TW202239766A - Anti-c5 antibodies and methods of use - Google Patents

Abstract

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

Description

Anti-C5 antibodies and methods of use

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

The complement system plays an important role in the clearance of immune complexes and in the immune response to infectious agents, foreign antigens, virus-infected cells and tumor cells. There are approximately 25 to 30 complement proteins, which are found as a complex collection of plasma proteins and membrane cofactors. Complement components achieve their immune defense functions by reacting with a series of intertwined and complex enzyme cleavage and membrane binding, resulting in a complement cascade that leads to the production of products with opsonic, immunomodulatory and cytolytic functions .

Currently, it is generally accepted that the complement system can be activated through three different pathways: the classical pathway, the lectin pathway and the alternative pathway. These pathways share many complements, and although they differ in their initial steps, they converge and share the same terminal complement components (C5 to C9) responsible for the activation and destruction of 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 mannose-binding lectin (MBL), H-ficolin (H-ficolin), M-ficolin Coagulin, L-ficolin, and C-type lectin CL-11. In contrast, the alternative pathway spontaneously undergoes a low degree of turnover activation, which can readily expand on foreign or other abnormal surfaces (bacteria, yeast, virus-infected cells or damaged tissue). These pathways converge where complement component C3 is cleaved by active proteolytic enzymes to yield C3a and C3b.

C3a is an anaphylatoxin, and C3b binds to bacteria and other cells, as well as certain viruses and immune complexes, and marks them for removal from circulation (known as the action of opsonins) . C3b also forms a complex with other components to form the C5 convertase, which cleaves C5 into C5a and C5b.

C5 is a 190 kDa protein found in normal serum at a concentration of approximately 80 μg/ml (0.4 μM). The C5 line is glycosylated, with about 1.5 to 3% of its mass being sugar. Mature C5 is a heterodimer of a 115 kDa alpha chain disulfide-bonded to a 75 kDa beta chain. C5 is synthesized as a single-chain precursor protein (pro-C5 precursor) of 1676 amino acids (see, eg, Patent Literature (PTL) 1 and PTL 2). The pro-C5 precursor is cleaved to generate the β-chain as the amino-terminal fragment and the α-chain as the carboxy-terminal fragment. The α-chain and β-chain polypeptide fragments are linked to each other by disulfide bonds and constitute the mature C5 protein.

When the complement pathway is activated, mature C5 is cleaved into C5a and C5b fragments. 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 comprises the remaining alpha chain disulfide bonded to the beta chain. About 20% of the 11 kDa mass of C5a is sugar.

C5a is another anaphylatoxin, C5b combines with C6, C7, C8 and C9 to form a membrane attack complex (membrane attack complex, MAC, C5b-9, terminal complement complex (membrane attack complex, TCC)) in 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 cytolysis of the target cell.

Based on the above, C3a and C5a are anaphylatoxins, which can trigger degranulation of mast cells, which release histamine and other mediators of inflammatory reactions, resulting in smooth muscle contraction, increased vascular permeability, white blood cell activation, and other inflammatory reactions. Including hypercellularity due to cell proliferation. C5a also acts as a chemotactic peptide, which is used to attract granulosa cells such as neutrophils, eosinophils, basophils and monocytes to the site of complement activation.

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

While a properly functioning complement system provides a robust defense against infectious microorganisms, inappropriate regulation or activation of complement has been implicated in the pathogenicity of a variety of diseases, including, for example: rheumatoid arthritis (RA); lupus nephritis; local Ischemia-reperfusion injury; paroxysmal nocturnal hemoglobinuria (PNH); atypical hemolytic uremic syndrome (aHUS); 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; Pauci-immune vasculitis; multiple sclerosis (MS); traumatic brain injury; and injuries resulting from myocardial infarction, cardiopulmonary bypass, and hemodialysis (see, eg, Non-Patent Literature (NPL) 1). Therefore, inhibiting excessive or uncontrolled activation of the complement cascade may provide clinical benefit to patients suffering from these diseases.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare blood disorder in which red blood cells are compromised so that they are destroyed more quickly than normal red blood cells. PNH results from the clonal proliferation of hematopoietic stem cells with somatic mutations in the PIG-A (phosphatidylinositol glycan class A) gene, which is located on the X chromosome. Mutations in PIG-A cause an initial block in the synthesis of glycosylphosphatidylinositol (GPI), a molecule required for the anchoring of many proteins to the cell surface. Thus, PNH hemocytes lack GPI-anchored proteins, including the complement regulatory proteins CD55 and CD59. Under normal conditions, these complement regulatory proteins prevent MAC formation on the cell surface, thus preventing red blood cell lysis. Deletion of GPI-anchored proteins leads to complement-mediated hemolytic responses in PNH.

PNH is characterized by hemolytic anemia (reduction in the number of red blood cells), hemoglobinuria (the appearance of hemoglobin in the urine, especially after sleep), and hemoglobinemia (the appearance of hemoglobin in the blood). Individuals with PNH are known to have paroxysms, defined herein as the incidence of dark urine. Hemolytic anemia is due to the intravascular destruction of red blood cells by complement components. Other known symptoms include dysphagia, fatigue, erectile dysfunction, thrombosis, and recurrent abdominal pain.

Eculizumab is a human monoclonal antibody against complement protein C5 and is the first approved therapy for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS) (see , such as NPL 2). Eculizumab inhibits the C5 convertase cleavage of C5 into C5a and C5b, which prevents the generation of the terminal complement complex C5b-9. Both C5a and C5b-9 lead to the phenomenon of terminal complement regulation that is characteristic of PNH and aHUS (see also PTL 3, PTL 4, PTL 5 and PTL 6).

Many reports have described anti-C5 antibodies, for example PTL7 describes an anti-C5 antibody that binds to the alpha chain of C5 but not to C5a, and prevents the activation of C5. Whereas PTL 8 describes an anti-C5 monoclonal antibody that inhibits the formation of C5a. On the other hand, PTL 9 describes an anti-C5 antibody that recognizes that the proteolytic site of C5 convertase is on the alpha chain of C5, and inhibits the conversion of C5 into C5a and C5b. PTL 10 describes an anti-C5 antibody with an affinity constant of at least ¹ x 107M ^-1 .

Antibodies (IgGs) bind to neonatal Fc receptors (FcRn) and have long plasma retention times. Binding of IgGs to FcRn is usually observed in an acidic environment (eg pH 6.0) and rarely observed in a neutral environment (eg pH 7.4). In general, IgGs are non-specifically incorporated into cells through endocytosis, and return to the cell surface through endosomal FcRn bound to the intracellular (endosomal) FcRn under the acidic environment of endosomes. Next, IgGs dissociate from FcRn in the neutral environment of plasma. IgGs not bound to FcRn are broken down in lysosomes. When the FcRn-binding ability of IgG in acidic environment is abolished by introducing mutations into its Fc region, IgG will not recycle from endosomes into plasma, resulting in significantly impaired plasma retention of IgG. In order to improve the plasma retention of IgGs, a method to increase its binding to FcRn in acidic environment has been reported, when the FcRn binding ability of IgG in acidic environment was enhanced by introducing an amino acid substitution into its Fc region , IgG is more efficiently recycled from endosomes to plasma, thus showing improved plasma retention. Meanwhile, it has also been reported that IgG with enhanced FcRn binding in neutral environment does not dissociate from FcRn in neutral environment plasma even when it returns to the cell surface by binding to FcRn in acidic environment, thus Its plasma retention remained unchanged, or should be said to be poor (see eg NPL 3; NPL 4; NPL 5).

Recently, antibodies that bind to antigens in a pH-dependent manner have been reported (see, eg, PTL 11 and PTL 12). These antibodies bind strongly to antigens in the neutral environment of plasma and dissociate from antigens in the acidic environment of the cell. After dissociation from the antigen, the antibody becomes re-associated with the antigen when recycled to the plasma by 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, and thus, when antigens are bound to antibodies, antigens usually exhibit prolonged plasma retention, resulting in an increase in the plasma concentration of antigen. On the other hand, it has been reported that the above-mentioned antibodies, which bind to antigens in a pH-dependent manner, can eliminate antigens from plasma more rapidly than typical antibodies because they are in the endosome during the FcRn-mediated recycling step. dissociated from the antigen. PTL 13 also describes in silico analysis showing that antibodies with pH-dependent binding to anti-C5 can prolong antigen knockdown. [citation list] [Patent Document]

[PTL 1] U.S. Patent No. 6,355,245 [PTL 2] U.S. 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 [PTL 8] WO 02/30985 [PTL 9] WO 2004/007553 [PTL 10] WO 2010/015608 [PTL 11] WO 2009/125825 [PTL 12] 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)

[technical problem]

One object of the present invention is to provide anti-C5 antibodies and methods of use thereof. [Solution to problem]

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

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

In a specific embodiment, the anti-C5 antibody of the present invention comprises (a) the VH of the sequence identification number: 1 and the VL of the sequence identification number: 11; (b) the VH and the sequence identification of the sequence identification number: 5 No.: VL of 15; (c) sequence identification number: VH of 4 and sequence identification number: VL of 14; (d) sequence identification number: VH of 6 and sequence identification number: VL of 16; (e) sequence identification number VH of :2 and VL of sequence identification number: 12; (f) VH of sequence identification number: 3 and VL of sequence identification number: 13; (g) VH of sequence identification number: 9 and VL of sequence identification number: 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 the antibody competition of VH and VL pair of the VL of sequence identification number: 20 and C5 is combined. In yet other embodiments, the anti-C5 antibody binds to C5 with a higher affinity at neutral pH compared to acidic pH. In yet other embodiments, the anti-C5 antibody binds to C5 with a higher affinity at pH 7.4 than at pH 5.8.

In some embodiments, the isolated anti-C5 antibody of the present 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 affinity of the antibody to C5 (SEQ ID NO: 39) is greater than the affinity of the antibody to the C5 mutant constituted by the E48A substitution of SEQ ID NO: 39; or (c) the antibody binds at pH 7.4 to No.: 39 amino acid sequence composed of the C5 protein, but at pH 7.4 did not bind to the C5 protein composed of the H72Y substitution sequence identification number: 39 amino acid sequence. In yet other embodiments, the antibody binds to C5 with a higher affinity at neutral pH compared to acidic pH. In yet other embodiments, the antibody binds to C5 with a 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 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, an isolated anti-C5 antibody of the invention is an antibody fragment that binds to C5. In some embodiments, the isolated anti-C5 antibody of the invention is a full length IgGl or IgG4 antibody.

In some embodiments, the isolated anti-C5 antibody of the present invention comprises (a) HVR-H3 comprising the amino acid sequence DX ₁ GYX ₂ X ₃ PTHAMX ₄ X ₅ , wherein X ₁ is G or A, X ₂ is V , Q or D, X ₃ is T or Y, X ₄ is Y or H, X ₅ is L or Y (SEQ ID NO: 128); (b) HVR-L3, including 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, X ₃ is A, T or H (SEQ ID NO: 131); and (c) HVR-H2, including the amino acid sequence X ₁ IX ₂ TGSGAX ₃ YX ₄ AX ₅ WX ₆ KG, where X ₁ is C, A or G, X ₂ is Y or F, X ₃ is T, D or E, X ₄ is Y, K or Q, X ₅ is S, D or E, X ₆ is A or V (SEQ ID NO: 127).

In some embodiments, the isolated anti-C5 antibody of the present invention comprises (a) HVR-H1, comprising the amino acid sequence SSYYX ₁ X ₂ , wherein X ₁ is M or V, and X ₂ is C or A (SEQ ID NO. : 126); (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, X ₂ is Y or F, and X ₃ 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, including amino acid The sequence DX ₁ GYX ₂ X ₃ PTHAMX ₄ X ₅ , wherein X ₁ is G or A, X ₂ is V, Q or D, X ₃ is T or Y, X ₄ is Y or H, X ₅ is L or Y ( Serial Identification Number: 128). In yet other 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, X ₂ is N, Q or G, X ₃ is G or S, X ₄ is D, K or S (SEQ ID NO: 129); (b) HVR-L2, including the amino acid sequence GASX ₁ X ₂ X ₃ S, wherein X ₁ is K, E or T , X ₂ is L or T, X ₃ is A, H, E or Q (SEQ ID NO: 130); and (c) 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, X ₃ is A, T or H (SEQ ID NO: 131).

In some embodiments, the isolated anti-C5 antibody of the present 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, X ₃ is G or S, X ₄ is D, K or S (SEQ ID NO: 129); (b) HVR-L2, including the amino acid sequence GASX ₁ X ₂ X ₃ S, wherein X ₁ is K, E or T, X ₂ is L or T, X ₃ is A, H, E or Q (SEQ ID NO: 130); and (c) HVR-L3, including 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, X ₃ is A, T or H (SEQ ID NO: 131).

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

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

The present invention provides an antibody, including the VH sequence of any one of the sequence identification numbers: 10, 106 to 110 and the VL sequence of any one of the sequence identification numbers: 20, 111 to 113.

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

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

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 are useful in the treatment of complement-regulated diseases or conditions involving excessive or uncontrolled C5 activation. The anti-C5 antibodies of the invention can be used to enhance the elimination of C5 from plasma.

The anti-C5 antibody of the present invention can be used in the manufacture of medicines. In some embodiments, the medicament is used to treat a complement-modulating disease or condition involving excessive or uncontrolled C5 activation. In some embodiments, the agent is to enhance the elimination of C5 from plasma.

The present invention also provides methods for treating individuals with complement-modulating diseases or conditions involving excessive or uncontrolled C5 activation. In some embodiments, the methods comprise administering to the individual an effective amount of an anti-C5 antibody of the invention. The invention also provides methods of enhancing the elimination of C5 from plasma in an individual. In some embodiments, the methods comprise administering to the individual an effective amount of an anti-C5 antibody of the invention to enhance elimination of C5 from plasma.

The techniques or procedures described or referenced herein are generally conventional methods well known and commonly used by those skilled in the art, such as, for example, as Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (eds. F.M. Ausubel et al. (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor, ed. (1995)), Harlow and Lane, ed. (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 (ed. J.E. Cellis, 1998) Academic Press; Animal Cell Culture (R.I. Freshney), edited, 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (eds. A. Doyle, J.B. Griffiths and D.G. Newell, 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (eds. D.M. Weir and C.C. Blackwell); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, ed., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., ed., 1994); Current Protocols in Immunology (J.E. Coligan et al., ed., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (ed. D. Catty., 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 the widely used method described in Cancer: Principles and Practice of Oncology (eds. V.T. DeVita et al., J.B. Lippincott Company, 1993).

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

In order to explain this specification, the following definitions will be used, and terms used in the singular may also include the plural and vice versa whenever appropriate. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth below shall control.

An "acceptor human framework" for the purposes herein is one comprising amine groups derived from a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework as defined below A framework for acid sequences. 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 comprise 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 1 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

"Affinity" refers to the aggregate strength of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). As used herein, unless otherwise stated, "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 to its partner Y can generally be expressed by a dissociation constant (Kd). Affinity can be measured by methods known in the art, including those described herein. Specific instructions and illustrative examples for measuring binding affinity are described below.

An "affinity matured" antibody is one that has one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody that does not possess such alterations, such The alteration results in an improvement in the affinity of the antibody for the antigen.

The terms "anti-C5 antibody" and "antibody that binds to C5" refer to an antibody that is capable of binding C5 with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent in targeting C5. In one embodiment, the extent to which an anti-C5 antibody binds to an irrelevant, non-C5 protein is less than about 10% of the binding of the antibody to C5, eg, as measured by radioimmunoassay (RIA). In specific embodiments, the antibody that binds to C5 has a concentration of 1 μM (micro M) 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 a dissociation constant (Kd) of 0.001 nM or less (eg, 10 ^-8 M or less, eg, from 10 ^-8 M to 10 ^-13 M, eg, from 10 ^-9 M to 10 ^-13 M). In certain embodiments, the anti-C5 antibody binds to an epitope of C5 that is conserved with C5 from a different species.

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

"Antibody fragment" refers to a molecule other than an intact antibody that includes a portion of an 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 (e.g., scFv); Formation of multispecific antibodies.

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

The term "chimeric" antibody refers to an antibody in which part of the heavy chain and/or light chain is from a specific source or species, and the remaining part of the heavy chain and/or light chain is from a different source or species.

The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subgroups (isotypes), eg, IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ . The heavy chain constant domains corresponding to different classes of immunoglobulins are called α (alpha), δ (delta), ε (epsilon), γ (gamma) and μ (mu), respectively.

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

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

An "effective amount" of an agent, such as a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time required, to achieve the desired therapeutic or prophylactic result.

The term "epitope" includes any determinant capable of being bound by an antibody. An epitope is the region of an antigen that is bound by an antibody targeting that antigen, and contains specific amino acids that directly contact the antibody. Epitope Epitopes may comprise chemically active surface clusters of molecules such as amino acids, sugar branches, phosphate groups, or sulfonyl groups, and may have specific three-dimensional structural properties, and/or specific charge properties. In general, antibodies specific for a particular target antigen will preferentially recognize epitopes on the target antigen in a complex mixture of proteins and/or macromolecules.

The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of the constant region. The term encompasses native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise stated 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 described 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 FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3 and FR4. Thus, HVR and FR sequences generally appear in a VH (or VL) in the following order: FR1-H1(LI)-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 having a structure substantially similar to that of a native antibody or having a heavy chain comprising an Fc region as defined herein.

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 the originally transformed cell and progeny derived therefrom, regardless of passage number. The nucleic acid content of the progeny may not be identical to that of the parental cells, but may contain mutations. Contemplated herein are mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell.

A "human antibody" is one having an amino acid sequence corresponding to an antibody produced by a human or a human cell or derived from a non-human source utilizing a human antibody repertoire or other human antibody coding sequence. This definition of a human antibody specifically excludes humanized antibodies that include non-human antigen-binding residues.

A "human consensus framework" is a framework representing the most frequently occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. In general, the selection of human immunoglobulin VL or VH sequences is derived from the subtype of variable domain sequences. Typically, the subtype of the sequence is that of Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one example, for VL, the subtype is subtype kappa I as in Kabat et al., supra. In one example, for VH, the subtype is subtype III as in Kabat et al., supra.

A "humanized" antibody refers to a chimeric antibody that includes amino acid residues from non-human HVRs as well as amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and usually two, variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and All or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally comprises 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 antibody variable domains that are hypervariable in sequence ("complementarity determining regions" or "CDRs"), and/or form structurally defined loops ("hypervariable loops") ), and/or each region comprising antigen contact residues ("antigen contacts"). In general, antibodies include six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein include: (a) occurrences at amino acid residues 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1), 53-55(H2 ) and the hypervariable loop of 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 95-102(H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service , NIH, Bethesda, MD (1991)) CDR; (c) occurs at amino acid residues 27c-36(L1), 46-55(L2), 89-96(L3), 30-35b(H1) , 47-58(H2) and 93-101(H3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)); and (d) (a), (b) and /or a combination of (c), comprising HVR amino acid residues 46-56(L2), 47-56(L2), 48-56(L2), 49-56(L2), 26-35(H1), 26-35b(H1), 49-65(H2), 93-102(H3) and 94-102(H3).

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

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

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

An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, antibodies are purified to greater than 95% or 99% purity by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse Phase HPLC) method for determination. For a review of methods for assessing antibody purity see, eg, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location other than 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), such nucleic acid molecules contained in a single vector or in separate vectors, and present in a host Such nucleic acid molecules at one or more locations in the cell.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., individual antibodies comprising the same population and/or binding to the same epitope, except, for example, comprising naturally occurring Mutations or possible variant antibodies that appear during the production of monoclonal antibodies (such variants usually exist in small amounts). Unlike the production of polyclonal antibodies, which usually contain different antibodies directed against different determinants (epitopes), monoclonal antibodies are prepared in which each monoclonal antibody is directed against a single epitope on the antigen. Thus, the modifier "monoclonal" refers to the characteristics of antibodies obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring any particular method to produce the antibodies. For example, monoclonal antibodies to be used in accordance with the present invention can be prepared by a variety of techniques including, but not limited to, fusion ), methods for transgenic animals of ), such methods and other exemplary methods for producing monoclonal antibodies are described herein.

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

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

The term "package insert" is used to refer to instructions usually included in commercial packages of therapeutic products, which contain information on indications, usage, dosage, administration, concomitant therapies, contraindications and/or information about such WARNINGS FOR USE OF THERAPY PRODUCTS.

"Percent (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent sequence identity, and not considering any conservative substitutions as In the case of partial sequence identity, the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the skill in the art, for example, 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. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been submitted with the user documentation to the US Copyright Office, Washington D.C., 20559, where it is registered under US Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available at Genentech, Inc., South San Francisco, California, or can be compiled from source code. ALIGN-2 programs should be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

In the case of using ALIGN-2 for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A over, and or relative to, a given amino acid sequence B (or it can be Expressed as a given amino acid sequence A having or comprising a specific % amino acid sequence identity over, and or relative to a given amino acid sequence B) calculated as: 100 multiplied by the fraction X/Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the alignment of A and B of the program, and where Y is the number of amino acid residues in B total. It will be understood that where 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 over B will not equal the % amino acid sequence identity of B over A sex. 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 preparation which is in such a form as to allow the biological activity of the active ingredients contained therein to be effective and which contains no additional ingredients which are unacceptably toxic to the subject to whom the formulation is administered.

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

The term "C5" as used herein encompasses any native C5 from any vertebrate source, including mammals such as primates (eg, humans and monkeys) and rodents (eg, mice and rats). Unless otherwise specified, the term "C5" refers to the human C5 protein having the amino acid sequence shown in SEQ ID NO: 39 and including the β (beta) chain sequence shown in SEQ ID NO: 40. The term encompasses "full length" unprocessed C5 as well as any form of C5 that results from intracellular processing. The term also encompasses 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 human C5 beta chain is shown in SEQ ID NO: 40. The amino acid sequences of the MG-1, MG-2 and MG1-MG2 domains of the β chain of exemplary human C5 are shown in SEQ ID NOs: 41, 42 and 43, respectively. Exemplary amino acid sequences of rhesus monkey and murine C5 are shown in SEQ ID NO: 44 and 105, respectively. Amino acid residues 1 to 19 of SEQ ID NO: 39, 40, 43, 44 and 105 correspond to message sequences which are removed during intracellular processing and therefore do not appear in the corresponding exemplary amino acid sequences.

