TWI610941B - Anti-c5 antibodies and methods of use - Google Patents

Abstract

The present invention provides an anti-C5 antibody and a method of using the same. In some embodiments, an isolated anti-C5 antibody of the invention binds to an epitope within the beta chain of C5 with a higher affinity at neutral pH than at acidic pH. The invention also provides an isolated nucleic acid encoding an anti-C5 antibody of the invention. The invention also provides a host cell comprising a nucleic acid of the invention. The present invention also provides a method for producing an antibody, comprising culturing a host cell of the present invention for producing the antibody. The invention further provides a method for manufacturing an anti-C5 antibody, which comprises immunizing an animal with a polypeptide including the MG1-MG2 domain of the β chain of C5. The anti-C5 antibody of the present invention can be used as a medicine.

Description

Anti-C5 antibodies and methods of use

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

The complement system plays an important role in the clearance of immune complexes and in the immune response to vector, foreign antigens, virus-infected cells and tumor cells. About 25 to 30 complement proteins are found as a complex collection of plasma proteins and membrane cofactors. Complement components achieve their immune defense functions by interacting with a series of complex and complex enzyme cleavage and membrane binding reactions. The complement cascade results in the production of products with opsonic, immunomodulatory and cytolytic functions. .

Currently, it is widely accepted that the complement system can be activated by three different pathways: the classical pathway, the lectin pathway, and the alternative pathway. These pathways share many complements, and although they differ in the initial steps, they meet 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 an antigen-antibody complex. Independently, the first step in activating the lectin pathway is the binding of specific lectins, such as mannan-binding lectin (MBL), H-ficolin, M-cellulose Lectin, L-fibrillin, and C-type lectin CL-11. In contrast, alternative pathways spontaneously experience low levels Turnover activation, which can easily expand on foreign or other abnormal surfaces (bacteria, yeast, virus-infected cells, or damaged tissue). These pathways converge at a position where the complement component C3 is cleaved by an active proteolytic enzyme to produce C3a and C3b.

C3a is an anaphylatoxin. C3b binds to bacteria and other cells. It also binds to specific viruses and immune complexes and marks them for removal from the circulation (known as the action of opsonin) . C3b also forms complexes with other components to form C5 invertase, which cuts C5 into C5a and C5b.

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

When the complement pathway is activated, mature C5 is cleaved into C5a and C5b fragments. C5a is cleaved by the C5 invertase from the alpha chain of C5 as an amine terminal fragment, including the first 74 amino acids of the alpha chain. The remainder of mature C5 is the fragment C5b, which contains the remaining alpha chain bound to the beta chain with a disulfide bond. Approximately 20% of the 11 kDa mass of C5a is sugar.

C5a is another allergic toxin. C5b combines with C6, C7, C8 and C9 to form a membrane attack complex. MAC, C5b-9, terminal complement complex (TCC)) are on the surface of target cells. When a sufficient amount of MAC is inserted into the target cell membrane, MAC holes are formed to regulate the rapid permeable cell lysis of the target cells.

In accordance with the foregoing, C3a and C5a are allergic toxins. They can trigger the degranulation of mast cells, which releases histamine and other mediators of inflammatory reactions, causing smooth muscle contraction, increased vascular permeability, white blood cell activation, and other inflammatory reactions. This includes hypercellularity due to cell proliferation. C5a can also be used as a chemotactic peptide, which is used to attract granular cells, such as: neutrophils, eosinophils, basophils, and monocytes to the site of complement activation.

C5a activity 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 anaphylatoxin activity and polymorphonuclear trending activity of unmodified C5a.

Although a properly functioning complement system provides a solid defense against infectious microorganisms, improper regulation or activation of complement has been implicated in the pathogenicity of a variety of diseases, including, for example: rheumatoid arthritis (RA); lupus nephritis; topical Ischemia-reperfusion injury; paroxysmal nocturnal hemoglobinuria (PNH); atypical hemolytic uremic syndrome (aHUS); dense sedimentary disease (DDD); macular degeneration (for example: age-related macular degeneration (AMD) ); Hemolysis, elevated liver enzymes, and low platelet (HELLP) syndrome; thrombotic thrombocytopenic purpura (TTP); spontaneous abortion; pauci-immune vasculitis; epidermolytic vesicular disease; recurrent abortion Multiple sclerosis (MS); traumatic brain injury; and injuries caused by myocardial infarction, cardiopulmonary bypass, and hemodialysis (see, for example, non-specialized Lee Literature (NPL) 1). Therefore, inhibiting excessive or uncontrolled activation of the complement cascade may provide clinical benefits to patients with these diseases.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare blood disease in which red blood cells are compromised and therefore destroyed faster 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. The PIG-A gene is located on the X chromosome. The mutation in PIG-A caused an initial obstacle to the synthesis of glycosylphosphatidylinositol (GPI), which is a molecule required for anchoring many proteins to the cell surface. Therefore, PNH blood cells lack GPI anchoring proteins, which include complement regulatory proteins CD55 and CD59. Under normal circumstances, these complement regulatory proteins prevent MAC from forming on the cell surface, thus preventing red blood cell lysis. Deletion of the GPI anchor protein causes complement to modulate the hemolytic response in PNH.

PNH is characterized by hemolytic anemia (decreased number of red blood cells), hemoglobinuria (hemoglobin appears in urine, particularly pronounced after sleep), and hemoglobinemia (hemoglobin appears in the blood). Individuals with PNH are known to have paroxysms, which are defined herein as the incidence of black urine. Hemolytic anemia is due to the intravascular destruction of red blood cells by complement components. Other known symptoms include difficulty swallowing, fatigue, erectile dysfunction, thrombosis, and recurrent abdominal pain.

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

Many reports have described anti-C5 antibodies, for example PTL7 describes anti-C5 antibodies that bind to the alpha chain of C5 but not to C5a and prevent the activation of C5. And 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 the proteolytic position of C5 convertase on the alpha chain of C5 and inhibits the conversion of C5 to C5a and C5b. PTL 10 describes an anti-C5 antibody having an affinity constant of at least 1 x 10 ⁷ M ^-1 .

Antibodies (IgGs) bind to nascent Fc receptors (FcRn) and have long plasma retention time. IgGs binding to FcRn is usually observed in an acidic environment (e.g. pH 6.0) and rarely observed in a neutral environment (e.g. pH 7.4). In general, IgGs are not specifically incorporated into cells by endocytosis, and return to the cell surface by endosomal FcRn that binds to the cells under the acidic environment of endosomes. Next, IgGs dissociate from FcRn in a neutral environment in plasma. IgGs that do not bind to FcRn are broken down in the lysosome. When the FcRn-binding ability of IgG in an acidic environment is eliminated by introducing mutations into its Fc region, IgG will not be recirculated from endosomes to plasma, resulting in significantly impaired plasma retention of IgG. In order to improve the plasma retention of IgGs, a method to increase the binding of IgGs to FcRn in an acidic environment has been reported. When the FcRn binding capacity of IgGs in an acidic environment is improved by introducing a monoamino acid into its Fc region, IgG is more efficiently recycled from endosomes to plasma, thus showing improved plasma retention. In the meantime, it has also been reported that IgG with enhanced FcRn binding under neutral environment will not dissociate from FcRn in plasma in neutral environment. Until it returns to the cell surface by binding to FcRn in an acidic environment, its plasma retention remains the same or should be poor (see, for example, NPL 3; NPL 4; NPL 5).

