JP2017226655A - Anti-c5 antibodies and methods of use - Google Patents

Abstract

PROBLEM TO BE SOLVED: 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

  The present invention relates to anti-C5 antibodies and methods of use thereof.

  The complement system plays a central role in the clearance of immune complexes and in the immune response against infectious agents, foreign antigens, virus-infected cells and tumor cells. There are about 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-bound events. The resulting complement cascade leads to the production of products with opsonin function, immunomodulatory function, and cytolytic function.

  It is now widely accepted that the complement system is activated through three different pathways: the classical pathway, the lectin pathway, and the alternative pathway. These pathways share many elements and are different in the early stages, but converge and share the same terminal complement components (C5 to C9) involved in target cell activation and destruction .

  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 C-type lectin CL-11. It is. In contrast, the alternative pathway spontaneously undergoes low levels of turnover activation, which is easily amplified on foreign or other abnormal surfaces (bacteria, yeast, virus-infected cells, or damaged tissue) obtain. These pathways converge at the point where complement component C3 is cleaved by the active protease to yield C3a and C3b.

  C3a is an anaphylatoxin. C3b binds not only to bacteria and other cells, but also to specific viruses and immune complexes and tags them for removal from the circulation (a role known as opsonin). C3b also forms a complex with other components to form C5 convertase, which cleaves C5 into C5a and C5b.

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

  Mature C5 is cleaved into C5a and C5b fragments during complement pathway activation. C5a is cleaved from the C5 alpha chain by the C5 convertase as an amino terminal fragment containing the first 74 amino acids of the alpha chain. The remaining part of mature C5 is fragment C5b, which contains the remainder of the α chain disulfide bonded to the β chain. About 20% of the 11 kDa mass of C5a is attributed to carbohydrates.

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

  As mentioned above, C3a and C5a are anaphylatoxins. They can induce mast cell degranulation, which releases histamine and other inflammatory mediators, which cause smooth muscle contraction, increased vascular permeability, leukocyte activation, and other inflammation It leads to a phenomenon 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 complement activation site.

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

  A properly functioning complement system provides strong protection against infectious microorganisms, but inappropriate regulation or activation of complement has been implicated in the pathogenesis of various 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 (eg, age-related macular) Degeneration (AMD)); HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome; thrombotic thrombocytopenic purpura (TTP); spontaneous fetal disappearance; epilepsy vasculitis; epidermolysis bullosa; Multiple sclerosis (MS); traumatic brain injury; and damage resulting from myocardial infarction, cardiopulmonary bypass and hemodialysis (see, eg, Non-Patent Document 1). Thus, suppression of over-activation or uncontrolled activation of the complement cascade can provide clinical benefits to patients with such diseases.

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

  PNH is characterized by hemolytic anemia (red blood cell count reduction), hemoglobinuria (presence of hemoglobin in the urine, especially after sleep), and hemoglobinemia (presence of hemoglobin in the bloodstream). Individuals suffering from PNH are known to have seizures defined herein as the appearance of dark urine. Hemolytic anemia is due to intravascular destruction of red blood cells by complement components. Other known symptoms include language impairment, fatigue, erectile dysfunction, thrombosis and recurrent abdominal pain.

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

Several reports describe anti-C5 antibodies. For example, Patent Document 7 describes an anti-C5 antibody that binds to the C5 α chain but not C5a and blocks C5 activation, while Patent Document 8 inhibits C5a formation. An anti-C5 monoclonal antibody was described. On the other hand, Patent Document 9 described an anti-C5 antibody that recognizes the proteolytic site for C5 convertase on the C5 α chain and inhibits the conversion of C5 to C5a and C5b. Patent document 10 described an anti-C5 antibody having an affinity constant of at least 1 × 10 ⁷ M ⁻¹ .

  The antibody (IgG) binds to the neonatal Fc receptor (FcRn) and has a long plasma residence time. Binding of IgG to FcRn is typically observed under acidic conditions (eg, pH 6.0) and rarely observed under neutral conditions (eg, pH 7.4). Typically, IgG is nonspecifically taken up into the cell via endocytosis and returns to the cell surface by binding to endosomal FcRn under acidic conditions within the endosome. IgG then dissociates from FcRn under neutral conditions in plasma. IgG that failed to bind to FcRn is degraded by lysosomes. If IgG's ability to bind FcRn under acidic conditions is lost by introducing mutations into its Fc region, IgG will not be recycled from endosomes into plasma, indicating that Leading to a significant decline. In order to improve the retention of IgG in plasma, methods have been reported to increase its FcRn binding under acidic conditions. If FcRn binding of IgG under acidic conditions is improved by introducing amino acid substitutions into its Fc region, IgG is more efficiently recycled from endosomes into plasma, and thereby improved plasma retention Showing gender. On the other hand, IgG with enhanced FcRn binding under neutral conditions is neutral in plasma even when it returns to the cell surface via its binding to FcRn under acidic conditions in endosomes. It has also been reported that it does not dissociate from FcRn under conditions and as a result its plasma retention remains unchanged or rather deteriorates (eg, Non-Patent Document 3; Non-Patent Document 4). ; See Non-Patent Document 5).

  Recently, antibodies that bind antigens in a pH-dependent manner have been reported (see, for example, US Pat. These antibodies bind strongly to the antigen under neutral conditions in plasma and dissociate from the antigen under acidic conditions in endosomes. After dissociating from the antigen, the antibody becomes able to bind to the antigen again when recycled to plasma via FcRn. Thus, a single antibody molecule can repeatedly bind to multiple antigen molecules. In general, the retention of antigen in plasma is much shorter than that of antibodies with the FcRn-mediated recycling mechanism described above. Thus, when an antigen binds to an antibody, the antigen typically exhibits prolonged plasma retention, which results in an increase in the plasma concentration of the antigen. On the other hand, the above-mentioned antibodies that bind to antigens in a pH-dependent manner dissociate from the antigen in the endosome during the FcRn-mediated recycling process and thus can remove antigens from plasma more rapidly than typical antibodies. It has been reported. U.S. Patent No. 6,057,034 also describes a computer modeling analysis that shows that antibodies that bind pH dependently to C5 can prolong antigen knockdown.

U.S. Patent No. 6,355,245 U.S. Patent No. 7,432,356 WO2005 / 074607 WO2007 / 106585 WO2008 / 069889 WO2010 / 054403 WO95 / 29697 WO02 / 30985 WO2004 / 007553 WO2010 / 015608 WO2009 / 125825 WO2011 / 122011 WO2011 / 111007

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

  An object of the present invention is to provide anti-C5 antibodies and methods of use thereof.

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

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

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

  In some embodiments, an isolated anti-C5 antibody of the 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 the C5 variant consisting of the E48A substitution of SEQ ID NO: 39; or (c) the antibody is SEQ ID NO: It binds to C5 protein consisting of 39 amino acid sequences at pH 7.4, but does not bind to C5 protein consisting of the amino acid sequence of SEQ ID NO: 39 having an H72Y substitution at pH 7.4. In a further embodiment, the antibody binds to C5 with higher affinity at neutral pH than at acidic pH. In a further embodiment, the antibody binds to C5 with higher affinity at pH 7.4 than at pH 5.8.

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

In some embodiments, an isolated anti-C5 antibody of the invention comprises (a) the amino acid sequence DX ₁ GYX ₂ X ₃ PTHAMX ₄ X ₅ (where X ₁ is G or A, X ₂ is V, Q or D, X ₃ is T or Y, X ₄ is Y or H, X ₅ is L or Y) (SEQ ID NO: 128), and (b) amino acid sequence QX ₁ TX ₂ VGSSYGNX ₃ ( Wherein X ₁ is S, C, N or T, X ₂ is F or K, X ₃ is A, T or H) (SEQ ID NO: 131), and (c) amino acid sequence X ₁ IX ₂ TGSGAX ₃ YX ₄ AX ₅ WX ₆ KG (where X ₁ is C, A or G, X ₂ is Y or F, X ₃ is T, D or E, 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 HVR-H2.

In some embodiments, an isolated anti-C5 antibody of the invention comprises (a) the amino acid sequence SSYYX ₁ X ₂ (where X ₁ is M or V and X ₂ is C or A) (SEQ ID NO: 126) HVR-H1 and (b) amino acid sequence X ₁ IX ₂ TGSGAX ₃ YX ₄ AX ₅ WX ₆ KG (where X ₁ is C, A or G, X ₂ is Y or F, X ₃ is T HVR-H2 including (SEQ ID NO: 127), (C) amino acid sequence, and D or E, X ₄ is Y, K or Q, X ₅ is S, D or E, and X ₆ is A or V) DX ₁ GYX ₂ X ₃ PTHAMX ₄ X ₅ (where X ₁ is G or A, X ₂ is V, Q or D, X ₃ is T or Y, X ₄ is Y or H, X ₅ is L or Y And HVR-H3 containing (SEQ ID NO: 128). In a further embodiment, the antibody comprises (a) the amino acid sequence X ₁ ASQX ₂ IX ₃ SX ₄ LA (where X ₁ is Q or R, X ₂ is N, Q or G, X ₃ is G or S, X ₄ HVR-L1 comprising (SEQ ID NO: 129); (b) amino acid sequence GASX ₁ X ₂ X ₃ S (where X ₁ is K, E or T, X ₂ is L Or T, X ₃ is A, H, E or Q) (SEQ ID NO: 130) and HVR-L2; (c) amino acid sequence QX ₁ TX ₂ VGSSYGNX ₃ (where X ₁ is S, C, N or T, X ₂ is F or K, and X ₃ is A, T or H) (SEQ ID NO: 131).

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

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

  In some embodiments, an isolated anti-C5 antibody of the invention comprises (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 10, 106-110 (B) a VL sequence having at least 95% sequence identity to any one amino acid sequence of SEQ ID NO: 20, 111-113; or (c) a VH sequence of (a) and a VL of (b) Contains an array. In a further embodiment, the antibody comprises the VH sequence of any one of SEQ ID NOs: 10, 106-110. In a further embodiment, the antibody comprises the VL sequence of any one of SEQ ID NOs: 20, 111-113.

  The present invention provides an antibody comprising the VH sequence of any one of SEQ ID NO: 10, 106-110 and the VL sequence of any one of SEQ ID NO: 20, 111-113.

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

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

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

  The anti-C5 antibody of the present invention may be for use as a medicament. The anti-C5 antibodies of the invention can be for use in the treatment of complement-mediated diseases or conditions with excessive or uncontrolled activation of C5. The anti-C5 antibodies of the invention can be for use in enhancing clearance of C5 from plasma.

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

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

FIG. 1 shows epitope binning of anti-C5 antibodies as described in Example 2.2. Antibodies classified into the same epitope bin are surrounded by bold lines. FIG.2A shows a BIACORE® sensorgram of anti-C5 antibody at pH 7.4 (solid line) and pH 5.8 (dashed line) to assess pH dependence as described in Example 3.2. Show. As described in Example 2.2, CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, and CFA0599 are antibodies classified as epitope C. FIG.2B shows a BIACORE® sensorgram of anti-C5 antibody at pH 7.4 (solid line) and pH 5.8 (dashed line) to assess pH dependence as described in Example 3.2. Show. 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. FIG. 3 is for a fragment derived from the C5β chain fused to a GST tag (SEQ ID NO: 40 amino acids 19-180, 161-340, 321-500, and 481-660) as described in Example 4.1. Western blot analysis is shown. CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675 are antibodies classified as epitope C. Anti-GST antibody is used as a positive control. The position of the GST fusion C5 fragment (46-49 kDa) is marked with an arrow. FIG. 4 shows a BIACORE® sensorgram of anti-C5 antibody against the MG1-MG2 domain of the C5β chain, as described in Example 4.3. The upper panel shows the results for CFA0305 (solid line), CFA0307 (dashed line), CFA0366 (dashed line), and eculizumab (dotted line). The middle panel shows the results for CFA0501 (solid line), CFA0599 (dashed line), CFA0538 (dashed line), and eculizumab (dotted line). The lower panel shows the results for CFA0666 (solid line), CFA0672 (dashed line), CFA0675 (dashed line), and eculizumab (dotted line). CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675 are antibodies classified as epitope C. Eculizumab is a control anti-C5 antibody. FIG. 5A shows a peptide fragment (SEQ ID NO: 40 amino acids 33-124, 45-124, 52-124, 33-111) derived from the MG1-MG2 domain fused to a GST tag, as described in Example 4.4. , 33-108, and 45-111). Anti-GST antibody is used as the antibody for the reaction. The position of the GST fusion C5 fragment (35-37 kDa) is marked with an arrow. FIG. 5B shows a peptide fragment derived from the MG1-MG2 domain fused to a GST tag as described in Example 4.4 (SEQ ID NO: 40 amino acids 33-124, 45-124, 52-124, 33-111). , 33-108, and 45-111). CFA0305 is used as an antibody for the reaction. FIG. 5C summarizes the binding reaction of anti-C5 antibodies to fragments derived from the C5β chain as described in Example 4.4. Fragments to which anti-C5 antibodies classified as epitope C (CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675) bind are shown in gray, and fragments to which they do not bind are shown in white. FIG. 6 shows Western blot analysis for C5 point mutants (E48A, D51A, and K109A, respectively) in which the β chain E48, D51, and K109 were replaced with alanine 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 C5 α chain (about 113 kDa) is marked with an arrow. In the right panel, CFA0305 (classified as epitope C, β chain binder) is used as an antibody for the reaction, and the position of the C5 β chain (approximately 74 kDa) is marked with an arrowhead. FIG. 7 presents a BIACORE® sensorgram showing the interaction of eculizumab-F760G4 (upper panel) or 305LO5 (lower panel) with the C5 variant as described in Example 4.6. Sensorgrams are C5-wt (thick solid line), C5-E48A (short dashed line), C5-D51A (long dashed line), and C5-K109A (narrow) on the sensor surface immobilized with eculizumab-F760G4 or 305LO5, respectively. (Solid line). Eculizumab is a control anti-C5 antibody. 305LO5 is a humanized antibody of CFA0305 (classified as Epitope C) as described in Example 2.3. FIG. 8 presents a BIACORE® sensorgram showing the interaction of 305LO5 with the C5 His mutant for assessing pH dependence, as described in Example 4.7. The sensorgrams are C5-wt (thick solid line), C5-H70Y (long dashed line), C5-H72Y (short dashed line), C5-H110Y (dotted line), and C5- It was obtained by injection of H70Y + H110Y (thin solid line). To assess pH dependent interactions, antibody / antigen complexes were dissociated at pH 7.4 followed by further dissociation at pH 5.8 (indicated by arrows). FIG. 9A shows inhibition of complement activated liposome lysis by anti-C5 antibodies as described in Example 5.1. The results of CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675 classified as epitope C as described in Example 2.2 are shown. FIG. 9B shows inhibition of complement activated liposome lysis by anti-C5 antibodies as described in Example 5.1. Results are shown for antibodies CFA0330 and CFA0341 classified as epitope B as described in Example 2.2. FIG. 10A shows inhibition of C5a production 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 FIG. 9A. FIG. 10B shows inhibition of C5a production 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 FIG. 9B. FIG. 11 shows inhibition of complement activated hemolysis by anti-C5 antibodies as described in Example 5.3. Complement was activated via the classical pathway. FIG. 12 shows the inhibition of complement activated hemolysis by anti-C5 antibodies as described in Example 5.4. Complement was activated via the alternative pathway. FIG. 13 shows the time course of plasma human C5 concentration after intravenous administration of human C5 alone or human C5 and anti-human C5 antibody in mice assessing 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. It is. FIG. 14 shows the time course of plasma anti-human C5 antibody concentration after intravenous administration of human C5 and anti-human C5 antibody in mice evaluating antibody pharmacokinetics as described in Example 6.3. As described in Example 2.2, CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675 are antibodies classified as epitope C, and CFA0330 and CFA0341 are antibodies classified as epitope B. It is. FIG. 15 shows inhibition of complement activated liposome lysis by anti-C5 antibodies as described in Example 9.1. Results for antibodies 305LO15-SG422, 305LO16-SG422, 305LO18-SG422, 305LO19-SG422, 305LO20-SG422, and 305LO20-SG115 are shown. FIG. 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. FIG. 17 shows inhibition of complement activated liposome lysis by anti-C5 antibodies as described in Example 9.1. Results for antibodies 305LO22-SG115, 305LO22-SG422, 305LO23-SG115, and 305LO23-SG422 are shown. FIG. 18 shows the inhibition of C5a production 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 FIG. FIG. 19 shows inhibition of C5a production 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 FIG. FIG. 20 shows the inhibition of complement activity in monkey plasma by anti-C5 antibodies as described in Example 9.3. Anti-C5 antibody was administered to cynomolgus monkeys, and complement activity in monkey plasma was measured by hemolysis assay. FIG. 21 shows wild-type C5 (WT) and C5 mutants (V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N, and E1437D) with an anti-C5 antibody (eclizumab) as described in Example 9.4. Inhibition of biological activity. FIG. 22 shows wild type C5 (WT) and C5 mutants (V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N, and E1437D with anti-C5 antibody (305 mutant) as described in Example 9.4. ) Inhibition of biological activity. FIG. 23 shows inhibition of complement activated liposome lysis by anti-C5 antibodies (BNJ441 and 305 variants) as described in Example 9.5. FIG. 24 shows the time course of plasma cynomolgus C5 concentration after intravenous administration of anti-human C5 antibody in cynomolgus monkeys assessing C5 clearance as described in Example 10.2. FIG. 25 shows the time course of plasma anti-human C5 antibody concentration after intravenous administration of anti-human C5 antibody in cynomolgus monkeys evaluating antibody pharmacokinetics as described in Example 10.3. FIGS. 26A and 26B show the crystal structure of 305 Fab bound to the human C5 (hC5) -MG1 domain, as described in Example 11.6. FIG. 26A shows an asymmetric unit. MG1 is shown in surface representation, and 305 Fab is shown as a ribbon (dark gray: heavy chain, light gray: light chain). FIG. 26B shows superimposed molecules 1 and 2 (dark gray: molecule 1, light gray: molecule 2). FIG. 27A shows the epitope of the 305 Fab contact region on the MG1 domain, as described in Example 11.6. FIG. 27A shows epitope mapping with the MG1 amino acid sequence (dark gray: closer than 3.0 mm, light gray: closer than 4.5 mm). FIG. 27B shows the epitope for the 305 Fab contact region on the MG1 domain, as described in Example 11.6. FIG. 27B shows epitope mapping in the crystal structure (dark gray spheres: closer than 3.0 mm, light gray bars: closer than 4.5 mm). FIG. 28A shows a close-up view of the interaction of E48, D51, and K109 (bar representation) with 305 Fab (surface representation) as described in Example 11.7. FIG. 28B shows the interaction between E48 and its surroundings (dark gray dotted line: hydrogen bond with Fab, light gray dotted line: hydrogen bond via water) as described in Example 11.7. FIG. 28C shows the interaction between D51 and its surroundings (dark gray dotted line: hydrogen bonding with Fab) as described in Example 11.7. FIG. 28D shows the interaction between K109 and its surroundings (dark gray dotted line: hydrogen bond with Fab, light gray dotted line: salt bridge with H-CDR3_D95) as described in Example 11.7. FIG. 29A shows a close-up view of the interaction of H70, H72, and H110 (bar representation) with 305 Fab (surface representation), as described in Example 11.8, in the same direction as FIG. 28A. FIG. 29B shows the interaction between H70 and its surroundings as described in Example 11.8. This histidine residue is shown in bar and mesh representations. Hydrogen bonds are indicated by dotted lines. FIG. 29C shows the interaction between H72 and its surroundings as described in Example 11.8. This histidine residue is shown in bar and mesh representations. Hydrogen bonds are indicated by dotted lines. FIG. 29D shows the interaction between H110 and its surroundings as described in Example 11.8. This histidine residue is shown in bar and mesh representations. The distance between H110 and H-CDR3_H100c is indicated by a dotted line.

DESCRIPTION OF THE EMBODIMENTS The techniques and procedures described or cited herein are generally well understood, 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 CC 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. Trabos, 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). Commonly used by those skilled in the art using conventional techniques.

I. Definitions Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill 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) provides general guidance to those skilled in the art for many of the terms used in this application. All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

  For purposes of interpreting this specification, the following definitions will apply and whenever applicable, terms used in the singular will also include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments 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 following definitions shall prevail.

  As used herein, an “acceptor human framework” refers to a light chain variable domain (VL) framework or heavy chain variable domain (VH) derived from a human immunoglobulin framework or human consensus framework as defined below. ) A framework containing the amino acid sequence of the framework. Acceptor human frameworks “derived from” a human immunoglobulin framework or a human consensus framework may contain the 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 human consensus framework sequence.

  “Affinity” refers to the total strength of a non-covalent interaction between a binding site of a molecule (eg, an antibody) and a binding partner (eg, an antigen) of the molecule. Unless otherwise indicated, “binding affinity” as used herein refers to intrinsic binding affinity that reflects a 1: 1 interaction between members of a binding pair (eg, an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented 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.

