CN109312326B - anti-C5 antibodies and methods of use - Google Patents

Abstract

The present invention provides anti-C5 antibodies and methods of using the same. In some embodiments, the isolated anti-C5 antibodies of the invention bind to epitopes within the beta chain of C5 with higher affinity at neutral pH than at acidic pH. The invention also provides isolated nucleic acids encoding anti-C5 antibodies of the invention. The present invention also provides host cells comprising the nucleic acids of the present invention. The present invention also provides a method of producing an antibody, the method comprising culturing a host cell of the present invention to produce the antibody. The present invention also provides a method of making an anti-C5 antibody, the method comprising immunizing an animal against a polypeptide comprising the MG1-MG2 domain of the beta chain of C5. The anti-C5 antibody of the present invention can be used as a medicine.

Description

Anti-C5 antibodies and methods of use

technical field

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

Background technique

The complement system plays a key role in the clearance of immune complexes and immune responses to infectious agents, foreign antigens, virus-infected cells, and tumor cells. There are about 25-30 complement proteins, which are found to be a complex collection of plasma proteins and membrane cofactors. Complement components achieve their immune defense functions by interacting in a complex series of enzymatic cleavage and membrane-binding events. The resulting complement cascade leads to the production of products with opsonin, immunomodulatory and bacteriolytic functions.

Currently, it is widely accepted that the complement system can be activated through three distinct pathways: the classical pathway, the lectin pathway and the alternative pathway. These pathways share many components, and although their initial steps differ, they converge and share the same late complement components (C5 to C9) responsible for activating and destroying target cells.

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

C3a is an anaphylatoxin. C3b binds to bacteria and other cells, as well as certain viruses and immune complexes, and marks it for removal from the circulation (known as the action of opsonins). C3b also forms complexes 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 approximately 80 μg/ml (0.4 μM). About 1.5-3% of the mass attributable to carbohydrates in C5 is glycosylated. Mature C5 is a heterodimer of a disulfide-linked 115 kDa alpha chain and a 75 kDa beta chain. C5 is synthesized as a single-chain precursor protein of 1676 amino acids (pro-C5 precursor) (see, eg, PTL1 and PTL2). The pro-C5 precursor is cleaved to generate the beta chain as the amino-terminal fragment and the alpha chain as the carboxy-terminal fragment. The alpha and beta chain polypeptide fragments are linked to each other via disulfide bonds and constitute the mature C5 protein.

Mature C5 is cleaved into C5a and C5b fragments during activation of the complement pathway. C5a is cleaved from the alpha chain of C5 by C5 convertase as an amino-terminal fragment comprising the first 74 amino acids of the alpha chain. The remainder of mature C5 is fragment C5b, which contains the remaining alpha and beta chains linked by disulfide bonds. About 20% of the 11 kDa molecular weight of C5a is attributed to carbohydrates.

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

As mentioned above, C3a and C5a are anaphylatoxins. It can trigger mast cell degranulation, which releases histamine and other inflammatory mediators, leading to smooth muscle contraction, increased vascular permeability, leukocyte activation, and other inflammatory phenomena, including cell proliferation leading to hypercellularity. C5a also acts as a chemotactic peptide, which is used to attract granulocytes such as neutrophils, eosinophils, basophils and monocytes to sites of complement activation.

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

A properly functioning complement system provides a powerful defense against infecting microorganisms, and inappropriate regulation or activation of complement is implicated in the pathogenesis of a variety of conditions including, for example, rheumatoidarthritis (RA); lupus nephritis (lupus nephritis); ischemia-reperfusion injury (ischemia-reperfusioninjury); paroxysmal nocturnal hemoglobinuria (PNH); atypical hemolytic uremic syndrome (aHUS); dense deposits Dense deposit disease (DDD); macular degeneration (eg, age-related macular degeneration (AMD)); hemolysis, elevated liver enzymes, and low platelets (HELLP) ) syndrome; thrombotic thrombocytopenic purpura (TTP); spontaneous abortion (spontaneous fetal loss); microimmune vasculitis (Pauci-immunvasculitis); epidermolysis bullosa (epidermolysis bullosa); recurrence multiple sclerosis (MS); traumatic brain injury; and caused by myocardial infarction, cardiopulmonary bypass, and hemodialysis damage (see, eg, NPL 1). Thus, inhibition of an excessive or uncontrolled complement activation cascade may provide clinical benefit to patients with such disorders.

Paroxysmal nocturnal hemoglobinuria (PNH) is an uncommon blood disorder in which red blood cells are damaged and thus destroyed more rapidly than normal red blood cells. PNH results from clonal expansion of hematopoietic stem cells with a somatic mutation of the PIG-A (glypican type A) gene located on the X chromosome. Mutations in PIG-A lead to an early block in the synthesis of glycosylphosphatidylinositol (GPI), a molecule required for anchoring many proteins to the cell surface. Thus, PNH blood cells lack GPI-anchored proteins, including the complement-regulatory proteins CD55 and CD59. Under normal conditions, these complement-regulatory proteins block the formation of MACs on the cell surface, thereby preventing erythrocyte lysis. In PNH, the absence of GPI-anchored proteins results in complement-mediated hemolysis.

PNH is characterized by hemolytic anemia (decreased number of red blood cells), hemoglobinuria (the presence of hemoglobin in the urine, especially after sleep), and hemoglobinemia (the presence of hemoglobin in the bloodstream). Individuals disturbed by PNH are known to have flare-ups, which are defined here as the onset of dark urine. Hemolytic anemia results from the intravascular destruction of red blood cells by complement components. Other known symptoms include dysphasia, fatigue, erectile dysfunction, thrombosis, and recurrent abdominal pain.

Eculizumab is a humanized monoclonal antibody directed against complement protein C5 and was the first approved for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic-uremic syndrome ( aHUS) (see, eg, NPL 2). Eculizumab inhibits the cleavage of C5 into C5a and C5b by C5 convertase, which prevents the production of the final complement complex C5b-9. Both C5a and C5b-9 lead to late complement-mediated events characterized as PNH and aHUS (see also, PTL 3, PTL 4, PTL 5 and PTL 6).

Several reports have described anti-C5 antibodies. For example, PTL 7 describes an anti-C5 antibody that binds the alpha chain of C5 but not C5a and blocks activation of C5, while PTL 8 describes an anti-C5 monoclonal antibody that inhibits C5a formation. On the other hand, PTL 9 describes an anti-C5 antibody that recognizes the proteolytic site of C5 convertase on the alpha chain of C5 and inhibits the conversion of C5 to C5a and C5b. PTL 10 describes anti-C5 antibodies with affinity constants of at least 1×10 ⁷ M ⁻¹ .

The antibody (IgG) binds to the neonatal Fc receptor (FcRn) and has a long plasma retention time. Binding of IgG to FcRn is typically observed under acidic conditions (eg, pH 6.0), and barely observable under neutral conditions (eg, pH 7.4). Typically, IgG enters cells nonspecifically via endocytosis and returns to the cell surface by binding to endosomal FcRn in endosomes under acidic conditions. Then, IgG dissociates from FcRn under neutral conditions in plasma. IgG that is not bound to FcRn is broken down in lysosomes. When IgG's FcRn binding ability under acidic conditions is eliminated by introducing mutations into the Fc region of IgG, IgG is not recycled from endosomes into plasma, which results in a significant impairment of plasma retention of IgG. In order to improve the plasma retention of IgG, methods to enhance its FcRn binding under acidic conditions have been reported. When IgG's FcRn binding under acidic conditions is improved by introducing amino acid substitutions into the Fc region of IgG, IgG is more efficiently recycled from endosomes to plasma, and thus shows improved plasma retention. At the same time, it has also been reported that IgG with enhanced FcRn binding under neutral conditions does not dissociate from FcRn under neutral conditions in plasma, even though it does not dissociate through its binding to FcRn under acidic conditions in endosomes Upon return to the cell surface, and thereafter its plasma retention remains unchanged, or deteriorates (see, eg, NPL 3; NPL4; NPL5).

More recently, antibodies that bind antigen in a pH-dependent manner have been reported (see, eg, PTL11 and PTL12). These antibodies bind antigen strongly under plasma neutral conditions and dissociate from antigen under endosomal acidic conditions. After dissociation from the antigen, the antibody becomes capable of rebinding to the antigen upon recycling to plasma via FcRn. Thus, a single antibody molecule can repeatedly bind to multiple antigen molecules. In general, the plasma retention of antigens is much shorter than that of antibodies with the FcRn-mediated recycling mechanism described above. Thus, when antigens bind to antibodies, antigens typically exhibit prolonged plasma retention, resulting in an increase in antigen plasma concentrations. On the other hand, it has been reported that the above-mentioned antibody that binds the antigen in a pH-dependent manner eliminates the antigen from the plasma more rapidly than typical antibodies because it interacts with the endosome during the FcRn-mediated recycling process. Antigen dissociation. PTL 13 also describes in silico modeling analysis showing that antibodies with pH-dependent binding to C5 can extend antigen knockdown.

Citation List

Patent Literature

PTL 1: US Patent No. 6,355,245

PTL 2: US Patent No. 7,432,356

PTL 3: WO 2005/074607

PTL 4: WO 2007/106585

PTL 5: WO 2008/069889

PTL 6: WO 2010/054403

PTL 7: WO 95/29697

PTL8: WO 02/30985

PTL 9: WO 2004/007553

PTL 10: WO 2010/015608

PTL 11: WO 2009/125825

PTL12: WO 2011/122011

PTL 13: WO 2011/111007

Non-patent literature

NPL 1: Holers et al., Immunol. Rev. 223:300-316 (2008)

NPL 2: Dmytrijuk et al, The Oncologist 13(9): 993-1000 (2008)

NPL 3: Yeung et al., J Immunol. 182(12):7663-7671 (2009)

NPL 4: Datta-Mannan et al., J Biol. Chem. 282(3): 1709-1717 (2007)

NPL 5: Dall'Acqua et al., J. Immunol. 169(9): 5171-5180 (2002)

SUMMARY OF THE INVENTION

technical problem

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

solution to the problem

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

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

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 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 and SEQ ID of SEQ ID NO: 6 VL of 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) SEQ ID NO: : VH of 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. In additional embodiments, the anti-C5 antibody binds C5 with higher affinity at neutral pH than at acidic pH. In additional embodiments, the anti-C5 antibody binds C5 with higher affinity at pH 7.4 than at pH 5.8.

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

In some embodiments, the isolated anti-C5 antibodies of the invention inhibit the activation of C5. In some embodiments, the isolated anti-C5 antibodies of the invention inhibit the activation of the 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, humanized, or chimeric antibody. In some embodiments, the isolated anti-C5 antibodies of the invention are antibody fragments that bind C5. In some embodiments, the isolated anti-C5 antibody of the invention is a full-length IgGl or IgG4 antibody.

In some embodiments, the isolated anti- _C5 antibody of the invention comprises ( _a ) HVR _- H3 comprising the amino _acid sequence _{DX1GYX2X3PTHAMX4X5} , wherein X1 is G or _A , _X2 is V, Q or D, _X3 is T or Y, _X4 is Y or H, X5 is L or Y (SEQ ID NO: 128), (b) HVR _- L3 comprising Amino _acid sequence _{QX1TX2VGSSYGNX3} , wherein X1 is _S , C, N or T, _X2 is F or K, _X3 is _A , T or H (SEQ ID NO: 131), and (c) HVR- H2, the HVR-H2 comprises the amino acid sequence X ₁ IX ₂ TGSGAX ₃ YX ₄ AX ₅ WX ₆ KG, wherein X ₁ is C, A or G, X ₂ is Y or F, X ₃ is T, D or E, _X4 is Y, K or Q, _X5 is S, D or E, and X6 is _A or V (SEQ ID NO: 127).

In some embodiments, the isolated anti-C5 antibody of the invention comprises (a) HVR-H1 comprising SSYYX ₁ X ₂ , wherein X ₁ is M or V and X ₂ is C or A (SEQ ID NO: 126), (b) HVR-H2 comprising the amino acid sequence X ₁ IX ₂ TGSGAX ₃ YX ₄ AX ₅ WX ₆ KG, wherein X ₁ is C, A or G and X ₂ is Y or F, _X3 is T, D or E, _X4 is Y, K or Q, _X5 is S, D or E, X6 is _A or V (SEQ ID NO: 127), and (c) HVR-H3 , the HVR _- H3 comprises the amino _acid sequence _{DX1GYX2X3PTHAMX4X5} , wherein _X1 is G or _A , _X2 is V, Q or D, _X3 is T or Y, and _X4 is _Y or H , _X5 is L or Y (SEQ ID NO: 128). In additional embodiments, the antibody comprises (a) HVR-L1 comprising the amino acid sequence X ₁ ASQX ₂ IX ₃ SX ₄ LA, wherein X ₁ is Q or R and X ₂ is N, Q or G, _X3 is G or S, _X4 is D, K or S (SEQ ID NO: 129); (b) HVR-L2 comprising the amino acid sequence GASX ₁ X ₂ X ₃ S, wherein X1 is K, E or T, _X2 is L or T, _X3 is _A , H, E or Q (SEQ ID NO: 130); and (c) HVR-L3 comprising the amino acid sequence QX ₁ TX ₂ VGSSYGNX ₃ , wherein 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, the isolated anti-C5 antibody of the invention comprises (a) HVR-L1 comprising the amino acid sequence X ₁ ASQX ₂ IX ₃ SX ₄ LA, wherein X ₁ is Q or R, X ₂ is N, Q or G, _X3 is G or S, _X4 is D, K or S (SEQ ID NO: 129); (b) HVR-L2 comprising the amino acid sequence GASX ₁ X _{2 X3S} , wherein X1 is K, E or T, _X2 is L or T, and _X3 is _A , H, E or Q (SEQ ID NO: 130); and (c) HVR-L3, the HVR -L3 comprises the amino acid sequence QX ₁ TX ₂ VGSSYGNX ₃ , wherein 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, the isolated anti-C5 antibody of the invention comprises heavy chain variable domain framework FR1 comprising the amino acid sequence of any one of SEQ ID NOs: 132-134; FR2 comprising SEQ ID NO: 132-134 The amino acid sequence of any of ID NOs: 135-136; FR3, comprising the amino acid sequence of any of SEQ ID NOs: 137-139; and FR4, comprising any of SEQ ID NOs: 140-141 amino acid sequence. In some embodiments, the isolated anti-C5 antibody of the invention comprises a light chain variable domain framework FRi comprising the amino acid sequence of any one of SEQ ID NOs: 142-143; FR2 comprising SEQ ID NO: 142-143 The amino acid sequence of any of ID NOs: 144-145; FR3 comprising the amino acid sequence of any of SEQ ID NOs: 146-147; and FR4 comprising the amino acid sequence of SEQ ID NO: 148.

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

The present invention provides antibodies comprising the VH sequence of any of SEQ ID NOs: 10, 106-110 and the VL sequence of any of SEQ ID NOs: 20, 111-113.

The present invention also provides isolated nucleic acids encoding the anti-C5 antibodies of the present invention. The present invention also provides host cells comprising the nucleic acids of the present invention. The present invention also provides a method of producing an antibody, the method comprising culturing a host cell of the present invention to produce the antibody.

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

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 can be used as a medicine. The anti-C5 antibodies of the invention can be used to treat complement-mediated diseases or disorders involving excessive or uncontrolled C5 activation. The anti-C5 antibodies of the invention can be used to enhance the clearance of C5 from plasma.

The anti-C5 antibody of the present invention can be used for the preparation of medicaments. In some embodiments, the medicament is for the treatment of complement-mediated diseases or disorders involving excessive or uncontrolled C5 activation. In some embodiments, the medicament is used to enhance the clearance of C5 from plasma.

The present invention also provides methods of treating individuals suffering from complement-mediated diseases or disorders involving excessive or uncontrolled C5 activation. 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 methods of 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.

Brief Description of Drawings

[figure 1]

Figure 1 shows epitope binning of anti-C5 antibodies, as described in Example 2.2. Antibodies grouped into the same epitope bin are boxed with bold lines.

[Figure 2A]

Figure 2A shows BIACORE (registered trademark) sensorgrams at pH 7.4 (solid line) and pH 5.8 (dashed line) for assessing pH-dependence of anti-C5 antibody, as described in Example 3.2. CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, and CFA0599 are antibodies grouped into Epitope C as described in Example 2.2.

[Figure 2B]

Figure 2B shows BIACORE (registered trademark) sensorgrams at pH 7.4 (solid line) and pH 5.8 (dashed line) for assessing pH-dependence of anti-C5 antibody, as described in Example 3.2. CFA0666, CFA0672, and CFA0675 are antibodies grouped into epitope C, and CFA0330 and CFA0341 are antibodies grouped into epitope B, as described in Example 2.2. 305LO5 is a humanized antibody to CFA0305, as described in Example 2.3.

[image 3]

Figure 3 shows Western blot analysis for C5 beta-chain derived fragments (amino acids 19-180, 161-340, 321-500, and 481-660 of SEQ ID NO: 40) fused to a GST-tag, as in Example 4.1 described in. CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675 are antibodies grouped into epitope C. Anti-GST antibody is a positive control. The position of the GST-fused C5 fragment (46-49 kDa) is marked with arrows.

[Figure 4]

Figure 4 shows a BIACORE (registered trademark) sensorgram of an anti-C5 antibody to the MG1-MG2 domains of the C5 beta-chain, as described in Example 4.3. The upper panel shows the results for CFA0305 (solid line), CFA0307 (dotted line), CFA0366 (dotted line), and eculizumab (dotted line). The middle panel shows the results for CFA0501 (solid line), CFA0599 (dotted line), CFA0538 (dotted line), and eculizumab (dotted line). The lower graph shows the results for CFA0666 (solid line), CFA0672 (dotted line), CFA0675 (dotted line), and eculizumab (dotted line). CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675 are antibodies grouped into epitope C. Eculizumab was the control anti-C5 antibody.

[Fig. 5A]

Figure 5A shows peptide fragments derived for the MG1-MG2 domain fused to a GST tag (amino acids 33-124, 45-124, 52-124, 33-111, 33-108, and 45-111 of SEQ ID NO: 40 ), as described in Example 4.4. Anti-GST antibodies were used as reaction antibodies. The position of the GST-fused C5 fragment (35-37 kDa) is marked with arrows.

[Figure 5B]

Figure 5B shows peptide fragments derived for the MG1-MG2 domain fused to a GST-tag (amino acids 33-124, 45-124, 52-124, 33-111, 33-108, and 45- 111), as described in Example 4.4. CFA0305 was used as the reaction antibody.

[Figure 5C]

Figure 5C summarizes the binding reaction of anti-C5 antibodies to C5 beta-chain derived fragments, as described in Example 4.4. Fragments that bind to the anti-C5 antibodies grouped into epitope C (CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675) are shown in grey and those that do not are shown in white.

[Image 6]

Figure 6 shows Western blot analysis for the C5 point mutant in which E48, D51, and K109 of the beta-strand were replaced by alanines (E48A, D51A, and K109A, respectively), as in Example 4.5 described in. In the left panel, eculizumab (anti-C5 antibody, α-chain binder) was used as the antibody for the reaction, and the position of the α-chain (about 113 kDa) of C5 was marked with an arrow. In the right panel, CFA0305 (grouped into epitope C, β-chain binder) was used as the antibody for reaction, and the position of the β-chain (about 74 kDa) of C5 is marked with an arrow.

[Figure 7]

Figure 7 shows a BIACORE (registered trademark) sensorgram showing the interaction of eculizumab-F760G4 (upper panel) or 305LO5 (lower panel) with the C5 mutant, as described in Example 4.6. Sensorgrams were obtained by injecting C5-wt (thick solid curve), C5-E48A (short dashed curve), C5-D51A (long dashed curve), and C5-K109A (thin solid curve) in immobilized eculizumab, respectively. -F760G4 or 305LO5 sensor surface obtained. Eculizumab was the control anti-C5 antibody. 305LO5 is a CFA0305 humanized antibody (grouped into Epitope C) as described in Example 2.3.

[Figure 8]

Figure 8 shows a BIACORE (registered trademark) sensorgram showing the interaction of 305LO5 with a C5 His mutant for assessing pH-dependence, as described in Example 4.7. The sensorgrams were obtained by dividing C5-wt (thick solid curve), C5-H70Y (long dashed curve), C5-H72Y (short dashed curve), C5-H110Y (dotted curve), and C5-H70Y+H110Y (thin solid curve), respectively. ) injections were obtained on the sensor surface immobilized with 305LO5. Antibody/antigen complexes were allowed to dissociate at pH 7.4, followed by further dissociation at pH 5.8 (indicated by arrows) to assess pH-dependent interactions.

[Fig. 9A]

Figure 9A shows inhibition of complement-activated lipolysis by anti-C5 antibody, as described in Example 5.1. Results are shown for CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675 grouped into epitope C, as described in Example 2.2.

[Figure 9B]

Figure 9B shows inhibition of complement-activated lipolysis by anti-C5 antibody, as described in Example 5.1. Results are shown for antibodies CFA0330 and CFA0341 grouped into epitope B, as described in Example 2.2.

[Fig. 10A]

Figure 10A shows inhibition of C5a production by anti-C5 antibodies, as described in Example 5.2. C5a concentrations were quantified in supernatants obtained during the liposome lysis assay described in Figure 9A.

[Figure 10B]

Figure 10B shows inhibition of C5a production by anti-C5 antibodies, as described in Example 5.2. C5a concentrations were quantified in supernatants obtained during the liposome lysis assay described in Figure 9B.

[Figure 11]

Figure 11 shows inhibition of complement-activated hemolysis by anti-C5 antibodies, as described in Example 5.3. Complement is activated via the classical pathway.

[Figure 12]

Figure 12 shows inhibition of complement-activated hemolysis by anti-C5 antibodies, as described in Example 5.4. Complement is activated via the alternative pathway.