"Treatment" (and its grammatical variants) as used herein refers to clinical intervention that attempts to alter the natural course of the disease in the individual being treated, and may be performed prophylactically or during the course of clinical pathology. Desired treatment effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliation of the disease state, and slowing or ameliorating prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or 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 binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies usually have a similar structure, each domain includes four conserved framework regions (FR) and three hypervariable regions (HVR) (see , eg Kindt et al., Kuby Immunology, 6 ^th ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a particular antigen can be isolated using the VH or VL domains from antibodies that bind that antigen to screen complementary VL or VH domain libraries, respectively. See, eg, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term "vector" as used herein refers to a nucleic acid molecule capable of multiplying another nucleic acid to which it has been linked. The term encompasses a vector that is an autonomously replicating nucleic acid structure, as well as a vector that is incorporated into the genome of a host cell into which it has been 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. COMPOSITION AND METHODS In one aspect, the invention is based, in part, on anti-C5 antibodies and uses thereof. In specific embodiments, antibodies that bind to C5 are provided. Antibodies of the invention are useful, for example, in the diagnosis and management of complement-regulated diseases or conditions involving excessive or uncontrolled C5 activation.

A. Exemplary Anti-C5 Antibodies In one aspect, the invention provides an isolated antibody that binds to C5. In specific implementations, an anti-C5 antibody of the invention binds to an epitope within the beta (beta) chain of C5. In specific implementations, the anti-C5 antibody binds to an epitope within the MG1-MG2 domain of the beta chain of C5. In a specific implementation, the anti-C5 antibody binds to an epitope within a fragment consisting of amino acids 19 to 180 of the beta chain of C5. In a specific implementation, the anti-C5 antibody binds to an epitope within the MG1 domain of the beta chain of C5 (amino acids 20 to 124 of SEQ ID NO: 40 (SEQ ID NO: 41 )). In a specific implementation, the anti-C5 antibody binds to an epitope within a fragment consisting of amino acids 33 to 124 of the beta chain of C5 (SEQ ID NO: 40). In another embodiment, the anti-C5 antibody does not bind to a fragment shorter than the fragment consisting of amino acids 33 to 124 of the beta chain of C5, for example, the amine of the beta chain of C5 (SEQ ID NO: 40). Fragments consisting of amino acids 45 to 124, 52 to 124, 33 to 111, 33 to 108 or 45 to 111.

In another aspect, the invention provides anti-C5 antibodies that exhibit pH-dependent binding properties. As used herein, the term "pH-dependent" means that the antibody exhibits "decreased binding to C5 at acidic pH compared to neutral pH" (both terms are used interchangeably for the purposes of this disclosure) . For example, an antibody "having pH-dependent binding properties" includes an antibody that binds to C5 with a higher affinity at neutral pH than at acidic pH. In particular embodiments, an antibody of the invention exhibits a pH of at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 at neutral pH as compared to at acidic pH. , 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more fold affinity to C5. In some embodiments, the antibody binds to C5 with a higher affinity at pH 7.4 than at pH 5.8. In yet other embodiments, the antibodies of the invention are at pH 7.4 at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 , 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more fold affinity to C5.

The "affinity" of an antibody for C5 is, for the purposes of this disclosure, expressed in terms of the KD of the antibody. The KD of an antibody refers to the equilibrium dissociation constant for the antibody-antigen interaction. The greater the KD value of an antigen binding to its antigen, the weaker its binding affinity for that particular antigen. Thus, as used herein, "higher affinity at neutral pH compared to acidic pH" (or the equivalent expression "pH-dependent binding") means that the antibody binds to C5 with a greater KD at acidic pH than at neutral pH. KD for binding to C5 at neutral pH. For example, in the context of the present invention, an antibody is considered to bind C5 at neutral pH as compared to acidic pH if its KD for binding to C5 at acidic pH is at least twice greater than the KD for binding to C5 at neutral pH. pH binds to C5 with higher affinity. Accordingly, the invention encompasses that the KD for binding to C5 at acidic pH is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more times the antibody. 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 greater.

In yet other embodiments, an antibody is considered to be at a neutral pH as compared to an acidic pH if the antibody binds to C5 with a KD at pH 5.8 that is at least two times greater than the antibody's KD for binding to C5 at pH 7.4. Binds to C5 with higher affinity. In some embodiments, provided antibodies have a KD that binds to C5 at pH 5.8 that is at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45 times greater than the KD of the antibody that binds to C5 at pH 7.4 , 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more times. In another embodiment, the KD value of the antibody at pH 7.4 may be 10 ⁻⁷ M, 10 ⁻⁸ M, 10 ⁻⁹ M, 10 ⁻¹⁰ M, 10 ⁻¹¹ M, 10 ⁻¹² M or less. In another embodiment, the KD value of the antibody at pH 5.8 may be 10 ⁻⁹ M, 10 ⁻⁸ M, 10 ⁻⁷ M, 10 ⁻⁶ M or greater.

The binding properties of an antibody to a specific antigen can also be expressed by the kd of the antibody. The kd of an antibody refers to the dissociation rate constant of the antibody for a specific antigen, expressed in the reciprocal of seconds (ie sec ^-1 ). An increased kd value indicates that the antibody binds weaker to its antigen. The invention thus encompasses antibodies that bind to C5 with a higher kd value at acidic pH compared to neutral pH. The present invention comprises binding to C5 at acidic pH with a kd at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 greater than the kd for binding to C5 at neutral pH , 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more times the antibody. In another embodiment, the kd value of the antibody may be 10 ^-2 1/s, 10 ^-3 1/s, 10 ^-4 1/s, 10 ^-5 1/s, 10 ^-6 1/s at neutral pH s or less. In another embodiment, the kd value of the antibody may be 10 ⁻³ 1/s, 10 ⁻² 1/s, 10 ⁻¹ 1/s or greater at acidic pH. The invention also encompasses antibodies that bind to C5 with a greater kd value at pH 5.8 compared to pH 7.4. The invention comprises that the kd binding to C5 at pH 5.8 is at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more times the antibody. In another embodiment, the kd value of the antibody at pH7.4 may be 10 ^-2 1/s, 10 ^-3 1/s, 10 ^-4 1/s, 10 ^-5 1/s, 10 ^-6 1/s s or less. In another embodiment, the antibody may have a kd value of 10 ⁻³ 1/s, 10 ⁻² 1/s, 10 ⁻¹ 1/s or greater at pH 5.8.

In a particular embodiment, "decreased binding to C5 at acidic pH compared to neutral pH" is defined as the ratio of the KD value for antibody binding to C5 at acidic pH to the KD value for antibody binding to C5 at neutral pH representation (or vice versa). For example, an antibody may be considered to have "reduced binding to C5 at acidic pH compared to neutral pH" for the purposes of the present invention if the antibody has an acidic/neutral KD ratio of 2 or greater. In some specific exemplary embodiments, the pH5.8/pH7.4 KD ratio of the antibodies of the invention may be 2 or greater. In some specific exemplary embodiments, the acidic/neutral KD ratio of the antibody of the present 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 greater. In a further example, for the purposes of the present invention, an antibody may be considered "at acidic pH with C5 relative to neutral pH" if the antibody has a pH 5.8/pH7. The combined reduction". In some specific exemplary embodiments, the pH5.8/pH7.4 KD ratio of the antibody of the present 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 KD value of the antibody at pH 7.4 may be 10 ⁻⁷ M, 10 ⁻⁸ M, 10 ⁻⁹ M, 10 ⁻¹⁰ M, 10 ⁻¹¹ M, 10 ⁻¹² M or less. In another embodiment, the KD value of the antibody at pH 5.8 may be 10 ⁻⁹ M, 10 ⁻⁸ M, 10 ⁻⁷ M, 10 ⁻⁶ M or greater.

In a particular example, "decreased binding to C5 at acidic pH compared to neutral pH" is defined as the ratio of the kd value for antibody binding to C5 at acidic pH to the kd value for antibody binding to C5 at neutral pH representation (or vice versa). For example, an antibody may be considered to have "reduced binding to C5 at acidic pH compared to neutral pH" for the purposes of the present invention if the antibody has an acidic/neutral kd ratio of 2 or greater. In some specific exemplary embodiments, antibodies of the invention may have a pH5.8/pH7.4 kd ratio of 2 or greater. In some specific exemplary embodiments, the acidic/neutral kd ratio of the antibody of the present 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 still other embodiments, the pH5.8/pH7.4 kd ratio of the antibody can be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more. In another embodiment, the kd value of the antibody at neutral pH may be 10 ^-2 1/s, 10 ^-3 1/s, 10 ^-4 1/s, 10 ^-5 1/s, 10 ^-6 1/s s or less. In yet another embodiment, the kd value of the antibody at pH7.4 may be 10 ⁻² 1/s, 10 ⁻³ 1/s, 10 ⁻⁴ 1/s, 10 ⁻⁵ 1/s, 10 ⁻⁶ 1/s s or less. In another embodiment, the kd value of the antibody at acidic pH may be 10 ⁻³ 1/s, 10 ⁻² 1/s, 10 ⁻¹ 1/s or greater. In yet another embodiment, the antibody may have a kd value of 10 ⁻³ 1/s, 10 ⁻² 1/s, 10 ⁻¹ 1/s or greater at pH 5.8.

As used herein, the term "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 , 6.2, 6.3, 6.4 and any of the pH values of 6.5. In certain aspects, the "acidic pH" is 5.8.

As used herein, the term "neutral pH" refers to a pH of 6.7 to about 10.0. The term "neutral pH" includes 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, Any pH value of 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 and 10.0. In certain aspects, "neutral pH" is 7.4.

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

In a specific embodiment, the anti-C5 antibody of the present invention binds to an epitope within the beta chain of C5, which consists of the MG1 domain (SEQ ID NO: 41). In a specific embodiment, the anti-C5 antibody of the present invention binds to an epitope in the beta chain of C5 (SEQ ID NO: 40), which includes at least one epitope selected from amino acids 47-57, 70-76 and 107 Fragment of the group consisting of -110. In a specific embodiment, the anti-C5 antibody of the present invention binds to an epitope within a fragment of the β chain of C5 (SEQ ID NO: 40), which includes at least one epitope selected from Thr47, Glu48, Ala49, Phe50, Asp51, Amino acids of the group consisting of Ala52, Thr53, Lys57, His70, Val71, His72, Ser74, Glu76, Val107, Ser108, Lys109 and His110. In a specific embodiment, the anti-C5 antibody of the present invention binds to an epitope within a fragment of the beta chain of C5 (SEQ ID NO: 40), which includes at least one epitope selected from Glu48, Asp51, His70, His72, Lys109 and Amino acids of the group consisting of His110. In specific embodiments, an anti-C5 antibody of the invention has reduced binding to a C5 mutant, wherein the C5 mutant has at least one amino acid substitution selected from Glu48, Asp51, Position of the group consisting of His72 and Lys109. In another embodiment, an anti-C5 antibody of the invention has reduced pH-dependent binding to a C5 mutant as compared to its pH-dependent binding to wild-type C5, wherein the C5 mutant has at least one amino acid substitution in Positions selected from the group consisting of His70, His72 and His110. In yet another embodiment, in the C5 mutant, amino acids at positions selected from Glu48, Asp51 and Lys109 are substituted with alanine, and amino acids at positions selected from His70, His72 and His110 The acid is replaced by tyrosine.

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

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

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

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

Without being bound by a particular theory, it is speculated that binding of an anti-C5 antibody to C5 is reduced (or nearly eliminated) when an amino acid residue on C5 is replaced by another amino acid, which represents the presence of that amine on C5. The amino acid residue is important for the interaction between the anti-C5 antibody and C5, and the antibody can recognize epitopes surrounding this amino acid on C5.

The present inventors have discovered that a population of anti-C5 antibodies that compete with another antibody or bind to the same epitope can exhibit pH-dependent binding properties. Among the amino acids, histidine, with a pKa value of about 6.0 to 6.5, can have different proton dissociation states between neutral and acidic pH. Thus, the 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 speculated that anti-C5 antibodies may recognize a conformational structure around the histidine residue on C5, which changes depending on pH. This presumably is consistent with the experimental results that the pH dependence of anti-C5 antibodies was reduced (or almost disappeared) when the histidine residue on C5 was replaced by another amino acid (i.e., at neutral pH). , an anti-C5 antibody with pH-dependent binding properties bound to the histidine mutant of C5 with a similar affinity to wild-type C5, whereas at acidic pH, the same antibody bound to wild-type C5 with a higher affinity. A histidine mutant that binds with affinity to C5).

In specific embodiments, an anti-C5 antibody of the invention binds to C5 from more than one species. In yet other embodiments, the anti-C5 antibody binds to C5 from human and non-human animals. In yet other embodiments, the anti-C5 antibody binds to antibodies from humans and monkeys (eg, cynomolgus, rhesus macaque, marmoset, chimpanzee, or baboon.

In one aspect, the invention provides anti-C5 antibodies that inhibit the activation of C5. In particular embodiments, anti-C5 antibodies are provided that prevent the cleavage of C5 to form C5a and C5b, thus preventing the generation of anaphylatoxin activity associated with C5a, and also preventing the generation of the C5b-9 membrane attack complex (MAC) associated with C5b combination. In specific embodiments, anti-C5 antibodies are provided that block the conversion of C5 to C5a and C5b by C5 convertase. In specific embodiments, anti-C5 antibodies are provided that block access of C5 convertase to the cleavage site on C5. In specific embodiments, anti-C5 antibodies are provided that block hemolytic activity resulting from activation of C5. In still other embodiments, the anti-C5 antibodies of the present invention inhibit the activation of C5 through the classical pathway and/or the alternative pathway.

In one aspect, the invention provides anti-C5 antibodies that inhibit the activation of C5 variants. The C5 variant refers to the variant of the C5 gene, which is caused by gene variation, such as mutation, polymorphism or allelic variation. Genetic variation may include deletions, substitutions or insertions of one or more nucleotides. C5 variants may include variations in one or more genes in C5. In certain embodiments, the C5 variant has a 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, R885H, for example, refers to a variant of a gene in which arginine at position 885 is replaced by histidine. In specific embodiments, an anti-C5 antibody of the invention inhibits activation of 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 present invention provides an anti-C5 antibody comprising at least one, two, three, four, five or six amines selected from (a) comprising any of SEQ ID NO: 45 to 54 (b) HVR-H2 including the amino acid sequence of any of the sequence identification numbers: 55 to 64; (c) including the amino acid sequence of any of the sequence identification numbers: 65 to 74 (d) HVR-L1 comprising the amino acid sequence of any of the sequence identification numbers: 75 to 84; (e) comprising the amino acid sequence of any of the sequence identification numbers: 85 to 94 HVR-L2; and (f) hypervariable regions (HVRs) of HVR-L3 comprising any amino acid sequence of SEQ ID NO: 95 to 104.

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

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

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

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

In one aspect, the present invention provides an anti-C5 antibody comprising at least one, two, three, four, five or six selected from (a) including sequence identification numbers: 45, 54, 117, 126 HVR-H1 of any amino acid sequence; (b) HVR-H2 including any amino acid sequence in sequence identification number: 55, 64, 118-120, 127; (c) including sequence identification number: HVR-H3 of any amino acid sequence in 65, 74, 121, 128; (d) HVR-L1 including any amino acid sequence in Sequence Identification Number: 75, 84, 122, 129; (e ) including the HVR-L2 of the amino acid sequence of any one of the sequence identification numbers: 85, 94, 123-124, 130; HVR of acid sequence HVR-L3.

In one aspect, the present invention provides an antibody comprising at least one, at least two or all three of the amino acid sequences selected from (a) comprising any of the sequence identification numbers: 45, 54, 117, 126 HVR-H1; (b) HVR-H2 including the amino acid sequence of any of the sequence identification numbers: 55, 64, 118-120, 127; and (c) including the sequence identification numbers: 65, 74, 121, 128 The VH HVR sequence of HVR-H3 of any amino acid sequence. In one embodiment, the antibody comprises HVR-H3, which comprises the amino acid sequence of any one of SEQ ID NO: 65, 74, 121, 128. In another embodiment, the antibody includes: HVR-H3, which includes the amino acid sequence of any one of the sequence identification numbers: 65, 74, 121, and 128, and HVR-L3, which includes the sequence identification numbers: 95, 104 , 125, 131 any amino acid sequence. In yet another embodiment, the antibody includes: HVR-H3, which includes the amino acid sequence of any one of the sequence identification numbers: 65, 74, 121, and 128, HVR-L3, which includes the sequence identification numbers: 95, 104, The amino acid sequence of any one of 125 and 131, and HVR-H2, which includes the amino acid sequence of any one of SEQ ID NO: 55, 64, 118-120, and 127. In yet another embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of any of Sequence Identification Numbers: 45, 54, 117, 126; (b) comprising Sequence Identification Numbers: 55, 64, 118 - HVR-H2 of any one of the amino acid sequences of 120 and 127; and (c) HVR-H3 comprising any of the amino acid sequences of SEQ ID NO: 65, 74, 121 and 128.

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

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

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

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

All possible combinations of the above substitutions are identified as the sequence identification numbers of the consensus sequences of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3: 126, 127, 128, 129, 130 and covered by 131.

In any of the above embodiments, the anti-C5 antibody is humanized. In one embodiment, the anti-C5 antibody comprises the HVR of any one of the above embodiments, and further comprises an acceptor human framework, for example, a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-C5 antibody includes HVR as in any of the above embodiments, and further includes VH or VL, which includes FR sequences, wherein the FR sequences are as follows. For the heavy chain variable domain, FR1 includes the amino acid sequence of any of the sequence identification numbers: 132 to 134, FR2 includes the amino acid sequence of any of the sequence identification numbers: 135 to 136, and FR3 includes the amino acid sequence of any of the sequence identification numbers: 137 Any amino acid sequence to 139, FR4 includes the sequence identification number: any amino acid sequence from 140 to 141. For the light chain variable domain, FR1 includes the amino acid sequence of any of the sequence identification numbers: 142 to 143, FR2 includes the amino acid sequence of any of the sequence identification numbers: 144 to 145, and FR3 includes the sequence identification number: 146 Any amino acid sequence to 147, FR4 includes the amino acid sequence of SEQ ID NO: 148.

In another aspect, an anti-C5 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92% of the amino acid sequence of any one of SEQ ID NO: 1 to 10 , 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some specific embodiments, VH sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, relative to the reference sequence , comprising substitutions (eg, conservative substitutions), insertions or deletions, but anti-C5 antibodies comprising that sequence retain the ability to bind to C5. In some specific embodiments, a total of 1 to 10 amino acids in any of SEQ ID NO: 1 to 10 are substituted, inserted and/or deleted. In some specific embodiments, substitutions, insertions or deletions occur in regions outside the HVRs (ie, in FRs). Optionally, the anti-C5 antibody comprises a VH sequence in any one of SEQ ID NO: 1 to 10 comprising post-translational modifications of that sequence. In a specific embodiment, the VH comprises one, two or three selected from: (a) HVR-H1 comprising the amino acid sequence of any of SEQ ID NO: 45 to 54, (b) comprising the sequence identified No.: HVR-H2 of any amino acid sequence of 55 to 64, and (c) HVR including HVR-H3 of any amino acid sequence of Sequence Identification No. 65 to 74.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises a light chain variable domain (VL), which has at least 90%, 91% of the amino acid sequence of any one of SEQ ID NO: 11 to 20 , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some specific embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, relative to the reference sequence , comprising substitutions (eg, conservative substitutions), insertions or deletions, but anti-C5 antibodies comprising that sequence retain the ability to bind to C5. In some specific embodiments, a total of 1 to 10 amino acids in any of SEQ ID NO: 11 to 20 are substituted, inserted and/or deleted. In some specific embodiments, substitutions, insertions or deletions occur in regions outside the HVRs (ie, in FRs). Optionally, the anti-C5 antibody comprises a VL sequence in any one of SEQ ID NO: 11 to 20 comprising post-translational modifications of that sequence. In a specific embodiment, VL comprises one, two or three selected from: (a) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NO: 75 to 84, (b) comprising Sequence Identification No.: HVR-L2 of any amino acid sequence of 85 to 94, and (c) HVR including HVR-L3 of any amino acid sequence of Sequence Identification No.: 95 to 104.

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 a VH sequence in any of SEQ ID NO: 1 to 10 and a VL sequence in any of SEQ ID NO: 11 to 20, respectively, comprising post-translational modifications of those sequences.

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

In another aspect, an anti-C5 antibody comprises a VH sequence having at least 90%, 91%, 92%, 93%, 94% of the amino acid sequence of any one of SEQ ID NO: 10, 106-110 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some specific embodiments, VH sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, relative to the reference sequence , comprising substitutions (eg, conservative substitutions), insertions or deletions, but anti-C5 antibodies comprising that sequence retain the ability to bind to C5. In some specific embodiments, a total of 1 to 10 amino acids in any of SEQ ID NO: 10, 106 to 110 are substituted, inserted and/or deleted. In some specific embodiments, substitutions, insertions or deletions occur in regions outside the HVRs (ie, in FRs). Optionally, the anti-C5 antibody comprises a VH sequence in any one of SEQ ID NO: 10, 106 to 110 comprising post-translational modifications of that sequence. In a specific embodiment, VH comprises one, two or three selected from: (a) HVR-H1 comprising any of the amino acid sequences of SEQ ID NO: 45, 54, 117, 126, (b ) HVR-H2 comprising the amino acid sequence of any of the sequence identification numbers: 55, 64, 118-120, and 127, and (c) comprising any of the amino acid sequences of the sequence identification numbers: 65, 74, 121, and 128 HVR of the acid sequence HVR-H3.

In another aspect, an anti-C5 antibody comprises a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 10 , 97%, 98%, 99%, or 100% sequence identity. In some specific embodiments, the VH sequence is the amino acid sequence of SEQ ID NO: 10. In some specific embodiments, VH sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, relative to the reference sequence , comprising substitutions (eg, conservative substitutions), insertions or deletions, but anti-C5 antibodies comprising that sequence retain the ability to bind to C5. In some specific embodiments, in SEQ ID NO: 10, a total of 1 to 10 amino acids are substituted, inserted and/or deleted. In some specific embodiments, substitutions, insertions or deletions occur in regions outside the HVRs (ie, in FRs). Alternatively, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 10, which comprises post-translational modifications of that sequence. In a specific embodiment, the VH comprises one, two or three selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 54, (b) the amine comprising SEQ ID NO: 64 HVR-H2 of the amino acid sequence, and (c) HVR of HVR-H3 comprising the amino acid sequence of SEQ ID NO: 74.