Recently, antibodies that bind to an antigen in a pH-dependent manner have been reported (see, for example, PTL 11 and PTL 12). These antibodies bind strongly to the antigen in a plasma neutral environment and dissociate from the antigen in an intracellular acidic environment. After dissociation from the antigen, when recycled to the plasma via FcRn, the antibody becomes able to bind to the antigen again, so a single antibody molecule can repeatedly bind to multiple antigen molecules. In general, the plasma retention of an antigen is much shorter than that of an antibody with the FcRn-regulated recycling mechanism described above, and therefore, when the antigen is bound to the antibody, the antigen usually shows prolonged plasma retention, resulting in an increase in the plasma concentration of the antigen. On the other hand, it has been reported that the above-mentioned antibodies, which bind to the antigen in a pH-dependent manner, can eliminate the antigen from the plasma more quickly than typical antibodies because they are in the intracellular body during the FcRn-regulated recycling step. Dissociation from the antigen. PTL 13 also describes computer simulation analysis showing that antibodies with pH-dependent binding to anti-C5 can prolong knockdown.

[Quote list]

[Patent Literature]

[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)

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

The present invention provides an anti-C5 antibody and a method of using the same.

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 the MG1-MG2 domain of the β chain of C5 (domian). In some embodiments, the isolated anti-C5 antibody of the present invention binds to an epitope in a fragment composed of amino acids 33-124 of the β chain (sequence identification number: 40) of C5. In some embodiments, an isolated anti-C5 antibody of the invention binds to an epitope in the β chain (sequence identification number: 40) of C5, which includes at least one selected from amino acids 47-57, 70-76 And fragments of groups 107-110. In some embodiments, the isolated anti-C5 antibody of the present invention binds to an epitope in a fragment of the β chain (sequence identification number: 40) of C5, which includes at least one selected from the sequence identification number: 40 Amino acid residues in a 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 higher affinity at pH 7.4 than at pH 5.8. In other examples, the isolated anti-C5 antibodies of the invention bind to the same epitope as the antibodies described in Table 2. In other examples, the antibody binds to the same epitope as the antibody described in Table 2 with higher affinity than at pH 5.8. In yet other embodiments, an isolated anti-C5 antibody of the invention binds to the same epitope as the antibodies described in Table 7 or 8. In still other examples, the antibody binds to the same epitope as the antibodies described in Table 7 or 8 with higher affinity than at pH 5.8.

In a specific embodiment, the anti-C5 antibody of the present invention includes a VH selected from (a) a sequence identification number: 1 and a VL sequence number: 11; (b) a VH and a sequence identification number: 5 Number: 15 VL; (c) sequence identification number: 4 of VH and sequence identification number: VL of 14; (d) sequence identification number: 6 of VH and sequence identification number: 16 of VL; (e) sequence identification number : 2 of VH and sequence identification number: 12 VL; (f) VH of sequence identification number: 3; VL of sequence identification number: 13; VL of sequence identification number: 9; VL of sequence identification number: 9; VL of sequence identification number: 19; (h) Sequence identification number: 7 of VH and sequence identification number: 17 of VL; (i) sequence identification number: 8 of VH and sequence identification number: 18 of VL; and (j) sequence identification number: 10 of VH and sequence identification number: 20 of VL of VH And VL pairs of antibodies compete with C5 binding. In yet other embodiments, the anti-C5 antibody binds to C5 with a higher affinity at a neutral pH compared to an acidic pH. In yet other embodiments, the anti-C5 antibody binds to C5 with 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 the amino acids D51 and K109 of C5 (sequence identification number: 39); (b ) The affinity of the antibody for C5 (sequence identification number: 39) is greater than the affinity of the antibody for the C5 mutant composed of the E48A substitution of sequence identification number: 39; or (c) the antibody binds to the sequence recognition at pH 7.4 C5 protein composed of amino acid sequence of No. 39, but did not bind to C5 protein composed of amino acid sequence of sequence identification number: 39 with H72Y substitution at pH 7.4. 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 higher affinity at pH 7.4 than at pH 5.8.

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

In some embodiments, the isolated anti-C5 antibodies of the invention include (a) HVR-H3, including the amino acid sequence DX ₁ GYX ₂ X ₃ PTHAMX ₄ X ₅ , where X ₁ is G or A, and X ₂ is V , Q or D, X ₃ is T or Y, X ₄ is Y or H, X ₅ is L or Y (sequence identification number: 128); (b) HVR-L3, including amino acid sequence QX ₁ TX ₂ VGSSYGNX ₃ , where X ₁ is S, C, N or T, X ₂ is F or K, X ₃ is A, T or H (sequence identification number: 131); and (c) HVR-H2, including 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, and X ₆ is A or V (sequence identification number: 127).

In some embodiments, the isolated anti-C5 antibodies of the invention include (a) HVR-H1, including the amino acid sequence SSYYX ₁ X ₂ , where X ₁ is M or V, and X ₂ is C or A (sequence identification number : 126); (b) HVR-H2, including amino acid sequence X ₁ IX ₂ TGSGAX ₃ YX ₄ AX ₅ WX ₆ KG, where X ₁ is C, A or G, X ₂ is Y or F, and X ₃ is T, D or E, X ₄ is Y, K or Q, X ₅ is S, D or E, X ₆ is A or V (sequence identification number: 127); and (c) HVR-H3, including amino acids Sequence DX ₁ GYX ₂ X ₃ PTHAMX ₄ X ₅ , where X ₁ is G or A, X ₂ is V, Q or D, X ₃ is T or Y, X ₄ is Y or H, X ₅ is L or Y ( Sequence ID: 128). In yet other embodiments, the antibody comprises (a) HVR-L1, including the amino acid sequence X ₁ ASQX ₂ IX ₃ SX ₄ LA, where X ₁ is Q or R, X ₂ is N, Q, or G, X ₃ G or S, X ₄ is D, K or S (sequence identification number: 129); (b) HVR-L2, including amino acid sequence GASX ₁ X ₂ X ₃ S, where X ₁ is K, E or T , X ₂ is L or T, X ₃ is A, H, E, or Q (sequence identification number: 130); and (c) HVR-L3, including the amino acid sequence QX ₁ TX ₂ VGSSYGNX ₃ , where X ₁ is S, C, N or T, X ₂ is F or K, and X ₃ is A, T or H (sequence identification number: 131).

In some embodiments, the isolated anti-C5 antibody of the present invention includes (a) HVR-L1, including the amino acid sequence X ₁ ASQX ₂ IX ₃ SX ₄ LA, where X ₁ is Q or R, and X ₂ is N, Q or G, X ₃ is G or S, X ₄ is D, K or S (sequence identification number: 129); (b) HVR-L2, including amino acid sequence GASX ₁ X ₂ X ₃ S, where X ₁ K, E or T, X ₂ is L or T, X ₃ is A, H, E or Q (sequence identification number: 130); and (c) HVR-L3, including the amino acid sequence QX ₁ TX ₂ VGSSYGNX ₃ , where X ₁ is S, C, N or T, X ₂ is F or K, and X ₃ is A, T or H (sequence identification number: 131).

In some embodiments, the isolated anti-C5 antibody of the present invention includes a heavy chain variable domain framework FR1, including an amino acid sequence of any one of sequence identification numbers: 132 to 134; FR2 includes a sequence identification number: 135 to 136 of which An amino acid sequence of any one; FR3 includes an amino acid sequence of any one of sequence identification numbers: 137 to 139; and FR4 includes an amino acid sequence of any one of sequence identification numbers: 140 to 141. In some embodiments, the isolated anti-C5 antibody of the present invention includes the light chain variable domain framework FR1, including an amino acid sequence of any one of sequence identification numbers: 142 to 143; FR2 includes a sequence identification number: 144 to 145 of which An amino acid sequence of any one; FR3 includes an amino acid sequence of any of sequence identification numbers: 146 to 147; and FR4 includes an amino acid sequence of sequence identification number: 148.