[Figure 13]

Figure 13 shows the time course of plasma human C5 concentrations following intravenous administration of human C5 alone or human C5 and anti-human C5 antibodies in mice assessing C5 clearance, as described in Example 6.2. CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672 and CFA0675 are antibodies grouped into epitope C, and CFA0330 and CFA0341 are antibodies grouped into epitope B as described in Example 2.2 .

[Figure 14]

Figure 14 shows the time course of plasma anti-human C5 antibody concentrations following intravenous administration of human C5 alone and anti-human C5 antibody in mice assessed for antibody pharmacokinetics, as described in Example 6.3. CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672 and CFA0675 are antibodies grouped into epitope C, and CFA0330 and CFA0341 are antibodies grouped into epitope B as described in Example 2.2 .

[Figure 15]

Figure 15 shows inhibition of complement-activated lipolysis by anti-C5 antibodies, as described in Example 9.1. Results are shown for antibodies 305LO15-SG422, 305LO16-SG422, 305LO18-SG422, 305LO19-SG422, 305LO20-SG422, and 305LO20-SG115.

[Figure 16]

Figure 16 shows inhibition of complement-activated lipolysis by anti-C5 antibodies, as described in Example 9.1. Results are shown for antibodies 305LO15-SG115 and 305LO23-SG429.

[Figure 17]

Figure 17 shows inhibition of complement-activated lipolysis by anti-C5 antibodies, as described in Example 9.1. Results are shown for antibodies 305LO22-SG115, 305LO22-SG422, 305LO23-SG115, and 305LO23-SG422.

[Figure 18]

Figure 18 shows inhibition of C5a production by anti-C5 antibodies, as described in Example 9.2. C5a concentrations were quantified in supernatants obtained during the liposome lysis assay described in Figure 15.

[Figure 19]

Figure 19 shows inhibition of C5a production by anti-C5 antibodies, as described in Example 9.2. C5a concentrations were quantified in supernatants obtained during the liposome lysis assay described in Figure 16.

[Figure 20]

Figure 20 shows inhibition of complement activity in monkey plasma by anti-C5 antibodies, as described in Example 9.3. Anti-C5 antibodies were administered into cynomolgus monkeys and complement activity in monkey plasma was measured in a hemolysis assay.

[Figure 21]

Figure 21 shows inhibition of biological activity of wild-type C5 (WT) and C5 variants (V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N, and E1437D) by anti-C5 antibody (eculizumab) , as described in Example 9.4.

[Figure 22]

Figure 22 shows inhibition of biological activity of wild-type C5 (WT) and C5 variants (V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N, and E1437D) by anti-C5 antibodies (305 variant), as As described in Example 9.4.

[Figure 23]

Figure 23 shows inhibition of complement-activated lipolysis by anti-C5 antibodies (BNJ441 and 305 variants), as described in Example 9.5.

[Figure 24]

Figure 24 shows the time course of plasma cynomolgus C5 concentrations following intravenous administration of anti-human C5 antibody in cynomolgus monkeys assessing C5 clearance, as described in Example 10.2.

[Figure 25]

Figure 25 shows the time course of plasma anti-human C5 antibody concentrations following intravenous administration of anti-human C5 antibody in cynomolgus monkeys evaluating antibody pharmacokinetics, as described in Example 10.3.

[Figure 26]

Figures 26A and 26B show the crystal structure of 305Fab bound to the human C5(hC5)-MG1 domain, as described in Example 11.6. Figure 26A shows an asymmetric cell. MG1 is shown in surface representation and 305Fab is shown as a ribbon (dark grey: heavy chain, light grey: light chain). Figure 26B shows molecules 1 and 2 superimposed (dark grey: molecule 1, light grey: molecule 2).

[Fig. 27A]

Figure 27A shows the epitope of the 305Fab contact region on the MG1 domain, as described in Example 11.6. Figure 27A shows epitope mapping in the MG1 amino acid sequence (dark grey: less than 3.0 Angstroms in proximity, light grey: less than 4.5 Angstroms in proximity).

[Fig. 27B]

Figure 27B shows the epitope of the 305 Fab contact region on the MG1 domain, as described in Example 11.6. Figure 27B shows epitope localization in the crystal structure (dark grey balls: less than 3.0 Angstroms in proximity, light grey sticks: less than 4.5 Angstroms in proximity).

[Fig. 28A]

Figure 28A shows a close-up view of the interaction of E48, D51 and K109 (stick representation) with 305Fab (surface representation), as described in Example 11.7.

[Figure 28B]

Figure 28B shows the interaction between E48 and its environment (dark grey dotted line: hydrogen bonding with Fab, light grey dotted line: water mediated hydrogen bonding) as described in Example 11.7.

[Figure 28C]

Figure 28C shows the interaction between D51 and its environment (dark grey dotted line: hydrogen bonding with Fab), as described in Example 11.7.

[Figure 28D]

Figure 28D shows the interaction between K109 and its environment (dark grey dotted line: hydrogen bond with Fab, light grey dotted line: salt bridge with H-CDR3_D95), as described in Example 11.7.

[Fig. 29A]

Figure 29A shows a close-up view of the interaction of H70, H72 and H110 (stick representation) with 305 Fab (surface representation), as described in Example 11.8, in the same orientation as in Figure 28A.

[Figure 29B]

Figure 29B shows the interaction between H70 and its environment, as described in Example 11.8. The histidine residues are indicated in stick and net notation. Hydrogen bonds are indicated by dotted lines.

[Figure 29C]

Figure 29C shows the interaction between H72 and its environment, as described in Example 11.8. The histidine residues are indicated in stick and net notation. Hydrogen bonds are indicated by dotted lines.

[Figure 29D]

Figure 29D shows the interaction between H110 and its environment, as described in Example 11.8. The histidine residues are indicated in stick and net notation. The distance between H110 and H-CDR3_H100c is shown as a dotted line.

specific implementation

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

I. Definitions

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

For the purpose of interpreting this application, the following definitions will apply and where appropriate, terms used in the singular will also include the plural and vice versa. It is to be understood that the techniques used herein are for the purpose of describing particular embodiments only and are not intended to be limiting. In the event of any conflict between any of the definitions given below and any document incorporated herein by reference, the definitions given below shall control.

An "acceptor human framework" for purposes herein is an amino acid comprising a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework The framework of the sequence, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it 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 sequence of the VL acceptor human framework is identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

"Affinity" refers to the sum of the forces of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by a 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 that has one or more changes in one or more hypervariable regions (HVRs) (compared to a parent antibody that does not have such changes) that result in the antibody's affinity for the antigen improve.

The terms "anti-C5 antibody" and "C5 binding antibody" refer to an antibody capable of binding C5 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent for targeting C5. In one embodiment, the binding of an anti-C5 antibody to an unrelated, non-C5 protein is less than about 10% of the binding of the antibody to C5, as measured, eg, by a radioimmunoassay (RIA). In certain embodiments, the C5-binding antibody has a dissociation constant (Kd) of: 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (eg, 10 ⁻⁸ M Hereinafter, for example, ^10-8 M to ^10-13 M, for example, ^10-9 M to ^10-13 M). In certain embodiments, the anti-C5 antibody binds to an epitope of C5 that is conserved among C5s derived from different species.

The term "antibody" is used herein in the broadest sense and includes various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, so long as they exhibit The desired antigen-binding activity is sufficient.

An "antibody fragment" refers to a molecule other than an intact antibody that comprises the portion of the intact antibody that binds the antigen to which the intact 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); specific antibodies.

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

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are mainly five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses ( _alotypes ), eg, _IgGi , IgG2, _IgG3 , _IgG4 , IgA ₁ , and IgA ₂ . The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (eg, radioisotopes of ^At211 , ^I131 , I125, ^y90 , ^Re186 , ^Re188 , ^Sm153 , ^Bi212 , ^p32 , ^{pb212 ,} and Lu); chemotherapeutic agents or drugs (eg, methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other chimeric agent); growth inhibitor ; enzymes and fragments thereof such as nucleic acid hydrolases; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various antineoplastic agents disclosed below or anticancer agents.

"Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with antibody allotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors (eg, B cells receptors); and B cell activation.

An "effective amount" of an agent (eg, a pharmaceutical formulation) refers to an effective amount for the dose and time necessary 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 targeting the antigen, and includes specific amino acids that are in direct contact with the antibody. Epitopic determinants can include chemically active surface clusters of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural characteristics, and/or specific charge characteristics. Typically, an antibody specific for a particular target antigen will preferentially recognize epitopes on the target antigen in a complex mixture of proteins and/or macromolecules.

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

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

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

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably to refer to a cell into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include transformed primary cells and progeny derived therefrom (regardless of the number of passages). The nucleic acid content of the progeny may not be identical to the parental cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the original transformed cell are included herein.

A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human or human cell or derived from an antibody of non-human origin utilizing human antibody repertoires or other human antibody coding sequences. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

A "human consensus framework" is a framework that represents the most common amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, human immunoglobulin VL or VH sequences are selected from a subset of variable domain sequences. Typically, a subset of sequences is as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup κI as in Kabat et al. above. In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al. above.

A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from a non-human HVR and amino acid residues from a human FR. In certain embodiments, a humanized antibody will comprise substantially all of at least one (and typically, two) variable domains, wherein all or substantially all of the HVRs (eg, CDRs) correspond to non-human antibodies HVRs, and all or substantially all of the FRs correspond to the FRs of the human antibody. A humanized antibody may optionally 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 been humanized.

The term "hypervariable region" or "HVR" as used herein refers to sequence hypervariability ("complementarity determining regions" or "CDRs") and/or formation of structurally defined loops ("hypervariable loops") in the variable domains of antibodies ") and/or regions containing residues that make contact with the antigen ("antigen contacts"). Typically, an antibody contains six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include: (a) occurring at amino acid residues 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1), 53-55(H2), and a hypervariable loop at 96-101 (H3) (Chothia, J. Mol. Biol. 196: 901-917 (1987)); (b) occurs at amino acid residues 24-34 (L1), 50-56 ( L2), 89-97(L3), 31-35b(H1), 50-65(H2), and CDRs at 95-102(H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service , NIH, Bethesda, MD (1991)); (c) occurs at amino acid residues 27c-36(L1), 46-55(L2), 89-96(L3), 30-35b(H1), 47-58 (H2), and antigenic contacts at 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)); and (d) (a), (b) and/or (c) a combination comprising HVR amino acid residues 46-56(L2), 47-56(L2), 48-56(L2), 49-56(L2), 26-35(H1), 26-35b ( H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

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

An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules including, but 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, and horses), primates (eg, humans and non-human primates such as monkeys), rabbits, and 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 natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, as determined by, eg, electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (eg, ion exchange or determined by reversed-phase HPLC). For a review of methods for assessing antibody purity, see, eg, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

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

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

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical and/or bind the same epitope, except where possible In addition to variant antibodies, eg, containing naturally-occurring mutations or produced during the preparation of monoclonal antibody preparations, such variants are usually present in small amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the attribute "monoclonal" indicates the nature of an antibody as being obtained from a substantially homogeneous population of antibodies, and is not considered to require that the antibody be prepared by any particular method. For example, monoclonal antibodies for use in accordance with the present invention can be prepared by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and the use of transgenic animals that contain all or a portion of the human immunoglobulin loci. Methods, such methods and other exemplary methods for making monoclonal antibodies are described herein.

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

"Native antibody" refers to a naturally occurring immunoglobulin molecule with a variety of structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, consisting of two identical light chains and two identical heavy chains bonded by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also known as the variable heavy chain domain or the heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3 ). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also known as the variable light chain domain or light chain variable domain, followed by the light chain constant (CL) domain . The light chains of antibodies can be assigned to one of two types, called kappa (kappa) and lambda (lambda), based on the amino acid sequence of their constant domains.

The term "package insert" is used to refer to instructions for use typically contained in commercial packages of therapeutic products, which contain indications, uses, dosages, administration, combination therapies, contraindications and/or warnings for use of such therapeutic products.

"Percent (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the alignment of the sequences and the introduction of gaps where necessary to achieve maximum percent sequence identity, without considering any conservative substitutions as The percentage of amino acid residues in the candidate sequence that are identical to amino acid residues in the reference polypeptide sequence after being part of the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways within the skill in the art, eg, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) 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. The author of the ALIGN-2 sequence comparison computer program is Genentech, Inc. and the source code has been filed with the user file in the US Copyright Office, Washington D.C., 20559, where it is registered under US Copyright Registration No. TXU510087. However, for purposes herein, % amino acid sequence identity values were generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or the program can be compiled from source code. ALIGN-2 programs should be compiled for UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not need to be changed.

Where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to or relative to a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence A to or relative to a given amino acid sequence A A given amino acid sequence B has or contains a specific % amino acid sequence identity) is calculated as follows: 100 times the score X/Y; where X is the same match scored by the sequence alignment program ALIGN-2 in the program alignment of A and B number of amino acid residues, and Y is the total number of amino acid residues in B. It is to be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless expressly stated otherwise, all % amino acid sequence identity values used herein were obtained using the ALIGN-2 computer program as described in the preceding paragraph.

The term "pharmaceutical formulation" refers to a formulation having a form that allows the biological activity of the active ingredient contained therein to be effective and which is free of unacceptable toxicity to the subject to which the formulation is to be administered of other components.

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

The term "C5" as used herein includes any native C5 from any vertebrate source, including mammals such as primates (eg, humans and monkeys) and rodents (eg, mice and rats). 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 containing the beta chain sequence set forth in SEQ ID NO:40. The term includes "full-length" unprocessed C5 as well as any form of C5 derived from processing in a cell. The term also includes naturally-occurring variants of C5, eg, splice variants or allelic variants. The amino acid sequence of an exemplary human C5 is shown in SEQ ID NO: 39 ("wild-type" or "WT" C5). The amino acid sequence of an exemplary beta chain of human C5 is shown in SEQ ID NO:40. The amino acid sequences of exemplary MG1, MG2 and MG1-MG2 domains of the beta chain of human C5 are shown in SEQ ID NOs: 41, 42 and 43, respectively. Exemplary cynomolgus monkey and murine C5 amino acid sequences are shown in SEQ ID NOs: 44 and 105, respectively. Amino acid residues 1-19 of SEQ ID NOs: 39, 40, 43, 44, and 105 correspond to signal sequences that are removed during processing in cells and are therefore not found in the corresponding exemplified amino acid sequences.

As used herein, "treatment" (and grammatical variants such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of the subject being treated, And can be done for prophylaxis or during the clinical course. Desirable effects of treatment include, but are not limited to, preventing disease occurrence or recurrence, reducing symptoms, eliminating any direct or indirect pathological consequences of disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating disease state, and eliminating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or to slow the progression of a disease.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in antibody antigen binding. The heavy and light chain variable domains (VH and VL, respectively) of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, eg, Kindt et al., ^{KubyImmunology} , 6th ed., WH Freeman & Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind a particular antigen can be isolated by screening a library of complementary VL or VH domains using the VH or VL domains, respectively, 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).

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of the host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".

II. Compositions and Methods

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

A. Exemplary Anti-C5 Antibodies

In one aspect, the invention provides isolated antibodies that bind C5. In certain embodiments, the anti-C5 antibodies of the invention bind to an epitope within the beta chain of C5. In certain embodiments, the anti-C5 antibody binds to an epitope within the MG1-MG2 domain of the beta chain of C5. In certain embodiments, the anti-C5 antibody binds to an epitope within a segment consisting of amino acids 19-180 of the beta chain of C5. In certain embodiments, the anti-C5 antibody binds to an epitope within the MG1 domain of the beta chain of C5 (amino acids 20-124 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 beta chain of C5 (SEQ ID NO: 40). In another embodiment, the antibody does not bind a fragment shorter than the fragment consisting of amino acids 33-124 of the beta chain of C5, eg, consisting of amino acids 45-124 of the beta chain of C5 (SEQ ID NO: 40), Fragments consisting of 52-124, 33-111, 33-108 or 45-111.

In another aspect, the present invention provides anti-C5 antibodies that exhibit pH-dependent binding properties. As used herein, the expression "pH-dependent binding" means that the antibody "shows reduced binding to C5 at acidic pH compared to its binding at neutral pH" (for the purposes of the present disclosure, the two expressions may be exchanged use). For example, antibodies that "have pH-dependent binding properties" include antibodies that bind C5 with higher affinity at neutral pH than at acidic pH. In certain embodiments, an antibody of the invention binds C5 with an affinity at neutral pH of at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 times higher. In some embodiments, the antibody binds C5 with higher affinity at pH 7.4 than at pH 5.8. In additional embodiments, the antibody binds C5 with an affinity at pH 7.4 of at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, at pH 5.8, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 times higher.

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

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

The binding properties of an antibody to a specific antigen can also be expressed as the kd of the antibody. The kd of an antibody refers to the dissociation rate constant of the antibody with respect to a particular antigen and is expressed in the reciprocal of the unit second (ie, sec ⁻¹ ). An increase in the kd value indicates weaker binding of the antibody to its antigen. The present invention thus includes antibodies that bind C5 with a higher kd value at acidic pH than at neutral pH. The invention includes antibodies that bind C5 with a kd at acidic pH that is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 times higher. In another embodiment, the antibody may have a kd value at neutral pH ^{of 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 antibody may have a kd value of ^10-3 1/s, ^10-2 1/s, ^10-1 1/s or more at acidic pH. The invention also includes antibodies that bind C5 with a higher kd at pH 5.8 than at pH 7.4. The invention includes antibodies whose kd for binding to C5 at pH 5.8 is at least 3, 5, 10, 15, 20, 25, 30, 35, 40 of the kd for binding of said antibody to C5 at pH 7.4 , 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 times higher. In another embodiment, the antibody may have a kd value at pH 7.4 of ^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 antibody may have a kd value at pH 5.8 of ^10-3 1/s, ^10-2 1/s, ^10-1 1/s or more.

In certain instances, "decreased binding to C5 at acidic pH compared to its binding at neutral pH" is expressed as the KD value of the antibody for binding to C5 at acidic pH versus the KD value for the antibody binding to C5 at neutral pH ratio (or vice versa). For example, for the present invention, if an antibody exhibits an acidic/neutral KD ratio of 2 or more, the antibody can be considered to exhibit "reduced binding to C5 at acidic pH compared to its binding at neutral pH". In certain exemplary embodiments, the antibodies of the invention have a pH5.8/pH7.4 KD ratio of 2 or greater. In certain exemplary embodiments, the acid/neutral KD ratio of an antibody of the invention may be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more. In another embodiment, the KD value of the antibody at neutral pH may be ^10-7 M, ^10-8 M, ^10-9 M, ^10-10 M ^{, 10-11} M, ^10-12 M or less. In another embodiment, the KD value of the antibody at acidic pH may be ^10-9 M, ^10-8 M, ^10-7 M, ^10-6 M or more. In another case, for the purposes of the present invention, if an antibody exhibits a pH5.8/pH7.4 KD ratio of 2 or more, the antibody can be considered to exhibit "combination with C5 at acidic pH versus its binding at neutral pH" than decrease". In certain exemplary embodiments, the pH5.8/pH7.4 KD ratio of the antibody of the invention may be 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more. In another embodiment, the antibody may have a KD value at pH 7.4 of ^10-7 M, ^10-8 M, ^10-9 M, ^10-10 M, ^10-11 M, ^10-12 M or less. In another embodiment, the KD value of the antibody at pH 5.8 may be ^10-9 M, ^10-8 M, ^10-7 M, ^10-6 M or more.

In some cases, "decreased binding to C5 at acidic pH compared to its binding at neutral pH" is expressed as the kd value of the antibody binding to C5 at acidic pH and the kd value of the antibody binding to C5 at neutral pH ratio (or vice versa). For example, for the present invention, if an antibody exhibits an acidic/neutral kd ratio of 2 or greater, the antibody can be considered to exhibit "reduced binding to C5 at acidic pH compared to its binding at neutral pH". In certain exemplary embodiments, the antibodies of the invention have a pH5.8/pH7.4 kd ratio of 2 or greater. In certain exemplary embodiments, the acid/neutral kd ratio of an antibody of the invention may be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more. In further exemplary embodiments, the pH5.8/pH7.4 kd ratio of the antibody of the invention may be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more. In another embodiment, the antibody may have a kd value at neutral pH of ^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 antibody may have a kd value at pH 7.4 of ^10-2 1/s, ^10-3 1/s, ^{10-4 1} /s, ^10-5 1/s, ^10-6 1/s the following. In another embodiment, the antibody may have a kd value of ^10-3 1/s, ^10-2 1/s, ^10-1 1/s or more at acidic pH. In another embodiment, the antibody may have a kd value at pH 5.8 of ^10-3 1/s ^{, 10-2} 1/s, ^10-1 1/s or more.

As used herein, the expression "acidic pH" refers to a pH of 4.0 to 6.5. The expression "acidic 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, 6.1, pH of any of 6.2, 6.3, 6.4 and 6.5. In particular aspects, the "acidic pH" is 5.8.

As used herein, the expression "neutral pH" refers to 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 particular aspects, the "neutral pH" is 7.4.

As represented herein, KD values as well as kd values 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 determined at 25°C or 37°C.