  An “affinity matured” antibody is one or more modifications that result in improved affinity of the antibody for the antigen in one or more hypervariable regions (HVRs) compared to the parent antibody without modification. Refers to an antibody with

The term “anti-C5 antibody” or “antibody that binds C5” is an antibody capable of binding C5 with sufficient affinity so that when the antibody targets C5, a diagnostic agent and / or treatment It refers to an antibody that is useful as an agent. In one embodiment, the degree of binding of the anti-C5 antibody to an irrelevant non-C5 protein is less than about 10% of the binding of the antibody to C5, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to C5 is ≦ 1 μM, ≦ 100 nM, ≦ 10 nM, ≦ 1 nM, ≦ 0.1 nM, ≦ 0.01 nM, or ≦ 0.001 nM (eg, 10 ⁻⁸ M or less, such as 10 ⁻⁸ M to 10 ^-13 M, for example, it has 10 ^-9 M~10 ^-13 M) dissociation constant of (Kd). In certain embodiments, the anti-C5 antibody binds to an epitope of C5 that is conserved among C5 from different species.

  As used herein, the term “antibody” is used in the broadest sense and is not limited thereto as long as it exhibits a desired antigen-binding activity, but is not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, Various antibody structures are included, including bispecific antibodies) and antibody fragments.

“Antibody fragment” refers to a molecule other than the complete antibody, including 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 ; diabodies; linear antibodies; single chain antibody molecules (eg, scFv And multispecific antibodies formed from antibody fragments.

  An “antibody that binds to the same epitope” as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay, and / or conversely, a reference antibody is These antibodies block the binding to their antigens. An exemplary competition assay is provided herein.

  The term “chimeric” antibody is one in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remaining portion of the heavy and / or light chain is derived from a different source or species. Refers to an antibody.

The “class” of an antibody refers to the type of constant domain or constant region provided on the heavy chain of the antibody. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of them 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.

The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents the function of cells and / or causes death or destruction of cells. Cytotoxic agents include, but are not limited to, radioisotopes (eg, At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² And radioisotopes of Lu); chemotherapeutic or chemotherapeutic agents (eg, methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalates Growth inhibitors; enzymes such as nucleolytic enzymes and fragments thereof; antibiotics; for example, small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin (fragments and / or mutations thereof) Toxins (including the body); and various anti-tumor or anti-cancer agents disclosed below.

  “Effector function” refers to a biological activity that depends on the antibody's isotype, resulting from the Fc region of the antibody. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity -mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (eg B cell receptors); and B cell activation.

  An “effective amount” of an agent (eg, a pharmaceutical formulation) refers to that amount at the required dose and over the required period of time that is effective to achieve the desired therapeutic or prophylactic result.

  The term “epitope” includes any determinant capable of being bound by an antibody. An epitope is a region of an antigen that is bound by an antibody that targets the antigen and includes specific amino acids that directly contact the antibody. Epitope determinants can include chemically active surface molecule groups such as amino acids, sugar side chains, phosphoryl groups or sulfonyl groups and have specific three-dimensional structural characteristics and / or specific charge characteristics Can do. 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.

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

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

  The terms “full-length antibody”, “complete antibody”, and “total antibody” are used interchangeably herein and have a structure substantially similar to or defined herein. An antibody having a heavy chain containing an Fc region.

  The terms “host cell”, “host cell line”, and “host cell culture” are used interchangeably and refer to a cell (including the progeny of such a cell) into which a foreign nucleic acid has been introduced. . Host cells include “transformants” and “transformed cells”, including the primary transformed cell and progeny derived from that cell regardless of the passage number. The progeny may not be completely identical in nucleic acid content with the parent cell, and may contain mutations. Also included herein are mutant progeny that have the same function or biological activity as was used when the original transformed cells were screened or selected.

  A “human antibody” is an antibody comprising an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or from a non-human source using a human antibody repertoire or other human antibody coding sequence. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen binding residues.

  A “human consensus framework” is a framework that indicates the most commonly occurring amino acid residues in a selected group 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 κI according to Kabat et al. In one embodiment, for VH, the subgroup is subgroup III by Kabat et al.

A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, typically two variable domains, in which all or substantially all HVRs (eg, CDRs) are non- It corresponds to that of a human antibody and all or substantially all FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody (eg, a non-human antibody) refers to an antibody that has undergone humanization.
The term “hypervariable region” or “HVR” as used herein is hypervariable in sequence (“complementarity determining region” or “CDR”) and / or structurally determined. Refers to each region of the variable domain of an antibody that forms a loop (“hypervariable loop”) and / or contains antigen contact residues (“antigen contact”). Usually, an antibody contains 6 HVRs: 3 in VH (H1, H2, H3) and 3 in VL (L1, L2, L3). Exemplary HVRs herein include the following:
(a) at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) The resulting hypervariable loop (Chothia, J. Mol. Biol. 196: 901-917 (1987));
(b) at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) The resulting CDR (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NIH, Bethesda, MD (1991));
(c) at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) Resulting antigen contact (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996));
(d) HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49 A combination of (a), (b), and / or (c), including -65 (H2), 93-102 (H3), and 94-102 (H3).

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

  An “immunoconjugate” is an antibody conjugated to one or more heterologous molecules (heterologous molecules include, but are not limited to, cytotoxic agents).

  An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domestic animals (eg, cattle, sheep, cats, dogs, horses), primates (eg, humans and non-human primates such as monkeys), rabbits, and , Including rodents (eg, mice and rats). In certain embodiments, the individual or subject is a human.

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

  An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from components of its original environment. An isolated nucleic acid includes a nucleic acid molecule contained within a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or on a chromosome different from the original chromosomal location. Exists in position.

  “Isolated nucleic acid encoding an anti-C5 antibody” refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, either in one vector or in separate vectors. It includes nucleic acid molecules that are on board and nucleic acid molecules that are present at one or more locations in a host cell.

  The term “monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies that make up the population are mutated antibodies that can occur (eg, mutated antibodies that contain naturally occurring mutations, or mutated antibodies that occur during the production of monoclonal antibody preparations. Are present in the same amount and / or bind to the same epitope. In contrast to polyclonal antibody preparations that typically include different antibodies to different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is against a single determinant on the antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention include, but are not limited to, hybridoma methods, recombinant DNA methods, phage display methods, transgenic animals containing all or part of a human immunoglobulin locus. Such methods and other exemplary methods for making monoclonal antibodies can be made by a variety of techniques, including methods utilizing the methods described herein.

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

  “Natural antibodies” refer to immunoglobulin molecules with various naturally occurring structures. For example, a native IgG antibody is a heterotetrameric glycoprotein of approximately 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 a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a constant light chain (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.

  The term “package insert” is usually included in commercial packages of therapeutic products and includes information about indications, usage, dosages, administration methods, combination therapies, contraindications, and / or warnings regarding the use of such therapeutic products. Used to refer to instructions for use.

  “Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence refers to any conservative substitutions after aligning the sequences and introducing gaps, if necessary, to obtain maximum percent sequence identity. Defined as the percentage ratio of amino acid residues in a candidate sequence that are identical to amino acid residues in a reference polypeptide sequence, when not considered part of the identity. Alignments for purposes of determining percent amino acid sequence identity are publicly available, such as various methods within the skill in the art, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software This can be achieved by using computer software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. However,% amino acid sequence identity values within the meaning of this specification are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is a work of Genentech, and its source code was submitted to the US Copyright Office (US Copyright Office, Wasington DC, 20559) along with the user documentation, and was registered under the US copyright registration number TXU510087. It is registered. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, and may be compiled from 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.

  In situations where ALIGN-2 is used for amino acid sequence comparison, the% amino acid sequence identity of a given amino acid sequence A to, or with, or against a given amino acid sequence B (or a given amino acid sequence) A given amino acid sequence A, which has or contains some% amino acid sequence identity to, with or to B) is calculated as follows: fraction X / Y Hundredfold. Where X is the number of amino acid residues scored by the sequence alignment program ALIGN-2 as identical matches in the A and B alignments of the program, 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, then the% amino acid sequence identity of A to B is not equal to the% amino acid sequence identity of B to A. Let's go. Unless stated otherwise, all% amino acid sequence identity values used herein are those obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

  The term “pharmaceutical formulation” refers to a preparation that is in a form such that the biological activity of the active ingredient contained therein can be effective and is unacceptable to the subject to which the formulation is administered. Refers to a preparation that does not contain additional toxic elements.

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

  The term “C5” as used herein refers to any natural form from any vertebrate source, including mammals such as primates (eg, humans and monkeys) and rodents (eg, mice and rats). Includes C5. Unless otherwise indicated, the term “C5” refers to the human C5 protein having the amino acid sequence set forth in SEQ ID NO: 39 and comprising the β chain sequence set forth in SEQ ID NO: 40. The term encompasses C5 that has not undergone "full length" processing 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 human C5 amino acid sequence is shown in SEQ ID NO: 39 ("wild type" or "WT" C5). An exemplary β chain amino acid sequence of human C5 is shown in SEQ ID NO: 40. The amino acid sequences of exemplary MG1, MG2, and MG1-MG2 domains of the human C5 β chain are shown in SEQ ID NOs: 41, 42, and 43, respectively. The amino acid sequences of exemplary cynomolgus monkey and mouse C5 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 signal sequences that are removed upon processing in the cell and are therefore missing from the corresponding exemplary amino acid sequence To do.

  As used herein, “treatment” (and grammatical derivatives thereof, eg, “treat”, “treating”, etc.) is clinical that is intended to alter the natural course of the individual being treated. Means intervention and can be carried out either for prevention or during the course of clinical pathology. Desirable effects of treatment include but are not limited to prevention of disease occurrence or recurrence, reduction of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, disease Includes reduced rate of progression, recovery or alleviation of disease state, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or slow the progression of disease.

  The term “variable region” or “variable domain” refers to the heavy or light chain domain of an antibody involved in binding the antibody to an antigen. Natural antibody heavy and light chain variable domains (VH and VL, respectively) are typically similar, each domain containing four conserved framework regions (FR) and three hypervariable regions (HVRs). It has a structure. (See, eg, Kindt et al. Kuby Immunology, 6th ed., W. H. Freeman and Co., page 91 (2007).) A single VH or VL domain will be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a particular antigen may be isolated by screening complementary libraries of VL or VH domains, respectively, using VH or VL domains from antibodies that bind to the antigen. See, eg, Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

  The term “vector” as used herein refers to a nucleic acid molecule capable of multiplying another nucleic acid to which it has been linked. The term includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of the host cell into which it has been introduced. Certain vectors can provide for the expression of nucleic acids to which they are operably linked. Such vectors are also referred to herein as “expression vectors”.

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

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

  In another aspect, the present invention provides anti-C5 antibodies that exhibit pH dependent binding properties. The expression “pH-dependent binding” as used herein indicates that the antibody exhibits “reduced binding to C5 at acidic pH compared to binding to C5 at neutral pH”. Means (for the purposes of this disclosure, both expressions may be used interchangeably). For example, antibodies having “pH-dependent binding properties” include antibodies that bind to C5 with higher affinity at neutral pH than at acidic pH. In certain embodiments, the antibodies of the invention are at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 at neutral pH than at acidic pH. , 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 fold, or more with high affinity. In some embodiments, the antibody binds to C5 with higher affinity at pH 7.4 than at pH 5.8. In a further embodiment, the antibody of the invention is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 at pH 7.4 than at pH 5.8. , 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 fold, or more with high affinity.

The “affinity” of an antibody for C5 is represented by the KD of the antibody for purposes of this disclosure. Antibody KD refers to the equilibrium dissociation constant of antibody-antigen interaction. The greater the KD value for an antibody that binds to an antigen, the weaker its binding affinity for that particular antigen. Thus, as used herein, the expression “higher affinity at neutral pH than at acidic pH” (or equivalent expression “pH-dependent binding”) means that the KD of an antibody that binds C5 at acidic pH is neutral. Means greater than the KD of an antibody that binds to C5 at 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 greater than the KD of an antibody that binds to C5 at neutral pH, the antibody will be neutral pH than at acidic pH. It is thought to bind to C5 with high affinity. Thus, the present invention is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, more than the KD of an antibody that binds to C5 at neutral pH. Antibodies that bind to C5 at acidic pH with KD of 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 fold or more are included. 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 M or less It can be. In another embodiment, the KD value of the antibody at acidic pH can be 10 ⁻⁹ M, 10 ⁻⁸ M, 10 ⁻⁷ M, 10 ⁻⁶ M, or higher.

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

The binding properties of an antibody to a particular antigen can also be expressed as the kd of the antibody. Antibody kd refers to the dissociation rate constant of an antibody for a particular antigen and is expressed in units of the reciprocal of seconds (ie, sec ⁻¹ ). An increase in the kd value means that the antibody has less binding to its antigen. Thus, the present invention encompasses antibodies that bind to C5 with a higher kd value at acidic pH than at neutral pH. The present invention is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, more than the kd of an antibody that binds to C5 at neutral pH. Antibodies that bind to C5 at acidic pH with kd 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 fold or more are included. In another embodiment, the kd value of the antibody at neutral pH is 10 ⁻² 1 / s, 10 ⁻³ 1 / s, 10 ⁻⁴ 1 / s, 10 ⁻⁵ 1 / s, 10 ⁻⁶ 1 / s. s or less. In another embodiment, the kd value of the antibody at acidic pH can be 10 ⁻³ 1 / s, 10 ⁻² 1 / s, 10 ⁻¹ 1 / s, or higher. The invention also encompasses antibodies that bind to C5 with a higher kd value at pH 5.8 than at pH 7.4. The present invention is at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, than the kd of the antibody that binds to C5 at pH 7.4. Antibodies that bind to C5 at pH 5.8 with kd 80, 85, 90, 95, 100, 200, 400, 1000, 10000 fold or more are included. In another embodiment, the kd value of the antibody at pH 7.4 is 10 ⁻² 1 / s, 10 ⁻³ 1 / s, 10 ⁻⁴ 1 / s, 10 ⁻⁵ 1 / s, 10 ⁻⁶ 1 / s. s or less. In another embodiment, the kd value of the antibody at pH 5.8 can be 10 ⁻³ 1 / s, 10 ⁻² 1 / s, 10 ⁻¹ 1 / s, or higher.

In a specific example, “reduced binding to C5 at acidic pH compared to binding to C5 at neutral pH” refers to the acidic pH relative to the KD value of an antibody that binds to C5 at neutral pH. It is expressed as the ratio of the KD value of an antibody that binds to C5 (or vice versa). For example, if an antibody exhibits an acidic / neutral KD ratio of 2 or greater, for the purposes of the present invention, the antibody is "reduced compared to binding to C5 at neutral pH, to C5 at acidic pH. It can be regarded as indicating "binding". In certain exemplary embodiments, the antibody of the invention has a pH 5.8 / pH 7.4 KD ratio of 2 or greater. In certain exemplary embodiments, the acidic / neutral KD ratio of the antibodies of the invention is 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 , 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more. In another embodiment, the KD value of the antibody at neutral pH is 10 ⁻⁷ M, 10 ⁻⁸ M, 10 ⁻⁹ M, 10 ⁻¹⁰ M, 10 ⁻¹¹ M, 10 ⁻¹² M or less It can be. In another embodiment, the KD value of the antibody at acidic pH can be 10 ⁻⁹ M, 10 ⁻⁸ M, 10 ⁻⁷ M, 10 ⁻⁶ M, or higher. In a further example, when an antibody exhibits a pH 5.8 / pH 7.4 KD ratio of 2 or greater, for the purposes of the present invention, the antibody is "reduced compared to binding to C5 at neutral pH, acidic pH. It can be considered that it shows "binding to C5". In certain exemplary embodiments, the antibodies of the invention have a pH 5.8 / pH 7.4 KD ratio of 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, It can be 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more. In another embodiment, the KD value of the antibody at pH 7.4 is 10 ⁻⁷ M, 10 ⁻⁸ M, 10 ⁻⁹ M, 10 ⁻¹⁰ M, 10 ⁻¹¹ M, 10 ⁻¹² M, or less. It can be. In another embodiment, the KD value of the antibody at pH 5.8 can be 10 ⁻⁹ M, 10 ⁻⁸ M, 10 ⁻⁷ M, 10 ⁻⁶ M, or higher.

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

  The expression “acidic pH” as used herein means a pH of 4.0 to 6.5. The expression `` acid pH '' includes 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, The pH value of any one of 6.1, 6.2, 6.3, 6.4, and 6.5 is included. In a particular aspect, the “acidic pH” is 5.8.

  As used herein, the expression “neutral pH” means a pH of 6.7 to about 10.0. The expression `` neutral pH '' includes 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.

  The KD and kd values expressed herein can be determined using surface plasmon resonance based biosensors to characterize antibody-antigen interactions. (See, eg, Example 3 herein.) KD and kd values can be measured at 25 ° C. or 37 ° C.

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

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

  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 a further embodiment, 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.

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

  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 having an H72Y substitution, wherein the C5 protein and 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.

  Without being bound by any particular theory, it is assumed that the binding of anti-C5 antibodies to C5 is reduced (or almost lost) when one amino acid residue on C5 is replaced with another amino acid. This means that the amino acid residue on C5 is important for the interaction between anti-C5 antibody and C5 and that the antibody can recognize an epitope around the amino acid residue on C5 It means that.

  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 having a pKa value of about 6.0 to 6.5 can have a proton dissociation state that varies between neutral and acidic pH. Therefore, histidine residues on C5 can contribute to the pH-dependent interaction between anti-C5 antibody and C5. Without being bound to a particular theory, it can be speculated that anti-C5 antibodies can recognize conformations around histidine residues on C5 that can change depending on the pH. This assumption may be consistent with the experimental results described below: when the histidine residue on C5 is replaced with another amino acid, the pH dependence of the anti-C5 antibody is reduced (or almost lost) (ie, Anti-C5 antibodies with pH-dependent binding properties bind C5 histidine mutants at neutral pH with similar affinity to wild-type C5, but at acidic pH the same antibody has higher affinity than wild-type C5. Binds to the histidine variant of C5).

  In certain embodiments, the anti-C5 antibodies of the invention bind to C5 from multiple species. In further embodiments, the anti-C5 antibody binds to C5 from human and non-human animals. In further embodiments, the anti-C5 antibody binds to C5 from humans and monkeys (eg, cynomolgus monkey, rhesus monkey, marmoset, chimpanzee, or baboon).

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

  In one aspect, the present invention provides an anti-C5 antibody that inhibits activation of a C5 variant. By C5 variant is meant a genetic variant of C5 resulting from a genetic variation such as mutation, polymorphism or allelic variation. A genetic variation can include a deletion, substitution or insertion of one or more nucleotides. A C5 variant can include one or more genetic variations of C5. In certain embodiments, the C5 variant has a biological activity similar to wild-type C5. Such a C5 variant can include at least one mutation selected from the group consisting of V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N, and E1437D. In the present specification, for example, R885H means a genetic mutation in which arginine at position 885 is replaced with histidine. In certain embodiments, the anti-C5 antibody of the invention comprises both wild type C5 and at least one C5 variant selected from the group consisting of V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N, and E1437D. Inhibits activation.

  In one aspect, the present invention provides (a) HVR-H1 comprising any one amino acid sequence of SEQ ID NOs: 45 to 54; (b) HVR- comprising any one amino acid sequence of SEQ ID NOs: 55 to 64 H2; (c) HVR-H3 comprising any one amino acid sequence of SEQ ID NO: 65-74; (d) HVR-L1 comprising any one amino acid sequence of SEQ ID NO: 75-84; (e) sequence HVR-L2 comprising any one amino acid sequence of No. 85-94; and (f) at least one selected from HVR-L3 comprising any one amino acid sequence of SEQ ID Nos: 95-104, Anti-C5 antibodies are provided that comprise 2, 3, 4, 5, or 6 hypervariable regions (HVRs).

  In one aspect, the present invention provides (a) HVR-H1 comprising any one amino acid sequence of SEQ ID NOs: 45 to 54; (b) HVR- comprising any one amino acid sequence of SEQ ID NOs: 55 to 64 H2; and (c) an antibody comprising at least one, at least two, or all three VH HVR sequences selected from HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 65-74 I will provide a. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 65-74. In another embodiment, the antibody comprises HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 65-74 and HVR-L3 comprising any one amino acid sequence of SEQ ID NOs: 95-104. In a further embodiment, the antibody comprises HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 65-74, HVR-L3 comprising any one amino acid sequence of SEQ ID NOs: 95-104, and SEQ ID NO: HVR-H2 containing any one amino acid sequence of 55-64. In a further embodiment, the antibody comprises (a) HVR-H1 comprising any one amino acid sequence of SEQ ID NO: 45-54; and (b) HVR- comprising any one amino acid sequence of SEQ ID NO: 55-64. H2 and (c) HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 65 to 74.

  In another aspect, the present invention provides (a) HVR-L1 comprising any one amino acid sequence of SEQ ID NO: 75-84; (b) HVR comprising any one amino acid sequence of SEQ ID NO: 85-94 An antibody comprising at least one, at least two, or all three VL HVR sequences selected from HVR-L3 comprising any one amino acid sequence of SEQ ID NOs: 95-104; I will provide a. In one embodiment, the antibody comprises (a) HVR-L1 comprising any one amino acid sequence of SEQ ID NO: 75-84; and (b) HVR- comprising any one amino acid sequence of SEQ ID NO: 85-94. L2; and (c) HVR-L3 comprising any one amino acid sequence of SEQ ID NO: 95-104.

  In another aspect, the antibody of the present invention comprises (a) a VH domain, (i) HVR-H1, comprising any one amino acid sequence of SEQ ID NOs: 45 to 54, (ii) SEQ ID NOs: 55 to At least one, at least two selected from HVR-H2 comprising any one amino acid sequence of 64, and (iii) HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 65-74, or A VH domain comprising all three VH HVR sequences; and (b) a VL domain, (i) HVR-L1, comprising any one amino acid sequence of SEQ ID NO: 75-84, (ii) SEQ ID NO: : HVR-L2 comprising any one amino acid sequence of 85 to 94, and (c) HVR-L3 comprising any one amino acid sequence of SEQ ID NO: 95 to 104, at least one, at least two VL domain, including one, or all three VL HVR sequences.

