex mixture of proteins and / or macromolecules.

[0055] The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl terminus of the heavy chain, except that the C-terminal lysine (Lys447) or glycine-lysine (residues 446-447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0056] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0057] The terms "full length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that includes an Fc region as defined herein.

[0058] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the originally transformed cell and progeny derived from that cell regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Also included herein are mutant progeny that have the same function or biological activity as that for which the original transformed cell was screened or selected.

[0059] A "human antibody" is an antibody with an amino acid sequence that corresponds to that of an antibody produced by a human or a human cell, or derived from a human antibody repertoire or other non-human source that uses human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0060] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Usually, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Usually, the subgroup of sequences is a subgroup 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 according to Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III according to Kabat et al., supra.

[0061] A "humanized" antibody refers to a chimeric antibody that contains amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody contains substantially all of at least one, and typically two, variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization. The term "hypervariable region" or "HVR" as used herein refers to each region of an antibody variable domain that is hypervariable in sequence (the "complementarity determining region" or "CDR") and / or forms structurally defined loops (the "hypervariable loops") and / or contains antigen contact residues (the "antigen contacts"). Typically, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein include the following: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NIH, Bethesda, MD (1991)); (c) antigenic contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)); and (d) combinations of (a), (b), and / or (c), comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

[0062] Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

[0063] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to, cytotoxic agents.

[0064] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0065] An "isolated" antibody is one that has been separated from the components of its original environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as measured by electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse-phase HPLC) methods. For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0066] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its original environment. Isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0067] "Isolated nucleic acid encoding an anti-C5 antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including nucleic acid molecules carried on a single vector or separate vectors, and including nucleic acid molecules present in one or more locations in a host cell.

[0068] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies that make up the population are identical and / or bind to the same epitope, except for possible variant antibodies (e.g., variant antibodies including naturally occurring variants or variant antibodies that arise during the manufacture of a monoclonal antibody preparation, which are usually present in small amounts). In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be made by a variety of techniques, including, but not limited to, hybridoma techniques, recombinant DNA techniques, phage display techniques, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, and such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0069] "Naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. Naked antibodies may be present in a pharmaceutical formulation.

[0070] "Native antibodies" refer to immunoglobulin molecules with various structures that occur naturally. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy domain or the 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 the variable light domain or the light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0071] The term "package insert" is used to refer to instructions typically included in commercial packaging of a therapeutic product that contain information about the indications, usage, dosage, method of administration, concomitant therapy, contraindications, and / or warnings regarding the use of such therapeutic product.

[0072] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence after aligning the sequences to obtain the maximum percent sequence identity and introducing gaps, if necessary, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetyx Corporation). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. The ALIGN-2 sequence comparison computer program is the property of Genentech, Inc., and its source code has been submitted with user documentation to the US Copyright Office, Washington DC, 20559, and is registered under US Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0073] In the context of using ALIGN-2 for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively, one may say a given amino acid sequence A has or contains a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in said program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0074] The term "pharmaceutical formulation" refers to a preparation in a form such that the biological activity of the active ingredients contained therein can be exerted, and which does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0075] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0076] As used herein, the term "C5" encompasses any native C5 from any vertebrate source, including mammals such as primates (e.g., humans and monkeys) and rodents (e.g., mice and rats). Unless otherwise indicated, the term "C5" refers to human C5 protein having the amino acid sequence set forth in SEQ ID NO:39 and including the β-chain sequence set forth in SEQ ID NO:40. The term encompasses "full-length" unprocessed C5 as well as any form of C5 that results from processing in a cell. The term also encompasses naturally occurring variants of C5, such as splice variants and allelic variants. An exemplary amino acid sequence of human C5 is set forth in SEQ ID NO:39 ("wild-type" or "WT" C5). An exemplary amino acid sequence of the β-chain of human C5 is set forth in SEQ ID NO:40. An exemplary amino acid sequence of the MG1, MG2, and MG1-MG2 domains of the β-chain of human C5 are set forth in SEQ ID NOs:41, 42, and 43, respectively. Exemplary cynomolgus and mouse C5 amino acid sequences are shown in SEQ ID NOs: 44 and 105, respectively. Amino acid residues 1-19 of SEQ ID NOs: 39, 40, 43, 44, and 105 correspond to a signal sequence that is removed during processing in the cell and is therefore missing from the corresponding exemplary amino acid sequences.

[0077] As used herein, "treatment" (and its grammatical derivatives, such as "treat", "treating", etc.) refers to a clinical intervention intended to modify the natural course of the individual being treated, and may be performed prophylactically or during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay disease onset or slow disease progression.

[0078] The term "variable region" or "variable domain" refers to the domain of an antibody's heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies usually have a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007).) One VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be isolated by screening a complementary library of VL or VH domains, respectively, with a VH or VL domain from an antibody that binds to the antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0079] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which it is introduced. A vector can effect expression of a nucleic acid to which it is operatively linked. Such vectors are also referred to herein as "expression vectors."

[0080] II. Compositions and Methods In one aspect, the present invention is based in part on anti-C5 antibodies and their uses. In certain embodiments, antibodies that bind to C5 are provided. The antibodies of the present invention are useful, for example, for the diagnosis or treatment of complement-mediated diseases or conditions involving excessive or uncontrolled activation of C5.

[0081] A. Exemplary Anti-C5 Antibodies In one aspect, the present invention provides an isolated antibody that binds to C5. In certain embodiments, the anti-C5 antibody of the present 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 19-180 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 20-124 (SEQ ID NO:41) of SEQ ID NO:40). In certain embodiments, the anti-C5 antibody binds to an epitope within a fragment consisting of amino acids 33-124 of the beta chain of C5 (SEQ ID NO:40). In another embodiment, the antibody does not bind to a fragment shorter than amino acids 33-124 of the β chain of C5, for example, a fragment consisting of amino acids 45-124, 52-124, 33-111, 33-108, or 45-111 of the β chain of C5 (SEQ ID NO:40).

[0082] In another aspect, the present invention provides an anti-C5 antibody that exhibits pH-dependent binding properties. The expression "pH-dependent binding" as used herein means that the antibody exhibits "reduced binding to C5 at acidic pH compared to binding to C5 at neutral pH" (for the purposes of this disclosure, both expressions can be used interchangeably). For example, an antibody with "pH-dependent binding properties" includes an antibody that binds to C5 with higher affinity at neutral pH than at acidic pH. In certain embodiments, the antibody of the present invention binds to C5 with at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 times or more higher affinity at neutral pH than at acidic pH. In some embodiments, the antibody binds to C5 with higher affinity at pH 7.4 than at pH 5.8. In further embodiments, the antibodies of the invention bind to C5 with at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more times greater affinity at pH 7.4 than at pH 5.8.

[0083] The "affinity" of an antibody to C5 is, for the purposes of this disclosure, expressed by the KD of the antibody. The KD of an antibody refers to the equilibrium dissociation constant of antibody-antigen interaction. The higher the KD value for an antibody that binds to an antigen, the weaker its binding affinity to 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 of an antibody that binds to C5 at acidic pH is higher than the KD of an antibody that binds to C5 at neutral pH. For example, in the context of the present invention, if the KD of an antibody that binds to C5 at acidic pH is at least two times higher than the KD of an antibody that binds to C5 at neutral pH, the antibody is considered to bind to C5 with higher affinity at neutral pH than at acidic pH. Thus, the invention encompasses antibodies that bind to C5 at acidic pH with a KD that is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more times greater than the KD of the antibody that binds to C5 at neutral pH. -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 In another embodiment, the K value of the antibody at acidic pH is 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M, or even higher.

[0084] In further embodiments, an antibody is considered to bind C5 with higher affinity at neutral pH than at acidic pH if the KD of the antibody that binds C5 at pH 5.8 is at least 2-fold greater than the KD of the antibody that binds C5 at pH 7.4. In some embodiments, the provided antibodies bind C5 at pH 5.8 with a KD that is at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more times greater than the KD of the antibody that binds C5 at pH 7.4. In another embodiment, the KD value of the antibody at pH 7.4 is 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 In another embodiment, the K value of the antibody at pH 5.8 is 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M, or even higher.

[0085] The binding properties of an antibody for a particular antigen can also be expressed in terms of the antibody's kd. The kd of an antibody refers to the dissociation rate constant of the antibody for a particular antigen, and is expressed as the reciprocal of seconds (i.e., sec -1 The kd value is expressed in units of 100 kd. An increased kd value means that the antibody binds weaker to its antigen. Thus, the present invention encompasses antibodies that bind to C5 at acidic pH with a higher kd value than at neutral pH. The present invention encompasses antibodies that bind to C5 at acidic pH with a kd that is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more times greater than the kd of the antibody that binds to C5 at neutral pH. 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 In another embodiment, the kd value of the antibody at acidic pH is 10 -3 1 / s, 10 -2 1 / s, 10 -1 The invention also encompasses antibodies that bind to C5 with a higher kd value at pH 5.8 than at pH 7.4. The invention encompasses antibodies that bind to C5 at pH 5.8 with a kd that is at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more times greater than the kd of the antibody that binds to C5 at pH 7.4. 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 In another embodiment, the kd value of the antibody at pH 5.8 is 10 -3 1 / s, 10 -2 1 / s, 10 -1 It can be 1 / s, or even higher.

[0086] In certain examples, "reduced binding to C5 at acidic pH compared to binding to C5 at neutral pH" is expressed as the ratio of the KD value of the antibody that binds to C5 at acidic pH to the KD value of the antibody that binds to C5 at neutral pH (or vice versa). For example, if 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 binding to C5 at neutral pH" for the purposes of the present invention. In certain exemplary embodiments, the pH5.8 / pH7.4 KD ratio of the antibody of the present invention is 2 or more. In certain exemplary embodiments, the acidic / neutral KD ratio of the antibody of the present invention can be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more. In another embodiment, the KD value of the antibody at neutral pH is 10 -7 M, 10-8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 In another embodiment, the K value of the antibody at acidic pH is 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M, or more. In a further example, if an antibody exhibits a pH5.8 / pH7.4 KD ratio of 2 or more, for purposes of the present invention, the antibody can be considered to exhibit "reduced binding to C5 at acidic pH compared to binding to C5 at neutral pH". In certain exemplary embodiments, the pH5.8 / pH7.4 KD ratio of the antibody of the present invention can be 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more. In another embodiment, the KD value of the antibody at pH 7.4 is 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 In another embodiment, the K value of the antibody at pH 5.8 is 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M, or even higher.

[0087] In certain examples, "reduced binding to C5 at acidic pH compared to binding to C5 at neutral pH" is expressed as the ratio of the kd value of the antibody that binds to C5 at acidic pH to the kd value of the antibody that binds to C5 at neutral pH (or vice versa). For example, if an antibody exhibits an acidic / neutral kd ratio of 2 or more, then for the purposes of the present invention, the antibody can be considered to exhibit "reduced binding to C5 at acidic pH compared to binding to C5 at neutral pH". In certain exemplary embodiments, the pH5.8 / pH7.4 kd ratio of the antibody of the present invention is 2 or more. In certain exemplary embodiments, the acidic / neutral kd ratio of the antibody of the present invention can be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more. In further exemplary embodiments, the pH 5.8 / pH 7.4 kd ratio of the antibodies of the invention can be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more. -2 1 / s, 10 -3 1 / s, 10 -4 1 / s, 10 -5 1 / s, 10 -6 1 / s, or less. In a further 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 In another embodiment, the kd value of the antibody at acidic pH is 10 -3 1 / s, 10 -2 1 / s, 10 -1 1 / s, or higher. In a further embodiment, the kd value of the antibody at pH 5.8 is 10 -3 1 / s, 10 -2 1 / s, 10 -1 It can be 1 / s, or even higher.

[0088] The expression "acidic pH" as used herein means a pH between 4.0 and 6.5. The expression "acidic pH" includes any one of the following pH values: 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 a particular aspect, the "acidic pH" is 5.8.

[0089] The expression "neutral pH" as used herein means a pH of 6.7 to about 10.0. The expression "neutral pH" includes any one of the following pH values: 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 a particular aspect, the "neutral pH" is 7.4.

[0090] The KD and kd values ​​expressed herein can be determined using a surface plasmon resonance-based biosensor to characterize antibody-antigen interactions. (See, for example, Example 3 herein.) The KD and kd values ​​can be measured at 25°C or 37°C.

[0091] In certain embodiments, the anti-C5 antibodies of the invention bind to an epitope within the beta chain of C5 consisting of the MG1 domain (SEQ ID NO: 41). In certain embodiments, the anti-C5 antibodies of the invention bind to an epitope within the beta chain of C5 (SEQ ID NO: 40) comprising at least one fragment selected from the group consisting of amino acids 47-57, 70-76, and 107-110. In certain embodiments, the anti-C5 antibodies of the invention bind to an epitope within a fragment of the beta chain of C5 (SEQ ID NO: 40) comprising 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. In certain embodiments, the anti-C5 antibody of the invention binds to an epitope within a fragment of the beta chain of C5 (SEQ ID NO:40) that comprises at least one amino acid selected from the group consisting of Glu48, Asp51, His70, His72, Lys109, and His110. In certain embodiments, the binding of the anti-C5 antibody of the invention to a C5 mutant is reduced compared to its binding to wild-type C5, wherein the C5 mutant has at least one amino acid substitution at a position selected from the group consisting of Glu48, Asp51, His72, and Lys109. In another embodiment, the pH-dependent binding of the anti-C5 antibody of the invention to a C5 mutant is reduced compared to its pH-dependent binding to wild-type C5, wherein the C5 mutant has at least one amino acid substitution at a position selected from the group consisting of His70, His72, and His110. In a further embodiment, in the C5 variant, an amino acid at a position selected from Glu48, Asp51, and Lys109 is substituted with an alanine, and an amino acid at a position selected from His70, His72, and His110 is substituted with a tyrosine.

[0092] In certain embodiments, the anti-C5 antibodies of the invention compete for binding to C5 with an antibody comprising a VH and VL pair selected from the following: (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) a VH of SEQ ID NO:5 and a VL of SEQ ID NO:15; (e) a VH of SEQ ID NO:4 and a VL of SEQ ID NO:14; (f) a VH of SEQ ID NO:6 and a VL of SEQ ID NO:16; (g) a VH of SEQ ID NO:2 and a VL of SEQ ID NO:12; (h) a VH of SEQ ID NO:3 and a VL of SEQ ID NO:13; (i) a VH of SEQ ID NO:9 and a VL of SEQ ID NO:19; (j) a VH of SEQ ID NO:7 and a VL of SEQ ID NO:17; (k) (l) a VH of SEQ ID NO:8 and a 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.

[0093] 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 additional embodiments, an anti-C5 antibody of the invention binds to C5 and contacts amino acid Lys109 (K109) of SEQ ID NO: 39. In further embodiments, an anti-C5 antibody of the invention binds to C5 and contacts amino acid Asp51 (D51) and amino acid Lys109 (K109) of SEQ ID NO: 39.

[0094] In a particular embodiment, the binding of an anti-C5 antibody of the invention to a C5 mutant is reduced compared to its binding to wild-type C5, wherein said C5 mutant has a Glu48Ala (E48A) substitution of SEQ ID NO: 39. In another embodiment, the pH-dependent binding of an anti-C5 antibody of the invention to a C5 mutant is reduced compared to its pH-dependent binding to wild-type C5, wherein said C5 mutant has a Glu48Ala (E48A) substitution of SEQ ID NO: 39.

[0095] In a further embodiment, the anti-C5 antibody binds to a C5 protein consisting of the amino acid sequence of SEQ ID NO:39 but does not bind to a C5 protein consisting of the amino acid sequence of SEQ ID NO:39 with an H72Y substitution, wherein the C5 protein and the H72Y substituted C5 protein are prepared and screened under the same conditions. In a further embodiment, the anti-C5 antibody binds to a C5 protein consisting of the amino acid sequence of SEQ ID NO:39 at pH 7.4 but does not bind to an H72Y substituted C5 protein at pH 7.4.

[0096] Without being bound to any particular theory, it can be speculated that the binding of an anti-C5 antibody to C5 is reduced (or almost lost) when a certain amino acid residue on C5 is replaced with another amino acid, which means that the amino acid residue on C5 is important for the interaction between the anti-C5 antibody and C5 and that the antibody can recognize an epitope around the amino acid residue on C5.

[0097] In the present invention, it has been found that a group of anti-C5 antibodies that compete with each other or bind to the same epitope can exhibit pH-dependent binding properties. Among amino acids, histidine, which has a pKa value of about 6.0 to 6.5, can 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 speculated that the anti-C5 antibody can recognize the three-dimensional structure around the histidine residue on C5, which can change depending on the pH. This speculation would be consistent with the experimental results described below: the pH dependence of anti-C5 antibodies is reduced (or almost lost) when the histidine residue on C5 is replaced by another amino acid (i.e., an anti-C5 antibody with pH-dependent binding properties binds to a histidine mutant of C5 with an affinity similar to wild-type C5 at neutral pH, but at acidic pH, the same antibody binds to a histidine mutant of C5 with a higher affinity than wild-type C5).

[0098] In certain embodiments, the anti-C5 antibody of the present invention binds to C5 from multiple species. In further embodiments, the anti-C5 antibody binds to C5 from humans and non-human animals. In further embodiments, the anti-C5 antibody binds to C5 from humans and monkeys (e.g., cynomolgus monkeys, rhesus monkeys, marmosets, chimpanzees, or baboons).

[0099] In one aspect, the present invention provides an anti-C5 antibody that inhibits the activation of C5. In certain embodiments, an anti-C5 antibody is provided that prevents the cleavage of C5 to form C5a and C5b, thereby preventing not only the generation of anaphylatoxin activity associated with C5a, but also the assembly of the C5b-9 membrane attack complex (MAC) associated with C5b. In certain embodiments, an anti-C5 antibody is provided that blocks the conversion of C5 to C5a and C5b by C5 convertase. In certain embodiments, an anti-C5 antibody is provided that blocks the access of C5 convertase to the cleavage site on C5. In certain embodiments, an anti-C5 antibody is provided that blocks the hemolytic activity caused by the activation of C5. In further embodiments, the anti-C5 antibody of the present invention inhibits the activation of C5 via the classical pathway and / or the alternative pathway.

[0100] In one aspect, the present invention provides an anti-C5 antibody that inhibits the activation of C5 mutants. C5 mutants refer to genetic variants of C5 resulting from genetic variations such as mutations, polymorphisms, or allelic variations. Genetic variations can include deletions, substitutions, or insertions of one or more nucleotides. C5 mutants can include one or more genetic variations of C5. In certain embodiments, C5 mutants have biological activity similar to wild-type C5. Such C5 mutants can include at least one mutation selected from the group consisting of V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N, and E1437D. For example, R885H herein refers to a genetic variation in which the arginine at position 885 is replaced with histidine. In certain embodiments, the anti-C5 antibodies of the present invention inhibit activation of both wild-type C5 and at least one C5 mutant selected from the group consisting of V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N, and E1437D.

[0101] In one aspect, the present invention provides an anti-C5 antibody comprising at least one, two, three, four, five, or six hypervariable regions (HVRs) selected from (a) HVR-H1 comprising any one of the amino acid sequences of SEQ ID NOs: 45 to 54; (b) HVR-H2 comprising any one of the amino acid sequences of SEQ ID NOs: 55 to 64; (c) HVR-H3 comprising any one of the amino acid sequences of SEQ ID NOs: 65 to 74; (d) HVR-L1 comprising any one of the amino acid sequences of SEQ ID NOs: 75 to 84; (e) HVR-L2 comprising any one of the amino acid sequences of SEQ ID NOs: 85 to 94; and (f) HVR-L3 comprising any one of the amino acid sequences of SEQ ID NOs: 95 to 104.