In certain embodiments, the anti-C5 antibodies of the invention bind to an epitope consisting of the MG1 domain within the beta chain of C5 (SEQ ID NO: 41). In certain embodiments, the anti-C5 antibodies of the invention bind to an epitope within the beta chain of C5 (SEQ ID NO: 40) comprising at least one fragment selected from the group consisting of: amino acids 47- 57, 70-76, and 107-110. In certain embodiments, the anti-C5 antibodies of the invention bind to an epitope within a fragment of the beta chain of C5 (SEQ ID NO: 40) comprising at least one amino acid selected from the group consisting of: Thr47 , Glu48, Ala49, Phe50, Asp51, Ala52, Thr53, Lys57, His70, Val71, His72, Ser74, Glu76, Val107, Ser108, LVs109, and His110. In certain embodiments, the anti-C5 antibodies of the invention bind to an epitope within a fragment of the beta chain of C5 (SEQ ID NO: 40) comprising at least one amino acid selected from the group consisting of Glu48 , Asp51, His70, His72, Lys109, and His110. In certain embodiments, an anti-C5 antibody of the invention binds to a C5 mutant with reduced binding to wild-type C5, wherein the C5 mutant has at least one at a position selected from the group consisting of Amino acid substitutions: Glu48, Asp51, His72, and Lys109. In another embodiment, an anti-C5 antibody of the invention has reduced pH-dependent binding to a C5 mutant having at least one selected Amino acid substitutions at positions from the group consisting of: His70, His72, and His110. In another embodiment, in the C5 mutant, the amino acid at a position selected from Glu48, Asp51 and Lys109 is replaced by alanine, and the amino acid at a position selected from His70, His72 and His110 is replaced by tyrosine acid.

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 VL of 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 and SEQ ID of SEQ ID NO: 5 VL of NO: 15; (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) SEQ ID NO: : VH of 2 and VL of SEQ ID NO: 12; (h) VH of SEQ ID NO: 3 and VL of SEQ ID NO: 13; (i) VH of SEQ ID NO: 9 and VL of SEQ ID NO: 19 (j) VH of SEQ ID NO:7 and VL of SEQ ID NO:17; (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 VL of SEQ ID NO:20.

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

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

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

Without being bound by a particular theory, it is expected that the binding of anti-C5 antibodies to C5 is diminished (or nearly lost) when one amino acid residue on C5 is replaced by another amino acid, implying that the The amino acid residues are critical for the interaction between the anti-C5 antibody and C5, and the antibody can recognize epitopes on C5 surrounding the amino acid residues.

It has been found in the present invention that a set of anti-C5 antibodies that compete with each other or bind to the same epitope can exhibit pH-dependent binding characteristics. Among amino acids, histidines with pKa values of about 6.0 to 6.5 can have different proton dissociation states between neutral and acidic pH. Thus, histidine residues on C5 may contribute to the pH-dependent interaction between anti-C5 antibodies and C5. Without being bound by a particular theory, it is expected that anti-C5 antibodies can recognize conformational structures near the histidine residues on C5 that are variable depending on pH. This expectation may be consistent with the experimental results described below: when the histidine residue on C5 is substituted for another amino acid, the pH-dependence of anti-C5 antibodies is diminished (or almost lost) (ie, in At acidic pH, an anti-C5 antibody with pH-dependent binding characteristics binds the histidine mutant of C5 with similar affinity to wild-type C5, while at acidic pH the same antibody binds with higher affinity than wild-type C5 Histidine mutant of C5).

In certain embodiments, the anti-C5 antibodies of the invention bind C5 from more than one species. In additional embodiments, the anti-C5 antibody binds C5 from human and non-human animals. In additional embodiments, the anti-C5 antibody binds C5 from humans and monkeys (eg, cynomolgus monkeys, macaques, marmosets, chimpanzees, or baboons).

In one aspect, the invention provides anti-C5 antibodies that inhibit the activation of C5. In certain embodiments, anti-C5 antibodies are provided that prevent cleavage of C5 to form C5a and C5b, thus preventing the production of anaphylatoxin activity associated with C5a, and preventing the C5b-9 membrane attack complex (MAC) associated with C5b assembly. In certain embodiments, anti-C5 antibodies are provided that block the conversion of C5 to C5a and C5b by C5 convertase. In certain embodiments, anti-C5 antibodies are provided that prevent C5 convertase from accessing a cleavage site on C5. In certain embodiments, anti-C5 antibodies are provided that block the hemolytic activity caused by activation of C5. In additional embodiments, the anti-C5 antibodies of the invention inhibit the activation of C5 via the classical pathway and/or the alternative pathway.

In one aspect, the invention provides anti-C5 antibodies that inhibit the activation of C5 variants. C5 variants refer to genetic variants of C5 due to genetic changes such as mutations, polymorphisms or allelic changes. Genetic changes can include deletions, substitutions or insertions of one or more nucleotides. A C5 variant may contain one or more genetic changes in C5. In certain embodiments, the C5 variant has biological activity similar to wild-type C5. Such a C5 variant may comprise at least one alteration selected from the group consisting of V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N, and E1437D. Herein, for example, R885H represents a genetic change in which the arginine at position 885 is replaced by histidine. In certain embodiments, the anti-C5 antibodies of the invention inhibit the activation of 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 .

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

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

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

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

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

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

In one aspect, the invention provides antibodies comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising SEQ ID NO:45 , the amino acid sequence of any one of 54, 117, 126; (b) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 55, 64, 118-120, 127; and (c) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 65, 74, 121, 128. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of any of SEQ ID NOs: 65, 74, 121, 128. In another embodiment, the antibody comprises HVR-H3 and HVR-L3, the HVR-H3 comprises the amino acid sequence of any one of SEQ ID NOs: 65, 74, 121, 128, the HVR-L3 comprises SEQ ID NO: 65, 74, 121, 128 The amino acid sequence of any of ID NO: 95, 104, 125, 131. In another embodiment, the antibody comprises HVR-H3, HVR-L3 and HVR-H2, the HVR-H3 comprises the amino acid sequence of any one of SEQ ID NOs: 65, 74, 121, 128, the HVR -L3 comprises the amino acid sequence of any one of SEQ ID NOs: 95, 104, 125, 131 and the HVR-H2 comprises the amino acid sequence of any one of SEQ ID NOs: 55, 64, 118-120, 127. In another embodiment, the antibody comprises (a) HVR-H1, said HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 45, 54, 117, 126; (b) HVR-H2, said HVR-H1 said HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 55, 64, 118-120, 127; and (c) HVR-H3 comprising SEQ ID NOs: 65, 74, 121, 128 The amino acid sequence of any one of them.

In another aspect, the invention provides antibodies comprising at least one, at least two, or all three of the VL HVR sequences selected from: (a) HVR-L1 comprising SEQ ID NO: The amino acid sequence of any one of 75, 84, 122, 129; (b) HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 85, 94, 123-124, 130; and (c) ) HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 95, 104, 125, 131. In one embodiment, the antibody comprises (a) HVR-L1, said HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 75, 84, 122, 129; (b) HVR-L2, said HVR-L1 HVR-L2 comprising the amino acid sequence of any of SEQ ID NOs: 85, 94, 123-124, 130; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NOs: 95, 104, 125, 131 any amino acid sequence.

In another aspect, the antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three of the VH HVR sequences selected from: (i) HVR-H1, the Said HVR-H1 comprises the amino acid sequence of any one of SEQ ID NOs: 45, 54, 117, 126, (ii) HVR-H2, said HVR-H2 comprises SEQ ID NOs: 55, 64, 118-120, 127 the amino acid sequence of any one, and (iii) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 65, 74, 121, 128; and (b) a VL domain, the VL structure The domain comprises at least one, at least two, or all three of the VL HVR sequences selected from: (i) HVR-L1 comprising the amino acids of any of SEQ ID NOs: 75, 84, 122, 129 Sequences, (ii) HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 85, 94, 123-124, 130, and (c) HVR-L3 comprising SEQ ID NO: 85, 94, 123-124, 130 The amino acid sequence of any of ID NO: 95, 104, 125, 131.

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

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

In certain embodiments, the substitutions are conservative substitutions, as described herein. In certain embodiments, any one or more of the following substitutions can be made in any combination: (a) in HVR-H1 (SEQ ID NO: 45), M5V or C6A; (b) in HVR-H2 ( SEQ ID NO:55), C1A or G, Y3F, T9D or E, Y11K or Q, S13D or E, or A15V; (c) in HVR-H3 (SEQ ID NO:65), G2A, V5Q or D , T6Y, Y12H, or L13Y; (d) in HVR-L1 (SEQ ID NO:75), Q1R, N5Q or G, G7S, D9K or S; (e) in HVR-L2 (SEQ ID NO:85) In, 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 identified by the consensus sequences of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3, respectively SEQ ID NOs: 126, 127, 128, 129, 130 and 131 covered.

In any of the above embodiments, the anti-C5 antibody is humanized. In one embodiment, the anti-C5 antibody comprises the HVR of 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 the HVR of any of the above embodiments, and further comprises a VH or VL comprising a FR sequence, wherein the FR sequence is as follows. For heavy chain variable domains, FR1 comprises the amino acid sequence of any of SEQ ID NOs: 132-134, FR2 comprises the amino acid sequence of any of SEQ ID NOs: 135-136, and FR3 comprises any of SEQ ID NOs: 137-139 The amino acid sequence of one, FR4 comprises the amino acid sequence of any one of SEQ ID NOs: 140-141. For the light chain variable domain, FR1 comprises the amino acid sequence of any one of SEQ ID NOs: 142-143, FR2 comprises the amino acid sequence of any one of SEQ ID NOs: 144-145, and FR3 comprises the amino acid sequence of any one of SEQ ID NOs: 146-147 The amino acid sequence of any one, FR4 comprises the amino acid sequence of SEQ ID NO:148.

In another aspect, the anti-C5 antibody comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid sequence of any one of SEQ ID NOs: 1-10, Heavy chain variable domain (VH) sequences of 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 relative to a reference sequence comprises Substitutions (eg, conservative substitutions), insertions, or deletions, but anti-C5 antibodies comprising the sequences retain the ability to bind C5. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in any of SEQ ID NOs: 1-10. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVR (ie, in FRs). Optionally, the anti-C5 antibody comprises the VH sequence in any one of SEQ ID NOs: 1-10, including post-translational modifications of said sequence. In particular embodiments, the VH comprises one, two or three HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 45-54, ( b) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 55-64, and (c) HVR-H3 comprising any one of SEQ ID NOs: 65-74 amino acid sequence.

In another aspect, there is provided an anti-C5 antibody, wherein the antibody comprises at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of any one of SEQ ID NOs: 11-20, Light chain variable domains (VL) of 96%, 97%, 98%, 99%, 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 relative to a reference sequence comprises Substitutions (eg, conservative substitutions), insertions, or deletions, but anti-C5 antibodies comprising the sequences retain the ability to bind C5. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in any of SEQ ID NOs: 11-20. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (ie, in a FR). Optionally, the anti-C5 antibody comprises the VL sequence in any of SEQ ID NOs: 11-20, including post-translational modifications of the sequence. In particular embodiments, the VL comprises one, two or three HVRs selected from: (a) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 75-84; (b) ) HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 85-94; and (c) HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 95-104 amino acid sequence.

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

In another aspect, the anti-C5 antibody comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid sequence of any one of SEQ ID NOs: 10, 106-110 %, 98%, 99%, or 100% sequence identity of heavy chain variable domain (VH) sequences. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity relative to a reference sequence comprises Substitutions (eg, conservative substitutions), insertions or deletions, but anti-C5 antibodies comprising the sequences retain the ability to bind C5. In certain embodiments, a total of 1 to 10 amino acids in any of SEQ ID NO: 10, 106-110 are substituted, inserted and/or deleted. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (ie, in a FR). Optionally, the anti-C5 antibody comprises the VH sequence of any of SEQ ID NO: 10, 106-110, including post-translational modifications of the sequence. In particular embodiments, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acids of any of SEQ ID NOs: 45, 54, 117, 126 Sequences, (b) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 55, 64, 118-120, 127, and (c) HVR-H3 comprising SEQ ID NO: 55, 64, 118-120, 127 The amino acid sequence of any of ID NOs: 65, 74, 121, 128.

In another aspect, the anti-C5 antibody comprises a VH sequence that is at least 90%, 91%, 92%, 93%, 94% identical to the amino acid sequence of any one of SEQ ID NOs: 10, 106-110, 95%, 96%, 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 relative to a reference sequence comprises Substitutions (eg, conservative substitutions), insertions or deletions, but anti-C5 antibodies comprising the sequences retain the ability to bind C5. In certain embodiments, a total of 1 to 10 amino acids in any of SEQ ID NO: 10, 106-110 are substituted, inserted and/or deleted. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (ie, in a FR). Optionally, the anti-C5 antibody comprises the VH sequence of any of SEQ ID NO: 10, 106-110, including post-translational modifications of the sequence. In particular embodiments, the VH comprises one, two or three HVRs selected from the group consisting of: (a) HVR-H1 comprising the HVR of any of SEQ ID NOs: 45, 54, 117, 126 Amino acid sequence, (b) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 55, 64, 118-120, 127, and (c) HVR-H3 comprising Amino acid sequence of any of SEQ ID NOs: 65, 74, 121, 128.

In another aspect, the anti-C5 antibody comprises a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid sequence of SEQ ID NO: 10 , 98%, 99%, or 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 relative to a reference sequence comprises Substitutions (eg, conservative substitutions), insertions or deletions, but anti-C5 antibodies comprising the sequences retain the ability to bind C5. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 10 are substituted, inserted and/or deleted. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (ie, in a FR). Optionally, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 10, including post-translational modifications of said sequence. In particular embodiments, the VH comprises one, two or three HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 54, (b) HVR-H2, The HVR-H2 comprises the amino acid sequence of SEQ ID NO:64, and (c) the HVR-H3 comprises the amino acid sequence of SEQ ID NO:74.

In another aspect, the anti-C5 antibody comprises a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid sequence of SEQ ID NO: 106 , 98%, 99%, or 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 relative to a reference sequence comprises Substitutions (eg, conservative substitutions), insertions or deletions, but anti-C5 antibodies comprising the sequences retain the ability to bind C5. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 106 are substituted, inserted and/or deleted. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (ie, in a FR). Optionally, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 106, including post-translational modifications of said sequence. In particular embodiments, the VH comprises one, two or three HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2, wherein The HVR-H2 comprises the amino acid sequence of SEQ ID NO:118, and (c) the HVR-H3 comprises the amino acid sequence of SEQ ID NO:121.

In another aspect, the anti-C5 antibody comprises a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid sequence of SEQ ID NO: 107 , 98%, 99%, or 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 relative to a reference sequence comprises Substitutions (eg, conservative substitutions), insertions or deletions, but anti-C5 antibodies comprising the sequences retain the ability to bind C5. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 107 are substituted, inserted and/or deleted. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (ie, in a FR). Optionally, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 107, including post-translational modifications of said sequence. In particular embodiments, the VH comprises one, two or three HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117 (b) HVR-H2, said HVR-H2 comprises the amino acid sequence of SEQ ID NO:119, and (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO:121.

In another aspect, the anti-C5 antibody comprises a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid sequence of SEQ ID NO: 108 , 98%, 99%, or 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 relative to a reference sequence comprises Substitutions (eg, conservative substitutions), insertions or deletions, but anti-C5 antibodies comprising the sequences retain the ability to bind C5. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 108 are substituted, inserted and/or deleted. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (ie, in a FR). Optionally, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 108, including post-translational modifications of said sequence. In particular embodiments, the VH comprises one, two or three HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117 (b) HVR-H2, said HVR-H2 comprises the amino acid sequence of SEQ ID NO:118, and (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO:121.

In another aspect, the anti-C5 antibody comprises a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid sequence of SEQ ID NO: 109 , 98%, 99%, or 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 relative to a reference sequence comprises Substitutions (eg, conservative substitutions), insertions or deletions, but anti-C5 antibodies comprising the sequences retain the ability to bind C5. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 109 are substituted, inserted and/or deleted. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (ie, in a FR). Optionally, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 109, including post-translational modifications of said sequence. In particular embodiments, the VH comprises one, two or three HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117 (b) HVR-H2, said HVR-H2 comprises the amino acid sequence of SEQ ID NO:118, and (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO:121.

In another aspect, the anti-C5 antibody comprises a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid sequence of SEQ ID NO: 110 , 98%, 99%, or 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 relative to a reference sequence comprises Substitutions (eg, conservative substitutions), insertions or deletions, but anti-C5 antibodies comprising the sequences retain the ability to bind C5. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 110 are substituted, inserted and/or deleted. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (ie, in a FR). Optionally, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 110, including post-translational modifications of said sequence. In particular embodiments, the VH comprises one, two or three HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117 (b) HVR-H2, said HVR-H2 comprises the amino acid sequence of SEQ ID NO:120, and (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO:121.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of any one of SEQ ID NOs: 20, 111-113 %, 96%, 97%, 98%, 99%, or 100% sequence identity of light chain variable domains (VL). In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity relative to a reference sequence comprises Substitutions (eg, conservative substitutions), insertions or deletions, but anti-C5 antibodies comprising the sequences retain the ability to bind C5. In certain embodiments, a total of 1 to 10 amino acids in any of SEQ ID NO: 20, 111-113 are substituted, inserted and/or deleted. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (ie, in a FR). Optionally, the anti-C5 antibody comprises the VL sequence of any of SEQ ID NO: 20, 111-113, including post-translational modifications of said sequence. In particular embodiments, VL comprises one, two or three HVR(a) HVR-L1 selected from the group consisting of amino acids of any of SEQ ID NOs: 75, 84, 122, 129 sequences; (b) HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 85, 94, 123-124, 130; and (c) HVR-L3 comprising SEQ ID The amino acid sequence of any of NO: 95, 104, 125, 131.

In another aspect, there is provided an anti-C5 antibody, wherein the antibody comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, VL of 98%, 99%, or 100% sequence identity. 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 relative to a reference sequence comprises Substitutions (eg, conservative substitutions), insertions or deletions, but anti-C5 antibodies comprising the sequences retain the ability to bind C5. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 20 are substituted, inserted and/or deleted. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (ie, in a FR). Optionally, the anti-C5 antibody comprises the VL sequence in SEQ ID NO: 20, including post-translational modifications of said sequence. In particular embodiments, VL comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 84; (b) HVR-L2, said HVR-L1 HVR-L2 comprises the amino acid sequence of SEQ ID NO:94; and (c) HVR-L3 comprises the amino acid sequence of SEQ ID NO:104.

In another aspect, there is provided an anti-C5 antibody, wherein the antibody comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, VL of 98%, 99%, or 100% sequence identity. In certain embodiments, 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 relative to a reference sequence comprises Substitutions (eg, conservative substitutions), insertions or deletions, but anti-C5 antibodies comprising the sequences retain the ability to bind C5. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 111 are substituted, inserted and/or deleted. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (ie, in a FR). Optionally, the anti-C5 antibody comprises the VL sequence in SEQ ID NO: 111, including post-translational modifications of said sequence. In particular embodiments, VL comprises one, two or three HVRs selected from the group consisting of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2, said HVR-L1 HVR-L2 comprises the amino acid sequence of SEQ ID NO:123; and (c) HVR-L3, which comprises the amino acid sequence of SEQ ID NO:125.

In another aspect, there is provided an anti-C5 antibody, wherein the antibody comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, VL of 98%, 99%, or 100% sequence identity. 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 relative to a reference sequence comprises Substitutions (eg, conservative substitutions), insertions or deletions, but anti-C5 antibodies comprising the sequences retain the ability to bind C5. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 112 are substituted, inserted and/or deleted. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (ie, in a FR). Optionally, the anti-C5 antibody comprises the VL sequence in SEQ ID NO: 112, including post-translational modifications of said sequence. In particular embodiments, VL comprises one, two or three HVRs selected from the group consisting of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2, said HVR-L1 HVR-L2 comprises the amino acid sequence of SEQ ID NO:123; and (c) HVR-L3, which comprises the amino acid sequence of SEQ ID NO:125.

In another aspect, there is provided an anti-C5 antibody, wherein the antibody comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, VL of 98%, 99%, or 100% sequence identity. 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 relative to a reference sequence comprises Substitutions (eg, conservative substitutions), insertions or deletions, but anti-C5 antibodies comprising the sequences retain the ability to bind C5. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 113 are substituted, inserted and/or deleted. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (ie, in a FR). Optionally, the anti-C5 antibody comprises the VL sequence in SEQ ID NO: 113, including post-translational modifications of said sequence. In particular embodiments, VL comprises one, two or three HVRs selected from the group consisting of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2, said HVR-L1 HVR-L2 comprises the amino acid sequence of SEQ ID NO:124; and (c) HVR-L3 comprises the amino acid sequence of SEQ ID NO:125.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises the VH of any of the embodiments provided above and the VL of any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences of any of SEQ ID NOs: 10, 106-110 and SEQ ID NOs: 20, 111-113, respectively, including post-translational translations of the sequences retouch. 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 additional embodiments, 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, an anti-C5 antibody is provided, wherein the antibody comprises a VH sequence comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 54, (b) HVR-H2, wherein Said HVR-H2 comprises the amino acid sequence of SEQ ID NO: 64, and (c) HVR-H3, said HVR-H3 comprises the amino acid sequence of SEQ ID NO: 74, and comprises the following VL sequence: (a) HVR-L1, said HVR-L1 comprises the amino acid sequence of SEQ ID NO: 84; (b) HVR-L2 comprises the amino acid sequence of SEQ ID NO: 94; and (c) HVR-L3 comprises the amino acid sequence of SEQ ID NO: 94 ID NO: 104.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises a VH sequence comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2, The HVR-H2 comprises the amino acid sequence of SEQ ID NO: 118, and (c) HVR-H3, the HVR-H3 comprises the amino acid sequence of SEQ ID NO: 121, and comprises the following VL sequence: (a) HVR-L1, The HVR-L1 comprises the amino acid sequence of SEQ ID NO: 122; (b) the HVR-L2 comprises the amino acid sequence of SEQ ID NO: 123; and (c) the HVR-L3 comprises the amino acids Sequence SEQ ID NO:125.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises a VH sequence comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2, The HVR-H2 comprises the amino acid sequence of SEQ ID NO: 119, and (c) HVR-H3, the HVR-H3 comprises the amino acid sequence of SEQ ID NO: 121, and comprises the following VL sequence: (a) HVR-L1, The HVR-L1 comprises the amino acid sequence of SEQ ID NO: 122; (b) the HVR-L2 comprises the amino acid sequence of SEQ ID NO: 123; and (c) the HVR-L3 comprises the amino acids Sequence SEQ ID NO:125.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises a VH sequence comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2, The HVR-H2 comprises the amino acid sequence of SEQ ID NO: 118, and (c) HVR-H3, the HVR-H3 comprises the amino acid sequence of SEQ ID NO: 121, and comprises the following VL sequence: (a) HVR-L1, The HVR-L1 comprises the amino acid sequence of SEQ ID NO: 122; (b) the HVR-L2 comprises the amino acid sequence of SEQ ID NO: 124; and (c) the HVR-L3 comprises the amino acids Sequence SEQ ID NO:125.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises a VH sequence comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2, Said HVR-H2 comprises the amino acid sequence of SEQ ID NO: 120, and (c) HVR-H3, said HVR-H3 comprises the amino acid sequence of SEQ ID NO: 121, and comprises the following VL sequence: (a) HVR-L1, The HVR-L1 comprises the amino acid sequence of SEQ ID NO: 122; (b) the HVR-L2 comprises the amino acid sequence of SEQ ID NO: 124; and (c) the HVR-L3 comprises the amino acids Sequence SEQ ID NO:125.