  In another aspect, the present invention provides (a) HVR-H1 comprising any one amino acid sequence of SEQ ID NO: 45-54; (b) HVR comprising any one amino acid sequence of SEQ ID NO: 55-64 -H2; (c) HVR-H3 comprising any one amino acid sequence of SEQ ID NO: 65-74; (d) HVR-L1, comprising any one amino acid sequence of SEQ ID NO: 75-84; (e) an antibody comprising HVR-L2 comprising any one amino acid sequence of SEQ ID NO: 85 to 94; and (f) HVR-L3 comprising any one amino acid sequence of SEQ ID NO: 95 to 104. provide.

  In one aspect, the present invention provides (a) HVR-H1 comprising any one amino acid sequence of SEQ ID NOs: 45, 54, 117, 126; (b) SEQ ID NOs: 55, 64, 118-120, 127 HVR-H2 comprising any one amino acid sequence; (c) HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 65, 74, 121, 128; (d) SEQ ID NOs: 75, 84, 122, HVR-L1 comprising any one amino acid sequence of 129; (e) HVR-L2 comprising any one amino acid sequence of 85, 94, 123 to 124, 130; and (f) SEQ ID NO: An anti-C5 antibody comprising at least one, two, three, four, five, or six HVRs selected from HVR-L3 comprising any one amino acid sequence of 95, 104, 125, 131 I will provide a.

  In one aspect, the present invention provides (a) HVR-H1 comprising any one amino acid sequence of SEQ ID NOs: 45, 54, 117, 126; (b) SEQ ID NOs: 55, 64, 118-120, 127 HVR-H2 comprising any one amino acid sequence; and (c) at least one, at least 2, selected from HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 65, 74, 121, 128 Antibodies are provided comprising one, or all three, VH HVR sequences. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 65, 74, 121, 128. In another embodiment, the antibody comprises HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 65, 74, 121, 128 and any one amino acid sequence of SEQ ID NOs: 95, 104, 125, 131. Includes HVR-L3. In a further embodiment, the antibody comprises HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 65, 74, 121, 128 and any one amino acid sequence of SEQ ID NOs: 95, 104, 125, 131. HVR-L3 and HVR-H2 comprising any one amino acid sequence of SEQ ID NOs: 55, 64, 118 to 120, 127 are included. In a further embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 45, 54, 117, 126; and (b) SEQ ID NOs: 55, 64, 118-120, 127. HVR-H2 comprising any one amino acid sequence; and (c) HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 65, 74, 121, 128.

  In another aspect, the present invention provides (a) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 75, 84, 122, 129; (b) SEQ ID NOs: 85, 94, 123-124, 130 HVR-L2 comprising any one amino acid sequence; and (c) at least one, at least 2, selected from HVR-L3 comprising any one amino acid sequence of SEQ ID NOs: 95, 104, 125, 131 Antibodies are provided that contain one or all three VL HVR sequences. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 75, 84, 122, 129; and (b) SEQ ID NOs: 85, 94, 123-124, 130. HVR-L2 comprising any one amino acid sequence; and (c) HVR-L3 comprising any one amino acid sequence of SEQ ID NOs: 95, 104, 125, 131.

  In another aspect, the antibody of the present invention comprises (a) a VH domain, (i) an HVR-H1, comprising the amino acid sequence of any one of SEQ ID NOs: 45, 54, 117, 126, (ii) a sequence No .: HVR-H2 comprising any one amino acid sequence of 55, 64, 118 to 120, 127, and (iii) HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 65, 74, 121, 128 A VH domain comprising at least one, at least two, or all three VH HVR sequences selected from; (b) a VL domain, (i) SEQ ID NOs: 75, 84, 122, 129 (Ii) SEQ ID NO: 85, 94, 123-124, HVR-L2 comprising any one amino acid sequence, and (c) SEQ ID NO: 95, And a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from HVR-L3 comprising any one amino acid sequence of 104, 125, 131.

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

  In certain embodiments, any one or more amino acids of the anti-C5 antibody described above are substituted at the following HVR positions: (a) In HVR-H1 (SEQ ID NO: 45), 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), positions 2, 5, At 6, 12, and 13; (d) at HVR-L1 (SEQ ID NO: 75) at positions 1, 5, 7, and 9; (e) at HVR-L2 (SEQ ID NO: 85) at position 4, At 5 and 6; and (f) at positions 2, 4, and 12 in HVR-L3 (SEQ ID NO: 95).

  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) in HVR-H1 (SEQ ID NO: 45); M5V or C6A; (b) HVR- In H2 (SEQ ID NO: 55), C1A or G, Y3F, T9D or E, Y11K or Q, S13D or E, or A15V; (c) In HVR-H3 (SEQ ID NO: 65), G2A, V5Q or D, T6Y, Y12H, or L13Y; (d) In HVR-L1 (SEQ ID NO: 75), Q1R, N5Q or G, G7S, D9K or S; (e) In HVR-L2 (SEQ ID NO: 85), K4T or E , L5T, or A6H, A6E, or A6Q; (f) In HVR-L3 (SEQ ID NO: 95), C2S, C2N, or C2T, F4K; or A12T or A12H.

  All possible combinations of the above substitutions are for SEQ ID NO: 126, 127, 128, 129, 130 for HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, respectively. And 131 consensus sequences.

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

  In another aspect, the anti-C5 antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% relative to the amino acid sequence of any one of SEQ ID NOs: 1-10 Heavy chain variable domain (VH) sequences with 98%, 99%, or 100% sequence identity. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity is relative to a reference sequence. , Including substitutions (eg, conservative substitutions), insertions, or deletions, an anti-C5 antibody comprising the sequence retains the ability to bind 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, substitutions, insertions, or deletions occur in a region outside of HVR (ie, within FR). Optionally, the anti-C5 antibody comprises a VH sequence in any one of SEQ ID NOs: 1-10, including those containing post-translational modifications of the sequence. In certain embodiments, VH comprises (a) HVR-H1 comprising any one amino acid sequence of SEQ ID NO: 45-54, (b) HVR comprising any one amino acid sequence of SEQ ID NO: 55-64. -H2, and (c) one, two, or three HVRs selected from HVR-H3 comprising any one amino acid sequence of SEQ ID NO: 65-74.

  In another aspect, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 with respect to any one amino acid sequence of SEQ ID NOs: 11-20 Anti-C5 antibodies are provided that comprise a light chain variable domain (VL) with% or 100% sequence identity. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity is relative to a reference sequence. , Including substitutions (eg, conservative substitutions), insertions, or deletions, an anti-C5 antibody comprising the sequence retains the ability to bind 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, substitutions, insertions, or deletions occur in a region outside of HVR (ie, within FR). Optionally, the anti-C5 antibody comprises a VL sequence in any one of SEQ ID NOs: 11-20, including those containing post-translational modifications of the sequence. In certain embodiments, the VL comprises (a) HVR-L1 comprising any one amino acid sequence of SEQ ID NO: 75-84, (b) HVR comprising any one amino acid sequence of SEQ ID NO: 85-94. -L2, and (c) one, two or three HVRs selected from HVR-L3 comprising any one amino acid sequence of SEQ ID NO: 95-104.

  In another aspect, an anti-C5 antibody is provided comprising VH in any of the above embodiments and VL in any of the above embodiments. In one embodiment, the antibody includes the VH and VL sequences in any one of SEQ ID NOs: 1-10 and any one of SEQ ID NOs: 11-20, including those containing post-translational modifications of the sequences Including.

  In another aspect, the anti-C5 antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96% with respect to the amino acid sequence of any one of SEQ ID NOs: 10, 106-110, Contains heavy chain variable domain (VH) sequences with 97%, 98%, 99%, or 100% sequence identity. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity is relative to a reference sequence. , Including substitutions (eg, conservative substitutions), insertions, or deletions, an anti-C5 antibody comprising the sequence retains the ability to bind 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, substitutions, insertions, or deletions occur in a region outside of HVR (ie, within FR). Optionally, the anti-C5 antibody comprises a VH sequence in any one of SEQ ID NOs: 10, 106-110, including those containing post-translational modifications of the sequence. In certain embodiments, VH comprises (a) HVR-H1, comprising an amino acid sequence of any one of SEQ ID NOs: 45, 54, 117, 126, (b) SEQ ID NOs: 55, 64, 118-120, 127 HVR-H2 comprising any one of the amino acid sequences, and (c) one, two selected from HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 65, 74, 121, 128, Or include three HVRs.

  In another aspect, the anti-C5 antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96% with respect to the amino acid sequence of any one of SEQ ID NOs: 10, 106-110, Includes VH sequences with 97%, 98%, 99%, or 100% sequence identity. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity is relative to a reference sequence. , Including substitutions (eg, conservative substitutions), insertions, or deletions, an anti-C5 antibody comprising the sequence retains the ability to bind 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, substitutions, insertions, or deletions occur in a region outside of HVR (ie, within FR). Optionally, the anti-C5 antibody comprises a VH sequence in any one of SEQ ID NOs: 10, 106-110, including those containing post-translational modifications of the sequence. In certain embodiments, VH comprises (a) HVR-H1, comprising an amino acid sequence of any one of SEQ ID NOs: 45, 54, 117, 126, (b) SEQ ID NOs: 55, 64, 118-120, 127 HVR-H2 comprising any one of the amino acid sequences, and (c) one, two selected from HVR-H3 comprising any one amino acid sequence of SEQ ID NOs: 65, 74, 121, 128, Or include three HVRs.

  In another aspect, the anti-C5 antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with respect to the amino acid sequence of SEQ ID NO: 10 Or a VH sequence with 100% sequence identity. In certain embodiments, the VH sequence is the amino acid sequence of SEQ ID NO: 10. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity is relative to a reference sequence. , Including substitutions (eg, conservative substitutions), insertions, or deletions, an anti-C5 antibody comprising the sequence retains the ability to bind 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, substitutions, insertions, or deletions occur in a region outside of HVR (ie, within FR). Optionally, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 10, including those containing post-translational modifications of the sequence. In certain embodiments, VH comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 54, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 64, and (c) of SEQ ID NO: 74. Contains one, two, or three HVRs selected from HVR-H3 containing amino acid sequences.

  In another aspect, the anti-C5 antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% relative to the amino acid sequence of SEQ ID NO: 106. Or a VH sequence with 100% sequence identity. In certain embodiments, the VH sequence is the amino acid sequence of SEQ ID NO: 106. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity is relative to a reference sequence. , Including substitutions (eg, conservative substitutions), insertions, or deletions, an anti-C5 antibody comprising the sequence retains the ability to bind 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, substitutions, insertions, or deletions occur in a region outside of HVR (ie, within FR). Optionally, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 106, including those containing post-translational modifications of the sequence. In certain embodiments, VH comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118, and (c) of SEQ ID NO: 121. Contains one, two, or three HVRs selected from HVR-H3 containing amino acid sequences.

  In another aspect, the anti-C5 antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% relative to the amino acid sequence of SEQ ID NO: 107. Or a VH sequence with 100% sequence identity. In certain embodiments, the VH sequence is the amino acid sequence of SEQ ID NO: 107. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity is relative to a reference sequence. , Including substitutions (eg, conservative substitutions), insertions, or deletions, an anti-C5 antibody comprising the sequence retains the ability to bind 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, substitutions, insertions, or deletions occur in a region outside of HVR (ie, within FR). Optionally, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 107, including those containing post-translational modifications of the sequence. In certain embodiments, VH comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 119, and (c) SEQ ID NO: 121. Contains one, two, or three HVRs selected from HVR-H3 containing amino acid sequences.

  In another aspect, the anti-C5 antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% relative to the amino acid sequence of SEQ ID NO: 108. Or a VH sequence with 100% sequence identity. In certain embodiments, the VH sequence is the amino acid sequence of SEQ ID NO: 108. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity is relative to a reference sequence. , Including substitutions (eg, conservative substitutions), insertions, or deletions, an anti-C5 antibody comprising the sequence retains the ability to bind 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, substitutions, insertions, or deletions occur in a region outside of HVR (ie, within FR). Optionally, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 108, including those containing post-translational modifications of the sequence. In certain embodiments, VH comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118, and (c) of SEQ ID NO: 121. Contains one, two, or three HVRs selected from HVR-H3 containing amino acid sequences.

  In another aspect, the anti-C5 antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% relative to the amino acid sequence of SEQ ID NO: 109. Or a VH sequence with 100% sequence identity. In certain embodiments, the VH sequence is the amino acid sequence of SEQ ID NO: 109. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity is relative to a reference sequence. , Including substitutions (eg, conservative substitutions), insertions, or deletions, an anti-C5 antibody comprising the sequence retains the ability to bind 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, substitutions, insertions, or deletions occur in a region outside of HVR (ie, within FR). Optionally, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 109, including those containing post-translational modifications of the sequence. In certain embodiments, VH comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118, and (c) of SEQ ID NO: 121. Contains one, two, or three HVRs selected from HVR-H3 containing amino acid sequences.

  In another aspect, the anti-C5 antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% relative to the amino acid sequence of SEQ ID NO: 110. Or a VH sequence with 100% sequence identity. In certain embodiments, the VH sequence is the amino acid sequence of SEQ ID NO: 110. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity is relative to a reference sequence. , Including substitutions (eg, conservative substitutions), insertions, or deletions, an anti-C5 antibody comprising the sequence retains the ability to bind 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, substitutions, insertions, or deletions occur in a region outside of HVR (ie, within FR). Optionally, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 110, including those containing post-translational modifications of the sequence. In certain embodiments, VH comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 120, and (c) of SEQ ID NO: 121. Contains one, two, or three HVRs selected from HVR-H3 containing amino acid sequences.

  In another aspect, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% relative to the amino acid sequence of any one of SEQ ID NOs: 20, 111-113 An anti-C5 antibody comprising a light chain variable domain (VL) with 99%, 99%, or 100% sequence identity is provided. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity is relative to a reference sequence. , Including substitutions (eg, conservative substitutions), insertions, or deletions, an anti-C5 antibody comprising the sequence retains the ability to bind 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, substitutions, insertions, or deletions occur in a region outside of HVR (ie, within FR). Optionally, the anti-C5 antibody comprises the VL sequence in any one of SEQ ID NOs: 20, 111-113, including those containing post-translational modifications of the sequence. In certain embodiments, VL comprises (a) HVR-L1, comprising any one amino acid sequence of SEQ ID NOs: 75, 84, 122, 129, (b) SEQ ID NOs: 85, 94, 123-124, 130. One, two, selected from HVR-L2 comprising any one amino acid sequence of: and (c) HVR-L3 comprising any one amino acid sequence of SEQ ID NO: 95, 104, 125, 131, Or include three HVRs.

  In another aspect, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 20 An anti-C5 antibody comprising VL with sequence identity is provided. In certain embodiments, the VL sequence is the amino acid sequence of SEQ ID NO: 20. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity is relative to a reference sequence. , Including substitutions (eg, conservative substitutions), insertions, or deletions, an anti-C5 antibody comprising the sequence retains the ability to bind 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, substitutions, insertions, or deletions occur in a region outside of HVR (ie, within FR). Optionally, the anti-C5 antibody comprises the VL sequence in SEQ ID NO: 20, including those containing post-translational modifications of the sequence. In certain embodiments, the VL comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 84, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 94, and (c) of SEQ ID NO: 104. Contains one, two, or three HVRs selected from HVR-L3 containing amino acid sequences.

  In another aspect, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 111 An anti-C5 antibody comprising VL with sequence identity is provided. In a particular embodiment, the VL sequence is the amino acid sequence of SEQ ID NO: 111. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity is relative to a reference sequence. , Including substitutions (eg, conservative substitutions), insertions, or deletions, an anti-C5 antibody comprising the sequence retains the ability to bind 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, substitutions, insertions, or deletions occur in a region outside of HVR (ie, within FR). Optionally, the anti-C5 antibody comprises the VL sequence in SEQ ID NO: 111, including those containing post-translational modifications of the sequence. In certain embodiments, VL comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123, and (c) of SEQ ID NO: 125. Contains one, two, or three HVRs selected from HVR-L3 containing amino acid sequences.

  In another aspect, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 112 An anti-C5 antibody comprising VL with sequence identity is provided. In certain embodiments, the VL sequence is the amino acid sequence of SEQ ID NO: 112. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity is relative to a reference sequence. , Including substitutions (eg, conservative substitutions), insertions, or deletions, an anti-C5 antibody comprising the sequence retains the ability to bind 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, substitutions, insertions, or deletions occur in a region outside of HVR (ie, within FR). Optionally, the anti-C5 antibody comprises the VL sequence in SEQ ID NO: 112, including those containing post-translational modifications of the sequence. In certain embodiments, VL comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123, and (c) of SEQ ID NO: 125. Contains one, two, or three HVRs selected from HVR-L3 containing amino acid sequences.

  In another aspect, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 113 An anti-C5 antibody comprising VL with sequence identity is provided. In certain embodiments, the VL sequence is the amino acid sequence of SEQ ID NO: 113. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity is relative to a reference sequence. , Including substitutions (eg, conservative substitutions), insertions, or deletions, an anti-C5 antibody comprising the sequence retains the ability to bind 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, substitutions, insertions, or deletions occur in a region outside of HVR (ie, within FR). Optionally, the anti-C5 antibody comprises the VL sequence in SEQ ID NO: 113, including those containing post-translational modifications of the sequence. In certain embodiments, VL comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 124, and (c) of SEQ ID NO: 125. Contains one, two, or three HVRs selected from HVR-L3 containing amino acid sequences.

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

  In one aspect, (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 54, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 64, and (c) HVR comprising the amino acid sequence of SEQ ID NO: 74 A VH sequence containing -H3, (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) SEQ ID NO: 104 An anti-C5 antibody is provided comprising a VL sequence containing HVR-L3 comprising an amino acid sequence.

  In another aspect, (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118, and (c) comprising the amino acid sequence of SEQ ID NO: 121 A VH sequence containing HVR-H3, and (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123, and (c) SEQ ID NO: 125 And an VL sequence containing HVR-L3 comprising the amino acid sequence of:

  In another aspect, (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 119, and (c) comprising the amino acid sequence of SEQ ID NO: 121 A VH sequence containing HVR-H3, and (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123, and (c) SEQ ID NO: 125 And an VL sequence containing HVR-L3 comprising the amino acid sequence of:

  In another aspect, (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118, and (c) comprising the amino acid sequence of SEQ ID NO: 121 A VH sequence containing HVR-H3, and (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 124, and (c) SEQ ID NO: 125 And an VL sequence containing HVR-L3 comprising the amino acid sequence of:

  In another aspect, (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 120, and (c) comprising the amino acid sequence of SEQ ID NO: 121 A VH sequence containing HVR-H3, and (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 124, and (c) SEQ ID NO: 125 And an VL sequence containing HVR-L3 comprising the amino acid sequence of:

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

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

  In a further aspect, the present invention provides an antibody that binds to the same epitope as an antibody provided herein. In a further aspect, the invention provides an antibody that binds to the same epitope as an antibody described in Table 7 or 8. In certain embodiments, an antibody is provided that binds to an epitope in a fragment consisting of amino acids 33-124 of the C5 beta chain (SEQ ID NO: 40). In certain embodiments, an antibody that binds to an epitope in the β 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 Provided. In certain embodiments, 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. An antibody is provided that binds to an epitope in a fragment of the β chain of C5 (SEQ ID NO: 40) comprising In another embodiment, the epitope of the anti-C5 antibody of the present invention is a conformational epitope.

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 an antibody fragment, such as, for example, an Fv, Fab, Fab ′, scFv, diabody, or F (ab ′) ₂ fragment. In another embodiment, the antibody is a full-length antibody, eg, a full IgG1 antibody or a full IgG4 antibody, or other antibody class or isotype as defined herein.

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

1. Antibody Affinity In certain embodiments, an antibody provided herein has a ≦ 1 μM, ≦ 100 nM, ≦ 10 nM, ≦ 1 nM, ≦ 0.1 nM, ≦ 0.01 nM or ≦ 0.001 nM (eg, 10 ⁻⁸ M or less, For example, it has a dissociation constant (Kd) of 10 ⁻⁸ M to 10 ⁻¹³ M, such as 10 ⁻⁹ M to 10 ⁻¹³ M.

In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, RIA is performed using the Fab version of the antibody of interest and its antigen. For example, Fab binding affinity of an antigen to an antigen is such that the Fab is equilibrated with a minimal concentration of ( ^125I ) -labeled antigen in the presence of an increasing series of unlabeled antigen, and then the bound antigen is coated with an anti-Fab antibody. Measured by catching on a plate. (See, eg, Chen et al., J. Mol. Biol. 293: 865-881 (1999)). To establish the measurement conditions, MICROTITER® multiwell plates (Thermo Scientific) were coated overnight with 5 μg / ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), then Block with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23 ° C.). In a non-adsorbed plate (Nunc # 269620), 100 pM or 26 pM [ ¹²⁵ I] -antigen (see, for example, the anti-VEGF antibody Fab- in Presta et al., Cancer Res. 57: 4593-4599 (1997)). Mix with serial dilutions of the desired Fab (as in the 12 assessment). The Fab of interest is then incubated overnight, which can be continued for a longer time (eg, about 65 hours) to ensure that equilibrium is achieved. The mixture is then transferred to a capture plate for incubation at room temperature (eg, 1 hour). The solution is then removed and the plate is washed 8 times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. When the plate is dry, 150 μl / well 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.