In another aspect, an anti-C5 antibody comprises a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 106 , 97%, 98%, 99%, or 100% sequence identity. In some specific embodiments, the VH sequence is the amino acid sequence of SEQ ID NO: 106. In some specific embodiments, VH sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, relative to the reference sequence , comprising substitutions (eg, conservative substitutions), insertions or deletions, but anti-C5 antibodies comprising that sequence retain the ability to bind to C5. In some specific embodiments, in SEQ ID NO: 106, a total of 1 to 10 amino acids are substituted, inserted and/or deleted. In some specific embodiments, substitutions, insertions or deletions occur in regions outside the HVRs (ie, in FRs). Alternatively, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 106 comprising post-translational modifications of that sequence. In a specific embodiment, the VH comprises one, two or three selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) the amine comprising SEQ ID NO: 118 HVR-H2 of the amino acid sequence, and (c) HVR of HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121.

In another aspect, an anti-C5 antibody comprises a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 107 , 97%, 98%, 99%, or 100% sequence identity. In some specific embodiments, the VH sequence is the amino acid sequence of SEQ ID NO: 107. In some specific embodiments, VH sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, relative to the reference sequence , comprising substitutions (eg, conservative substitutions), insertions or deletions, but anti-C5 antibodies comprising that sequence retain the ability to bind to C5. In some specific embodiments, in SEQ ID NO: 107, a total of 1 to 10 amino acids are substituted, inserted and/or deleted. In some specific embodiments, substitutions, insertions or deletions occur in regions outside the HVRs (ie, in FRs). Alternatively, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 107 comprising post-translational modifications of that sequence. In a specific embodiment, the VH comprises one, two or three selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) the amine comprising SEQ ID NO: 119 HVR-H2 of the amino acid sequence, and (c) HVR of HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121.

In another aspect, an anti-C5 antibody comprises a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 108 , 97%, 98%, 99%, or 100% sequence identity. In some specific embodiments, the VH sequence is the amino acid sequence of SEQ ID NO: 108. In some specific embodiments, VH sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, relative to the reference sequence , comprising substitutions (eg, conservative substitutions), insertions or deletions, but anti-C5 antibodies comprising that sequence retain the ability to bind to C5. In some specific embodiments, in SEQ ID NO: 108, a total of 1 to 10 amino acids are substituted, inserted and/or deleted. In some specific embodiments, substitutions, insertions or deletions occur in regions outside the HVRs (ie, in FRs). Alternatively, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 108 comprising post-translational modifications of that sequence. In a specific embodiment, the VH comprises one, two or three selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) the amine comprising SEQ ID NO: 118 HVR-H2 of the amino acid sequence, and (c) HVR of HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121.

In another aspect, an anti-C5 antibody comprises a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 109 , 97%, 98%, 99%, or 100% sequence identity. In some specific embodiments, the VH sequence is the amino acid sequence of SEQ ID NO: 109. In some specific embodiments, VH sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, relative to the reference sequence , comprising substitutions (eg, conservative substitutions), insertions or deletions, but anti-C5 antibodies comprising that sequence retain the ability to bind to C5. In some specific embodiments, in SEQ ID NO: 109, a total of 1 to 10 amino acids are substituted, inserted and/or deleted. In some specific embodiments, substitutions, insertions or deletions occur in regions outside the HVRs (ie, in FRs). Alternatively, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 109 comprising post-translational modifications of that sequence. In a specific embodiment, the VH comprises one, two or three selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) the amine comprising SEQ ID NO: 118 HVR-H2 of the amino acid sequence, and (c) HVR of HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121.

In another aspect, an anti-C5 antibody comprises a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 110 , 97%, 98%, 99%, or 100% sequence identity. In some specific embodiments, the VH sequence is the amino acid sequence of SEQ ID NO: 110. In some specific embodiments, VH sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, relative to the reference sequence , comprising substitutions (eg, conservative substitutions), insertions or deletions, but anti-C5 antibodies comprising that sequence retain the ability to bind to C5. In some specific embodiments, in SEQ ID NO: 110, a total of 1 to 10 amino acids are substituted, inserted and/or deleted. In some specific embodiments, substitutions, insertions or deletions occur in regions outside the HVRs (ie, in FRs). Alternatively, the anti-C5 antibody comprises the VH sequence in SEQ ID NO: 110 comprising post-translational modifications of that sequence. In a specific embodiment, the VH comprises one, two or three selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117, (b) the amine comprising SEQ ID NO: 120 HVR-H2 of the amino acid sequence, and (c) HVR of HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some specific embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, relative to the reference sequence , comprising substitutions (eg, conservative substitutions), insertions or deletions, but anti-C5 antibodies comprising that sequence retain the ability to bind to C5. In some specific 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 some specific embodiments, substitutions, insertions or deletions occur in regions outside the HVRs (ie, in FRs). Optionally, the anti-C5 antibody comprises a VL sequence in any of SEQ ID NO: 20, 111-113 comprising post-translational modifications of that sequence. In a specific embodiment, VL comprises one, two or three selected from: (a) HVR-L1 comprising any of the amino acid sequences of SEQ ID NO: 75, 84, 122, 129, (b ) HVR-L2 comprising the amino acid sequence of any of the sequence identification numbers: 85, 94, 123-124, 130, and (c) comprising any of the amino acid sequences of the sequence identification numbers: 95, 104, 125, 131 HVR of acid sequence HVR-L3.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises a VL having at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 20 , 96%, 97%, 98%, 99%, or 100% sequence identity. In some specific embodiments, the VL sequence is the amino acid sequence of SEQ ID NO: 20. In some specific embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, relative to the reference sequence , comprising substitutions (eg, conservative substitutions), insertions or deletions, but anti-C5 antibodies comprising that sequence retain the ability to bind to C5. In some specific embodiments, in SEQ ID NO: 20, a total of 1 to 10 amino acids are substituted, inserted and/or deleted. In some specific embodiments, substitutions, insertions or deletions occur in regions outside the HVRs (ie, in FRs). Alternatively, the anti-C5 antibody comprises the VL sequence in SEQ ID NO: 20 comprising post-translational modifications of that sequence. In a specific embodiment, VL comprises one, two or three selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 84, (b) the amine comprising SEQ ID NO: 94 HVR-L2 of the amino acid sequence, and (c) HVR of HVR-L3 comprising the amino acid sequence of SEQ ID NO: 104.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises a VL having at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 111 , 96%, 97%, 98%, 99%, or 100% sequence identity. In some specific embodiments, the VL sequence is the amino acid sequence of SEQ ID NO: 111. In some specific embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, relative to the reference sequence , comprising substitutions (eg, conservative substitutions), insertions or deletions, but anti-C5 antibodies comprising that sequence retain the ability to bind to C5. In some specific embodiments, in SEQ ID NO: 111, a total of 1 to 10 amino acids are substituted, inserted and/or deleted. In some specific embodiments, substitutions, insertions or deletions occur in regions outside the HVRs (ie, in FRs). Alternatively, the anti-C5 antibody comprises the VL sequence in SEQ ID NO: 111 comprising post-translational modifications of that sequence. In a specific embodiment, VL comprises one, two or three selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (b) the amine comprising SEQ ID NO: 123 HVR-L2 of the amino acid sequence, and (c) HVR of HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises a VL having at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 112 , 96%, 97%, 98%, 99%, or 100% sequence identity. In some specific embodiments, the VL sequence is the amino acid sequence of SEQ ID NO: 112. In some specific embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, relative to the reference sequence , comprising substitutions (eg, conservative substitutions), insertions or deletions, but anti-C5 antibodies comprising that sequence retain the ability to bind to C5. In some specific embodiments, in SEQ ID NO: 112, a total of 1 to 10 amino acids are substituted, inserted and/or deleted. In some specific embodiments, substitutions, insertions or deletions occur in regions outside the HVRs (ie, in FRs). Alternatively, the anti-C5 antibody comprises the VL sequence in SEQ ID NO: 112 comprising post-translational modifications of that sequence. In a specific embodiment, VL comprises one, two or three selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (b) the amine comprising SEQ ID NO: 123 HVR-L2 of the amino acid sequence, and (c) HVR of HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises a VL having at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 113 , 96%, 97%, 98%, 99%, or 100% sequence identity. In some specific embodiments, the VL sequence is the amino acid sequence of SEQ ID NO: 113. In some specific embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, relative to the reference sequence , comprising substitutions (eg, conservative substitutions), insertions or deletions, but anti-C5 antibodies comprising that sequence retain the ability to bind to C5. In some specific embodiments, in SEQ ID NO: 113, a total of 1 to 10 amino acids are substituted, inserted and/or deleted. In some specific embodiments, substitutions, insertions or deletions occur in regions outside the HVRs (ie, in FRs). Alternatively, the anti-C5 antibody comprises the VL sequence in SEQ ID NO: 113 comprising post-translational modifications of that sequence. In a specific embodiment, VL comprises one, two or three selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (b) the amine comprising SEQ ID NO: 124 HVR-L2 of the amino acid sequence, and (c) HVR of HVR-L3 comprising 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 a VH sequence in any of SEQ ID NO: 10, 106 to 110 and a VL sequence in any of SEQ ID NO: 20, 111 to 113, respectively, comprising the sequence of any of those sequences Post-translational modification. In one embodiment, the antibody comprises a VH sequence of SEQ ID NO: 10, and a VL sequence of SEQ ID NO: 20. In one embodiment, the antibody comprises a VH sequence of SEQ ID NO: 106, and a VL sequence of SEQ ID NO: 111. In another embodiment, the antibody comprises a VH sequence of SEQ ID NO: 107, and a VL sequence of SEQ ID NO: 111. In yet another embodiment, the antibody comprises a VH sequence of SEQ ID NO: 108, and a VL sequence of SEQ ID NO: 111. In another embodiment, the antibody comprises a VH sequence of SEQ ID NO: 109, and a VL sequence of SEQ ID NO: 111. In another embodiment, the antibody comprises a VH sequence of SEQ ID NO: 109, and a VL sequence of SEQ ID NO: 112. In another embodiment, the antibody comprises a VH sequence of SEQ ID NO: 109, and a VL sequence of SEQ ID NO: 113. In another embodiment, the antibody comprises a VH sequence of SEQ ID NO: 110, and a 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) comprising the amine of SEQ ID NO: 64 HVR-H2 of the amino acid sequence, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 74, and a VL sequence comprising (a) HVR comprising the amino acid sequence of SEQ ID NO: 84 -L1; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 94; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 104.

In another 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) comprising the amino acid sequence of SEQ ID NO: 118 HVR-H2 of amino acid sequence, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121, and VL sequence comprising (a) comprising the amino acid sequence of SEQ ID NO: 122 HVR-L1; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125.

In another 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) comprising the amino acid sequence of SEQ ID NO: 119 HVR-H2 of amino acid sequence, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121, and VL sequence comprising (a) comprising the amino acid sequence of SEQ ID NO: 122 HVR-L1; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125.

In another 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) comprising the amino acid sequence of SEQ ID NO: 118 HVR-H2 of amino acid sequence, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121, and VL sequence comprising (a) comprising the amino acid sequence of SEQ ID NO: 122 HVR-L1; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 124; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125.

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

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

In another aspect, the invention provides an antibody that binds to the same epitope as an anti-C5 antibody provided herein. For example, in some specific embodiments, provided antibodies bind to the same epitope as the antibodies described in Table 2. As shown in the working examples below, all anti-C5 antibodies described in Table 2 were sorted into the same epitope bin of C5 and exhibited pH-dependent binding properties.

In yet another aspect, the invention provides an antibody that binds to the same epitope as an antibody provided herein. In yet another aspect, the invention provides an antibody that binds to the same epitope as the antibody described in Table 7 or 8. In some specific embodiments, an antibody is provided that binds to an epitope within a fragment consisting of amino acids 33 to 124 of the beta (beta) chain of C5 (SEQ ID NO: 40). In some specific embodiments, an antibody is provided that binds to an epitope in the beta chain of C5 (SEQ ID NO: 40) comprising at least one selected from amino acids 47-57, 70-76 and 107 Fragment of the group consisting of -110. In some specific embodiments, an antibody is provided that binds to an epitope within a fragment of the β chain of C5 (SEQ ID NO: 40), which includes at least one selected from Thr47, Glu48, Ala49, Phe50, Asp51, Amino acids of the group consisting of 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 yet another aspect, the anti-C5 antibody according to any of the above embodiments is a monoclonal antibody, including chimeric, humanized or human antibodies. In one embodiment, the anti-C5 antibody is an antibody fragment, eg, Fv, Fab, Fab', scFv, diabody, or F(ab') ₂ fragment. In another embodiment, the antibody is a full length antibody, eg, a full IgGl or IgG4 antibody or other antibody class or isotype as defined herein.

In yet another aspect, an anti-C5 antibody according to any of the above embodiments may incorporate any of the features, singly or in combination, as described in Sections 1-7 below:

1. Antibody Affinity In certain specific embodiments, the antibodies provided herein have an affinity of 1 µM (micro M) or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less , a dissociation constant of 0.01 nM or less, or 0.001 nM or less (eg, 10 ^-8 M or less, eg, from 10 ^-8 M to 10 ^-13 M, eg, from 10 ^-9 M to 10 ^-13 M) (Kd).

In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, RIA is performed in Fab format of the antibody of interest and its antigen. Solution binding of Fab to antigen is measured, for example, by equilibrating the Fab with a minimal concentration of (125I)-labeled antigen in the presence of a titration series of unlabeled antigen, followed by capturing the bound antigen on an anti-Fab antibody-coated dish Affinity (see, eg, Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish assay conditions, MICROTITER (registered trademark) multiwell plates (Thermo Scientific) were coated overnight with 5 micrograms/milliliter (μg/ml) anti-Fab antibody (Cappel Labs) for capture in 50 mM sodium carbonate (pH 9.6), Blocking was then performed with 2% (w/v) bovine serum albumin in PBS at room temperature (approximately 23 degrees C (°C)) for 2 to 5 hours. In non-adsorbent plates (Nunc #269620), mix 100 pM or 26 pM [125I]-antigen with serial dilutions of the Fab of interest (eg, with Presta et al., Cancer Res. 57:4593-4599 (1997) against VEGF Antibodies, Fab-12 assessed in agreement) mixed. The Fab of interest is then cultured overnight; however, culture can be continued for a longer period of time (eg, about 65 hours) to ensure equilibrium is reached. Thereafter, the mixture is transferred to a capture plate for incubation at room temperature (eg, 1 hour). Then the solution was removed and the plate was washed 8 times with PBS containing 0.1% polysorbate 20 (TWEEN-20 (registered trademark)). When the disk had dried, 150 μl/well of scintillation fluid (MICROSCINT-20 ^™ ; Packard) was added and the disk was counted for 10 minutes on a TOPCOUNT ^™ gamma counter (Packard). Concentrations of each Fab giving less than or equal to 20% of maximal binding were chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using BIACORE (registered trademark) surface plasmon resonance assay. For example, an assay using BIACORE(registered trademark)-2000 or BIACORE(registered trademark)-3000 (BIACORE(registered trademark), Inc., Piscataway, NJ) at 25°C with an immobilized antigen CM5 chip at about 10 response units (RU). In one embodiment, N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) activated carboxymethylated polydextrose biosensor chip (CM5, BIACORE (registered trademark), Inc.). Antigen was diluted to 5 μg/ml (approximately 0.2 μM (microM)). Following injection of antigen, 1M 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 containing 0.05% polysorbate 20 (TWEEN-20 ^™ ) surfactant at 25°C at a flow rate of approximately 25 μl/min. (PBST). Association rates (kon) and dissociation rates (koff) 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). Equilibrium dissociation constants (Kd) were calculated as the ratio koff/kon. See, eg, Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 10 ⁶ M ⁻¹ s ⁻¹ according to the surface plasmon resonance assay described above, the on-rate is determined using the fluorescence quenching technique measured at 20 nM in PBS, pH 7.2, at 25° C. An increase or decrease in the fluorescence emission intensity (excitation=295nm; emission=340nm, 16nm bandpass) of an anti-antigen antibody (Fab format) in a spectrometer, such as a spectrophotometer equipped with a stop-flow device ( Aviv Instruments) or an 8000 series SLM-AMINCO ^™ spectrophotometer (ThermoSpectronic) with agitated cuvettes to measure the increasing concentration of the antigen in the presence.

2. Antibody Fragments In some specific embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') ₂ , Fv, and scFv fragments, and other fragments as described below. For a review of specific antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For a review of specific scFv fragments see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. (Springer-Verlag, New York), pp. 269-315 (1994); 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 with increased in vivo half-life, see US Patent No. 5,869,046.

A diobody is an antibody fragment with two antigen binding sites, which may be bivalent or bispecific, see for example 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). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

A single domain antibody is an antibody fragment that includes all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In some specific embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, eg, US Patent No. 6,248,516).

Antibody fragments can be formed by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies, and production by recombinant host cells (eg, E. coli or phage), as described herein.

3. Chimeric and humanized antibodies In some specific embodiments, the antibodies provided herein are chimeric antibodies. Specific chimeric antibodies are described, eg, 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 includes non-human variable regions (eg, variable regions derived from mouse, rat, rodent, rabbit, or a non-human primate such as monkey) and human constant regions. In yet another example, a chimeric antibody is a "class switched" antibody, wherein the class or subclass has been changed from that of the parent antibody. A chimeric antibody comprises an antigen-binding fragment thereof.

In some specific embodiments, chimeric antibodies are humanized antibodies. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, eg, CDRs (or portions thereof) are derived from non-human antibodies and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally also will comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity sex.

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

Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using a "best fit" approach (see, e.g., Sims et al., J. Immunol. 151:2296 (1993); derived from a particular subtype The framework regions of the consensus sequences of human antibodies for light or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); and Presta et al., J . Immunol. 151:2623 (1993); Human mature (somatically mutated) or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR databases (see, e.g., 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 some specific embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using a variety of well-known techniques. 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 Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or part of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly within 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, please refer to Lonberg, Nat. Biotech. 23:1117-1125 (2005). Please also refer to, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE ^™ technology; U.S. Patent No. 5,770,429, which describes HuMab(R) technology; U.S. Patent No. 7,041,870, which describes KM MOUSE(R) technology, and U.S. Patent No. Publication No. US 2007/0061900, which describes VELOCIMOUSE (registered trademark) technology). The human variable regions of intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

Human antibodies can also be generated by fusionoma-based methods. Human myeloma and mouse-human fusion myeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor, J. Immunol. 133:3001 (1984); Brodeur et al., Monoclonal antibody Production Techniques and Applications, pp. 51-63 (Dekker, Inc., New York, 1987); and Boerner et al. , J. Immunol. 147:86 (1991). Human antibodies produced by human B cell fusion tumor technology are also described in Li et al., Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006). Other methods include those described, for example, in U.S. Pat. No. 7,189,826 (describing production of monoclonal human IgM antibodies by fusionoma cell lines) and Ni, Xiandai Mianyixue 26(4):265-268 (2006) (describing human-human fusionoma cell lines ) method. Human fusion tumor 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 variable domain sequences of Fv strains selected from human phage display databases. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody databases are described below.

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

In a specific phage display method, the repertoires of VH and VL genes are respectively selected by polymerase chain reaction (PCR) and randomly recombined in the phage database, which can then be obtained by such as Winter et al., Ann . Rev. Immunol. 12:433-455 (1994) in which antigen-binding phage were screened. Phage typically present antibody fragments in the form of single-chain Fv (ScFv) fragments or Fab fragments. Libraries from immune sources provide high-affinity antibodies to immunogens without the need to construct fusionomas. Alternatively, natural repertoires can be cloned (e.g., from humans) to provide a single source of antibodies against a broad range of non-self antigens as well as self antigens without any immunization, as in Griffiths et al., EMBO J, 12:725-734 (1993). Finally, natural repertoires can also be generated synthetically by transplanting unrearranged V gene segments from stem cells, using PCR primers containing random sequences to encode the hypervariable CDR3 region, and performing in vitro rearrangements, As described by Hoogenboom, J. Mol. Biol. 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Patent No. 5,750,373, and U.S. Patent Publication Nos. 0292936 and 2009/0002360.

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

6. Multispecific Antibodies In some specific 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 some specific embodiments, one of the binding specificities is for C5 and the other is for any other antigen. In some specific embodiments, bispecific antibodies can bind to two different epitopes of C5. Bispecific antibodies can also be used to localize cytotoxic agents to C5-expressing cells. Bispecific antibodies can be produced as full-length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression (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). Multi-specific antibodies can also be produced by the following methods: engineering electrostatic guidance for the preparation of antibody Fc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or more antibodies or fragments ( See, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science 229:81 (1985)); use of leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol. 148 (5 ):1547-1553 (1992)); use of "diabody" technology to generate bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)) and the use of single-chain Fv (scFv) diabodies (see, for example, Gruber et al., J. Immunol. 152:5368 (1994)); and the preparation of trispecific antibodies, such as, for example, Tutt et al., J. Immunol. 147:60 (1991).

Also included herein are engineered antibodies having three or more functional antigen-binding sites, including "Octopus antibodies" (see, eg, US 2006/0025576).

Antibodies or fragments herein also include "dual acting FAbs" or "DAFs" that include an antigen binding site that binds to C5 as well as a different antigen (see, eg, US 2008/0069820).

7. Antibody variants In some specific 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 antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions, and/or insertions and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions and substitutions can be made into the final construct provided that the final construct possesses the desired characteristics, eg, antigen binding.

a. Substitution, Insertion, and Deletion Variants In certain specific embodiments, antibody variants having one or more amino acid substitutions are provided. Positions of interest for substitution mutagenesis include HVR and FR. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions". More substantial changes are provided in Table 1 under the heading "Exemplary Substitutions" and are described further below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into an antibody of interest and the products screened for a desired activity, eg, retained/improved antibody binding, reduced immunogenicity, or improved ADCC or CDC. Table 1 original residue exemplary substitution preferred substitution Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Gln Asp (D) Glu;Asn Glu Cys (C) Ser; Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; 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; Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Trp (W) Tyr; Tyr Tyr (Y) Trp; Phe; Thr; Phe Val (V) Ile; Leu; Met; Phe; Ala; Leu

Amino acids can be classified according to common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral and hydrophilic: 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 will exchange a member of one of these classes for another.

One class of substitutional variants involves substituting one or more hypervariable region residues of a parent antibody (eg, a humanized or human antibody). In general, the resulting variant selected for further study will have a modification (e.g., improvement) relative to the parental antibody in a particular biological property (e.g., increased affinity, reduced immunogenicity) and/or will have Certain biological properties of the parent antibody are substantially retained. Exemplary substitutional variants are affinity matured antibodies, which can be conveniently produced, for example, 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 particular biological activity (eg, binding affinity).