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

In another aspect, the invention provides antibodies that bind the same epitope as the anti-C5 antibodies provided herein. For example, in certain embodiments, antibodies are provided that bind the same epitope as the antibodies described in Table 2. As demonstrated by the working examples below, all anti-C5 antibodies described in Table 2 were grouped into the same epitope box for C5 and displayed pH-dependent binding characteristics.

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

In another aspect of the invention, the anti-C5 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, eg, an Fv, Fab, Fab', scFv, diabody, or F(ab') ₂ fragment. In another embodiment, the antibody is a full-length antibody, eg, an intact IgGl or IgG4 antibody or other antibody class or isotype as defined herein.

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

1. Antibody Affinity

In certain embodiments, the antibodies provided herein have a dissociation constant (Kd) of: 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (eg ^, 10- ⁸ M or less, eg, ^{10 -8 M} to 10 ^-13 M, eg, 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 form of the antibody of interest and its antigen. For example, solution binding affinity of a Fab for an antigen is measured by equilibrating the Fab with a minimal concentration of ( ¹²⁵ I)-labeled antigen in the presence of a titration series of unlabeled antigen, followed by capture on an anti-Fab antibody-coated plate Bound antigen (see, eg, Chen et al., J. Mol. Biol. 293:865-881 (1999)). To determine the assay conditions, MICROTITER (registered trademark) multi-well plates (Thermo Scientific) were coated overnight with 5 μg/ml of capture anti-Fab antibody (CappelLabs) in 50 mM sodium carbonate (pH 9.6), and then used in PBS 2% (w/v) fetal bovine serum albumin in 2 to 5 hours at room temperature (about 23°C). In non-adsorbing plates (Nunc #269620), 100 pM or 26 pM [ ¹²⁵ I]-antigen was mixed with serial dilutions of the Fab of interest (eg, with Presta et al, Cancer Res. 57: 4593-4599 (1997) ) Anti-VEGF antibody, Fab-12 was assessed in agreement). The Fabb of interest is then incubated overnight; however, the incubation can be continued for longer periods (eg, about 65 hours) to ensure equilibrium is achieved. Thereafter, the mixture is transferred to a capture plate for incubation at room temperature (eg, for one hour). The solution was then removed and the plate was washed eight times with 0.1% polysorbate 20 (TWEEN-20 (registered trademark)) in PBS. When the plate had dried, 150 μl/well of scintillator (MICROSCINT-20 ^™ ; Packard) was added and the plate was counted on a TOPCOUNT ^™ gamma counter (Packard) for ten minutes. The concentration of each Fab that resulted in maximal binding of less than or equal to 20% was selected for competitive binding assays.

According to another embodiment, Kd is measured using BIACORE (registered trademark) surface plasmon resonance assay. For example, use BIACORE (registered trademark)-2000 or BIACORE (registered trademark)-3000 (BIACORE (registered trademark), Inc., Piscataway, NJ) at ~10 response units (RU) using an immobilized antigen CM5 chip at 25°C. measurement. In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE (registered trademark), Inc. ) was treated with N-ethyl-N'-(3-diethyl) according to the provider's instructions. Methylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) activation. Antigen was diluted to 5 μg/ml (~0.2 μM) with 10 mM sodium acetate pH 4.8 before injection at a flow rate of 5 μl/min to achieve approximately 10 response units (RU) of coupled protein. Following injection of antigen, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) were injected into PBS with 0.05% polysorbate 20 (TWEEN-20 ^™ ) surfactant at 25°C at a flow rate of about 25 μl/min (PBST). Association rates ( _kon ) and dissociation were calculated by simultaneously fitting association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE (registered trademark) Evaluation Software version 3.2) rate (k _off ). The equilibrium dissociation constant (Kd) was calculated as the ratio of _koff / _kon . See, eg, Chen et al., J. Mol. Biol. 293:865-881 (1999). If the binding rate measured by the above surface plasmon resonance assay exceeds 10 ⁶ M ^-1 s ^-1 , the binding rate can be determined as in a spectrometer such as a spectrophotometer equipped with a stop-flow device (Aviv Instruments) or in an 8000-Series SLM-AMINCO ^™ Spectrophotometer with Stirred Chamber (ThermoSpectronic) using 20 nM anti- Fluorescence quenching technique (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) for increase or decrease in fluorescence emission intensity of antigen-antibody (Fab format).

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 a review of scFv fragments, see, eg, Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, edited by Rosenburg and Moore, (Springer-Verlag, New York), pp. 269-315 (1994); see also, WO 93 /16185; and US Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab') ₂ fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see US Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites, which can be bivalent or bispecific. See, eg, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993 ). Tri- and quaternary antibodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).

Single domain antibodies are antibody fragments comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. 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 can be prepared by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies and by the preparation of recombinant host cells (eg, E. coli or phage), as described herein.

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 non-human variable regions (eg, variable regions derived from mouse, rat, hamster, rabbit, or non-human primates such as monkeys) and human constant regions. In a further example, a chimeric antibody is a "class-switched" antibody, wherein the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parental non-human antibody. Typically, a humanized antibody comprises one or more variable domains, wherein HVRs, eg, CDRs (or portions thereof) are derived from non-human antibodies, and FRs (or portions thereof) are derived from human antibody sequences. The humanized antibody optionally further comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (eg, the antibody from which the HVR residues are derived), eg, to restore or improve antibody specificity or Affinity.

Humanized antibodies and methods of making them are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008), and are further described, for example, in Riechmann et al., Nature 332: 323-329 (1988); Queen et al, Proc. Nat'lAcad. 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) (describes specificity determining regions ( SDR) transplantation); Padlan, Mol. Immunol. 28:489-498 (1991) (described "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (described "FR shuffling"); and Osbourn et al, Methods 36: 61-68 (2005) and Klimka et al, Br. J. Cancer, 83: 252-260 (2000) (describe "directed selection" for FR shuffling method).

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

4. Human Antibodies

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

Human antibodies can be prepared by administering immunogens to transgenic animals that have been modified to produce fully human antibodies or whole antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or part of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present outside the chromosome or are randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, for example, US Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE ^™ technology; US Patent No. 5,770,429, which describes HUMAB (registered trademark) technology; US Patent No. 7,041,870, which describes KM MOUSE (registered trademark) technology, and US Patent Application Publication No. US 2007/0061900, which describes VELOCIMOUSE (registered trademark) technology). Human variable regions from intact antibodies produced by such animals can be further modified, eg, by combining with different human constant regions.

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

Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from human phage display libraries. Such variable domain sequences can then be combined with the desired human constant domains. The following describes techniques for selecting human antibodies from antibody libraries.

5. Antibodies Derived from the Library

Antibodies of the invention can be isolated by screening combinatorial libraries of antibodies having the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening the libraries for antibodies with desired binding properties. Such methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described in, 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., HumanPress, 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 some phage display methods, VH and VL gene repertoires are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phage, as described in Winter et al., Ann. Rev. . Immunol. 12: 433-455 (1994). Phages typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, naive repertoires (eg, from humans) can be cloned to provide a single source of antibodies to multiple non-self-antigens as well as self-antigens without any immunization, as in Griffiths et al., EMBO J, 12:725- 734 (1993). Finally, natural libraries can also be prepared synthetically by cloning unrearranged V-gene segments from leukocytes and using PCR primers containing random sequences to encode hypervariable CDR3 regions and achieve rearrangement in vitro, as described by Hoogenboom, J. . Mol. Biol. 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: US Patent No. 5,750,373 and US Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936 and 2009 0002360.

In this context, an antibody or antibody fragment isolated from a human antibody library is considered to be 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 for at least two different sites. In certain embodiments, one binding specificity is for C5 and the other is for another antigen. In certain embodiments, bispecific antibodies can bind to two different epitopes of C5. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing C5. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see, Milstein and Cuello, Nature 305:537 (1983)), WO 93 /08829, and Traunecker et al., EMBO J. 10:3655 (1991)), and "knob-in-hole" engineering (see, eg, US Pat. No. 5,731,168). Multispecific antibodies can also be prepared by engineering electrostatic targeting of Fc-heterodimeric molecules used to prepare antibodies (WO2009/089004A1); cross-linking two or more antibodies or fragments (see, eg, US Pat. No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); use of leucine zippers to prepare bispecific antibodies (see, eg, Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)); using "Diabody" technology for the preparation of bispecific antibody fragments (see, eg, Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); and the use of single-chain Fv (scFv) bis Aggregates (see, eg, Gruber et al, J. Immunol. 152:5368 (1994)); and trispecific antibodies are prepared as described, eg, in Tutt et al, J. Immunol. 147:60 (1991).

Also included herein are engineered antibodies with more than three functional antigen binding sites, including "octopus antibodies" (see, eg, US 2006/0025576).

Antibodies or fragments herein also include "dual-acting FAbs" or "DAFs" that contain an antigen binding site that binds C5 as well as a different antigen (see, eg, US2008/0069820).

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to increase the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from the antibody amino acid sequence, and/or insertions into and/or substitutions of residues within the antibody amino acid sequence. Any combination of deletions, insertions and substitutions can be made to obtain the final construct, provided that the final construct has the desired characteristics, eg, antigen-binding.

a. Substitution, insertion and deletion variants

In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVR and FR. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions". Further changes are provided in Table 1 under the heading "Exemplary Substitutions" and as described further below with respect to amino acid side chain classification. Amino acid substitutions can be introduced into antibodies of interest and products screened for the desired activity (eg, maintained/increased antigen binding, decreased immunogenicity, or increased ADCC or CDC).

[Table 1]

initial residue Exemplary permutation Preferred replacement Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gin; His; Asp, Lys; Arg Gln Asp(D) Glu; Asn Glu Cys(C) Ser; Ala Ser Gln(Q) Asn;Glu Asn Glu(E) Asp;Gln Asp Gly(G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Lcu; Val; Met; Ala; Phc; Norleucine Leu Leu(L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids can be grouped into: (1) Hydrophobicity: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions require exchanging a member of one of these groups for a member of the other group.

A substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (eg, a humanized or human antibody). Typically, the resulting variant selected for further study will have an altered (eg, increased) certain biological property (eg, increased affinity, decreased immunogenicity) relative to the parent antibody and/or will be substantially Certain biological properties of the parent antibody are preserved. Exemplary substitutional variants are affinity matured antibodies, which can be routinely prepared, eg, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and variant antibodies are displayed on phage and screened for specific biological activity (eg, binding affinity).

Alterations (eg, substitutions) can be made in the HVR, eg, to increase antibody affinity. Such alterations can be made in HVR "hot spots", ie, codon-encoded residues that are mutated with high frequency during the somatic maturation process (see, eg, Chowdhury, Methods Mol. Biol. 207 : 179-196 (2008)), and/or contact residues of the antigen, the resulting variant VH or VL was tested for binding affinity. Affinity maturation by construction of secondary libraries and reselection therefrom has been described, for example, in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)) middle. 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, strand shuffling, or oligonucleotide-directed mutagenesis). . Secondary libraries are then generated. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity includes HVR-directed approaches, in which several HVR residues (eg, 4-6 residues simultaneously) are randomized. HVR residues involved in antigen binding can be specifically identified, eg, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

In certain embodiments, substitutions, insertions or deletions may occur within one or more HVRs, so long as such changes do not significantly reduce the ability of the antibody to bind antigen. For example, conservative changes that do not significantly reduce binding affinity (eg, conservative substitutions described herein) can be made in HVR. Such alterations may, for example, contact the exterior of residues of the antigen in the HVR. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unchanged, or contains no more than one, two or three amino acid substitutions.

A method that can be used to identify antibody residues or regions that can be targeted for mutagenesis is known as "alanine scanning mutagenesis," as described by Cunningham, Science 244: 1081-1085 (1989). In this method, a residue or group of target residues (eg, charged residues such as arg, asp, his, lys, and glu) are identified and neutralized by neutral or negatively charged amino acids (eg, alanine or polyalanine) substitution to determine whether it affects the interaction of the antibody with the antigen. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition, the crystal structure of the antigen-antibody complex is used to determine the point of contact between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or excluded as candidates for substitution. Variants can be screened to determine whether they possess desired properties.

Amino acid sequence insertions include amino- and/or carboxy-terminal fusions ranging from one residue in length to polypeptides containing more than a hundred residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with N-terminal methionyl residues. Other insertional variants of the antibody molecule include fusing the N- or C-terminus of the antibody 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 are altered to increase or decrease the degree to which the antibody is glycosylated. Adding or lacking glycosylation sites to an antibody can be readily accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

When an antibody contains an Fc region, the carbohydrate attached to it can be varied. Native antibodies produced by mammalian cells typically contain branched, bi-branched (biantennary) oligosaccharides, usually N-linked to Asn297 of the CH2 domain of the Fc region. See, eg, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include a variety of carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucoidans linked to GlcNAc in the "stem" of the bifurcated oligosaccharide structure sugar. In some embodiments, modification of the oligosaccharides in the antibodies of the invention can be performed to generate antibody variants with specific improved properties.

In one embodiment, antibody variants are provided that have carbohydrate structures lacking fucose linked (directly or indirectly) to the Fc region. For example, the amount of fucose in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297 relative to the sum of all sugar structures (eg complexes, hybrids and high mannose structures) linked to Asn297, As measured by MALDI-TOF mass spectrometry, as described in WO2008/077546, eg. Asn297 refers to the aspartic acid residue located near position 297 (Eu numbering of Fc region residues) in the Fc region; however, due to small sequence variations in antibodies, Asn297 may also be located about +// upstream or downstream of position 297 - at 3 amino acids, ie between positions 294 and 300. Such fucosylated variants may have enhanced ADCC function. See, eg, US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Example variants disclosed involving "defucosylated" or "fucose-deficient" antibodies include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2002/031140; Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include proteofucosylation deficient Lecl3CHO cells (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US 2003/0157108, Presta , L; and WO2004/056312, Adams et al., especially Example 11), as well as knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, For example, Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda et al., Biotechnol. Bioeng. 94(4):680-688 (2006); and WO2003/085107).

Antibody variants are also provided with bisected oligosaccharides, eg, wherein the bifurcated oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or increased ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide linked to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

c. Fc region variants

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants. Fc region variants may comprise human Fc region sequences (eg, human IgGl, IgG2, IgG3, or IgG4 Fc regions) comprising amino acid modifications (eg, substitutions) at one or more amino acid positions.

In certain embodiments, the present invention contemplates antibody variants that possess some, but not all, effector functions that make them important for the in vivo half-life of the antibody while certain effector functions (eg, complement and ADCC) are not necessary or harmful applications are ideal candidates. In vitro and/or in vivo cytotoxicity assays can be performed to confirm the reduction/elimination of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcyR binding (and thus likely lacks ADCC activity), but retains FcRn binding ability. The primary cells mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression 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 for assessing ADCC activity of molecules of interest are described in US Pat. No. 5,500,362 (see, eg, Hellstrom et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and In Hellstrom et al., Proc. Nat'lAcad. Sci. USA 82: 1499-1502 (1985); US Pat. No. 5,821,337 (see Bruggemann et al., J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, nonradioactive assays can be used (see, eg, ACTI ^™ Nonradioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96 (registered trademark) nonradioactive cell Toxicity assay (Promega, Madison, WI)). Useful effector cells for this assay include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of a molecule of interest can be assessed in vivo, eg, in animal models as described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, eg, C1q and C3c binding ELISA in WO2006/029879 and WO2005/100402. To assess complement activation, CDC assays can be performed (see, 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)). FcRn binding and in vivo clearance/half-life assays can also be performed using methods known in the art (see, eg, Petkova et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

Antibodies with reduced effector function include antibodies with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at more than two of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutation in which residues 265 and 297 are substituted to alanines body (US Patent No. 7,332,581).

Certain antibody variants are described that have increased or decreased binding to FcRs. (See, eg, US Patent No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).)

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

In some embodiments, changes are made in the Fc region that result in altered (ie, increased or decreased) C1q binding and/or complement-dependent cytotoxicity (CDC), eg, as in US Pat. No. 6,194,551 , WO 1999/51642, and Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

Such antibodies are described in US2005/0014934 (Hinton et al.) with increased half-life and increased interaction with the neonatal Fc receptor (FcRn) responsible for the transport of maternal IgG to the fetus (J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). The antibody comprises an Fc region having one or more substitutions therein that increase binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the following 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, eg, substitution of Fc region residue 434 (US Pat. No. 7,371,826).

See also, Duncan, Nature 322:738-40 (1988); US Patent No. 5,648,260; US Patent No. 5,624,821; and WO 1994/29351 for other examples of Fc region variants.

d. Cysteine-engineered antibody variants

In certain embodiments, it may be desirable to prepare cysteine-engineered antibodies, eg, "thioMAbs," in which one or more residues of the antibody are replaced with cysteine residues. In particular embodiments, the substituted residues occur at the access site of the antibody. By replacing the residue with a cysteine, a reactive thiol group is thus placed at the antibody's access site and can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drugs moieties, thereby producing immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues may be substituted for a cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and of the Fc region of the heavy chain S400 (EU number). Cysteine engineered antibodies can be produced as described, eg, in US Pat. No. 7,521,541.

e. Antibody Derivatives

In certain embodiments, the antibodies provided herein can be further modified to contain additional non-proteinaceous moieties known in the art and readily available. Moieties suitable 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, polyvinylpyrrolidone, poly-1 , 3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly( n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (eg, glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde can be advantageous in preparation due to its stability in water. The polymers can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, it can be the same or different molecules. Generally, the amount and/or type of polymer due to derivatization can be determined based on considerations including, but not limited to, the specific property or function of the antibody to be improved, whether the antibody derivative is useful for therapy under defined conditions, and the like.

In another embodiment, conjugates of antibodies and non-protein moieties are provided that can be selectively heated by exposure to radiation. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength and includes, but is not limited to, wavelengths that do not damage normal cells, but heat the non-protein moiety to a temperature at which cells adjacent to the antibody-non-protein moiety are killed.

B. Recombinant Methods and Compositions

Antibodies can be prepared using recombinant methods and compositions, eg, as described in US Pat. No. 4,816,567. In one embodiment, an isolated nucleic acid is provided that encodes an anti-C5 antibody described herein. Such nucleic acid may encode an amino acid sequence comprising the VL of the antibody and/or an amino acid sequence comprising the VH of the antibody (eg, the light and/or heavy chain of the antibody). In another embodiment, one or more vectors (eg, expression vectors) comprising such nucleic acids are provided. In another embodiment, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (eg, transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) ) a first vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VL, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VH. In one embodiment, the host cell is eukaryotic, eg, Chinese Hamster Ovary (CHO) cells or lymphocytes (eg, Y0, NSO, Sp20 cells). In one embodiment, a method of making an anti-C5 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expressing the antibody as described above, and optionally removing the antibody from the host The cells (or host cell culture medium) recover the antibody.

For recombinant production of anti-C5 antibodies, nucleic acid encoding the antibody, eg, as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional methods (eg, using oligonucleotide probes capable of binding specifically to genes encoding antibody heavy and light chains).

Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be made in bacteria, especially when glycosylation and Fc effector functions are not required. For the expression of antibody fragments and polypeptides in bacteria, see, eg, US Pat. Nos. 5,648,237, 5,789,199 and 5,840,523. (See also, Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describe the expression of antibody fragments in E. coli). After expression, the antibody can be isolated from the bacterial cell paste into a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including that the glycosylation pathway has been "humanized" to result in production of partially or fully human sugars Fungal and yeast strains of antibodies with sylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006).

Suitable host cells for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Various baculovirus strains have been identified that can be used with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts. See, eg, US Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 (which describe PLANTIBODIES ^™ technology for the production of antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for suspension culture can be useful. Other examples of useful mammalian host cell lines are the SV40 (COS-7) transformed monkey kidney CV1 cell line; the human embryonic kidney cell line (293 or as in eg Graham et al., J. Gen Virol. 36:59 (1977) ) 293 cells); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells, as described, for example, 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 mouse hepatocytes (BRL 3A); Human lung cells (W138); Human hepatocytes (Hep G2); Mouse mammary tumor (MMT060562); TRI cells; MRC5 cells; and FS4 cells as described, eg, in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982). Other useful mammalian host cells Lines include Chinese Hamster Ovary (CHO) cells, including DHFR ^- CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NSO and Sp2/0. For a review of certain mammalian host cell lines suitable for use in antibody production, see, eg, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255- 268 (2003).