According to another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, a measurement method using BIACORE®-2000 or BIACORE®-3000 (BIACORE®, Inc., Piscataway, NJ) has an antigen of approximately 10 response units (RU). Performed at 25 ° C using a fixed CM5 chip. In one embodiment, the carboxymethylated dextran biosensor chip (CM5, BIACORE®, Inc.) is N-ethyl-N ′-(3-dimethylaminopropyl) -carbodiimide hydrochloride (in accordance with the supplier's instructions). EDC) and N-hydroxysuccinimide (NHS). The antigen is brought to 5 μg / ml (approximately 0.2 μM) using 10 mM sodium acetate, pH 4.8 before being injected at a flow rate of 5 μl / min to achieve approximately 10 reaction units (RU) of protein binding. Diluted. After the injection of antigen, 1M ethanolamine is injected to block unreacted groups. For kinetic measurements, a 2-fold serial dilution of Fab (0.78 nM) in PBS (PBST) containing 0.05% polysorbate 20 (TWEEN-20 ™) surfactant at 25 ° C. and a flow rate of approximately 25 μl / min. ~ 500 nM) is injected. The association rate (k _on ) and dissociation rate (k _off ) can be determined by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir association model (BIACORE® evaluation software version 3.2). Calculated. The equilibrium dissociation constant (Kd) is calculated as the ratio k _off / k _on . See, for example, Chen et al., J. Mol. Biol. 293: 865-881 (1999). When the on-rate exceeds 10 ⁶ M ^-1 s ^-1 by the surface plasmon resonance assay described above, the on-rate is measured using a spectrophotometer (eg, a stop flow spectrophotometer (Aviv Instruments) or a 8000 series SLM- Fluorescence emission intensity at 25 ° C. (excitation) of 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2 in the presence of increasing concentrations of antigen as measured in an AMINCO ™ spectrophotometer (ThermoSpectronic)) = 295 nm; emission = 340 nm, bandpass 16 nm) can be determined by using a fluorescence quenching technique that measures the increase or decrease.

2. Antibody Fragments 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 specific antibody fragments, see Hudson et al., Nat. Med. 9: 129-134 (2003). For reviews of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a discussion of Fab and F (ab ′) ₂ fragments that contain salvage receptor binding epitope residues and have increased in vivo half-life.

  A diabody is an antibody fragment with two antigen binding sites, which may be bivalent or bispecific. For example, EP 404,097; WO1993 / 01161; Hudson et al., Nat. Med. 9: 129-134 (2003); Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) See). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).

  Single domain antibodies are antibody fragments that comprise all or part of an antibody heavy chain variable domain, or all or part of a light chain variable domain. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, eg, US Pat. No. 6,248,516).

  Antibody fragments include, but are not limited to, a variety of antibodies, including proteolytic digestion of complete antibodies, production by recombinant host cells (eg, E. coli or phage) as described herein. It can be made by the method of.

3. Chimeric and humanized antibodies In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in US Pat. 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 (eg, 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 switch” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies also include antigen binding fragments thereof.

  In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized in order to reduce human immunogenicity 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 HVR (eg, CDR (or portion thereof)) is derived from a non-human antibody and FR (or portion thereof) is derived from a human antibody sequence. Derived from. A humanized antibody optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are non-human antibodies (eg, antibodies derived from HVR residues, eg, to restore or improve antibody specificity or affinity). ) With the corresponding residue from

  Humanized antibodies and methods for their production are reviewed in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008), and also see, for example, Riechmann et al., Nature 332: 323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86: 10029-10033 (1989); US Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36 : 25-34 (2005) (specify 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 Osbourn et al., Methods 36: 61-68 (2005) and Klimka et al., Br. J. Cancer 83: 252 -260 (2000) (which describes a “guide selection” approach for FR shuffling).

  Human framework regions that can be used for humanization are selected using, but not limited to: the “best fit” method (see Sims et al., J. Immunol. 151: 2296 (1993)). Framework regions; framework regions derived from consensus sequences of human antibodies of specific subgroups of light or heavy chain variable regions (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992 ) And Presta et al., J. Immunol. 151: 2623 (1993)); human maturation (somatic mutation) framework region or human germline framework region (eg, Almagro and Fransson, Front. Biosci. 13). : 1619-1633 (2008)); and framework regions derived from FR library screening (Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

4). Human Antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be 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).

  Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce fully human antibodies or fully antibodies with human variable regions in response to an antigen challenge (load). Such animals typically include all or part of a human immunoglobulin locus, which all or part of the human immunoglobulin locus replaces the endogenous immunoglobulin locus or is extrachromosomal or It exists in a state of being randomly incorporated in the chromosome of the animal. 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). Also, for example, US Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE ™ technology; US Pat. No. 5,770,429 describing HUMAB® technology; US Patent No. describing KM MOUSE® technology See also 7,041,870; and US Patent Application Publication No. 2007/0061900 describing the VELOCIMOUSE® technology. Human variable regions from intact antibodies produced by such animals may be further modified, for example, in combination with different human constant regions.

  Human antibodies can also be made by methods based on hybridomas. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have already been described. (Eg, Kozbor, J. Immunol. 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Dekker, Inc., New York, 1987)); and Boerner et al. , J. Immunol. 147: 86 (1991).) Human antibodies 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, for example, US Pat. 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) (human- Human hybridoma is described). 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). ing.

  Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with the desired human constant domain. A technique for selecting a human antibody from an antibody library is described below.

5. Library-derived antibodies Antibodies of the present invention may be isolated by screening combinatorial libraries for antibodies with one or more desired 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 properties. 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, for example, McCafferty et al., Nature 348: 552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks, Meth. Mol. Biol. 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338 (2): 299-310 (2004); Lee et al., J. Mol. Biol. 340 (5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101 (34): 12467-12472 (2004); Lee et al., J Immunol. Methods 284 (1-2): 119-132 (2004).

  In certain phage display methods, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which is then used in Winter et al. ., Ann. Rev. Immunol. 12: 433-455 (1994) can be screened for antigen-binding phages. 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, as described in Griffiths et al., EMBO J, 12: 725-734 (1993), a naïve repertoire can be cloned (eg, from a human) without extensive immunization and without immunization. It is also possible to provide a single source of antibodies to self antigens. Finally, the naive library is a clone of the pre-rearranged V-gene segment from stem cells, as described in Hoogenboom, J. Mol. Biol. 227: 381-388 (1992), which encodes the hypervariable CDR3 region. However, it can also be made synthetically by using PCR primers containing random sequences to achieve reconstitution in vitro. Patent documents describing human antibody phage libraries include, for example: US Pat. No. 5,750,373, and US Patent Application Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007. / 0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

  An antibody or antibody fragment isolated from a human antibody library is considered herein a human antibody or human antibody fragment.

6). Multispecific Antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies (eg, bispecific antibodies). Multispecific antibodies are monoclonal antibodies that have binding specificities 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, the bispecific antibody may bind to two different epitopes of C5. Bispecific antibodies may be used to localize cytotoxic agents to cells that express C5. Bispecific antibodies can be prepared as full length antibodies or as antibody fragments.

  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 (Milstein and Cuello, Nature 305: 537 (1983), WO93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and “knob-in-hole” technology (see, eg, US Pat. No. 5,731,168). Multispecific antibodies manipulate the electrostatic steering effect to create Fc heterodimeric molecules (WO2009 / 089004A1); cross-link two or more antibodies or fragments (US patents) No. 4,676,980 and Brennan et al., Science 229: 81 (1985)); generating antibodies with two specificities using a leucine zipper (Kostelny et al., J. Immunol. 148 (5): 1547-1553 (1992)); generation of bispecific antibody fragments using “diabody” technology (Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) And) using a single chain Fv (scFv) dimer (see Gruber et al., J. Immunol. 152: 5368 (1994)); and, for example, Tutt et al., J. Immunol. 147: 60 (1991) to prepare trispecific antibodies.

  Modified antibodies with more than two functional antigen binding sites, including “Octopus antibodies” are also included herein (see, eg, US Patent Application Publication No. 2006/0025576).

  As used herein, an antibody or fragment also includes a “dual-acting Fab” or “DAF” that includes one antigen-binding site that binds C5 to another different antigen (eg, US Patent Application Publication No. 2008/0069820). Issue).

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 to the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletion from the amino acid sequence of the antibody, and / or insertion into the amino acid sequence of the antibody, and / or substitution of residues in the amino acid sequence of the antibody. Given that the final construct is provided with the desired characteristics (eg, antigen binding), any combination of deletions, insertions, and substitutions can be made to arrive at the final construct.

a. Substitution, insertion, and deletion variants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include HVR and FR. Conservative substitutions are shown under the heading “Preferred substitutions” in Table 1. More substantial changes are provided under the heading “Exemplary substitutions” in Table 1 and are detailed below with reference to the class of amino acid side chains. Amino acid substitutions may be introduced into the antibody of interest, and the product may be screened for the desired activity, eg, retention / improved antigen binding, decreased immunogenicity, or improved ADCC or CDC. Good.

Amino acids can be grouped according to common side chain properties:
(1) Hydrophobicity: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile);
(2) Neutral hydrophilicity: cysteine (Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln);
(3) Acidity: Aspartic acid (Asp), glutamic acid (Glu);
(4) Basicity: histidine (His), lysine (Lys), arginine (Arg);
(5) Residues that affect chain orientation: glycine (Gly), proline (Pro); and
(6) Aromaticity: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe).

  Non-conservative substitutions refer to exchanging one member of these classes for another class.

  One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (eg, a humanized or human antibody). Usually, the resulting mutants selected for further study are modified (eg, improved) in specific biological properties compared to the parent antibody (eg, increased affinity, decreased immunogenicity). And / or substantially retain certain biological properties of the parent antibody. An exemplary substitution variant is an affinity matured antibody, which can be made as appropriate using, for example, phage display-based affinity maturation techniques (eg, those described herein). Briefly, one or more HVR residues are mutated and the mutated antibody is displayed on a phage and screened for a specific biological activity (eg, binding affinity).

  Modifications (eg, substitutions) can be made in HVRs, eg, to improve antibody affinity. Such modifications are HVR “hot spots”, ie, residues encoded by codons that undergo mutations frequently during the somatic maturation process (eg, Chowdhury, Methods Mol. Biol. 207: 179-196). (See 2008)) and / or at residues that contact the antigen and the resulting mutant VH or VL can be tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, by 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 variable genes selected for maturation by any of a variety of methods (eg, 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-oriented approach that randomizes several HVR residues (eg, 4-6 residues at a time). HVR residues involved in antigen binding can be specifically identified using, for example, alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

  In certain embodiments, substitutions, insertions, or deletions can be made within one or more HVRs as long as such modifications do not substantially reduce the antibody's ability to bind antigen. For example, conservative modifications that do not substantially reduce binding affinity (eg, conservative substitutions as provided herein) can be made in HVRs. Such modifications can be, for example, outside of the antigen contact residues of HVR. In certain embodiments of the above mutated VH and VL sequences, each HVR is unaltered or contains as few as 1, 2 or 3 amino acid substitutions.

  A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is described by Cunningham, Science 244: 1081-1085 (1989), referred to as "alanine scanning mutagenesis". It is. In this method, a residue or group of target residues (eg, charged residues such as arginine, aspartic acid, histidine, lysine, and glutamic acid) are identified and neutral or negatively charged amino acids (eg, alanine or Polyalanine) to determine whether the antibody-antigen interaction is affected. Additional substitutions can be introduced at amino acid positions that have shown functional sensitivity to this initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex may be analyzed to identify contact points between the antibody and the antigen. Such contact residues and neighboring residues may be targeted as substitution candidates or excluded from substitution candidates. Variants can be screened to determine if they contain the desired property.

  Amino acid sequence insertions, as well as insertions of single or multiple amino acid residues within the sequence, are within the length of a polypeptide comprising from 1 to 100 residues or more at the amino and / or carboxyl terminus. Includes fusion. Examples of terminal insertions include antibodies with a methionyl residue at the N-terminus. Other insertional variants of the antibody molecule include those in which the N- or C-terminus of the antibody is fused to an enzyme (eg, for ADEPT) or a polypeptide that increases the serum half-life of the antibody.

b. Glycosylation variants In certain embodiments, the antibodies provided herein have been modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to the antibody can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

  If the antibody contains an Fc region, the carbohydrate added thereto may be modified. Natural-type antibodies produced by mammalian cells typically contain a branched biantennary oligosaccharide, which is usually added by N-linkage to Asn297 in the CH2 domain of the Fc region. See, for example, 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 added to GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modification of oligosaccharides in the antibodies of the invention may be made to create antibody variants with certain improved properties.

  In one embodiment, antibody variants having carbohydrate structures lacking fucose added (directly or indirectly) to the Fc region are provided. 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 the sum of all sugar structures (eg, complex, hybrid, and high mannose structures) added to Asn297 as measured by MALDI-TOF mass spectrometry as described, for example, in WO2008 / 077546 In contrast, it is determined by calculating the average amount of fucose in the sugar chain in Asn297. Asn297 represents an asparagine residue located around position 297 in the Fc region (EU numbering of Fc region residues). However, due to the slight sequence diversity between the antibodies, Asn297 may be located ± 3 amino acids upstream or downstream of position 297, ie, between positions 294-300. Such fucosylated mutants may have improved ADCC function. See, for example, US Patent Application Publication No. 2003/0157108 (Presta, L.); 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to “defucosylated” or “fucose-deficient” antibody variants are US2003 / 0157108; WO2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / US2004 / 0110282; US2004 / 0109865; WO2003 / 085119; WO2003 / 084570; WO2005 / 035586; WO2005 / 035778; WO2005 / 03052; WO2002 / 031140; Okazaki et al., J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells that lack protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249: 533-545 (1986); US2003 / 0157108, Presta, L; and WO2004 / 056312, Adams et al., In particular Example 11) and knockout cell lines such as the alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (eg Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004); Kanda et al., Biotechnol. Bioeng. 94 (4): 680-688 (2006); and WO2003 / 085107).

  Further provided are antibody variants having a bisected oligosaccharide, for example, a biantennary oligosaccharide added to the Fc region of an antibody is 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.); US Pat. No. 6,602,684 (Umana et al.); And US2005 / 0123546 (Umana et al.). Yes. 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.).

c. Fc region variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants. An Fc region variant may comprise a human Fc region sequence (eg, a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (eg, substitution) at one or more amino acid positions.

  In certain embodiments, antibody variants with some, but not all, effector functions are also within the scope of the present invention, which effector functions with respect to its in vivo half-life, Certain effector functions (such as complement and ADCC) are good candidates for application when they are unnecessary or harmful. In vitro and / or in vivo cytotoxicity measurements can be performed to confirm a decrease / deficiency of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding measurements can be performed to ensure that antibodies lack FcγR binding (and thus are more likely to lack ADCC activity) while maintaining FcRn binding ability. NK cells, which are primary cells that mediate 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 (assays) for assessing ADCC activity of a molecule of interest include US Pat. No. 5,500,362 (eg, 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); US Pat. No. 5,821,337 (Bruggemann et al., J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, non-radioactive assays may be used (eg, ACT1 ™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96 ™ non -radioactive cytotoxicity assays (see Promega, Madison, WI). Effector cells useful for such measurement methods include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or in addition, the ADCC activity of the molecule of interest can be determined in vivo in an animal model as described, for example, in Clynes et al., Proc. Nat'l Acad. Sci. USA 95: 652-656 (1998). It may be evaluated. Also, C1q binding measurements may be performed to confirm that the antibody cannot bind to C1q and thus lacks CDC activity. See, for example, the C1q and C3c binding ELISA in WO2006 / 029879 and WO2005 / 100402. CDC measurements may also be performed to assess complement activation (eg, 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)). Furthermore, determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (eg, Petkova et al., Int'l. Immunol. 18 (12): 1759- 1769 (2006)).

  Antibodies with reduced effector function include those with one or more substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Pat. No. 6,737,056). Such Fc variants include amino acid positions 265, 269, 270, 297, and 327, including so-called “DANA” Fc variants (US Pat. No. 7,332,581) with substitution of residues 265 and 297 to alanine. Includes Fc variants with two or more substitutions.

  Specific antibody variants with improved or attenuated binding to FcR have been described. (See US Pat. No. 6,737,056; WO2004 / 056312 and Shields et al., J. Biol. Chem. 9 (2): 6591-6604 (2001).)

  In certain embodiments, the antibody variant has one or more amino acid substitutions that improve ADCC (eg, substitutions at positions 298, 333, and / or 334 (residues in the EU numbering) of the Fc region). Includes accompanying Fc region.

  In some embodiments, modified (ie, improved) as described, for example, in US Pat. No. 6,194,551, WO1999 / 51642, and Idusogie et al., J. Immunol. 164: 4178-4184 (2000). Modifications are made in the Fc region that result in C1q binding and / or complement dependent cytotoxicity (CDC), which are either attenuated or attenuated.

  Increased half-life and role in transferring neonatal Fc receptors (FcRn: maternal IgGs to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Antibodies with improved binding to Immunol. 24: 249 (1994))) are described in US Patent Application Publication No. 2005/0014934 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that improve the binding of the Fc region to FcRn. Such Fc variants include 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 at one or more substitutions (eg, substitution of Fc region residue 434 (US Pat. No. 7,371,826)).

  See also Duncan, Nature 322: 738-40 (1988); US Pat. No. 5,648,260; US Pat. No. 5,624,821; and WO1994 / 29351 for other examples of Fc region variants.

d. Cysteine engineered antibody variants In certain embodiments, it may be desirable to create a cysteine engineered antibody (eg, “thioMAb”) in which one or more residues of the antibody are replaced with cysteine residues. In certain embodiments, the residue undergoing substitution occurs at an accessible site in the antibody. By replacing those residues with cysteine, a reactive thiol group is placed at an accessible site of the antibody, and the reactive thiol group can be attached to the other moiety (drug moiety or linker-drug moiety). Etc.) may be used to create immunoconjugates as described in further detail herein. In certain embodiments, any one or more of the following residues may be replaced with cysteine: light chain V205 (Kabat numbering); heavy chain A118 (EU numbering); and heavy chain Fc region S400. (EU numbering). Cysteine engineered antibodies may be generated, for example, as described in US Pat. No. 7,521,541.

e. Antibody Derivatives In certain embodiments, the antibodies provided herein may be further modified to include additional non-protein moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly 1,3 Dioxolane, poly1,3,6, trioxane, ethylene / maleic anhydride copolymer, polyamino acid (either homopolymer or random copolymer), and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, Polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (eg, glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its water stability. The polymer may have any molecular weight and may or may not be branched. The number of polymers added to the antibody can vary, and if two or more polymers are added, they can be the same molecule or different molecules. In general, the number and / or type of polymers used for derivatization is not limited to these, but is specific to the antibody's particular characteristics or function to be improved, under conditions where the antibody derivative is under defined conditions. It can be determined based on considerations such as whether or not to use in therapy.

  In another embodiment, a conjugate of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation is provided. 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, but is not limited to such, as it does not harm normal cells but heats the non-protein part to a temperature that kills cells in close proximity to the antibody-non-protein part. Includes wavelength.

B. Recombinant Methods and Configurations Antibodies can be produced using recombinant methods and configurations, for example, as described in US Pat. No. 4,816,567. In one aspect, an isolated nucleic acid encoding an anti-C5 antibody described herein is provided. Such a nucleic acid may encode an amino acid sequence comprising an antibody VL and / or an amino acid sequence comprising a VH (eg, an antibody light and / or heavy chain). In further embodiments, one or more vectors (eg, expression vectors) comprising such nucleic acids are provided. In a further aspect, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the antibody VL and an amino acid sequence comprising the antibody VH, or (2) an amino acid comprising the antibody VL. A first vector comprising a nucleic acid encoding sequence and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the antibody VH are included (eg, transformed). In one embodiment, the host cell is eukaryotic (eg, Chinese hamster ovary (CHO) cells) or lymphoid cells (eg, Y0, NS0, SP20 cells)). In one embodiment, culturing a host cell comprising a nucleic acid encoding the antibody as described above under conditions suitable for expression of the anti-C5 antibody, and optionally, the antibody is transformed into a host cell (or host cell culture medium). A method for producing an anti-C5 antibody is provided.

  For recombinant production of anti-C5 antibodies, nucleic acid encoding the antibody (eg, as described above) is isolated and transferred to one or more vectors for further cloning and / or expression in a host cell. insert. Such nucleic acids will be readily isolated and sequenced using conventional procedures (eg, oligonucleotide probes that can specifically bind to the genes encoding the antibody heavy and light chains). By using).

  Suitable host cells for cloning or expressing the antibody-encoding vector include prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, particularly where glycosylation and Fc effector function are not required. See, eg, US Pat. Nos. 5,648,237, 5,789,199, and 5,840,523 for expression of antibody fragments and polypeptides in bacteria. (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 from the bacterial cell paste in a soluble fraction and can be further purified.

  In addition to prokaryotes, true species such as filamentous fungi or yeast, including fungal and yeast strains in which the glycosylation pathway is “humanized”, resulting in the production of antibodies with partial or complete human glycosylation patterns. Nuclear microorganisms 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).