[0102] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising any one of the amino acid sequences of SEQ ID NOs: 45 to 54; (b) HVR-H2 comprising any one of the amino acid sequences of SEQ ID NOs: 55 to 64; and (c) HVR-H3 comprising any one of the amino acid sequences of SEQ ID NOs: 65 to 74. In one embodiment, the antibody comprises HVR-H3 comprising any one of the amino acid sequences of SEQ ID NOs: 65 to 74. In another embodiment, the antibody comprises HVR-H3 comprising any one of the amino acid sequences of SEQ ID NOs: 65 to 74 and HVR-L3 comprising any one of the amino acid sequences of SEQ ID NOs: 95 to 104. In a further embodiment, the antibody comprises HVR-H3 comprising any one of the amino acid sequences of SEQ ID NOs: 65 to 74, HVR-L3 comprising any one of the amino acid sequences of SEQ ID NOs: 95 to 104, and HVR-H2 comprising any one of the amino acid sequences of SEQ ID NOs: 55 to 64. In a further aspect, the antibody comprises (a) HVR-H1 having an amino acid sequence of any one of SEQ ID NOs: 45 to 54; (b) HVR-H2 having an amino acid sequence of any one of SEQ ID NOs: 55 to 64; and (c) HVR-H3 having an amino acid sequence of any one of SEQ ID NOs: 65 to 74.

[0103] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from (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) HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 95 to 104. In one embodiment, the antibody comprises (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) HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 95 to 104.

[0104] In another aspect, the antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (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) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 65 to 74; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (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 (c) HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 95 to 104.

[0105] In another aspect, the present invention provides an antibody comprising: (a) HVR-H1 comprising any one of the amino acid sequences of SEQ ID NOs: 45 to 54; (b) HVR-H2 comprising any one of the amino acid sequences of SEQ ID NOs: 55 to 64; (c) HVR-H3 comprising any one of the amino acid sequences of SEQ ID NOs: 65 to 74; (d) HVR-L1 comprising any one of the amino acid sequences of SEQ ID NOs: 75 to 84; (e) HVR-L2 comprising any one of the amino acid sequences of SEQ ID NOs: 85 to 94; and (f) HVR-L3 comprising any one of the amino acid sequences of SEQ ID NOs: 95 to 104.

[0106] In one aspect, the present invention provides an anti-C5 antibody comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising any one of the amino acid sequences of SEQ ID NOs: 45, 54, 117, 126; (b) HVR-H2 comprising any one of the amino acid sequences of SEQ ID NOs: 55, 64, 118-120, 127; (c) HVR-H3 comprising any one of the amino acid sequences of SEQ ID NOs: 65, 74, 121, 128; (d) HVR-L1 comprising any one of the amino acid sequences of SEQ ID NOs: 75, 84, 122, 129; (e) HVR-L2 comprising any one of the amino acid sequences of SEQ ID NOs: 85, 94, 123-124, 130; and (f) HVR-L3 comprising any one of the amino acid sequences of SEQ ID NOs: 95, 104, 125, 131.

[0107] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising any one of the amino acid sequences of SEQ ID NOs: 45, 54, 117, 126; (b) HVR-H2 comprising any one of the amino acid sequences of SEQ ID NOs: 55, 64, 118-120, 127; and (c) HVR-H3 comprising any one of the amino acid sequences of SEQ ID NOs: 65, 74, 121, 128. In one embodiment, the antibody comprises HVR-H3 comprising any one of the amino acid sequences of SEQ ID NOs: 65, 74, 121, 128. In another embodiment, the antibody comprises HVR-H3 comprising any one of the amino acid sequences of SEQ ID NOs: 65, 74, 121, 128 and HVR-L3 comprising any one of the amino acid sequences of SEQ ID NOs: 95, 104, 125, 131. In a further embodiment, the antibody comprises an HVR-H3 comprising any one of the amino acid sequences of SEQ ID NOs: 65, 74, 121, 128, an HVR-L3 comprising any one of the amino acid sequences of SEQ ID NOs: 95, 104, 125, 131, and an HVR-H2 comprising any one of the amino acid sequences of SEQ ID NOs: 55, 64, 118-120, 127. In a further embodiment, the antibody comprises (a) an HVR-H1 comprising any one of the amino acid sequences of SEQ ID NOs: 45, 54, 117, 126; (b) an HVR-H2 comprising any one of the amino acid sequences of SEQ ID NOs: 55, 64, 118-120, 127; and (c) an HVR-H3 comprising any one of the amino acid sequences of SEQ ID NOs: 65, 74, 121, 128.

[0108] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising any one of the amino acid sequences of SEQ ID NOs: 75, 84, 122, 129; (b) HVR-L2 comprising any one of the amino acid sequences of SEQ ID NOs: 85, 94, 123-124, 130; and (c) HVR-L3 comprising any one of the amino acid sequences of SEQ ID NOs: 95, 104, 125, 131. In one embodiment, the antibody comprises (a) HVR-L1 comprising any one of the amino acid sequences of SEQ ID NOs: 75, 84, 122, 129; (b) HVR-L2 comprising any one of the amino acid sequences of SEQ ID NOs: 85, 94, 123-124, 130; and (c) HVR-L3 comprising any one of the amino acid sequences of SEQ ID NOs: 95, 104, 125, 131.

[0109] In another aspect, the antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH domains selected from (i) HVR-H1 comprising any one of the amino acid sequences of SEQ ID NOs: 45, 54, 117, and 126; (ii) HVR-H2 comprising any one of the amino acid sequences of SEQ ID NOs: 55, 64, 118 to 120, and 127; and (iii) HVR-H3 comprising any one of the amino acid sequences of SEQ ID NOs: 65, 74, 121, and 128. (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising any one of the amino acid sequences of SEQ ID NOs: 75, 84, 122, 129, (ii) HVR-L2 comprising any one of the amino acid sequences of SEQ ID NOs: 85, 94, 123 to 124, 130, and (c) HVR-L3 comprising any one of the amino acid sequences of SEQ ID NOs: 95, 104, 125, 131.

[0110] In another aspect, the present invention provides an antibody comprising: (a) an HVR-H1 comprising any one of the amino acid sequences of SEQ ID NOs: 45, 54, 117, 126; (b) an HVR-H2 comprising any one of the amino acid sequences of SEQ ID NOs: 55, 64, 118-120, 127; (c) an HVR-H3 comprising any one of the amino acid sequences of SEQ ID NOs: 65, 74, 121, 128; (d) an HVR-L1 comprising any one of the amino acid sequences of SEQ ID NOs: 75, 84, 122, 129; (e) an HVR-L2 comprising any one of the amino acid sequences of SEQ ID NOs: 85, 94, 123-124, 130; and (f) an HVR-L3 comprising any one of the amino acid sequences of SEQ ID NOs: 95, 104, 125, 131.

[0111] In certain embodiments, any one or more amino acids of the above-mentioned anti-C5 antibodies are substituted at the following HVR positions: (a) in HVR-H1 (SEQ ID NO: 45), at positions 5 and 6; (b) in HVR-H2 (SEQ ID NO: 55), at positions 1, 3, 9, 11, 13, and 15; (c) in HVR-H3 (SEQ ID NO: 65), at positions 2, 5, 6, 12, and 13; (d) in HVR-L1 (SEQ ID NO: 75), at positions 1, 5, 7, and 9; (e) in HVR-L2 (SEQ ID NO: 85), at positions 4, 5, and 6; and (f) in HVR-L3 (SEQ ID NO: 95), at positions 2, 4, and 12.

[0112] In certain embodiments, the substitutions provided herein are conservative substitutions. In certain embodiments, any one or more of the following substitutions may be made in any combination: (a) M5V or C6A in HVR-H1 (SEQ ID NO: 45); (b) C1A or G, Y3F, T9D or E, Y11K or Q, S13D or E, or A15V in HVR-H2 (SEQ ID NO: 55); (c) G2A, V5Q or D, T6Y, Y12H, or L13Y in HVR-H3 (SEQ ID NO: 65); (d) Q1R, N5Q or G, G7S, D9K or S in HVR-L1 (SEQ ID NO: 75); (e) K4T or E, L5T, or A6H, A6E, or A6Q in HVR-L2 (SEQ ID NO: 85); (f) In HVR-L3 (SEQ ID NO:95), C2S, C2N, or C2T, F4K; or A12T or A12H.

[0113] All possible combinations of the above substitutions are encompassed in the consensus sequences 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.

[0114] In any of the above-mentioned embodiments, the anti-C5 antibody is humanized. In one embodiment, the anti-C5 antibody comprises the HVR of any of the above-mentioned embodiments, and further comprises an acceptor human framework (e.g., a human immunoglobulin framework or a human consensus framework). In another embodiment, the anti-C5 antibody comprises the HVR of any of the above-mentioned embodiments, and further comprises a VH or VL comprising a FR sequence as follows: For the heavy chain variable domain, FR1 comprises any one of the amino acid sequences of SEQ ID NOs: 132-134, FR2 comprises any one of the amino acid sequences of SEQ ID NOs: 135-136, FR3 comprises any one of the amino acid sequences of SEQ ID NOs: 137-139, and FR4 comprises any one of the amino acid sequences of SEQ ID NOs: 140-141. For the light chain variable domain, FR1 comprises any one of the amino acid sequences of SEQ ID NOs: 142 to 143, FR2 comprises any one of the amino acid sequences of SEQ ID NOs: 144 to 145, FR3 comprises any one of the amino acid sequences of SEQ ID NOs: 146 to 147, and FR4 comprises the amino acid sequence of SEQ ID NO: 148.

[0115] In another aspect, the anti-C5 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-10. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-C5 antibody comprising the sequence retains the ability to bind to C5. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in any one of SEQ ID NOs: 1-10. In certain embodiments, the substitutions, insertions, or deletions occur in the regions outside the HVR (i.e., in the FR). Optionally, the anti-C5 antibody comprises a VH sequence in any one of SEQ ID NOs: 1 to 10, including those containing post-translational modifications of said sequences. In certain embodiments, the VH comprises one, two, or three HVRs selected from (a) HVR-H1 comprising an amino acid sequence in any one of SEQ ID NOs: 45 to 54, (b) HVR-H2 comprising an amino acid sequence in any one of SEQ ID NOs: 55 to 64, and (c) HVR-H3 comprising an amino acid sequence in any one of SEQ ID NOs: 65 to 74. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0116] In another aspect, an anti-C5 antibody is provided, comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 11-20. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-C5 antibody comprising the sequence retains the ability to bind to C5. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in any one of SEQ ID NOs: 11-20. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the HVR (i.e., in the FR). Optionally, the anti-C5 antibody comprises a VL sequence in any one of SEQ ID NOs: 11 to 20, including those containing post-translational modifications of said sequences. In certain embodiments, the VL comprises one, two, or three HVRs selected from (a) HVR-L1 comprising an amino acid sequence in any one of SEQ ID NOs: 75 to 84, (b) HVR-L2 comprising an amino acid sequence in any one of SEQ ID NOs: 85 to 94, and (c) HVR-L3 comprising an amino acid sequence in any one of SEQ ID NOs: 95 to 104. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0117] In another aspect, an anti-C5 antibody is provided, comprising a VH of any of the above embodiments, and a VL of any of the above embodiments. In one embodiment, the antibody comprises a VH and VL sequence in any one of SEQ ID NOs: 1-10 and 11-20, respectively, including post-translational modifications of said sequences. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0118] In another aspect, the anti-C5 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 10, 106-110. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-C5 antibody comprising the sequence retains the ability to bind to C5. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in any one of SEQ ID NOs: 10, 106-110. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-C5 antibody comprises a VH sequence in any one of SEQ ID NOs: 10, 106-110, including post-translational modifications of said sequences. In certain embodiments, the VH comprises one, two, or three HVRs selected from (a) HVR-H1 comprising an amino acid sequence in any one of SEQ ID NOs: 45, 54, 117, 126, (b) HVR-H2 comprising an amino acid sequence in any one of SEQ ID NOs: 55, 64, 118-120, 127, and (c) HVR-H3 comprising an amino acid sequence in any one of SEQ ID NOs: 65, 74, 121, 128. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the heavy or light chain N-terminus to pyroglutamic acid by pyroglutamylation.

[0119] In another aspect, the anti-C5 antibody comprises a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 10, 106-110. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-C5 antibody comprising the sequence retains the ability to bind to C5. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in any one of SEQ ID NOs: 10, 106-110. In certain embodiments, the substitutions, insertions, or deletions occur in the regions outside the HVR (i.e., in the FR). Optionally, the anti-C5 antibody comprises a VH sequence in any one of SEQ ID NOs: 10, 106 to 110, including those containing post-translational modifications of said sequences. In certain embodiments, the VH comprises one, two, or three HVRs selected from (a) HVR-H1 comprising any one of the amino acid sequences of SEQ ID NOs: 45, 54, 117, 126, (b) HVR-H2 comprising any one of the amino acid sequences of SEQ ID NOs: 55, 64, 118 to 120, 127, and (c) HVR-H3 comprising any one of the amino acid sequences of SEQ ID NOs: 65, 74, 121, 128. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0120] In another aspect, the anti-C5 antibody comprises a VH sequence 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: 10. In certain embodiments, the VH sequence is the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-C5 antibody comprising the sequence retains the ability to bind to C5. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 10. In certain embodiments, the substitutions, insertions, or deletions occur in the regions outside the HVRs (i.e., in the FRs). Optionally, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 10, including post-translational modifications of said sequence. In certain embodiments, the VH comprises one, two, or three HVRs selected from (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-H3 comprising the amino acid sequence of SEQ ID NO: 74. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the heavy or light chain N-terminus to pyroglutamic acid by pyroglutamylation.

[0121] In another aspect, the anti-C5 antibody comprises a VH sequence 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: 106. In certain embodiments, the VH sequence is the amino acid sequence of SEQ ID NO: 106. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-C5 antibody comprising the sequence retains the ability to bind to C5. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 106. In certain embodiments, the substitutions, insertions, or deletions occur in the regions outside the HVRs (i.e., in the FRs). Optionally, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 106, including post-translational modifications of said sequence. In certain embodiments, the VH comprises one, two, or three HVRs selected from (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-H3 comprising the amino acid sequence of SEQ ID NO: 121. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the heavy or light chain N-terminus to pyroglutamic acid by pyroglutamylation.

[0122] In another aspect, the anti-C5 antibody comprises a VH sequence 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: 107. In certain embodiments, the VH sequence is the amino acid sequence of SEQ ID NO: 107. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-C5 antibody comprising the sequence retains the ability to bind to C5. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 107. In certain embodiments, the substitutions, insertions, or deletions occur in the regions outside the HVRs (i.e., in the FRs). Optionally, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 107, including post-translational modifications of said sequence. In certain embodiments, the VH comprises one, two, or three HVRs selected from (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-H3 comprising the amino acid sequence of SEQ ID NO: 121. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the heavy or light chain N-terminus to pyroglutamic acid by pyroglutamylation.

[0123] In another aspect, the anti-C5 antibody comprises a VH sequence 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: 108. In certain embodiments, the VH sequence is the amino acid sequence of SEQ ID NO: 108. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-C5 antibody comprising the sequence retains the ability to bind to C5. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 108. In certain embodiments, the substitutions, insertions, or deletions occur in the regions outside the HVRs (i.e., in the FRs). Optionally, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 108, including post-translational modifications of said sequence. In certain embodiments, the VH comprises one, two, or three HVRs selected from (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-H3 comprising the amino acid sequence of SEQ ID NO: 121. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the heavy or light chain N-terminus to pyroglutamic acid by pyroglutamylation.

[0124] In another aspect, the anti-C5 antibody comprises a VH sequence 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: 109. In certain embodiments, the VH sequence is the amino acid sequence of SEQ ID NO: 109. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-C5 antibody comprising the sequence retains the ability to bind to C5. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 109. In certain embodiments, the substitutions, insertions, or deletions occur in the regions outside the HVRs (i.e., in the FRs). Optionally, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 109, including post-translational modifications of said sequence. In certain embodiments, the VH comprises one, two, or three HVRs selected from (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-H3 comprising the amino acid sequence of SEQ ID NO: 121. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the heavy or light chain N-terminus to pyroglutamic acid by pyroglutamylation.

[0125] In another aspect, the anti-C5 antibody comprises a VH sequence 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: 110. In certain embodiments, the VH sequence is the amino acid sequence of SEQ ID NO: 110. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-C5 antibody comprising the sequence retains the ability to bind to C5. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 110. In certain embodiments, the substitutions, insertions, or deletions occur in the regions outside the HVRs (i.e., in the FRs). Optionally, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 110, including post-translational modifications of said sequence. In certain embodiments, the VH comprises one, two, or three HVRs selected from (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-H3 comprising the amino acid sequence of SEQ ID NO: 121. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the heavy or light chain N-terminus to pyroglutamic acid by pyroglutamylation.

[0126] In another aspect, an anti-C5 antibody is provided that comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 20, 111-113. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-C5 antibody comprising the sequence retains the ability to bind to C5. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in any one of SEQ ID NOs: 20, 111-113. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-C5 antibody comprises a VL sequence in any one of SEQ ID NOs: 20, 111-113, including post-translational modifications of said sequences. In certain embodiments, the VL comprises one, two, or three HVRs selected from (a) HVR-L1 comprising an amino acid sequence in any one of SEQ ID NOs: 75, 84, 122, 129, (b) HVR-L2 comprising an amino acid sequence in any one of SEQ ID NOs: 85, 94, 123-124, 130, and (c) HVR-L3 comprising an amino acid sequence in any one of SEQ ID NOs: 95, 104, 125, 131. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the heavy or light chain N-terminus to pyroglutamic acid by pyroglutamylation.

[0127] In another aspect, an anti-C5 antibody is provided, comprising a VL 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. In certain embodiments, the VL sequence is the amino acid sequence of SEQ ID NO:20. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-C5 antibody comprising the sequence retains the ability to bind to C5. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO:20. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., in the FR). Optionally, the anti-C5 antibody comprises the VL sequence in SEQ ID NO: 20, including post-translational modifications of said sequence. In certain embodiments, the VL comprises one, two, or three HVRs selected from (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. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the heavy or light chain N-terminus to pyroglutamic acid by pyroglutamylation.

[0128] In another aspect, an anti-C5 antibody is provided, comprising a VL 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. In certain embodiments, the VL sequence is the amino acid sequence of SEQ ID NO:111. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-C5 antibody comprising the sequence retains the ability to bind to C5. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO:111. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., in the FR). Optionally, the anti-C5 antibody comprises the VL sequence in SEQ ID NO: 111, including post-translational modifications of said sequence. In certain embodiments, the VL comprises one, two, or three HVRs selected from (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. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the heavy or light chain N-terminus to pyroglutamic acid by pyroglutamylation.

[0129] In another aspect, an anti-C5 antibody is provided, comprising a VL 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. In certain embodiments, the VL sequence is the amino acid sequence of SEQ ID NO: 112. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-C5 antibody comprising the sequence retains the ability to bind to C5. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 112. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., in the FR). Optionally, the anti-C5 antibody comprises the VL sequence in SEQ ID NO: 112, including post-translational modifications of said sequence. In certain embodiments, the VL comprises one, two, or three HVRs selected from (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. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the heavy or light chain N-terminus to pyroglutamic acid by pyroglutamylation.

[0130] In another aspect, an anti-C5 antibody is provided, comprising a VL 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. In certain embodiments, the VL sequence is the amino acid sequence of SEQ ID NO: 113. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-C5 antibody comprising the sequence retains the ability to bind to C5. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 113. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., in the FR). Optionally, the anti-C5 antibody comprises the VL sequence in SEQ ID NO: 113, including post-translational modifications of said sequence. In certain embodiments, the VL comprises one, two, or three HVRs selected from (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. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the heavy or light chain N-terminus to pyroglutamic acid by pyroglutamylation.