Polyclonal antibodies are preferably prepared in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. It may be useful to use bifunctional or derivatizing reagents, for example, maleimidobenzoic acid succinimidyl ester (conjugated via cysteine residues), N-hydroxysuccinimidyl (via lysine residues) amino acid residues), glutaraldehyde, succinic anhydride, SOCl ₂ , or R ¹ N=C=NR, where R and R ¹ are different alkyl groups, to associate the relevant antigen with the immunogen in the species to be immunized Sexual proteins (eg, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor) are conjugated.

Animals (usually non-human mammals) by combining, for example, 100 μg or 5 μg of protein or conjugate (rabbit or mouse, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites ) is immunized against an antigen, immunogenic conjugate or derivative. One month later, animals are boosted with 1/5 to 1/10 of the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. After 7 to 14 days, the animals are bled and the sera are assayed for antibody titers. Animals are boosted until titer plateaus. Preferably, the animals are boosted with a conjugate of the same antigen conjugated to a different protein and/or via a different cross-linking agent. Conjugates can also be prepared in recombinant cell culture as protein fusions. In addition, aggregating agents such as alum are useful for enhancing immune responses.

Monoclonal antibodies are obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies that make up the population except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts Are the same. Thus, the attribute "monoclonal" refers to an antibody that is not characterized as a mixture of discrete antibodies.

For example, monoclonal antibodies can be prepared using the hybridoma method first described by Kohler et al., Nature 256(5517):495-497 (1975). In the hybridoma method, a mouse or other suitable host animal (eg, a hamster) is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro.

The immunological agent will typically comprise an antigenic protein or fusion variant thereof. Typically, peripheral blood lymphocytes (PBL) are used if cells of human origin are desired, or spleen cells or lymph node cells if non-human mammalian origin is desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusion reagent 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. Typically, rat or mouse myeloma cell lines are employed. The hybridoma cells thus prepared are seeded and cultured in a suitable medium, preferably containing one or more substances that inhibit the growth or survival of the non-fused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the medium used for the hybridoma will typically contain hypoxanthine, aminopterin, and thymidine (HAT cultures base), which are substances that prevent the growth of HGPRT-deficient cells.

Preferred immortalized myeloma cells are those cells that fuse efficiently, support stable high levels of antibody production by the antibody-producing cells of choice, and are sensitive to a medium such as HAT medium. Of these, preferred are murine myeloma cell lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and from US type cultures SP-2 cells (and derivatives thereof, eg, X63-Ag8-653) from the American Type Culture Collection, Manassas, Virginia USA. Human myeloma and mouse-human hybrid myeloma 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).

For the production of monoclonal antibodies against the antigen, the medium in which the hybridoma cells are cultured is assayed. 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 assays are known in the art. For example, binding affinity can be determined by Scatchard analysis of Munson, Anal. Biochem. 107(1):220-239 (1980).

After identification of hybridoma cells producing antibodies with the desired specificity, affinity and/or activity, clones can be subcloned by limiting dilution and grown by standard methods (Goding, supra). Media suitable for this purpose include, for example, D-MEM or RPMI-1640 media. In addition, hybridoma cells can be cultured as tumors in mammals in vivo.

Monoclonal antibodies secreted by subclones are suitably separated from culture medium, ascites fluid or serum by conventional immunoglobulin purification methods such as, for example, Protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography .

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

C. Determination

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

1. Binding Assays and Other Assays

In one aspect, for example, the antibody of the present invention is tested for antigen-binding activity by known methods such as ELISA, Western blot, BIACORE (registered trademark), 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 a competing antibody is present in excess, it blocks (eg, reduces) binding of the reference antibody to C5 by at least 10%, 15%, 20%, 25%, 30%, 35% , 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or higher. In some cases, binding is inhibited by at least 80%, 85%, 90%, 95% or more. In certain embodiments, such an epitope (eg, a linear or conformational epitope) for binding by such a competing antibody is bound by an anti-C5 antibody described herein (eg, an anti-C5 antibody described in Table 2) The epitope is the same. Detailed exemplary methods for mapping antibody-bound epitopes are provided in Morris "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ) (1996).

In an exemplary competition assay, immobilized C5 is incubated in a solution comprising a first labeled (reference) antibody, which binds C5, and a second unlabeled antibody, which is tested for the The second unlabeled antibody competes with the first antibody for its ability to bind to C5. The secondary antibody can be present in the hybridoma supernatant. As a control, immobilized C5 was incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the primary antibody to C5, excess unbound antibody is removed and the amount of label bound to immobilized C5 is measured. If the amount of label bound to immobilized C5 is significantly reduced in the test sample relative to the control sample, this indicates that the secondary antibody competes with the primary antibody for binding to C5. See, Harlow and Lane, Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) (1988).

In another exemplary competition assay, the BIACORE (registered trademark) assay is used to determine the ability of a test anti-C5 antibody to compete with a second (reference) anti-C5 antibody for binding to C5. In another aspect of operating a BIACORE (registered trademark) instrument (eg, BIACORE (registered trademark) 3000) according to the manufacturer's recommendations, C5 protein is captured on a CM5BIACORE (registered trademark) chip using standard techniques known in the art to thereby A C5-coated surface was produced. Typically, 200-800 response units of C5 are coupled to the chip (this amount readily provides measurable levels of binding and is readily saturable by the concentration of test antibody used). The two antibodies to be evaluated for their ability to compete with each other (ie, the test antibody and the reference antibody) are mixed in a suitable buffer at a 1 : 1 molar ratio of binding sites to prepare a test mixture. When calculating concentrations based on binding sites, the molecular weight of the test or reference antibody is assumed to be the total molecular weight of the corresponding antibody divided by the number of C5 binding sites on the antibody. The concentration of each antibody (ie, test antibody and reference antibody) in the test mixture should be high enough to readily saturate the binding site for the antibody captured on the C5 molecule on the BIACORE (registered trademark) chip. The test and reference antibodies in the mixture are at the same molar concentration (based on binding), typically 1.00 to 1.5 [mu]M (based on binding site). Separate solutions containing only the test antibody and only the reference antibody were also prepared. The test and reference antibodies in these solutions should be in the same buffer and at the same concentrations and conditions as in the test mixture. The test mixture containing test and reference antibodies was passed over a C5-coated BIACORE (registered trademark) chip and the total amount of binding was recorded. The chip is then treated to remove bound test or reference antibody without destroying C5 bound to the chip. Typically this is done by treating the chip with 30 mM HCl for 60 seconds. Solutions of individual test antibodies were then passed over the C5-coated surface and the amount of binding recorded. The chip was processed again to remove all bound antibody without destroying C5 bound to the chip. A solution of the reference antibody alone was then passed over the C5-coated surface and the amount of binding recorded. The maximum theoretical binding of the mixture of test and reference antibodies is next calculated, which is the sum of the bindings of each antibody (ie, test and reference) individually passing over the C5 surface. If the actual recorded binding of the mixture is less than this theoretical maximum, the test and reference antibodies compete with each other for binding to C5. Thus, typically, a competitive test anti-C5 antibody is one that binds C5 in the BIACORE (registered trademark) blocking assay above such that during the assay and in the presence of a reference anti-C5 antibody, the recorded binding is the test antibody and 80% to 0.1% (eg, 80% > to 4%) of maximum theoretical binding (as defined above) for the combination of reference antibodies, specifically 75% to 0.1% (eg, 75% to 4%) of maximum theoretical binding 4%), and more specifically 70% to 0.1% (eg, 70% to 4%) of maximum theoretical binding.

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 the group consisting of antibodies CFA0341 and CFA0330. In some embodiments, the anti-C5 antibody competes for binding to C5 with an antibody selected from the group consisting of CFA0538, CFA0501, CFA0599, CFA0307, CFA0366, CFA0675, and CFA0672. 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 the VH and VL pair of antibody CFA0305 or 305LO5.

In additional embodiments, the anti-C5 antibody binds 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 the group consisting of CFA0538, CFA0501, CFA0599, CFA0307, CFA0366, CFA0675, and CFA0672. In some embodiments, the anti-C5 antibody competes with antibody CFA0666 for binding to C5. In additional embodiments, the anti-C5 antibody binds C5 with higher affinity at pH 7.4 than at pH 5.8.

In additional embodiments, the anti-C5 antibody binds 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 the VH and VL pair of antibody CFA0305 or 305LO5. In additional embodiments, the anti-C5 antibody binds C5 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 the group consisting of: VH of SEQ ID NO:22 and VL of SEQ ID NO:26, or SEQ ID NO : VH of 21 and VL of SEQ ID NO:25. In some embodiments, the anti-C5 antibody competes for binding to C5 with an antibody comprising a VH and VL pair selected from: (a) VH of SEQ ID NO:5 and VL of SEQ ID NO:15; (b) SEQ ID NO : VH of 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 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 VL of SEQ ID NO: 11; (g) VH and SEQ ID NO: 9 VL of 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 for binding to C5 with an antibody comprising the VH of SEQ ID NO:23 and the VL of SEQ ID NO:27. In some embodiments, the anti-C5 antibody competes for binding to C5 with an antibody comprising the VH of SEQ ID NO:7 and the VL of SEQ ID NO:17.

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 VL of 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 and SEQ ID of SEQ ID NO: 5 VL of NO: 15; (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) SEQ ID NO: : VH of 2 and VL of SEQ ID NO: 12; (h) VH of SEQ ID NO: 3 and VL of SEQ ID NO: 13; (i) VH of SEQ ID NO: 9 and VL of SEQ ID NO: 19 (j) VH of SEQ ID NO: 7 and VL of SEQ ID NO: 17; (k) VH of SEQ ID NO: 8 and VL of SEQ ID NO: 18; (1) VH and SEQ ID NO: 23 VL of ID NO:27; and (m) VH of SEQ ID NO:10 and VL of SEQ ID NO:20.

In certain embodiments, an anti-C5 antibody of the invention competes for binding to C5 with an antibody comprising a VH and VL pair selected from: (a) VH of SEQ ID NO: 22 and VL of 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 and SEQ ID of SEQ ID NO: 4 VL of NO: 14; (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) SEQ ID NO: : VH of 3 and VL of SEQ ID NO: 13; (h) VH of SEQ ID NO: 9 and VL of SEQ ID NO: 19; (i) VH of SEQ ID NO: 7 and VL of SEQ ID NO: 17 (j) VH of SEQ ID NO:8 and VL of SEQ ID NO:18; (k) VH of SEQ ID NO:23 and VL of SEQ ID NO:27.

In certain embodiments, the anti-C5 antibodies of the invention are combined with an anti-C5 antibody comprising a VH selected from the group consisting of 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 Antibodies to the VH and VL pair compete for binding to C5.

In additional embodiments, the anti-C5 antibody binds C5 with higher affinity at neutral pH than at acidic pH. In certain embodiments, the anti-C5 antibody binds C5 with higher affinity at neutral pH than at acidic pH and competes for binding to C5 with an antibody comprising a VH and VL pair selected from: (a) SEQ ID NO : VH of 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 and SEQ ID of SEQ ID NO: 3 VL of 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) SEQ ID NO: : VH of 8 and VL of SEQ ID NO: 18; and (j) VH of SEQ ID NO: 10 and VL of SEQ ID NO: 20. In additional embodiments, the anti-C5 antibody binds C5 with higher affinity at pH 7.4 than at pH 5.8.

In some embodiments, the anti-C5 antibody binds C5 with higher affinity at neutral pH than at acidic pH and competes for binding to C5 with an antibody comprising a VH and VL pair selected from: (a) SEQ ID NO: VH of 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 VL of SEQ ID NO: 12; (e) VH of SEQ ID NO: 3 and VL of SEQ ID NO: 13; (f) VH and SEQ ID NO: 1 VL of ID NO: 11; (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) SEQ ID NO: 17 VH of NO:8 and VL of SEQ ID NO:18. In additional embodiments, the anti-C5 antibody binds C5 with higher affinity at pH 7.4 than at pH 5.8.

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

In certain embodiments, it is possible to determine whether an anti-C5 antibody of the invention binds a particular epitope as follows: C5 point mutants in which amino acids on C5 (other than alanine) are replaced by alanine in 293 cells Expressed and tested for binding of the anti-C5 antibody to the C5 mutant via ELISA, Western blot or BIACORE (registered trademark); wherein the binding of the anti-C5 antibody to the C5 mutant was significantly reduced or eliminated relative to its binding to wild-type C5 Anti-C5 antibodies are indicated to bind to an epitope on C5 comprising the amino acid. In certain embodiments, the amino acid to be replaced with alanine on C5 is selected from the group consisting of Glu48, Asp51, His70, His72, Lys109, and His110 of the beta chain of C5 (SEQ ID NO: 40). In additional embodiments, the amino acid on C5 to be replaced with alanine is Asp51 or Lys109 of the beta chain of C5 (SEQ ID NO: 40).

In another embodiment, whether an anti-C5 antibody with pH-dependent binding characteristics binds to a particular epitope can be determined by substituting a histidine residue on C5 with another amino acid (eg, tyrosine ) C5 point mutants were expressed in 293 cells, and binding of anti-C5 antibodies to C5 mutants was tested via ELISA, Western blot or BIACORE (registered trademark); wherein the binding of anti-C5 antibodies to wild-type C5 at acidic pH Binding to the C5 mutant was significantly diminished relative to its binding at acidic pH, indicating that the anti-C5 antibody binds an epitope on C5 containing the histidine residue. In additional embodiments, the binding of the anti-C5 antibody to wild-type C5 at neutral pH is not significantly diminished relative to its binding to the C5 mutant at neutral pH. In certain embodiments, the histidine residue on C5 to be replaced with another amino acid is selected from the group consisting of His70, His72, and His110 of the beta strand of C5 (SEQ ID NO: 40). In another embodiment, the histidine residue His70 is replaced with tyrosine.

2. Activity Assay

In one aspect, assays are provided for identifying biologically active anti-C5 antibodies. Biological activities can include, for example, inhibiting activation of C5, preventing C5 from being cleaved to form C5a and C5b, preventing C5 convertase from accessing cleavage sites on C5, blocking hemolytic activity caused by activation of C5, and the like. Antibodies having such in vivo and/or in vitro biological activities are also provided.

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

In certain embodiments, whether a test antibody inhibits cleavage of C5 into C5a and C5b is determined by the methods described, eg, in Isenman et al., J Immunol. 124(1):326-331 (1980). In another embodiment, this is determined by a method for the specific detection of cleaved C5a and/or C5b protein (eg, ELISA or Western blot). A test antibody is identified as an antibody capable of inhibiting C5 cleavage when a reduction in the amount of C5 cleavage products (ie, C5a and/or C5b) is detected in the presence of (or after contact with) the test antibody. In certain embodiments, the concentration and/or physiological activity of C5a can be determined by methods, eg, chemotaxis assays, RIA or ELISA (see, eg, Ward and Zvaifler J. Clin. Invest. 50(3): 606-616 (1971)).

In certain embodiments, whether a test antibody prevents C5 convertase from accessing C5 is determined by a method for detecting protein interactions between C5 convertase and C5 (eg, ELISA or BIACORE (registered trademark)). The test antibody is identified as an antibody capable of preventing the C5 convertase from accessing C5 when the interaction is diminished in the presence of (or after contact with) the test antibody.

In certain embodiments, C5 activity can be measured as a function of its cytolytic capacity in a subject's body fluids. The cytolytic capacity of C5 or its reduction can be measured by methods known in the art, eg, conventional hemolytic assays such as Kabat and Mayer (eds.), Experimental Immunochemistry, 2nd Edition, 135-240, Springfield, IL, CC Thomas (1961), pp. 135-139, the hemolysis assay, or a conventional variation of the assay, such as the chicken erythrocyte described in, for example, Hillmen et al., N. Engl. J. Med. 350(6): 552-559 (2004). Hemolysis method. In certain embodiments, the CH50eq assay is used to quantify C5 activity or inhibition thereof. The CH50eq assay is a method for measuring total classical complement activity in serum. The test is a lysis assay that uses antibody-sensitized red blood cells as the activator of the classical complement pathway, and different dilutions of the test serum are used to determine the amount required to produce 50% lysis (CH50). The percent hemolysis can be determined, for example, using a spectrophotometer. The CH50eq assay provides an indirect measure of final complement complex (TCC) formation since TCC itself is directly responsible for the measured hemolysis. Inhibition of C5 activation can also be detected and/or measured using the methods given and exemplified in the working examples. Using these or other suitable types of assays, candidate antibodies capable of inhibiting C5 activation can be screened. In certain embodiments, inhibiting C5 activation comprises: at least a 5%, 10%, 15%, 20%, 25%, 30% reduction in C5 activation in an assay compared to the effect of a negative control under similar conditions, 35%, or more than 40%. In some embodiments, it refers to inhibiting C5 activation by at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more.

D. Immunoconjugates

The present invention also provides immunoconjugates comprising an anti-C5 antibody herein conjugated to one or more cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitors, toxins ( For example, protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof), or radioisotopes.

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 maytansinoids (see US Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); auristatins such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see US Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); dolastatin; calicheamicin or derivatives thereof (see US Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al. Res. 58: 2925-2928 (1998)); anthracyclines such as daunomycin or doxorubicin (see 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; Taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel ) and ortataxel; 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 (from Pseudomonas aeruginosa), Ricin A chain, Acacia toxin A chain, Capsule root toxin A chain, α-sarcinus, Aleurites fordii protein, caryophyllin (dianthin) protein, Phytolaca Americana protein (PAPI, PAPII and PAP-S), bitter gourd (momordica charantia) inhibitor, jatrophin, crotonin, sapaonaria officinalis inhibitor, white tree Toxins, mitogellin, aspergillus constricted, phenomycin, enomycin, and tricothecene.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the preparation of radioconjugates. Examples include At ²¹¹ , I ¹³¹ , I ¹²⁵ , y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and radioisotopes of Lu. When the radioconjugate is used for detection, it may contain radioactive atoms for scintillation studies, such as tc99m or I123, or spin labeling for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), Such as iodine-123 (again), iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Conjugates of antibody and cytotoxic agent can be prepared using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate ( SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminosulfane hydrochloride (IT), imino Bifunctional derivatives of esters (such as dimethyladipic acid HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), diazido compounds (such as bis( p-azidobenzoyl)hexamethylenediamine), di-diazo derivatives (such as di-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) ), and di-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, 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 chelating agent for conjugating radionuclides to antibodies. See WO94/11026. The linker may be a "cleavable linker" that facilitates the release of cytotoxic drugs from cells. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al., Cancer Res. 52: 127-131 (1992); US Patent No. 5,208,020).

Immunoconjugates or ADCs herein are expressly contemplated, but not limited to such conjugates prepared using cross-linking reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC , MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, Thio-EMCS, Thio-GMBS, Thio-KMUS, Thio-MBS, Thio-SIAB, Thio-SMCC and Thio- SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (eg, from Pierce Biotechnology, Inc., Rockford, IL., U.S.A).

E. Methods and compositions for diagnosis and detection

In certain embodiments, any of the anti-C5 antibodies provided herein can be used to detect the presence of C5 in a biological sample. As used herein, the term "detection" includes quantitative detection or qualitative detection. In certain embodiments, biological samples include cells or tissues, such as serum, whole blood, plasma, biopsy samples, tissue samples, cell suspensions, saliva, sputum, oral fluid, cerebrospinal fluid, amniotic fluid, ascites, milk, colostrum , mammary gland secretion, lymph fluid, urine, sweat, tears, gastric fluid, synovial fluid, peritoneal fluid, ocular lens fluid and mucus.

In one embodiment, an anti-C5 antibody for use in a diagnostic or detection method is provided. In another aspect, a method of detecting the presence of C5 in a biological sample is provided. In certain embodiments, the method comprises contacting a biological sample with an anti-C5 antibody as described herein under conditions that allow the anti-C5 antibody to bind to C5, and detecting between the anti-C5 antibody and C5 form a complex. Such methods may be in vitro or in vivo methods. In one embodiment, an anti-C5 antibody is used to select subjects suitable for treatment with an anti-C5 antibody, eg, wherein C5 is a biomarker for selection of patients.

In another embodiment, the present invention provides a method of selecting an individual with a complement-mediated disease or a disorder involving excessive or uncontrolled C5 activation suitable for therapy comprising an anti-C5 antibody of the present invention . In certain embodiments, the method comprises: (a) detecting a genetic change in C5 derived from an individual, and (b) when a genetic change is detected in C5 derived from the individual, selecting the individual as a A therapy comprising an anti-C5 antibody of the invention is suitable. In another embodiment, the present invention provides a method of selecting a therapy for an individual suffering from a complement-mediated disease or disorder involving excessive or uncontrolled C5 activation. In certain embodiments, the method comprises: (a) detecting a genetic change in C5 derived from an individual, and (b) when a genetic change is detected in C5 derived from the individual, selecting a C5 comprising the present invention Anti-C5 antibody therapy is used for the individual.

In another embodiment, the present invention provides a method of treating an individual suffering from a complement-mediated disease or a disorder involving excessive or uncontrolled C5 activation. In certain embodiments, the method comprises: (a) detecting a genetic change in C5 derived from an individual, (b) when a genetic change is detected in C5 derived from the individual, selecting the individual as a suitable in a therapy comprising an anti-C5 antibody of the invention, and (c) administering an anti-C5 antibody of the invention to the individual.

In another embodiment, the invention provides an anti-C5 antibody of the invention for use in the treatment of an individual suffering from a complement-mediated disease or a disorder involving excessive or uncontrolled C5 activation. In certain embodiments, the individual is treated with an anti-C5 antibody of the invention when a genetic change is detected in C5 derived from the individual.