  Those derived from multicellular organisms (invertebrates and vertebrates) are also suitable host cells for the expression of glycosylated antibodies. Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified that are used for mating with insect cells, particularly for transformation of Spodoptera frugiperda cells.

  Plant cell cultures can also be used as hosts. See, for example, US Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (described PLANTIBODIES ™ technology for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension will be useful. Other examples of useful mammalian host cell lines include monkey kidney CV1 strain (COS-7) transformed with SV40; human embryonic kidney strain (Graham et al., J. Gen Virol. 36:59 (1977 293 or 293 cells described above); hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells described in Mather, Biol. Reprod. 23: 243-251 (1980)); monkey kidney cells (CV1 ); African green monkey kidney cells (VERO-76); Human cervical cancer cells (HELA); Canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); Human lung cells (W138); Human hepatocytes ( Hep G2); mouse breast cancer (MMT 060562); TRI cells (for example, described in Mather et al., Annals NY Acad. Sci. 383: 44-68 (1982)); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR ^- CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); and Y0, Includes myeloma cell lines such as NS0 and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268. (2003).

Polyclonal antibodies are preferably produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. Related antigens to proteins that are immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, bifunctional substances or derivatizing agents, such as , Maleimidobenzoylsulfosuccinimide ester (conjugation via cysteine residue), N-hydroxysuccinimide (via lysine residue), glutaraldehyde, succinic anhydride, SOCl ₂ , or R ¹ N = C = NR (where R And R ¹ are different alkyl groups) may be useful to conjugate.

  Animals (usually non-human mammals) may, for example, inject 100 μg or 5 μg protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and inject the solution into multiple sites. To immunize against an antigen, immunogenic conjugate, or derivative. One month later, the animals are boosted with an initial amount of 1/5 to 1/10 peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later, the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer reaches a plateau. Preferably, the animal is boosted with the same antigen but conjugated to another protein and / or conjugated via another cross-linking reagent. Conjugates can also be prepared as protein fusions in recombinant cell culture. In order to enhance the immune response, an aggregating agent such as alum is also preferably used.

  Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are naturally occurring potential mutations and / or post-translational modifications (e.g., The same except for isomerization and amidation). Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies.

  For example, monoclonal antibodies can be made using the hybridoma method first described in Kohler et al. Nature 256 (5517): 495-497 (1975). In the hybridoma method, the ability to immunize a mouse or other suitable host animal, such as a hamster, as described herein above to produce or produce an antibody that specifically binds to the protein used for immunization. Induces certain lymphocytes. Alternatively, lymphocytes can be immunized in vitro.

  The immunizing agent typically includes an antigenic protein or a fusion variant thereof. In general, peripheral blood lymphocytes (PBL) are used when cells of human origin are desired, and spleen cells or lymph node cells are used when non-human mammalian sources are desired. The lymphocytes are then fused with an immortal 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).

  Immortal cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are utilized. The hybridoma cells thus produced are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for hybridomas typically contains hypoxanthine, aminopterin, a substance that prevents the growth of HGPRT-deficient cells, And thymidine (HAT medium).

  Preferred immortalized myeloma cells are cells that fuse efficiently, assist in the production of stable high levels of antibody by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. . Among these, mouse myeloma strains, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center in San Diego, California, USA, and from the American Type Culture Collection in Manassas, Virginia, USA SP-2 cells (and derivatives thereof such as X63-Ag8-653) are preferred. Human myeloma cell lines and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor et al., J Immunol. 133 (6): 3001-3005 (1984); Brodeur et al Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987), pp. 51-63).

  Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and measurement methods 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).

  After hybridoma cells producing antibodies 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.

  Monoclonal antibodies secreted by subclones are obtained from culture media, ascites fluid, or serum by conventional immunoglobulin purification methods such as protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. , Properly separated.

  The antibody 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 C5 beta chain (SEQ ID NO: 40). In one embodiment, the antigen is a polypeptide comprising the MG1-MG2 domain of the C5 β chain (SEQ ID NO: 43). In one embodiment, the antigen is a polypeptide comprising the MG1 domain of the C5 β chain (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 C5 beta chain. In one embodiment, the antigen is a polypeptide comprising a region corresponding to amino acids at positions 33-124 of the C5 beta chain. 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 C5 beta chain (SEQ ID NO: 40). In one embodiment, the antigen is at least 1 selected from the group consisting of Thr47, Glu48, Ala49, Phe50, Asp51, Ala52, Thr53, Lys57, His70, Val71, His72, Ser74, Glu76, Val107, Ser108, Lys109, and His110. A polypeptide comprising a fragment of the β chain of C5 containing one amino acid. In one embodiment, the antigen is a polypeptide comprising a C5 β chain fragment comprising at least one amino acid selected from the group consisting of Glu48, Asp51, His70, His72, Lys109, and His110. Antibodies produced by immunizing an animal against an antigen are also included in the present invention. The antibody can incorporate any of the features described in “Exemplary anti-C5 antibodies” above, alone or in combination.

C. Measurement method (assay)
The anti-C5 antibodies provided herein are identified, screened, or revealed for physical / chemical properties and / or biological activity by various assays known in the art. May be.

1. Binding Assays and Other Assays In one aspect, the antibodies of the present invention are tested for their antigen binding activity by known methods such as ELISA, Western blot, BIACORE® and the like.

  In another aspect, competition assays can be used to identify antibodies that compete with the anti-C5 antibodies described herein for binding to C5. In certain embodiments, when such competing antibodies are present in excess, this results in at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% binding of the reference antibody to C5. Block (eg, reduce)%, 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 competing antibodies are the same epitope (eg, linear or steric) that is bound by the anti-C5 antibodies described herein (eg, the anti-C5 antibodies described in Table 2). To the structural epitope). A detailed exemplary method for mapping the epitope to which an antibody binds is provided by Morris, “Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ) (1996).

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

  In another exemplary competitive assay, BIACORE® analysis is used to determine the ability of a test anti-C5 antibody to compete for binding to C5 by a second (reference) anti-C5 antibody. In a further aspect of operating a BIACORE® instrument (eg, BIACORE® 3000) according to the manufacturer's recommendations, C5 protein can be converted to CM5 BIACORE® using standard techniques known in the art. ) Capture on chip and create C5 coated surface. Typically, 200-800 resonance units of C5 are bound to the chip (an amount that provides an easily measurable level of binding but is easily saturable at the concentration of test antibody used). Two antibodies to be evaluated for their ability to compete with each other (ie, test antibody and reference antibody) are mixed in a suitable buffer at a 1: 1 molar ratio of binding sites to produce a test mixture. When calculating the concentration based on the binding site, the molecular weight of the test or reference antibody is the total molecular weight of the corresponding antibody divided by the number of C5 binding sites on the antibody. The concentration of each antibody (i.e., test antibody and reference antibody) in the test mixture must be high enough to easily saturate the binding site of the antibody on the C5 molecule captured on the BIACORE® chip. I must. The test antibody and reference antibody in the mixture are at the same molar concentration (based on binding), typically 1.00-1.5 micromolar (based on binding site). Separate solutions containing only the test antibody and only the reference antibody are also prepared. The test antibody and reference antibody 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 antibody and the reference antibody is passed over a C5 coated BIACORE® chip and the total amount of binding is recorded. The chip is then treated to remove bound test or reference antibody without damaging C5 bound to the chip. Typically this is done by treating the chip with 30 mM HCl for 60 seconds. The test antibody alone solution is then passed over a C5 coated surface and the amount of binding is recorded. Again, the chip is treated to remove all of the bound antibody without damaging the C5 bound to the chip. A solution of reference antibody alone is then passed over the C5 coated surface and the amount of binding is recorded. Next, the theoretical maximum of the binding of the test antibody and reference antibody mixture is calculated, which is the sum of the binding of each antibody (i.e., test and reference) when passed alone over the C5 surface. is there. If the actual recorded binding of the mixture is less than this theoretical maximum, the test antibody and reference antibody are competing with each other for binding to C5. Thus, in general, competing test anti-C5 antibodies have a binding that is recorded during the BIACORE® blocking assay described above in the presence of a reference anti-C5 antibody combined with the combined test antibody. Between 80% and 0.1% of the antibody's theoretical binding maximum (as defined above) (eg, 80%>-4%), especially between 75% and 0.1% of the theoretical binding maximum (For example, 75% to 4%), more specifically, binding to C5 in the measurement method to be between 70% and 0.1% of the theoretical maximum of binding (for example, 70% to 4%). An antibody.

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

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

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

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

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

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

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

  In certain embodiments, an anti-C5 antibody of the invention is selected from VH of SEQ ID NO: 1 and VL of SEQ ID NO: 11, or VH of SEQ ID NO: 10 and VL of SEQ ID NO: 20 for binding to C5. Compete with antibodies containing VH and VL pairs.

  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 binds to 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 : (A) VH of SEQ ID NO: 1 and VL of SEQ ID NO: 11; (b) VH of SEQ ID NO: 5 and VL of SEQ ID NO: 15; (c) VH of SEQ ID NO: 4 and SEQ ID NO: 14 (D) VH of SEQ ID NO: 6 and VL of SEQ ID NO: 16; (e) VH of SEQ ID NO: 2 and VL of SEQ ID NO: 12; (f) VH of SEQ ID NO: 3 and SEQ ID NO: 13 (G) VH of SEQ ID NO: 9 and VL of SEQ ID NO: 19; (h) VH of SEQ ID NO: 7 and VL of SEQ ID NO: 17; (i) VH of SEQ ID NO: 8 and SEQ ID NO: 18 VL; and (j) VH of SEQ ID NO: 10 and VL of SEQ ID NO: 20. In a further embodiment, the anti-C5 antibody binds to C5 with higher affinity at pH 7.4 than at pH 5.8.

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

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

  In certain embodiments, whether an anti-C5 antibody of the invention binds to a particular epitope can be determined as follows: C5 point mutants in which the amino acid of C5 (except alanine) is replaced with alanine Was tested in 293 cells and tested for binding of anti-C5 antibody to the C5 variant by ELISA, Western blot or BIACORE®; where compared to binding of anti-C5 antibody to wild-type C5. A substantial decrease or disappearance of binding of the anti-C5 antibody to the C5 variant indicates that the anti-C5 antibody binds to an epitope comprising the amino acid on C5. In certain embodiments, the amino acid on C5 that is substituted with alanine is selected from the group consisting of Glu48, Asp51, His70, His72, Lys109, and His110 of the β chain of C5 (SEQ ID NO: 40). In a further embodiment, the amino acid on C5 that is substituted with alanine is Asp51 or Lys109 in the β chain of C5 (SEQ ID NO: 40).

  In another embodiment, whether an anti-C5 antibody having pH-dependent binding properties binds to a particular epitope can be determined as follows: a histidine residue on C5 is another amino acid (eg, tyrosine ) -Substituted C5 point mutants are expressed in 293 cells and the binding of anti-C5 antibodies to the C5 mutants is tested by ELISA, Western blot or BIACORE®; A substantial decrease in binding of anti-C5 antibody to wild-type C5 at acidic pH compared to binding of C5 antibody to the C5 variant indicates that the anti-C5 antibody has an epitope containing the histidine residue on C5. Indicates to combine. In a further embodiment, binding of the anti-C5 antibody to wild type C5 at neutral pH is not substantially reduced compared to binding of the anti-C5 antibody to the C5 variant at neutral pH. In certain embodiments, the histidine residue on C5 that is substituted with another amino acid is selected from the group consisting of His70, His72, and His110 of the C5 β chain (SEQ ID NO: 40). In a further embodiment, the histidine residue His70 is replaced with tyrosine.

2. Activity Measuring Method In one aspect, a measuring method for identifying that of an anti-C5 antibody having biological activity is provided. Biological activity includes, for example, inhibiting C5 activation, preventing C5 cleavage to form C5a and C5b, preventing access of C5 convertase to the cleavage site on C5, It may include blocking hemolytic activity caused by C5 activation. Also provided are antibodies having such biological activity in vivo and / or in vitro.

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

  In certain embodiments, whether a test antibody inhibits 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). judge. In another embodiment, this is determined by methods for specifically detecting cleaved C5a and / or C5b proteins, such as ELISA or Western blot. A test antibody can inhibit C5 cleavage if a decrease in the amount of C5 cleavage product (i.e., C5a and / or C5b) is detected in the presence of (or after contact with) the test antibody Identified as an antibody. In certain embodiments, the concentration and / or physiological activity of C5a can be measured by methods such as chemotaxis, RIA, or ELISA (eg, Ward and Zvaifler J. Clin. Invest. 50 ( 3): 606-616 (1971)).

  In certain embodiments, a method for detecting protein interactions between C5 convertase and C5, such as ELISA or BIACORE®, determines whether the test antibody blocks access of C5 convertase to C5. ,judge. 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 C5 convertase access to C5.

  In certain embodiments, C5 activity can be measured as a function of its cytolytic ability in a subject's body fluid. The cytolytic ability of C5 or its decrease can be measured by methods well known in the art, for example, conventional hemolysis methods such as Kabat and Mayer (ed.), Experimental Immunochemistry, 2nd edition, 135-240. , Springfield, IL, CC Thomas (1961), pages 135-139, or conventional variations of the method, such as Hillmen et al., N. Engl. J. Med. 350 (6 ): Measured by chicken erythrocyte hemolysis as described in 552-559 (2004). In certain embodiments, C5 activity or inhibition thereof is quantified using a CH50eq assay. The CH50eq assay is a method for measuring total classical complement activity in serum. This test is a lytic assay that uses antibody-sensitized red blood cells as activators of the classical complement pathway and a variety of test sera to determine the amount needed to give 50% lysis (CH50). Use a dilution of The percentage of hemolysis can be determined, for example, using a spectrophotometer. The CH50eq assay provides an indirect measure of TCC formation since the direct cause of hemolysis measured is the terminal complement complex (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 is at least 5%, 10%, 15%, 20%, 25%, 30% of C5 activation in the assay compared to the effect of a negative control under similar conditions. %, 35%, or 40% or more decrease. In some embodiments, it is at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more of C5 activation Refers to inhibition of

D. Immunoconjugates The invention also includes one or more cytotoxic agents (eg, chemotherapeutic or chemotherapeutic agents, growth inhibitors, toxins (eg, protein toxins of bacterial, fungal, plant or animal origin, enzymatically active). Provided is an immunoconjugate comprising an anti-C5 antibody herein conjugated to a toxin, or fragment thereof) or radioisotope).

  In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) wherein the antibody is conjugated to one or more drugs including, but not limited to: There are: maytansinoids (see US Pat. Nos. 5,208,020, 5,416,064, and European Patent No. 0,425,235 B1); for example, monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (US Pat. And auristatin such as US Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, And 5,877,296; see Hinman et al., Cancer Res. 53: 3336-3342 (1993); and Lode et al., Cancer Res. 58: 2925-2928 (1998)); daunomycin or Anthracyclines such as 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 US Pat. No. 6,630,579); methotrexate; vindesine; docetaxel, paclitaxel, larotaxel, tesetaxel, And taxanes such as Ortaxel; Trichothecene; and CC1065.

  In another embodiment, the immunoconjugate comprises an antibody described herein conjugated to an enzymatically active toxin or fragment thereof including, but not limited to: diphtheria A chain. , Non-binding active fragment of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modesin A chain, alpha-sarcin, Aleurites fordii protein, diantine Protein, Phytolacca americana protein (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, gelonin, mitogellin, Restrictocin, phenomycin, enomycin, and trichothecene.

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

  Antibody and cytotoxic agent conjugates can be made using a variety of bifunctional protein linking agents. For example, N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (Eg dimethyl adipimidate HCl), active esters (eg disuccinimidyl suberate), aldehydes (eg glutaraldehyde), bis-azide compounds (eg bis (p-azidobenzoyl) hexanediamine), bis- Diazonium derivatives (eg bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (eg toluene 2,6-diisocyanate), and bis-active fluorine compounds (eg 1,5-difluoro-2,4-dinitrobenzene) It is. For example, a ricin immunotoxin 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 chelator for conjugation of radionuclides to antibodies. See WO94 / 11026. The linker may be a “cleavable linker” that facilitates the release of the cytotoxic agent within the cell. For example, acid labile linkers, peptidase sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al., Cancer Res. 52: 127-131 (1992); US Pat. No. 5,208,020) are used. obtain.

  The immunoconjugates or ADCs herein are commercially available (eg, from Pierce Biotechnology, Inc., Rockford, IL., USA) 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) Conjugates prepared using cross-linking reagents including but not limited to benzoate are explicitly considered, but not limited thereto.

E. Methods and compositions for diagnosis and detection In certain embodiments, any of the anti-C5 antibodies provided herein are useful for detecting the presence of C5 in a biological sample. As used herein, the term “detection” encompasses quantitative or qualitative detection. In certain embodiments, the biological sample is a cell or tissue, eg, serum, whole blood, plasma, biopsy sample, tissue sample, cell suspension, saliva, sputum, oral fluid, cerebrospinal fluid, amniotic fluid, ascites Breast milk, colostrum, mammary secretions, lymph, urine, sweat, tears, gastric juice, synovial fluid, ascites, ophthalmic lens fluid, and mucus.

  In one embodiment, an anti-C5 antibody for use in a diagnostic or detection method is provided. In a further aspect, a method for detecting the presence of C5 in a biological sample is provided. In certain embodiments, the method comprises contacting a biological sample with an anti-C5 antibody described herein under conditions that permit binding of the anti-C5 antibody to C5, and the anti-C5 antibody and C5. Detecting whether a complex has formed between them. 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 compatible with treatment with the anti-C5 antibody, for example when C5 is a biomarker for selecting patients.

  In another embodiment, a method of selecting an individual having a complement-mediated disease or condition with excessive or uncontrolled activation of C5 as suitable for a therapy comprising an anti-C5 antibody of the invention. Provided. In certain embodiments, the method comprises: (a) detecting a genetic variation in C5 from an individual; and (b) if a genetic variation is detected in C5 from the individual, Selecting to be suitable for therapies involving C5 antibodies. In another embodiment, a method is provided for selecting a therapy for an individual having a complement-mediated disease or condition with excessive or uncontrolled activation of C5. In certain embodiments, the method comprises: (a) detecting a genetic mutation in C5 from an individual; and (b) detecting the genetic mutation in C5 from the individual, if the genetic mutation is detected Selecting for the individual a treatment comprising.

  In another embodiment, a method of treating an individual having a complement-mediated disease or condition with excessive or uncontrolled activation of C5 is provided. In certain embodiments, the method comprises: (a) detecting a genetic variation in C5 from an individual; (b) if a genetic variation is detected in C5 from the individual, the individual is treated with an anti-C5 of the present invention. Selecting as suitable for a therapy comprising the antibody, and (c) administering the anti-C5 antibody of the invention to the individual.

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

  In another embodiment, for selecting an individual having a complement-mediated disease or condition with excessive or uncontrolled activation of C5 as suitable for a therapy comprising an anti-C5 antibody of the invention In vitro use of genetic mutations in C5 is provided. In certain embodiments, an individual is selected as suitable for treatment if a genetic variation is detected in C5 from the individual. In another embodiment, the use of genetic mutations in C5 in vitro to select treatments for individuals with complement-mediated diseases or conditions with excessive or uncontrolled activation of C5 Is provided. In certain embodiments, a treatment comprising an anti-C5 antibody of the invention is selected for an individual when a genetic variation is detected in C5 from the individual.

  Some patients with genetic mutations in C5 have been reported to be unresponsive to treatments that contain existing anti-C5 antibodies (Nishimura et al., N. Engl. J. Med. 370 : 632-639 (2014)). It is recommended that such patients be treated using a therapy comprising an anti-C5 antibody of the present invention. This is because the anti-C5 antibody of the present invention has an inhibitory activity not only against the activation of C5 mutants but also wild-type C5, as demonstrated in Examples described later.

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

  Exemplary disorders that can be diagnosed using 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) (eg, atypical hemolytic uremic syndrome (aHUS)); dense deposit disease (DDD); optic neuromyelitis (NMO); multifocal motor neuropathy (MMN); Systemic sclerosis (MS); systemic sclerosis; macular degeneration (eg, 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; Shiga toxin Escherichia coli related hemolytic uremic syndrome; typical or infectious hemolytic uremic syndrome (tHUS); C3 thread Anti-neutrophil cytoplasmic antibody (ANCA) -related vasculitis; humoral and vascular transplant rejection; acute antibody-mediated rejection (AMR); graft dysfunction; myocardial infarction; allograft; Hereditary angioedema; Dermatomyositis; Graves' disease; Atherosclerosis; Alzheimer's disease (AD); Huntington's disease; Creutzfeldt-Jakob disease; Parkinson's disease; Cancer; Wounds; Septic shock; Diabetic eye disease; retinopathy of prematurity; glomerulonephritis; membranous nephritis; immunoglobulin A nephropathy; adult respiratory distress syndrome (ARDS); chronic obstructive pulmonary disease (COPD); cystic fibrosis; Anemia; Paroxysmal cold hemoglobinuria; Anaphylactic shock; Allergy; Osteoporosis; Osteoarthritis; Hashimoto's thyroiditis; Type I diabetes; Psoriasis; Pemphigus; Autoimmune hemolytic anemia (AIHA); Reduced purpura (ITP); Goodpasture syndrome; Degos disease; Antiphospholipid antibody syndrome (APS); Fulminant APS (CAPS); Cardiovascular disease; Myocarditis; Cerebrovascular disorder; Peripheral vascular disorder; Mesentery / intestinal vascular disorder; vasculitis; Henoch-Schönlein purpura nephritis; Takayasu disease; dilated cardiomyopathy; diabetic vascular disorder; Kawasaki disease (arteritis); venous gas embolization (VGE); Post-restenosis; rotational atherectomy; membranous nephropathy; Guillain-Barre syndrome (GBS); Fisher syndrome; antigen-induced arthritis; synovitis; viral infection; bacterial infection; Includes damage due to infarctions, cardiopulmonary bypass and hemodialysis.