[0131] In another aspect, an anti-C5 antibody is provided, comprising a VH in any of the above-mentioned embodiments, and a VL in any of the above-mentioned embodiments. In one embodiment, the antibody comprises a VH and VL sequence in any one of SEQ ID NOs: 10, 106-110, and any one of SEQ ID NOs: 20, 111-113, including post-translational modifications of said sequences. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the heavy or light chain N-terminus to pyroglutamic acid by pyroglutamylation. In one embodiment, the antibody comprises a VH sequence of SEQ ID NO: 10 and a VL sequence of SEQ ID NO: 20. In one embodiment, the antibody comprises a VH sequence of SEQ ID NO: 106 and a VL sequence of SEQ ID NO: 111. In another embodiment, the antibody comprises a VH sequence of SEQ ID NO: 107 and a VL sequence of SEQ ID NO: 111. In a further embodiment, the antibody comprises a VH sequence of SEQ ID NO: 108 and a 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.

[0132] In one aspect, an anti-C5 antibody is provided comprising a VH sequence comprising (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:54, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:64, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:74, and a VL sequence comprising (a) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:84, (b) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:94, and (c) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:104.

[0133] In another aspect, an anti-C5 antibody is provided comprising a VH sequence containing (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:117, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:118, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:121, and a VL sequence containing (a) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:122, (b) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:123, and (c) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:125.

[0134] In another aspect, an anti-C5 antibody is provided comprising a VH sequence containing (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:117, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:119, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:121, and a VL sequence containing (a) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:122, (b) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:123, and (c) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:125.

[0135] In another aspect, an anti-C5 antibody is provided comprising a VH sequence containing (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:117, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:118, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:121, and a VL sequence containing (a) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:122, (b) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:124, and (c) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:125.

[0136] In another aspect, an anti-C5 antibody is provided comprising a VH sequence containing (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:117, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:120, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:121, and a VL sequence containing (a) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:122, (b) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:124, and (c) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:125.

[0137] In certain embodiments, an anti-C5 antibody of the invention comprises a VH of any of the above-mentioned embodiments and a heavy chain constant region comprising the amino acid sequence of any one of SEQ ID NOs: 33, 34, 35, 114, 115, and 116. In certain embodiments, an anti-C5 antibody of the invention comprises a VL of any of the above-mentioned embodiments and a light chain constant region comprising the amino acid sequence of any one of SEQ ID NOs: 36, 37, and 38.

[0138] In another aspect, the present invention provides an antibody that binds to the same epitope as the anti-C5 antibody provided herein. For example, in a specific embodiment, an antibody that binds to the same epitope as the antibody described in Table 2 is provided. As demonstrated by the examples below, all of the anti-C5 antibodies described in Table 2 are classified into the same epitope bin of C5 and exhibit pH-dependent binding characteristics.

[0139] In a further aspect, the present invention provides antibodies that bind to the same epitope as the antibodies provided herein. In a further aspect, the present invention provides antibodies that bind to the same epitope as the antibodies described in Tables 7 or 8. In a particular embodiment, an antibody is provided that binds to an epitope in a fragment consisting of amino acids 33-124 of the beta chain of C5 (SEQ ID NO: 40). In a particular embodiment, an antibody is provided that binds to an epitope in the beta chain of C5 (SEQ ID NO: 40) comprising at least one fragment selected from the group consisting of amino acids 47-57, 70-76, and 107-110. In certain embodiments, antibodies are provided that bind to an epitope in a fragment of the beta chain of C5 (SEQ ID NO: 40) that comprises 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. In another embodiment, the epitope of an anti-C5 antibody of the invention is a conformational epitope.

[0140] In a further aspect of the invention, the anti-C5 antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized, or human antibody. In one embodiment, the anti-C5 antibody is, for example, an Fv, Fab, Fab', scFv, diabody, or F(ab') 2 In another embodiment, the antibody is a full length antibody, e.g., a full IgG1 antibody or a full IgG4 antibody, or any other antibody class or isotype as defined herein.

[0141] In further aspects, an anti-C5 antibody according to any of the above embodiments may incorporate, either alone or in combination, any of the features described in items 1-7 below.

[0142] 1. Antibody affinity In certain embodiments, the antibodies provided herein have a concentration of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10-8 M or less, e.g. 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 It has a dissociation constant (Kd) of 1 M.

[0143] In one embodiment, Kd is measured by radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is determined by measuring the binding affinity of the Fab to the antigen at the lowest concentration ( 125 I) Fab is equilibrated with labeled antigen, and then bound antigen is captured by a plate coated with anti-Fab antibody. (See, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999)). To establish the measurement conditions, MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and then blocked with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM of [ 125I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., as in the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight, although the incubation can be continued for longer (e.g., about 65 hours) to ensure that equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed and the plate is washed 8 times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μl / well of scintillant (MICROSCINT-20™, Packard) is added and the plate is counted for 10 minutes in a TOPCOUNT™ gamma counter (Packard). The concentration of each Fab that gives 20% or less of maximum binding is selected for use in the competitive binding assay.

[0144] In another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, measurements using a BIACORE®-2000 or BIACORE®-3000 (BIACORE®, Inc., Piscataway, NJ) are performed at 25° C. using a CM5 chip with approximately 10 response units (RU) of antigen immobilized. In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE®, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8, before being injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of protein binding. After injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. with a flow rate of approximately 25 μl / min. The binding rate (k on ) and dissociation rate (k off ) is calculated by simultaneously fitting the association and dissociation sensorgrams with a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2). The equilibrium dissociation constant (Kd) is k off / k on The on-rate is calculated as a ratio of 10 to 10 by the surface plasmon resonance assay described above. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). 6 M -1 s -1If the on-rate exceeds , the on-rate can be determined by using a fluorescence quenching technique to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, band pass 16 nm) of 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2 at 25°C in the presence of increasing concentrations of antigen as measured in a spectrometer (e.g., a stopped-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) using a stirred cuvette).

[0145] 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, as well as other fragments described below. For a review of certain antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp.269-315 (1994); in addition, WO93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. Fab and F(ab') fragments that contain salvage receptor binding epitope residues and have extended in vivo half-lives are also known. 2 For a discussion of fragments, see US Pat. No. 5,869,046.

[0146] Diabodies are antibody fragments with two antigen-binding sites, which may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); 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).

[0147] A single domain antibody is an antibody fragment that contains all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

[0148] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of whole antibodies and production by recombinant host cells (e.g., E. coli or phages), as described herein.

[0149] 3. Chimeric and humanized antibodies In certain embodiments, the antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate, such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In a further example, a chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from that of the parent antibody. Chimeric antibodies also include antigen-binding fragments thereof.

[0150] In certain embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity in humans while maintaining the specificity and affinity of the parent non-human antibody. Usually, a humanized antibody comprises one or more variable domains, in which the HVRs (e.g., CDRs (or portions thereof)) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. The humanized antibody optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0151] Humanized antibodies and methods for making them are reviewed in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008) and also see, e.g., Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing resurfacing); Dall'Acqua et al., Methods 36:43-60 (2005) (describing FR shuffling); and further described in Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer 83:252-260 (2000) (describing a "guide selection" approach for FR shuffling).

[0152] Human framework regions that may be used for humanization include, but are not limited to: framework regions selected using the "best-fit" method (see Sims et al., J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of a particular subgroup of light or heavy chain variable regions (see Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992) and Presta et al., J. Immunol. 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (Baca et al., J. Biol. Chem. 272:10678-10684 (2008)). (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0153] 4. Human antibodies In certain embodiments, the antibody provided herein is a human antibody. Human antibodies can be produced by various techniques known in the art. Human antibodies are reviewed in van Dijk and van de Winkel, Curr. Opin. Pharma. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0154] Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been engineered to produce fully human antibodies or complete antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or a portion of a human immunoglobulin locus, which either replaces an endogenous immunoglobulin locus or is present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin locus is usually 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, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HUMAB® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology; and U.S. Patent Application Publication No. 2007 / 0061900, which describes VELOCIMOUSE® technology. The human variable regions from complete antibodies generated by such animals may be further modified, for example, by combining with different human constant regions.

[0155] 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, e.g., 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 generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue 26(4):265-268 (2006) (describing human-human hybridomas). 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).

[0156] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0157] 5. Library-derived Antibodies Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics. Such methods are reviewed in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described in, e.g., 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).

[0158] In a particular phage display method, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol. 12:433-455 (1994). Phages typically display 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, naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies to a wide range of non-self and self antigens without immunization, as described in Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can be made synthetically by cloning pre-rearranged V-gene segments from stem cells and using PCR primers that encode the hypervariable CDR3 regions and contain random sequences to achieve rearrangement in vitro, as described in Hoogenboom, J. Mol. Biol. 227:381-388 (1992). Patent literature describing human antibody phage libraries includes, for example: U.S. Patent No. 5,750,373, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0159] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0160] 6. Multispecific antibodies In certain embodiments, the antibody provided herein is a multispecific antibody (e.g., a bispecific antibody). A multispecific antibody is a monoclonal antibody that has binding specificity at at least two different sites. In certain embodiments, one of the binding specificities is for C5 and the other is for any other antigen. In certain embodiments, a bispecific antibody may bind to two different epitopes of C5. A bispecific antibody may be used to localize cytotoxic agents to cells expressing C5. A bispecific antibody may be prepared as a full-length antibody or as an antibody fragment.

[0161] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983), WO93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)), and the "knob-in-hole" technology (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can be made by engineering electrostatic steering effects to create Fc heterodimeric molecules (WO2009 / 089004A1); cross-linking two or more antibodies or fragments (see U.S. Pat. No. 4,676,980 and Brennan et al., Science 229:81 (1985)); using leucine zippers to generate antibodies with two specificities (see Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)); using "diabody" technology to create bispecific antibody fragments (see Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (Gruber et al., J. Immunol. 152:5368 (1994)). see above); and by preparing trispecific antibodies, for example, as described in Tutt et al., J. Immunol. 147:60 (1991).

[0162] Engineered antibodies with three or more functional antigen binding sites are also included herein, including "octopus antibodies" (see, eg, US Patent Application Publication No. 2006 / 0025576).

[0163] The antibody or fragment herein also includes a "dual-acting Fab" or "DAF" that contains one antigen-binding site that binds to C5 and another distinct antigen (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820).

[0164] 7. Antibody Variants In certain embodiments, amino acid sequence variants of the antibodies provided herein are also 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 the antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct, provided that the final construct possesses the desired characteristics (e.g., antigen binding).

[0165] Substitution, insertion, and deletion mutants In certain embodiments, antibody variants are provided that have one or more amino acid substitutions. Target sites for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions". More substantial changes are provided in Table 1 under the heading of "exemplary substitutions" and are detailed below with reference to classes of amino acid side chains. Amino acid substitutions may be introduced into the antibody of interest, and the products may be screened for desired activity, such as, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0166] [Table 1]

[0167] Amino acids can be divided into groups according to common side chain properties: (1) Hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile); (2) neutral hydrophilic: cysteine ​​(Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln); (3) Acidic: aspartic acid (Asp), glutamic acid (Glu); (4) Basic: histidine (His), lysine (Lys), arginine (Arg); (5) residues that affect chain orientation: glycine (Gly), proline (Pro); and (6) Aromatic: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe).

[0168] Non-conservative substitutions refer to the exchange of a member of one of these classes for one from another class.

[0169] One type of substitutional variant involves the substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Usually, the resulting variant selected for further study will have a modification (e.g., an improvement) in a particular biological property compared to the parent antibody (e.g., increased affinity, decreased immunogenicity) and / or will substantially retain a particular biological property of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, for example, using phage display-based affinity maturation techniques (e.g., those described herein). Briefly, one or more HVR residues are mutated, and the mutated antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0170] Modifications (e.g., substitutions) can be made in HVRs, for example, to improve the affinity of the antibody. Such modifications can be made in "hot spots" of HVRs, i.e., residues encoded by codons where mutations occur frequently during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or residues that contact antigens, and the resulting mutated VH or VL can be tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al., in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. This library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves an HVR-directed approach, where several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0171] In certain embodiments, substitution, insertion, or deletion may be made within one or more HVRs, as long as such modification does not substantially reduce the ability of the antibody to bind to antigen.For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs.Such modifications may, for example, be outside the antigen contact residues of HVRs.In certain embodiments of the above mutant VH and VL sequences, each HVR is unmodified or contains only one, two, or three amino acid substitutions.

[0172] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is that described by Cunningham, Science 244:1081-1085 (1989), called "alanine scanning mutagenesis". In this method, a residue or group of target residues (e.g., charged residues such as arginine, aspartic acid, histidine, lysine, and glutamic acid) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and it is determined whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to this initial substitution. Alternatively or additionally, a crystal structure of the antigen-antibody complex can be analyzed to identify contact points between the antibody and the antigen. Such contact residues and neighboring residues can be targeted as replacement candidates or excluded from replacement candidates. Mutants can be screened to determine whether they contain the desired properties.

[0173] Amino acid sequence insertions include fusions ranging in length from one residue to polypeptides containing 100 or more residues at the amino and / or carboxyl termini, as well as single or multiple amino acid residue insertions within the sequence. An example of a terminal insertion includes an antibody with a methionyl residue at the N-terminus. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody of an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0174] B glycosylation mutants In certain embodiments, the antibodies provided herein have been modified to increase or decrease the extent to which the antibody is glycosylated. Adding or deleting glycosylation sites to an antibody can be conveniently accomplished by modifying the amino acid sequence to create or remove one or more glycosylation sites.

[0175] If the antibody comprises an Fc region, the carbohydrate attached thereto may be modified. Natural antibodies produced by mammalian cells typically comprise branched, biantennary oligosaccharides, which are usually attached by N-linkage to Asn297 in the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides include, for example, various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the invention may be made to generate antibody variants with specific improved properties.

[0176] In one embodiment, antibody variants are provided that have carbohydrate structures that lack fucose added (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1%-80%, 1%-65%, 5%-65% or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycostructures (e.g., complex, hybrid, and high mannose structures) added to Asn297, as measured by MALDI-TOF mass spectrometry, e.g., as described in WO2008 / 077546. Asn297 represents an asparagine residue located at about position 297 of the Fc region (EU numbering of Fc region residues). However, due to slight sequence variability between multiple antibodies, Asn297 can also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants can have improved ADCC function. See, for example, U.S. Patent Application Publication Nos. 2003 / 0157108 (Presta, L.); 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108; WO2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO2003 / 085119; WO2003 / 084570; WO2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which lack protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US2003 / 0157108, Presta, L; and WO2004 / 056312, Adams et al., especially Example 11) and knockout cell lines, such as alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda et al., Biotechnol. Bioeng. 94(4):680-688 (2006); and WO2003 / 085107).

[0177] Further provided are antibody variants having bisected oligosaccharides, for example, bisected oligosaccharides added to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US2005 / 0123546 (Umana et al.). Also provided are antibody variants having at least one galactose residue in the oligosaccharide added to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087 (Patel et al.); WO1998 / 58964 (Raju, S.); and WO1999 / 22764 (Raju, S.).

[0178] c. Fc region mutants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0179] In certain embodiments, antibody variants with some, but not all, effector functions are also contemplated by the present invention, which make the antibody a desirable candidate for applications where its in vivo half-life is important, but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / lack of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) while retaining FcRn binding. NK cells, the primary cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see, Bruggemann et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be used (see, e.g., ACT1™ non-radioactive cytotoxicity assays for flow cytometry (CellTechnology, Inc. Mountain View, Calif.); and CytoTox 96® non-radioactive cytotoxicity assays (Promega, Madison, Wis.)).Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that described in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be performed to confirm that the antibody is unable to bind C1q and thus lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. CDC measurements may also be performed to assess complement activation (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg et al., Blood 101:1045-1052 (2003); and Cragg et al., Blood 103:2738-2743 (2004)). In addition, FcRn binding and in vivo clearance / half-life determinations may also be performed using methods known in the art (see, e.g., Petkova et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0180] Antibodies with reduced effector function include those with one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0181] Certain antibody variants with improved or diminished binding to FcRs have been described (see U.S. Pat. No. 6,737,056; WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0182] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC (e.g., substitutions at positions 298, 333, and / or 334 (residues in EU numbering) of the Fc region).

[0183] In some embodiments, modifications are made in the Fc region that result in altered (i.e., either improved or attenuated) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO1999 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0184] Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn, responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) have been described in U.S. Patent Application Publication No. 2005 / 0014934 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (e.g., substitution at Fc region residue 434 (U.S. Patent No. 7,371,826)).

[0185] For other examples of Fc region variants, see also Duncan, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO1994 / 29351.

[0186] d. Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to create cysteine ​​engineered antibodies (e.g., "thioMAbs") in which one or more residues of an antibody are substituted with a cysteine ​​residue. In certain embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are placed at accessible sites of the antibody, which may be used to conjugate the antibody to other moieties (such as drug moieties or linker-drug moieties) to create immunoconjugates as further detailed herein. In certain embodiments, any one or more of the following residues may be substituted with a cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine ​​engineered antibodies may be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0187] e. Antibody derivative In certain embodiments, the antibodies provided herein may be further modified to include additional non-protein moieties that are 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, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly 1,3 dioxolane, poly 1,3,6, trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody can vary, and if more than one polymer is attached, they can be the same molecule or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations such as, but 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.

[0188] In another embodiment, a conjugate is provided between an antibody and a non-protein moiety that can be selectively heated by exposure to radiation. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation can be of any wavelength, including but not limited to wavelengths that heat the non-protein moiety to a temperature that is not harmful to normal cells but that kills cells in close proximity to the antibody-non-protein moiety.

[0189] B. Recombinant Methods and Constructs Antibodies can be produced using recombinant methods and constructs, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid is provided that encodes an anti-C5 antibody described herein. Such a nucleic acid may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In a further embodiment, a host cell comprising such a nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., is transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, 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 eukaryotic (e.g., a Chinese hamster ovary (CHO) cell) or a lymphoid cell (e.g., a Y0, NS0, SP20 cell)). In one aspect, a method of producing an anti-C5 antibody is provided, comprising culturing a host cell comprising nucleic acid encoding the antibody as described above under conditions suitable for expression of the anti-C5 antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0190] For recombinant production of an anti-C5 antibody, nucleic acid encoding the antibody (such as, for example, those described above) is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes capable of specifically binding to genes encoding the antibody heavy and light chains).

[0191] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody may be isolated in a soluble fraction from the bacterial cell paste, and may be further purified.

[0192] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of antibodies with partial or fully human glycosylation patterns, are suitable cloning or expression hosts for antibody-encoding vectors. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0193] Host cells derived from multicellular organisms (invertebrate and vertebrate) are also suitable for the expression of glycosylated antibodies. Examples of invertebrate cells include plants and insect cells. Numerous baculovirus strains have been identified for conjugation with insect cells, particularly for transformation of Spodoptera frugiperda cells.

[0194] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing the PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0195] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that have been adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (293 or 293 cells, e.g., as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, e.g., as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary carcinoma (MMT 060562); TRI cells (e.g., as described in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)). Other useful mammalian host cell lines include DHFR cells, -Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0196] Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. The relevant antigen is conjugated to a protein that is immunogenic in the species being immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, with a bifunctional substance or derivatizing agent, such as maleimidobenzoyl sulfosuccinimide ester (conjugation via cysteine ​​residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCl. 2 , or R 1 N=C=NR, where R and R 1 are different alkyl groups).

[0197] An animal (usually a non-human mammal) is immunized against the antigen, immunogenic conjugate, or derivative, for example, by combining 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animal is boosted with 1 / 5 to 1 / 10 of the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. After 7 to 14 days, the animal is bled and the serum is assayed for antibody titer. The animal is boosted until the titer reaches a plateau. The animal is preferably boosted with the same antigen but conjugated to a different protein and / or via a different cross-linking reagent. Conjugates can also be prepared as protein fusions in recombinant cell culture. Agglutinating agents such as alum are also suitably used to enhance the immune response.