Alterations (eg, substitutions) can be made in the HVR, eg, to improve antibody affinity. Such modifications can be made in HVR "hotspots", residues encoded by codons that undergo mutation at high frequency during somatic cell maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179- 196 (2008), and/or residues that contact the antigen, and test the binding affinity of the obtained variant VH or VL. Affinity maturation by construction and reselection from a second database has been done in, for example, Hoogenboom et al. , Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001) described. In some embodiments of affinity maturation, by any of a variety of methods One (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis) introduces diversity into the variable genes selected for maturation. A second database is then generated. The database is then screened to identify genes with the desired Affinity of any antibody variant. Another method of introducing diversity involves an HVR-directed approach, in which several HVR residues are randomized (for example, 4 to 6 residues at a time). HVR residues involved in antigen binding can be Specifically identified, eg using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are particularly frequently targeted.

In some specific embodiments, substitutions, insertions or deletions may occur within one or more HVRs, so long as such modifications do not substantially reduce the ability of the antibody to bind antigen. For example, conservative modifications (eg, conservative substitutions as provided herein) can be made in HVRs that do not substantially reduce binding affinity. Such modifications may, for example, be outside of the antigen contacting residues in the HVR. In certain examples of the variant VH and VL sequences provided above, each HVR is either unmodified, or contains no more than one, two or three amino acid substitutions.

One method that can be used to identify residues or regions of an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham, Science 244:1081-1085 (1989). In this method, a residue or group of residues (e.g., charged residues such as arg, asp, his, lys, and glu) can be identified and neutralized or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether it affects the interaction of the antibody with the antigen. Further substitutions can be introduced at amino acid positions showing functional susceptibility to initial substitution. Alternatively, or additionally, the crystal structure of the antigen-antibody complex identifies contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain the desired property.

Amino acid sequence insertions include amino- and/or carboxy-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, and within-sequences of single or multiple amino acid residues insert. Examples of terminal inserts include antibodies with an N-terminal methionyl residue. Other insertional variants of the antibody molecule comprise fusions to the N- or C-terminus of the antibody to an enzyme (eg, ADEPT) or polypeptide that increases the serum half-life of the antibody.

b. Glycosylation variants In some specific embodiments, the antibodies provided herein are modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to antibodies can be conveniently accomplished by modifying the amino acid sequence such that one or more glycosylation sites are created or removed.

When the antibody includes an Fc region, the sugars attached to it can be modified. Native antibodies produced by mammalian cells typically comprise branched, biantennary oligosaccharides attached, typically via an N-linkage, to Asn297 of the CH2 domain of the Fc region, see, e.g., Wright et al., TIBTECH 15: 26-32 (1997). Oligosaccharides may comprise sugars such as mannose, N-acetylglucosamine (GIcNAc), galactose, and sialic acid, as well as fucose attached to the GIcNAc in the "backbone" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the invention may be modified to produce antibody variants with particular improved properties.

In one embodiment, antibody variants are provided that have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies may be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. The amount of fucose was calculated by calculating the average amount of fucose at Asn297 within the sugar chain relative to the sum of all glycosyl structures attached to Asn297 as measured by MALDI-TOF mass spectrometry (e.g., complexes, hybrids and high mannose structures) are determined, for example, as described in WO 2008/077546. Asn297 refers to an asparagine residue located in the Fc region at approximately position 297 (Eu numbering of Fc region residues); however, due to minor sequence variations in antibodies, Asn297 can also be located approximately +/- 3 positions from position 297 Amino acid upstream or downstream, ie between positions 294 and 300. Such fucosylated variants may have improved ADCC function, please refer to, for example, US Patent Publication Nos. 2003/0157108 (Presta, L.); 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: 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; ; 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 Lecl3 CHO cells deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US 2003/0157108, Presta, L; and WO2004/056312, Adams et al., especially in Example 11), and knockout cell lines such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells ( See, eg, Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda et al., Biotechnol. Bioeng. 94(4):680-688 (2006); and WO2003/085107).

Also provided are antibody variants having bisected oligosaccharides, eg, wherein a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by a GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, eg, 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 attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, eg, in WO 1997/30087 (Patel et al); WO 1998/58964 (Raju, S); and WO 1999/22764 (Raju, S).

c. Fc region variants In some specific 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 can include human Fc region sequences (eg, human IgGl, IgG2, IgG3, or IgG4 Fc regions) that include amino acid modifications (eg, substitutions) at one or more amino acid positions.

In some specific embodiments, the present invention contemplates antibody variants with some but not all effector functions, making them ideal candidates for applications where the in vivo half-life of the antibody is important , however certain effector functions such as complement and ADCC are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be performed to confirm reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks Fcγ(gamma)R binding (and thus likely lacks ADCC activity), but retains FcRn binding ability. The main cells that regulate ADCC, NK cells, express only FcyRIII, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). A non-limiting example of an in vitro assay for assessing ADCC activity of a molecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986 )) and Hellstrom et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); US Pat. 1987)). Alternatively, nonradioactive assays can be used (see, e.g., ACTI™ Nonradioactive Cytotoxicity Assay for Flow Cytometry (Cell Technology, Inc. Mountain View, CA); and CytoTox 96(R) Nonradioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays 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, e.g., in an animal model, such as in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998) disclosed. Clq binding assays can also be performed to determine that the antibody is unable to bind Clq and thus lacks CDC activity. Please refer, for example, to Clq and C3c binding ELISAs in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg et al., Blood 101:1045-1052 (2003); and Cragg et al., Blood 103:2738-2743 (2004)). 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 those having substitutions in one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc mutants comprise two or more Fc mutants with substitutions in amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA "Fc mutants (US Patent No. 7,332,581).

Specific antibody variants with improved or reduced binding to FcRs have been described. (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, antibody variants comprise an Fc region with one or more amino acid substitutions that increase ADCC, e.g., at positions 298, 333, and/or 334 (EU numbering of residues) of the Fc region. replace.

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

Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al.), are described in US2005/0014934 (Hinton et al. , J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). Those antibodies include an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants are comprised of residues in the Fc region: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, One or more of 413, 424, or 434 have those substituted, eg, of Fc region residue 434 (US Pat. No. 7,371,826).

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

d) Cysteine engineered antibody variants In some specific embodiments, it may be desirable to generate cysteine engineered antibodies, e.g., "thioMAbs," in which one or more residues of the antibody are substituted with cysteine residues . In particular embodiments, the substituted residue is present at an antibody accessible position. By substituting those residues with cysteine, a reactive thiol group is placed in an accessible position of the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or link drug moieties, to create immunoconjugates Linkages, as further described herein. In some specific embodiments, any one or more of the following residues may be substituted by cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 of the Fc region of the heavy chain (EU number). Cysteine engineered antibodies can be produced, eg, as described in US Pat. No. 7,521,541.

(poly-1,3,6-trioxane)、乙烯/順丁烯二酸酐共聚物、聚胺基酸(同聚物或隨機共聚物)，及聚葡萄糖或聚(n-乙烯基吡咯啶酮)聚乙二醇、聚丙二醇同聚物、聚環氧丙烷/環氧乙烷共聚物、聚氧乙烷化多元醇(例如，丙三醇)、聚乙烯醇、及其混合物。聚乙二醇丙醛可在生產中具有優勢由於其在水中的穩定性。多聚物可具有任一分子量，且可為分支或無分支的。附著於抗體的多聚物的數目可不同，且如果附著多於一個多聚物，它們可以是相同或不同的分子。一般而言，用於衍生化的多聚物的數量及/或類型可根據，包含但不限於，考慮待改善的抗體的特別的性質或功能、抗體衍生物是否將在定義的條件下用於療法中等加以決定。 e) Antibody Derivatives In certain specific embodiments, the antibodies provided herein can be further modified to include additional non-protein moieties known in the art and readily available. Moieties suitable for derivatization of antibodies include, but are not limited to, water soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, polydextrose, polyvinyl alcohol, polyvinylpyrrolidone, Poly-1,3-dioxolane, poly-1,3,6-three 㗁

(poly-1,3,6-trioxane), ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and polydextrose or poly(n-vinylpyrrolidone) Polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyols (eg, glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water. 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, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined according to, including but not limited to, consideration of the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used under defined conditions, Therapy is determined in the middle.

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

B. Recombinant Methods and Compositions Antibodies can be produced using recombinant methods and compositions, eg, as described in US 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-C5 antibody described herein is provided. Such nucleic acids can encode amino acid sequences comprising the VL and/or amino acid sequences comprising the VH of the antibody (eg, the light and/or heavy chains of the antibody). In yet another embodiment, one or more vectors (eg, expression vectors) comprising such nucleic acids are provided. In yet another embodiment, a host cell comprising such nucleic acid is provided. In this embodiment, the host cell comprises (eg, has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2 ) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is a eukaryote, eg, Chinese Hamster Ovary (CHO) cells or lymphocytes (eg, Y0, NSO, Sp20 cells). In one embodiment, there is provided a method of preparing an anti-C5 antibody, wherein the method comprises culturing a host cell as provided above comprising a nucleic acid encoding an antibody under conditions suitable for expression of the antibody, and optionally extracting the antibody from the host cell (or host cell culture medium) to recover the antibody.

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

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

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

Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A variety of baculovirus strains have been identified that can be used with insect cells, particularly for the 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 (describing PLANTIBODIES™ technology for antibody production in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be used. Other examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 cell line (COS-7); the human embryonic kidney cell line (293 or as, e.g., Graham et al., J. Gen Virol. 36:59 (1977) described in 293 cells); baby hamster kidney cells (BHK); mouse Sertoli cells (such as, for example, TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey Kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells Cells (Hep G2); Mouse Mammary Tumor (MMT 060562); TRI cells as described, for example, in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, which comprise DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cells For a review of specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

Polyclonal antibodies are preferably produced by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant in animals. Using bifunctional or derivatizing reagents (e.g., maleimidobenzoyl sulfosuccinimide ester (coupled via a cysteine residue), N-hydroxysuccinimide ( It is useful to couple the relevant antigen to the protein via a lysine residue), glutaraldehyde, succinic anhydride, SOCl ₂ , or R ¹ N=C=NR, wherein R and R ¹ are different alkyl groups), The protein is immune to the species to be immunized (eg keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin or soybean trypsin inhibitor).

By combining, for example, 100 micrograms (µg) or 5 micrograms of protein or conjugate (for rabbits and mice, respectively) with 3 volumes of Freund's complete adjuvant, and injecting the solution intradermally at multiple sites An animal (usually a non-human mammal) is immunized against the antigen, immunogenic conjugate or derivative. One month later, animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple locations. On days 7-14 after the booster injection, the animals were bled to determine the antibody titer concentration in the serum. Animals are boosted until a titer plateaus is reached. Preferably, the animal is boosted with a conjugate of the same antigen, but conjugated to a different protein and/or via a different cross-linking agent. Conjugates can also be produced in recombinant cell culture as protein fusions. Coagulants such as alum are also suitable for enhancing the immune response.

Monoclonal antibodies can be obtained from a population of antibodies that is substantially homogeneous, i.e., that the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerization, amidation, ). Thus, the modifier "monoclonal" indicates the identity of the antibody, which is not a mixture of dispersed antibodies.

For example, monoclonal antibodies can be generated using the fusionoma method first described by Kohler et al., Nature 256(5517): 495-497 (1975). In the fusionoma approach, mice or other suitable host animals, such as hamsters, are immunized as described herein to induce lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing protein. Alternatively, lymphocytes can be immunized in vitro.

Immunization reagents will generally comprise a protein of the antigen or a fusion variant thereof. Generally, peripheral blood lymphocytes (PBL) are used if cells of human origin are desired, or spleen cells or lymph node cells are used if cells of non-human mammalian origin are desired. The lymphocytes are then fused with an immortal cell line using a suitable fusing agent, such as polyethylene glycol, to form a fusionoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103).

Immortal cell lines are usually transformed mammalian cells, especially myeloma cells of rodent, bovine and human origin. Typically, rat or mouse myeloma cell lines are used. The fusionoma cells thus prepared are seeded and grown in a suitable medium, which preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium of the fusionoma will usually contain hypoxanthine, aminopterin, and thymine (HAT medium), which prevent Growth of HGPRT-deficient cells.

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

To determine whether the monoclonal antibody against the antigen is produced in the growth medium of fusion tumor cells. Preferably, the binding specificity of monoclonal antibodies produced by fusion tumor cells is detected by immunoprecipitation or by in vitro binding (e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)) To be measured. Such techniques and assays are well known in the art. Binding affinity can be determined by Scatchard analysis of Munson, Anal Biochem. 107(1):220-239 (1980).

After identification of fusionoma cells with the desired specificity, affinity and/or activity, these colonies can be sub-selected by limiting dilution procedures and grown using standard methods (Goding, supra). Media suitable for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, fusionoma cells can be grown in vivo as tumors in mammals.

Monoclonal antibodies secreted by subclonal strains are isolated from culture medium, ascites by conventional immunoglobulin purification procedures such as, for example, protein A-agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. fluid) or serum is suitably separated.

Antibodies can be produced by immunizing an appropriate host animal against an antigen. In one embodiment, the antigen is a polypeptide including full-length C5. In one embodiment, the antigen is a polypeptide comprising the beta (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 the 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 β 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 selected from Thr47, Glu48, Ala49, Phe50, Asp51, Ala52, Thr53, Lys57, His70, Val71, His72, Ser74, Glu76 , Val107, Ser108, Lys109 and the amino acid of the group consisting of 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. The invention also includes antibodies produced by immunizing animals against the above antigens. Antibodies may incorporate any of the features, singly or in combination, as described above under "Exemplary anti-C5 antibodies".

C. Determination The physical/chemical properties and/or biological activities of the anti-C5 antibodies provided herein can be identified, screened or characterized by various 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 its 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 some specific embodiments, when such competing antibody is present in excess, it blocks (e.g., reduces) the binding of the reference antibody to C5 by at least 10%, 15%, 20%, 25%, 30%, 35%, 40% %, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more. In some instances, binding is inhibited by at least 80%, 85%, 90%, 95% or more. In some specific embodiments, such competing antibodies bind to the same epitope (e.g., linear or conformational epitope) that is bound by an anti-C5 antibody described herein (e.g., an antibody as described in Table 2). ). Detailed exemplary methods for mapping epitopes bound by antibodies 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 that includes a first labeled (reference) antibody that binds to C5, and a second unlabeled antibody that is tested for its ability to compete with the first antibody for binding to C5. labeled antibody. Secondary antibodies may be present in the fusion tumor supernatant. As a control group, immobilized C5 were incubated in a solution including the first labeled antibody but not the second labeled antibody. After incubation under conditions permissive for primary antibody binding to C5, excess unbound antibody is removed and the amount of label bound to immobilized C5 is measured. If the amount of label bound to immobilized C5 is substantially reduced in the test sample relative to the control sample, this indicates that the second 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 ability of a tested anti-C5 antibody to compete for binding to C5 is determined by a second (reference) anti-C5 antibody using the BIACORE (registered trademark) assay. In yet another aspect, C5 protein is captured on a CM5 BIACORE (registered trademark) chip using known standard techniques using a BIACORE(registered trademark) apparatus (e.g., BIACORE(registered trademark) 3000) according to the manufacturer's recommendations to Fabricate a C5-coated surface. Typically 200 to 800 resonance units of C5 will be attached to the chip (this amount gives an easily measurable degree of binding, but can be easily saturated by the concentration of test antibody used). Two antibodies (ie, test and reference antibodies) to be assessed for their ability to compete with each other are mixed at a 1:1 molar ratio of binding sites in an appropriate buffer to generate a test mixture. When calculating concentrations on the basis of binding sites, the molecular weight of a 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 that antibody. The concentration of each antibody (ie, test and reference antibodies) in the test mixture should be high enough to readily saturate the binding sites of the antibodies on the C5 molecule captured on the BIACORE (registered trademark) chip. The antibodies in the mixture have the same molar concentration (on a binding basis), usually between 1.00 and 1.5 micromolar (on a binding site basis). Different solutions containing individual test antibodies and individual reference antibodies were also prepared. The test and reference antibodies in these solutions should be in the same buffer and under the same concentration and conditions as the test mix. A test mixture containing test and reference antibodies was passed over a C5-coated BIACORE (registered trademark) chip, and the total amount bound was recorded. The wafer is then processed in a manner that removes bound test or reference antibody without destroying wafer-bound C5. Typically, this is done by treating the wafer with 30 mM HCl for 60 seconds. A solution of the test antibody alone was then passed over the C5-coated surface and the amount of binding recorded. The wafer was processed again to remove all bound antibody without disrupting wafer-bound C5. A solution of 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 was then calculated as the sum of the binding of each antibody (ie, test and reference) when passing only the C5 surface. If the actual recorded cocktail binding is less than this theoretical maximum, then the test and reference antibodies compete with each other for binding to C5. Thus, in general, competition test anti-C5 antibodies bind to C5 in the BIACORE (registered trademark) blocking assay described above, such that the recorded binding is 80% of the maximum theoretical binding during the assay and in the presence of a reference anti-C5 antibody and 0.1% (e.g., 80% to 4%), specifically between 75% and 0.1% (e.g., 75% to 4%) of maximum theoretical binding, more specifically the combined test and reference antibody Between 70% and 0.1% (eg 70% to 4%) of the maximum theoretical binding (as defined above).

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

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

In yet other embodiments, the anti-C5 antibody binds to C5 with a higher affinity at neutral pH compared to acidic pH. In some specific 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 yet other embodiments, the anti-C5 antibody binds to C5 with a higher affinity at pH 7.4 than at pH 5.8.

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

In some specific embodiments, the anti-C5 antibody of the present invention comprises: (a) the VH of the sequence identification number: 1 and the VL of the sequence identification number: 11; (b) the VH of the sequence identification number: 22 and Sequence identification number: VL of 26; (c) VH of Sequence identification number: 21 and VL of Sequence identification number: 25; (d) VH of Sequence identification number: 5 and VL of sequence identification number: 15; (e) Sequence Identification number: VH of 4 and VL of sequence identification number: 14; (f) VH of sequence identification number: 6 and VL of sequence identification number: 16; (g) VH of sequence identification number: 2 and VL of sequence identification number: 12 (h) VH of sequence identification number: 3 and VL of sequence identification number: 13; (i) VH of sequence identification number: 9 and VL of sequence identification number: 19; (j) VL of sequence identification number: 7 VH and VL of Sequence Identification Number: 17; (k) VH of Sequence Identification Number: 8 and VL of Sequence Identification Number: 18; (l) VH of Sequence Identification Number: 23 and VL of Sequence Identification Number: 27; and ( m) Sequence identification number: VH of 10 and VL of sequence identification number: 20 The antibodies of the VH and VL pair compete for binding to C5.

In some specific embodiments, the anti-C5 antibody of the present invention comprises: (a) the VH of the sequence identification number: 22 and the VL of the sequence identification number: 26; (b) the VH of the sequence identification number: 21 and Sequence ID: VL of 25; (c) VH of Sequence ID: 5 and VL of Sequence ID: 15; (d) VH of Sequence ID: 4 and VL of Sequence ID: 14; (e) Sequence VH of sequence identification number: 6 and VL of sequence identification number: 16; (f) VH of sequence identification number: 2 and VL of sequence identification number: 12; (g) VH of sequence identification number: 3 and VL of sequence identification number: 13 (h) VH of sequence identification number: 9 and VL of sequence identification number: 19; (i) VH of sequence identification number: 7 and VL of sequence identification number: 17; (j) VL of sequence identification number: 8 VH and sequence identification number: VL of 18; (k) VH of sequence identification number: 23 and VL of sequence identification number: 27 The antibody of the VH and VL pair competes for binding to C5.

In some specific embodiments, the anti-C5 antibody of the present invention is selected from the VH of sequence identification number: 1 and the VL of sequence identification number: 11, or the VH of sequence identification number: 10 and the sequence identification number: 20 The VH of the VL and the antibody to the VL compete for binding to C5.

In yet other embodiments, the anti-C5 antibody binds to C5 with a higher affinity at neutral pH compared to acidic pH. In some specific embodiments, the anti-C5 antibody binds to C5 with a higher affinity at neutral pH than at acidic pH, and is associated with a VH selected from: (a) SEQ ID NO: 1 and SEQ ID NO: VL of 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) SEQ ID NO: 6 (e) VH of sequence identification number: 2 and VL of sequence identification number: 12; (f) VH of sequence identification number: 3 and VL of sequence identification number: 13; ( g) VH of sequence identification number: 9 and VL of sequence identification number: 19; (h) VH of sequence identification number: 7 and VL of sequence identification number: 17; (i) VH and sequence identification of sequence identification number: 8 No.: VL of 18; and (j) VH of SEQ ID NO: 10 and VL of SEQ ID NO: 20 The antibody of the VH and VL pair competes for binding to C5. In yet other embodiments, the anti-C5 antibody binds to C5 with a higher affinity at pH 7.4 than at pH 5.8.

In some embodiments, the anti-C5 antibody binds to C5 with higher affinity at neutral pH than at acidic pH, and binds to a VH comprising: (a) a VH of SEQ ID NO: 5 and a VH of SEQ ID NO: 15 VL; (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 sequence identification number: VL of 12; (e) VH of sequence identification number: 3 and VL of sequence identification number: 13; (f) VH of sequence identification number: 1 and VL of sequence identification number: 11; (g) Sequence identification number: VH of 9 and VL of sequence identification number: 19; (h) VH of sequence identification number: 7 and VL of sequence identification number: 17; and (i) VH and sequence identification number of sequence identification number: 8 : VH of VL of 18 and antibodies against VL compete for binding to C5. In yet other embodiments, the anti-C5 antibody binds to C5 with a higher affinity at pH 7.4 than at pH 5.8.

In some embodiments, the anti-C5 antibody binds to C5 with a higher affinity at neutral pH than at acidic pH, and is associated with a VH comprising: a VH of SEQ ID NO: 1 and a VL of SEQ ID NO: 11, or SEQ ID NO: 10 for VH and SEQ ID NO: 20 for VL The antibodies of the VH and VL pair compete for binding to C5. In yet other embodiments, the anti-C5 antibody binds to C5 with a higher affinity at pH 7.4 than at pH 5.8.