In certain embodiments, a labeled anti-C5 antibody is provided. Labels are detected directly through labels or moieties that are directly detected (eg, fluorescent labels, chromogenic labels, high electron density labels, chemiluminescent labels, and radioactive labels) and indirectly through, for example, enzymatic reactions or intermolecular interactions. Moieties (eg, enzymes or ligands). Exemplary labels include, but are not limited to: 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, firefly luciferase and bacterial luciferase (US Pat. No. 4,737,456), luciferase, luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline Enzymes that oxidize dye precursors using phosphatase, β-galactosidase, glucoamylase, lysozyme, monosaccharide oxidase (eg glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase), hydrogen peroxide (eg HRP, lactoperoxy) Da Heterocyclic oxidases such as uricase and xanthine oxidase, biotin / avidin, spin label, bacteriophage label, stable free radicals, and the like.

F. Pharmaceutical Formulations The anti-C5 antibody pharmaceutical formulations described herein can be prepared by administering an antibody having a desired purity to one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A Ed. (1980)) and is prepared in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, including but not limited to the following: phosphate, citric acid Buffers such as acid salts 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); small molecule (less than about 10 residues) polypeptide; serum albumin, gelatin, or Proteins such as immunoglobulins; polyvinyl Hydrophilic polymers such as loridone; 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; Nonionic surfactants such as sugars; salt-forming counterions such as sodium; metal complexes (eg, Zn-protein complexes); and / or polyethylene glycol (PEG), such as mannitol, trehalose, sorbitol. Exemplary pharmaceutically acceptable carriers herein further include a soluble neutral active hyaluronidase glycoprotein (sHASEGP) (eg, rHuPH20 (HYLENEX®, Baxter International, Inc.) PH-20 hyaluronidase glycoprotein) and other interstitial drug dispersants. Certain exemplary sHASEGPs and their methods of use (including rHuPH20) are described in US Patent Application Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinase.

  Exemplary lyophilized antibody formulations are described in US Pat. No. 6,267,958. Aqueous antibody formulations include those described in US Pat. No. 6,171,586 and WO2006 / 044908, the latter formulations containing histidine-acetate buffer.

  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 suitably present in combination in amounts that are effective for the purpose intended.

  The active ingredients are incorporated into microcapsules prepared by, for example, droplet formation (coacervation) techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin microcapsules and poly (methyl methacrylate) microcapsules, respectively). Alternatively, it may be incorporated into colloidal drug delivery systems (eg, liposomes, albumin spherules, microemulsions, nanoparticles, and nanocapsules) or macroemulsions. Such an approach is disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

  Sustained release formulations may be prepared. Suitable examples of sustained release formulations include a semi-permeable matrix of solid hydrophobic polymer containing antibodies, which matrix is in the form of shaped articles such as films or microcapsules.

  Formulations used for in vivo administration are usually sterile. Aseptic conditions are easily achieved, for example, by filtration through sterile filtration membranes.

G. Therapeutic methods and therapeutic compositions Any of the anti-C5 antibodies provided herein may be used in therapeutic methods.

  In one aspect, anti-C5 antibodies are provided for use as pharmaceuticals. In a further aspect, anti-C5 antibodies are provided for use in the treatment of complement-mediated diseases or conditions with excessive or uncontrolled activation of C5. In certain embodiments, anti-C5 antibodies are provided for use in therapeutic methods. In certain embodiments, the present invention is a method of treating an individual having a complement-mediated disease or condition with excessive or uncontrolled activation of C5, wherein the individual has an effective amount of an anti-C5 antibody. An anti-C5 antibody is provided for use in a method comprising the step of administering 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.

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

  In a further embodiment, the present invention provides an anti-C5 antibody for use in enhancing clearance of C5 from plasma. In certain embodiments, the present invention is a method of enhancing C5 clearance from plasma in an individual comprising administering to the individual an effective amount of an anti-C5 antibody to enhance C5 clearance from plasma. Anti-C5 antibodies are provided for use in the methods. 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.

  In a further aspect, the present invention provides an anti-C5 antibody for use in inhibiting the accumulation of C5 in plasma. In certain embodiments, the present invention is a method for inhibiting the accumulation of C5 in plasma in an individual comprising the step of administering to the individual an effective amount of an anti-C5 antibody to inhibit the accumulation of C5 in plasma. Anti-C5 antibodies are provided for use in the methods. 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. An “individual” according to any of the above embodiments is preferably a human.

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

  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 with excessive or uncontrolled activation of C5. In a further embodiment, the medicament is a method of treating a complement-mediated disease or condition involving excessive activation or uncontrolled activation of C5, comprising complementing with excessive activation or uncontrolled activation of C5. For use in a method comprising administering an effective amount of a pharmaceutical agent to an individual having a body-mediated disease or condition. 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.

  In a further embodiment, the medicament is for enhancing C5 clearance from plasma. In a further embodiment, the medicament is used in a method for enhancing C5 clearance from plasma in an individual comprising administering an effective amount of the medicament to the individual to enhance clearance of C5 from plasma. Is for. 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 aspects may be a human.

  In a further embodiment, the medicament is for inhibiting the accumulation of C5 in plasma. In a further embodiment, the medicament is used in a method of inhibiting the accumulation of C5 in plasma in an individual, the method comprising administering to the individual an effective amount of the medicament to inhibit the accumulation of C5 in plasma. Is for. 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. An “individual” according to any of the above aspects may be a human.

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

  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 a complement-mediated disease or condition with such 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 aspects may be a human.

  In a further aspect, the present invention provides a method for enhancing C5 clearance 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 C5 clearance 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, an “individual” is a human.

  In a further aspect, the present invention provides a method for inhibiting the accumulation of C5 in plasma in an individual. In one embodiment, the method includes administering to the individual an effective amount of an anti-C5 antibody to inhibit the accumulation of C5 in 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. In one embodiment, an “individual” is a human.

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

  In a further aspect, the present invention provides a pharmaceutical formulation comprising any of the anti-C5 antibodies provided herein (eg, 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 pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the anti-C5 antibodies provided herein and at least one additional therapeutic agent.

  In a further aspect, the pharmaceutical formulation is for treating a complement-mediated disease or condition with excessive or uncontrolled activation of C5. In a further embodiment, the pharmaceutical formulation is for enhancing C5 clearance 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 C5 activation. In a further embodiment, the pharmaceutical formulation is for inhibiting activation of C5. In one embodiment, C5-mediated cytotoxicity is suppressed by inhibiting C5 activation. In one embodiment, the pharmaceutical formulation is administered to an individual having a complement-mediated disease or condition with excessive or uncontrolled activation of C5. An “individual” according to any of the above embodiments is preferably a human.

  In one aspect, the individual has wild type C5. In another aspect, the individual has a C5 variant. In certain embodiments, the C5 variant has a biological activity similar to wild-type C5. Such a C5 variant may comprise at least one mutation selected from the group consisting of V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N, and E1437D. In the present specification, for example, R885H means a genetic mutation in which arginine at position 885 is replaced with histidine.

  In a further aspect, the present invention includes a step of mixing any anti-C5 antibody provided herein with a pharmaceutically acceptable carrier, eg, a medicament for use in any of the above therapeutic methods or Methods are provided for preparing pharmaceutical formulations. In one embodiment, the method for preparing a pharmaceutical or pharmaceutical formulation further comprises the step of adding at least one additional therapeutic agent to the pharmaceutical or pharmaceutical formulation.

  In certain embodiments, the complement-mediated disease or condition with 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) (eg, atypical hemolytic uremic syndrome (aHUS)); dense deposit disease (DDD); optic neuromyelitis (NMO); multifocal motor neuropathy (MMN); multiple sclerosis (MS); systemic sclerosis; macular degeneration (eg, 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 Toxic E. coli-related hemolytic uremic syndrome; typical or infectious hemolytic uremic syndrome (tHUS); C3 glomerulonephritis; anti-neutrophil cytoplasmic antibody (ANCA) -related 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); Disease; Creutzfeldt-Jakob Disease; Parkinson's Disease; Cancer; Wound; Septic Shock; Spinal Cord Injury; Uveitis; Diabetic Eye Disease; Premature Retinopathy; Glomerulonephritis; Membranous Nephritis; Adult respiratory distress syndrome (ARDS); chronic obstructive pulmonary disease (COPD); cystic fibrosis; hemolytic anemia; paroxysmal cold hemoglobinuria; anaphylactic shock; allergy; osteoporosis; Hashimoto's thyroiditis; type I diabetes; psoriasis; pemphigus; autoimmune hemolytic anemia (AIHA); idiopathic thrombocytopenic purpura (ITP); Goodpascher syndrome; Degos disease; antiphospholipid antibody syndrome (APS); Fulminant APS (CAPS); cardiovascular disease; myocarditis; cerebrovascular disorder; peripheral vascular disorder; renal vascular disorder; mesenteric / intestinal vascular disorder; vasculitis; Henoch-Schönlein purpura nephritis; Dilated vascular disorders; Kawasaki disease (arteritis); venous gas embolization (VGE); restenosis after stent placement; rotational atherectomy; membranous nephropathy; Guillain-Barre syndrome (GBS); Fisher syndrome; antigen-induced arthritis; synovitis; viral infection; bacterial infection; fungal infection; and damage resulting from myocardial infarction, cardiopulmonary bypass and hemodialysis.

  In certain embodiments, the complement-mediated disease or condition is an ocular disease state. In a further embodiment, the ocular disease state is macular degeneration. In a further embodiment, the macular degeneration is AMD. In a further embodiment, the AMD is dry AMD.

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

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

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

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

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

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

  The antibodies of the invention can be used in therapy, either alone or in combination with other agents. For example, an antibody of the invention may be co-administered with at least one additional therapeutic agent.

  Combination therapy as described above includes combination administration (two or more therapeutic agents included in the same or separate formulations) and individual administration, where administration of the antibody of the present invention is an additional treatment. Prior to, simultaneously with and / or subsequent to administration of the agent. In one embodiment, administration of the anti-C5 antibody and administration of the additional therapeutic agent are within about one month, or within about 1, 2, or 3 weeks of each other, or about 1, 2, 3, 4, 5, or 6 Done within days.

  The antibodies (and any additional therapeutic agent) of the invention may be administered by any suitable means, including parenteral, pulmonary, and nasal administration, and intralesional administration if desired for local treatment. Can be administered. Parenteral injection includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is short or long term. Various dosing schedules are within the scope of this specification, including, but not limited to, single doses or repeated doses over various time points, bolus doses, and pulse infusions.

  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 are the specific disorder being treated, the particular mammal being treated, the clinical symptoms of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the administration Schedule, and other factors known to healthcare professionals. The antibody need not be, but is optionally 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 usually at the same dose and route of administration as described herein, or at about 1 to 99% of the doses described herein, or any dose determined empirically / clinically appropriate. And in any route.

  For the prevention or treatment of disease, an appropriate dose of the antibody of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will determine the type of disease being treated, It will depend on the type, severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, drug history, patient clinical history and response to antibodies, 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, from about 1 μg / kg to 15 mg / kg (eg, 0.1 mg / kg to 10 mg, either by one or more separate doses or by continuous infusion) / kg) antibody may be the first candidate dose for administration to a patient. One typical daily dose may vary from about 1 μg / kg to over 100 mg / kg, depending on the factors described above. For repeated administrations over several days or longer, depending on the situation, treatment is usually maintained until a desired suppression of disease symptoms occurs. One exemplary dose of antibody is in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses (or any combination thereof) of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg may be administered to the patient. Such doses may be administered intermittently, such as every week or every 3 weeks (eg, so that the patient receives about 2 to about 20, or such as about 6 doses of antibody). One or more low doses may be administered after the high initial loading dose. The progress of this therapy is easily monitored by conventional techniques and measurements.

  It will be appreciated that any of the formulations or therapeutic methods described above may be practiced with the immunoconjugates of the invention in place of or in addition to anti-C5 antibodies.

H. Products In another aspect of the invention, products are provided that include equipment useful for the treatment, prevention, and / or diagnosis of the disorders described above. The product includes a container and a label or package insert on or attached to the container. Preferred containers include, for example, bottles, vials, syringes, IV solution bags and the like. The containers may be formed from various materials such as glass and plastic. The container may hold the composition alone, in combination with another composition that is effective for the treatment, prevention, and / or diagnosis of symptoms, and with a sterile access port. (For example, the container may be a solution bag or vial for intravenous administration having a stopper that can be pierced by a hypodermic needle). At least one effective agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is to be used to treat the selected condition. The product further comprises: (a) a first container with a composition comprising an antibody of the present invention contained therein; and (b) a second container, A second container may be included with the composition containing an additional cytotoxic agent or otherwise therapeutic agent contained therein. The product 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 product further comprises a second (or pharmaceutically acceptable buffer, such as water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. A third) container may be included. Other buffers, diluents, filters, needles, and syringes may further include equipment desirable from other commercial or user standpoints.

  It will be appreciated that any of the products described above may include an immunoconjugate of the invention instead of or in addition to an anti-C5 antibody.

Examples The following are examples of the methods and compositions of the present invention. It will be understood that various other aspects may be implemented in light of the above general description.

Example 1
Preparation of C5 1.1. Expression and purification of recombinant human and cynomolgus monkey C5 Recombinant human C5 (NCBI GenBank accession number: NP_001726.2, SEQ ID NO: 39) was transiently transferred using the FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA). It was expressed in sex. 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, after which the salt concentration and pH were adjusted to 80 mM NaCl and pH 6.4, respectively. The resulting sample was applied to an SP Sepharose HP cation exchange column (GE healthcare, Uppsala, Sweden) and eluted with a NaCl gradient. Fractions containing human C5 were pooled and applied to a CHT ceramic hydroxyapatite column (Bio-Rad Laboratories, Hercules, CA, USA). The human C5 eluate was then applied to a Superdex 200 gel filtration column (GE healthcare, Uppsala, Sweden). Fractions containing human C5 were pooled and stored at -150 ° C.

  Expression and purification of recombinant cynomolgus monkey C5 (NCBI GenBank accession number: XP — 005580972, SEQ ID NO: 44) was performed in the same manner as the human counterpart.

1.2. Purification of Cynomolgus C5 (cynoC5) from Plasma Plasma samples from cynomolgus monkey were applied to SSL7-agarose (Invivogen, San Diego, Calif., USA) and subsequently eluted with 100 mM Na acetate, pH 3.5. Fractions containing cynoC5 were immediately neutralized and subjected to peptide M agarose (Invivogen, San Diego, CA, USA) and a protein A HP column (GE healthcare, Uppsala, Sweden) linked in tandem. 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.

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

  12-16 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 2 months. One week after the final immunization, spleen and blood were collected from the immunized rabbit. Antigen-specific B cells are stained with labeled antigen, sorted with FCM cell sorter (FACS aria III, BD), at a density of 1 cell / well, 25,000 cells / well of EL4 cells (European Collection of Cell Cultures) and A 96-well plate was inoculated with 20-fold diluted activated rabbit T cell conditioned medium and cultured for 7-12 days. EL4 cells were treated with mitomycin C (Sigma, catalog number M4287) for 2 hours and washed 3 times beforehand. Activated rabbit T-cell conditioned medium contains rabbit thymocytes, RPMI containing phytohemagglutinin M (Roche, catalog number 11082132-001), phorbol 12-myristate 13-acetate (Sigma, catalog number P1585) and 2% FBS. Prepared by culturing in -1640. After culture, the B cell culture supernatant was collected for further analysis and the pellet was stored frozen.

  An ELISA assay was used to test the specificity of the antibodies in the B cell culture supernatant. Streptavidin (GeneScript, catalog number Z02043) was coated on 384 well MAXISorp (Nunc, catalog number 164688) at 50 nM in PBS for 1 hour at room temperature. The plates were then blocked with Blocking One (Nacalai Tesque, catalog number 03953-95) diluted 5-fold. Human or monkey C5 was labeled with NHS-PEG4-biotin (PIERCE, catalog number 21329), added to the blocked ELISA plate, incubated for 1 hour and washed. B cell culture supernatant was added to the ELISA plate and washed by incubation for 1 hour. Binding was detected by adding goat anti-rabbit IgG-horseradish peroxidase (BETHYL, catalog number A120-111P) followed by ABTS (KPL, catalog number 50-66-06).

An ELISA assay was used to assess the pH-dependent binding of antibodies to C5. Goat anti-rabbit IgG-Fc (BETHYL, catalog number A120-111A) diluted to 1 μg / ml in PBS (-) is added to 384 well MAXISorp (Nunc, catalog number 164688), incubated for 1 hour at room temperature, 5 Blocked with Blocking One (Nacalai Tesque, catalog number 03953-95) diluted twice. After incubation, the plate was washed and B cell culture supernatant was added. Plates were incubated for 1 hour, washed, and 500 pM biotinylated human C5 or monkey C5 was added and incubated for 1 hour. After incubation, the plates are washed and at room temperature with either pH 7.4 MES buffer (20 mM MES, 150 mM NaCl and 1.2 mM CaCl ₂ ) or pH 5.8 MES buffer (20 mM MES, 150 mM NaCl and 1 mM EDTA). Incubated for 1 hour. After incubation, biotinylated C5 binding was detected by adding streptavidin-horseradish peroxidase conjugate (Thermo Scientific, catalog number 21132) followed by ABTS (KPL, catalog number 50-66-06).

  The Octet RED384 system (Pall Life Sciences) was used to assess the affinity and pH dependent binding of antibodies to C5. The antibody secreted in the B cell culture supernatant is loaded onto a protein A biosensor chip (Pall Life Sciences) and immersed in 50 nM human or monkey C5 in MES buffer at pH 7.4 for binding kinetics Was analyzed. The dissociation kinetics were analyzed in both pH7.4 MES buffer and pH5.8 MES buffer.

  A total of 41,439 B cell lines were screened for affinity and pH dependent binding to human or monkey C5, and 677 cell lines were selected and designated CFA0001-0677. RNA of selected cell lines was purified from cryopreserved cell pellets using the ZR-96 Quick-RNA kit (ZYMO RESEARCH, catalog number R1053). DNA encoding the antibody heavy chain variable region in the selected cell line is 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. I let you. 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 existing humanized anti-C5 antibody eculizumab heavy and light chain genes (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 VL (EcuL, SEQ ID NO: 32) was The encoding DNA was fused in-frame to DNA encoding the k0 light chain constant region (SEQ ID NO: 37). Each fused coding sequence was cloned into an expression vector. The antibody was expressed in FreeStyle ™ 293-F cells (Invitrogen) and purified from the culture supernatant to assess functional activity. The neutralizing activity of the antibodies was evaluated by testing for inhibition of complement activity using the liposome lysis assay as described in Example 5.1.

2.2. Epitope binning by sandwich ELISA Anti-C5 antibodies with high affinity, pH dependence or neutralizing activity were selected for further analysis. Selected antibodies were classified into various epitope bins that bind to the same or overlapping epitopes of the C5 protein using a sandwich ELISA assay. Unlabeled capture antibody was diluted to 1 μg / ml with PBS (−) and added to a 384 well MAXISorp plate (Nunc, catalog number 164688). Plates were incubated for 1 hour at room temperature and blocked with Blocking One (Nacalai Tesque, catalog number 03953-95) diluted 5-fold. 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 antibody (1 μg / mL, biotinylated with NHS-PEG4-biotin) was added. After 1 hour incubation, binding of biotinylated antibody is detected by adding streptavidin-horseradish peroxidase conjugate (Thermo Scientific, catalog number 21132) followed by ABTS (KPL, catalog number 50-66-06) did.

  All anti-C5 antibodies were used as both capture and detection antibodies and were paired exhaustively. As shown in FIG. 1, antibodies that compete with each other were classified into seven epitope bins: CFA0668, CFA0334, and CFA0319 are classified as epitope A, and CFA0647, CFA0589, CFA0341, CFA0639, CFA0635, CFA0330, and CFA0318 are epitope B CFA0538, CFA0501, CFA0599, CFA0307, CFA0366, CFA0305, CFA0675, CFA0666 and CFA0672 are classified as epitope C, eculizumab and CFA0322 are classified as epitope D, CFA0329 is classified as epitope E, CFA0359 and CFA0217 are epitopes Classified as F, CFA0579, CFA0328 and CFA0272 were classified as epitope G. FIG. 1 shows the epitope binning of several anti-C5 chimeric antibodies. The VH and VL sequences of anti-C5 antibodies classified as epitope C are shown in Table 2.

2.3. Humanization and optimization Humanization of the variable regions of several anti-C5 antibodies was performed to reduce the potential immunogenicity of the antibodies. The complementarity determining region (CDR) of the anti-C5 rabbit antibody was grafted to a homologous human antibody framework (FR) using a conventional CDR grafting technique (Nature 321: 522-525 (1986)). Genes encoding humanized VH and VL were synthesized and respectively combined with modified human IgG4 CH (SG402, SEQ ID NO: 35) and human CL (SK1, SEQ ID NO: 38), and each of the combined sequences was expressed as an expression vector. Cloned into.