[0198] Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for potential naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of separate antibodies.

[0199] For example, monoclonal antibodies can be produced using the hybridoma method first 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 herein above to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro.

[0200] The immunizing agent typically includes an antigen protein or a fusion variant thereof. In general, peripheral blood lymphocytes (PBLs) are used if cells of human origin are desired, and spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103).

[0201] Immortalized cell lines are usually transformed mammalian cells, especially myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are used. The hybridoma cells thus produced are seeded and grown in a suitable culture medium, preferably 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 phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), substances that prevent the growth of HGPRT-deficient cells.

[0202] Preferred immortalized myeloma cells are those that fuse efficiently, support stable high-level production of antibody by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred are mouse myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California, USA, and SP-2 cells (and their derivatives, such as X63-Ag8-653) available from the American Type Culture Collection, Manassas, Virginia, USA. For the production of human monoclonal antibodies, human myeloma cell lines and mouse-human heteromyeloma cell lines have also been described (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).

[0203] The culture medium in which the hybridoma cells are growing is assayed for the production of monoclonal antibodies against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. For example, binding affinity can be determined by the Scatchard analysis of Munson, Anal Biochem. 107(1):220-239 (1980).

[0204] After the hybridoma cells that produce the antibody of the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. Hybridoma cells can also be grown in vivo as tumors in mammals.

[0205] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0206] Antibodies may be produced by immunizing a suitable host animal against the 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 amino acids at positions 19-180 of the beta chain of C5. In one embodiment, the antigen is a polypeptide comprising a region corresponding to amino acids at positions 33-124 of the beta chain of C5. In one embodiment, the antigen is a polypeptide comprising at least one fragment 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 a polypeptide comprising a fragment of the beta chain of C5, comprising 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. In one embodiment, the antigen is a polypeptide comprising a fragment of the beta chain of C5, comprising at least one amino acid selected from the group consisting of Glu48, Asp51, His70, His72, Lys109, and His110. Antibodies produced by immunizing animals against the antigen are also included in the present invention. The antibody can incorporate any of the features described above in "Exemplary anti-C5 antibodies," either alone or in combination.

[0207] C. Assays The anti-C5 antibodies provided herein may be identified, screened, or characterized for their physical / chemical properties and / or biological activity by a variety of assays known in the art.

[0208] 1. Binding and other assays In one aspect, the antibodies of the invention are tested for their antigen binding activity by known methods, such as ELISA, Western blot, BIACORE®, and the like.

[0209] In another aspect, a competitive assay can be used to identify antibodies that compete with the anti-C5 antibodies described herein for binding to C5. In certain embodiments, when such a competing antibody is present in excess, it blocks (e.g., reduces) the 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. In some embodiments, binding is inhibited by at least 80%, 85%, 90%, 95%, or more. In certain embodiments, such a competing antibody binds to the same epitope (e.g., linear or conformational epitope) as that bound by the anti-C5 antibodies described herein (e.g., the anti-C5 antibodies described in Table 2). Detailed exemplary methods for mapping epitopes to which antibodies bind are provided in Morris, "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ) (1996).

[0210] In an exemplary competitive assay, immobilized C5 is incubated in a solution containing a first labeled (reference) antibody that binds to C5 and a second unlabeled antibody that is tested for its ability to compete with the first antibody for binding to C5. The second antibody can be present in a 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 label bound to 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. See Harlow and Lane, Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) (1988).

[0211] In another exemplary competitive assay, BIACORE® analysis 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 a further aspect, a BIACORE® instrument (e.g., BIACORE® 3000) is operated according to the manufacturer's recommendations, C5 protein is captured on a CM5 BIACORE® chip using standard techniques known in the art to create a C5-coated surface. Typically, 200-800 resonance units of C5 are coupled to the chip (an amount that results in a readily measurable level of binding, but is readily saturable with the concentration of test antibody used). The two antibodies (i.e., test antibody and reference antibody) to be evaluated for their ability to compete with each other are mixed in a suitable buffer in a 1:1 molar ratio of binding sites to generate a test mixture. When calculating the concentration based on binding sites, the molecular weight of the test antibody or reference antibody is the total molecular weight of the corresponding antibody divided by the number of C5 binding sites on that antibody. The concentration of each antibody (i.e., test antibody and reference antibody) in the test mixture should be high enough to easily saturate the binding sites of that antibody on the C5 molecules captured on the BIACORE® chip. The test and reference antibodies in the mixture are at the same molar concentration (based on binding), typically 1.00-1.5 micromolar (based on binding sites). Separate solutions containing only the test antibody and only the reference antibody are also prepared. The test and reference antibodies in these solutions should be in the same buffer as the test mixture and at the same concentration and conditions. The test mixture containing the test and reference antibodies is passed over a BIACORE® chip coated with C5, and the total amount of binding is recorded. The chip is then treated to remove any bound test or reference antibody without damaging the C5 bound to the chip. Typically, this is done by treating the chip with 30 mM HCl for 60 seconds. The solution of only the test antibody is then passed over the C5-coated surface, and the amount of binding is recorded. The chip is again treated to remove all of the bound antibody without damaging the chip-bound C5, and then a solution of reference antibody alone is passed over the C5-coated surface and the amount of binding recorded.The theoretical maximum binding of the mixture of test and reference antibodies is then calculated, which is the sum of the binding of each antibody (i.e., test and reference) when passed alone over the C5 surface. If the actual recorded binding of the mixture is less than this theoretical maximum, then the test and reference antibodies are competing with each other for binding to C5. Thus, in general, a competing test anti-C5 antibody is one that binds to C5 in the BIACORE® blocking assay described above such that, in the presence of the reference anti-C5 antibody, the recorded binding during the assay is between 80% and 0.1% (e.g., 80%>-4%) of the theoretical maximum binding (as defined above) of the combined test and reference antibodies, particularly between 75% and 0.1% (e.g., 75%-4%) of the theoretical maximum binding, more particularly between 70% and 0.1% (e.g., 70%-4%) of the theoretical maximum binding.

[0212] In certain embodiments, the anti-C5 antibody of the present 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 for binding to C5 with an antibody selected from CFA0538, CFA0501, CFA0599, CFA0307, ​​CFA0366, CFA0675, and CFA0672. In some embodiments, the anti-C5 antibody competes for binding to C5 with antibody CFA0329. In some embodiments, the anti-C5 antibody competes for binding to C5 with antibody CFA0666.

[0213] In certain embodiments, an anti-C5 antibody of the invention competes for binding to C5 with an antibody comprising the VH and VL pair of antibody CFA0305 or 305LO5.

[0214] In further embodiments, the anti-C5 antibody binds to C5 with higher affinity at neutral pH than at acidic pH. In certain embodiments, the anti-C5 antibody of the present invention competes with an antibody comprising a VH and VL pair selected from CFA0538, CFA0501, CFA0599, CFA0307, ​​CFA0366, CFA0675, and CFA0672 for binding to C5. In some embodiments, the anti-C5 antibody competes with antibody CFA0666 for binding to C5. In further embodiments, the anti-C5 antibody binds to C5 with higher affinity at pH 7.4 than at pH 5.8.

[0215] In further embodiments, the anti-C5 antibody binds to C5 with higher affinity at neutral pH than at acidic pH. In certain embodiments, the anti-C5 antibody of the present invention competes with an antibody comprising the VH and VL pair of antibody CFA0305 or 305LO5 for binding to C5. In further embodiments, the anti-C5 antibody binds to C5 with higher affinity at pH 7.4 than at pH 5.8.

[0216] In certain embodiments, the anti-C5 antibody of the present invention competes for binding to C5 with an antibody comprising a VH and VL pair selected from a VH of SEQ ID NO:22 and a VL of SEQ ID NO:26, or a VH of SEQ ID NO:21 and a VL of SEQ ID NO:25. In some embodiments, the anti-C5 antibody competes for binding to C5 with an antibody comprising a VH and VL pair selected from the following: (a) a VH of SEQ ID NO:5 and a VL of SEQ ID NO:15; (b) a VH of SEQ ID NO:4 and a VL of SEQ ID NO:14; (c) a VH of SEQ ID NO:6 and a VL of SEQ ID NO:16; (d) a VH of SEQ ID NO:2 and a VL of SEQ ID NO:12; (e) a VH of SEQ ID NO:3 and a VL of SEQ ID NO:13; (f) a VH of SEQ ID NO:1 and a VL of SEQ ID NO:11; (g) a VH of SEQ ID NO:9 and a VL of SEQ ID NO:19; (h) a VH of SEQ ID NO:7 and a VL of SEQ ID NO:17; and (i) a VH of SEQ ID NO:8 and a VL of SEQ ID NO:18. In some embodiments, the anti-C5 antibody competes for binding to C5 with an antibody comprising a VH of SEQ ID NO:23 and a VL of SEQ ID NO:27. In some embodiments, the anti-C5 antibody competes for binding to C5 with an antibody comprising a VH of SEQ ID NO:7 and a VL of SEQ ID NO:17.

[0217] In certain embodiments, the anti-C5 antibodies of the invention compete for binding to C5 with an antibody comprising a VH and VL pair selected from the following: (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) a VH of SEQ ID NO:5 and a VL of SEQ ID NO:15; (e) a VH of SEQ ID NO:4 and a VL of SEQ ID NO:14; (f) a VH of SEQ ID NO:6 and a VL of SEQ ID NO:16; (g) a VH of SEQ ID NO:2 and a VL of SEQ ID NO:12; (h) a VH of SEQ ID NO:3 and a VL of SEQ ID NO:13; (i) a VH of SEQ ID NO:9 and a VL of SEQ ID NO:19; (j) a VH of SEQ ID NO:7 and a VL of SEQ ID NO:17; (k) (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.

[0218] In certain embodiments, the anti-C5 antibodies of the invention compete for binding to C5 with an antibody comprising a VH and VL pair selected from the following: (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) a VH of SEQ ID NO:5 and a VL of SEQ ID NO:15; (d) a VH of SEQ ID NO:4 and a VL of SEQ ID NO:14; (e) a VH of SEQ ID NO:6 and a VL of SEQ ID NO:16; (f) a VH of SEQ ID NO:2 and a VL of SEQ ID NO:12; (g) a VH of SEQ ID NO:3 and a VL of SEQ ID NO:13; (h) a VH of SEQ ID NO:9 and a VL of SEQ ID NO:19; (i) a VH of SEQ ID NO:7 and a VL of SEQ ID NO:17; (j) a VH of SEQ ID NO:8 and a VL of SEQ ID NO:18; (k) a VH of SEQ ID NO:23 and a VL of SEQ ID NO:27.

[0219] In certain embodiments, the anti-C5 antibody of the present invention competes for binding to C5 with an antibody comprising a VH and VL pair selected from a VH of SEQ ID NO:1 and a VL of SEQ ID NO:11, or a VH of SEQ ID NO:10 and a VL of SEQ ID NO:20.

[0220] In a further embodiment, the anti-C5 antibody binds to C5 with greater affinity at neutral pH than at acidic pH. In certain embodiments, the anti-C5 antibody binds to C5 with greater affinity at neutral pH than at acidic pH and competes for binding to C5 with an antibody comprising a VH and VL pair selected from the following: (a) a VH of SEQ ID NO:1 and a VL of SEQ ID NO:11; (b) a VH of SEQ ID NO:5 and a VL of SEQ ID NO:15; (c) a VH of SEQ ID NO:4 and a VL of SEQ ID NO:14; (d) a VH of SEQ ID NO:6 and a VL of SEQ ID NO:16; (e) a VH of SEQ ID NO:2 and a VL of SEQ ID NO:12; (f) a VH of SEQ ID NO:3 and a VL of SEQ ID NO:13; (g) a VH of SEQ ID NO:9 and a VL of SEQ ID NO:19; (h) a VH of SEQ ID NO:7 and a VL of SEQ ID NO:17; (i) a VH of SEQ ID NO:8 and a VL of SEQ ID NO:18; and (j) a VH of SEQ ID NO:10 and a VL of SEQ ID NO:20. In a further embodiment, the anti-C5 antibody binds to C5 with greater affinity at pH 7.4 than at pH 5.8.

[0221] In some embodiments, the anti-C5 antibody binds to C5 with greater affinity at neutral pH than at acidic pH and competes for binding to C5 with an antibody comprising a VH and VL pair selected from the following: (a) a VH of SEQ ID NO:5 and a VL of SEQ ID NO:15; (b) a VH of SEQ ID NO:4 and a VL of SEQ ID NO:14; (c) a VH of SEQ ID NO:6 and a VL of SEQ ID NO:16; (d) a VH of SEQ ID NO:2 and a VL of SEQ ID NO:12; (e) a VH of SEQ ID NO:3 and a VL of SEQ ID NO:13; (f) a VH of SEQ ID NO:1 and a VL of SEQ ID NO:11; (g) a VH of SEQ ID NO:9 and a VL of SEQ ID NO:19; (h) a VH of SEQ ID NO:7 and a VL of SEQ ID NO:17; and (i) a VH of SEQ ID NO:8 and a VL of SEQ ID NO:18. In a further embodiment, the anti-C5 antibody binds to C5 with greater affinity at pH 7.4 than at pH 5.8.

[0222] In some embodiments, the anti-C5 antibody binds C5 with higher affinity at neutral pH than at acidic pH and competes for binding to C5 with an antibody comprising a VH and VL pair selected from the following: a VH of SEQ ID NO: 1 and a VL of SEQ ID NO: 11, or a VH of SEQ ID NO: 10 and a VL of SEQ ID NO: 20. In further embodiments, the anti-C5 antibody binds C5 with higher affinity at pH 7.4 than at pH 5.8.

[0223] In certain embodiments, whether an anti-C5 antibody of the present invention binds to a particular epitope can be determined as follows: a C5 point mutant in which amino acids of C5 (except alanine) are replaced with alanine is expressed in 293 cells, and the binding of an anti-C5 antibody to the C5 mutant is tested by ELISA, Western blot, or BIACORE®; where a substantial reduction or abolishment of binding of the anti-C5 antibody to the C5 mutant compared to binding of the anti-C5 antibody to wild-type C5 indicates that the anti-C5 antibody binds to an epitope that includes the amino acid on C5. In certain embodiments, the amino acid on C5 that is replaced with alanine is selected from the group consisting of Glu48, Asp51, His70, His72, Lys109, and His110 of the β-strand of C5 (SEQ ID NO:40). In further embodiments, the amino acid on C5 that is replaced with alanine is Asp51 or Lys109 of the β-strand of C5 (SEQ ID NO:40).

[0224] In another embodiment, whether an anti-C5 antibody with pH-dependent binding properties binds to a particular epitope can be determined as follows: a C5 point mutant in which a histidine residue on C5 is replaced with another amino acid (e.g., tyrosine) is expressed in 293 cells, and the binding of an anti-C5 antibody to the C5 mutant is tested by ELISA, Western blot, or BIACORE®; where a substantial reduction in the binding of the anti-C5 antibody to wild-type C5 at acidic pH compared to the binding of the anti-C5 antibody to the C5 mutant at acidic pH indicates that the anti-C5 antibody binds to an epitope that includes the histidine residue on C5. In a further embodiment, the binding of the anti-C5 antibody to wild-type C5 at neutral pH is not substantially reduced compared to the binding of the anti-C5 antibody to the C5 mutant at neutral pH. In a particular embodiment, the histidine residue on C5 that is replaced with another amino acid is selected from the group consisting of His70, His72, and His110 of the β-chain of C5 (SEQ ID NO:40). In a further embodiment, the histidine residue His70 is substituted with tyrosine.

[0225] 2.Activity measurement method In one aspect, an assay method is provided for identifying an anti-C5 antibody that has biological activity. Biological activity may include, for example, inhibiting the activation of C5, preventing the cleavage of C5 to form C5a and C5b, blocking the access of C5 convertase to the cleavage site on C5, blocking the hemolytic activity caused by the activation of C5, etc. Also provided are antibodies that have such biological activity in vivo and / or in vitro.

[0226] In certain embodiments, the antibodies of the invention are tested for such biological activities.

[0227] In certain embodiments, whether the test antibody inhibits the cleavage of C5 into C5a and C5b is determined, for example, by the method described in Isenman et al., J Immunol. 124(1):326-331 (1980). In another embodiment, this is determined by a method for specifically detecting cleaved C5a and / or C5b protein, for example, ELISA or Western blot. If a decrease in the amount of cleavage products of C5 (i.e., C5a and / or C5b) is detected in the presence of (or after contact with) the test antibody, the test antibody is identified as an antibody that can inhibit the cleavage of C5. In certain embodiments, the concentration and / or physiological activity of C5a can be measured, for example, by a method such as chemotaxis assay, RIA, or ELISA (see, for example, Ward and Zvaifler J. Clin. Invest. 50(3):606-616 (1971)).

[0228] In certain embodiments, whether a test antibody blocks the access of C5 convertase to C5 is determined by a method for detecting protein interaction between C5 convertase and C5, such as ELISA or BIACORE®. If the interaction is reduced in the presence of (or after contact with) the test antibody, the test antibody is identified as an antibody that can block the access of C5 convertase to C5.

[0229] In certain embodiments, C5 activity can be measured as a function of its cytolytic ability in the body fluid of a subject.C5 cytolytic ability or its reduction can be measured by methods well known in the art, for example, by conventional hemolysis assay, for example, the hemolysis assay described in Kabat and Mayer (eds.), Experimental Immunochemistry, 2nd ed., 135-240, Springfield, IL, CC Thomas (1961), pp. 135-139, or by a conventional modification of said assay, for example, chicken erythrocyte hemolysis assay, as described in Hillmen et al., N. Engl. J. Med. 350(6): 552-559 (2004).In certain embodiments, C5 activity or its inhibition is quantified using CH50eq assay.CH50eq assay is a method for measuring the total classical complement activity in serum. This test is a lysis assay that uses antibody-sensitized red blood cells as activators of the classical complement pathway and various dilutions of test serum to determine the amount required to give 50% lysis (CH50). The percentage of hemolysis can be determined, for example, using a spectrophotometer. The CH50eq assay provides an indirect measure of terminal complement complex (TCC) formation, since the direct cause of the hemolysis measured is the TCC itself. Inhibition of C5 activation can also be detected and / or measured using the methods described and exemplified in the Examples. These or other suitable types of assays can be used to screen candidate antibodies that can inhibit C5 activation. In certain embodiments, inhibition of C5 activation includes at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% or more decrease in C5 activation in the assay compared to the effect of a negative control under similar conditions. In some embodiments, it refers to at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more inhibition of C5 activation.

[0230] D. Immunoconjugates The invention also provides immunoconjugates comprising an anti-C5 antibody herein conjugated to one or more cytotoxic agents (e.g., a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin of bacterial, fungal, plant or animal origin, an enzymatically active toxin, or fragments thereof), or a radioactive isotope).

[0231] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more drugs, including but not limited to: maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064, and European Patent No. 0,425,235 B1); auristatins, such as the monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatins; calicheamicin or a derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342; (1993); and see Lode et al., Cancer Res. 58:2925-2928 (1998)); anthracyclines such as daunomycin or doxorubicin (Kratz et al., Current 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 U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

[0232] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana protein (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0233] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the production 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 used for detection, the radioconjugate may contain a radioactive atom (e.g., tc99m or I123) for scintigraphic examinations, or a spin label (e.g., again iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron) for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI).

[0234] Conjugates of antibodies and cytotoxic agents can be made using a variety of bifunctional protein linking agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (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, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionuclides to antibodies. See WO94 / 11026. The linker can be a "cleavable linker" that facilitates the release of the cytotoxic drug inside the cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020) can be used.

[0235] The immunoconjugates or ADCs herein expressly contemplate, but are not limited to, conjugates prepared using cross-linking reagents including, but not limited to, 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), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA).