In some specific embodiments, whether an anti-C5 antibody of the invention binds to a specific epitope can be determined by expressing a C5 point mutation in which an amino acid on C5 (except alanine) is replaced by alanine in 293 cells and the binding of the anti-C5 antibody to the C5 mutant was tested by ELISA, Western blot, or BIACORE (registered trademark); wherein the anti-C5 antibody substantially reduced the binding of the C5 mutant compared to its binding to wild-type C5 or disappearing binding represents binding of the anti-C5 antibody to an epitope that includes that amino acid on C5. In some specific embodiments, the amino acid to be substituted by alanine on C5 is selected from the group consisting of Glu48, Asp51, His70, His72, Lys109 and His110 of the β chain of C5 (SEQ ID NO: 40). In yet other embodiments, the amino acid on C5 to be substituted by alanine is Asp51 or Lys109 of the β chain of C5 (SEQ ID NO: 40).

In another example, whether an anti-C5 antibody with pH-dependent binding properties binds to a particular epitope can be determined by expressing a histidine residue on C5 in 293 cells by another amino acid (e.g., , tyrosine) substituted C5 point mutants, and the binding of anti-C5 antibodies to C5 mutants was tested by ELISA, Western blot or BIACORE (registered trademark); The substantially reduced binding of the anti-C5 antibody to wild-type C5 at acidic pH represents binding of the anti-C5 antibody to an epitope that includes that histidine residue on C5. In yet other embodiments, the binding of the anti-C5 antibody to wild-type C5 at neutral pH is not substantially reduced compared to its binding to the C5 mutant at neutral pH. In some specific embodiments, the histidine residue on C5 to be substituted with another amino acid is selected from the group consisting of His70, His72 and His110 of the β chain of C5 (SEQ ID NO: 40). In yet another embodiment, the histidine residue His70 is substituted with tyrosine.

2. Activity Assay In one aspect, an assay is provided to identify the biological activity associated with the anti-C5 antibody. Biological activities may include, for example, inhibiting the activation of C5, preventing the cleavage of C5 to form C5a and C5b, blocking the access of C5 convertase to the cleavage site on C5, blocking the hemolytic activity caused by the activation of C5, and the like. Antibodies having such biological activity in vivo and/or in vitro are also provided.

In some specific embodiments, the antibodies of the invention are tested for such biological activities.

In some specific embodiments, whether the test antibody inhibits the cleavage of C5 into C5a and C5b is determined as described, eg, in Isenman et al., J Immunol. 124(1):326-331 (1980). In another embodiment, this is determined by a method that specifically detects cleaved C5a and/or C5b protein, such as ELISA or Western blot. A test antibody is considered to be an antibody that inhibits cleavage of C5 when a decrease in the amount of the cleavage products of C5 (C5a and/or C5b) is detected in the presence of (or following contact with) the test antibody. In some specific embodiments, the concentration and/or physiological activity of C5a can be measured by, for example, chemotaxis assays, RIA or ELISA methods (please refer to, for example, Ward and Zvaifler J. Clin. Invest. 50(3 ):606-616 (1971)).

In some specific embodiments, whether the test antibody blocks access of C5 convertase to C5 is determined by a method for detecting protein interaction between C5 convertase and C5, for example, ELISA or BIACORE registered trademark. In cases where the interaction is reduced in the presence of (or following contact with) the test antibody, the test antibody is considered to be an antibody that blocks C5 convertase access to C5.

In some specific embodiments, C5 activity is measurable as a function of its cytolytic capacity in a body fluid of a subject. It can be performed by methods known in the art, for example, conventional hemolytic assays, such as Kabat and Mayer (eds.), Experimental Immunochemistry, 2nd Edition, 135-240, Springfield, IL, CC Thomas (1961), pages 135-139 The hemolysis assay described above, or a routine variation of this assay, such as the chicken erythrocyte hemolysis method described in Hillmen et al. (2004), N. Engl. J. Med. 350(6): 552-559 (2004), measures The cytolytic ability of C5 or its decrease. In some specific 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. This test is a lytic assay that utilizes antibody-sensitized erythrocytes as activators of the classical complement pathway and uses various dilutions of test serum to determine the amount required to achieve 50% lysis (CH50). Percent hemolysis can be determined, for example, using a spectrophotometer. The CH50eq assay provides an indirect measure of terminal complement complex (TCC) formation, as TCC itself is directly responsible for the measured hemolysis. Inhibition of C5 activation can also be detected and/or measured using the methods exemplified and exemplified in the working examples. Using these or other types of assays, candidate antibodies capable of inhibiting the activation of C5 can be screened. In some specific embodiments, inhibition of C5 activation is comprised in an assay of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or a 40%, or greater reduction in C5 activity. In some embodiments, it represents inhibition of C5 activation by at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or more .

D. Immunoconjugates The invention also provides immunoconjugates comprising an anti-C5 antibody herein conjugated to one or more cytotoxic agents, e.g., chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., bacterial, fungal, plant or animal origin protein toxins, enzymatic toxins, or fragments thereof) or radioactive isotopes.

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, U.S. Pat. No. 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) (please refer to, U.S. Patent Nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin (dolastatin ); calicheamicin or its derivatives (see, U.S. Pat. 1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); anthracyclines such as daunomycin (daunomycin) or doxorubicin (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 U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, Iarotaxel, tesetaxel, and ortataxe; trichothecene; and CC1065.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain ( From Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleutites fordii (Aleutites fordii) toxin protein, carnation (dianthin) toxin protein, pokeweed (Phytolaca americana) protein (PAPI, PAPII and PAP-S), bitter melon (Momordica charantia) inhibitor, jatropha toxin (curcin ), crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, Enomycin and trichothecene.

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

A variety of bifunctional protein coupling agents can be used to form conjugates of antibodies and cytotoxic agents, such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4 -(N-maleiminomethyl)cyclohexane-I-carboxylate (SMCC), iminothiolane (IT), imidate (e.g. dimethyl adipylimidate hydrochloride) Bifunctional derivatives of bis-functional derivatives, active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azides (such as bis(p-azidobenzoyl)hexanedi amines), dinitrogen derivatives (such as bis(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bisactive fluorine compounds (such as 1,5- difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. Please refer to WO94/11026. A linker may be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell. For example, acid-labile, peptidase-sensitive, photolabile, dimethyl, or disulfide-containing linkers can be used (Chari et al., Cancer Res. 52:127-131 (1992 ); U.S. Patent No. 5,208,020).

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

E. Methods and Compositions for Diagnosis and Detection In some specific embodiments, any of the anti-C5 antibodies provided herein are useful for detecting the presence of C5 in a biological sample. The word "detection", as used herein, encompasses quantitative or qualitative detection. In some specific embodiments, biological samples include cells or tissues, such as serum, whole blood, plasma, section samples, tissue samples, cell suspensions, saliva, sputum, oral fluid, cerebrospinal fluid, amniotic fluid, ascites, milk , colostrum, breast secretions, lymph, urine, sweat, tears, gastric juice, joint fluid, peritoneal fluid, intraocular fluid and mucus.

In one embodiment, an anti-C5 antibody for use in a method of diagnosis or detection is provided. In yet another aspect, a method of detecting the presence of C5 in a biological sample is provided. In some specific embodiments, the method comprises contacting the biological sample with an anti-C5 antibody as described herein under conditions that allow the anti-C5 antibody to bind to C5, and detecting whether a complex is formed between the anti-C5 antibody and Between C5. Such methods can be in vitro or in vivo methods. In one embodiment, anti-C5 antibodies are used to select subjects suitable for anti-C5 antibody therapy, eg, where C5 is a biomarker for patient selection.

In another embodiment, a method of selecting an individual with a complement-modulating disease or condition involving excessive or uncontrolled C5 activation as suitable for therapy comprising an anti-C5 antibody of the invention is provided. In some specific embodiments, the method comprises (a) detecting a genetic variation in C5 derived from an individual, and (b) when a genetic variation in C5 derived from an individual is detected, selecting a C5 suitable for comprising the present invention Individuals on anti-C5 antibody therapy. In another embodiment, a method of selecting a therapy for an individual with a complement-regulated disease or condition involving excessive or uncontrolled C5 activation is provided. In some specific embodiments, the method comprises (a) detecting a genetic variation in C5 derived from an individual and (b) when a genetic variation in C5 derived from an individual is detected, selecting for the individual a C5 comprising Anti-C5 antibody therapy.

In another embodiment, a method of treating an individual with a complement-regulated disease or condition involving excessive or uncontrolled C5 activation is provided. In some specific embodiments, the method comprises (a) detecting a genetic variation in C5 derived from an individual, (b) when a genetic variation in C5 derived from an individual is detected, selecting a C5 suitable for inclusion in the present invention The subject of anti-C5 antibody therapy and (c) administering an anti-C5 antibody of the invention to the subject.

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

In another embodiment, the in vivo use of genetic variation of C5 is provided for selecting individuals with complement-regulated diseases or conditions involving excessive or uncontrolled C5 activation as suitable for therapy comprising an anti-C5 antibody of the invention By. In some specific embodiments, when a genetic variation in C5 from an individual is detected, an individual is selected for therapy. In another embodiment, the in vivo use of a genetic variation of C5 for selection of therapy for an individual with a complement-regulated disease or condition involving excessive or uncontrolled C5 activation is provided. In some specific embodiments, when a genetic variation in C5 derived from the individual is detected, the individual is selected for therapy comprising an anti-C5 antibody of the invention.

It has been reported that some patients with genetic variation in C5 show poor response to therapy involving pre-existing anti-C5 antibodies (Nishimura et al., N. Engl. J. Med. 370:632-639 (2014)). Such patients are advised to be treated with a therapy comprising an anti-C5 antibody of the present invention due to the inhibitory activity of this antibody on the activation of the C5 variant as well as the activation of wild-type C5, as shown in the Examples below.

Detection of genetic variation in C5 can be performed by methods known in the art. Such methods may include sequencing, PCR, RT-PCR, and hybridization-based methods such as southern blot analysis or northern blot analysis, but are not limited thereto. A C5 variant may comprise at least one genetic variation. The genetic variation can be selected from the group consisting of V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N and E1437D. Herein, R885H, for example, represents a genetic variation in which arginine at position 885 is substituted for histidine. In some specific embodiments, the C5 variant has similar biological activity to wild-type C5.

Exemplary diseases that can be diagnosed using the antibodies of the invention include rheumatoid arthritis (RA); systemic lupus erythematosus (SLE); lupus nephritis; ischemia reperfusion injury (IRI); ; paroxysmal nocturnal hemoglobinuria (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; AMD); hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome; thrombotic thrombocytopenic purpura (TTP); spontaneous fetal loss; epidermolysis bullosa; relapse fetal loss; pre-eclampsia; traumatic brain injury; myasthenia gravis; cold agglutinin disease; Sjogren's syndrome; dermatomyositis; bullous pemphigoid ; phototoxic reaction; Shiga toxin E. coli-related hemolytic uremic syndrome (Shiga toxin E. coli-related hemolytic uremic syndrome); typical or infectious hemolytic uremic syndrome (tHUS); C3 glomerulonephritis; ANCA-associated vasculitis; humoral and vascular graft rejection; acute antibody-mediated rejection (AMR); graft dysfunction; myocardial infarction; allograft transplantation; sepsis; coronary artery disease; Hereditary angioedema; dermatomyositis; Graves' disease; atherosclerosis; Alzheimer's disease (AD); Huntington's disease (Huntington's disease ); Creutzfeld-Jacob; Parkinson's disease; cancer; wounds; septic shock; spinal cord injury; uveitis; diabetic eye disease; retinopathy of prematurity; glomerulonephritis ); Membranous nephritis; Immunoglobulin A nephropathy; Adult respiratory distress syndrome (ARDS); Chronic obstructive pulmonary disease (COPD); Cystic fibrosis; Hemolytic Anemia; paroxysmal cold hemoglobinuria; anaphylactic shock; allergy; osteoporosis; osteoarthritis; Hashimoto's thyroiditis; type 1 diabetes Psoriasis; Pemphigus; Autoimmune hemolytic anemia (AIHA); Idiopathic thrombocytopenic purpura (ITP); Gupastier syndrome (Goodpasture syndrome); Degos disease; antiphospholipid syndrome (APS); catastrophic antiphospholipid syndrome (catastrophic APS, CAPS); cardiovascular disease; myocarditis; cerebrovascular disorder ; peripheral vascular disease; renal vascular disease; mesenteric/enteric vascular disorder; vasculitis; Henoch-Schonlein purpura nephritis; Takayasu's disease); dilated cardiomyopathy; diabetic vascular disease; Kawasaki's disease (arteritis); venous gas embolus (VGE); restenosis following stent placement; rotational porridge Plaque-like Rotational atherectomy; membranous nephropathy; Guillain-Barresyndrome (GBS); Fisher syndrome; antigen-induced arthritis; synovial inflammation; viral infection; bacterial infection; fungal infection and injuries resulting from myocardial infarction, cardiopulmonary bypass, and hemodialysis.

In some specific embodiments, labeled anti-C5 antibodies are provided. Labels include, but are not limited to, labels or moieties that are detected directly (e.g., fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels) and indirectly (e.g., by enzymatic reactions or molecular interactions). Moieties, like enzymes or ligands. Exemplary labels include, but are not limited to, radioactive isotopes ³² P, ¹⁴ C, ¹²⁵ I, ³ H and ¹³¹ I, fluorophores such as rare earth chelates or luciferin and its derivatives, rhodamine and its Derivatives, dansyl, umbellifeixme, luceriferase, eg, firefly luciferase and bacterial luciferase (US Patent No. 4,737,456), luciferin , 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, beta-galactosidase, glucoamylase, lytic enzyme, carbohydrate oxidase, e.g. glucose oxidase , galactose oxidase and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, combined with enzymes that utilize hydrogen peroxide to oxidize dye precursors such as HRP, lactoperoxidase, or micro Peroxidase (microperoxidase), biotin/avidin, spin label, phage label, stable free radical, etc.

F. Pharmaceutical formulations Pharmaceutical formulations of anti-C5 antibodies as described herein are prepared by combining such antibodies having 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 as a lyophilized formulation or as an aqueous solution. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine ; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexahydroxyquat ammonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butanol or benzyl alcohol; alkyl parabens such as Methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; Proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, spermine monosaccharides, disaccharides, and other sugars, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (eg Zn-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include drug interstitial dispersants, such as soluble neutral-active hyaluronidase glycoprotein (sHASEGP), for example, 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 Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

Exemplary lyophilized antibody formulations are described in US Patent No. 6,267,958. Aqueous antibody formulations include those described in US Patent No. 6,171,586 and WO 2006/044908, the latter formulations comprising a histidine-acetate buffer.

The formulations herein may also contain more than one active ingredient, preferably those with complementary activities that do not adversely affect each other, if required for the particular indication being treated. Such active ingredients are suitably present in combination in amounts effective for the intended use.

The active ingredient can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively, In colloidal drug delivery systems such as 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 comprise semipermeable matrices of solid hydrophobic polymers containing the antibody in the form of shaped articles such as films or microcapsules.

Formulations to be used for in vivo administration are generally sterile. Sterility is readily achieved, for example, by filtration through sterile filtration membranes.

G. Treatment Methods and Compositions 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 yet another aspect, anti-C5 antibodies for use in the treatment of complement-regulated diseases or conditions involving excessive or uncontrolled C5 activation are provided. In some specific embodiments, anti-C5 antibodies for use in methods of treatment are provided. In some specific embodiments, the invention provides an anti-C5 antibody for use in a method of treating an individual having a complement-regulated disease or condition involving excessive or uncontrolled C5 activation, the method comprising administering to the individual an effective amount of Anti-C5 antibody. In such an embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. The "individual" according to any of the above embodiments is preferably a human being.

When the antibody is a soluble protein, binding of the antibody to its antigen can result in an increased half-life of the antigen in plasma (i.e., reduced elimination of the antigen from plasma), since the antibody itself has a longer half-life in plasma and acts as an antigen a. This results from the recycling of antigen-antibody complexes by FcRn through the endosomal pathway in cells (Roopenian, Nat. Rev. Immunol. 7(9):715-725 (2007)). However, antibodies with pH-dependent binding properties, which bind to their antigen in a neutral extracellular environment and then release it into the acidic intracellular compartment upon entry into the cell, are expected to be more effective in antigen neutralization and clearance Has superior properties compared to its counterparts that bind in a pH-independent manner (Igawa et al., Nat. Biotech.. 28(11):1203-1207 (2010); Devanaboyina et al., mAbs 5(6 ):851-859 (2013); WO 2009/125825).

In yet other embodiments, the invention provides an anti-C5 antibody for use in enhancing the elimination of C5 from plasma. In some specific embodiments, the invention provides an anti-C5 antibody for use in a method of enhancing elimination of C5 from plasma in an individual, the method comprising administering to the individual an effective amount of the anti-C5 antibody to enhance elimination of C5 from plasma. In one embodiment, an anti-C5 antibody enhances the elimination of C5 from plasma compared to conventional anti-C5 antibodies that do not have pH-dependent binding properties. According to any of the above embodiments, the "individual" is preferably a human being.

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

The anti-C5 antibody of the present invention can inhibit the activation of C5. In yet other embodiments, the invention provides an anti-C5 antibody for use in inhibiting the activation of C5. In some specific embodiments, the invention provides an anti-C5 antibody for use in a method of inhibiting C5 activation in an individual, the method comprising administering to the individual an effective amount of the anti-C5 antibody to inhibit the activation of C5. In one embodiment, C5-mediated cytotoxicity is inhibited by inhibiting the activation of C5. According to any of the above embodiments, the "individual" is preferably a human being.

In yet another aspect, the present invention provides the use of an anti-C5 antibody in the production or preparation of a medicament. In one embodiment, the medicament is for the treatment of a complement-regulated disease or condition involving excessive or uncontrolled C5 activation. In yet another embodiment, the medicament is for use in a method of treating a complement-modulating disease or condition involving excessive or uncontrolled C5 activation comprising adding to a complement-modulating disease or condition involving excessive or uncontrolled C5 activation The subject is administered an effective amount of the drug. In such an embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. The "individual" according to any of the above embodiments is preferably a human being.

In yet another embodiment, the medicament is to enhance the elimination of C5 from plasma. In yet another embodiment, the medicament is a method for enhancing elimination of C5 from plasma in an individual, the method comprising administering to the individual an effective amount of the medicament to enhance elimination of C5 from plasma. In one embodiment, an anti-C5 antibody enhances the elimination of C5 from plasma compared to conventional anti-C5 antibodies that do not have pH-dependent binding properties. According to any of the above embodiments, an "individual" may be a human being.

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

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

In yet another aspect, the present invention provides methods for treating complement-regulated diseases or conditions involving excessive or uncontrolled C5 activation. In one embodiment, the method comprises administering to an individual having said complement-modulating disease or condition involving excessive or uncontrolled C5 activation an effective amount of an anti-C5 antibody. In such an 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 being.

In yet another aspect, the invention provides a method for enhancing the elimination 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 elimination of C5 from plasma. In one embodiment, an anti-C5 antibody enhances the elimination of C5 from plasma compared to conventional anti-C5 antibodies that do not have pH-dependent binding properties. In one embodiment, an "individual" is a human being.

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

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

In yet another aspect, the 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, a pharmaceutical formulation includes any anti-C5 antibody provided herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical formulation includes any of the anti-C5 antibodies provided herein and at least one additional therapeutic agent.

In yet another aspect, the pharmaceutical formulation is for the treatment of a complement-regulated disease or condition involving excessive or uncontrolled C5 activation. In yet another embodiment, the pharmaceutical formulation is for enhancing the elimination of C5 from plasma. In one embodiment, an anti-C5 antibody enhances the elimination of C5 from plasma compared to conventional anti-C5 antibodies that do not have pH-dependent binding properties. In yet 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 a result of the formation of antigen-antibody complexes. In another embodiment, an anti-C5 antibody inhibits the accumulation of C5 in plasma, compared to conventional anti-C5 antibodies that do not have pH-dependent binding properties. The anti-C5 antibody of the present invention can inhibit the activation of C5. In yet another embodiment, the pharmaceutical formulation is for inhibiting the activation of C5. In one embodiment, C5-mediated cytotoxicity is inhibited by inhibiting the activation of C5. In one embodiment, the pharmaceutical formulation is administered to an individual with a complement-modulating disease or condition involving excessive or uncontrolled C5 activation. The "individual" according to any of the above embodiments is preferably a human being.

In one aspect, the individual has wild-type C5. In another aspect, the individual has the C5 variant. In some specific embodiments, the C5 variant has similar biological activity to wild-type C5. Such C5 variants may comprise at least one variation selected from the group consisting of V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N and E1437D. Herein, R885H, for example, represents a genetic variation in which arginine at position 885 is substituted for histidine.

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

In some specific embodiments, the complement modulating disease or condition involving excessive or uncontrolled C5 activation is selected from the group consisting of: rheumatoid arthritis (RA); systemic lupus erythematosus (SLE); lupus lupus nephritis; ischemia reperfusion injury (IRI); asthma; paroxysmal nocturnal hemoglobinuria (PNH); hemolytic uremic syndrome (HUS) (eg, SARS hemolytic uremic syndrome (aHUS); dense deposit disease (DDD); neuromyelitis optica (NMO); multifocal motor neuropathy (MMN); multiple sclerosis (multiple sclerosis) sclerosis, MS); systemic sclerosis; macular degeneration (eg, age-related macular degeneration (AMD)); hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome; thrombotic thrombocytopenic purpura (thrombotic thrombocytopenic purpura, TTP); spontaneous fetal loss; epidermolysis bullosa; recurrent fetal loss; pre-eclampsia; traumatic brain injury; myasthenia gravis; (Sjogren's syndrome); dermatomyositis; bullous pemphigoid; phototoxic reaction; Shiga toxin E. coli-related hemolytic uremic syndrome (Shiga toxin E. coli-related hemolytic uremic syndrome); typical or infectious hemolytic uremic syndrome (tHUS); C3 glomerulonephritis; antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis; humoral and vascular graft rejection; acute antibody-mediated rejection (antibody mediated rejection, AMR); graft dysfunction; myocardial infarction; allograft transplantation; sepsis; coronary artery disease; hereditary angioedema; dermatomyositis; Graves' disease; atherosclerosis atherosclerosis; Alzheimer's disease (AD); Huntington's disease; Creutzfeld-Jacob; Parkinson's disease; cancer; wounds; septic shock; spinal cord injury; uveitis; diabetic eye disease; retinopathy of prematurity; kidney Glomerulonephritis; Membranous nephritis; Immunoglobulin A nephropathy; Adult respiratory distress syndrome (ARDS); Chronic obstructive pulmonary disease (COPD); Hemolytic anemia; paroxysmal cold hemoglobinuria; anaphylactic shock; allergy; osteoporosis; osteoarthritis; Hashimoto's thyroiditis ); type I diabetes; psoriasis; pemphigus; autoimmune hemolytic anemia (AIHA); idiopathic thrombocytopenic purpura (ITP); Goodpasture syndrome; Degos disease; antiphospholipid syndrome (APS); catastrophic antiphospholipid syndrome (CAPS); cardiovascular disease; myocarditis; cerebrovascular disorder; peripheral vascular disease; renal vascular disease; mesenteric/enteric vascular disorder; vasculitis; Henoch-Schonlein purpura nephritis ); Takayasu's disease; dilated cardiomyopathy; diabetic vascular disease; Kawasaki's disease (arteritis); venous thromboembolism (venous gas embolus, VGE); ng stent placement); rotational atherectomy; membranous nephropathy; Guillain-Barre syndrome (GBS); Fisher syndrome; antigen-induced arthritis; membrane inflammation; viral infection; bacterial infection; fungal infection; and injury caused by myocardial infarction, cardiopulmonary bypass, and hemodialysis.