  In order to identify mutations and mutation combinations that improve the binding properties of several lead antibodies, many mutations and mutation combinations were considered. Next, multiple mutations were introduced into the humanized variable region to increase binding affinity for C5 at neutral pH or to reduce binding affinity for C5 at acidic pH. Thus, one of the optimized variants, 305LO5 (VH, SEQ ID NO: 10; VL, SEQ ID NO: 20; HVR-H1, SEQ ID NO: 54; HVR-H2, SEQ ID NO: 64; HVR-H3 SEQ ID NO: 74; HVR-L1, SEQ ID NO: 84; HVR-L2, SEQ ID NO: 94; and HVR-L3, SEQ ID NO: 104) were generated from CFA0305.

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

Example 3
Determination of binding characteristics 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 from the conditioned medium expressing the antibody was performed by conventional methods using protein A. Further gel filtration was performed as needed.

3.2. Evaluation of pH dependence The kinetic parameters of the anti-C5 antibody against recombinant human C5 were evaluated at 37 ° C. at pH 7.4 and pH 5.8 using a BIACORE® T200 instrument (GE Healthcare). ProA / G (Pierce) was immobilized on a CM4 sensor chip using an amine coupling kit (GE Healthcare) according to the settings recommended by GE Healthcare. Antibodies and analytes were diluted 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 ). Each antibody was captured on the sensor surface by ProA / G. Antibody capture levels were generally 60-90 resonance units (RU). Next, recombinant human C5 was injected at concentrations of 10 and 20 nM or 20 and 40 nM, followed by dissociation. The surface was regenerated using 25 mM NaOH. Kinetic parameters at both pH conditions were determined by fitting sensorgrams with a 1: 1 binding model using BIACORE® T200 Evaluation software, version 2.0 (GE Healthcare). Sensorgrams for all antibodies are shown in FIGS. 2A and 2B. The antibody binding rate (ka), dissociation rate (kd), and binding affinity (KD) are shown in Table 3. All antibodies except 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) compare at pH 5.8 over pH 7.4 Fast dissociation rate.

3.3. Cross-reactivity study To observe the cross-reactivity of anti-C5 antibodies against human C5 (hC5) and cynomolgus monkey C5 (cynoC5), BIACORE® kinetic analysis was performed. 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 reaction rate parameters for hC5 shown in Table 4 are the results of Example 3.2. All anti-C5 antibodies except CFA0672 showed similar KD to hC5 and cynoC5. The KD for cynoC5 of CFA0672 was 8 times less than that for hC5.

Example 4
Epitope mapping of anti-C5 antibody 4.1. Anti-C5 MAb Binding to C5β Chain Derived Peptides Anti-C5 monoclonal antibodies (MAbs) were tested for binding to C5β chain derived peptides in Western blot analysis. C5 peptides fused to GST tag (pGEX-4T-1, GE Healthcare Life Sciences, 28-9545-49): 19-180, 161-340, 321-500, and 481-660 were transformed into E. coli. ) (DH5α, TOYOBO, DNA-903). After incubation for 5 hours at 37 ° C. with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), E. coli samples were collected and centrifuged at 20000 × g for 1 minute to obtain a pellet. This 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) (FIG. 3). Arrow indicates GST-fused C5 peptide (46-49 kDa). Anti-C5 MAbs: CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675 bound to C5 19-180 (FIG. 3).

4.2. Expression and purification of human C5 MG1-MG2 domain (1-225) Recombinant MG1-MG2 domain (SEQ ID NO: 43) of human C5 β-chain was isolated from the FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA). It was expressed transiently using Conditioned media expressing the MG1-MG2 domain was diluted with 1/2 volume of milliQ water and subsequently 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 an SP Sepharose HP cation exchange column (GE healthcare, Uppsala, Sweden) and eluted with a NaCl gradient. Fractions containing the MG1-MG2 domain were collected from the eluate and subsequently applied to a Superdex 75 gel filtration column (GE healthcare, Uppsala, Sweden) equilibrated with 1 × PBS. Thereafter, fractions containing the MG1-MG2 domain were pooled and stored at -80 ° C.

4.3. Ability to bind to MG1-MG2 domain
The binding capacity of anti-C5 antibody to the MG1-MG2 domain was measured using the same assay setup described in Example 3.2, except that the measurement was only performed under pH 7.4 conditions. MG1-MG2 domains were injected at a concentration of 20 nM and 40 nM. As shown in FIG. 4, all antibodies except eculizumab-F760G4 showed an increased binding response, indicating that these antibodies are MG1-MG2 binders. A known α chain binder, eculizumab-F760G4, did not show binding to the MG1-MG2 domain.

4.4. Anti-C5 MAb Binding to C5 MG1-MG2 Domain Derived Peptides Anti-C5 MAbs were tested for binding to MG1-MG2 domain derived peptides in Western blot analysis. C5 peptides fused to the GST tag: 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 collected after 5 hours incubation with 1 mM IPTG at 37 ° C. and centrifuged at 20000 × g for 1 minute to obtain a pellet. This pellet was suspended in sample buffer (2ME +) and used for Western blot analysis. The expression of C5-derived peptide was confirmed by anti-GST antibody (FIG. 5A). CFA0305 bound only to peptides 33-124 (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 response of anti-C5 MAbs to C5-derived peptides.

4.5. Binding of anti-C5 MAb to C5 mutant
Three amino acid residues in the C5 beta chain: E48, D51, and K109 were predicted to be involved in the binding of C5 and anti-C5 MAb by crystal structure analysis, so anti-C5 MAb was analyzed by Western blot analysis. Were tested for binding to human C5 point mutants. 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 FIG. While eculizumab bound to the wild type (WT) C5 alpha chain and three C5 point mutants, CFA0305 binds strongly to the WT C5 beta chain and weakly to the E48A C5 mutant, with D51A and K109A C5 It did not bind to the mutant β chain. This indicates that these three amino acid residues are involved in the antibody / antigen interaction. Table 5 outlines 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 into the same epitope C, the binding patterns differ slightly between antibodies, indicating that the binding regions of C5 to these anti-C5 MAbs are close to each other but not identical Suggests that.

4.6. BIACORE® binding analysis between anti-C5 antibodies and C5 variants BIACORE® binding to test whether residues E48, G51, and K109 are actually involved in the antibody / antigen interaction Analysis was performed. Three C5 variants: E48A, G51A, and K109A were prepared as described in Example 4.5. A culture supernatant sample containing mutant C5 overexpressed in FS293 cells was prepared to 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 give 4 μg / ml The final sample concentration of mutant C5.

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

  As shown in FIG. 7, all three C5 mutants were able to bind to eculizumab with a similar binding profile compared to wild type C5. For 305LO5, all three mutants showed a lower binding response to 305LO5 compared to wild type C5. The D51A and K109A mutants reduced C5 binding by 305LO5 to baseline levels.

4.7. Identification of His residues on C5 that contribute to the pH-dependent interaction between anti-C5 antibody and C5. Crystal structure analysis shows that three histidine residues on human C5 are located at the antibody / antigen interface. Revealed. 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 examine which His residues on the antibody / antigen interface contribute to the pH-dependent interaction between anti-C5 antibody and C5, BIACORE® binding analysis was performed. Three human C5 mutants with a single His mutation (H70Y, H72Y, and H110Y) and one mutant with a double His mutation (H70Y + H110Y) were generated as follows: H70, H72, and A single His mutant in which any one of H110 was replaced with tyrosine, and a double His mutant 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 mutant to 305LO5, a pH-dependent anti-C5 antibody, were measured by a modified BIACORE® assay as described in Example 4.6. Briefly, an additional dissociation step at pH 5.8 was added to the BIACORE® assay to assess the pH-dependent dissociation between antibodies and antigens from complexes formed at pH 7.4. Was incorporated immediately after the dissociation phase at pH 7.4. The dissociation rate at pH 5.8 was determined by processing the data and performing fitting using Scrubber 2.0 (BioLogic Software) curve fitting software.

  As shown in FIG. 8, single and double His mutations (H70 + H110) at C70 H70 or H110 did not affect C5 binding to 305LO5 at neutral pH. On the other hand, a single His mutation at H72 showed a marked decrease in C5 binding to 305LO5. The dissociation rates of C5 His mutant 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 tested C5 antigens. Compared to C5-wt, a single His mutation at H70 showed a nearly 2-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 in both H70 and H110 had a relatively large effect on pH-dependent binding, with a dissociation rate at pH 5.8 that was almost 3 times slower than C5-wt.

Example 5
5. Inhibitory activity of anti-C5 antibody on C5 activation 5.1. Inhibition of complement-activated liposome lysis by anti-C5 MAbs Anti-C5 MAbs were tested for inhibition of complement activity by the liposome lysis assay. 30 μL of normal human serum (6.7%) (Biopredic, SER018) was mixed with 20 μL of diluted MAb in a 96-well plate and incubated on a shaker at 25 ° C. for 30 minutes. Liposomes sensitized with antibodies to dinitrophenyl (Autokit CH50, Wako, 995-40801) were transferred to each well and the plates were placed on a shaker at 25 ° C. for 2 minutes. 50 μL of substrate solution (Autokit CH50) was added to each well and mixed by shaking at 25 ° C. for 2 minutes. The final mixture was incubated at 37 ° C. for 40 minutes, after which the OD at 340 nm of the mixture was measured. The percent liposome lysis was defined as 100 × [(OD _MAb —OD _{serum and liposome background} )] / [( _No OD _MAb— OD _{serum and liposome background} )]. FIG. 9A shows that 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 (FIG. 9B).

5.2. Inhibition of C5a production by anti-C5 MAbs To confirm that anti-C5 MAbs inhibit C5 cleavage into C5a and C5b, anti-C5 MAbs were tested for C5a production 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 inhibited C5a production in the supernatant in a dose-dependent manner (FIGS. 10A and 10B).

5.3. Inhibition of complement-activated hemolysis by anti-C5 MAbs Anti-C5 MAbs were tested for inhibition of classical complement activity in hemolysis assays. Chicken red blood cells (cRBC) (Innovative research, IC05-0810 ) washing the at 0.5 mM MgCl ₂ and 0.15mM gelatin / veronal-buffered saline containing _{CaCl 2 (GVB ++) (Boston} BioProducts, IBB-300X), Thereafter, sensitization was performed at 4 ° C. for 15 minutes using 1 μg / ml anti-chicken RBC antibody (Rockland 103-4139). The cells were then washed with GVB ++ and suspended at 5 × 10 ⁷ cells / ml in the same buffer. In another 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 minutes. Next, 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 minutes. After incubation, the plates were centrifuged at 1000 xg and 4 ° C for 2 minutes. To measure OD at 415 nm using a reference wavelength of 630 nm, the supernatant (100 μl) was transferred to the well of a flat bottom 96 well microtest plate. Percent hemolysis was defined as 100 × [(OD _MAb —OD _{serum and cRBC} )] / [( _No OD _MAb— OD _{serum and cRBC background} )]. FIG. 11 shows that anti-C5 MAbs: CFA0305 and 305LO5 inhibited cRBC hemolysis.

5.4. Inhibition of the alternative complement pathway by anti-C5 MAbs The hemolysis assay for the alternative pathway was performed in a manner similar to that of the classical pathway. Blood collected 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 RBC (rRBC). rRBC was washed with GVB supplemented with ₂ mM MgCl ₂ and 10 mM EGTA and suspended at 7 × 10 ⁸ cells / ml in the same buffer. In a round bottom 96-well microtest plate, 40 μl of normal human serum (25%) (Biopredic, SER019) was mixed with 40 μl of diluted MAb and incubated on a shaker at 37 ° C. for 30 minutes. Next, 20 μl of rRBC suspension was added to the wells containing the serum and the antibody mixture was incubated at 37 ° C. for 60 minutes. After incubation, the plates were centrifuged at 1000 xg and 4 ° C for 2 minutes. To measure OD at 415 nm using a reference wavelength of 630 nm, the supernatant (70 μl) was transferred to a well on a flat bottom 96 well microtest plate. FIG. 12 shows that anti-C5 MAbs: CFA0305 and CFA0672 inhibited rRBC hemolysis, indicating that these antibodies inhibit the alternative complement pathway.

Example 6
6. Pharmacokinetic study of anti-C5 monoclonal antibody using 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 after human C5 (Calbiochem) alone or human C5 and anti-human C5 antibodies were administered to C57BL / 6 mice (In Vivos or Biological Resource Centre, Singapore) were evaluated. Human C5 solution (0.01 mg / ml) or mixed solution containing human C5 and anti-human C5 antibody (0.01 mg / ml and 2 mg / ml, respectively (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)) at a dose of 10 ml / kg in the tail vein did. In this case, since anti-human C5 antibody exists in excess relative to human C5, almost all human C5 is considered to be bound to the antibody. Blood was collected 5 minutes, 7 hours, 1 day, 2 days, 3 days, and 7 days after administration. The collected blood was immediately centrifuged at 14,000 rpm and 4 ° C. for 10 minutes to separate plasma. The separated plasma was stored at −80 ° C. in a freezer until assay. The anti-human C5 antibodies used are as follows: CFA0305-F760G4, CFA0307-F760G4, CFA0330-F760G4, CFA0341-F760G4, CFA0366-F760G4, CFA0501-F760G4, CFA0538-F760G4, CFA0599-F760G4, CFA0666 , CFA0672-F760G4, and CFA0675-F760G4.

6.2. Measurement of total human C5 concentration in plasma by electrochemiluminescence (ECL) assay Total human C5 concentration in mouse plasma was measured by ECL.

  For plasma samples in which only CFA0330-F760G4, CFA0341-F760G4, or human C5 is present, 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 allowed to stand at 4 ° C. overnight to prepare an anti-human C5 solid-phase plate. A standard curve sample and a mouse plasma sample diluted 100 times or more with 1 μg / ml injected antibody (CFA0330-F760G4 or CFA0341-F760G4) were prepared and incubated at 37 ° C. for 30 minutes. Thereafter, the sample was dispensed onto an anti-human C5 solid-phase plate and allowed to stand at room temperature for 1 hour. Subsequently, a SULFO-TAG labeled anti-human IgG antibody (Meso Scale Discovery) was added, and the mixture was reacted at room temperature for 1 hour for washing. Immediately thereafter, Read Buffer T (x4) (Meso Scale Discovery) was dispensed, and measurement was performed using Sector Imager 2400 (Meso Scale Discovery).

  For plasma samples with CFA0305-F760G4, CFA0307-F760G4, CFA0366-F760G4, CFA0501-F760G4, CFA0538-F760G4, CFA0599-F760G4, CFA0666-F760G4, CFA0672-F760G4, or CFA0672-F760G4, . Anti-human C5 antibody (CFA0329-F939G4; VH, SEQ ID NO: 23 and VL, SEQ ID NO: 27) is dispensed into an untreated MULTI-ARRAY 96 well plate (Meso Scale Discovery) and left at 4 ° C. overnight. Thus, an anti-human C5 solid phase plate was prepared. A sample for a calibration curve and a mouse plasma sample diluted 100 times or more with an acidic solution (pH 5.5) were prepared and incubated at 37 ° C. for 30 minutes. Thereafter, the sample was dispensed onto an anti-human C5 solid-phase plate and allowed to stand at room temperature for 1 hour. Subsequently, SULFO-TAG-labeled anti-human C5 antibody (CFA0300-F939G4; VH, SEQ ID NO: 24 and VL, SEQ ID NO: 28) was added, reacted at room temperature for 1 hour, and washed. Immediately thereafter, Read Buffer T (x4) (Meso Scale Discovery) was dispensed, and measurement was performed using Sector Imager 2400 (Meso Scale Discovery).

  The human C5 concentration was calculated using analysis software SOFTmax PRO (Molecular Devices) based on the response of the calibration curve. FIG. 13 shows the time course of plasma human C5 concentration after intravenous administration measured by this method. The data is plotted as the remaining percentage compared to the plasma human C5 concentration at the 5 minute time point.

6.3. Measurement of plasma anti-human C5 antibody concentration by ECL assay The anti-human C5 antibody concentration in mouse plasma was measured by ECL. Dispense anti-human IgG (γ chain specific) F (ab ′) 2 antibody fragment (Sigma) or anti-human IgG κ chain antibody (Antibody Solutions) into an untreated MULTI-ARRAY 96 well plate (Meso Scale Discovery), The plate was allowed to stand at 4 ° C. overnight to prepare an anti-human IgG immobilized plate. A sample for a calibration curve and a mouse plasma sample diluted 100 times or more were prepared. Thereafter, the sample was dispensed onto an anti-human IgG solid phase plate and allowed to stand at room temperature for 1 hour. Subsequently, biotinylated anti-human IgG antibody (Southernbiotech) or SULFO-TAG labeled anti-human IgG Fc antibody (Southernbiotech) was added, reacted at room temperature for 1 hour, and washed. Thereafter, only when a biotinylated anti-human IgG antibody was used, streptavidin labeled with SULFO-TAG (Meso Scale Discovery) was added, reacted at room temperature for 1 hour, and washed. Immediately thereafter, Read Buffer T (x4) (Meso Scale Discovery) was dispensed, and measurement was performed using Sector Imager 2400 (Meso Scale Discovery). The anti-human C5 antibody concentration was calculated using analysis software SOFTmax PRO (Molecular Devices) based on the response of the calibration curve. FIG. 14 shows the time course of plasma anti-human C5 antibody concentration after intravenous administration, measured by this method. Data is plotted as the percentage remaining compared to the plasma anti-human C5 antibody concentration at the 5 minute time point.

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 non-CFA0675-F760G4) Anti-human C5 antibodies (CFA0330-F760G4 and CFA0341-F760G4) were tested in vivo and the resulting plasma anti-human C5 antibody concentration and plasma human C5 concentration were compared. As shown in FIG. 14, antibody exposure was comparable. On the other hand, the clearance of human C5 administered simultaneously with the pH-dependent anti-human C5 antibody was accelerated compared to the clearance of human C5 administered simultaneously with the pH-independent anti-human C5 antibody (FIG. 13).

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

  The antibody was expressed in HEK293 cells co-transfected with a combination of heavy and light chain expression vectors and purified with protein A.

Example 8
Binding characterization of anti-C5 antibody (305 variant) The kinetic parameters of anti-C5 antibody against recombinant human C5 were measured at 37 ° C using a BIACORE® T200 instrument (GE Healthcare) under the following three different conditions: (1) Both binding and dissociation were pH 7.4, (2) both binding and dissociation were pH 5.8, and (3) binding was pH 7.4, dissociation was pH 5.8. ProA / G (Pierce) was immobilized on the CM1 sensor chip using an amine coupling kit (GE Healthcare) according to the settings recommended by GE Healthcare. For conditions (1) and (3), dilute antibody and analyte in ACES pH7.4 buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl ₂ , 0.05% Tween 20, 0.005% NaN ₃ ) 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 ). Each antibody was captured on the sensor surface by ProA / G. Antibody capture levels were generally 60-90 resonance units (RU). Next, recombinant human C5 was 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 a sensorgram with a 1: 1 binding model, The dissociation rate under condition (3) was measured by fitting a sensorgram with a 1: 1 MCK dissociation model. The pH dependence of all antibodies was shown as the ratio of the dissociation rates of conditions (2) and (1).

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

The binding affinity of anti-C5 antibodies (BNJ441, eculizumab, and 305 variants) to recombinant human C5 at pH 7.4 and pH 5.8 was measured at 37 ° C. using a BIACORE® T200 instrument (GE Healthcare). The effect of pH on antigen binding was evaluated. A 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. Diluted antibody with the analyte, 20mM ACES, 150mM NaCl, in any of _{1.2mM CaCl 2, 0.05% Tween 20} , and in ACES pH 7.4 buffer containing 0.005% NaN ₃ or ACES pH 5.8 buffer did. The antibody was captured on the sensor surface using an anti-Fc method. The capture level was generally 50-80 resonance units (RU). Recombinant human C5 was prepared by 3-fold serial dilution starting from 27 nM for pH 7.4 assay conditions and 135 nM for pH 5.8 assay conditions. The surface was 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).

  The binding affinity (KD) of BNJ441, eculizumab, and 305 mutants for recombinant human C5 at pH 7.4 and pH 5.8 is shown in Table 10. The 305 mutant showed a ratio of 800 (KD at pH 5.8) / (KD at pH 7.4) / 93 (KD at pH 5.8) / (KD at pH 7.4). ) Was 8 times higher than BNJ441, which only showed a ratio.

Example 9
9. Inhibitory activity of anti-C5 antibody (305 mutant) on C5 activation 9.1. Inhibition of complement-activated liposome lysis by anti-C5 MAbs Anti-C5 MAbs were tested for inhibition of complement activity by the 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 for 30 minutes at room temperature. Liposome solution (Autokit CH50, Wako, 995-40801) sensitized with an antibody against dinitrophenyl was transferred to each well and placed on a shaker at 37 ° C. for 2 minutes. 50 μL of substrate solution (Autokit CH50) was added to each well and mixed by shaking at 37 ° C. for 2 minutes. The final mixture was incubated at 37 ° C. for 40 minutes, after which the OD at 340 nm was measured. The percent liposome lysis was defined as 100 × [(OD _MAb —OD _{serum and liposome background} )] / [( _No OD _MAb— OD _{serum and liposome background} )]. FIG. 15 shows that 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 (FIG. 16).