[0236] E. Methods and Compositions for Diagnostics and Detection In certain embodiments, any of the anti-C5 antibodies provided herein is useful for detecting the presence of C5 in biological samples.The term "detection" as used herein includes quantitative or qualitative detection.In certain embodiments, biological samples include cells or tissues, such as serum, whole blood, plasma, biopsy sample, tissue sample, cell suspension, saliva, sputum, oral fluid, cerebrospinal fluid, amniotic fluid, ascites, breast milk, colostrum, mammary gland secretion, lymph, urine, sweat, tears, gastric juice, synovial fluid, ascites, ophthalmic lens fluid, and mucus.

[0237] In one embodiment, anti-C5 antibody is provided for use in diagnostic or detection method. In a further aspect, a method is provided for detecting the presence of C5 in a biological sample. In a particular embodiment, the method comprises contacting a biological sample with an anti-C5 antibody described herein under conditions that allow the binding of the anti-C5 antibody to C5, and detecting whether a complex is formed between the anti-C5 antibody and C5. Such a method can be an in vitro method or an in vivo method. In one embodiment, the anti-C5 antibody is used to select subjects that are suitable for treatment with the anti-C5 antibody, for example, when C5 is a biomarker for selecting patients.

[0238] In another embodiment, a method is provided for selecting an individual having a complement-mediated disease or condition involving excessive or uncontrolled activation of C5 as suitable for a therapy comprising the anti-C5 antibody of the present invention. In a particular embodiment, the method comprises (a) detecting a genetic mutation in C5 from the individual, and (b) selecting the individual as suitable for a therapy comprising the anti-C5 antibody of the present invention if a genetic mutation is detected in C5 from the individual. In another embodiment, a method is provided for selecting a therapy for an individual having a complement-mediated disease or condition involving excessive or uncontrolled activation of C5. In a particular embodiment, the method comprises (a) detecting a genetic mutation in C5 from the individual, and (b) selecting a therapy comprising the anti-C5 antibody of the present invention for the individual if a genetic mutation is detected in C5 from the individual.

[0239] In another embodiment, a method for treating an individual having a complement-mediated disease or condition involving excessive or uncontrolled activation of C5 is provided. In a particular embodiment, the method includes (a) detecting a genetic mutation in C5 from the individual, (b) selecting the individual as suitable for a therapy comprising an anti-C5 antibody of the present invention if a genetic mutation is detected in C5 from the individual, and (c) administering the anti-C5 antibody of the present invention to the individual.

[0240] In another embodiment, an anti-C5 antibody of the present invention is provided for use in treating an individual having a complement-mediated disease or condition involving excessive or uncontrolled activation of C5. In a particular embodiment, an individual is treated with an anti-C5 antibody of the present invention when a genetic mutation is detected in C5 from the individual.

[0241] In another embodiment, an in vitro use of genetic mutations in C5 is provided to select an individual having a complement-mediated disease or condition involving excessive or uncontrolled activation of C5 as suitable for a therapy comprising the anti-C5 antibody of the present invention. In a particular embodiment, an individual is selected as suitable for a therapy when a genetic mutation is detected in C5 from the individual. In another embodiment, an in vitro use of genetic mutations in C5 is provided to select a therapy for an individual having a complement-mediated disease or condition involving excessive or uncontrolled activation of C5. In a particular embodiment, a therapy comprising the anti-C5 antibody of the present invention is selected for the individual when a genetic mutation is detected in C5 from the individual.

[0242] It has been reported that some patients with genetic mutations in C5 show poor response to existing therapies containing anti-C5 antibodies (Nishimura et al., N. Engl. J. Med. 370:632-639 (2014)). It is recommended that such patients be treated with a therapy containing the anti-C5 antibody of the present invention. This is because the anti-C5 antibody of the present invention has inhibitory activity against the activation of not only wild-type C5 but also C5 mutants, as demonstrated in the Examples below.

[0243] The detection of genetic mutations in C5 can be carried out by using methods known in the prior art. Such methods can include, but are not limited to, sequencing, PCR, RT-PCR, and hybridization-based methods, such as Southern blot or Northern blot. C5 mutants can include at least one genetic mutation. The genetic mutation can be selected from the group consisting of V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N, and E1437D. For example, R885H herein refers to the genetic mutation in which the arginine at position 885 is replaced with histidine. In certain embodiments, C5 mutants have similar biological activity to wild-type C5.

[0244] Exemplary disorders that may be diagnosed using the antibodies of the invention include rheumatoid arthritis (RA); systemic lupus erythematosus (SLE); lupus nephritis; ischemia-reperfusion injury (IRI); asthma; paroxysmal nocturnal hemoglobinuria (PNH); hemolytic uremic syndrome (HUS) (e.g., atypical hemolytic uremic syndrome (aHUS)); dense deposit disease (DDD); neuromyelitis optica (NMO); multifocal motor neuropathy (MMN); multiple sclerosis (MS); systemic sclerosis; macular degeneration (e.g., age-related macular degeneration (AMD)); HELLP (hemolysis, elevated liver enzymes, and low platelets syndrome; thrombotic thrombocytopenic purpura (TTP); spontaneous abortion; epidermolysis bullosa; habitual abortion; preeclampsia; traumatic brain injury; myasthenia gravis; cold agglutinin disease; Sjogren's syndrome; dermatomyositis; bullous pemphigoid; phototoxic reactions; Shiga toxin E. coli-associated hemolytic uraemic syndrome; typical or infectious hemolytic uraemic syndrome (tHUS); C3 glomerulonephritis; antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis; humoral and Vascular transplant rejection;Acute antibody-mediated rejection (AMR);Graft dysfunction;Myocardial infarction;Allograft;Sepsis;Coronary artery disease;Hereditary angioedema;Dermatomyositis;Graves' disease;Atherosclerosis;Alzheimer's disease (AD);Huntington's disease;Creutzfeldt-Jakob disease;Parkinson's disease;Cancer;Wound;Septic shock;Spinal cord injury;Uveitis;Diabetic eye disease;Retinopathy of prematurity;Glomerulonephritis;Membranous nephritis;Immuno 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 I diabetes; psoriasis; pemphigus; autoimmune hemolytic anemia (AIHA); idiopathic thrombocytopenic purpura (ITP); Goodpasture's syndrome; Degos disease; antiphospholipid syndrome (A PS);Fulminant APS (CAPS);Cardiovascular disease;Myocarditis;Cerebrovascular disease;Peripheral vascular disease;Renal vascular disease;Mesenteric / intestinal vascular disease;Vaccitis;Henoch-Schönlein purpura nephritis;Takayasu's disease;Dilated cardiomyopathy;Diabetic vascular disease;Kawasaki disease (arteritis);Venous gas embolism (VGE), restenosis after stent placement;Rotational atherectomy;Membranous nephropathy;Guillain-Barré syndrome (GBS);Fisher syndrome;Antigen-induced arthritis;Synovitis;viral infections; bacterial infections; fungal infections; and injuries resulting from myocardial infarction, cardiopulmonary bypass, and hemodialysis.

[0245] In certain embodiments, a labeled anti-C5 antibody is provided. Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels) and indirectly detectable moieties (e.g., enzymes or ligands) through, for example, enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, the following: radioisotopes 32 P, 14 C. 125 I, 3 H and 131 I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, monosaccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases such as uricase and xanthine oxidase linked to enzymes that oxidize dye precursors using hydrogen peroxide (e.g., HRP, lactoperoxidase, or microperoxidase), biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0246] F. Pharmaceutical Formulations Pharmaceutical formulations of the anti-C5 antibodies described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing the antibody having the desired purity with any one or more pharma- ceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, the following: buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (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, etc.; small (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 dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmacologic carriers herein further include interstitial drug dispersing agents, such as soluble neutral activated hyaluronidase glycoproteins (sHASEGPs) (e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.)). Certain exemplary sHASEGPs and methods of their use (including rHuPH20) are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968.In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases, such as a chondroitinase.

[0247] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0248] The formulations herein may contain more than one active ingredient as necessary for the particular indication being treated. Those with complementary activities that do not adversely affect each other are preferred. Such active ingredients are present in suitable combinations in amounts that are effective for the intended purpose.

[0249] The active ingredient may be incorporated into microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules, and poly(methyl methacrylate) microcapsules, respectively) prepared, for example, by droplet formation (coacervation) techniques or by interfacial polymerization, into colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or into macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0250] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg, films, or microcapsules.

[0251] Preparations to be used for in vivo administration are typically sterile. Sterility is readily accomplished, for example, by filtration through sterile filtration membranes.

[0252] G. Therapeutic Methods and Compositions Any of the anti-C5 antibodies provided herein may be used in therapeutic methods.

[0253] In one aspect, an anti-C5 antibody is provided for use as a medicament. In a further aspect, an anti-C5 antibody is provided for use in the treatment of a complement-mediated disease or condition involving excessive or uncontrolled activation of C5. In a particular embodiment, an anti-C5 antibody is provided for use in a method of treatment. In a particular embodiment, the invention provides an anti-C5 antibody for use in a method of treating an individual having a complement-mediated disease or condition involving excessive or uncontrolled activation of C5, the method comprising administering to the individual an effective amount of an anti-C5 antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. An "individual" according to any of the above embodiments is preferably a human.

[0254] When an antigen is a soluble protein, the binding of an antibody to the antigen can result in an increased plasma half-life of the antigen (i.e., reduced clearance of the antigen from plasma) because the antibody itself has a relatively long plasma half-life and acts as a carrier of the antigen. This is due to the recycling of the antigen-antibody complex by FcRn via the endosomal pathway in cells (Roopenian, Nat. Rev. Immunol. 7(9):715-725 (2007)). However, antibodies with pH-dependent binding properties, which bind antigens in the neutral extracellular environment but release antigens into acidic endosomal compartments after entering cells, are expected to have superior attributes in terms of antigen neutralization and clearance compared to their counterparts that bind in a pH-independent manner (Igawa et al., Nat. Biotech.. 28(11):1203-1207 (2010); Devanaboyina et al., mAbs 5(6):851-859 (2013); WO 2009 / 125825).

[0255] In a further embodiment, the present invention provides an anti-C5 antibody for use in enhancing the clearance of C5 from plasma. In a particular embodiment, the present invention provides an anti-C5 antibody for use in a method of enhancing the clearance of C5 from plasma in an individual, the method comprising administering to the individual an effective amount of an anti-C5 antibody to enhance the clearance of C5 from plasma. In one embodiment, the anti-C5 antibody enhances the clearance 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.

[0256] In a further embodiment, the present invention provides an anti-C5 antibody for use in suppressing the accumulation of C5 in plasma. In a particular embodiment, the present invention provides an anti-C5 antibody for use in a method of suppressing the accumulation of C5 in plasma in an individual, the method comprising administering to the individual an effective amount of an anti-C5 antibody to suppress the accumulation of C5 in plasma. In one embodiment, the accumulation of C5 in plasma is the result of the formation of an antigen-antibody complex. In another embodiment, the anti-C5 antibody suppresses the accumulation of C5 in plasma compared to a conventional anti-C5 antibody that does not have pH-dependent binding properties. The "individual" according to any of the above embodiments is preferably a human.

[0257] The anti-C5 antibody of the present invention can inhibit the activation of C5. In a further embodiment, the present invention provides an anti-C5 antibody for use in inhibiting the activation of C5. In a particular embodiment, the present invention provides an anti-C5 antibody for use in a method of inhibiting the activation of C5 in an individual, the method 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 suppressed by inhibiting the activation of C5. An "individual" according to any of the above embodiments is preferably a human.

[0258] In a further aspect, the present invention provides the use of an anti-C5 antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of a complement-mediated disease or condition involving excessive or uncontrolled activation of C5. In a further embodiment, the medicament is for use in a method of treating a complement-mediated disease or condition involving excessive or uncontrolled activation of C5, comprising administering an effective amount of the medicament 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 at least one additional therapeutic agent. An "individual" according to any of the above embodiments is preferably a human.

[0259] In a further embodiment, the medicament is for enhancing the clearance of C5 from plasma. In a further embodiment, the medicament is for use in a method of enhancing the clearance of C5 from plasma in an individual, the method comprising administering to the individual an effective amount of the medicament to enhance the clearance of C5 from plasma. In one embodiment, the anti-C5 antibody enhances the clearance of C5 from plasma compared to a conventional anti-C5 antibody that does not have pH-dependent binding properties. The "individual" according to any of the above embodiments may be a human.

[0260] In a further embodiment, the medicament is for suppressing accumulation of C5 in plasma. In a further embodiment, the medicament is for use in a method of suppressing accumulation of C5 in plasma in an individual, comprising administering to the individual an effective amount of the medicament to suppress accumulation of C5 in plasma. In one embodiment, accumulation of C5 in plasma is the result of formation of an antigen-antibody complex. In another embodiment, the anti-C5 antibody suppresses accumulation of C5 in plasma compared to a conventional anti-C5 antibody that does not have pH-dependent binding properties. The "individual" according to any of the above embodiments may be a human.

[0261] The anti-C5 antibody of the present invention can inhibit the activation of C5. In a further embodiment, the medicament is for inhibiting the activation of C5. In a further 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 suppressed by inhibiting the activation of C5. The "individual" according to any of the above embodiments may be a human.

[0262] In a further aspect, the present 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 such 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 at least one additional therapeutic agent. An "individual" according to any of the above embodiments may be a human.

[0263] In a further aspect, the present invention provides a method for enhancing the clearance of C5 from plasma in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-C5 antibody to enhance the clearance of C5 from plasma. In one embodiment, the anti-C5 antibody enhances the clearance 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.

[0264] In a further aspect, the present invention provides a method for suppressing the accumulation of C5 in plasma in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-C5 antibody to suppress the accumulation of C5 in plasma. In one embodiment, the accumulation of C5 in plasma is the result of the formation of an antigen-antibody complex. In another embodiment, the anti-C5 antibody suppresses 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.

[0265] The anti-C5 antibody of the present invention can inhibit the activation of C5. In a further aspect, the present invention provides a method for inhibiting the activation of C5 in an individual. In one embodiment, the method comprises administering to an individual an effective amount of an anti-C5 antibody to inhibit the activation of C5. In one embodiment, the cytotoxicity mediated by C5 is suppressed by inhibiting the activation of C5. In one embodiment, the "individual" is a human.

[0266] In a further aspect, the present invention provides pharmaceutical formulations comprising any of the anti-C5 antibodies provided herein (e.g., for use in any of the therapeutic methods described above). In one embodiment, the pharmaceutical formulation comprises any of the anti-C5 antibodies provided herein and a pharma- ceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the anti-C5 antibodies provided herein and at least one additional therapeutic agent.

[0267] In a further aspect, the pharmaceutical formulation is for treating a complement-mediated disease or condition involving excessive or uncontrolled activation of C5. In a further embodiment, the pharmaceutical formulation is for enhancing the clearance of C5 from plasma. In one embodiment, the anti-C5 antibody enhances the clearance of C5 from plasma compared to a conventional anti-C5 antibody that does not have pH-dependent binding properties. In a further embodiment, the pharmaceutical formulation is for inhibiting the accumulation of C5 in plasma. In one embodiment, the accumulation of C5 in plasma is the result of the formation of an antigen-antibody complex. 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 a further 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 having 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.

[0268] In one aspect, the individual has wild-type C5. In another aspect, the individual has C5 mutant. In certain embodiments, C5 mutant has similar biological activity to wild-type C5. Such C5 mutant can include at least one mutation selected from the group consisting of V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N and E1437D. For example, R885H refers to the genetic mutation in which the arginine at position 885 is replaced with histidine.

[0269] In a further aspect, the present invention provides a method for preparing a medicament or pharmaceutical formulation, e.g., for use in any of the above-mentioned therapeutic methods, comprising mixing any of the anti-C5 antibodies provided herein with a pharma- ceutically acceptable carrier. In one embodiment, the method for preparing a medicament or pharmaceutical formulation further comprises adding at least one additional therapeutic agent to the medicament or pharmaceutical formulation.

[0270] 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; ischemia-reperfusion injury (IRI); asthma; paroxysmal nocturnal hemoglobinuria (PNH); hemolytic uremic syndrome (HUS) (e.g., atypical hemolytic uremic syndrome (aHUS)); dense deposit disease (DDD); neuromyelitis optica (NMO); multifocal motor neuropathy (MMN); multiple sclerosis (MS); systemic sclerosis; macular degeneration (e.g., age-related macular degeneration (AMD)); HELLP (hemolysis, elevated liver enzymes, and low platelets syndrome; thrombotic thrombocytopenic purpura (TTP); spontaneous abortion; epidermolysis bullosa; habitual abortion; preeclampsia; traumatic brain injury; myasthenia gravis; cold agglutinin disease; Sjogren's syndrome; dermatomyositis; bullous pemphigoid; phototoxic reaction; Shiga toxin E. coli-associated hemolytic uremic syndrome; typical or infectious hemolytic uremic syndrome (tHUS); C3 glomerulonephritis; antineutrophil cytoplasmic antibody (ANCA)-associated Vasculitis;humoral and vascular transplant rejection;acute antibody-mediated rejection (AMR);graft dysfunction;myocardial infarction;allograft;sepsis;coronary artery disease;hereditary angioedema;dermatomyositis;Graves' disease;atherosclerosis;Alzheimer's disease (AD);Huntington's disease;Creutzfeldt-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 I diabetes mellitus;psoriasis;pemphigus;autoimmune hemolytic anemia (AIHA);idiopathic thrombocytopenic purpura (IT P);Goodpasture's syndrome;Degos disease;Antiphospholipid syndrome (APS);Fulminant APS (CAPS);Cardiovascular disease;Myocarditis;Cerebrovascular disease;Peripheral vascular disease;Renal vascular disease;Mesenteric / intestinal vascular disease;Vaccitis;Henoch-Schönlein purpura nephritis;Takayasu's disease;Dilated cardiomyopathy;Diabetic vascular disease;Kawasaki disease (arteritis);Venous gas embolism (VGE), restenosis after stent placement;Rotational atherectomy;Membranous nephropathy; Guillain-Barré syndrome (GBS); Fisher syndrome; antigen-induced arthritis; synovitis; viral infections; bacterial infections; fungal infections; and damage resulting from myocardial infarction, cardiopulmonary bypass, and hemodialysis.

[0271] In certain embodiments, the complement-mediated disease or condition is an ophthalmic disease condition. In further embodiments, the ophthalmic disease condition is macular degeneration. In further embodiments, the macular degeneration is AMD. In further embodiments, the AMD is dry AMD.

[0272] In certain embodiments, the complement-mediated disease or condition is PNH.

[0273] In a particular embodiment, the complement-mediated disease or condition is myocardial infarction.

[0274] In a particular embodiment, the complement-mediated disease or condition is RA.

[0275] In certain embodiments, the complement-mediated disease or condition is osteoporosis or osteoarthritis.

[0276] In certain embodiments, the complement-mediated disease or condition is inflammation.

[0277] In certain embodiments, the complement-mediated disease or condition is cancer.

[0278] The antibodies of the invention can be used in therapy either alone or in combination with other agents, for example, the antibodies of the invention can be co-administered with at least one additional therapeutic agent.

[0279] Combination therapy as described above includes combined administration (two or more therapeutic agents in the same or separate formulations) and separate administration, where administration of an antibody of the invention may precede, be concurrent with, and / or be subsequent to administration of the additional therapeutic agent. In one embodiment, administration of the anti-C5 antibody and administration of the additional therapeutic agent occur within about one month, or within about 1, 2, or 3 weeks, or within about 1, 2, 3, 4, 5, or 6 days of each other.

[0280] The antibodies of the invention (and any additional therapeutic agents) may be administered by any suitable means, including parenteral, pulmonary, and nasal administration, and, if desired for localized treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing may be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single or repeated doses over various time periods, bolus administration, and pulse infusion.