In some embodiments, the isolated anti-C5 antibody of the present invention comprises (a) a VH sequence, which has at least 95% sequence identity to an amino acid sequence of any of sequence identification numbers: 10, 106 to 110; b) VL sequence, which has at least 95% of the amino acid sequence of any one of the sequence identification numbers: 20, 111 to 113 Sequence identity; or (c) a VH sequence as described in (a) and a VL sequence as described in (b). In yet other embodiments, the antibody includes a VH sequence of any of sequence identification numbers: 10, 106 to 110. In yet other embodiments, the antibody includes a VL sequence of any of sequence identification numbers: 20, 111 to 113.

The invention provides an antibody comprising a VH sequence of any one of sequence identification numbers: 10, 106 to 110 and a VL sequence of any one of 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 a host cell comprising a nucleic acid of the invention. The invention also provides a method for producing an antibody, which comprises culturing a host cell of the invention such that the antibody is produced.

The invention further provides a method for producing an anti-C5 antibody. In some embodiments, the method comprises immunizing an animal with a polypeptide comprising the MG1-MG2 domain of the β chain of C5 (sequence identification number: 43). In some embodiments, the method comprises immunizing the animal with a polypeptide comprising a region of amino acids corresponding to positions 33 to 124 of the β chain (sequence identification number: 40) of C5. In some embodiments, the method comprises immunizing an animal with a polypeptide comprising at least one amino acid 47-57, 70-76, and 107-110 fragment selected from the β chain (sequence identification number: 40) of C5 . In some embodiments, the method includes immunizing an animal with a polypeptide including a fragment of the β chain (sequence identification number: 40) of C5, which includes at least one selected from Glu48, Asp51, His70, His72, Lys109, and His110. Amino acids.

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

The anti-C5 antibody of the present invention can be used as a medicine. The anti-C5 antibodies of the invention can be used to treat complement-regulated diseases or conditions involving excessive or uncontrolled C5 activation. The anti-C5 antibody of the present 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 a medicament. In some embodiments, the medicament is used to treat a complement-regulated disease or condition involving excessive or uncontrolled C5 activation. In some embodiments, the drug is used to enhance the elimination of C5 from plasma.

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

[Figure 1]

Figure 1 shows the epitope binning of the anti-C5 antibody, as described in Example 2.2. Antibodies classified into the same epitope compartment are framed by thick lines.

[Figure 2A]

Figure 2A shows a BIACORE (registered trademark) sensing map of the 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 in Example 2.2 As described.

[Figure 2B]

Figure 2B shows a BIACORE (registered trademark) sensor map of the 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.

[Figure 3]

Figure 3 shows the Western Blot for the β-chain extended fragment (sequence identification number: 40 amino acids 19 to 180, 161 to 340, 321 to 500 and 481 to 660) fused to GST-labeled C5 Analysis, as described in Example 4.1. CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675 are antibodies classified into epitope C. The anti-GST antibody is a positive control group, and the positions of GST-fused C5 fragments (46 to 49 kDa) are indicated by arrows.

[Figure 4]

Figure 4 shows a BIACORE (registered trademark) sensing map of the MG1-MG2 domain of the β chain of C5 by an anti-C5 antibody, as described in Example 4.3. The upper column shows the results of CFA0305 (solid line), CFA0307 (dashed line), CFA0366 (dashed line-dotted line), and eculizumab (dotted line). The middle column shows the results of CFA0501 (solid line), CFA0599 (dashed line), CFA0538 (dashed line-dotted line), and eculizumab (dotted line). The lower column shows the results of CFA0666 (solid line), CFA0672 (dashed line), CFA0675 (dashed line-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 from the fused GST-tagged MG1-MG2 domain (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 ) Analysis of western dot ink, as described in Example 4.4. An anti-GST antibody was used as a reactive antibody, and the position of the C5 fragment (35 to 37 kDa) of the GST fusion was indicated by an arrow.

[Figure 5B]

Figure 5B shows peptide fragments derived from the fused GST-tagged MG1-MG2 domain (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 ) Analysis of western dot ink, as described in Example 4.4. CFA0305 was used as the reactive antibody.

[Figure 5C]

Figure 5C summarizes the binding reaction of the anti-C5 antibody to the β-chain extended fragment of C5, as described in Example 4.4. Fragments that bind to anti-C5 antibodies (CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675) classified as epitope C are shown in gray, and fragments that do not bind to them are shown in white.

[Figure 6]

Figure 6 shows Western blot analysis of C5 point mutants of E48, D51, and K109 in the beta chain that were substituted with alanine (E48A, D51A, and K109A, respectively), as described in Example 4.5. In the left column, eculizumab (anti-C5 antibody, α Chain conjugate) was used as a reactive antibody and the position of the alpha chain (about 113 kDa) of C5 was indicated by an arrow. In the right column, CFA0305 (classified to epitope C, β chain conjugate) is used as a reactive antibody and the position of the β chain (about 74 kDa) of C5 is indicated by an arrow.

[Figure 7]

Figure 7 presents a BIACORE (registered trademark) sensor map showing the interaction of eculizumab-F760G4 (upper bar) or 305LO5 (lower bar) with the C5 mutant, as described in Example 4.6. The sensor map is injected with C5-wt (coarse curve), C5-E48A (short dashed curve), C5-D51A (long dashed curve), and C5-K109A (fine curve) in the eculizumab-F760G4 or 305LO5 on the surface of the sensor. Eculizumab is a control group of anti-C5 antibodies, and 305LO5 is a humanized antibody of CFA0305 (classified to epitope C), as described in Example 2.3.

[Figure 8]

Figure 8 presents a BIACORE (registered trademark) sensor map showing the interaction of 305LO5 with the His mutant of C5 to assess pH dependence, as described in Example 4.7. The sensing map is by injecting C5-wt (rough curve), C5-H70Y (long dashed curve), C5-H72Y (short dashed curve), C5-H110Y (point curve), and C5-H70Y + H110Y ( (Fine curve) was obtained on the surface of the sensor to which 305LO5 was fixed. The antibody / antigen complex can be dissociated at pH 7.4, followed by further dissociation at pH 5.8 (indicated by the arrow) to assess pH-dependent interactions.

[Figure 9A]

Figure 9A shows inhibition of complement-activated liposome cell lysis by anti-C5 antibodies, as described in Example 5.1. The results show CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675 are classified as epitope C, as described in Example 2.2.

[Figure 9B]

Figure 9B shows inhibition of complement-activated liposome cell lysis by anti-C5 antibodies, as described in Example 5.1. The results showed that antibodies CFA0330 and CFA0341 were classified as epitope B, as described in Example 2.2.

[Figure 10A]

Figure 10A shows inhibition of C5a production by anti-C5 antibodies, as described in Example 5.2. C5a concentration was quantified in the supernatant obtained in a liposome lysis assay as described in Figure 9A.

[Figure 10B]

Figure 10B shows inhibition of C5a production by anti-C5 antibodies, as described in Example 5.2. C5a concentration was quantified in the supernatant obtained in the liposomal cell lysis assay described in Figure 9B.

[Figure 11]

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

[Figure 12]

Figure 12 shows the inhibition of complement activation by an anti-C5 antibody, as described in Example 5.4. Complement is activated by alternative pathways.

[Figure 13]

Figure 13 shows the plasma human C5 concentration after intravenous administration of human C5 or human C5 alone and anti-human C5 antibodies after assessment of C5 abolished mice. The time course is 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 after intravenous administration of human C5 and anti-human C5 antibodies to mice evaluated for 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.

[Figure 15]

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

[Figure 16]

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

[Figure 17]

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

[Figure 18]

Figure 18 shows inhibition of C5a production by anti-C5 antibodies, as described in Example 9.2. C5a concentration was quantified in the supernatant obtained in the liposomal cell lysis assay as described in FIG. 15.