In some specific embodiments, the complement-modulated disease or condition is an ocular condition. In yet other embodiments, the eye condition is macular degeneration. In yet other embodiments, the macular degeneration is age-related macular degeneration (AMD). In yet other embodiments, the AMD is dry AMD.

In some specific embodiments, the complement-modulated disease or condition is paroxysmal nocturnal hemoglobinuria (PNH).

In some specific embodiments, the complement-modulated disease or condition is myocardial infarction.

In some specific embodiments, the complement-modulated disease or condition is rheumatoid arthritis (RA).

In some specific embodiments, the complement-modulated disease or condition is osteoporosis or osteoarthritis.

In some specific embodiments, the complement-modulated disease or condition is inflammation.

In some specific embodiments, the complement-modulated disease or condition is cancer.

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

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

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

Antibodies of the invention will be formulated, dosed and administered in a manner consistent with good medical practice. Factors considered in this context include the particular condition to be treated, the particular mammal to be treated, the clinical state of the individual patient, the cause of the condition, the location of the agent being delivered, the method of administration, the timing of administration, and known to the medical practitioner. other factors. Antibodies need not, but are optionally, formulated with one or more agents currently used to prevent or treat the condition in question. Effective amounts of other agents depend on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used at the same dosage, and using the route of administration as described herein, or at about 1 to 99% of the dosage described herein, or at any dosage and whichever route is empirically/clinically determined to be suitable.

For the prevention or treatment of disease, the appropriate dose of the 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 and course of the disease , whether the antibody is administered for prophylactic or therapeutic purposes, previous therapy, 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 as a one-time treatment or over a series of treatments. Depending on the type and severity of the disease, about 1 microgram/kg (μg/kg) to 15 milligrams/kg (mg/kg) (eg, 0.1 mg/kg-10 mg/kg) of the antibody can be used for the treatment of patients The initial candidate dose is administered, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations for several days or longer, depending on the condition, the treatment will generally be continued until a desired suppression of disease symptoms occurs. An exemplary dosage of antibody would be in the range of about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered at intervals, eg, every week or every three weeks (eg, such that the patient receives from about 2 to about 20, or eg, about 6 doses of the antibody). An initial higher loading dose followed by one or more lower doses may be administered. The progress of this therapy is readily monitored by conventional methods and assays.

It should be understood that any of the above formulations or treatment methods can be implemented using the immunoconjugate of the present invention instead of or in addition to the anti-C5 antibody.

F. Products In another aspect of the present invention, there is provided an article of manufacture comprising materials useful for treating, preventing and/or diagnosing the aforementioned disorders. Articles of manufacture include containers and labels or package inserts on or associated with the containers. Suitable containers include, for example, bottles, vials, syringes, IV solution packs, and the like. The container can be made of various materials such as glass or plastic. The container contains a composition which is effective for the treatment of a condition, alone or in combination with another composition effective for the treatment, prophylaxis and/or diagnosis of a condition, and which can have a sterile access Mouth (for example, the container may 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 states that the composition is intended to treat a particular condition. Additionally, the article of manufacture may comprise (a) a first container comprising a composition therein, wherein the composition comprises an antibody of the invention; and (b) a second container comprising a composition therein, wherein the composition comprises additional cells Toxic or other therapeutic agents. The article of manufacture in this embodiment of the present invention may further include a package insert indicating that the composition can be used to treat a specific condition. Alternatively or additionally, 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. From a commercial and user standpoint, it can also encompass other materials as desired, including other buffers, diluents, filters, needles, and syringes.

It is to be understood that any of the preparations described above may comprise an immunoconjugate of the invention in place of or in addition to the anti-C5 antibody. [Example]

The following are examples of methods and compositions of the invention. It is 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 rhesus 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, then applied to a Q-sepharose FF or Q-sepharose HP anion exchange column (GE healthcare, Uppsala, Sweden), followed by NaCl gradient elution. Fractions containing human C5 were pooled, followed by adjustment of salt concentration and pH to 80 mM NaCl and pH 6.4, respectively. The obtained sample was applied to SP-sepharose HP cation exchange column (GE healthcare, Uppsala, Sweden) and eluted with NaCl gradient. Fractions containing human C5 were pooled and applied to a CHT ceramic hydroxyapatite column (Bio-Rad Laboratories, Hercules, CA, USA). The human C5 eluate was then applied to a Superdex 200 colloidal filtration column (GE healthcare, Uppsala, Sweden). Fractions containing human C5 were pooled and stored at -150°C.

Expression and purification of recombinant rhesus monkey C5 (NCBI GenBank accession number: XP_005580972, SEQ ID NO: 44) was performed in the same manner as the human part.

1.2. Purification of rhesus monkey C5 (cynoC5) from plasma Plasma samples from rhesus monkeys were applied to SSL7-agarose (Invivogen, San Diego, CA, USA), followed by elution with 100 mM sodium acetate, pH 3.5. Fractions containing cynoC5 were immediately neutralized and applied in tandem to protein A HP columns (GE healthcare, Uppsala, Sweden) and peptide M sepharose (Invivogen, San Diego, CA, USA). The flow-through fraction was then applied to a Superdex 200 gel filtration column (GE healthcare, Uppsala, Sweden). Fractions containing cynoC5 were collected and stored at -80 degrees C (°C). [Example 2]

Production of anti-C5 antibody 2.1. Antibody screening Prepare, select and measure anti-C5 antibodies in the following manner:

12- to 16-week-old NZW rabbits were immunized intradermally with human C5 and/or monkey C5 (50-100 micrograms ([mu]g)/dose/rabbit). Repeat this dose 4-5 times over a period of 2 months. One week after the final immunization, the spleen and blood of the immunized rabbits were collected. Antigen-specific B cells are stained with labeled antigens and sorted by an FCM cell sorter (FACS aria III, BD) in 96-well plates at a density of 1 cell/well along with 25,000 cells/well of EL4 cells Medium (European Collection of Cell Cultures), and the activated rabbit T cell conditioned medium was diluted 20 times, and cultured for 7-12 days. EL4 cells were pre-treated with Mitomycin C (Sigma, Cat No. M4287) for 2 hours and washed 3 times. By culturing rabbit thymocytes in a medium containing phytohemagglutinin-M (Roche, Cat No. 1 1082132-001), phorbol 12-myristate 13-acetate (Sigma, Cat No. P1585) and 2 Rabbit T cell conditioned medium was prepared in RPMI-1640 with % FBS. After incubation, B cell culture supernatants were collected for further analysis and pellets were cryopreserved.

An ELISA assay was used to test the specificity of antibodies in B cell culture supernatants. Coat biotin (GeneScript, Cat No. Z02043) with 50 nM PBS on 384-well MAXISorp (Nunc, Cat No. 164688) and place at room temperature for 1 hour. The disc was then blocked with 5-fold diluted Blocking One (Nacalai Tesque, Cat No. 03953-95). Human or monkey C5 was labeled with NHS-PEG4-Biotin (PIERCE, Cat No. 21329) and added to blocked ELISA plates, incubated for 1 hour and washed. The B cell culture supernatant was added to the ELISA dish, 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. Add goat anti-rabbit IgG-Fc (BETHYL, Cat No. A120-111A) diluted to 1 microgram/milliliter (µg/ml) with PBS (-) to 384-well MAXISorp (Nunc, Cat No. 164688), in Incubate at room temperature for 1 hour, and block with Blocking One (Nacalai Tesque, Cat No. 03953-95) diluted 5 times. After incubation, plates were washed and B cell culture supernatant was added. Plates were incubated for 1 hour, washed, and 500 pM biotinylated human or monkey C5 was added and incubated for 1 hour. After incubation, plates were washed and incubated with pH 7.4 MES buffer (20 mM MES, 150 mM NaCl, and 1.2 mM CaCl ₂ ) or pH 5.8 MES buffer (20 mM MES, 150 mM NaCl, and 1 mM EDTA). Incubate at room temperature for 1 hour. After incubation, the binding of biotinylated C5 was detected by biotin-horseradish peroxidase conjugate (Thermo Scientific, Cat No. 21132) followed by ABTS (KPL, Cat No. 50-66-06) .

The Octet RED384 system (Pall Life Sciences) was used to assess affinity and pH-dependent binding of antibodies to C5. Antibodies secreted from B cell culture supernatants were loaded onto Protein A biosensing probes (Pall Life Sciences) and dipped into 50 nM human or monkey C5 in pH 7.4 MES buffer to analyze binding kinetics . Dissociation kinetics were analyzed in both pH 7.4 MES buffer as well as 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 lines were selected and designated CFA0001-0677. RNA from selected cell lines was purified from cryopreserved pellets using the ZR-96 Quick-RNA Kit (ZYMO RESEARCH, Cat No. R1053). The DNA encoding the heavy chain variable region of the selected cell line was amplified by reverse transcription PCR and recombined with the DNA encoding the heavy chain constant region of F760G4 (SEQ ID NO: 33) or F939G4 (SEQ ID NO: 34). The DNA encoding the antibody light chain variable region was amplified by reverse transcription PCR, and recombined with the DNA encoding the kOMTC light chain constant region (SEQ ID NO: 36). Separately, the heavy chain and light chain genes of the existing humanized anti-C5 antibody, eculizumab (EcuH-G2G4, SEQ ID NO: 29 and EcuL-k0, SEQ ID NO: 30), were synthesized. DNA encoding VH (EcuH, SEQ ID NO: 31) was fused in-frame to DNA encoding modified human IgG4 CH (F760G4, SEQ ID NO: 33), and DNA encoding VL (EcuL , SEQ ID NO: 32) fused in-frame to the DNA encoding the k0 light chain constant region (SEQ ID NO: 37). The coding sequences for the respective fusions are transferred into expression vectors. Antibodies were expressed in FreeStyle ^™ 293-F cells (Invitrogen) and purified from culture supernatants to assess functional activity. Neutralizing activity of antibodies was assessed by testing for inhibition of complement activity using the liposome lysis assay as described in Example 5.1.

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

All anti-C5 antibodies were used as capture and detection antibodies and were paired comprehensively. As shown in Figure 1, competing antibodies were classified into seven epitope bins: CFA0668, CFA0334, and CFA0319 were classified into epitope A, CFA0647, CFA0589, CFA0341, CFA0639, CFA0635, CFA0330 and CFA0318 were classified into epitope B, CFA0538, CFA0501, CFA0599, CFA0307, CFA0366, CFA0305, CFA0675, CFA0666 and CFA0672 were classified into epitope C, eculizumab and CFA0322 were classified into epitope D, CFA0329 was grouped into epitope E, CFA0359 and CFA0217 were grouped into epitope F, and CFA0579, CFA0328 and CFA0272 were grouped into epitope G. Figure 1 shows some epitope compartments of the anti-C5 chimeric antibody. Table 2 lists the sequences of the VH and VL of anti-C5 antibodies classified to epitope C. [Table 2] Anti-C5 antibodies classified to epitope C

2.3. Humanization and optimization To reduce the potential immunogenicity of the antibody, humanization of some variable regions of the anti-C5 antibody was performed. The complementarity determining regions (CDRs) of the anti-C5 rabbit antibody were grafted onto the homologous human antibody framework (FR) by the conventional CDR grafting method (Nature 321:522-525 (1986)). The genes encoding humanized VH and VL were synthesized and recombined with modified human IgG4 CH (SG402, SEQ ID NO: 35) and human CL (SK1, SEQ ID NO: 38) respectively, and each recombinant sequence was transferred in 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 lead antibodies. Multiple mutations were then introduced into the humanized variable region to enhance the binding affinity for C5 at neutral pH or to decrease the binding affinity for C5 at acidic pH. One of the optimized variants, 305LO5 (VH, SEQ ID NO: 10; VL, SEQ ID NO: 20; HVR-H1, SEQ ID NO: 54; HVR-H2, SEQ ID NO: 64; HVR-H3 , SEQ ID NO: 74; HVR-L1, SEQ ID NO: 84; HVR-L2, SEQ ID NO: 94; and HVR-L3, SEQ ID NO: 104), thus generated from CFA0305.

Antibodies were expressed and purified by protein A in HEK293 cells co-transfected with a mixture of heavy and light chain expression vectors. [Example 3]

Binding characteristics of anti-C5 antibodies 3.1. Expression and purification of recombinant antibodies Recombinant antibodies were transiently expressed using the FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA). Purification using Protein A from conditioned medium expressing the antibody was performed by conventional methods. Further colloid filtration was performed as needed.

3.2. Evaluation of pH dependence Kinetic parameters of anti-C5 antibody against recombinant human C5 were evaluated using BIACORE (registered trademark) T200 instrument (GE Healthcare) at pH 7.4 and pH 5.8 at 37 degrees C (°C). ProA/G (Pierce) was immobilized on a CM4 sensing wafer using an amine coupling kit (GE Healthcare) according to the GE Healthcare recommended setup. Antibodies and analytes were diluted in respective electrophoresis buffers, ACES pH7.4 and pH5.8 (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl ₂ , 0.05% Tween 20, 0.005% NaN ₃ ). Individual antibodies were captured on the sensing surface by ProA/G. The extent of antibody capture is typically 60-90 resonance units (RU). Next, recombinant human C5 was injected at concentrations of 10 and 20 nM or 20 and 40 nM, followed by dissociation. The surface was regenerated with 25 mM NaOH. The sensorgrams were fitted with a 1:1 binding model using BIACORE (registered trademark) T200 evaluation software, version 2.0 (GE Healthcare) to determine kinetic parameters under two pH conditions. Figures 2A and 2B show the sensorgrams for all antibodies. Table 3 lists the on-rate (ka), off-rate (kd) and binding affinity (KD) of the antibodies. All antibodies except CFA0330 (VH, SEQ ID NO: 21 and VL, SEQ ID NO: 25) and A0341 (VH, SEQ ID NO: 22 and VL, SEQ ID NO: 26) were compared at pH 7.4 , showing a relatively fast dissociation rate at pH 5.8. [Table 3] Kinetic parameters of anti-C5 antibody at pH7.4 and pH5.8

[實施例4] 3.3. Cross-reactivity test In order to observe the cross-reactivity of the anti-C5 antibody to human C5 (hc5) and rhesus monkey C5 (cynoC5), a BIACORE (registered trademark) kinetic assay was performed. The assay setup was the same as described in Example 3.2, recombinant cynoC5 was injected at concentrations of 2, 10 and 50 nM. Kinetic parameters were determined by the same data fitting as described in Example 3.2. Table 4 lists the binding kinetics and affinities at pH 7.4. The kinetic parameters for hC5 shown in Table 4 are the results of Example 3.2. All anti-C5 antibodies except CFA0672 showed similar KD for hC5 and cynoC5. The KD of CFA0672 is 8-fold weaker for cynoC5 than 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 Antibody 4.1. Binding of anti-C5 MAbs to C5 β chain-derived peptides Anti-C5 monoclonal antibodies (MAbs) were tested for binding to β-chain derived peptides of C5 in a western blot assay. Expression of C5 peptides fused to GST markers (pGEX-4T-1, GE Healthcare Life Sciences, 28-9545-49) in E. coli (DH5α (DH5alpha), TOYOBO, DNA-903): 19-180, 161- 340, 321-500 and 481-660. E. coli were harvested after incubation with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37°C (°C) for 5 hours and centrifugation at 20,000 x g for 1 minute to obtain a pellet. sample. The precipitate was suspended in sample buffer solution (2ME+) (Wako, 191-13272) and used for western blot analysis. The expression of each peptide was confirmed with an anti-GST antibody (Abcam, ab9085) (Fig. 3). Arrows indicate GST-fused C5 peptide (46-49 kDa). Anti-C5 MAbs: CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672 and CFA0675, bind to 19-180 of C5 (Fig. 3).

4.2. Expression and purification of MG1-MG2 domain (1-225) of human C5 The recombinant MG1-MG2 domain of the β chain of human C5 (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 and then applied to a Q-Sepharose FF anion exchange column (GE healthcare, Uppsala, Sweden). The fraction passing through 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 applied to a Superdex 75 gel filtration column (GE healthcare, Uppsala, Sweden) equilibrated with 1× PBS. Fractions containing MG1-MG2 domains were then pooled and stored at -80°C.

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

4.4. Binding of anti-C5 MAbs to C5 MG1-MG2 domain-derived peptides Anti-C5 Mabs were tested for binding to MG1-MG2 domain-derived peptides in a western blot assay. C5 peptides fused to GST tag were expressed in E. coli: 33-124, 45-124, 52-124, 33-111, 33-108 and 45-111 (SEQ ID NO: 40). E. coli samples were collected after incubation with 1 mM IPTG at 37° C. for 5 hours and centrifugation at 20000 x g for 1 minute to obtain a pellet. The precipitate was suspended in sample buffer solution (2ME+) and used for western blot analysis. Expression of C5-derived peptide was confirmed with anti-GST antibody (Fig. 5A). CFA0305 bound only to the 33-124 peptide (Fig. 5B). CFA0305 binds to the beta chain of recombinant human C5 (rhC5) (approximately 70 kDa), which was used as a control. Figure 5C summarizes anti-C5 MAb responses to C5-derived peptides.

4.5. Binding of anti-C5 MAb to C5 mutant Since three amino acid residues in the β (beta) chain of C5 were predicted by crystal structure analysis: E48, D51 and K109, involved in the relationship between C5 and anti-C5-MAb Binding of C5 MAbs to human C5 point mutants was therefore analyzed in Western blot assays. C5 point mutants in which any of E48, D51 and K109 were substituted with alanine were expressed in FS293 cells by lipofection. Media was harvested 5 days after lipofection and later used for western blotting. SDS-PAGE was carried out under reducing conditions, and the results are shown in Figure 6. Eculizumab binds to the α (alpha) chain of wild-type (WT) C5 and three C5 point mutants, whereas CFA0305 binds strongly to the β chain of WT C5, weakly to the β chain of the E48A C5 mutant, and does not bind to β chain of D51A and K109A C5 mutants, which means that these 3 amino acid residues are involved in antibody/antigen interaction. Table 5 shows a summary of Western blot analysis of anti-C5 MAbs (CFA0305, CFA0307, CFA0366, CFA0501 , CFA0538, CFA0599, CFA0666, CFA0672 and CFA0675). Anti-C5 MAbs were classified to the same epitope C, but the binding pattern was slightly different between antibodies, suggesting that the binding regions of C5 anti-C5 MAbs are close to each other but not identical. [Table 5] Summary of anti-C5 MAb responses to C5 mutants

4.6. BIACORE (registered trademark) binding analysis of anti-C5 antibody and C5 mutant To test that residues E48, G51 and K109 are indeed involved in antibody/antigen interactions, BIACORE (registered trademark) binding assays were performed. Three C5 mutants were prepared: E48A, G51A and K109A, as described in Example 4.5. Culture supernatant samples containing mutant C5 overexpressed in FS293 cells were prepared at 40 micrograms per milliliter (µg/ml) of mutant C5. For BIACORE (registered trademark) binding analysis, the sample was diluted 10 times with BIACORE (registered trademark) electrophoresis buffer (ACES pH7.4, 10 mg/ml BSA, 1 mg/ml carboxymethyl dextran) to mutant The final sample concentration of C5 was 4 micrograms per milliliter (µg/ml).

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

As shown in Figure 7, all three C5 mutants can bind to eculizumab with similar binding profiles as wild-type C5. Regarding 305LO5, all three mutants showed lower binding responses to 305LO5 compared to wild-type C5. 305LO5 reduced the binding of D51A and K109A mutant C5 to baseline levels.

4.7. Identification of His residues on C5 that contribute to the pH-dependent interaction between anti-C5 antibodies and C5 Crystal structure analysis revealed that the 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 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 a mutant with double His mutations (H70Y+H110Y) were prepared by expressing H70, H72 and H110 in FS293 cells by lipofection A single His mutant in which either is substituted with tyrosine, and a double-His mutant in which both H70 and H110 are substituted with tyrosine. The antigen binding properties of the C5 His mutants to 305LO5, a pH-dependent anti-C5 antibody, were determined by the modified BIACORE (registered trademark) assay as described in Example 4.6. Briefly, an additional dissociation phase at pH 5.8 was added in the BIACORE (registered trademark) assay immediately after the dissociation phase at pH 7.4 to assess the pH dependence between antibody and antigenic source from complexes formed at pH 7.4 sexual dissociation. Scrubber 2.0 (BioLogic Software) curve fitting software was used to process and fit the data to determine the dissociation rate at pH 5.8.

[實施例5] As shown in Figure 8, C5 single His mutants at H70 or H110 and double His mutants (H70+H110) did not affect the binding of C5 to 305LO5 at neutral pH. Meanwhile, a single His mutant at H72 showed significantly reduced binding of C5 to 305LO5. The dissociation rates of C5 His mutant and C5-wt protein at pH 5.8 are shown in Table 6. As shown in Table 6, among the C5 antigens tested, C5-wt showed the fastest dissociation from 305LO5 at pH 5.8. The single His mutant at H70 showed an almost twice slower off-rate at pH 5.8 and the single His mutant at H110 caused a slightly slower off-rate at pH 5.8 compared to C5-wt. The double-His mutants at H70 and H110 had a greater effect on pH-dependent binding, with an almost 3-fold slower off-rate than C5-wt at pH 5.8. [Table 6] His mutant pair binding to 305LO5 pH5.8 dissociation rate value

[Example 5]

Inhibitory Activity of Anti-C5 Antibodies on C5 Activation 5.1. Inhibition of Complement-Activating Liposome Cytolysis by Anti-C5 MAbs Inhibition of complement activity by anti-C5 MAbs was tested by liposome lysis assay. Thirty microliters of normal human serum (6.7%) (Biopredic, SER018) was mixed with 20 microliters (µL) of diluted MAb in a 96-well plate and incubated for 30 minutes at 25°C on a shaker. Liposomes sensitized with an antibody against dinitrophenyl (Autokit CH50, Wako, 995-40801) were transferred to each well and the plates were placed on a shaker at 25 degrees C (°C) for 2 minutes. 50 microliters of matrix solution (Autokit CH50) was added to each well and mixed by shaking at 25°C for 2 minutes. The final mixture was incubated at 37°C for 40 minutes, after which the OD of the mixture was measured at 340 nm. The percentage of liposome cytolysis was defined as 100x [(OD _MAb - OD _{serum and liposome background} )]/[(OD _{no MAb} - OD _{serum and liposome background} )]. Figure 9A shows that anti-C5 Mabs: CFA0305, 0307, 0366, 0501, 0538, 0599, 0666, 0672 and 0675, inhibit liposome cytolysis. Two pH-independent antibodies, CFA0330 and 0341, also inhibited cell lysis (Fig. 9B).