[0281] The antibodies of the invention are formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the schedule of administration, and other factors known to medical practitioners. The antibodies are optionally, but not necessarily, formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are typically used in the same dosages and routes of administration as described herein, or about 1 to 99% of the dosages described herein, or in any dosage and by any route as empirically / clinically determined to be appropriate.

[0282] For the prevention or treatment of disease, the appropriate dose of the antibody of the invention (when used alone or with one or more other additional therapeutic agents) will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, medical history, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, for example, about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of antibody may be the initial candidate dose for administration to the patient, whether by one or more separate administrations or by continuous infusion. One typical daily dose may range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. In the case of repeated administration over several days or longer, depending on the circumstances, treatment is usually maintained until a desired suppression of disease symptoms occurs. One exemplary dose of the antibody is in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example, every week or every three weeks (e.g., such that the patient receives about 2 to about 20, or, for example, about 6 doses of the antibody). A high initial loading dose may be followed by one or more lower doses. The progress of this therapy is easily monitored by conventional techniques and measurements.

[0283] It will be understood that any of the above-described formulations or therapeutic methods may be practiced using an immunoconjugate of the invention in place of, or in addition to, an anti-C5 antibody.

[0284] H.Product In another aspect of the invention, an article of manufacture is provided that includes materials useful for the treatment, prevention, and / or diagnosis of the above-mentioned disorders. The article of manufacture includes a container and a label or package insert on or associated with the container. Preferred containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The containers may be formed from a variety of materials, such as glass or plastic. The container may hold the composition alone or in combination with another composition effective for the treatment, prevention, and / or diagnosis of a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is for use in treating a selected condition. The article of manufacture may further include (a) a first container with a composition comprising the antibody of the invention contained therein; and (b) a second container with a composition comprising an additional cytotoxic or otherwise therapeutic agent contained therein. The article of manufacture in this aspect of the invention may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further include a second (or third) container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other equipment desirable from a commercial or user standpoint, such as other buffers, diluents, filters, needles, and syringes.

[0285] It will be understood that any of the above-mentioned products may contain an immunoconjugate of the invention instead of, or in addition to, an anti-C5 antibody. EXAMPLES

[0286] The following are examples of the methods and compositions of the present invention. In light of the general description above, it will be understood that various other embodiments may be practiced.

[0287] Example 1 Preparation of C5 1.1. Expression and purification of recombinant human and cynomolgus C5 Recombinant human C5 (NCBI GenBank accession number: NP_001726.2, SEQ ID NO: 39) was transiently expressed using the FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA). Conditioned medium expressing human C5 was diluted with an equal volume of milliQ water and then applied to a Q Sepharose FF or Q Sepharose HP anion exchange column (GE healthcare, Uppsala, Sweden) followed by elution with a NaCl gradient. Fractions containing human C5 were pooled, and then the salt concentration and pH were adjusted to 80 mM NaCl and pH 6.4, respectively. The resulting sample was applied to a SP Sepharose HP cation exchange column (GE healthcare, Uppsala, Sweden) and eluted with a NaCl gradient. Fractions containing human C5 were pooled and subjected 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 pooled and stored at -150°C.

[0288] Expression and purification of recombinant cynomolgus C5 (NCBI GenBank Accession Number: XP_005580972, SEQ ID NO: 44) was performed in the same manner as the human counterpart.

[0289] 1.2. Purification of cynomolgus C5 (cynoC5) from plasma Plasma samples from cynomolgus monkeys were applied to SSL7-agarose (Invivogen, San Diego, CA, USA) and subsequently eluted with 100 mM Na-acetate, pH 3.5. Fractions containing cynoC5 were immediately neutralized and subjected to a Protein A HP column (GE healthcare, Uppsala, Sweden) coupled in tandem with peptide M agarose (Invivogen, San Diego, CA, USA). The flow-through fraction was then applied to a Superdex 200 gel filtration column (GE healthcare, Uppsala, Sweden). Fractions containing cynoC5 were pooled and stored at -80°C.

[0290] Example 2 Preparation of anti-C5 antibody 2.1. Antibody Screening Anti-C5 antibodies were prepared, selected, and assayed as follows.

[0291] Twelve to 16 week old NZW rabbits were immunized intradermally with human C5 and / or monkey C5 (50-100 μg / dose / rabbit). This dose was repeated 4-5 times over a period of 2 months. One week after the final immunization, spleens and blood were collected from the immunized rabbits. Antigen-specific B cells were stained with labeled antigens, sorted with an FCM cell sorter (FACS aria III, BD), and seeded at a density of 1 cell / well into 96-well plates with 25,000 cells / well of EL4 cells (European Collection of Cell Cultures) and 20-fold diluted activated rabbit T cell conditioned medium, and cultured for 7-12 days. EL4 cells were treated with mitomycin C (Sigma, Cat. No. M4287) for 2 h and washed three times beforehand. Activated rabbit T cell conditioned medium was prepared by culturing rabbit thymocytes in RPMI-1640 containing phytohemagglutinin M (Roche, Cat. No. 11082132-001), phorbol 12-myristate 13-acetate (Sigma, Cat. No. P1585) and 2% FBS. After culture, B cell culture supernatants were collected for further analysis and pellets were frozen and stored.

[0292] ELISA assays were used to test the specificity of antibodies in B cell culture supernatants. Streptavidin (GeneScript, Cat. No. Z02043) was coated on 384-well MAXISorp (Nunc, Cat. No. 164688) at 50 nM in PBS for 1 h at room temperature. Plates were then blocked with Blocking One (Nacalai Tesque, Cat. No. 03953-95) diluted 5-fold. Human or monkey C5 was labeled with NHS-PEG4-biotin (PIERCE, Cat. No. 21329) and added to the blocked ELISA plates, incubated for 1 h and washed. B cell culture supernatants were added to the ELISA plates, incubated for 1 h and washed. Binding was detected by adding goat anti-rabbit IgG-horseradish peroxidase (BETHYL, Cat. No. A120-111P) followed by ABTS (KPL, Cat. No. 50-66-06).

[0293] An ELISA assay was used to assess pH-dependent binding of antibodies to C5. Goat anti-rabbit IgG-Fc (BETHYL, Cat. No. A120-111A) diluted to 1 μg / ml in PBS(-) was added to a 384-well MAXISorp (Nunc, Cat. No. 164688), incubated at room temperature for 1 h, and blocked with 5-fold diluted Blocking One (Nacalai Tesque, Cat. No. 03953-95). After incubation, the plate was washed and B cell culture supernatant was added. The plate was incubated for 1 h, washed, and 500 pM biotinylated human C5 or monkey C5 was added and incubated for 1 h. After incubation, the plate was washed and blocked with MES buffer at pH 7.4 (20 mM MES, 150 mM NaCl and 1.2 mM CaCl 2) or MES buffer pH 5.8 (20 mM MES, 150 mM NaCl and 1 mM EDTA) for 1 h at room temperature. After incubation, binding of biotinylated C5 was detected by adding streptavidin-horseradish peroxidase conjugate (Thermo Scientific, Cat. No. 21132) followed by ABTS (KPL, Cat. No. 50-66-06).

[0294] The affinity and pH-dependent binding of antibodies to C5 was evaluated using the Octet RED384 system (Pall Life Sciences). Antibodies secreted into B cell culture supernatants were loaded onto a Protein A biosensor chip (Pall Life Sciences) and soaked in 50 nM human or monkey C5 in MES buffer, pH 7.4, to analyze binding kinetics. Dissociation kinetics were analyzed in both MES buffer, pH 7.4 and MES buffer, pH 5.8.

[0295] 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 lines was purified from cryopreserved cell pellets using the ZR-96 Quick-RNA Kit (ZYMO RESEARCH, Cat. No. R1053). DNA encoding the antibody heavy chain variable region in the selected cell lines was amplified by reverse transcription PCR and recombined with DNA encoding the F760G4 (SEQ ID NO: 33) or F939G4 (SEQ ID NO: 34) heavy chain constant region. DNA encoding the antibody light chain variable region was amplified by reverse transcription PCR and recombined with DNA encoding the k0MTC light chain constant region (SEQ ID NO: 36). Separately, the 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 to DNA encoding modified human IgG4 CH (F760G4, SEQ ID NO:33), and DNA encoding VL (EcuL, SEQ ID NO:32) was fused in frame to DNA encoding k0 light chain constant region (SEQ ID NO:37). Each of the fused coding sequences was cloned into an expression vector. Antibodies were expressed in FreeStyle™ 293-F cells (Invitrogen) and purified from culture supernatants to assess functional activity. Neutralizing activity of the antibodies was assessed by testing inhibition of complement activity using a liposome lysis assay as described in Example 5.1.

[0296] 2.2. Epitope binning by sandwich ELISA Anti-C5 antibodies with high affinity, pH-dependence or neutralizing activity were selected for further analysis. The selected antibodies were sorted into various epitope bins that bind to the same or overlapping epitopes of C5 protein using sandwich ELISA assay. Unlabeled capture antibodies were diluted to 1 μg / ml in PBS(-) and added to 384-well MAXISorp plates (Nunc, Cat. No. 164688). The plates were incubated for 1 hour at room temperature and blocked with 5-fold diluted Blocking One (Nacalai Tesque, Cat. No. 03953-95). The plates were incubated for 1 hour, washed, and 2 nM human C5 was added and incubated for 1 hour. After incubation, the plates were washed and labeled detection antibodies (1 μg / mL, biotinylated with NHS-PEG4-biotin) were added. After 1 h of incubation, binding of biotinylated antibodies was detected by adding streptavidin-horseradish peroxidase conjugate (Thermo Scientific, Cat. No. 21132) followed by ABTS (KPL, Cat. No. 50-66-06).

[0297] All anti-C5 antibodies were used as both capture and detection antibodies and were paired comprehensively. As shown in Figure 1, the competing antibodies were classified into seven epitope bins: CFA0668, CFA0334 and CFA0319 were classified into epitope A; CFA0647, CFA0589, CFA0341, CFA0639, CFA0635, CFA0330 and CFA0318 were classified into epitope B; CFA0538, CFA0501, CFA0599, CFA030 and CFA0318 were classified into epitope C; 7, CFA0366, CFA0305, CFA0675, CFA0666 and CFA0672 were classified as epitope C, eculizumab and CFA0322 were classified as epitope D, CFA0329 was classified as epitope E, CFA0359 and CFA0217 were classified as epitope F, and CFA0579, CFA0328 and CFA0272 were classified as epitope G. Figure 1 shows the epitope binning of several anti-C5 chimeric antibodies. The VH and VL sequences of the anti-C5 antibodies classified as epitope C are shown in Table 2.

[0298] [Table 2]

[0299] 2.3. Humanization and Optimization Humanization of the variable regions of some anti-C5 antibodies was performed to reduce the potential immunogenicity of the antibodies. The complementarity determining regions (CDRs) of anti-C5 rabbit antibodies were grafted onto homologous human antibody frameworks (FRs) using conventional CDR grafting techniques (Nature 321:522-525 (1986)). Genes encoding humanized VH and VL were synthesized and combined with modified human IgG4 CH (SG402, SEQ ID NO:35) and human CL (SK1, SEQ ID NO:38), respectively, and each of the combined sequences was cloned into an expression vector.

[0300] Many mutations and combinations of mutations were examined to identify mutations and combinations of mutations that improve the binding properties of some lead antibodies. Then, multiple mutations were introduced into the humanized variable region to enhance the binding affinity to C5 at neutral pH or to decrease the binding affinity to C5 at acidic pH. Thus, one of the optimized mutants, 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), was generated from CFA0305.

[0301] Antibodies were expressed in HEK293 cells co-transfected with a mixture of heavy and light chain expression vectors and purified by Protein A.

[0302] Example 3 Binding characterization of anti-C5 antibodies 3.1. Expression and purification of recombinant antibodies Recombinant antibodies were transiently expressed using the FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA). Purification of the antibodies from conditioned medium was performed by conventional methods using Protein A. Gel filtration was further performed if necessary.

[0303] 3.2. Evaluation of pH Dependence Kinetic parameters of anti-C5 antibodies against recombinant human C5 were evaluated at pH 7.4 and pH 5.8 at 37°C using a BIACORE® T200 instrument (GE Healthcare). ProA / G (Pierce) was immobilized onto a CM4 sensor chip using an amine coupling kit (GE Healthcare) according to the settings recommended by GE Healthcare. Antibodies and analytes were incubated in their 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 ) in 100 mM NaOH. Each antibody was captured on the sensor surface by ProA / G. The capture level of the antibodies was generally 60-90 resonance units (RU). Recombinant human C5 was then injected at concentrations of 10 and 20 nM or 20 and 40 nM, followed by dissociation. The surface was regenerated with 25 mM NaOH. The kinetic parameters at both pH conditions were determined by fitting the sensorgrams with a 1:1 binding model using BIACORE® T200 Evaluation software, version 2.0 (GE Healthcare). The sensorgrams of all antibodies are shown in Figures 2A and 2B. The binding rates (ka), dissociation rates (kd), and binding affinities (KD) of the antibodies are shown in Table 3. All antibodies, except for CFA0330 (VH, SEQ ID NO:21 and VL, SEQ ID NO:25) and CFA0341 (VH, SEQ ID NO:22 and VL, SEQ ID NO:26), showed relatively faster dissociation rates at pH 5.8 than at pH 7.4.

[0304] [Table 3]

[0305] 3.3. Cross-reactivity study BIACORE® kinetic analysis was performed to observe the cross-reactivity of anti-C5 antibodies against human C5 (hC5) and cynomolgus monkey C5 (cynoC5). The assay setup was the same as described in Example 3.2. Recombinant cynoC5 was injected at concentrations of 2, 10, and 50 nM. The 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 shown in Table 4. The kinetic parameters for hC5 shown in Table 4 are the results of Example 3.2. All anti-C5 antibodies except CFA0672 showed similar KDs for hC5 and cynoC5. The KD of CFA0672 for cynoC5 was 8-fold weaker than that for hC5.

[0306] [Table 4]

[0307] Example 4 Epitope mapping of anti-C5 antibodies 4.1. Binding of anti-C5 MAbs to peptides derived from the C5 β chain Anti-C5 monoclonal antibodies (MAbs) were tested for binding to C5 β-chain derived peptides in Western blot analysis. C5 peptides: 19-180, 161-340, 321-500, and 481-660 fused to a GST tag (pGEX-4T-1, GE Healthcare Life Sciences, 28-9545-49) were expressed in Escherichia coli (E. coli) (DH5α, TOYOBO, DNA-903). E. coli samples were harvested after 5 h incubation with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37°C and centrifuged at 20000×g for 1 min to obtain a pellet. The pellet was suspended in sample buffer (2ME+) (Wako, 191-13272) and used for Western blot analysis. The expression of each peptide was confirmed using an anti-GST antibody (Abcam, ab9085) (Figure 3). The arrow indicates the GST-fused C5 peptide (46-49 kDa). Anti-C5 MAbs: CFA0305, CFA0307, ​​CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675 bound to 19-180 of C5 (Figure 3).

[0308] 4.2. Expression and purification of the MG1-MG2 domain (1-225) of human C5 Recombinant MG1-MG2 domains of the β-chain of human C5 (SEQ ID NO: 43) were transiently expressed using the FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA). Conditioned medium expressing the MG1-MG2 domains 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 flow-through fraction from the anion exchange column was adjusted to pH 5.0, applied to a SP Sepharose HP cation exchange column (GE healthcare, Uppsala, Sweden) and eluted with a NaCl gradient. Fractions containing the MG1-MG2 domains were collected from the eluate and subsequently subjected to a Superdex 75 gel filtration column (GE healthcare, Uppsala, Sweden) equilibrated with 1x PBS. The fractions containing the MG1-MG2 domains were then pooled and stored at -80°C.

[0309] 4.3. Binding Ability to MG1-MG2 Domain The binding ability of anti-C5 antibodies to the MG1-MG2 domain was measured using the same assay setup as described in Example 3.2, except that measurements were only performed under pH 7.4 conditions. The MG1-MG2 domain was injected at concentrations of 20 nM and 40 nM. As shown in Figure 4, all antibodies except eculizumab-F760G4 showed an increased binding response, indicating that these antibodies are MG1-MG2 binders. Eculizumab-F760G4, a known α-chain binder, did not show binding to the MG1-MG2 domain.

[0310] 4.4. Binding of anti-C5 MAbs to peptides derived from the MG1-MG2 domain of C5 Anti-C5 MAbs were tested for binding to peptides derived from the MG1-MG2 domain in Western blot analysis. C5 peptides fused to GST tags: 33-124, 45-124, 52-124, 33-111, 33-108, and 45-111 (SEQ ID NO: 40) were expressed in E. coli. E. coli samples were harvested after 5 h incubation with 1 mM IPTG at 37° C. and centrifuged at 20000×g for 1 min to obtain a pellet. The pellet was suspended in sample buffer (2ME+) and used for Western blot analysis. Expression of C5-derived peptides was confirmed by anti-GST antibody (FIG. 5A). CFA0305 bound only to the 33-124 peptide (FIG. 5B). CFA0305 bound to the β-chain (approximately 70 kDa) of recombinant human C5 (rhC5), which was used as a control. FIG. 5C summarizes the responses of anti-C5 MAbs to C5-derived peptides.

[0311] 4.5. Binding of anti-C5 MAbs to C5 mutants Because three amino acid residues in the β-chain of C5, E48, D51, and K109, were predicted by crystal structure analysis to be involved in the binding of C5 to anti-C5 MAb, anti-C5 MAb was tested for binding to human C5 point mutants in Western blot analysis. C5 point mutants in which any one of E48, D51, and K109 was replaced with alanine were expressed in FS293 cells by lipofection. Culture medium was collected 5 days after lipofection and then used for Western blot. SDS-PAGE was performed under reducing conditions. The results are shown in Figure 6. Eculizumab bound to the α-chain of wild-type (WT) C5 and the three C5 point mutants, whereas CFA0305 bound strongly to the β-chain of WT C5, weakly to the E48A C5 mutant, and did not bind to the β-chain of the D51A and K109A C5 mutants. This indicates that these three amino acid residues are involved in antibody / antigen interaction. Table 5 shows a summary of the Western blot analysis of anti-C5 MAbs (CFA0305, CFA0307, ​​CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675). Although these anti-C5 MAbs are classified as the same epitope C, the binding patterns differ slightly between the antibodies, suggesting that the binding regions of C5 for these anti-C5 MAbs are close to each other but not identical.

[0312] [Table 5]

[0313] 4.6. BIACORE® BINDING ANALYSIS OF ANTI-C5 ANTIBODIES AND C5 MUTANTS To test whether residues E48, G51, and K109 are indeed involved in the antibody / antigen interaction, BIACORE® binding analysis was performed. Three C5 mutants: E48A, G51A, and K109A were prepared as described in Example 4.5. Culture supernatant samples containing mutant C5 overexpressed in FS293 cells were prepared at 40 μg / ml mutant C5. For BIACORE® binding analysis, samples were diluted 10-fold with BIACORE® running buffer (ACES pH 7.4, 10 mg / ml BSA, 1 mg / ml carboxymethyl dextran) to a final sample concentration of 4 μg / ml mutant C5.

[0314] The interaction of anti-C5 antibodies with the three C5 mutants was evaluated on a BIACORE® T200 instrument (GE Healthcare) at 37° C. using the assay conditions described in Example 3.2. ACES pH 7.4 buffer containing 10 mg / ml BSA, 1 mg / ml carboxymethyl dextran was used as the running buffer. Eculizumab-F760G4 and 305LO5 were captured on different flow cells by the Fc fragment-specific antibody monoclonal mouse anti-human IgG (GE Healthcare). Flow cell 1 was used as the reference surface. Wild-type and mutant C5 proteins were injected over the sensor surface at a concentration of 4 μg / ml and allowed to interact with the captured antibodies. At the end of each analysis cycle, the sensor surface was washed with 3M MgCl 2 The results were analyzed using Bia Evaluation software, version 2.0 (GE Healthcare). The curves of the reference flow cell (flow cell 1) and a blank injection of running buffer were subtracted from the curve of the flow cell with captured antibody.