[Figure 19]

Figure 19 shows inhibition of C5a production by anti-C5 antibodies, as described in Example 9.2. C5a concentration was quantified in the supernatant obtained in the liposomal cell lysis assay as described in FIG. 16.

[Figure 20]

Figure 20 shows inhibition of complement activity in monkey plasma by anti-C5 antibodies, as described in Example 9.3. Anti-C5 antibody was administered to a cynomolgus monkey, and complement activity in monkey plasma was measured in a hemolytic response assay.

[Figure 21]

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

[Figure 22]

Figure 22 shows 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 As described.

[Figure 23]

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

[Figure 24]

Figure 24 shows the time course of plasma rhesus C5 concentrations after intravenous administration of anti-human C5 antibodies after assessment of C5 eliminated rhesus monkeys, as described in Example 10.2.

[Figure 25]

Figure 25 shows the time course of plasma anti-human C5 antibody concentrations following intravenous administration of anti-human C5 antibodies after rheological monkeys for pharmacokinetic evaluation, as described in Example 10.3.

[Figure 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 asymmetric units, MG1 is shown as a surface schematic, and 305 Fab is shown as a ribbon (dark gray: heavy chain, light gray: light chain). Figure 26B shows overlapping molecules 1 and 2 (dark gray: molecule 1, light gray: molecule 2).

[Figure 27A]

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

[Figure 27B]

Figure 27B shows the epitope of the 305 Fab contact region on the MG1 domain, as described in Example 11.6. Figure 27B shows the epitope localization in the crystal structure (dark gray globular: closer than 3.0Å, light gray rod: closer than 4.5Å).

[Figure 28A]

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

[Fig. 28B]

Figure 28B shows the interaction of E48 and its surroundings (dark gray dotted line: hydrogen bonding with Fab, light gray dotted line: water-regulated 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 bonding 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 the interaction of H70, H72, and H110 (rod images) 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. This histidine residue is indicated by a rod-shaped and reticulated image, and hydrogen bonds are indicated by dotted lines.

[Figure 29C]

Figure 29C shows the interaction of H72 and its surroundings, as in the example 11.8. This histidine residue is indicated by a rod-shaped and reticulated image, 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. This histidine residue is indicated by rod-shaped and reticulated images. The distance between H110 and H-CDR3_H100c is indicated by a dotted line.

The techniques or procedures described or referenced herein are generally conventional methods that are well known and commonly used by those skilled in the art, for example, such as Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (edited by FMAusubel et al. (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJMacPherson, BDHames Edited by GRTaylor (1995)), Edited by Harlow and Lane (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (edited by RIFreshney, (1987)); Oligonucleotide Synthesis (edited by MJGait, 1984); Methods in Molecular Biology Humana Press; Cell Biology: A Laboratory Notebook (edited by JECellis, 1998) Academic Press; Edited by Animal Cell Culture (RIFreshney, 1987); Introduction to Cell and Tissue Culture (JPMather and PERoberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (edited by A. Doyle, JBGriffiths, and DGNewell 1993-8) J.Wiley And Sons; Handbook of Experimental Immunology (edited by DMWeir and CCBlackwell); Gene Transfer Vectors for Mammalian Cells (edited by JMMiller and MPCalos, 1987); PCR: The Polymerase Chain Reaction, (edited by Mullis et al., 1994); Current Protocols in Immunology (edited by JEColigan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CAJaneway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (Editor D. Catty., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (Editor P. Shepherd and C. Dean, Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow) And D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (edited by M. Zanetti and JD Capra, Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (edited by VTDeVita et al., JBLippincott Company , 1993) is widely used.

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, NY1994) and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, NY 1992) provides a generalization of many terms used in this application by those skilled in the art guide. The references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety.

For the purpose of explaining this specification, the following definitions will be used, and the terms used in the singular can also include the plural as long as appropriate, and vice versa. 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 of a conflict between any of the definitions described below and any literature incorporated herein by reference, the definitions set forth below will prevail.

A "receptor human framework" for the purposes herein is an amine group comprising a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework as defined below Framework for acid sequences. A receptor human framework that is "derived from" a human immunoglobulin framework or a human consensus framework may include its identical amino acid sequence, or it may include amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 Or less, 3 or less, or 2 or less. In some embodiments, the VL receptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

"Affinity" refers to the total strength of a non-covalent interaction between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless stated otherwise, "binding affinity" as used herein refers to intrinsic binding affinity, which reflects a 1: 1 interaction between members of a binding pair (eg, an antibody and an antigen). The affinity of molecule X to its partner Y can be generally expressed by the dissociation constant (Kd). Affinity can be measured by methods known in the art, including those described herein. For measuring binding affinity The detailed description and exemplary embodiments will be described below.

An "affinity mature" antibody refers to an antibody that has one or more changes in one or more hypervariable regions (HVRs) compared to a parent antibody that does not have such changes. The change 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 so that the antibody can be used as a diagnostic and / or therapeutic agent in target C5. In one embodiment, for example, as measured by a radioimmunoassay (RIA), the degree of binding of an anti-C5 antibody to an unrelated non-C5 protein is less than about 10% of the antibody's binding to C5. In a specific embodiment, the antibody that binds to C5 has 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 0.001 Dissociation constant (Kd) of nM or less (for example, 10 ^-8 M or less, for example, from 10 ^-8 M to 10 ^-13 M, for example, from 10 ^-9 M to 10 ^-13 M). In particular embodiments, the anti-C5 antibody binds to the epitope of C5, which is conserved from C5 from 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 (eg, bispecific antibodies), and antibody fragments, as long as they exhibit Desired antigen binding activity.

An "antibody fragment" refers to a molecule that includes a portion of an intact antibody, in addition to the intact antibody, which binds to 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 ; diabody; linear antibodies; single chain antibody molecules (e.g., scFv); and from antibody fragments Multispecific antibodies formed.

"Antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody and its antigen in a competition assay, and / or conversely, the reference antibody binds the antibody to Antigen binding is blocked. An exemplary competition assay is provided herein.

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

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

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

"Effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary depending on the isotype of the antibody. 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 (such as B cell receptors); and B cell activation.

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

The term "antigenic determinant" includes any determinant capable of being bound by an antibody. An epitope is a region of an antigen bound by an antibody that targets the antigen, and contains a specific amino acid that directly contacts the antibody. An epitope may comprise a chemically active surface cluster of molecules (such as amino acids, sugar branches, phosphates, or sulfonyl groups), and may have specific three-dimensional structural characteristics, and / or specific charge characteristics. In general, antibodies specific for a specific target antigen will preferentially recognize epitopes on the target antigen in a complex mixture of proteins and / or macromolecules.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of the constant region. This 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 carboxy terminus of the heavy chain. However, the Fc region C-terminal lysine (Lys447) may be present or absent. Unless otherwise stated herein, the numbering of amino acid residues in the Fc region or constant region is in accordance with the EU numbering system, also known as the EU index, such as 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 highly variable region (HVR) residues. The FR of the variable domain is generally composed of four FR domains: FR1, FR2, FR3, and ER4. Therefore, HVR and FR sequences generally appear in 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 and refer to an antibody that has a structure that is substantially similar to the structure of a native antibody or that has 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 an exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include the cells that were originally transformed and the progeny derived from them, regardless of the number of passages. The progeny may not have exactly the same nucleic acid content as the parent cell, but may contain mutations. Included herein are progeny of mutants having the same function or biological activity as those screened or selected in initially transformed cells.

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

A "human consensus framework" is a framework that represents the amino acid residue most commonly found in the selection of human immunoglobulin VL or VH framework sequences. In general, the selection of human immunoglobulin VL or VH sequences is from a subtype of variable domain sequence. Generally, the subtype of a sequence is the subtype in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subtype is the subtype kappa I in supra, as in Kabat et al. In one embodiment, for VH, the subtype is subtype III in Kapra et al., Supra.