5.2. Inhibition of C5a production by anti-C5 MAb Anti-C5 MAbs were tested for C5a production during liposomal lysis to confirm that anti-C5 MAbs inhibit 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 dose-dependently inhibited C5a production in the supernatants (Figures 10A and 10B).

5.3. Inhibition of the hemolytic response to complement activation by anti-C5 MAb Inhibition of classical complement activity by anti-C5 MAb was tested in a hemolytic assay. Chicken red blood cells (cRBCs) (Innovative research, IC05-0810) were washed with gelatin/veronal buffered saline containing _0.5 mM MgCl and 0.15 mM CaCl ₍ GVB++) (Boston BioProducts, IBB-300X), Then sensitize with 1 microgram/milliliter (µg/ml) anti-chicken RBC antibody (Rockland 103-4139) for 15 minutes at 4°C. Cells were then washed with GVB++ and suspended in the same buffer at 5x10 ⁷ cells/ml. Mix 50 microliters (µl) of normal human serum (20%) (Biopredic, SER019) with 50 µl of diluted Mab in separate round-bottom 96-well microtiter plates and incubate at 37°C on a shaker 30 minutes. Next, 60 microliters of the sensitized cRBC supernatant was added to the serum-containing wells, and the antibody cocktail was incubated at 37°C for 30 minutes. After incubation, the plates were centrifuged at 1000 xg for 2 minutes at 4°C. The supernatant (100 microliters) was transferred to wells of a flat-bottomed 96-well microtiter plate to measure the OD at 415 nm referenced to 630 nm. The percentage of hemolytic response was defined as 100×[(OD _MAb – OD _{serum and cRBC} )]/[(OD _{no MAb} – OD _{serum and cRBC background} )]. Figure 11 shows that anti-C5 Mabs: CFA0305 and 305LO5, inhibit the hemolytic response of cRBC.

5.4. Inhibition of the Alternative Complement Pathway by Anti-C5 MAb The alternative pathway hemolytic response assay was performed in a similar manner to the classical pathway hemolytic response assay. Blood collected from New Zealand white rabbits (InVivos) was mixed with the same volume of Alsever's solution (Sigma, A3551), and the mixed solution 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. Mix 40 microliters (µl) of normal human serum (25%) (Biopredic, SER019) with 40 microliters of diluted Mab in a round-bottom 96-well microtiter plate and incubate at 37°C on a shaker for 30 minutes . Next, 20 microliters of rRBC supernatant was added to the serum-containing wells, and the antibody cocktail was incubated at 37°C for 60 minutes. After incubation, the plates were centrifuged at 1000 xg for 2 minutes at 4°C. The supernatant (70 microliters) was transferred to wells of a flat-bottomed 96-well microtiter plate to measure the OD at 415 nm referenced to 630 nm. Figure 12 shows that anti-C5 Mabs: CFA0305 and CFA0672, inhibit the hemolytic response of rRBC, which means 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 testing using C57BL/6 mice The in vivo kinetics of human C5 (Calbiochem) and anti-human C5 antibody were assessed after single administration of human C5 or human C5 and anti-human C5 antibody in C57BL/6 mice (In Vivos or Biological Resource Centre, Singapore). Human C5 solution (0.01 mg/ml) or a solution containing human C5 and anti-human C5 antibody (0.01 mg/ml and 2 mg/ml (CFA0305-F760G4, CFA0307-F760G4, CFA0366-F760G4, CFA0501-F760G4, CFA0538-F760G4 , CFA0599-F760G4, CFA0666-F760G4, CFA0672-F760G4 and CFA0675-F760G4) or 0.2 mg/ml (CFA0330-F760G4 and CFA0341-F760G4), respectively) were administered into the tail vein at a dose of 10 ml/kg. In this case, anti-human C5 antibody was present in excess compared to human C5, so it can be assumed that almost every human C5 was bound to the antibody. Blood was collected 5 minutes, 7 hours, 1 day, 2 days, 3 days and 7 days after administration. The collected blood was immediately centrifuged at 14,000 rpm and 4°C for 10 minutes to separate the plasma. Separated plasma was stored in a -80°C refrigerator until assayed.使用之抗人類C5抗體為上述的CFA0305-F760G4、CFA0307-F760G4、CFA0330-F760G4、CFA0341-F760G4、CFA0366-F760G4、CFA0501-F760G4、CFA0538-F760G4、CFA0599-F760G4、CFA0666-F760G4、CFA0672-F760G4及CFA0675 -F760G4.

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

In cases where CFA0330-F760G4, CFA0341-F760G4, or human C5 alone were present in plasma samples, the following method was used. The anti-human C5 antibody (Santa Cruz) was dispensed on MULTI-ARRAY 96-well bare plates (Meso Scale Discovery) and left to stand overnight at 4°C to prepare anti-human C5-immobilized plates. Prepare calibration curve samples and mouse plasma samples diluted 100-fold or more with the injected antibody (CFA0330-F760G4 or CFA0341-F760G4) at 1 microgram/ml (µg/ml), and incubate at 37°C for 30 minutes. Next, the samples were dispensed onto anti-human C5 immobilized plates and allowed to stand at room temperature for 1 hour. Next, SULFO-TAG-labeled anti-human IgG antibody (Meso Scale Discovery) was added to react at room temperature for 1 hour, and washed. Immediately thereafter, Read Buffer T (x4) (Meso Scale Discovery) was allocated and measured using Sector Imager 2400 (Meso Scale Discovery).

In the case of CFA0305-F760G4, CFA0307-F760G4, CFA0366-F760G4, CFA0501-F760G4, CFA0538-F760G4, CFA0599-F760G4, CFA0666-F760G4, CFA0672-F760G4, or CFA0501-F760G4, CFA0538-F760G4, in plasma samples the following method was used. The anti-human C5 antibody (CFA0329-F939G4; VH, SEQ ID NO: 23 and VL, SEQ ID NO: 27) was distributed on a MULTI-ARRAY 96-well bare plate (Meso Scale Discovery) and left standing overnight at 4°C to prepare Plates immobilized with anti-human C5. Prepare calibration curve samples and mouse plasma samples diluted 100 times or more with acidic solution (pH 5.5), and incubate at 37°C for 30 minutes. Next, the samples were dispensed onto anti-human C5 immobilized plates and allowed to stand at room temperature for 1 hour. Next, SULFO-TAG-labeled anti-human C5 antibody (CFA0300-F939G4; VH, SEQ ID NO: 24 and VL, SEQ ID NO: 28) was added to react at room temperature for 1 hour, and washed. Immediately thereafter, Read Buffer T (x4) (Meso Scale Discovery) was allocated and measured using Sector Imager 2400 (Meso Scale Discovery).

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

6.4. Effect of pH-dependent anti-human C5 antibody binding on elimination of human C5 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-F706G74 and CFA) Dependence anti-human C5 antibody CFA0330-F760G4 and CFA0341-F760G4), and compare the plasma anti-human C5 antibody concentration and plasma human C5 concentration. As shown in Figure 14, antibody exposures were comparable. Meanwhile, the elimination of human C5 was accelerated with simultaneous administration of a pH-dependent anti-human C5 antibody compared to a pH-independent anti-human C5 antibody (Fig. 13). [Example 7]

Optimization of anti-C5 monoclonal antibody (305 variant) Some mutations were introduced into the optimized variable region of 305LO5 of the anti-C5 antibody to further improve its properties and generate optimized variable regions 305LO15, 305LO16, 305LO18, 305LO19, 305LO20, 305LO22, and 305LO23. VH of 305 variants The amino acid sequences of VL and VL are listed in Tables 7 and 8, respectively. Gene encoding humanized VH with modified human IgG1 CH variant SG115 (SEQ ID NO: 114) and modified human IgG4 CH variant SG422 (SEQ ID NO: 115) or SG429 (SEQ ID NO: 116) combined. The gene encoding humanized VL binds to human CL (SK1, SEQ ID NO: 38). Separately, the heavy chain 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 transformed into an expression vector.

Antibodies were expressed and purified by protein A in HEK239 cells co-transfected with a combination of heavy and light chain expression vectors. [Table 7] VH amino acid sequence of 305 variants Antibody VH HVR-H1 HVR-H2 HVR-H3 305LO5 Serial Identification Number: 10 Serial Identification Number: 54 SSYYVA Serial Identification Number: 64 AIYTGSGATYKASWAKG Serial Identification Number: 74 DGGYDYPTHAMHY 305LO15 Serial Identification Number: 106 Serial Identification Number: 117 SSYYMA Serial Identification Number: 118 AIFTGSGAEYKAEWAKG Serial Identification Number: 121 DAGYDYPTHAMHY 305LO16 Serial Identification Number: 107 Serial Identification Number: 117 SSYYMA Serial Identification Number: 119 AIFTGSGAEYKAEWVKG Serial Identification Number: 121 DAGYDYPTHAMHY 305LO18 Serial Identification Number: 108 Serial Identification Number: 117 SSYYMA Serial Identification Number: 118 AIFTGSGAEYKAEWAKG Serial Identification Number: 121 DAGYDYPTHAMHY 305LO19 Serial Identification Number: 109 Serial Identification Number: 117 SSYYMA Serial Identification Number: 118 AIFTGSGAEYKAEWAKG Serial Identification Number: 121 DAGYDYPTHAMHY 305LO20 Serial Identification Number: 109 Serial Identification Number: 117 SSYYMA Serial Identification Number: 118 AIFTGSGAEYKAEWAKG Serial Identification Number: 121 DAGYDYPTHAMHY 305LO22 Serial Identification Number: 109 Serial Identification Number: 117 SSYYMA Serial Identification Number: 118 AIFTGSGAEYKAEWAKG Serial Identification Number: 121 DAGYDYPTHAMHY 305LO23 Serial Identification Number: 110 Serial Identification Number: 117 SSYYMA Serial Identification Number: 120 GIFTGSGATYKAEWAKG Serial Identification Number: 121 DAGYDYPTHAMHY [Table 8] VL amino acid sequence of 305 variants305 variant Antibody VL HVR-L1 HVR-L2 HVR-L3 305LO5 Serial Identification Number: 20 Serial Identification Number: 84 QASQNIGSSLA Serial Identification Number: 94 GASKTHS Serial Identification Number: 104 QSTKVGSSYGNH 305LO15 Serial Identification Number: 111 Serial Identification Number: 122 RASQGISSSLA Serial Identification Number: 123 GASETES Serial Identification Number: 125 QNTKVGSSYGNT 305LO16 Serial Identification Number: 111 Serial Identification Number: 122 RASQGISSSLA Serial Identification Number: 123 GASETES Serial Identification Number: 125 QNTKVGSSYGNT 305LO18 Serial Identification Number: 111 Serial Identification Number: 122 RASQGISSSLA Serial Identification Number: 123 GASETES Serial Identification Number: 125 QNTKVGSSYGNT 305LO19 Serial Identification Number: 111 Serial Identification Number: 122 RASQGISSSLA Serial Identification Number: 123 GASETES Serial Identification Number: 125 QNTKVGSSYGNT 305LO20 Serial Identification Number: 112 Serial Identification Number: 122 RASQGISSSLA Serial Identification Number: 123 GASETES Serial Identification Number: 125 QNTKVGSSYGNT 305LO22 Serial Identification Number: 113 Serial Identification Number: 122 RASQGISSSLA Serial Identification Number: 124 GASTTQS Serial Identification Number: 125 QNTKVGSSYGNT 305LO23 Serial Identification Number: 113 Serial Identification Number: 122 RASQGISSSLA Serial Identification Number: 124 GASTTQS Serial Identification Number: 125 QNTKVGSSYGNT [Example 8]

Binding properties of anti-C5 antibody (305 variant) Utilize BIACORE (registered trademark) T200 equipment (GE Healthcare) at 37 degrees C to evaluate the kinetic parameters of the anti-C5 antibody against recombinant human C5 under three different conditions; ( 1) Both bind and dissociate at pH 7.4, (2) both bind and dissociate at pH 5.8, and (3) bind at pH 7.4 but dissociate at pH 5.8. ProA/G (Pierce) was immobilized on the CM1 sensing wafer using an amine coupling kit (GE Healthcare) according to the GE Healthcare recommended setup. Dilute the antibodies and analytes of conditions (1) and (3) in ACES pH7.4 buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl ₂ , 0.05% Tween 20, 0.005% NaN ₃ ), and condition (2) Antibodies and analytes were diluted in ACES pH5.8 buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl ₂ , 0.05% Tween 20, 0.005% NaN ₃ ). Individual antibodies were captured on the sensing surface by ProA/G. The extent of antibody capture is typically 60-90 resonance units (RU). Next, recombinant human C5, prepared by three-fold serial dilution, was injected at 3 to 27 nM or 13.3 to 120 nM, followed by dissociation. The surface was regenerated with 25 mM NaOH. Utilize BIACORE (registered trademark) T200 evaluation software, version 2.0 (GE Healthcare) to fit (fit) the sensory map with 1:1 binding model to determine the kinetic parameters under conditions (1) and (2), and MCK A 1:1 dissociation of the model was fitted to the sensorgram to determine the dissociation rate under condition (3). The pH dependence of all antibodies is expressed as the ratio of off-rates under conditions (2) and (1).

The on-rate (ka), off-rate (kd), binding affinity (KD) and pH dependence are listed in Table 9. All antibodies showed faster off-rates at pH 5.8 than at pH 7.4, and their pH dependence was approximately 20-fold. [Table 9] Kinetic parameters of anti-C5 antibody under the conditions of pH7.4 and pH5.8

Use BIACORE (registered trademark) T200 equipment (GE Healthcare) to measure the binding affinity of anti-C5 antibodies (BNJ441, eculizumab and 305 variants) to recombinant human C5 at pH 7.4 and pH 5.8 at 37 degrees C to evaluate Effect of pH on Antigen Binding. Goat anti-human IgG (Fc) polyclonal antibody (KPL #01-10-20) was immobilized on a CM4 sensing chip using an amine coupling kit (GE Healthcare) according to the supplier'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 surface of the sensing wafer using the anti-Fc method, typically to the extent of 50-80 resonance units (RU). Recombinant human C5 was prepared by 3-fold serial dilutions starting from 27 nM in the assay conditions at pH 7.4 or by 3-fold serial dilutions starting from 135 nM in the assay conditions at pH 5.8 . Regenerate the surface 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).

[實施例9] The binding affinities (KD) of BNJ441, eculizumab and 305 variants to recombinant human C5 at pH 7.4 and pH 5.8 are listed in Table 10. The 305 variant showed a ratio of (KD at pH 5.8)/(KD at pH 7.4) of about 800, which was 8 times higher than that of BNJ441, whose ratio of (KD at pH 5.8)/(KD at pH 7.4) was only 93. [Table 10]

[Example 9]

Inhibitory Activity of Anti-C5 Antibody (305 Variant) on C5 Activation 9.1. Inhibition of Complement-Activating Liposome Cytolysis by Anti-C5 MAbs Inhibition of complement activity by anti-C5 MAbs was tested by liposome lysis assay. 30 μl of normal human serum (6.7%) (Biopredic, SER019) was mixed with 20 μl of diluted MAb in a 96-well plate and incubated on a shaker at room temperature for 30 minutes. Liposome solutions sensitized with antibodies against dinitrophenyl (Autokit CH50, Wako, 995-40801) were transferred to each well and the plates were placed on a shaker at 37°C for 2 minutes. 50 microliters of matrix solution (Autokit CH50) was added to each well and mixed by shaking at 37°C for 2 minutes. The final mixture was incubated at 37°C for 40 minutes before measuring the OD at 340 nm. The percentage of liposome cytolysis was defined as 100x [(OD _MAb - OD _{serum and liposome background} )]/[(OD _{no MAb} - 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 cytolysis. Two antibodies with Fc variants: 305LO15-SG115 and 305LO23-SG429, also inhibited liposome cytolysis (Fig. 16).

The anti-C5 MAb was tested for inhibition of recombinant human C5 (SEQ ID NO: 39). In a 96-well plate, 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 microgram/milliliter (µg/mL)) and placed in Incubate for 1 hour at 37°C on a shaker. Liposomes (Autokit CH50) were transferred to each well and placed on a shaker at 37°C for 2 minutes. 50 microliters of matrix solution (Autokit CH50) was added to each well and mixed by shaking at 37°C for 2 minutes. The final mixture was incubated at 37°C for 180 minutes before measuring the OD at 340 nm. The percentage of liposome cytolysis is as defined above. Figure 17 shows that anti-C5 Mabs: 305LO22-SG115, 305LO22-SG422, 305LO23-SG115 and 305LO23-SG422 inhibit liposome cytolysis.

9.2. Inhibition of C5a production by anti-C5 MAb Anti-C5 MAbs were tested for C5a production during liposomal lysis to confirm that anti-C5 MAbs inhibit 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 supernatants in a dose-dependent manner (Figures 18 and 19).

9.3. Measurement of complement activity in rhesus plasma Inhibition of complement activity by anti-C5 MAb was measured in rhesus plasma. Anti-C5 Mab was administered internally to monkeys (20 mg/kg), and plasma samples were collected periodically until day 56. Chicken red blood cells (cRBCs) (Innovative research, IC05-0810) were washed with gelatin/veronal buffered saline containing _0.5 mM MgCl and 0.15 mM CaCl ₍ GVB++) (Boston BioProducts, IBB-300X), Then sensitize with 1 microgram/milliliter (µg/ml) anti-chicken RBC antibody (Rockland 103-4139) for 15 minutes at 4°C. Cells were then washed with GVB++ and suspended in the same buffer at 1×10 ⁸ cells/ml. Monkey plasma was incubated with sensitized cRBCs at 37°C for 20 minutes in separate round-bottom 96-well microtiter dishes. After incubation, the plates were centrifuged at 1000 xg for 2 minutes at 4°C. The supernatant was transferred to the wells of a flat-bottomed 96-well microtiter plate to measure the OD at 415 nm referenced to 630 nm. The percentage of hemolytic response was defined as 10Ox [( _{after OD administration} - OD _{plasma and cRBC background} )]/[( _{before OD administration} - 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. Inhibition of biological activity of C5 variants by anti-C5 MAbs Anti-C5 MAbs were tested for inhibition of recombinant human C5 variants: V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N and E1437D. It has been reported that PNH patients with the R885H mutation in C5 respond poorly 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 supernatants were used in subsequent studies. In a 96-well plate, mix 10 µl of C5-deficient human serum (Sigma, C1163) with 20 µl of diluted Mab and 20 µl of recombinant C5 variant (2-3 micrograms/milliliter (µg/mL)) The cell culture medium was mixed and incubated on a shaker at 37°C for 0.5 hours. Liposomes (Autokit CH50) were transferred to each well and placed on a shaker at 37°C for 2 minutes. 50 microliters of matrix solution (Autokit CH50) was added to each well and mixed by shaking at 37°C for 2 minutes. The final mixture was incubated at 37°C for 90 minutes before measuring the OD at 340 nm. The percentage of liposome cytolysis is as defined above. Figure 21 shows that the anti-C5 MAb (eculizumab) did not inhibit the R885H C5 variant, but did inhibit the other variants tested. Figure 22 shows that the anti-C5 Mab (305 variant) inhibits all variants of C5 tested.

9.5. Inhibition of Complement-Activated Liposome Cytolysis by Anti-C5 MAb Inhibition of complement activity by anti-C5 Mab was tested by liposome lysis assay. 30 μl of normal human serum (6.7%) (Biopredic, SER019) was mixed with 20 μl of diluted MAb in a 96-well plate and incubated on a shaker at room temperature for 30 minutes. A solution of liposomes sensitized with an antibody against dinitrophenyl (Autokit CH50, Wako, 995-40801) was transferred to each well and the plate was placed on a shaker at 25°C for 2 minutes. 50 microliters of matrix solution (Autokit CH50) was added to each well and mixed by shaking at 25°C for 2 minutes. The final mixture was incubated at 37°C for 45 minutes before measuring the OD at 340 nm. Percent inhibition of liposome cytolysis was defined as 100x [(OD _MAb - OD _{serum and liposome background} )]/[(OD _{no MAb} - OD _{serum and liposome background} )]. Figure 13 shows that anti-C5 Mabs: 305LO15-SG422, 305LO16-SG422, 305LO18-SG422, 305LO19-SG422, 305LO20-SG422 and 305LO20-SG115, inhibited liposome cytolysis. Figure 23 shows that the anti-C5 MAb, BNJ441 and the 305 variant inhibit liposome cytolysis, and the 305 variant has stronger inhibitory activity than BNJ441. [Example 10]

Pharmacokinetic study of anti-C5 monoclonal antibody (305 variant) in rhesus monkeys 10.1. In vivo testing using rhesus monkeys The in vivo kinetics of the anti-human C5 antibody was evaluated after administration of the anti-human C5 antibody to rhesus monkeys (Shin Nippon Biomedical Laboratories, Ltd., Japan). A solution (2.5 mg/ml) of anti-human C5 antibody was administered into the cephalic vein of the forearm by infusion for 30 minutes at a dose of about 8 ml/kg each. Before administration (pre-administration) and 5 minutes, 7 hours, 1 day, 2 days, 3 days, 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days and 56 days after administration Collect blood. The collected blood was immediately centrifuged at 1,700xg and 4°C for 10 minutes to separate the plasma. Separated plasma was stored in a refrigerator at or below -70°C until assayed. Anti-human C5 antibody was prepared as described in Example 7.