  Anti-C5 MAb was tested for inhibition of recombinant human C5 (SEQ ID NO: 39). 10 μL of C5-deficient human serum (Sigma, C1163) was mixed with 20 μL of diluted MAb and 20 μL of recombinant C5 (0.1 μg / mL) in a 96-well plate and incubated on a shaker at 37 ° C. for 1 hour. Liposomes (Autokit CH50) were transferred to each well and placed on a shaker at 37 ° C. for 2 minutes. 50 μL of substrate solution (Autokit CH50) was added to each well and mixed by shaking at 37 ° C. for 2 minutes. The final mixture was incubated at 37 ° C. for 180 minutes, after which the OD at 340 nm was measured. The percent liposome lysis was defined as above. FIG. 17 shows that anti-C5 MAbs: 305LO22-SG115, 305LO22-SG422, 305LO23-SG115, and 305LO23-SG422 inhibited liposome lysis.

9.2. Inhibition of C5a production by anti-C5 MAb To confirm that anti-C5 MAb inhibits C5 cleavage into C5a and C5b, anti-C5 MAb was tested for production 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 inhibited C5a production in the supernatant in a dose-dependent manner (FIGS. 18 and 19).

9.3. Measurement of Complement Activity in Cynomolgus Monkey Plasma Anti-C5 MAbs were tested for inhibition of complement activity in cynomolgus plasma. Anti-C5 MAb was administered to monkeys (20 mg / kg) and plasma samples were taken periodically until day 56. Chicken red blood cells (cRBC) (Innovative research, IC05-0810 ) washing the at 0.5 mM MgCl ₂ and 0.15mM gelatin / veronal-buffered saline containing _{CaCl 2 (GVB ++) (Boston} BioProducts, IBB-300X), Thereafter, sensitization was performed at 4 ° C. for 15 minutes using 1 μg / ml anti-chicken RBC antibody (Rockland 103-4139). The cells were then washed with GVB ++ and suspended at 1 × 10 ⁸ cells / ml in the same buffer. In another round bottom 96-well microtest plate, monkey plasma was incubated with sensitized cRBC for 20 minutes at 37 ° C. After incubation, the plates were centrifuged at 1000 xg and 4 ° C for 2 minutes. To measure OD at 415 nm using a reference wavelength of 630 nm, the supernatant was transferred to a well on a flat bottom 96 well microtest plate. Percent hemolysis was defined as 100 × [( _post- OD _{administration—} OD _{plasma and cRBC background} )] / [( _pre- OD _{administration—} OD _{plasma and cRBC background} )]. FIG. 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. It is shown that.

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

9.5. Inhibition of complement-activated liposome lysis by anti-C5 MAbs Anti-C5 MAbs were tested for inhibition of complement activity by the 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 for 30 minutes at room temperature. Liposome solution (Autokit CH50, Wako, 995-40801) sensitized with an antibody against dinitrophenyl was transferred to each well and placed on a shaker at 25 ° C. for 2 minutes. 50 μL of substrate solution (Autokit CH50) was added to each well and mixed by shaking at 25 ° C. for 2 minutes. The final mixture was incubated at 37 ° C. for 45 minutes, after which the OD at 340 nm was measured. The percent inhibition of liposome lysis was defined as 100 × [(OD _MAb —OD _{serum and liposome background} )] / [( _No OD _MAb— OD _{serum and liposome background} )]. FIG. 23 shows that anti-C5 MAbs BNJ441 and 305 mutants inhibited liposome lysis and that 305 mutants have stronger inhibitory activity than BNJ441.

Example 10
Pharmacokinetic study of anti-C5 monoclonal antibody (305 mutant) in cynomolgus monkeys 10.1. In vivo test using cynomolgus monkey The in vivo kinetics of anti-human C5 antibody after administration of anti-human C5 antibody to cynomolgus monkey (Shin Nihon Kagaku, Japan) was evaluated. An anti-human C5 antibody solution (2.5 mg / ml) was administered once by a 30 minute infusion at a dose of about 8 ml / kg into the cephalic vein of the forearm. Blood 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 Were collected. 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 assayed. Anti-human C5 antibody was prepared as described in Example 7.

10.2. Measurement of total cynomolgus monkey C5 concentration in plasma by ELISA assay Total cynomolgus monkey C5 concentration in cynomolgus monkey plasma was measured by ELISA. Anti-human C5 antibody (in-house antibody prepared using the method described in Example 2) was dispensed into Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and allowed to stand overnight at 4 ° C to obtain an anti-cynomolgus monkey C5 solid phase. Prepared plates. A standard curve sample and a cynomolgus monkey plasma sample diluted 20000 times with 0.4 μg / ml injected antibody were prepared and incubated at 37 ° C. for 60 minutes. Thereafter, the sample was dispensed onto an anti-cynomolgus monkey C5 solid phase plate and allowed to stand at room temperature for 1 hour. Next, an HRP-labeled anti-human IgG antibody (SouthernBiotech) was added, and the mixture was reacted at room temperature for 30 minutes for washing. ABTS ELISA HRP substrate (KPL) was then added. The signal was measured with a plate reader at a wavelength of 405 nm. The cynomolgus monkey C5 concentration was calculated using analysis software SOFTmax PRO (Molecular Devices) based on the response of the calibration curve. FIG. 24 shows the time course of plasma cynomolgus monkey C5 concentration after intravenous administration, measured by this method. Data are plotted as the percentage remaining compared to plasma cynomolgus C5 concentration at the pre-dose 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 antibody.

10.3. Measurement of plasma anti-human C5 antibody concentration by ELISA assay Anti-human C5 antibody concentration in cynomolgus monkey plasma was measured by ELISA. Anti-human IgG kappa chain antibody (Antibody Solutions) was dispensed into Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and allowed to stand at 4 ° C. overnight 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. Thereafter, the sample was dispensed onto an anti-human IgG solid phase plate and allowed to stand at room temperature for 1 hour. Next, an HRP-labeled anti-human IgG antibody (SouthernBiotech) was added, and the mixture was reacted at room temperature for 30 minutes for washing. ABTS ELISA HRP substrate (KPL) was then added. The signal was measured with a plate reader at a wavelength of 405 nm. The anti-human C5 antibody concentration was calculated using analysis software SOFTmax PRO (Molecular Devices) based on the response of the calibration curve. FIG. 25 shows the time course of plasma anti-human C5 antibody concentration after intravenous administration, measured by this method. 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) It exhibited a longer half-life compared to the pH-independent anti-human C5 antibody.

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

11.2. Preparation of mutant Fab fragments
Fab fragment of one of the optimized variants from 305 was digested by restriction digestion with papain (Roche Diagnostics, catalog number 1047825), followed by a protein A column (MabSlect SuRe, GE Healthcare), cation Prepared by conventional methods loading on exchange columns (HiTrap SP HP, GE Healthcare) and gel filtration columns (Superdex200 16/60, GE Healthcare). Fractions containing Fab fragments were pooled and stored at -80 ° C.

11.3. Preparation of Complex of 305 Mutant Fab and Human C5-MG1 Domain Purified recombinant human C5-MG1 domain was mixed with the purified 305 mutant Fab fragment in a 1: 1 molar ratio. 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.

11.4. Crystallization The purified complex was concentrated to about 10 mg / mL and crystallized at 4 ° C. by a sitting drop vapor diffusion method combined with a seeding method. The reservoir solution consisted of 0.2M magnesium formate anhydrous, 15.0% w / v polyethylene glycol 3350. This succeeded in obtaining plate crystals in 2 to 3 days. The crystals were immersed in a solution of 0.2M magnesium formate anhydride, 25.0% w / v polyethylene glycol 3350, and 20% glycerol.

11.5. Data collection and structure determination
X-ray diffraction data was measured with BL32XU of SPring-8. During the measurement, keep the crystal in a frozen state by always placing it in a nitrogen flow of -178 ° C, and rotate the crystal by 1.0 ° at a time using the MX-225HS CCD detector (RAYONIX) connected to the beam line. A total of 180 X-ray diffraction images were collected. Determination of cell parameters, indexing of diffraction spots, and processing of diffraction data obtained from diffraction images were performed 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 diffraction intensity data up to a resolution of 2.11 mm was obtained. Crystallographic data statistics are shown in Table 11.

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

11.6. Overall structure of complex of mutant Fab and C5-MG1 domain
The optimized mutant Fab fragment from 305 (“305 Fab”) binds to human C5-MG1 domain (“MG1”) in a 1: 1 ratio, and the asymmetric unit of this crystal structure is shown in FIG. 26A. As such, it contained two complexes, molecule 1 and molecule 2. As shown in FIG. 26B, molecules 1 and 2 can be superposed well with a RMSD at the Cα atom position of 0.717 at all residues. The drawings described below were created using molecule 1.

  In FIGS. 27A and 27B, the epitope of the 305 Fab contact region is mapped in the MG1 amino acid sequence and in the crystal structure, respectively. The epitope includes an amino acid residue of MG1 that contains one or more atoms located within a distance of 4.5 cm from any part of 305 Fab in the crystal structure. In addition, epitopes within 3.0 mm are highlighted in FIG. 27A.

11.7. Interaction of E48, D51, and K109 As described in Examples 4.5 and 4.6, anti-C5 MAbs containing the 305 antibody series were isolated in three human C5 point mutants by Western blot and BIACORE® binding analysis. We tested for binding to certain E48A, D51A, and K109A. The 305 mutant bound strongly to WT C5, but only weakly to the E48A C5 mutant and not to the D51A and K109A mutants. As shown in FIG. 28A, the crystal structure of the complex of 305 Fab and MG1 is that these three amino acids E48, D51, and K109 are all within a distance of 3.0 mm from 305 Fab, and the Fab and some hydrogen It was clarified that a bond was formed. Considered in more detail, the K109 residue of MG1 is buried in a groove formed at the Fab heavy chain interface, by three hydrogen bonds with H-CDR3_G97, H-CDR3_Y100, and H-CDR3_T100b, and The salt bridge with H-CDR3_D95 strongly interacts with Fab (FIG. 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 (FIG. 28C). These indicate that both K109 and D51 of C5 are critical residues for binding of the 305 antibody series. On the other hand, E48 is located relatively close to the surface and has only one hydrogen bond with the Fab, which will contribute its contribution to antibody binding below that of K109 and E51. This suggests (FIG. 28B). These relationships are consistent with the results of Western blots and BIACORE® binding analysis of human C5 variants (Examples 4.5 and 4.6). Supplement: The amino acid residue numbering of the Fab 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)

11.8. Interaction of human C5 H70, H72, and H110 with the 305 antibody series Crystal structure analysis showed that three histidine residues on human C5, namely H70, H72, and H110, as shown in FIGS. 27A and 29A, It was clarified that it is contained in the epitope of 305 mutant Fab. To investigate the contribution of these histidine residues to the pH-dependent protein-protein interaction between human C5 and 305 mutant Fabs, we used BIACORE with human C5 mutants H70Y, H72Y, H110Y, and H70Y + H110Y. (R) binding analysis was performed (Example 4.7). H72Y resulted in complete loss of binding of 305 mutant Fab to C5. This residue of C5 is located in the pocket formed by the CDR2 loop of the heavy chain of 305 Fab and the loop of MG1 (L73, S74, and E76), as shown in Figure 29C, filling this space tightly. ing. Furthermore, the H72 residue of C5 forms a hydrogen bond with H-CDR2_Y58. The H72Y mutation is expected to be unacceptable because there is not enough space to accommodate the relatively bulky side chain of tyrosine. In addition, the hydrogen bond with H-CDR2_Y58 cannot be maintained. Regarding the contribution of H70 and H110 to the pH dependence, the H70Y and H110Y mutations resulted in 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 C5 H70 is protonated at pH 5.8, a conformational change occurs at the corresponding part of the interaction interface of MG1, and this hydrogen bond is It is thought to be destroyed (FIG. 29B). For H110, this protonation of the C5 residue is expected to cause charge repulsion to 305 Fab, which can be enhanced by protonation of the adjacent histidine residue H-CDR3_H100c (FIG. 29D).

  Although the foregoing invention has been described in detail with illustrative examples and illustrations for purposes of clarity of understanding, the descriptions and illustrations herein should not be construed as limiting the scope of the invention. Absent. The disclosures of all patent and scientific literature cited herein are hereby expressly incorporated herein by reference in their entirety.

Claims (29)

  An antibody that binds C5 for use in the treatment of complement-mediated diseases or conditions with excessive or uncontrolled activation of C5, with higher affinity at neutral pH than at acidic pH An antibody that binds to an epitope within the β chain of C5.   An antibody that binds to C5, for use in enhancing clearance of C5 from plasma, that binds to an epitope within the β chain of C5 with higher affinity at neutral pH than at acidic pH.   Use of an antibody that binds C5 in the manufacture of a medicament for treating a complement-mediated disease or condition with excessive or uncontrolled activation of C5, wherein the antibody is more potent at acidic pH. Use, which also binds to an epitope within the β chain of C5 with high affinity at neutral pH.   Use of an antibody that binds to C5 in the manufacture of a medicament to enhance clearance of C5 from plasma, wherein the antibody is an epitope within the β chain of C5 with higher affinity at neutral pH than at acidic pH Use to bind to.   A method of treating an individual having a complement-mediated disease or condition with excessive or uncontrolled activation of C5, comprising the step of administering to the individual an effective amount of an antibody that binds C5, A method wherein the antibody binds to an epitope within the β chain of C5 with higher affinity at neutral pH than at acidic pH.   A method of enhancing the clearance of C5 from plasma in an individual comprising the step of administering to the individual an effective amount of an antibody that binds C5 to enhance the clearance of C5 from plasma, wherein the antibody is acidic A method that binds to an epitope within the β chain of C5 with higher affinity at neutral pH than at pH. The antibody is 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 the C5 variant consisting of the E48A substitution of SEQ ID NO: 39;
(c) The antibody binds to C5 protein consisting of the amino acid sequence of SEQ ID NO: 40 at pH 7.4, but does not bind to C5 protein consisting of the amino acid sequence of SEQ ID NO: 39 having the H72Y substitution at pH 7.4. An antibody, use, or method for use according to any one of claims 1-6. For the binding of the antibody to C5,
(a) VH of SEQ ID NO: 1 and VL of SEQ ID NO: 11;
(b) VH of SEQ ID NO: 5 and VL of SEQ ID NO: 15;
(c) VH of SEQ ID NO: 4 and VL of SEQ ID NO: 14;
(d) VH of SEQ ID NO: 6 and VL of SEQ ID NO: 16;
(e) VH of SEQ ID NO: 2 and VL of SEQ ID NO: 12;
(f) VH of SEQ ID NO: 3 and VL of SEQ ID NO: 13;
(g) VH of SEQ ID NO: 9 and VL of SEQ ID NO: 19;
(h) VH of SEQ ID NO: 7 and VL of SEQ ID NO: 17;
(i) VH of SEQ ID NO: 8 and VL of SEQ ID NO: 18; and
(j) VH of SEQ ID NO: 10 and VL of SEQ ID NO: 20
7. An antibody, use or method for use according to any one of claims 1 to 6 which competes with an antibody comprising a VH and VL pair selected from.   9. The antibody, use or method for use according to any one of claims 1 to 8, wherein said antibody binds to an epitope within the MG1-MG2 domain of the C5 beta chain.   The antibody for use according to any one of claims 1 to 9, wherein the antibody binds to an epitope within a fragment consisting of amino acids 33-124 of the β chain of C5 (SEQ ID NO: 40). Or the method.   The antibody binds to an epitope within the β 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. An antibody, use, or method for use according to any one of 1-10.   The antibody binds to an epitope within a fragment of the C5 β chain (SEQ ID NO: 40) comprising at least one amino acid selected from the group consisting of Glu48, Asp51, His70, His72, Lys109, and His110. Item 12. The antibody, use, or method for use according to any one of Items 1-11.   13. An antibody, use, or method for use according to any one of claims 1 to 12, wherein the antibody binds to the same epitope as an antibody described in Tables 2, 7, or 8.   14. An antibody, use or method for use according to any one of claims 1 to 13, wherein the antibody inhibits C5 activation.   15. An antibody, use or method for use according to any one of claims 1 to 14, wherein said antibody inhibits activation of C5 variant R885H.   16. An antibody, use or method for use according to any one of claims 1 to 15, wherein the antibody is a monoclonal antibody.   The antibody, use, or method for use according to any one of claims 1 to 16, wherein the antibody is a human antibody, a humanized antibody or a chimeric antibody.   18. An antibody, use or method for use according to any one of claims 1 to 17, wherein the antibody is an antibody fragment that binds to C5. Said antibody is (a) amino acid sequence DX ₁ GYX ₂ X ₃ PTHAMX ₄ X ₅ (where X ₁ is G or A, X ₂ is V, Q or D, X ₃ is T or Y, X ₄ is Y or H, X ₅ is L or Y) (SEQ ID NO: 128) and HVR-H3 comprising, (b) the amino acid sequence QX ₁ TX ₂ VGSSYGNX ₃ (where X ₁ is S, C, N, or T, X ₂ is F or K, X ₃ is A, T or H) (SEQ ID NO: 131) and (c) amino acid sequence X ₁ IX ₂ TGSGAX ₃ YX ₄ AX ₅ WX ₆ KG (here X ₁ is C, A or G, X ₂ is Y or 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 The antibody, use, or method for use according to any one of claims 1 to 18, comprising HVR-H2 comprising (SEQ ID NO: 127). The antibody comprises (a) HVR-H1 comprising the amino acid sequence SSYYX ₁ X ₂ (where X ₁ is M or V, X ₂ is C or A) (SEQ ID NO: 126), and (b) 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 or F, X ₃ is T, D or E, X ₄ is Y, K or Q, HVR-H2 containing (SEQ ID NO: 127) and (c) amino acid sequence DX ₁ GYX ₂ X ₃ PTHAMX ₄ X ₅ (where X ₅ is S, D or E, X ₆ is A or V) ₁ is G or A, X ₂ is V, Q or D, X ₃ is T or Y, X ₄ is Y or H, X ₅ is L or Y) (SEQ ID NO: 128) and HVR-H3 An antibody, use, or method for use according to any one of claims 1-18. Said antibody is (a) amino acid sequence X ₁ ASQX ₂ IX ₃ SX ₄ LA (where X ₁ is Q or R, X ₂ is N, Q or G, X ₃ is G or S, X ₄ is D, K HVR-L1 comprising (SEQ ID NO: 129); and (b) amino acid sequence GASX ₁ X ₂ X ₃ S (where X ₁ is K, E or T, X ₂ is L or T, X ₃ is A, H, E or Q) (SEQ ID NO: 130) and HVR-L2; (c) amino acid sequence QX ₁ TX ₂ VGSSYGNX ₃ (where X ₁ is S, C, N or T, X ₂ is F or K, X ₃ is a, T or H) (SEQ ID NO: 131) further comprises a HVR-L3 comprising an antibody for use according to claim 20, use or method, . Said antibody is (a) amino acid sequence X ₁ ASQX ₂ IX ₃ SX ₄ LA (where X ₁ is Q or R, X ₂ is N, Q or G, X ₃ is G or S, X ₄ is D, K HVR-L1 comprising (SEQ ID NO: 129); and (b) amino acid sequence GASX ₁ X ₂ X ₃ S (where X ₁ is K, E or T, X ₂ is L or T, X HVR-L2 comprising (SEQ ID NO: 130); (c) amino acid sequence QX ₁ TX ₂ VGSSYGNX ₃ (where X ₁ is S, C, N or T, ₃ is A, H, E or Q) X ₂ is F or K, X ₃ is a, T or H) (SEQ ID NO: 131) and a HVR-L3 comprising, for use according to any one of claims 1 to 18 Antibody, use, or method.   The antibody comprises a heavy chain variable domain framework FR1 comprising any one amino acid sequence of SEQ ID NOs: 132 to 134; and a heavy chain variable domain framework FR2 comprising any one amino acid sequence of SEQ ID NOs: 135 to 136 A heavy chain variable domain framework FR3 comprising any one amino acid sequence of SEQ ID NO: 137-139; and a heavy chain variable domain framework FR4 comprising any one amino acid sequence of SEQ ID NO: 140-141. 21. The antibody, use or method for use according to claim 20, further comprising:   The antibody comprises a light chain variable domain framework FR1 comprising any one amino acid sequence of SEQ ID NO: 142-143; and a light chain variable domain framework FR2 comprising any one amino acid sequence of SEQ ID NO: 144-145. And further comprising: a light chain variable domain framework FR3 comprising the amino acid sequence of any one of SEQ ID NOs: 146-147; and a light chain variable domain framework FR4 comprising the amino acid sequence of SEQ ID NO: 148. Antibodies, uses, or methods for use as described in.   The antibody comprises (a) a VH sequence having at least 95% sequence identity to any one amino acid sequence of SEQ ID NOs: 10, 106 to 110; (b) any of SEQ ID NOs: 20, 111 to 113 19. A VL sequence having at least 95% sequence identity to any one amino acid sequence; or (c) a VH sequence of (a) and a VL sequence of (b). Antibodies, uses, or methods for use as described in.   26. The antibody, use, or method for use according to claim 25, wherein the antibody comprises a VH sequence of any one of SEQ ID NOs: 10, 106-110.   26. The antibody, use, or method for use according to claim 25, wherein the antibody comprises a VL sequence of any one of SEQ ID NOs: 20, 111-113.   28. The use according to claim 26 or 27, wherein the antibody comprises a VH sequence of any one of SEQ ID NO: 10, 106-110 and a VL sequence of any one of SEQ ID NO: 20, 111-113. Antibody, use, or method.   29. The antibody, use, or method for use according to any one of claims 1-28, wherein the antibody is a full-length IgG1 antibody or a full-length IgG4 antibody.

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