A "humanized" antibody refers to a chimeric antibody that includes an amino acid residue from a non-human HVR and an amino acid residue from a human FR. In particular embodiments, a humanized antibody will include at least one and usually two variable domains of all, where all or substantially all HVRs (eg, CDRs) correspond to those of non-human antibodies, and All or substantially all FRs correspond to those of human antibodies. A humanized antibody optionally includes 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.

As used herein, the term "highly variable region" or "HVR" refers to the highly variable ("complementarity determining region" or "CDR") sequence of an antibody variable domain and / or to form a structurally defined loop ("highly variable loop" ), And / or each region comprising an antigen contact residue ("antigen contact"). In general, antibodies include six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include: (a) Appears at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2 ) And 96-101 (H3) (Chothia, J. Mol. Biol. 196: 901-917 (1987)) highly variable loops; (b) appears at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NIH, Bethesda, MD (1991)); (c) Appears 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 (c) combination, including 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 the variable domain (eg, FR residues) 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.

"Individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g. cattle, sheep, cats, dogs and horses), primates (e.g. human and non-human primates, e.g. monkeys), rabbits and rodents (e.g. mice and rats ). In a particular embodiment, the individual or subject is a human.

An "isolated" antibody is one that has been isolated from a component of its natural environment. In some embodiments, the antibody is 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 reversed-phase Phase HPLC) method. For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848: 79-87 (2007).

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

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

The term "single antibody" as used herein refers to an antibody obtained from a family of substantially homogeneous antibodies, that is, individual antibodies that include the same group and / or bind to the same epitope, except for example: containing naturally occurring Mutations or possible variant antibodies that appear during the production of monoclonal antibodies (such variants are usually present in smaller amounts). Unlike the preparation of multiple strains of antibodies that usually contain different antibodies directed against different epitopes (antigenics), each monoclonal antibody produced by a monoclonal antibody is directed against a single determinant on the antigen. Thus, the modifier "single strain" refers to antibody characteristics obtained from a substantially homogeneous antibody population and is not to be construed as requiring the production of antibodies by any particular method. For example, the monoclonal antibodies to be used according to the present invention can be prepared by a variety of techniques, including but not limited to fusion tumor technology, recombinant DNA method, phage display method, and the use of all or part of a human immunoglobulin locus (loci ) Methods of transgenic animals, such methods, and other exemplary methods for making monoclonal antibodies will be described herein.

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

"Native antibody" refers to a naturally occurring immunoglobulin molecule having a different structure. For example, a natural IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N to C terminus, each light chain has a variable region (VL), also known as a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. Based on the amino acid sequence of their constant domain, the light chain of an antibody can be classified into one of two types, called κ (kappa) and λ (lambda).

The term "package insert" is used to refer to the instructions normally included in commercial packaging of therapeutic products, which contain information on indications, usage, dosages, administration, combination therapies, contraindications and / or information about such Warnings for the use of therapeutic products.

"Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the sequence that is aligned and, if necessary, introduced a gap to achieve the maximum percent sequence identity without any conservative substitutions When the sequence identity is considered to be the same, the percentage of amino acid residues in the candidate sequence that are the same as the amino acid residues in the reference polypeptide sequence. Alignment can be achieved in various ways within the technical field for the purpose of determining percent amino acid sequence identity, 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 algorithm required to achieve maximum alignment over the full length of the compared sequences. For the purposes of this document, however, the% amino acid sequence identity values were generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the original code has been submitted with the user documentation to the US Copyright Office, Washington D.C., 20559, which is registered with the US Copyright Office under the US Copyright Registration Number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from source code. The ALIGN-2 program 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 change.

In the case of using ALIGN-2 for amino acid sequence comparison, the given amino acid sequence A is higher than, or the% amino acid sequence identity to a given amino acid sequence B (or it may be Expressed as a given amino acid sequence A, which has or includes a specific% amino acid sequence identity ratio, and / or relative to a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, Where X is the number of amino acid residues that were scored the same match by the sequence alignment program ALIGN-2 in the A and B alignments of the program, and where Y is the amino acid residue of B total. It should be understood that when the length of the amino acid sequence A is not equal to the length of the amino acid sequence B, the% amino acid sequence identity of A over B will not be equal to the same% amino acid sequence of B over A Sex. Unless explicitly stated otherwise, all% amino acid sequence identity values used herein were obtained using the ALIGN-2 computer program as described in the previous paragraph.

The term "pharmaceutical formula" refers to a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective, and it does not contain additional ingredients that have unacceptable toxicity to the subject to which the formula is to be administered.

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

The term "C5" as used herein encompasses any natural C5 from any vertebrate source, including mammals such as primates (e.g., humans and monkeys) and rodents (e.g., mice and rats). Unless otherwise stated, the term "C5" refers to a human C5 protein having an amino acid sequence represented by sequence identification number: 39 and a β (beta) chain sequence represented by sequence identification number: 40. This term encompasses "full length" untreated C5 as well as any form of C5 produced by intracellular processing. The term also encompasses naturally occurring variants of C5, such as splice variants or allelic variants. The amino acid sequence of an exemplary human C5 is shown in sequence identification number: 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 an exemplary human C5 are shown by sequence identification numbers: 41, 42, and 43, respectively. The amino acid sequences of exemplary rhesus monkeys and murine C5 are shown as sequence identification numbers: 44 and 105, respectively. The sequence identification numbers: amino acid residues 1 to 19 of 39, 40, 43, 44 and 105 correspond to the message sequence, which were removed during intracellular processing and therefore did not appear in the corresponding exemplary amino acid sequence.

As used herein, "treatment" (and its grammatical variants) refers to clinical interventions that attempt to alter the natural course of a treated individual and can be performed for prevention or during the course of clinical pathology. Desired treatment effects include, but are not limited to, preventing the occurrence or recurrence of the disease, reducing symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the state of the disease, and alleviating or improving Prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or to slow the progression of a disease.

The term "variable region" or "variable domain" refers to a domain involved in the heavy or light chain of an antibody that binds an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) usually have similar structures, and each domain includes four conserved framework regions (FR) and three highly variable regions (HVR) (see For example, 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 specificity for antigen binding. In addition, antibodies that bind to a specific antigen can be isolated using a VH or VL domain from an antibody that binds that antigen to screen complementary VL or VH domain libraries, respectively. See, for example, 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 reproducing another nucleic acid to which it is linked. This term encompasses vectors that serve as autonomously replicating nucleic acid structures, as well as vectors incorporated into the genome of a host cell into which it has been introduced. Specific vectors can indicate the performance of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors".

II. Composition and method

In one aspect, the invention is based, in part, on anti-C5 antibodies and their use. In a specific embodiment, an antibody that binds to C5 is provided. Antibodies of the invention are useful, for example, in the diagnosis and management of diseases or conditions that are regulated by complement or overregulated C5 activation.

A. Exemplary anti-C5 antibodies

In one aspect, the invention provides an isolated antibody that binds to C5. In a specific implementation, an anti-C5 antibody of the invention binds to an epitope within the β (beta) chain of C5. In a specific implementation, an anti-C5 antibody binds to the β chain of C5 Epitope in the MG1-MG2 domain. In a specific implementation, the anti-C5 antibody binds to an epitope within a fragment consisting of amino acids 19 to 180 of the β chain of C5. In a specific implementation, the anti-C5 antibody binds to an epitope within the MG1 domain (sequence identification number: 40 (sequence identification number: 41) of amino acids 20 to 124) of the β chain of C5. In a specific implementation, the anti-C5 antibody binds to an epitope within a fragment consisting of amino acids 33 to 124 of the β chain of C5 (sequence identification number: 40). In another embodiment, the anti-C5 antibody does not bind to a fragment shorter than the fragment consisting of the amino acids 33 to 124 of the beta chain of C5, for example, the beta chain amine of C5 (sequence identification number: 40) Based on 45 to 124, 52 to 124, 33 to 111, 33 to 108 or 45 to 111.