10.2. Measurement of total rhesus monkey C5 plasma concentration by ELISA analysis The concentration of total rhesus C5 in rhesus plasma was measured by ELISA. The anti-human C5 antibody (in-house antibody generated using the method described in Example 2) was dispensed on Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International), and left at 4°C overnight to prepare immobilized anti-constant Disk of rhesus monkey C5. Prepare a calibration curve sample diluted 20,000 times with 0.4 microgram/ml (µg/ml) antibody injection and a rhesus monkey plasma sample, and incubate at 37°C for 60 minutes. Next, the samples were dispensed onto plates immobilized with anti-rhesus monkey C5 and allowed to stand at room temperature for 1 hour. Next, HRP-labeled anti-human IgG antibody (Southernbiotech) was added to react at room temperature for 30 minutes, and washed. Next, ABTS ELISA HRP Substrate (KPL) was added. Signals were measured at a wavelength of 405 nm by a plate reader. According to the response of the calibration curve, the concentration of anti-rhesus monkey C5 was calculated using the analysis software SOFTmax PRO (Molecular Devices). The time course of plasma rhesus monkey C5 concentration after intravenous administration measured by this method is shown in FIG. 24 . Data are plotted as percent remaining compared to plasma rhesus C5 concentrations before administration. pH-dependent anti-human C5 antibodies (305LO15-SG422, 305LO15-SG115, 305LO16-SG422, 305LO18-SG422, 305LO19-SG422, SG422, 305LO23-SG422 and 305LO23-SG115) showed decreased accumulation of plasma C5.

10.3. Measurement of anti-human C5 antibody plasma concentration by ELISA analysis The concentration of anti-human C5 antibody in plasma of rhesus monkeys was measured by ELISA. Anti-human IgG kappa (kappa) chain antibody (Antibody Solutions) was dispensed on unc-ImmunoPlate MaxiSorp (Nalge Nunc International), and left standing overnight at 4°C to prepare anti-human IgG-immobilized plates. Prepare calibration curve samples diluted 100 times or more and rhesus monkey plasma samples. Next, the samples were dispensed onto anti-human IgG-immobilized plates and allowed to stand at room temperature for 1 hour. Next, HRP-labeled anti-human IgG antibody (Southernbiotech) was added to react at room temperature for 30 minutes, and washed. Next, ABTS ELISA HRP Substrate (KPL) was added. The signal was measured by a disk reader at a wavelength of 405 nm. According to the response of the calibration curve, the concentration of anti-human C5 antibody was calculated using the analysis software SOFTmax PRO (Molecular Devices). The time course of plasma anti-human C5 antibody concentration after intravenous administration measured by this method is shown in FIG. 25 . pH-dependent anti-human C5 antibodies (305LO15-SG422, 305LO15-SG115, 305LO16-SG422, 305LO18-SG422, 305LO19-SG422, SG422, 305LO23-SG422 and 305LO23-SG115) showed a longer half-life. [Example 11]

X-ray crystal structure analysis of 305 variant Fab and human C5-MG1 domain complex 11.1. Expression and purification of MG1 domain (20-124) of human C5 Using the pGEX-4T-1 vector (GE healthcare), the MG1 domain (SEQ ID NO: 39 amino acid residues 20-124). Protein expression was induced with 0.1 Mm isopropyl β(beta)-D-1-thiogalactoside (IPTG) at 25°C for 5 hours. Bacterial cell pellets were lysed with Bugbuster (Merck) supplemented with lysonase (Merck) and complete protease inhibitor cocktail, followed by purification of GST-MG1 from the soluble fraction using GSTrap columns (GE healthcare) according to the supplier's instructions. The GST marker was cleaved with thrombin (Sigma), and the resulting MG1 domain was further purified with a Superdex 75 colloidal filtration column (GE healthcare). Fractions containing the MG1 domain were pooled and stored at -80°C.

11.2. Preparation of Fab fragments of 305 variants Fab fragments from one of the 305 optimized variants were prepared by conventional methods using papain (Roche Diagnostics, Cat No. 1047825), followed by loading on a protein A column (MabSelect SuRe, GE Healthcare) to remove Fc Fragment, cation exchange column (HiTrap SP HP, GE Healthcare) and colloidal 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 complex 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 at 4°C by the sitting drop vapor diffusion method combined with the seeding method. The storage solution consisted of 0.2 M magnesium formate dehydrate, 15.0% w/v polyethylene glycol 3350. This successfully produced disc-shaped crystals within a few days, which were soaked in a solution of 0.2 M magnesium formate dehydrate, 25.0% w/v polyethylene glycol 3350, and 20% glycerol.

a; R _merge= ∑ hkl∑ j| Ij( hkl) － 〈 I( hkl)〉|/∑ hkl∑ j| I( hkl)|，其中 Ij( hkl)及〈 I( hkl)〉分別為測量的強度 j及以 hkl指數反射的平均強度。 b; R因子= ∑ hkl| F _calc( hkl)| － | F _obs( hkl)|/∑ hkl| F _obs( hkl)|，其中 F _obs及 F _calc分別為觀測及計算的結構因子振幅。 c; R _free是以5%的隨機預留反射計算。 11.5. Data Collection and Structure Determination X-ray diffraction data were measured at SPring-8 by BL32XU. During the measurement, the crystal was continuously placed in a nitrogen stream at -178°C to maintain a frozen state, and the crystal was rotated 1.0° each time using an MX-225HS CCD detector (RAYONIX) attached to the ray to collect A total of 180 X-ray diffraction images. Using 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 determine the cell parameters, index the diffraction points, and process the diffraction data from the obtained diffraction image, and finally obtain the diffraction intensity data with a resolution of 2.11 Angstroms (Å). The statistics of the crystal data are shown in Table 11. [Table 11] X-ray data collection and improvement statistics

a; R _merge = ∑ hkl ∑ j | Ij ( hkl ) - 〈 I ( hkl )〉|/∑ hkl ∑ j | I ( hkl )|, where Ij ( hkl ) and 〈 I ( hkl )〉 are measured Intensity j and the average intensity reflected by the hkl index. b; R factor = ∑ hkl | F _calc ( hkl )| - | F _obs ( hkl )|/∑ hkl | F _obs ( hkl )|, where F _obs and F _calc are the observed and calculated structure factor amplitudes, respectively. c; R _free is calculated based on 5% random reserved reflection.

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

11.6. The overall structure of the 305 variant Fab and the C5-MG1 domain complex The Fab piece of the variant optimized from 305 ("305 Fab") binds to the human C5-MG1 domain ("MG1") in a 1:1 ratio, and the asymmetric unit of the crystal structure contains 2 complexes, molecule 1 and 2, as depicted in Figure 26A. Molecules 1 and 2 can be aligned with Cα (alpha) atoms in all residues at 0.717 Angstrom (Å) RMSD, as shown in Figure 26B. The diagrams discussed below were prepared using Molecule 1.

In Figures 27A and 27B, the epitope in the contact region of 305 Fab is located in the amino acid sequence and crystal structure of MG1, respectively. The epitope comprises amino acid residues of MG1 that contain one or more atoms within 4.5 Angstroms (Å) of any part of the 305 Fab in the crystal structure. In addition, epitopes within 3.0 Angstroms (Å) are highlighted in Figure 27A.

11.7. Interaction of E48, D51 and K109 Anti-C5 Mabs comprising the 305 antibody series were tested for binding to three human C5 point mutants E48, D51 and K109 by Western blot and BIACORE (registered trademark) binding assays as described in Examples 4.5 and 4.6. While the 305 variants bound strongly to WT C5, they only weakly bound to the E48A C5 mutant, and did not bind to the D51A and K109A mutants. The crystal structure of the 305 Fab and MG1 complex reveals that the three amino acids E48, D51 and K109 are all within 3.0 Angstroms (Å) of the 305 Fab, which form several hydrogen bonds with the Fab, as shown in Figure 28A Show. Examined in more detail, the K109 residue of MG1 is buried in a groove formed at the interface of the heavy chain of the Fab, and is connected by three hydrogen bonds to H-CDR3_G97, H-CDR3_Y100 and H-CDR3_T100b and by a salt bridge Tightly interact with Fab with H-CDR3_D95 (Fig. 28D). D51 is located between the heavy chains of MG1 and 305 Fab and forms two hydrogen bonds with H-CDR1_Ser32 and H-CDR2_Ser54 to fill the space (Fig. 28C). These K109 and D51 representing C5 are important residues for the binding of the 305 antibody series. On the other hand, E48 is closer to the surface and forms only one hydrogen bond with Fab, suggesting that it contributes less to antibody binding than K109 and E51 (Fig. 28B). These relationships are consistent with the results of Western blot and BIACORE (registered trademark) binding assays of human C5 mutants (Examples 4.5 and 4.6). Supplementary Note: The residue numbering of Fab amino acids is according to 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 and 305 antibody series Crystal structure analysis revealed that three histidine residues on human C5, namely H70, H72 and H110, are contained in the epitope of the 305 variant Fab, as shown in Figures 27A and 29A. BIACORE (registered trademark) binding assays were performed to investigate the contribution of these histidine residues to pH-dependent protein-protein interactions between human C5 and the 305 variant Fab using the human C5 mutant H70Y , H72Y, H110Y and H70Y+H110Y (Example 4.7). H72Y caused a complete loss of binding of the 305 variant Fab to C5. This residue of C5 is located in the pocket formed by the CDR2 loop of the heavy chain of the 305 Fab and the loop of MG1 (L73, S74 and E76), and tightly fills this space, as shown in Figure 29C. In addition, the H72 residue of C5 forms a hydrogen bond with H-CDR2_Y58. The H72Y mutation is not expected to be tolerated, as there is not enough room to accommodate the bulky side chain of tyrosine, and the hydrogen bond to H-CDR2_Y58 cannot be maintained. Regarding the contribution of H70 and H110 to the 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, which is thought to be broken at pH 5.8, when protonation of H70 at C5 leads to a conformational change in the corresponding part of the interaction interface of MG1 (Fig. 29B). As for H110, protonation of this C5 residue is expected to cause charge repulsion towards the 305 Fab, which can be enhanced by protonation of the adjacent histidine residue H-CDR3_H100c (Fig. 29D).

While the foregoing invention has been described in detail by way of illustration and example for purposes of clarity of understanding, such description and examples should not be construed as limiting the scope of the invention. The contents of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

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[Picture 1] Figure 1 shows the epitope binning of anti-C5 antibody, as described in Example 2.2. Antibodies sorted into the same epitope bin are framed in bold. [Figure 2A] Figure 2A shows BIACORE (registered trademark) sensorgrams of anti-C5 antibody at pH 7.4 (solid line) and pH 5.8 (dashed line) to assess pH dependence, as described in Example 3.2. CFA0305, CFA0307, CFA0366, CFA0501, CFA0538 and CFA0599 are antibodies classified into epitope C, as described in Example 2.2. [Figure 2B] Figure 2B shows BIACORE (registered trademark) sensorgrams of anti-C5 antibody at pH 7.4 (solid line) and pH 5.8 (dashed line) to assess pH dependence, as described in Example 3.2. CFA0666, CFA0672 and CFA0675 are antibodies classified into epitope C, and CFA0330 and CFA0341 are antibodies classified into epitope B, as described in Example 2.2. 305LO5 is a humanized antibody to CFA0305, as described in Example 2.3. [Picture 3] Figure 3 shows the Western Blot for the extended fragment of the β chain of C5 fused with the GST marker (SEQ ID NO: 40 amino acids 19 to 180, 161 to 340, 321 to 500 and 481 to 660) Analysis was as described in Example 4.1. CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675 are antibodies classified into epitope C. Anti-GST antibody is the positive control group, and the position of the GST-fused C5 fragment (46 to 49 kDa) is indicated by the arrow. [Picture 4] Figure 4 shows the BIACORE (registered trademark) sensing map of the anti-C5 antibody for the MG1-MG2 domain of the beta chain of C5, as described in Example 4.3. The upper column shows the results for CFA0305 (solid line), CFA0307 (dashed line), CFA0366 (dashed-dotted line) and eculizumab (dotted line). The middle column shows the results for CFA0501 (solid line), CFA0599 (dashed line), CFA0538 (dashed-dotted line) and eculizumab (dotted line). The lower column shows the results for CFA0666 (solid line), CFA0672 (dashed line), CFA0675 (dashed-dotted line) and eculizumab (dotted line). CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675 are antibodies classified into epitope C, and Eculizumab is a control group of anti-C5 antibodies. [Figure 5A] Figure 5A shows peptide fragments derived against the MG1-MG2 domain fused to a GST tag (SEQ ID NO: 40 amino acids 33 to 124, 45 to 124, 52 to 124, 33 to 111, 33 to 108 and 45 to 111 ) Western blot analysis, as described in Example 4.4. An anti-GST antibody was used as the reactive antibody, and the position of the GST-fused C5 fragment (35 to 37 kDa) is indicated by an arrow. [Figure 5B] Figure 5B shows peptide fragments derived against the MG1-MG2 domain fused to the GST tag (SEQ ID NO: 40 amino acids 33 to 124, 45 to 124, 52 to 124, 33 to 111, 33 to 108 and 45 to 111 ) Western blot analysis, as described in Example 4.4. CFA0305 was used as the reactive antibody. [Figure 5C] Figure 5C summarizes the binding response of an anti-C5 antibody to a beta chain-derived fragment of C5, as described in Example 4.4. Fragments that bind to anti-C5 antibodies classified in epitope C (CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675) are indicated in gray, and fragments that do not bind to them are indicated in white. [Picture 6] Figure 6 shows Western blot analysis for C5 point mutants with alanine substitutions at E48, D51 and K109 of the beta chain (E48A, D51A and K109A, respectively), as described in Example 4.5. In the left column, eculizumab (anti-C5 antibody, alpha chain conjugate) was used as the reacting antibody and the position of the alpha chain of C5 (about 113 kDa) is indicated by an arrow. In the right column, CFA0305 (classified to epitope C, beta chain binder) was used as the reacting antibody and the position of the beta chain of C5 (approximately 74 kDa) is indicated by an arrow. [Figure 7] Figure 7 presents BIACORE (registered trademark) sensorgrams showing the interaction of eculizumab-F760G4 (upper panel) or 305LO5 (lower panel) with C5 mutants, as described in Example 4.6. The sensor maps were obtained by injecting C5-wt (thick curve), C5-E48A (short dashed curve), C5-D51A (long dashed curve) and C5-K109A (thin curve) into immobilized eculizumab-F760G4 or 305LO5 sensor surface obtained. Eculizumab was a control group of anti-C5 antibody, and 305LO5 was a humanized antibody of CFA0305 (classified to epitope C), as described in Example 2.3. [Picture 8] Figure 8 presents a BIACORE (registered trademark) sensorgram showing the interaction of 305LO5 with a His mutant of C5 to assess pH dependence, as described in Example 4.7. The sensor map was obtained by respectively injecting C5-wt (thick curve), C5-H70Y (long dashed curve), C5-H72Y (short dashed curve), C5-H110Y (dotted curve) and C5-H70Y+H110Y ( Thin curve) obtained on the surface of the sensor with 305LO5 immobilized. Antibody/antigen complexes can be dissociated at pH 7.4, followed by further dissociation at pH 5.8 (arrows) to assess pH-dependent interactions. [Figure 9A] Figure 9A shows inhibition of complement-activated liposome lysis by anti-C5 antibody, as described in Example 5.1. The results showed that CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672 and CFA0675 were classified to epitope C, as described in Example 2.2. [Figure 9B] Figure 9B shows inhibition of complement activated liposome cytolysis by anti-C5 antibody, as described in Example 5.1. The results showed that antibodies CFA0330 and CFA0341 were classified to epitope B, as described in Example 2.2. [Figure 10A] Figure 10A shows inhibition of C5a production by anti-C5 antibody, as described in Example 5.2. C5a concentrations were quantified in the supernatants obtained in the liposome lysis assay as described in Figure 9A. [Figure 10B] Figure 10B shows inhibition of C5a production by anti-C5 antibody, as described in Example 5.2. C5a concentrations were quantified in the supernatants obtained in the liposome lysis assay described in Figure 9B. [Picture 11] Figure 11 shows inhibition of hemolytic response to complement activation by anti-C5 antibody, as described in Example 5.3. Complement is activated by the classical pathway. [Picture 12] Figure 12 shows the inhibition of hemolytic response to complement activation by anti-C5 antibody, as described in Example 5.4. Complement is activated through the alternative pathway. [Picture 13] Figure 13 shows the time course of plasma human C5 concentrations following intravenous administration of human C5 alone or human C5 and an anti-human C5 antibody in mice assessed for C5 depletion, as described in Example 6.2. CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675 are antibodies classified to epitope C, and CFA0330 and CFA0341 are antibodies classified to 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 and anti-human C5 antibody in mice to assess antibody pharmacokinetics, as described in Example 6.3. CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675 are antibodies classified to epitope C, and CFA0330 and CFA0341 are antibodies classified to epitope B, as described in Example 2.2. [Picture 15] Figure 15 shows inhibition of complement activated liposome cytolysis by anti-C5 antibody, as described in Example 9.1. Results are shown for antibodies 305LO15-SG422, 305LO16-SG422, 305LO18-SG422, 305LO19-SG422, 305LO20-SG422 and 305LO20-SG115. [Picture 16] Figure 16 shows inhibition of complement activated liposome cytolysis by anti-C5 antibody, as described in Example 9.1. Results are shown for antibodies 305LO15-SG115 and 305LO23-SG429. [Picture 17] Figure 17 shows inhibition of complement activated liposome cytolysis by anti-C5 antibody as described in Example 9.1. Results are shown for antibodies 305LO22-SG115, 305LO22-SG422, 305LO23-SG115 and 305LO23-SG422. [Picture 18] Figure 18 shows inhibition of C5a production by anti-C5 antibody, as described in Example 9.2. C5a concentrations were quantified in the supernatants obtained in the liposome lysis assay as described in FIG. 15 . [Picture 19] Figure 19 shows inhibition of C5a production by anti-C5 antibody, as described in Example 9.2. C5a concentrations were quantified in the supernatants obtained in the liposome lysis assay as described in FIG. 16 . [Picture 20] Figure 20 shows inhibition of complement activity in monkey plasma by anti-C5 antibody, as described in Example 9.3. Anti-C5 antibodies were administered to rhesus monkeys (cynomolgus monkeys), and complement activity in monkey plasma was measured in a hemolytic response assay. [Picture 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 . [Picture 22] Figure 22 shows the inhibition of the biological activity of wild-type C5 (WT) and C5 variants (V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N and E1437D) by anti-C5 antibody (305 variant), as in Example 9.4 mentioned. [Picture 23] Figure 23 shows inhibition of complement activated liposome cytolysis by anti-C5 antibodies (BNJ441 and 305 variants), as described in Example 9.5. [Picture 24] Figure 24 shows the time course of plasma rhesus monkey C5 concentrations following intravenous administration of anti-human C5 antibodies in rhesus monkeys assessing C5 depletion, as described in Example 10.2. [Picture 25] Figure 25 shows the time course of plasma anti-human C5 antibody concentrations following intravenous administration of anti-human C5 antibodies in rhesus monkeys to assess pharmacokinetics, as described in Example 10.3. [Picture 26] Figures 26A and 26B show the crystal structure of the 305 Fab bound to the human C5(hC5)-MG1 domain, as described in Example 11.6. Figure 26A shows the asymmetric unit, MG1 is shown as a surface schematic, and the 305 Fab is shown as a ribbon (dark grey: heavy chain, light grey: light chain). Figure 26B shows overlapping molecules 1 and 2 (dark gray: molecule 1, light gray: molecule 2). [Figure 27A] Figure 27A shows epitopes located in the 305 Fab contact region on the MG1 domain, as described in Example 11.6. Figure 27A shows the epitope mapping in the MG1 amino acid sequence (dark gray: closer than 3.0 Å (Angstrom), light gray: closer than 4.5 Å). [Figure 27B] Figure 27B shows epitopes located in the 305 Fab contact region on the MG1 domain, as described in Example 11.6. Figure 27B shows the epitope positioning in the crystal structure (dark gray spheres: closer than 3.0 Å, light gray sticks: closer than 4.5 Å). [Figure 28A] Figure 28A shows a close up view of E48, D51 and K109 (stick image) interacting with 305 Fab (surface image), as described in Example 11.7. [Figure 28B] Figure 28B shows the interaction of E48 and its surroundings (dark gray dotted line: hydrogen bonding with Fab, light gray dotted line: water mediated hydrogen bonding), as described in Example 11.7. [Figure 28C] Figure 28C shows the interaction of D51 and its surroundings (dark gray dotted line: hydrogen bonding with Fab), as described in Example 11.7. [Figure 28D] Figure 28D shows the interaction of K109 and its surroundings (dark gray dotted line: hydrogen bond with Fab, light gray dotted line: salt bridge with H-CDR3_D95), as described in Example 11.7. [Figure 29A] Figure 29A shows a close-up view of H70, H72 and H110 (rod image) interacting with 305 Fab (surface image), as described in Example 11.8, in the same orientation as Figure 28A. [Figure 29B] Figure 29B shows the interaction of H70 and its surroundings, as described in Example 11.8. The histidine residues are indicated by stick and mesh images, and hydrogen bonds are indicated by dotted lines. [Figure 29C] Figure 29C shows the interaction of H72 and its surroundings, as described in Example 11.8. The histidine residues are indicated by stick and mesh images, and hydrogen bonds are indicated by dotted lines. [Figure 29D] Figure 29D shows the interaction of H110 and its surroundings, as described in Example 11.8. The histidine residues are indicated by stick and mesh images. The distance between H110 and H-CDR3_H100c is indicated by a dotted line.

none.

Claims (3)

A use of an anti-C5 antibody for the manufacture of a drug for treating paroxysmal nocturnal hemoglobinuria (PNH), wherein the antibody includes: (a) VH sequence with sequence identification number: 106 and VL sequence with sequence identification number: 111; (b) sequence identification number: VH sequence of 107 and sequence identification number: VL sequence of 111; (c) VH sequence with sequence identification number: 108 and VL sequence with sequence identification number: 111; (d) VH sequence with sequence identification number: 109 and VL sequence with sequence identification number: 111; (e) VH sequence with sequence identification number: 109 and VL sequence with sequence identification number: 112; (f) the VH sequence of sequence identification number: 109 and the VL sequence of sequence identification number: 113; or (g) VH sequence of sequence identification number: 110 and VL sequence of sequence identification number: 113. The use of the anti-C5 antibody as claimed in claim 1, wherein the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 114 and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 38 Area. The use of the anti-C5 antibody as described in claim 1 or 2, wherein the antibody is a full-length IgG1 antibody or a full-length IgG4 antibody.

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