In another embodiment, the present invention provides the in vitro use of genetic alterations in C5 for selection as suitable for therapy comprising an anti-C5 antibody of the present invention, suffering from a complement-mediated disease or involving excessive or inadequate Individuals with controlled C5 activation disorders. In certain embodiments, the individual is selected as suitable for the therapy when a genetic change is detected in C5 derived from the individual. In another embodiment, the present invention provides the in vitro use of genetic alterations in C5 for the selection of therapy for an individual suffering from a complement-mediated disease or a disorder involving excessive or uncontrolled C5 activation disease. In certain embodiments, a therapy comprising an anti-C5 antibody of the invention is selected for use in said individual when a genetic change is detected in C5 derived from said individual.

It has been reported that some patients with genetic changes in C5 show poor responses to therapies comprising existing anti-C5 antibodies (Nishimura et al., N. Engl. J. Med. 370:632-639 (2014)). It is recommended that such patients be treated with a therapy comprising the anti-C5 antibodies of the invention, as such antibodies have inhibitory activity against both C5 variants as well as activation of wild-type C5, as demonstrated in the examples below.

Genetic changes in C5 can be detected by using methods known in the art. Such methods may include, but are not limited to, sequencing, PCR, RT-PCR, and hybridization-based methods such as southern blotting or northern blotting. A C5 variant may contain at least one genetic change. The genetic change can be selected from the group consisting of V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N and E1437D. Here, for example, R885H means a genetic change in which the arginine at position 885 is replaced by histidine. In certain embodiments, the C5 variant has biological activity similar to wild-type C5.

Exemplary disorders that can be diagnosed using the antibodies of the invention include: rheumatoid arthritis (RA); systemic lupus erythematosus (SLE); lupus nephritis; ischemia-reperfusion injury (IRI); asthma; paroxysmal nocturnal hemoglobin Urine (PNH); hemolytic uremic syndrome (HUS) (eg, atypical hemolytic uremic syndrome (aHUS)); dense deposit disease (DDD); neuromyelitis optica (NMO); multifocal motor neuropathy (MMN); multiple sclerosis (MS); systemic sclerosis; macular degeneration (eg, age-related macular degeneration (AMD)); hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome; thrombotic thrombocytopenia TTP; spontaneous abortion; epidermolysis bullosa; recurrent abortion; pre-eclampsia; traumatic brain injury; myasthenia gravis; cold agglutinin disease; Sjogren's syndrome Dermatomyositis; Bullous Pemphigoid; Phototoxicity; Shiga toxin E. coli-related hemolytic uremic syndrome; Typical or Infectious Hemolytic uremic syndrome (typical or infectious hemolytic uremic syndrome (tHUS)); C3 glomerulonephritis; anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis; humoral and vascular graft rejection; acute antibody mediated rejection (AMR); graft dysfunction; myocardial infarction; allograft; sepsis; coronary artery disease; hereditary angioedema; dermatomyositis; Graves' disease; atherosclerosis Sclerosis; Alzheimer's disease (AD); Huntington's disease; Creutzfeld-Jacob disease; Parkinson's disease; cancer; trauma; septic shock shock); spinal cord injury; uveitis; diabetic eye disease; retinopathy of prematurity; glomerulonephritis; membranous nephritis; immunoglobulin A nephropathy; adult respiratory distress syndrome (ARDS); chronic obstructive pulmonary disease (COPD); Cystic fibrosis; hemolytic anemia; paroxysmal cold hemoglobinuria; anaphylactic shock; allergy; osteoporosis; osteoarthritis; Hashimoto's thyroiditis; type 1 diabetes; psoriasis; pemphigus sores; autoimmune hemolytic anemia (AIHA); idiopathic thrombocytopenic purpura (ITP); Goodpasture syndrome; Degos disease s disease); antiphospholipid syndrome (APS); catastrophic APS (CAPS)); cardiovascular disease; myocarditis; cerebrovascular disorders; peripheral vascular disease; renal vascular disease; mesenteric/intestinal vascular disease; vasculitis; Henoch-Schonlein purpura nephritis; Takayasu's disease; Dilated cardiomyopathy; Diabetic vascular disease; Kawasaki's disease (arteritis); Venous gas embolism (VGE), stent placement posterior restenosis; rotational atherectomy; membranous nephropathy; Guillain-Barre syndrome (GBS); Fisher syndrome; antigen-induced arthritis; Membrane inflammation; viral infection; bacterial infection; fungal infection; and injury from myocardial infarction, cardiopulmonary bypass, and hemodialysis.

In certain embodiments, labeled anti-C5 antibodies are provided. Labels include, but are not limited to, labels or moieties that are directly detected (such as fluorescent, chromophore, electron density, chemiluminescent, and radioactive labels), and moieties that are indirectly detected such as enzymes or ligands, eg, by enzymatic reactions or molecular interactions . Exemplary labels include, but are not limited to, radioisotopes32P, ^14C , ^125I , ^{3H , and131I ,} fluorophores such as rare earth chelates or fluorescein and derivatives thereof, rhodamine and derivatives thereof, dansyl, Umbelliferone, luciferase, eg, firefly luciferase and bacterial luciferase (US Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP) , alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, sugar oxidase, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic Oxidases such as uricase and xanthine oxidase, coupled with hydrogen peroxide-utilizing enzymes to oxidize dye precursors such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, Spin tags, phage tags, stabilized free radicals, etc.

F. Pharmaceutical Preparations

Pharmaceutical formulations of anti-C5 antibodies as described herein are prepared by combining the antibody with the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th Edition, Osol, A . Ed. (1980)) and prepared in the form of lyophilized preparations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations used, and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine Acids; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethylbisammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens acid esters such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) ) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, aspartic acid, histidine, arginine, or lysine Amino acids; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metals complexes (eg, Zn-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutical carriers herein also include interstitial drug dispersants such as soluble neutrally active hyaluronidase glycoprotein (sHASEGP), eg, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX (registered) trademark), Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanase enzymes 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 WO 2006/044908, the latter formulations comprising histidine-acetic acid buffer.

When desired, the formulations herein may also contain more than one active ingredient for a particular indication for treatment, preferably those having complementary activities that do not adversely affect each other. Such active ingredients are suitably present in the combination in an amount effective for the intended use.

The active ingredient can be encapsulated, for example, in microcapsules prepared by coacervation techniques or by interfacial polymerization such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems ( For example, in liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th Edition, Osol, A. Ed. (1980).

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies in the form of tangible articles, eg, films or microcapsules.

Formulations useful for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

G. METHODS AND COMPOSITIONS OF TREATMENT

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

In one aspect, an anti-C5 antibody for use as a medicament is provided. In a further aspect, anti-C5 antibodies are provided for use in the treatment of complement-mediated diseases or disorders involving excessive or uncontrolled C5 activation. In certain embodiments, anti-C5 antibodies for use in methods of treatment are provided. In certain embodiments, the invention provides anti-C5 antibodies for use in a method of treating an individual having a complement-mediated disease or a disorder involving excessive or uncontrolled C5 activation, the method comprising administering to the individual An effective amount of anti-C5 antibody is administered. 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, binding of the antibody to its antigen can result in prolonged half-life of the antigen in plasma (ie, reduced clearance of the antigen from the plasma) because the antibody itself has a longer half-life in plasma and acts as a carrier for the antigen . This is due to the recycling of antigen-antibody complexes by FcRn via the endosomal pathway in cells (Roopenian, Nat. Rev. Immunol. 7(9):715-725 (2007)). However, antibodies with pH-dependent binding properties (which bind their antigen in a neutral extracellular environment and release the antigen into the acidic endosomal compartment upon entry into the cell) are expected to be relatively relatively neutral in antigen neutralization and clearance Better properties for their counterparts that bind in a pH-independent manner (Igawa et al., Nat. Biotech.. 28(11): 1203-1207 (2010); Devanaboyina et al., mAbs5(6): 851-859 ( 2013); WO2009/125825).

In another embodiment, the present invention provides anti-C5 antibodies for enhancing the clearance of C5 from plasma. In certain embodiments, the present invention provides an anti-C5 antibody for use in a method of enhancing the clearance of C5 from plasma in an individual, the method comprising administering to the individual an effective amount of an anti-C5 antibody to enhance the clearance of C5 from plasma Clear. In one embodiment, the anti-C5 antibody enhances the clearance of C5 from plasma compared to conventional anti-C5 antibodies that do not have pH-dependent binding characteristics. An "individual" according to any of the above embodiments is preferably a human.

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

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

In another aspect, the present invention provides the use of an anti-C5 antibody in the manufacture or formulation of a medicament. In one embodiment, the medicament is for the treatment of complement-mediated diseases or disorders involving excessive or uncontrolled C5 activation. In another embodiment, the medicament is for use in a method of treating a complement-mediated disease or a disorder involving excessive or uncontrolled C5 activation, the method comprising treating patients with a complement-mediated disease or involving excessive C5 activation or uncontrolled C5 activation disorder is administered an effective amount of the drug. 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 another embodiment, the medicament is used to enhance the clearance of C5 from plasma. In another embodiment, the medicament is used in a method of enhancing the clearance of C5 from plasma in an individual, the method comprising administering to the individual an effective amount of the medicament to enhance the clearance of C5 from plasma. In one embodiment, the anti-C5 antibody enhances the clearance of C5 from plasma compared to conventional anti-C5 antibodies that do not have pH-dependent binding characteristics. An "individual" according to any of the above embodiments may be a human.

In another embodiment, the medicament is used to inhibit the accumulation of C5 in plasma. In another embodiment, the medicament is for use in a method of inhibiting the accumulation of C5 in plasma in an individual, the method comprising administering to the individual an effective amount of a medicament to inhibit the accumulation of C5 in plasma. In one embodiment, the accumulation of C5 in plasma is the result of the formation of antigen-antibody complexes. In another embodiment, the anti-C5 antibody inhibits the accumulation of C5 in plasma compared to conventional anti-C5 antibodies that do not have pH-dependent binding characteristics. An "individual" according to any of the above embodiments may be a human.

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

In another aspect, the present invention provides methods for treating complement-mediated diseases or disorders involving excessive or uncontrolled C5 activation. In one embodiment, the method comprises administering an effective amount of an anti-C5 antibody to an individual suffering from the complement-mediated disease or disorder involving excessive or uncontrolled C5 activation. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. An "individual" according to any of the above embodiments may be a human.

In another aspect, the present invention provides methods for enhancing the clearance of C5 from plasma in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-C5 antibody to enhance clearance of C5 from plasma. In one embodiment, the anti-C5 antibody enhances the clearance of C5 from plasma compared to conventional anti-C5 antibodies that do not have pH-dependent binding characteristics. In one embodiment, an "individual" is a human.

In another aspect, the present invention provides methods for inhibiting the accumulation of C5 in plasma in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-C5 antibody to inhibit the accumulation of C5 in plasma. In one embodiment, the accumulation of C5 in plasma is the result of the formation of antigen-antibody complexes. In another embodiment, the anti-C5 antibody inhibits the accumulation of C5 in plasma compared to conventional anti-C5 antibodies that do not have pH-dependent binding characteristics. In one embodiment, an "individual" is a human.

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

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

In another aspect, the pharmaceutical formulation is for use in the treatment of complement-mediated diseases or disorders involving excessive or uncontrolled C5 activation. In another embodiment, the pharmaceutical formulation is used to enhance the clearance of C5 from plasma. In one embodiment, the anti-C5 antibody enhances the clearance of C5 from plasma compared to conventional anti-C5 antibodies that do not have pH-dependent binding characteristics. In another embodiment, the pharmaceutical formulation is used to inhibit the accumulation of C5 in plasma. In one embodiment, the accumulation of C5 in plasma is the result of the formation of antigen-antibody complexes. In another embodiment, the anti-C5 antibody inhibits the accumulation of C5 in plasma compared to conventional anti-C5 antibodies that do not have pH-dependent binding characteristics. The anti-C5 antibody of the present invention can inhibit the activation of C5. In another embodiment, the pharmaceutical formulation is used to inhibit the activation of C5. In one embodiment, C5-mediated cytotoxicity is inhibited by inhibiting the activation of C5. In another embodiment, the pharmaceutical formulation is administered to an individual suffering from a complement-mediated disease or involving excessive or uncontrolled C5 activation. 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 biological activity similar to wild-type C5. Such a C5 variant may comprise at least one variation selected from the group consisting of V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N, and E1437D. Here, for example, R885H means a genetic change in which the arginine at position 885 is replaced by histidine.

In another aspect, the present invention provides a method for preparing a medicament or pharmaceutical formulation, the method comprising admixing any of the anti-C5 antibodies provided herein with a pharmaceutically acceptable carrier, eg, for use in any of the above methods of treatment. In one embodiment, the method for preparing a medicament or pharmaceutical formulation further comprises adding at least one additional therapeutic agent to the medicament or pharmaceutical formulation.

In certain embodiments, the complement-mediated disease or disorder involving excessive or uncontrolled C5 activation 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 Deposition disease (DDD); neuromyelitis optica (NMO); multifocal motor neuropathy (MMN); multiple sclerosis (MS); systemic sclerosis; macular degeneration (eg, age-related macular degeneration (AMD)); hemolysis , elevated liver enzymes, and low platelet (HELLP) syndrome; thrombotic thrombocytopenic purpura (TTP); spontaneous abortion; epidermolysis bullosa; recurrent abortion; preeclampsia; traumatic brain injury; Myasthenia gravis; cold agglutinin disease; Sjogren's syndrome; dermatomyositis; bullous pemphigoid; phototoxicity; Shiga toxin E. coli-associated hemolytic uremic syndrome; typical or infectious hemolytic Uremic syndrome (tHUS); C3 glomerulonephritis; anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis; humoral and vascular graft rejection; acute antibody-mediated rejection (AMR); graft function Disorders; Myocardial Infarction; Allograft; Sepsis; Coronary Artery Disease; Hereditary Angioedema; Dermatomyositis; Graves' Disease; Atherosclerosis; Alzheimer's Disease (AD); Huntington's Disease; CJJ Second disease; Parkinson's disease; cancer; trauma; septic shock; spinal cord injury; uveitis; diabetic eye disease; retinopathy of prematurity; glomerulonephritis; membranous nephritis; immunoglobulin A nephropathy; adult respiratory distress syndrome Chronic Obstructive Pulmonary Disease (COPD); Cystic Fibrosis; Hemolytic Anemia; Paroxysmal Cold Hemoglobinuria; Anaphylactic Shock; Allergy; Osteoporosis; Osteoarthritis; Hashimoto's Thyroiditis; I type diabetes; psoriasis; pemphigus; autoimmune hemolytic anemia (AIHA); idiopathic thrombocytopenic purpura (ITP); APS); catastrophic APS (CAPS); cardiovascular disease; myocarditis; cerebrovascular disorders; peripheral vascular disease; renal vascular disease; mesenteric/intestinal vascular disease; vasculitis; ; diabetic vascular disease; Kawasaki disease (arteritis); venous gas embolism (VGE), restenosis after stent placement; rotational atherectomy; membranous nephropathy; Guillain-Barré syndrome (GBS); Fisher syndrome ; antigen-induced arthritis; synovial inflammation; viral infections; bacterial infections; fungal infections; and injuries from myocardial infarction, cardiopulmonary bypass, and hemodialysis.

In certain embodiments, the complement-mediated disease or disorder is an ocular disease or disorder. In another embodiment, the ocular disorder is macular degeneration. In another embodiment, the macular degeneration is AMD. In another embodiment, the AMD is a dry form of AMD.

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

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

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

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

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

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

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

Such combination therapy described above includes combined administration (wherein two or more therapeutic agents are contained in the same or separate formulations) and separate administration, in which case administration of the antibody of the invention may occur with the additional therapeutic agent or Before, at the same time, and/or after administration of the agent. In one embodiment, the administration of the anti-C5 antibody and the administration of the additional therapeutic agent occur within about one month of each other, or within about one week, two weeks or three weeks, or within about one day, two days, three days, Within four, five or six days.

The antibodies of the invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular administration, intravenous administration, intraarterial administration, intraperitoneal administration, or subcutaneous administration. Administration may be by any suitable route, eg, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is brief or chronic. Various dosing regimens are contemplated herein, including, but not limited to, a single administration or multiple administrations at multiple time points, bolus administration, and pulsatile infusion.

Antibodies of the invention can be formulated, administered and administered in a manner consistent with good medical practice. Factors considered in this context include the specific disease being treated, the specific mammal being treated, the individual patient's clinical condition, etiology, site of drug delivery, method of administration, timing of administration, and other factors known to medical practitioners . The antibody need not, but optionally, be formulated with one or more agents currently used to prevent or treat the disease of interest. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disease or treatment, and other factors discussed above. These are generally used at the same dose and route of administration as described herein, or at about 1 to 99% of the dose described herein, or at any dose and any route as determined empirically/clinically as appropriate.

For the prevention or treatment of disease, the appropriate dosage of an antibody of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity of the disease degree and course of disease, whether the antibody is being administered for prophylactic or therapeutic purposes, previous treatment, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitably administered to the patient at one time or in a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (eg, 0.1 mg/kg-10 mg/kg) of antibody may be an initial candidate dose for administration to a patient, whether, for example, via a single or Multiple separate administrations, again by continuous infusion. A typical daily dose may range from about 1 μg/kg to over 100 mg/kg, depending on the factors mentioned above. For repeated administration over a period of several days or longer, depending on the condition, treatment can generally be continued until the desired suppression of disease symptoms occurs. An exemplary dose of the antibody is from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, eg, weekly or every three weeks (eg, such that the patient receives from about two to about twenty, or eg, about six doses of the antibody). An initial higher loading dose can be administered, followed by one or more lower doses. The course of treatment can be readily monitored by conventional techniques and assays.

It is to be understood that any of the above formulations or methods of treatment can be performed using the immunoconjugates of the invention (in place of or in addition to anti-C5 antibodies).

H. Products

In another aspect of the present invention, there is provided an article of manufacture comprising materials useful in the treatment, prevention and/or diagnosis of the aforementioned diseases. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container can be made of a variety of materials such as glass or plastic. The container holds a composition, alone or in combination with another composition effective for treating, preventing and/or diagnosing a condition, and the container may have a sterile access port (eg, the container This can be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is used to treat the condition of choice. Furthermore, the article of manufacture may comprise (a) a first container containing a composition therein, wherein the composition comprises an antibody of the invention; and (b) a second container containing a composition therein, wherein the composition Cytotoxic or other therapeutic agents are also included. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and Dextrose solution. It may also contain other materials required by commercial or user needs, including other buffers, diluents, fillers, needles and syringes.

It is to be understood that any of the above preparations can include an immunoconjugate of the invention in place of, or in addition to, an anti-C5 antibody, an immunoconjugate of the invention.

Example

The following are examples of methods and compositions of the present invention. It is to be understood that various other embodiments may be practiced given the general description provided above.

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 expressed using the FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA). Conditioned medium expressing human C5 was diluted with an equal volume of milliQ water and applied to Q-sepharose FF or Q-sepharose HP anion exchange columns (GEhealthcare, Uppsala, Sweden), followed by NaCl gradient elution. Fractions containing human C5 were pooled and then the salt concentration and pH were adjusted to 80 mM NaCl and pH 6.4, respectively. The resulting samples were applied to SP-sepharose HP cation exchange columns (GEhealthcare, Uppsala, Sweden) and eluted with a NaCl gradient. Fractions containing human C5 were pooled and passed through a CHT ceramic hydroxyapatite column (Bio-Rad Laboratories, Hercules, CA, USA). The human C5 eluate was then applied to a Superdex200 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 monkey C5 (cynoC5) from plasma

Plasma samples from cynomolgus monkeys were applied to SSL7-agarose (Invivogen, San Diego, CA, USA) before elution with 100 mM sodium acetate pH 3.5. Fractions containing cynoC5 were immediately neutralized and passed through a Protein A HP column (GE healthcare, Uppsala, Sweden) with Peptide M Sepharose (Invivogen, San Diego, CA, USA). The flow through fractions were 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

Generation of anti-C5 antibodies

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). Repeat this dose 4-5 times over a 2-month period. One week after the final immunization, spleens and blood were collected from the immunized rabbits. Antigen-specific B-cells were stained with labeled antigen, sorted with an FCM cell sorter (FACS aria III, BD), and mixed with EL4 cells (European Collection of 25,000 cells/well at a density of one cell/well). Cell Cultures) were plated in 96-well plates with 20-fold diluted activated rabbit T-cell conditioned medium and cultured for 7-12 days. EL4 cells were treated with mitomycin C (Sigma, cat. no. M4287) for 2 hours and washed 3 times in advance. By culturing in RPMI-1640 containing lectin-M (Roche, cat. no. 1 1082132-001), phorbol 12-myristate 13-acetate (Sigma, cat. no. P1585) and 2% FBS Rabbit thymocytes were used to prepare activated rabbit T-cell conditioned medium. After incubation, B-cell culture supernatants were collected for further analysis and cell pellets were cryopreserved.

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

An ELISA assay was used to assess pH-dependent binding of antibodies to C5. Goat anti-rabbit IgG-Fc (BETHYL, cat. no. A120-111A) diluted to 1 μg/ml in PBS(-) was added to 384-well MAXISorp (Nunc, cat. no. 164688), incubated for 1 hour at room temperature, and diluted with 5 Blocking One (Nacalai Tesque, cat. no. 03953-95) closed at times. After incubation, plates were washed and B-cell culture supernatants were added. Plates were incubated for 1 hour, washed, and 500 pM of biotinylated human or monkey C5 was added and incubated for 1 hour. After incubation, plates were washed and incubated with pH 7.4 MES buffer (20 mM MES, 150 mM NaCl and 1.2 mM _CaCl2 ) or pH 5.8 MES buffer (20 mM MES, 150 mM NaCl and 1 mM EDTA) for 1 hour at room temperature. After incubation, biotinylated C5 was detected by streptavidin-horseradish peroxidase conjugate (Thermo Scientific, cat. no. 21132) followed by ABTS (KPL, cat. no. 50-66-06) combination.