In another aspect, the invention provides an anti-C5 antibody that exhibits pH-dependent binding properties. As used herein, the term "pH-dependent" means that the antibody displays "a decrease in the binding of acidic pH to C5 compared to neutral pH" (both terms are used interchangeably for the purposes of this disclosure) . For example, an antibody "having pH-dependent binding properties" comprises an antibody that binds to C5 with a higher affinity at neutral pH than at acidic pH. In specific embodiments, the antibodies of the invention have a neutral pH at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, compared to at acidic pH. , 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10,000 or more times the affinity to C5. In some embodiments, the antibody binds to C5 with higher affinity at pH 7.4 than at pH 5.8. In yet other embodiments, the antibody of the invention has a pH of at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 compared to pH 5.8. , 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10,000, or more times bind to C5.

The "affinity" of an antibody to C5, for the purposes of this disclosure, is expressed in terms of the KD of the antibody. The KD of an antibody refers to the equilibrium dissociation constant of the antibody-antigen interaction. The greater the KD value of an antigen bound to its antigen, the weaker its binding affinity for that particular antigen. Therefore, as used herein, "higher affinity at neutral pH compared to acidic pH" (or the equivalent expression "pH-dependent binding") means that the KD of the antibody that binds to C5 at acidic pH is greater than that of the antibody in medium KD bound to C5 at pH. For example, in the context of the present invention, if the KD of an antibody that binds to C5 at an acidic pH is at least twice greater than the KD of an antibody that binds to C5 at a neutral pH, the antibody is considered to be neutral compared to an acidic pH. The pH binds to C5 with higher affinity. Therefore, the invention comprises a KD that binds to C5 at acidic pH is at least greater than 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, KD that binds to C5 at neutral pH. 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10,000 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 an acidic pH may be 10 ^-9 M, 10 ^-8 M, 10 ^-7 M, 10 ^-6 M or more.

In still other embodiments, if the KD of the antibody bound to C5 at pH 5.8 is at least twice greater than the KD of the antibody bound to C5 at pH 7.4, the antibody is considered to be at a neutral pH compared to an acidic pH Binding to C5 with higher affinity. In some embodiments, the KD of the provided antibody that binds to C5 at pH 5.8 is at least greater than 3, 5, 10, 15, 20, 25, 30, 35, 40, 45 of the KD that the antibody binds to C5 at pH 7.4. , 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10,000 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 more.

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 off-rate constant of the antibody for a specific antigen, expressed in reciprocal seconds (ie, sec ^-1 ). An increase in the kd value indicates a weaker binding of the antibody to its antigen. The invention therefore includes antibodies that bind to C5 at higher kd values at acidic pH compared to neutral pH. The invention includes that the kd bound to C5 at acidic pH is at least greater than 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, and kd bound to C5 at neutral pH. , 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10,000 or more times. 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 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 more at an acidic pH. The invention also includes antibodies that bind to C5 with a larger kd value at pH 5.8 than at pH 7.4. The invention includes that the kd bound to C5 at pH 5.8 is at least greater than 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10,000 or more times the antibody. In another embodiment, the kd value of the antibody at pH 7.4 may be 10 ^-2 1 / s, 10 ^-3 1 / s, 10 ^-4 1 / s, 10 ^-5 1 / s, 10 ^-6 1 / s or less. In another embodiment, the kd value of the antibody at pH 5.8 may be 10 ^-3 1 / s, 10 ^-2 1 / s, 10 ^-1 1 / s or more.

In a specific implementation, "compared with neutral pH, the binding of acidic pH to C5 is reduced" is the ratio of the KD value of antibody binding to C5 at acidic pH to the KD value of antibody binding to C5 at neutral pH Means (or vice versa). For example, for the purposes of the present invention, if an antibody has an acidic / neutral KD ratio of 2 or greater, the antibody can be considered as "reducing the binding of C5 to acidic pH at acidic pH compared to neutral pH." In some specific exemplary embodiments, a pH 5.8 / pH 7.4 KD ratio of an antibody of the invention may be 2 or greater. In some specific exemplary embodiments, the acidic / neutral KD ratio of the antibodies 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, 10,000 or more. In another embodiment, the KD value of the antibody at neutral pH may be 10 ^-7 M, 10 ^-8 M, 10 ^-9 M, 10 ^-10 M, 10 ^-11 M, 10 ^-12 M or less. In another embodiment, the KD value of the antibody at an acidic pH may be 10 ^-9 M, 10 ^-8 M, 10 ^-7 M, 10 ^-6 M or more. In a further example, for the purposes of the present invention, if the antibody has a pH 5.8 / pH7.4 KD ratio of 2 or greater, then the antibody can be considered "as compared to neutral pH, acidic pH and C5 The combination is reduced. " In some specific exemplary embodiments, the pH 5.8 / pH 7.4 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, 10,000 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 more.

In a specific example, "combined with acidic pH and C5 is reduced compared to neutral pH" as the ratio of the kd value of antibody binding to C5 at acidic pH to the kd value of antibody binding to C5 at neutral pH Means (or vice versa). For example, for the purposes of the present invention, if an antibody has an acidic / neutral kd ratio of 2 or greater, then the antibody can be considered as "reduced in binding to C5 at acidic pH compared to neutral pH". In some specific exemplary embodiments, the antibody of the present invention may have a pH 5.8 / pH 7.4 kd ratio of 2 or greater. In some specific exemplary embodiments, the acid / 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, 10,000 or more. In still other embodiments, the pH 5.8 / pH 7.4 kd ratio of the antibody 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, 10,000 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 or less. In yet another embodiment, the kd value of the antibody at pH 7.4 may be 10 ^-2 1 / s, 10 ^-3 1 / s, 10 ^-4 1 / s, 10 ^-5 1 / s, 10 ^-6 1 / s or less. In another embodiment, the kd value of the antibody at an acidic pH may be 10 ^-3 1 / s, 10 ^-2 1 / s, 10 ^-1 1 / s or more. In yet another embodiment, the kd value of the antibody at pH 5.8 may be 10 ^-3 1 / s, 10 ^-2 1 / s, 10 ^-1 1 / s or more.

As used herein, the term "acidic pH" refers to a pH of 4.0 to 6.5. The term "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 6.5. In a particular aspect, 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, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, PH of any of 9.8, 9.9, and 10.0. In a particular aspect, the "neutral pH" is 7.4.

The KD value and kd value, as indicated herein, can be measured using surface plasmon resonance based biosensors to characterize antibody-antigen interactions (see, for example, Example 3 in the text). KD value and kd value can be measured at 25 ° C (° C) or 37 ° C.

In a specific embodiment, the anti-C5 antibody of the present invention binds to an epitope within the β chain of C5, which consists of the MG1 domain (sequence identification number: 41). In a specific embodiment, the anti-C5 antibody of the present invention binds to an epitope within the β chain (sequence identification number: 40) of C5, which includes at least one selected from amino acids 47-57, 70-76, and 107 A fragment of the group 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 (sequence identification number: 40) of C5, which includes at least one selected from Thr47, Glu48, Ala49, Phe50, Asp51, Ala52, Thr53, Lys57, His70, Va171, His72, Ser74, Glu76, Va1107, Ser108, Lys109 and His110 group of amino acids. In a specific embodiment, the anti-C5 antibody of the present invention binds to an epitope within a fragment of the β chain (sequence identification number: 40) of C5, which includes at least one selected from Glu48, Asp51, His70, His72, Lys109, and Amino acids in the group of His110. In a specific embodiment, the anti-C5 antibody of the present invention has reduced binding to a C5 mutant compared to its binding to wild-type C5, wherein the C5 mutant has at least one amino acid substitution selected from Glu48, Asp51, The position of the group consisting of His72 and Lys109. In another embodiment, the pH-dependent binding of an anti-C5 antibody of the invention to a C5 mutant is reduced compared to its pH-dependent binding to wild-type C5, wherein the C5 mutant has At least one amino acid is substituted at a position selected from the group consisting of His70, His72, and His110. In yet another embodiment, in the C5 mutant, the amino acids selected at positions selected from Glu48, Asp51, and Lys109 are substituted with alanine, and the amino groups selected at positions selected from His70, His72, and His110 The acid is replaced by tyrosine.