[0315] As shown in Figure 7, all three C5 mutants were able to bind eculizumab with similar binding profiles compared to wild-type C5. With respect to 305LO5, all three mutants showed lower binding responses to 305LO5 compared to wild-type C5. The D51A and K109A mutants reduced the binding of C5 by 305LO5 to baseline levels.

[0316] 4.7. Identification of His residues on C5 that contribute to the pH-dependent interaction between anti-C5 antibodies and C5 Crystal structure analysis revealed 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 His residues on the antibody / antigen interface contribute to the pH-dependent interaction between anti-C5 antibodies and C5, BIACORE® binding analysis was performed. Three human C5 mutants with single His mutations (H70Y, H72Y, and H110Y) and one mutant with double His mutations (H70Y+H110Y) were generated as follows: single His mutants in which any one of H70, H72, and H110 was replaced with tyrosine, and double His mutants in which both H70 and H110 were replaced with tyrosine were expressed in FS293 cells by lipofection. The antigen binding properties of the C5 His mutants to the pH-dependent anti-C5 antibody 305LO5 were measured by a modified BIACORE® assay as described in Example 4.6. Briefly, to evaluate the pH-dependent dissociation between the antibody and antigen from the complex formed at pH 7.4, an additional dissociation step at pH 5.8 was incorporated into the BIACORE® assay immediately after the dissociation phase at pH 7.4. The dissociation rate at pH 5.8 was determined by processing and fitting the data using Scrubber 2.0 (BioLogic Software) curve fitting software.

[0317] As shown in FIG. 8, single His mutations and double His mutations (H70+H110) at H70 or H110 of C5 did not affect the binding of C5 to 305LO5 at neutral pH. Meanwhile, a single His mutation at H72 showed a significant decrease in the binding of C5 to 305LO5. The dissociation rates of C5 His mutants and C5-wt protein at pH 5.8 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. Compared to C5-wt, a single His mutation at H70 showed a nearly two-fold slower dissociation rate at pH 5.8, and a single His mutation at H110 resulted in a slightly slower dissociation rate at pH 5.8. Double His mutations at both H70 and H110 had a relatively large effect on pH-dependent binding, with the dissociation rate at pH 5.8 being nearly three-fold slower than C5-wt.

[0318] [Table 6]

[0319] Example 5 Inhibitory activity of anti-C5 antibodies against C5 activation 5.1. Inhibition of complement-activated liposome lysis by anti-C5 MAb Anti-C5 MAbs were 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 MAbs in a 96-well plate and incubated on a shaker at 25° C. for 30 min. 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 min. 50 μL of substrate solution (Autokit CH50) was added to each well and mixed by shaking at 25° C. for 2 min. The final mixture was incubated at 37° C. for 40 min, after which the OD of the mixture was measured at 340 nm. Percent liposome lysis was calculated as 100×[(OD MAb - O.D. 血清およびリポソームバックグラウンド)] / [(OD MAbなし - O.D. 血清およびリポソームバックグラウンド ) was defined as the pH-independent antibody that inhibited liposome lysis. Figure 9A shows that the anti-C5 MAbs CFA0305, CFA0307, ​​CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675 inhibited liposome lysis. Two pH-independent antibodies CFA0330 and CFA0341 also inhibited the lysis (Figure 9B).

[0320] 5.2. Inhibition of C5a production by anti-C5 MAb To confirm that anti-C5 MAbs inhibit the cleavage of C5 into C5a and C5b, anti-C5 MAbs were tested for C5a generation upon liposome lysis. C5a levels in the supernatant from the liposome lysis assay were quantified using a C5a ELISA kit (R&D systems, DY2037). All MAbs dose-dependently inhibited C5a generation in the supernatant (Figures 10A and 10B).

[0321] 5.3. Inhibition of complement-activated hemolysis by anti-C5 MAb Anti-C5 MAbs were tested for inhibition of classical complement activity in a hemolytic assay. Chicken red blood cells (cRBCs) (Innovative research, IC05-0810) were incubated with 0.5 mM MgCl 2 and 0.15 mM CaCl 2 The cells were then washed with gelatin / veronal buffered saline (GVB++) (Boston BioProducts, IBB-300X) containing 1 μg / ml of chicken RBC antibody (Rockland 103-4139) for 15 min at 4° C. The cells were then washed with GVB++ and 5×10 cells were soaked in the same buffer. 7cRBCs were suspended at 1000xg / 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 on a shaker at 37°C for 30 min. Then, 60 μl of sensitized cRBC suspension was added to the wells containing the serum and the antibody mixture was incubated at 37°C for 30 min. After incubation, the plate was centrifuged at 1000xg for 2 min at 4°C. Supernatants (100 μl) were transferred to wells of a flat-bottom 96-well microtest plate for measuring OD at 415 nm with a reference wavelength of 630 nm. Percent hemolysis was calculated as 100x[(OD MAb - O.D. 血清およびcRBC )] / [(OD MAbなし - O.D. 血清およびcRBCバックグラウンド Figure 11 shows that the anti-C5 MAbs CFA0305 and 305LO5 inhibited hemolysis of cRBCs.

[0322] 5.4. Inhibition of the alternative complement pathway by anti-C5 MAb The hemolysis assay for the alternative pathway was performed in a similar manner to the classical pathway hemolysis assay. Blood drawn from New Zealand white rabbits (InVivos) was mixed with an equal volume of Alsever's solution (Sigma, A3551), and the mixture was used as rabbit RBCs (rRBCs). rRBCs were diluted with 2 mM MgCl 2 and washed with GVB supplemented with 10 mM EGTA, and then soaked in the same buffer with 7 × 10 cells. 8rRBCs were suspended at 1000 / ml. 40 μl of normal human serum (25%) (Biopredic, SER019) was mixed with 40 μl of diluted MAb in a round-bottom 96-well microtest plate and incubated on a shaker for 30 min at 37° C. 20 μl of rRBC suspension was then added to the wells containing the serum and the antibody mixture was incubated for 60 min at 37° C. After incubation, the plate was centrifuged at 1000×g for 2 min at 4° C. Supernatants (70 μl) were transferred to wells on a flat-bottom 96-well microtest plate for measuring OD at 415 nm with a reference wavelength of 630 nm. Figure 12 shows that anti-C5 MAbs: CFA0305 and CFA0672 inhibited hemolysis of rRBCs, indicating that these antibodies inhibit the alternative complement pathway.

[0323] Example 6 Pharmacokinetic study of anti-C5 monoclonal antibody with human C5 in mice 6.1. In vivo studies using C57BL / 6 mice The in vivo kinetics of human C5 and anti-human C5 antibodies were assessed after administration of human C5 (Calbiochem) alone or human C5 and anti-human C5 antibodies to C57BL / 6 mice (In Vivos or Biological Resource Centre, Singapore). Human C5 solution (0.01 mg / ml) or a mixed solution containing human C5 and anti-human C5 antibody (0.01 mg / ml and 2 mg / ml (CFA0305-F760G4, CFA0307-F760G4, CFA0366-F760G4, CFA0501-F760G4, CFA0538-F760G4, CFA0599-F760G4, CFA0666-F760G4, CFA0672-F760G4, and CFA0675-F760G4) or 0.2 mg / ml (CFA0330-F760G4 and CFA0341-F760G4), respectively) was administered once into the tail vein at a dose of 10 ml / kg. In this case, anti-human C5 antibodies are present in excess relative to human C5, so it is believed that almost all human C5 is bound to the antibodies. Blood samples were 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 at 4°C for 10 minutes to separate plasma. The separated plasma was stored in a freezer at -80°C until assay. The anti-human C5 antibodies used were as follows: CFA0305-F760G4, CFA0307-F760G4, CFA0330-F760G4, CFA0341-F760G4, CFA0366-F760G4, CFA0501-F760G4, CFA0538-F760G4, CFA0599-F760G4, CFA0666-F760G4, CFA0672-F760G4, and CFA0675-F760G4 as described above.

[0324] 6.2. Measurement of plasma total human C5 concentration by electrochemiluminescence (ECL) assay Total human C5 concentrations in mouse plasma were measured by ECL.

[0325] For plasma samples containing only CFA0330-F760G4, CFA0341-F760G4, or human C5, the following method was used. Anti-human C5 antibody (Santa Cruz) was dispensed into an untreated MULTI-ARRAY 96-well plate (Meso Scale Discovery) and left to stand overnight at 4°C to prepare an anti-human C5 solid-phase plate. Calibration curve samples and mouse plasma samples diluted 100-fold or more with 1 μg / ml of injected antibody (CFA0330-F760G4 or CFA0341-F760G4) were prepared and incubated at 37°C for 30 minutes. The samples were then dispensed into the anti-human C5 solid-phase plate and left to stand at room temperature for 1 hour. SULFO-TAG-labeled anti-human IgG antibody (Meso Scale Discovery) was then added, reacted at room temperature for 1 hour, and washed. Immediately afterwards, Read Buffer T (x4) (Meso Scale Discovery) was dispensed and measurements were performed using a Sector Imager 2400 (Meso Scale Discovery).

[0326] For plasma samples containing CFA0305-F760G4, CFA0307-F760G4, CFA0366-F760G4, CFA0501-F760G4, CFA0538-F760G4, CFA0599-F760G4, CFA0666-F760G4, CFA0672-F760G4, or CFA0675-F760G4, the following method was used: Anti-human C5 antibody (CFA0329-F939G4; VH, SEQ ID NO: 23 and VL, SEQ ID NO: 27) was dispensed into an untreated MULTI-ARRAY 96-well plate (Meso Scale Discovery) and left to stand overnight at 4°C to prepare an anti-human C5 solid-phase plate. A calibration curve sample and a mouse plasma sample diluted 100-fold 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 an anti-human C5 solid-phase plate and allowed to stand at room temperature for 1 hour. SULFO-TAG-labeled anti-human C5 antibody (CFA0300-F939G4; VH, SEQ ID NO: 24 and VL, SEQ ID NO: 28) was then added, reacted at room temperature for 1 hour, and washed. Immediately after that, Read Buffer T (x4) (Meso Scale Discovery) was dispensed, and measurements were performed using a Sector Imager 2400 (Meso Scale Discovery).

[0327] The human C5 concentration was calculated using the analysis software SOFTmax PRO (Molecular Devices) based on the response of the calibration curve. The time course of the plasma human C5 concentration after intravenous administration measured by this method is shown in Figure 13. The data are plotted as the remaining percentage compared to the plasma human C5 concentration at 5 minutes.

[0328] 6.3. Measurement of plasma anti-human C5 antibody concentrations by ECL assay Anti-human C5 antibody concentration in mouse plasma was measured by ECL. Anti-human IgG (γ-chain specific) F(ab')2 antibody fragment (Sigma) or anti-human IgG κ-chain antibody (Antibody Solutions) was dispensed into an untreated MULTI-ARRAY 96-well plate (Meso Scale Discovery) and left to stand overnight at 4°C to prepare an anti-human IgG solid-phase plate. A calibration curve sample and a mouse plasma sample diluted 100-fold or more were prepared. The samples were then dispensed into the anti-human IgG solid-phase plate and left to stand at room temperature for 1 hour. Next, a biotinylated anti-human IgG antibody (Southernbiotech) or a SULFO-TAG-labeled anti-human IgG Fc antibody (Southernbiotech) was added, reacted at room temperature for 1 hour, and washed. Then, only when a biotinylated anti-human IgG antibody was used, a SULFO-TAG-labeled streptavidin (Meso Scale Discovery) was added, reacted at room temperature for 1 hour, and washed. Immediately after, Read Buffer T (x4) (Meso Scale Discovery) was dispensed and measurements were performed using a Sector Imager 2400 (Meso Scale Discovery). The anti-human C5 antibody concentration was calculated using the analysis software SOFTmax PRO (Molecular Devices) based on the response of the calibration curve. Figure 14 shows the time course of the plasma anti-human C5 antibody concentration after intravenous administration measured by this method. The data are plotted as the remaining percentage compared to the plasma anti-human C5 antibody concentration at 5 minutes.

[0329] 6.4. Effect of pH-Dependent Anti-Human C5 Antibody Binding on Human C5 Clearance 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-F760G4) and pH-independent anti-human C5 antibodies (CFA0330-F760G4 and CFA0341-F760G4) were tested in vivo and the resulting plasma anti-human C5 antibody and plasma human C5 concentrations were compared. As shown in FIG. 14, the antibody exposure was comparable. On the other hand, the clearance of human C5 administered simultaneously with a pH-dependent anti-human C5 antibody was accelerated compared to the clearance of human C5 administered simultaneously with a pH-independent anti-human C5 antibody (FIG. 13).

[0330] Example 7 Optimization of anti-C5 monoclonal antibody (305 variant) Several mutations were introduced into the optimized variable region of anti-C5 antibody 305LO5 to further improve its properties, generating optimized variable regions 305LO15, 305LO16, 305LO18, 305LO19, 305LO20, 305LO22, and 305LO23. The amino acid sequences of the VH and VL of these 305 variants are shown in Tables 7 and 8, respectively. The gene encoding the humanized VH was combined with the modified human IgG1 CH variant SG115 (SEQ ID NO: 114), and the modified human IgG4 CH variant SG422 (SEQ ID NO: 115) or SG429 (SEQ ID NO: 116). The gene encoding the humanized VL was combined with human CL (SK1, SEQ ID NO: 38). Separately, heavy and light chain genes encoding the humanized anti-C5 antibody BNJ441 (BNJ441H, SEQ ID NO: 149; BNJ441L, SEQ ID NO: 150) were synthesized and each was cloned into an expression vector.

[0331] Antibodies were expressed in HEK293 cells co-transfected with a combination of heavy and light chain expression vectors and protein A purified.

[0332] [Table 7]

[0333] [Table 8]

[0334] Example 8 Binding characterization of anti-C5 antibody (305 mutant) The kinetic parameters of anti-C5 antibodies against recombinant human C5 were evaluated at 37°C using a BIACORE® T200 instrument (GE Healthcare) under three different conditions: (1) pH 7.4 for both binding and dissociation, (2) pH 5.8 for both binding and dissociation, and (3) pH 7.4 for binding and pH 5.8 for dissociation. ProA / G (Pierce) was immobilized on a CM1 sensor chip using an amine coupling kit (GE Healthcare) according to the settings recommended by GE Healthcare. For conditions (1) and (3), the antibodies and analytes were dissolved 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 for condition (2), they 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) in 100 mM NaOH. Each antibody was captured on the sensor surface by ProA / G. The antibody capture level was generally 60-90 resonance units (RU). Recombinant human C5 was then prepared by 3-fold serial dilution and injected at 3-27 nM or 13.3-120 nM, followed by dissociation. The surface was regenerated with 25 mM NaOH. Using BIACORE® T200 Evaluation software, version 2.0 (GE Healthcare), the kinetic parameters under conditions (1) and (2) were determined by fitting the sensorgrams with a 1:1 binding model, and the dissociation rate under condition (3) was measured by fitting the sensorgrams with a 1:1 MCK dissociation model. The pH dependence of all antibodies was expressed as the ratio of the dissociation rates under conditions (2) and (1).

[0335] The association rates (ka), dissociation rates (kd), binding affinities (KD), and pH dependencies are shown in Table 9. All antibodies showed faster dissociation rates at pH 5.8 than at pH 7.4, and their pH dependencies were approximately 20-fold.

[0336] [Table 9]

[0337] The binding affinity of anti-C5 antibodies (BNJ441, eculizumab, and 305 variant) to recombinant human C5 at pH 7.4 and pH 5.8 was measured at 37°C using a BIACORE® T200 instrument (GE Healthcare) to assess the effect of pH on 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 immobilized in 20 mM ACES, 150 mM NaCl, 1.2 mM CaCl 2 , 0.05% Tween 20, and 0.005% NaN 3Antibodies were captured to the sensor surface using the anti-Fc method. Capture levels were typically 50-80 resonance units (RU). Recombinant human C5 was prepared by a 3-fold serial dilution starting at 27 nM for pH 7.4 assay conditions and 135 nM for pH 5.8 assay conditions. Surfaces were regenerated with 20 mM HCl, 0.01% Tween 20. Data were processed and fitted with a 1:1 binding model using BiaEvaluation 2.0 software (GE Healthcare).

[0338] The binding affinity (KD) of BNJ441, eculizumab, and the 305 variant for recombinant human C5 at pH 7.4 and pH 5.8 is shown in Table 10. The 305 variant exhibited a (KD at pH 5.8) / (KD at pH 7.4) ratio of nearly 800, which was 8-fold higher than BNJ441, which exhibited a (KD at pH 5.8) / (KD at pH 7.4) ratio of only 93.

[0339] [Table 10]

[0340] Example 9 Inhibitory activity of anti-C5 antibody (305 mutant) on C5 activation 9.1. Inhibition of Complement-Activated Liposome Lysis by Anti-C5 MAb Anti-C5 MAbs were tested for inhibition of complement activity by liposome lysis assay. 30 μL of normal human serum (6.7%) (Biopredic, SER019) was mixed with 20 μL of diluted MAbs in a 96-well plate and incubated on a shaker at room temperature for 30 min. A liposome solution sensitized with antibodies against dinitrophenyl (Autokit CH50, Wako, 995-40801) was transferred to each well and placed on a shaker at 37°C for 2 min. 50 μL of substrate solution (Autokit CH50) was added to each well and mixed by shaking at 37°C for 2 min. The final mixture was incubated at 37°C for 40 min, after which the OD at 340 nm was measured. Percent liposome lysis was calculated as 100×[(OD MAb - O.D. 血清およびリポソームバックグラウンド )] / [(OD MAbなし - O.D. 血清およびリポソームバックグラウンド ) was defined as the number of C5 MAbs that inhibited liposome lysis. Figure 15 shows that the anti-C5 MAbs 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 (Figure 16).

[0341] Anti-C5 MAbs were 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 incubated on a shaker at 37° C. for 1 h. Liposomes (Autokit CH50) were transferred to each well and placed on a shaker at 37° C. for 2 min. 50 μL of substrate solution (Autokit CH50) was added to each well and mixed by shaking at 37° C. for 2 min. The final mixture was incubated at 37° C. for 180 min, after which the OD at 340 nm was measured. Percent liposome lysis was defined as above. FIG. 17 shows that the anti-C5 MAbs: 305LO22-SG115, 305LO22-SG422, 305LO23-SG115, and 305LO23-SG422 inhibited liposome lysis.

[0342] 9.2. Inhibition of C5a production by anti-C5 MAbs To confirm that anti-C5 MAbs inhibit the cleavage of C5 into C5a and C5b, anti-C5 MAbs were tested for the generation of C5a during liposome lysis. C5a levels in the supernatant from the liposome lysis assay were quantified using a C5a ELISA kit (R&D systems, DY2037). All MAbs dose-dependently inhibited C5a generation in the supernatant (Figures 18 and 19).