The Octet RED384 system (Pall Life Sciences) was used to assess antibody affinity and pH-dependent binding to C5. Antibodies secreted in B-cell culture supernatants were loaded onto Protein A biosensor tips (Pall LifeSciences) and immersed in 50 nM human or monkey C5 in pH 7.4 MES buffer to analyze binding kinetics. Dissociation kinetics were analyzed in pH 7.4 MES buffer and pH 5.8 MES buffer.

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

2.2. Epitope boxing by sandwich ELISA

Anti-C5 antibodies with high affinity, pH-dependent or neutralizing activity were selected for further analysis. A sandwich ELISA assay was used to group selected antibodies into distinct epitope boxes that bind the same or overlapping epitopes of the C5 protein. Unlabeled capture antibody was diluted to 1 μg/ml with PBS(-) and added to a 384-well MAXISorp plate (Nunc, cat. no. 164688). Plates were incubated for 1 hour at room temperature and blocked with a 5-fold dilution of Blocking One (Nacalai Tesque, cat. no. 03953-95). Plates were incubated for 1 hour, washed, and 2 nM of human C5 was added and incubated for 1 hour. After incubation, plates were washed and labeled detection antibody (1 μg/mL, biotinylated with NHS-PEG4-biotin) was added. After 1 hour incubation, biotinylation was detected by streptavidin-horseradish peroxidase conjugate (Thermo Scientific, cat. no. 21132) followed by ABTS (KPL, cat. no. 50-66-06) binding of the antibody.

All anti-C5 antibodies were used as both capture and detection antibodies and were fully paired. As shown in Figure 1, competing antibodies were grouped into 7 epitope boxes: CFA0668, CFA0334 and CFA0319 were grouped into epitope A, CFA0647, CFA0589, CFA0341, CFA0639, CFA0635, CFA0330 and CFA0318 were grouped into epitope B, CFA0538, CFA0501, CFA0599, CFA0307, CFA0366, CFA0305, CFA0675, CFA0666 and CFA0672 into epitope C, eculizumab and CFA0322 into epitope D, and CFA0329 into epitope C In epitope E, CFA0359 and CFA0217 were grouped into epitope F, and CFA0579, CFA0328 and CFA0272 were grouped into epitope G. Figure 1 shows the epitope binning of some anti-C5 chimeric antibodies. The VH and VL sequences of anti-C5 antibodies grouped into epitope C are listed in Table 2.

[Table 2]

Anti-C5 antibodies grouped into epitope C

2.3. Humanization and optimization

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

Several mutations and combinations of mutations were tested to identify mutations and combinations of mutations that improved the binding properties of some of the lead antibodies. Multiple mutations were then introduced into the humanized variable regions to enhance the binding affinity for C5 at neutral pH or decrease the binding affinity for C5 at acidic pH. An optimized variant was thus generated from CFA0305, 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).

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

Example 3

Binding characterization of anti-C5 antibodies

3.1. Expression and purification of recombinant antibodies

Recombinant antibodies were transiently expressed using the FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA). Purification from conditioned media expressing antibodies was performed using protein A using conventional methods. If necessary, further gel filtration was performed.

3.2. Assessing pH dependence

Kinetic parameters of anti-C5 antibodies against recombinant human C5 were evaluated at pH 7.4 and pH 5.8 using a BIACORE (registered trademark) T200 instrument (GE Healthcare) at 37°C. 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 into corresponding running buffers ACES pH 7.4 and pH 5.8 (20 mM ACES, 150 mM NaCl, 1.2 mM _CaCl2 , 0.05% Tween20, 0.005% NaN3 ₎ . Each antibody was captured on the sensor surface by ProA/G. Antibody capture levels are typically 60-90 response units (RU). Then, 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 the two pH conditions were determined by fitting sensorgrams using a 1:1 binding model using BIACORE (registered trademark) T200 Evaluation software, version 2.0 (GE Healthcare). Sensorgrams of all antibodies are shown in Figures 2A and 2B. The on-rate (ka), off-rate (kd) and binding affinity (KD) of the antibodies are listed in Table 3. Dissociation at pH 5.8 exhibited by 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) The rates are relatively faster than at pH 7.4.

[table 3]

Kinetic parameters of anti-C5 antibody at pH 7.4 and pH 5.8

3.3. Cross-reactivity check

In order to observe the cross-reactivity of anti-C5 antibodies to human C5 (hC5) and cynomolgus monkey C5 (cynoC5), BIACORE (registered trademark) kinetic analysis was performed. The assay setup was as described in Example 3.2, recombinant cynoC5 was injected at concentrations of 2, 10 and 50 nM. Kinetic parameters were determined by the same data fit as described in Example 3.2. The binding kinetics and affinities at pH 7.4 are listed in Table 4. The kinetic parameters for hC5 given in Table 4 are the results of Example 3.2. All anti-C5 antibodies except CFA0672 showed comparable KDs for hC5 and cynoC5. The KD of CFA0672 for cynoC5 is 8-fold weaker than that for hC5.

[Table 4]

Binding kinetics and affinity of anti-C5 antibody to hC5 and cynoC5 at pH 7.4

Example 4

Epitope Mapping of Anti-C5 Antibodies

4.1. Binding of anti-C5 MAbs to C5 beta-chain derived peptides

Anti-C5 monoclonal antibodies (MAbs) were tested for binding to C5 beta-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 in E. coli (DH5α, TOYOBO , DNA-903) expression. E. coli samples were harvested after incubation with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 5 hours at 37° C. and centrifuged at 20000×g for 1 min to obtain the pellet. The pellet was suspended with sample buffer solution (2ME+) (Wako, 191-13272) and used for Western blot analysis. The expression of each peptide was confirmed using an anti-GST antibody (Abeam, ab9085) (Fig. 3). Arrows indicate C5 peptide (46-49 kDa) fused to GST. Anti-C5 MAbs: CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675, bind 19-180 of C5 (Figure 3).

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

The recombinant MG1-MG2 domain of human C5 beta-chain (SEQ ID NO: 43) was transiently expressed using the FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA). Conditioned medium expressing MG1-MG2 domains was diluted with 1/2 volume of milliQ water before being 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 and applied to a SP-sepharose HP cation exchange column (GE healthcare, Uppsala, Sweden) and eluted with a NaCl gradient. Fractions containing MG1-MG2 domains were collected from the eluate and then passed through a Superdex 75 gel filtration column (GE healthcare, Uppsala, Sweden) equilibrated with 1x PBS. Fractions containing MG1-MG2 domains were then pooled and stored at -80°C.

4.3. Ability to bind to MG1-MG2 domains

The binding capacity of anti-C5 antibodies to the MG1-MG2 domain was measured using the same assay setup as described in Example 3.2, except that the measurement was performed only at pH 7.4. The MG1-MG2 domains were injected at concentrations of 20 nM and 40 nM. As shown in Figure 4, all antibodies except eculizumab-F760G4 showed increased binding responses, indicating that these antibodies are MG1-MG2 binders. Eculizumab-F760G4, a known alpha-chain binder, did not show binding to the MG1-MG2 domains.

4.4. Binding of anti-C5MAbs to C5MG1-MG2 domain-derived peptides

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

4.5. Binding of anti-C5 MAbs to C5 mutants

Because three amino acid residues in the C5 beta-chain, E48, D51 and K109, were predicted by crystal structure analysis to be involved in the binding between C5 and anti-C5-MAb, the anti-C5 MAb was tested in Western blot analysis with human C5 dots Binding of mutants. A C5 point mutant in which any of E48, D51 and K109 was substituted with alanine was expressed in FS293 cells by lipofection. Medium was harvested 5 days after lipofection before being used for Western blotting. SDS-PAGE was performed under reducing conditions. The results are shown in Figure 6. Eculizumab binds to the α-chain of wild-type (WT) C5 and the three C5 point mutants, whereas CFA0305 binds strongly to the β-chain of WT C5, binds weakly to the β-chain of the E48A C5 mutant, and does not bind to the β-chain of the E48A C5 mutant. Binding to the β-chain of the D51A and K109A C5 mutants indicated that these 3 amino acid residues are involved in the antibody/antigen interaction. Table 5 gives an overview of western blot analysis of anti-C5 MAbs (CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672 and CFA0675). The anti-C5 MAbs were grouped into the same epitope C, but the binding patterns differed slightly between the antibodies, indicating that the binding regions of C5 for anti-C5 MAbs were not identical to each other adjacent to each other.

[table 5]

Overview of the response of anti-C5 MAbs to C5 mutants

4.6. BIACORE (registered trademark) binding analysis of anti-C5 antibody and C5 mutant

To test whether residues E48, G51 and K109 are actually involved in antibody/antigen interactions, a BIACORE (registered trademark) binding assay was performed. Three C5 mutants were prepared: E48A, G51A and K109A, as described in Example 4.5. A culture supernatant sample was prepared containing mutant C5 overexpressed in FS293 cells at 40 μg/ml. For BIACORE (registered trademark) binding assay, samples were diluted 10x with BIACORE (registered trademark) running buffer (ACES pH7.4, 10 mg/ml BSA, 1 mg/ml carboxymethyl dextran) to the final sample of mutant C5 The concentration was 4 μg/ml.

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

As shown in Figure 7, all three C5 mutants were able to bind eculizumab with similar binding characteristics compared to wild-type C5. For 305LO5, all three mutants showed lower binding responses to 305LO5 compared to wild-type C5. The D51A and K109A mutants attenuated 305LO5 binding to C5 to baseline levels.

4.7. Identification of His residues on C5 that contribute to pH-dependent interactions between anti-C5 antibodies and C5

Crystal structure analysis revealed that three histidine residues on human C5 are located at the antibody/antigen interface. Histidine residues with a typical pKa of about 6.0 are known to contribute to pH-dependent protein-protein interactions (Igawa et al., Biochim Biophys Acta 1844(11):1943-1950 (2014)). To investigate which His residue on the antibody/antigen interface contributes to the pH-dependent interaction between the anti-C5 antibody and C5, a BIACORE (registered trademark) binding assay was performed. Three human C5 mutants with single His mutations (H70Y, H72Y and H110Y) and mutants with double His mutations (H70Y+H110Y) were prepared by lipofection by replacing any of H70, H72 and H110 with A single His mutant of tyrosine and a double His mutant with both H70 and H110 replaced by tyrosine were expressed in FS293 cells. The antigen binding properties of the C5His mutant to 305LO5 (pH-dependent anti-C5 antibody) were determined by a modified BIACORE (registered trademark) assay, as described in Example 4.6. Briefly, an additional dissociation phase at pH 5.8 was incorporated into the BIACORE (registered trademark) assay, immediately following the dissociation phase at pH 7.4, to assess the efflux from complexes formed at pH 7.4. pH-dependent dissociation between antibody and antigen. The dissociation rate at pH 5.8 was determined by processing and fitting the data using Scrubber 2.0 (BioLogic Software) curve fitting software.

As shown in Figure 8, C5 single-His mutation and double-His mutation (H70+H110) at H70 or H110 did not affect the binding of C5 to 305LO5 at neutral pH. Meanwhile, a single His mutation at H72 showed a marked impairment of C5 binding to 305LO5. The dissociation rates of the C5His mutant and C5-wt protein at pH 5.8 are shown in Table 6. As shown in Table 6, of the C5 antigens tested, at pH 5.8, C5-wt showed the fastest dissociation from 305LO5. Compared to C5-wt, the single-His mutation at H70 showed an almost twice slower dissociation rate at pH 5.8 and the single-His mutation at H110 resulted in a slightly slower dissociation rate at pH 5.8. Double His mutation at H70 and H110 resulted in a larger effect on pH-dependent binding, with the dissociation rate at pH 5.8 almost three times slower than that of C5-wt.

[Table 6]

pH 5.8 off-rate value of C5 His mutant bound to 305LO5

Example 5

Inhibitory activity of anti-C5 antibodies on C5 activation

5.1. Anti-C5 MAb inhibits complement-activated liposome lysis

Anti-C5 MAbs were tested for inhibition of complement activity by a lipolysis 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 at 25°C for 30 min on a shaker. Liposomes sensitized with an antibody against dinitrophenyl (Autokit CH50, Wako, 995-40801) were transferred to each well and the plate was placed on a shaker for 2 min at 25°C. 50 μL of substrate solution (Autokit CH50) was added to each well and mixed with shaking for 2 min at 25°C. The final mixture was incubated at 37°C for 40 minutes, after which the OD of the mixture at 340 nm was measured. The percentage of liposome solubilization was defined as 100×[(OD _MAb -OD _{serum and liposome background} )]/[(OD _MAb-free- OD _{serum and liposome background} )]. Figure 9A shows that anti-C5 Mabs: CFA0305, 0307, 0366, 0501, 0538, 0599, 0666, 0672, and 0675, inhibit liposome lysis. Two pH-independent antibodies, CFA0330 and 0341, also inhibited lysis (Figure 9B).

5.2. Anti-C5MAb inhibits C5a production

The anti-C5 MAb was tested for C5a production during lipolysis to confirm that the anti-C5 MAb inhibits the cleavage of C5 into C5a and C5b. C5a levels in supernatants from liposome lysis assays were quantified using a C5a ELISA kit (R&D systems, DY2037). All MAbs inhibited C5a production in the supernatant in a dose-dependent manner (Figures 10A and 10B).

5.3. Anti-C5 MAbs inhibit complement-activated hemolysis

Anti-C5MAbs were tested for inhibition of classical complement activity in a hemolytic assay. Chicken red blood cells (cRBC) (Innovative research, IC05-0810) were washed with gelatin/barbital buffered saline (Boston BioProducts, IBB-300X) containing 0.5 mM MgCl ₂ and 0.15 mM CaCl ₂ (GVB++) followed by 1 μg 1/ml of anti-chicken RBC antibody (Rockland 103-4139) was sensitized at 4°C for 15 minutes. Cells were then washed with GVB++ and suspended in the same buffer at ^5x107 cells/ml. In a separate round bottom 96-well microtest plate, 50 μL of normal human serum (20%) (Biopredic, SER019) was mixed with 50 μL of the diluted Mab and incubated for 30 min at 37°C on a shaker. 60 μL of the sensitized cRBC suspension was then added to the serum-containing wells and the antibody mixture was incubated for 30 min at 37°C. After incubation, the plates were centrifuged at 1000xg for 2 minutes at 4°C. The supernatant (100 μL) was transferred to the wells of a flat-bottomed 96-well microtest plate for measurement of OD at 415 nm with a reference wavelength of 630 nm. Percent hemolysis was defined as 100×[(OD _MAb -OD _{serum and cRBC} )]/[(OD _MAb-free- OD _{serum and cRBC background} )]. Figure 11 shows that anti-C5 Mabs: CFA0305 and 305LO5, inhibit hemolysis of cRBCs.

5.4. Inhibition of the alternative complement pathway by anti-C5MAbs

The hemolysis assay for the alternative pathway was performed in a similar manner to the classical pathway hemolysis assay. Blood collected from New Zealand white rabbits (InVivos) was mixed with the same volume of Alsever solution (Sigma, A3551), and the mixture was used as rabbit RBC (rRBC). rRBCs were washed with GVB supplemented with 2 mM MgCl ₂ and 10 mM EGTA and suspended in the same buffer at 7x10 ⁸ cells/ml. In a round bottom 96-well microtest plate, 40 [mu]L of normal human serum (25%) (Biopredic, SER019) was mixed with 40 [mu]L of the diluted Mab and incubated on a shaker for 30 minutes at 37[deg.]C. 20 μL of the rRBC suspension was then added to the serum-containing wells and the antibody mixture was incubated for 60 minutes at 37°C. After incubation, the plates were centrifuged at 1000xg for 2 minutes at 4°C. The supernatant (70 μL) was transferred to wells in a flat-bottomed 96-well micro-assay plate for measurement of OD at 415 nm with a reference wavelength of 630 nm. Figure 12 shows that anti-C5 Mabs: CFA0305 and CFA0672, inhibit hemolysis of rRBCs, indicating that these antibodies inhibit the alternative complement pathway.

Example 6

Pharmacokinetic Study of Anti-C5 Monoclonal Antibody and Human C5 in Mice

6.1. In vivo tests using C57BL/6 mice

The in vivo kinetics of human C5 (Calbiochem) and anti-human C5 antibodies were assessed following administration of human C5 alone or human C5 with anti-human C5 antibody to C57BL/6 mice (In Vivos or Biological Resource Centre, Singapore). Human C5 solution (0.01 mg/ml) or containing human C5 and anti-human C5 antibodies (0.01 mg/ml and 2 mg/ml, respectively (CFA0305-F760G4, CFA0307-F760G4, CFA0366-F760G4, CFA0501-F760G4, CFA0538 - A solution of a mixture of F760G4, CFA0599-F760G4, CFA0666-F760G4, CFA0672-F760G4 and CFA0675-F760G4) or 0.2 mg/ml (CFA0330-F760G4 and CFA0341-F760G4)) was administered in a single dose to the tail vein at a dose of 10 ml/kg middle. In this case, the anti-human C5 antibody is present in excess relative to human C5, and it is therefore believed that almost every human C5 binds to the antibody. Blood was collected at 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 for 10 minutes at 4°C to separate plasma. Separated plasma was stored in a -80°C freezer until assay. The anti-human C5 antibodies used were CFA0305-F760G4, CFA0307-F760G4, CFA0330-F760G4, CFA0341-F760G4, CFA0366-F760G4, CFA0501-F760G4, CFA0538-F760G4, CFA0599-F766-F760G6, CFA0599-F762-F760G4, CFA0599-F760G6 as described above F760G4 and CFA0675-F760G4.

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

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

In the presence of CFA0330-F760G4, CFA0341-F760G4 or human C5 alone in plasma samples, the following method was used. Anti-human C5 antibody (Santa Cruz) was dispensed onto MULTI-ARRAY 96-well bare plates (Meso Scale Discovery) and left at 4°C overnight to prepare anti-human C5 immobilized plates. Calibration curve samples and mouse plasma samples diluted more than 100-fold with 1 μg/ml of injected antibody (CFA0330-F760G4 or CFA0341-F760G4) were prepared and incubated at 37°C for 30 minutes. Subsequently, samples were dispensed onto anti-human C5 immobilized plates and allowed to stand at room temperature for 1 hour. Then, SULFO-TAG-labeled anti-human IgG antibody (Meso Scale Discovery) was added to react at room temperature for one hour, and washed. Read buffer T(x4) (Meso Scale Discovery) was then dispensed immediately and measured using a Sector Imager2400 (Meso Scale Discovery).

The following method was used in the presence of CFA0305-F760G4, CFA0307-F760G4, CFA0366-F760G4, CFA0501-F760G4, CFA0538-F760G4, CFA0599-F760G4, CFA0666-F760G4, CFA0672-F760G4 or CFA060G4 in the plasma sample. Anti-human C5 antibodies (CFA0329-F939G4; VH, SEQ ID NO: 23, and VL, SEQ ID NO: 27) were dispensed onto MULTI-ARRAY 96-well bare plates (Meso Scale Discovery) and left at 4°C overnight , to prepare plates immobilized with anti-human C5. Calibration curve samples and mouse plasma samples diluted more than 100-fold with an acidic solution (pH 5.5) were prepared and incubated at 37°C for 30 minutes. Subsequently, samples were dispensed onto anti-human C5 immobilized plates and allowed to stand at room temperature for 1 hour. Then, SULFO-TAG-labeled anti-human C5 antibody (CFA0300-F939G4; VH, SEQ ID NO: 24, and VL, SEQ ID NO: 28) was added to react at room temperature for one hour, and washed. Read buffer T(x4) (Meso Scale Discovery) was then dispensed immediately and measured using a Sector Imager 2400 (Meso Scale Discovery).

Based on the response from the calibration curve, human C5 concentrations were calculated using the analytical software SOFTmax PRO (Molecular Devices). The time course of plasma human C5 concentrations after intravenous administration measured by this method is shown in FIG. 13 . Data are plotted as percent remaining compared to plasma human C5 concentration at 5 minutes.

6.3. Measurement of anti-human C5 antibody plasma concentrations by ECL assay

The concentration of anti-human C5 antibody in mouse plasma was measured by ECL. Dispense anti-human IgG (gamma-chain specific) F(ab')2 antibody fragment (Sigma) or anti-human IgG kappa chain antibody (Antibody Solutions) onto MULTI-ARRAY 96-well bare plates (Meso Scale Discovery) and left at 4°C overnight to prepare anti-human IgG-immobilized plates. Prepare calibration curve samples and mouse plasma samples diluted more than 100-fold. Subsequently, the samples were dispensed onto anti-human IgG-immobilized plates and allowed to stand at room temperature for 1 hour. Then, biotinylated anti-human IgG antibody or SULFO-TAG-labeled anti-human IgG Fc antibody (Southernbiotech) was added to react at room temperature for one hour, and washed. Subsequently, only when biotinylated anti-human IgG antibodies were used, SULFO-TAG-labeled streptavidin (Meso Scale Discovery) was added to react at room temperature for one hour, and washed. Read buffer T(x4) (Meso Scale Discovery) was then dispensed immediately and measured using a Sector Imager 2400 (Meso Scale Discovery). Anti-human C5 antibody concentrations were calculated using the analytical software SOFTmax PRO (Molecular Devices) based on the responses from the calibration curve. The time course of plasma anti-human C5 antibody concentrations after intravenous administration measured by this method is shown in FIG. 14 . Data are plotted as percent remaining compared to plasma anti-human C5 antibody concentration at 5 minutes.