In a specific embodiment, the anti-C5 antibody and an antibody of the present invention compete for binding to C5. The antibody includes: (a) a VH selected from the sequence identification number: 1 and a VL selected from the sequence identification number: 11; (b) a sequence Identification number: VH of 22 and sequence identification number: VL of 26; (c) Sequence identification number: VH of 21 and sequence identification number: VL of 25; (d) Sequence identification number: VH of 5 and sequence identification number: 15 (E) the sequence identification number: VH of 4 and the sequence identification number: VL of 14; (f) the sequence identification number: VH of 6 and the sequence identification number: VL of 16; (g) the sequence identification number: 2 VH and sequence identification number: 12 VL; (h) sequence identification number: 3 VH and sequence identification number: 13 VL; (i) sequence identification number: 9 VH and sequence identification number: 19 VL; (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 of 23 and sequence identification number : VL of 27; and (m) sequence identification number: VH of 10 and sequence identification number: VH and VL pair of VL of 20.

In a specific embodiment, an anti-C5 antibody of the invention binds C5 and contacts the amino acid Asp51 (D51) of sequence identification number: 39. In yet other embodiments, an anti-C5 antibody of the invention binds C5 and contacts the amino acid Lys109 (K109) of sequence identification number: 39. In yet another embodiment, an anti-C5 antibody of the invention binds C5 and contacts the amino acid Asp51 (D51) and amino acid Lys109 (K109) of sequence identification number: 39.

In a specific embodiment, the anti-C5 antibody of the present invention has reduced binding to a C5 mutant compared to its binding to wild-type C5, wherein the C5 mutant has a Glu48Ala (E48A) substitution with sequence identification number: 39. In another embodiment, the pH-dependent binding of an anti-C5 antibody of the present invention to a C5 mutant is reduced compared to its pH-dependent binding to wild-type C5, wherein the C5 mutant has a sequence identification number: 39 of Glu48Ala (E48A).

In yet another embodiment, the anti-C5 antibody binds to the C5 protein consisting of the amino acid sequence of sequence identification number: 39, but does not bind to the C5 protein consisting of the amino acid sequence of sequence identification number: 39 with H72Y substitution. The C5 protein is composed of C5 protein and H72Y substituted C5 protein under the same conditions. In yet another embodiment, the anti-C5 antibody binds to the C5 protein consisting of the amino acid sequence of sequence identification number: 39 at pH 7.4, but does not bind to the sequence identification number with H72Y substitution at pH 7.4. : 39 C5 protein consisting of amino acid sequences.

Without being limited to a particular theory, it can be speculated that when a monoamino acid residue on C5 is replaced by another amino acid, the binding of the anti-C5 antibody to C5 decreases (or almost disappears), which represents the amine on C5 The amino acid residues are important for anti-C5 antibodies and the interaction between C5, and the antibody can recognize epitopes around the amino acid on C5.

The present invention has discovered that a group of anti-C5 antibodies that compete with or bind to the same epitope of another antibody can exhibit pH-dependent binding properties. Among amino acids, histidine has a pKa value of about 6.0 to 6.5, and may have different proton dissociation states between neutral and acidic pH. Therefore, histidine residues on C5 can contribute to pH-dependent interactions between anti-C5 antibodies and C5. Unaffected Limited to a specific theory, it can be speculated that anti-C5 antibodies can recognize the conformational structure around histidine residues on C5, whose changes depend on pH. This is speculated to be consistent with the experimental results that when the histidine residue on C5 is replaced with another amino acid, the pH dependence of the anti-C5 antibody decreases (or almost disappears) (that is, at neutral pH Anti-C5 antibodies with pH-dependent binding properties bind to C5 histidine mutants with similar affinity to wild-type C5 binding, and at acidic pH, the same antibodies are higher than those binding to wild-type C5. Histidine mutant with affinity binding to C5).

In a particular embodiment, 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 humans and monkeys (eg, cynomolgus, rhesus macaque, marmoset, chimpanzee, or baboon.

In one aspect, the invention provides an anti-C5 antibody that inhibits C5 activation. In a specific embodiment, an anti-C5 antibody is provided that prevents C5 from cleavage to form C5a and C5b, and thus prevents the production of allergic toxin activity associated with C5a, and also prevents the C5b-9 membrane attack complex (MAC) associated with C5b. combination. In a specific embodiment, an anti-C5 antibody is provided that prevents C5 convertase from converting C5 to C5a and C5b. In a specific embodiment, an anti-C5 antibody is provided that prevents the C5 convertase from approaching the cleavage site on C5. In a specific embodiment, an anti-C5 antibody is provided which hinders the hemolytic activity caused by the activation of C5. In yet other embodiments, the anti-C5 antibodies of the invention inhibit C5 activation by the classical and / or alternative pathways.

In one aspect, the invention provides an anti-C5 antibody that inhibits C5 Variant activation. A C5 variant refers to a variant of the C5 gene, which is caused by a mutation in a gene, such as a mutation, polymorphism, or an allele. Variations in a gene can include deletions, substitutions, or insertions of one or more nucleotides. C5 variants can include variations in one or more genes in C5. In a particular embodiment, the C5 variant has a biological activity similar to that of wild-type C5. Such C5 variants may include at least one mutation selected from the group consisting of V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N, and E1437D. Here, R885H, for example, refers to a mutation in a gene whose arginine at position 885 is replaced by histidine. In a specific embodiment, the anti-C5 antibody of the invention inhibits the 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 invention provides an anti-C5 antibody comprising at least one, two, three, four, five or six amines selected from (a) including any of sequence identification numbers: 45 to 54 HVR-H1 of amino acid sequence; (b) HVR-H2 including amino acid sequence of any one of sequence identification number: 55 to 64; (c) amino acid sequence of any one of amino acid sequence of 65 to 74 HVR-H3 of the sequence; (d) HVR-L1 including the amino acid sequence of any one of the sequence identification number: 75 to 84; (e) HVR-L1 including the amino acid sequence of any of the sequence identification number: 85 to 94 HVR-L2; and (f) Highly variable regions (HVRs) of HVR-L3 including amino acid sequences of sequence identification numbers: 95 to 104.

In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three selected from (a) HVR-H1 including an amino acid sequence of any of sequence identification numbers: 45 to 54 ; (B) Including serial identification numbers: 55 to 64 HVR-H2 of any of the amino acid sequences; and (c) a VH HVR sequence of HVR-H3 of any of the amino acid sequences of sequence identification numbers: 65 to 74. In one embodiment, the antibody includes HVR-H3, which includes an amino acid sequence of any of sequence identification numbers: 65 to 74. In another embodiment, the antibody includes HVR-H3, which includes an amino acid sequence of any one of sequence identification numbers: 65 to 74, and HVR-L3, which includes an amine of any one of sequence identification numbers: 95 to 104. Amino acid sequence. In yet another embodiment, the antibody includes HVR-H3, which includes an amino acid sequence of any one of sequence identification numbers: 65 to 74, and HVR-L3, which includes an amino group of any one of sequence identification numbers: 95 to 104 Acid sequence, and HVR-H2, which includes an amino acid sequence of any of sequence identification numbers: 55 to 64. In yet another embodiment, the antibody includes (a) an HVR-H1 comprising an amino acid sequence of any one of the sequence identification numbers: 45 to 54; (b) an amino group comprising any one of the sequence identification numbers: 55 to 64 HVR-H2 of the acid sequence; and (c) HVR-H3 of the amino acid sequence including any of sequence identification numbers: 65 to 74.