[0343] 9.3. Measurement of complement activity in cynomolgus monkey plasma Anti-C5 MAbs were tested for inhibition of complement activity in cynomolgus monkey plasma. Anti-C5 MAbs were administered to monkeys (20 mg / kg) and plasma samples were collected periodically up to day 56. Chicken red blood cells (cRBCs) (Innovative research, IC05-0810) were incubated with 0.5 mM MgCl 2 and 0.15 mM CaCl 2The cells were then washed with gelatin / veronal buffered saline (GVB++) (Boston BioProducts, IBB-300X) containing 1 μg / ml of anti-chicken RBC antibody (Rockland 103-4139) for 15 min at 4° C. The cells were then washed with GVB++ and aliquots of 1×10 cells were placed in the same buffer. 8 In a separate round-bottom 96-well microtest plate, monkey plasma was incubated with the sensitized cRBCs for 20 min at 37°C. After incubation, the plates were centrifuged at 1000 x g for 2 min at 4°C. Supernatants were transferred to wells on a flat-bottom 96-well microtest plate for measuring OD at 415 nm with a reference wavelength of 630 nm. Percent hemolysis was calculated as 100 x [(OD 投与後 - O.D. 血漿およびcRBCバックグラウンド )] / [(OD 投与前 - O.D. 血漿およびcRBCバックグラウンド Figure 20 shows that anti-C5 MAbs 305LO15-SG422, 305LO15-SG115, 305LO16-SG422, 305LO18-SG422, 305LO19-SG422, 305LO20-SG422, 305LO20-SG115, and 305LO23-SG115 inhibited complement activity in plasma.

[0344] 9.4. Inhibition of Biological Activity of C5 Mutants by Anti-C5 MAbs Anti-C5 MAbs were tested for inhibition of recombinant human C5 mutants: V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N, and E1437D. It has been reported that PNH patients with the R885H mutation in C5 show poor response to eculizumab (see, e.g., Nishimura et al., New Engl. J. Med. 370:632-639 (2014)). Each of the human C5 mutants was expressed in FS293 cells, and the supernatant was used for the following studies. 10 μL of C5-deficient human serum (Sigma, C1163) was mixed with 20 μL of diluted MAb and 20 μL of recombinant C5 mutant (2-3 μg / mL)-containing cell culture medium in a 96-well plate and incubated on a shaker at 37°C for 0.5 h. Liposomes (Autokit CH50) were transferred to each well and placed on a shaker for 2 min at 37° C. 50 μL of substrate solution (Autokit CH50) was added to each well and mixed by shaking for 2 min at 37° C. The final mixture was incubated at 37° C. for 90 min, after which the OD at 340 nm was measured. Percent liposome lysis was defined as above. Figure 21 shows that anti-C5 MAb (eculizumab) did not inhibit the R885H C5 mutant, but inhibited the other mutants tested. Figure 22 shows that anti-C5 MAb (305 mutant) inhibited all C5 mutants tested.

[0345] 9.5. Inhibition of Complement-Activated Liposome Lysis by Anti-C5 MAb Anti-C5 MAbs were tested for inhibition of complement activity by liposome lysis assay. 30 μL of normal human serum (6.7%) (Biopredic, SER019) was mixed with 20 μL of diluted MAb in a 96-well plate and incubated on a shaker at room temperature for 30 min. A liposome solution sensitized with antibodies against dinitrophenyl (Autokit CH50, Wako, 995-40801) was transferred to each well and placed on a shaker at 25° C. for 2 min. 50 μL of substrate solution (Autokit CH50) was added to each well and mixed by shaking at 25° C. for 2 min. The final mixture was incubated at 37° C. for 45 min, after which the OD at 340 nm was measured. The percent inhibition of liposome lysis was calculated as 100×[(OD MAb - O.D. 血清およびリポソームバックグラウンド )] / [(OD MAbなし - O.D. 血清およびリポソームバックグラウンド Figure 23 shows that anti-C5 MAb BNJ441 and the 305 mutant inhibited liposome lysis, and that the 305 mutant had stronger inhibitory activity than BNJ441.

[0346] Example 10 Pharmacokinetic study of anti-C5 monoclonal antibody (305 variant) in cynomolgus monkeys 10.1. In vivo studies using cynomolgus monkeys The in vivo kinetics of anti-human C5 antibody after administration to cynomolgus monkeys (Shin Nippon Kagaku Co., Ltd., Japan) was evaluated. Anti-human C5 antibody solution (2.5 mg / ml) was administered once via 30-minute infusion into the cephalic vein of the forearm at a dose of approximately 8 ml / kg. Blood was collected before administration, and 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×g and 4° C. for 10 minutes to separate plasma. The separated plasma was stored in a freezer at −70° C. or lower until assay. Anti-human C5 antibody was prepared as described in Example 7.

[0347] 10.2. Measurement of Plasma Total Cynomolgus C5 Concentrations by ELISA Assay The total cynomolgus C5 concentration in cynomolgus plasma was measured by ELISA. Anti-human C5 antibody (in-house antibody produced using the method described in Example 2) was dispensed into Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and left to stand overnight at 4°C to prepare an anti-cynomolgus C5 solid-phase plate. A calibration curve sample and a cynomolgus plasma sample diluted 20,000 times with 0.4 μg / ml of injected antibody were prepared and incubated at 37°C for 60 minutes. The samples were then dispensed into an anti-cynomolgus C5 solid-phase plate and left to stand at room temperature for 1 hour. Then, an HRP-labeled anti-human IgG antibody (SouthernBiotech) was added, reacted at room temperature for 30 minutes, and washed. Then, ABTS ELISA HRP substrate (KPL) was added. The signal was measured at a wavelength of 405 nm using a plate reader. Cynomolgus C5 concentrations were calculated using the analysis software SOFTmax PRO (Molecular Devices) based on the response of the calibration curve. The time course of plasma cynomolgus C5 concentrations after intravenous administration measured by this method is shown in FIG. 24. Data are plotted as the remaining percentage compared to the plasma cynomolgus C5 concentration at the pre-administration time point. 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 lower plasma C5 accumulation compared to pH-independent anti-human C5 antibodies.

[0348] 10.3. Measurement of plasma anti-human C5 antibody concentrations by ELISA assay The anti-human C5 antibody concentration in cynomolgus monkey plasma was measured by ELISA. Anti-human IgG κ chain antibody (Antibody Solutions) was dispensed into Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and left to stand overnight at 4 °C to prepare an anti-human IgG solid-phase plate. A calibration curve sample and a cynomolgus monkey plasma sample diluted 100 times or more were prepared. The sample was then dispensed into the anti-human IgG solid-phase plate and left to stand at room temperature for 1 hour. Next, HRP-labeled anti-human IgG antibody (SouthernBiotech) was added, reacted at room temperature for 30 minutes, and washed. Then, ABTS ELISA HRP substrate (KPL) was added. The signal was measured at a wavelength of 405 nm using a plate reader. The anti-human C5 antibody concentration was calculated using the analysis software SOFTmax PRO (Molecular Devices) based on the response of the calibration curve. Figure 25 shows the time course of the anti-human C5 antibody concentration in plasma after intravenous administration measured by this method. The 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) exhibited a longer half-life compared to the pH-independent anti-human C5 antibodies.

[0349] Example 11 X-ray crystal structure analysis of the complex between 305 mutant Fab and human C5-MG1 domain 11.1. Expression and purification of the MG1 domain of human C5 (20-124) The MG1 domain (amino acid residues 20-124 of SEQ ID NO: 39) fused to a GST tag via a thrombin-cleavable linker (GST-MG1) was expressed in E. coli BL21 DE3 pLysS strain (Promega) using the pGEX-4T-1 vector (GE healthcare). Protein expression was induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 5 h at 25°C. Bacterial cell pellets were lysed with Bugbuster (Merck) supplemented with lysonase (Merck) and complete protease inhibitor cocktail (Roche), and GST-MG1 was subsequently purified from the soluble fraction using a GSTrap column (GE healthcare) according to the manufacturer's instructions. The GST tag was cleaved with thrombin (Sigma) and the resulting MG1 domain was further purified on a Superdex 75 gel filtration column (GE healthcare). Fractions containing the MG1 domain were pooled and stored at -80°C.

[0350] 11.2. Preparation of Fab fragments of 305 mutants Fab fragments of one of the optimized variants from 305 were prepared by conventional methods using restriction digestion with papain (Roche Diagnostics, Cat. No. 1047825), followed by loading onto a Protein A column (MabSlect SuRe, GE Healthcare) to remove the Fc fragment, a cation exchange column (HiTrap SP HP, GE Healthcare), and a gel filtration column (Superdex200 16 / 60, GE Healthcare). Fractions containing the Fab fragment were pooled and stored at -80°C.

[0351] Preparation of a Complex of 11.3.305 Mutant Fab with Human C5-MG1 Domain Purified recombinant human C5-MG1 domain was mixed with purified 305 mutant Fab fragment at a molar ratio of 1:1. The complex was purified by gel filtration chromatography (Superdex200 10 / 300 increase, GE Healthcare) using a column equilibrated with 25 mM HEPES pH 7.5, 100 mM NaCl.

[0352] 11.4.Crystallization The purified complex was concentrated to approximately 10 mg / mL and crystallized at 4 °C by the sitting-drop vapor diffusion method combined with the seeding method. The reservoir solution consisted of 0.2 M magnesium formate anhydrous, 15.0% w / v polyethylene glycol 3350. This allowed plate-shaped crystals to be successfully obtained within 2 to 3 days. The crystals were immersed in a solution of 0.2 M magnesium formate anhydrous, 25.0% w / v polyethylene glycol 3350, and 20% glycerol.

[0353] 11.5. Data collection and structure determination X-ray diffraction data were measured at BL32XU of SPring-8. During the measurements, the crystal was kept frozen under a nitrogen flow at −178° C., and a total of 180 X-ray diffraction images were collected by rotating the crystal 1.0° at a time using an MX-225HS CCD detector (RAYONIX) connected to the beamline. The cell parameters were determined, the diffraction spots were indexed, and the diffraction data obtained from the diffraction images were processed using the Xia2 program (J. Appl. Cryst. 43:186-190 (2010)), the XDS package (Acta. Cryst. D66:125-132 (2010)), and Scala (Acta. Cryst. D62:72-82 (2006)), and finally the diffraction intensity data was obtained to a resolution of 2.11 Å. The crystallographic data statistics are shown in Table 11.

[0354] [Table 11]

[0355] The structure was determined by molecular replacement 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 derived from the published human C5 crystal structure (PDB code: 3CU7, Nat.Immunol. 9:753-760 (2008)). The model was constructed with the program Coot (Acta Cryst. D66:486-501 (2010)) and refined with the program Refmac5 (Acta Cryst. D67:355-367 (2011)). The crystallographic confidence factor (R) of the diffraction intensity data from 25-2.11 Å was 20.42%, and the free R value was 26.44%. The structure refinement statistics are shown in Table 11.

[0356] Overall structure of the complex between 11.6.305 mutant Fab and the C5-MG1 domain The Fab fragment of an optimized variant derived from 305 ("305 Fab") bound to the human C5-MG1 domain ("MG1") in a 1:1 ratio, and the asymmetric unit of the crystal structure contained two complexes, molecule 1 and molecule 2, as shown in Figure 26A. Molecules 1 and 2 are well superimposable with an RMSD of 0.717 Å for Cα atom positions at all residues, as shown in Figure 26B. The figures described below were generated using molecule 1.

[0357] In Figures 27A and 27B, epitopes in the 305 Fab contact region are mapped in the MG1 amino acid sequence and in the crystal structure, respectively. The epitopes include amino acid residues of MG1 that include one or more atoms located within 4.5 Å of any part of the 305 Fab in the crystal structure. Additionally, epitopes within 3.0 Å are highlighted in Figure 27A.

[0358] 11.7.Interaction of E48, D51, and K109 Anti-C5 MAbs including the 305 antibody series were tested for binding to three human C5 point mutants, E48A, D51A, and K109A, by Western blot and BIACORE® binding analysis as described in Examples 4.5 and 4.6. The 305 mutants bound strongly to WT C5, but only weakly to the E48A C5 mutant and did not bind to the D51A and K109A mutants. The crystal structure of the complex of 305 Fab with MG1 revealed that the three amino acids, E48, D51, and K109, are all within 3.0 Å of the 305 Fab and form several hydrogen bonds with the Fab, as shown in Figure 28A. In more detail, the K109 residue of MG1 is buried in a groove formed at the interface of the heavy chain of Fab, and strongly interacts with Fab through three hydrogen bonds with H-CDR3_G97, H-CDR3_Y100, and H-CDR3_T100b, as well as through a salt bridge with H-CDR3_D95 (Figure 28D). D51 is located between MG1 and the heavy chain of 305 Fab, and forms two hydrogen bonds with H-CDR1_Ser32 and H-CDR2_Ser54 to fill the space (Figure 28C). These indicate that both K109 and D51 of C5 are crucial residues for the binding of the 305 antibody series. On the other hand, E48 is located relatively close to the surface and forms only one hydrogen bond with Fab, suggesting that its contribution to antibody binding would be less than that of K109 and E51 (Figure 28B). These relationships are consistent with the results of Western blot and BIACORE® binding analysis of human C5 variants (Examples 4.5 and 4.6). Note: Fab amino acid residue numbering is based on the Kabat numbering scheme. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0359] 11.8. Interaction of H70, H72, and H110 of human C5 with the 305 antibody series Crystal structure analysis revealed that three histidine residues on human C5, namely H70, H72, and H110, are included in the epitope of the 305 mutant Fab, as shown in Figure 27A and Figure 29A. To investigate the contribution of these histidine residues to the pH-dependent protein-protein interaction between human C5 and the 305 mutant Fab, BIACORE® binding analysis was performed with human C5 mutants H70Y, H72Y, H110Y, and H70Y+H110Y (Example 4.7). H72Y resulted in a complete loss of binding of the 305 mutant Fab to C5. This residue of C5 is located within the pocket formed by the CDR2 loop of the heavy chain of the 305 Fab and the loop of MG1 (L73, S74, and E76), as shown in Figure 29C, and fills this space tightly. In addition, the H72 residue of C5 forms a hydrogen bond with H-CDR2_Y58. The H72Y mutation is expected to be poorly tolerated, as there is not enough space to accommodate the relatively bulky side chain of tyrosine. It also cannot maintain a hydrogen bond with H-CDR2_Y58. Regarding the contribution of H70 and H110 to the pH dependence, the H70Y and H110Y mutations led to a relatively slow dissociation of the 305 mutant Fab from C5 at pH 5.8. H70 forms an intramolecular hydrogen bond with T53 of MG1, but when H70 of C5 is protonated at pH 5.8, a conformational change occurs at the corresponding part of the interaction interface of MG1, disrupting this hydrogen bond (Figure 29B). As for H110, the protonation of this C5 residue is expected to cause a charge repulsion towards the 305 Fab, which can be enhanced by the protonation of the adjacent histidine residue H-CDR3_H100c (Figure 29D).

[0360] The foregoing invention has been described in detail by way of illustration and example for purposes of facilitating a clear understanding, but the descriptions and illustrations herein should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated herein by reference in their entireties.

Claims

1. (a) Amino acid sequence DX 1 GYX 2 X 3 PTHAMX 4 X 5 (where X 1 is G, A, X 2 is V, Q or D, X 3 is T, Y, X 4 is Y, H, or X 5 is L or Y) (SEQ ID NO: 128); and (b) HVR-H3 comprising the amino acid sequence QX 1 TX 2 VGSSYGNX 3 (where X 1 is S, C, N or T, X 2 is F, K, X 3 is A, T or H) (SEQ ID NO: 131); and (c) an HVR-L3 comprising the 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, F, X 3 is T, D or E, X 4 is Y, K or Q, X 5 is S, D or E, X 6 and HVR-H2 comprising HVR-H3 comprising HVR-H4 comprising HVR-H5 comprising HVR-H6 comprising HVR-H7 comprising HVR-H8 comprising HVR-H9 comprising HVR-H10 comprising HVR-H11 comprising HVR-H20 comprising HVR-H11 comprising HVR-H12 comprising HVR-H20 comprising HVR-H1 ...20 comprising HVR-H11 comprising HVR-H12 comprising H

2. (a) Amino acid sequence SSYYX 1 X 2 (where X 1 is M, V, X 2 is C or A) (SEQ ID NO: 126); and (b) HVR-H1 comprising the 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, F, X 3 is T, D or E, X 4 is Y, K or Q, X 5 is S, D or E, X 6 is A or V) (SEQ ID NO: 127), and (c) an HVR-H2 having the amino acid sequence DX 1 GYX 2 X 3 PTHAMX 4 X 5 (where X 1 is G, A, X 2 is V, Q or D, X 3 is T, Y, X 4 is Y, H, or X 5 and HVR-H3 comprising HVR-H5 comprising HVR-H6 comprising HVR-H7 comprising HVR-H8 comprising HVR-H9 comprising HVR-H10 comprising HVR-H11 comprising HVR-H20 comprising HVR-H3 comprising HVR-H4 comprising HVR-H5 comprising HVR-H6 comprising HVR-H7 comprising HVR-H8 comprising HVR-H9 comprising HVR-H10 comprising HVR-H11 comprising HVR-H20 comprising HVR-H11

3. (a) Amino acid sequence X 1 ASQX 2 IX 3 SX 4 LA (where X 1 is Q, R, X 2 is N, Q or G, X 3 is G, S, or X 4 is D, K or S) (SEQ ID NO: 129); and (b) an HVR-L1 comprising the amino acid sequence GASX 1 X 2 X 3 S (where X 1 is K, E or T, X 2 is L, T, X 3 is A, H, E or Q) (SEQ ID NO: 130); and (c) an HVR-L2 comprising the amino acid sequence QX 1 TX 2 VGSSYGNX 3 (where X 1 is S, C, N or T, X 2 is F, K, X 3 The antibody of claim 2, further comprising HVR-L3 comprising the following SEQ ID NO: 131:

4. (a) Amino acid sequence X 1 ASQX 2 IX 3 SX 4 LA (where X 1 is Q, R, X 2 is N, Q or G, X 3 is G, S, or X 4 is D, K or S) (SEQ ID NO: 129); and (b) an HVR-L1 comprising the amino acid sequence GASX 1 X 2 X 3 S (where X 1 is K, E or T, X 2 is L, T, X 3 is A, H, E or Q) (SEQ ID NO: 130); and (c) an HVR-L2 comprising the amino acid sequence QX 1 TX 2 VGSSYGNX 3 (where X 1 is S, C, N or T, X 2 is F, K, X 3 and HVR-L3 comprising:

5. The antibody of claim 2, further comprising a heavy chain variable domain framework FR1 comprising any one of the amino acid sequences of SEQ ID NOs: 132 to 134; FR2 comprising any one of the amino acid sequences of SEQ ID NOs: 135 to 136; FR3 comprising any one of the amino acid sequences of SEQ ID NOs: 137 to 139; and FR4 comprising any one of the amino acid sequences of SEQ ID NOs: 140 to 141.

6. The antibody of claim 4, further comprising a light chain variable domain framework FR1 comprising any one of the amino acid sequences of SEQ ID NOs: 142-143; FR2 comprising any one of the amino acid sequences of SEQ ID NOs: 144-145; FR3 comprising any one of the amino acid sequences of SEQ ID NOs: 146-147; and FR4 comprising the amino acid sequence of SEQ ID NO:

148.

7. An isolated antibody that binds to C5, comprising: (a) a VH sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 10, 106-110; (b) a VL sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 20, 111-113; or (c) a VH sequence of (a) and a VL sequence of (b).

8. The antibody of claim 7, comprising any one of the VH sequences of SEQ ID NOs: 10, 106 to 110.

9. The antibody of claim 7, comprising any one of the VL sequences of SEQ ID NOs: 20, 111 to 113.

10. An antibody comprising any one of the VH sequences of SEQ ID NOs: 10, 106-110 and any one of the VL sequences of SEQ ID NOs: 20, 111-113.

11. The antibody of any one of claims 1 to 10, which is a full length IgG1 antibody or a full length IgG4 antibody.

12. An isolated nucleic acid encoding an antibody according to any one of claims 1 to 11.

13. A host cell comprising the nucleic acid of claim 12.

14. 14. A method for producing an antibody, comprising culturing the host cell of claim 13 so that the antibody is produced.

15. A pharmaceutical formulation comprising the antibody of any one of claims 1 to 11 and a pharma- ceutically acceptable carrier.

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