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

In vivo testing of pH-dependent anti-human C5 antibodies (CFA0305-F760G4, CFA0307-F760G4, CFA0366-F760G4, CFA0501-F760G4, CFA0538-F760G4, CFA0599-F760G4, CFA0666-F760G4, CFA0672-F760G4) and CFA060G4 Dependent anti-human C5 antibodies (CFA0330-F760G4 and CFA0341-F760G4), and the resulting plasma anti-human C5 antibody concentrations and plasma human C5 concentrations were compared. As shown in Figure 14, antibody exposures were comparable. At the same time, the clearance of human C5 co-administered with the pH-dependent anti-human C5 antibody was accelerated compared to the pH-independent anti-human C5 antibody ( FIG. 13 ).

Example 7

Optimized anti-C5 monoclonal antibody (305 variant)

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

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

[Table 7]

VH amino acid sequence of the 305 variant

[Table 8]

VL amino acid sequence of the 305 variant

Example 8

Binding characterization of anti-C5 antibody (305 variant)

Kinetic parameters of anti-C5 antibodies against recombinant human C5 were assessed under three different conditions using a BIACORE (registered trademark) T200 instrument (GE Healthcare) at 37°C; (1) both association and dissociation were at pH 7.4, (2) ) both bound and dissociated at pH 5.8, and (3) bound at pH 7.4 and dissociated at pH 5.8. ProA/G (Pierce) was immobilized onto the CM1 sensor chip using an amine coupling kit (GE Healthcare) according to the settings recommended by GE Healthcare. Antibodies and analytes of conditions (1) and (3) were diluted in ACES pH7.4 buffer (20 mM ACES, 150 mM NaCl, 1.2 mM _CaCl2 , 0.05% Tween20, 0.005% NaN3) and condition ( ₂ ) The antibodies and analytes were diluted in ACES pH 5.8 buffer (20 mM ACES, 150 mM NaCl, 1.2 mM _CaCl2 , 0.05% Tween ₂₀ , 0.005% NaN3). Each antibody was captured on the sensor surface by ProMG. Antibody capture levels are typically 60-90 response units (RU). Recombinant human C5 was then injected at 3 to 27 nM or 13.3 to 120 nM (prepared by three-fold serial dilution) prior to dissociation. The surface was regenerated with 25 mM NaOH. Kinetic parameters for conditions (1) and (2) were determined by fitting the sensorgrams with a 1:1 binding model and the dissociation rate for condition (3) was determined by fitting the sensorgrams with a 1:1 dissociation for the MCK model , wherein BIACORE (registered trademark) T200 Evaluation software, version 2.0 (GE Healthcare) was used. The pH dependence of all antibodies is shown as the ratio of the dissociation rates for conditions (2) and (1).

On-rate (ka), off-rate (kd), binding affinity (KD) and pH dependence are listed in Table 9. All antibodies exhibited dissociation rates at pH 5.8 that were faster than at pH 7.4 and were pH-dependent about 20-fold.

[Table 9]

Kinetic parameters of anti-C5 antibody variants at pH 7.4 and pH 5.8

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 determined at 37°C using a BIACORE (registered trademark) T200 instrument (GE Healthcare), to assess the effect of pH on antigen binding. Goat anti-human IgG (Fc) polyclonal antibody (KPL#01-10-20) was immobilized on the CM4 sensor chip using an amine coupling kit (GE Healthcare) according to the manufacturer's recommended settings. Antibodies and analytes were diluted in ACES pH 7.4 buffer or ACES pH 5.8 buffer containing 20 mM ACES, 150 mM _NaCl , 1.2 mM _CaCl2 , 0.05% Tween 20 and 0.005% NaN3. Antibodies are captured on the sensor surface using an anti-Fc method, typically at capture levels of 50-80 response units (RU). Recombinant human C5 was prepared by three-fold serial dilution starting from 27 nM (for pH 7.4 assay conditions) or 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 affinities (KD) of BNJ441, eculizumab and the 305 variant for recombinant human C5 at pH 7.4 and pH 5.8 are shown in Table 10. The 305 variant showed a (KD at pH 5.8)/(KD at pH 7.4) ratio of almost 800, while BNJ441 showed a (KD at pH 5.8)/(KD at pH 7.4) ratio of only 93 8 times higher.

[Table 10]

Example 9

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

9.1. Anti-C5MAb inhibits complement-activated lipolysis

Anti-C5MAbs were tested for inhibition of complement activity by a lipolysis 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 min at room temperature. The liposome solution sensitized with an antibody against dinitrophenyl (Autokit CH50, Wako, 995-40801) was transferred to each well and placed on a shaker for 2 min at 37°C. 50 μL of substrate solution (Autokit CH50) was added to each well and mixed with shaking for 2 min at 37°C. The final mixture was incubated at 37°C for 40 minutes after which the OD at 340nm was measured. The percentage of liposome solubilization was defined as 100×[(OD _MAb -OD _{serum and liposome background} )]/[(OD _MAb-free- OD _{serum and liposome background} )]. Figure 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, were also shown to inhibit liposome lysis (Figure 16).

The 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 for 1 hour at 37°C. Liposomes (Autokit CH50) were transferred to each well and placed on a shaker for 2 min at 37°C. 50 μL of substrate solution (Autokit CH50) was added to each well and mixed with shaking for 2 min at 37°C. The final mixture was incubated at 37°C for 180 minutes after which the OD at 340nm was measured. The percentage of liposome solubilization is as defined above. Figure 17 shows that anti-C5 Mabs: 305LO22-SG115, 305LO22-SG422, 305LO23-SG115 and 305LO23-SG422, inhibit liposome lysis.

9.2. Anti-C5MAb inhibits C5a production

The anti-C5 MAb was tested for C5a production during lipolysis to confirm that the anti-C5 MAb inhibits the cleavage of C5 into C5a and C5b. C5a levels in supernatants from liposome lysis assays were quantified using a C5a ELISA kit (R&D systems, DY2037). All MAbs inhibited C5a production in the supernatant in a dose-dependent manner (Figures 18 and 19).

9.3. Measurement of complement activity in cynomolgus monkey plasma

Anti-C5MAbs were tested for inhibition of complement activity in cynomolgus monkey plasma. Anti-C5 MAbs were administered intramuscularly to monkeys (20 mg/kg), and plasma samples were collected periodically until day 56. Chicken red blood cells (cRBC) (Innovativeresearch, IC05-0810) were washed with gelatin/barbital buffered saline (Boston BioProducts, IBB-300X) containing 0.5 mM MgCl ₂ and 0.15 mM CaCl ₂ (GVB++), followed by 1 μg/ ml of anti-chicken RBC antibody (Rockland 103-4139) was sensitized for 15 minutes at 4°C. Cells were then washed with GVB++ and suspended in the same buffer at ^1x108 cells/ml. Monkey plasma was incubated with sensitized cRBCs at 37°C for 20 minutes in separate round-bottomed 96-well microtest plates. After incubation, the plates were centrifuged at 1000xg for 2 minutes at 4°C. The supernatant was transferred to wells on a flat-bottom 96-well microtest plate for measurement of OD at 415 nm with a reference wavelength of 630 nm. Percent hemolysis was defined as 100×[( _post -OD _{plasma and cRBC background} )]/[( _pre- OD-OD _{plasma and cRBC background} )]. Figure 20 shows that anti-C5 Mabs: 305LO15-SG422, 305LO15-SG115, 305LO16-SG422, 305LO18-SG422, 305LO19-SG422, 305LO20-SG422, 305LO20-SG115 and 305LO23-SG115 inhibit complement activity in plasma.

9.4. Anti-C5 MAbs inhibit the biological activity of C5 variants

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 responses to eculizumab (see, eg, Nishimura et al., New Engl. J. Med. 370:632-639 (2014)). Individual human C5 variants were expressed in FS293 cells, and the supernatants were used for subsequent studies. 10 μL of C5-deficient human serum (Sigma, C1163) was mixed with 20 μL of diluted MAb and 20 μL of cell culture medium containing recombinant C5 variants (2-3 μg/mL) in 96-well plates and incubated at 37°C at 37°C. Incubate on a shaker for 0.5 hours. Liposomes (Autokit CH50) were transferred to each well and placed on a shaker for 2 min at 37°C. 50 μL of substrate solution (Autokit CH50) was added to each well and mixed with shaking for 2 min at 37°C. The final mixture was incubated at 37°C for 90 minutes after which the OD at 340nm was measured. The percentage of liposome solubilization is as defined above. Figure 21 shows that the anti-C5 MAb (eculizumab) did not inhibit the R885H C5 variant, but inhibited other variants tested. Figure 22 shows that the anti-C5 MAb (305 variant) inhibited all C5 variants tested.

9.5. Anti-C5 MAb inhibits complement-activated lipolysis

Anti-C5MAbs were tested for inhibition of complement activity by a lipolysis 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 min at room temperature. The liposome solution sensitized with an antibody against dinitrophenyl (Autokit CH50, Wako, 995-40801) was transferred to each well and placed on a shaker for 2 min at 25°C. 50 μL of substrate solution (Autokit CH50) was added to each well and mixed with shaking for 2 min at 25°C. The final mixture was incubated at 37°C for 45 minutes after which the OD at 340nm was measured. Percent inhibition of liposome lysis was defined as 100×[(OD _MAb -OD _{serum and liposome background} )]/[(OD _MAb-free- OD _{serum and liposome background} )]. Figure 23 shows that the anti-C5 MAb, BNJ441 and 305 variants inhibit liposome lysis, and that the inhibitory activity of the 305 variant is stronger than that of BNJ441.

Example 10

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

10.1. In vivo testing using cynomolgus monkeys

The in vivo kinetics of anti-human C5 antibodies were assessed following administration of anti-human C5 antibodies in cynomolgus monkeys (Shin Nippon Biomedical Laboratories, Ltd., Japan). A solution of anti-human C5 antibody (2.5 mg/ml) was administered in a single dose of approximately 8 ml/kg into the cephalic vein of the forearm by 30 minute infusion. Blood was collected before administration and at 5 minutes, 7 hours, 1 day, 2 days, 3 days, 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days and 56 days after administration. The collected blood was immediately centrifuged at 1,700 xg for 10 minutes at 4°C to separate plasma. Separated plasma was stored in a freezer below -70°C until assayed. Anti-human C5 antibodies were prepared as described in Example 7.

10.2. Measurement of Cynomolgus C5 Total Plasma Concentration by ELISA Assay

The total concentration of cynomolgus C5 in cynomolgus monkey plasma was measured by ELISA. Anti-human C5 antibody (internal antibody generated using the method described in Example 2) was dispensed onto a Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and left at 4°C overnight to prepare anti-cynomolgus C5-immobilized flat. Calibration curve samples and cynomolgus monkey plasma samples diluted 20000-fold with 0.4 μg/ml of injected antibody were prepared and incubated at 37°C for 60 minutes. Subsequently, samples were dispensed onto anti-cynomolgus C5 immobilized plates and allowed to stand at room temperature for 1 hour. Then, HRP-labeled anti-human IgG antibody (SouthernBiotech) was added to react at room temperature for 30 minutes, and washed. Subsequently, ABTSELISA HRP substrate (KPL) was added. The signal was measured by a microplate reader at a wavelength of 405 nm. Based on the response from the calibration curve, cynomolgus C5 concentrations were calculated using the analysis software SOFTmax PRO (Molecular Devices). The time course of plasma cynomolgus C5 concentrations after intravenous administration measured by this method is shown in FIG. 24 . Data are plotted as percent remaining compared to pre-administration plasma cynomolgus C5 concentrations. Compared with pH-independent anti-human C5 antibodies, 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.

10.3. Measurement of anti-human C5 antibody plasma concentrations by ELISA assay

The concentration of anti-human C5 antibody in cynomolgus monkey plasma was measured by ELISA. Anti-human IgG kappa chain antibody (Antibody Solutions) was dispensed onto Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and left at 4°C overnight to prepare anti-human IgG immobilized plates. Prepare calibration curve samples and cynomolgus monkey plasma samples diluted more than 100-fold. Subsequently, the samples were dispensed onto anti-human IgG-immobilized plates and allowed to stand at room temperature for 1 hour. Then, HRP-labeled anti-human IgG antibody (SouthernBiotech) was added to react at room temperature for 30 minutes, and washed. Subsequently, ABTS ELISA HRP substrate (KPL) was added. The signal was measured by a microplate reader at a wavelength of 405 nm. Anti-human C5 antibody concentrations were calculated using the analytical software SOFTmax PRO (Molecular Devices) based on the responses from the calibration curve. The time course of plasma anti-human C5 antibody concentrations after intravenous administration measured by this method is shown in FIG. 25 . Compared with pH-independent anti-human C5 antibodies, pH-dependent anti-human C5 antibodies (305LO15-SG422, 305LO15-SG115, 305LO16-SG422, 305LO18-SG422, 305LO19-SG422, 305LO20-SG422, 305LO20-SG115, 305LO22-SG422, 305LO23-SG422 and 305LO23-SG115) exhibited longer half-lives.

Example 11

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

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

Using the pGEX-4T-1 vector (GE healthcare), the MG1 domain (amino acid residues 20-124 of SEQ ID Bacillus strain BL21DE3pLysS (Promega). Protein expression was induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 5 hours at 25°C. Bacterial cell pellets were lysed with Bugbuster (Merck) supplemented with lysonase (Merck) and complete protease inhibitor cocktail (Roche), followed by analysis of GST-MG1 from the soluble fraction using GSTrap columns (GE healthcare) according to the manufacturer's instructions. purification. The GST tag was cleaved with thrombin (Sigma) and the resulting MG1 domains were further purified with Superdex 75 gel filtration columns (GE healthcare). The MG1 domain containing fractions were pooled and stored at -80°C.

11.2. Preparation of Fab fragments of the 305 variant

The Fab fragment from one of the optimized variants of 305 was prepared by conventional methods using limited digestion with papain (Roche Diagnostics, cat. no. 1047825) before loading on a protein A column (MabSlect SuRe, GE Healthcare) to remove Fc Fragments were obtained and loaded onto a cation exchange column (HiTrap SP HP, GE Healthcare) and a gel filtration column (Superdex200 16/60, GE Healthcare). Fractions containing Fab fragments were pooled and stored at -80°C.

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

The purified recombinant human C5-MG1 domains were mixed with purified 305 variant Fab fragments 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 by drop vapor diffusion method combined with seeding method at 4°C. The mother liquor consisted of 0.2M anhydrous magnesium formate, 15.0% w/v polyethylene glycol 3350. This successfully produced platelet-like crystals within a few days. The crystals were soaked in a solution of 0.2M magnesium formate anhydrous, 25.0% w/v polyethylene glycol 3350 and 20% glycerol.

11.5. Data collection and structure determination

X-ray diffraction data were obtained at SPring-8 by BL32XU. During the measurement, the crystals were kept frozen in a nitrogen flow at -178°C, and a total of 180 X-ray diffraction images were collected using an MX-225HS CCD detector (RAYONIX) connected to the beamline, while the crystals were Rotate 1.0 degrees. Using the Xia2 program (J.Appl.Cryst.43:186-190 (2010), XDS package (Acta.Cryst.D66:125-132 (2010)) and Scala (Acta.Cryst.D62:72-82 (2006) )) to perform cell parameter determination, indexing of diffraction spots and processing of diffraction data obtained from diffraction images, and finally to obtain diffraction intensity data with a resolution of up to 2.11 Angstroms. Crystallographic data statistics are shown in Table 11.

[Table 11]

X-ray data collection and statistical refinement

a; R _merge =∑hk/∑j|Ij(hkl)-<I(hkl)>|/∑hk/∑j|Ij(hkl)|, where Ij(hkl) and <I(hkl)> are the measurements, respectively The intensity of j and the average intensity of the reflection with index hkl.

b; R factor= _∑hkl | _Fcacl (hkl)|-|Fobs(hkl)|/ _∑hkl |Fobs(hkl)|, where _Fobs and _Fcalc are the observed and calculated mechanism factor amplitudes, respectively.

c; R _free is calculated using a randomly picked 5% reflection.

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

11.6.305 Overall structure of variant Fab and C5-MG1 domain complex

The Fab fragment from the optimized variant of 305 ("305Fab") binds the human C5-MG1 domain ("MG1") in a 1:1 ratio, and the asymmetric unit of the crystal structure contains two complexes, molecules 1 and 2 , as shown in Figure 26A. Molecules 1 and 2 were well aligned with 0.717 Angstroms RMSD using the Cα atomic positions in all residues, as shown in Figure 26B. The graphs discussed below were prepared using Molecule 1.

In Figures 27A and 27B, the epitopes of the 305Fab contact region are mapped in the MGl amino acid sequence and in the crystal structure, respectively. The epitope includes the amino acid residue of MG1 containing one or more atoms within a 4.5 Angstrom distance from any portion of 305Fab in the crystal structure. In addition, epitopes within 3.0 Angstroms are highlighted in Figure 27A.

11.7. Interaction of E48, D51 and K109

Anti-C5 MAbs including the 305 antibody series were tested for binding to three human C5 point mutants E48A, D51A and K109A by Western blot and BIACORE (registered trademark) binding assays as described in Examples 4.5 and 4.6. Although the 305 variants bound strongly to WT C5, they bound only weakly to the E48A C5 mutant and not to the D51A and K109A mutants. The crystal structures of the 305Fab and MG1 complex revealed that the three amino acids E48, D51 and K109 were all within a distance of 3.0 Angstroms from the 305Fab, forming several hydrogen bonds with the Fab, as shown in Figure 28A. According to a more detailed study, the K109 residue of MG1 is embedded in the groove formed in the interface of the heavy chain of the Fab and is bound by three hydrogen bonds with H-CDR3_G97, H-CDR3_Y100 and H-CDR3_T100b and with H-CDR3_D95 The salt bridge interacts tightly with the Fab (FIG. 28D). D51 is located between the heavy chains of MG1 and 305Fab 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 the binding of the 305 antibody series. On the other hand, E48 is located next to the surface and forms only one hydrogen bond with the Fab, indicating that its contribution to antibody binding is smaller than that of K109 and E51 (Figure 28B). These relationships are consistent with the results of Western blot and BIACORE (registered trademark) binding analysis of human C5 mutants (Examples 4.5 and 4.6). Additional note: The residue numbering of Fab amino acids 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 H70, H72 and H110 of human C5 with the 305 antibody series

Crystal structure analysis revealed that three histidine residues on human C5, ie, H70, H72 and H110, were included in the epitope of the 305 variant Fab, as shown in Figures 27A and 29A. BIACORE (registered trademark) binding assay was performed to investigate the contribution of these histidine residues to pH-dependent protein-protein interactions between human C5 and 305 variant Fabs using human C5 mutants H70Y, H72Y, H110Y and H70Y+H110Y (Example 4.7). H72Y resulted in a complete loss of binding of the 305 variant Fab to C5. This C5 residue is located in the pocket formed by the CDR2 loop of the 305 Fab heavy chain and the loops of MG1 (L73, S74 and E76) and this space is tightly packed, as shown in Figure 29C. Furthermore, the H72 residue of C5 forms a hydrogen bond with H-CDR2_Y58. It is expected that the H72Y mutation will not be tolerated because there is not enough room for the bulkier side chain of tyrosine. The hydrogen bond with H-CDR2_Y58 could not be maintained either. Regarding the contribution of H70 and H110 to pH dependence, the H70Y and H110Y mutations resulted in slower dissociation of the 305 variant Fab from C5 at pH 5.8. H70 forms an intramolecular hydrogen bond with T53 of MG1 that is believed to be disrupted at pH 5.8 when protonation of H70 of C5 induces a conformational change in the corresponding part of the interaction interface of MG1 (FIG. 29B). For H110, protonation of this C5 residue is expected to cause charge repulsion to 305Fab, which can be enhanced by protonation of the adjacent histidine residue H-CDR3_H100c (FIG. 29D).

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

Claims (4)

1. Use of an anti-C5 antibody in the manufacture of a pharmaceutical formulation for the treatment of a complement-mediated disease or disorder involving excessive or uncontrolled activation of C5, wherein the antibody comprises:

(a) the VH sequence of SEQ ID NO 106 and the VL sequence of SEQ ID NO 111;

(b) the VH sequence of SEQ ID NO. 107 and the VL sequence of SEQ ID NO. 111;

(c) the VH sequence of SEQ ID NO 108 and the VL sequence of SEQ ID NO 111;

(d) the VH sequence of SEQ ID NO:109 and the VL sequence of SEQ ID NO: 111;

(e) the VH sequence of SEQ ID NO:109 and the VL sequence of SEQ ID NO: 112;

(f) the VH sequence of SEQ ID NO:109 and the VL sequence of SEQ ID NO: 113; or

(g) The VH sequence of SEQ ID NO. 110 and the VL sequence of SEQ ID NO. 113.

2. Use of an anti-C5 antibody in the manufacture of a pharmaceutical formulation for enhancing clearance of C5 from plasma, wherein the antibody comprises:

(a) the VH sequence of SEQ ID NO 106 and the VL sequence of SEQ ID NO 111;

(b) The VH sequence of SEQ ID NO. 107 and the VL sequence of SEQ ID NO. 111;

(c) the VH sequence of SEQ ID NO 108 and the VL sequence of SEQ ID NO 111;

(d) the VH sequence of SEQ ID NO:109 and the VL sequence of SEQ ID NO: 111;

(e) the VH sequence of SEQ ID NO:109 and the VL sequence of SEQ ID NO: 112;

(f) the VH sequence of SEQ ID NO:109 and the VL sequence of SEQ ID NO: 113; or

(g) The VH sequence of SEQ ID NO. 110 and the VL sequence of SEQ ID NO. 113.

3. The use of claim 1 or 2, wherein the antibody comprises a heavy chain variable region comprising SEQ ID NO: 114 and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 38, or a light chain constant region of the amino acid sequence of seq id no.

4. The use of claim 1 or 2, wherein the antibody is a full length IgG1 antibody or a full length IgG4 antibody.

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