In another aspect, the invention provides an antibody comprising at least one, at least two, or all three selected from (a) HVR-L1 comprising an amino acid sequence of any of sequence identification numbers: 75 to 84 ; (B) HVR-L2 including an amino acid sequence of any one of the sequence identification number: 85 to 94; and (c) HVR-L3 including an amino acid sequence of any of the sequence identification number: 95 to 104 VL HVR sequence. In one embodiment, the antibody includes (a) an HVR-L1 comprising an amino acid sequence of any one of the sequence identification number: 75 to 84; (b) an amino acid comprising any of an amino acid sequence of the sequence identification number: 85 to 94 HVR-L2 of the sequence; and (c) HVR-L3 of an amino acid sequence including a sequence identification number: 95 to 104.

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

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

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

In one aspect, the invention provides an antibody comprising at least one, at least two, or all three selected from (a) an amino acid sequence comprising any of the sequence identification numbers: 45, 54, 117, 126 HVR-H1; (b) HVR-H2 including the amino acid sequence of any of 55, 64, 118-120, and 127; and (c) Sequence ID: 65, 74, 121, 128 VH HVR sequence of HVR-H3 of any amino acid sequence. In one embodiment, the antibody includes HVR-H3, which includes an amino acid sequence of any of sequence identification numbers: 65, 74, 121, 128. In another embodiment, the antibody includes: HVR-H3, which includes an amino acid sequence of any one of sequence identification numbers: 65, 74, 121, 128, and HVR-L3, which includes a sequence identification number: 95, 104 , 125, 131 amino acid sequence. In another embodiment, the antibody includes: HVR-H3, which includes an amino acid sequence of any one of sequence identification numbers: 65, 74, 121, 128, and HVR-L3, which includes a sequence identification number: 95, 104, The amino acid sequence of any one of 125, 131, and HVR-H2, which includes the amino acid sequence of any one of sequence identification numbers: 55, 64, 118-120, and 127. In yet another embodiment, the antibody includes (a) HVR-H1 including an amino acid sequence of any one of sequence identification numbers: 45, 54, 117, 126; (b) including sequence identification numbers: 55, 64, 118 -HVR-H2 of the amino acid sequence of any one of 120, 127; and (c) HVR-H3 of the amino acid sequence of any one of sequence identification numbers: 65, 74, 121, 128.

In another aspect, the invention provides an antibody comprising at least one, at least two, or all three selected from (a) an amino acid sequence 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 sequence identification numbers: 95, 104, 125, 131 Amino acid sequence VL HVR sequence of HVR-L3. In one embodiment, the antibody includes (a) HVR-L1 including an amino acid sequence of any one of sequence identification numbers: 75, 84, 122, 129; (b) including sequence identification numbers: 85, 94, 123- HVR-L2 of the amino acid sequence of any one of 124, 130; and (c) HVR-L3 of the amino acid sequence of any one of the sequence identification numbers: 95, 104, 125, 131.

In another aspect, the antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three selected from (i) including a sequence identification number: any of 45, 54, 117, 126 HVR-H1 of the amino acid sequence of No. 1 (ii) includes the sequence identification number: HVR-H2 of the amino acid sequence of any of 55, 64, 118-120, and 127, and (iii) includes the sequence identification number: 65, 74, 121, 128 VH HVR sequences of the HVR-H3 amino acid sequence; and (b) a VL domain comprising at least one, at least two or all three selected from (i) including Sequence identification number: HVR-L1 comprising the amino acid sequence of any of the amino acid sequences of 75, 84, 122, 129, (ii) including the sequence identification number: any of 85, 94, 123-124, 130 The HVR-L2 of amino acid sequence and (c) include the VL HVR sequence of HVR-L3 of amino acid sequence of any of sequence identification numbers: 95, 104, 125, 131.

In another aspect, the invention provides an antibody comprising: (a) HVR-H1 including an amino acid sequence of any one of the sequence identification numbers: 45, 54, 117, and 126; (b) including a sequence identification number: 55, 64, 118-120, 127 HVR-H2 of the amino acid sequence; (c) includes the sequence identification number: 65, 74, 121, 128 of the amino acid sequence of HVR-H3; (d) HVR-L1 including amino acid sequence of any one of sequence identification number: 75, 84, 122, 129; (e) amine including any of sequence number: 85, 94, 123-124, 130 HVR-L2 of the amino acid sequence; and (f) HVR-L3 including an amino acid sequence of any one of sequence identification numbers: 95, 104, 125, and 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 (sequence identification number: 45), at positions 5 and 6; (b) in HVR-H2 (serial identification number: 55) at positions 1, 3, 9, 11, 13, and 15; (c) in HVR-H3 (serial identification number: 65) at position 2, 5, 6, 12, and 13; (d) in HVR-L1 (serial identification number: 75), in positions 1, 5, 7, and 9; (e) in HVR-L2 (serial identification number: 85) In positions 4, 5, and 6; and (f) in HVR-L3 (serial identification number: 95) at positions 2, 4, and 12.

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

All possible combinations of the above substitutions are the sequence identification numbers of the common 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 (humanized). In one embodiment, the anti-C5 antibody includes the HVR as in any of the above embodiments, and further includes a receptor human framework, such as 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 a FR sequence, wherein the FR sequence is as follows. For the heavy chain variable domain, FR1 includes the amino acid sequence of any one of the sequence identification number: 132 to 134, FR2 includes the amino acid sequence of any one of the sequence identification number: 135 to 136, and FR3 includes the sequence identification number: 137 The amino acid sequence of any one of 139 to 139, and FR4 includes the amino acid sequence of any one of sequence identification numbers: 140 to 141. For the light chain variable domain, FR1 includes an amino acid sequence of any one of sequence identification numbers: 142 to 143, FR2 includes an amino acid sequence of any one of sequence identification numbers: 144 to 145, and FR3 includes a sequence identification number: 146 The amino acid sequence of any one of 147, FR4 includes the amino acid sequence of sequence identification number: 148.

In another aspect, the primary anti-C5 antibody includes a heavy chain variable domain (VH) sequence having at least 90%, 91%, and 92% of the amino acid sequence of any of sequence identification numbers: 1 to 10. , 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some specific embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity relative to a reference sequence Anti-C5 antibodies that include substitutions (eg, conservative substitutions), insertions or deletions, but that include the sequence retain the ability to bind to C5. In some specific embodiments, in any of the sequence identification numbers: 1 to 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 a region outside the HVR (ie, in the FR). Optionally, the anti-C5 antibody comprises a VH sequence of any of sequence identification numbers: 1 to 10, which contains a post-translational modification of that sequence. In a specific implementation In the example, VH includes one, two, or three selected from: (a) HVR-H1 including an amino acid sequence of any one of 45 to 54, (b) including a sequence identification number: 55 to The HVR-H2 of the amino acid sequence of any one of 64 and (c) includes the HVR of the HVR-H3 of the amino acid sequence of any one of the sequence identification numbers: 65 to 74.

In another aspect, an anti-C5 antibody is provided, wherein the antibody includes a light chain variable domain (VL), which has at least 90%, 91% for the amino acid sequence of any of sequence identification numbers: 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 a reference sequence Anti-C5 antibodies that include substitutions (eg, conservative substitutions), insertions or deletions, but that include the sequence retain the ability to bind to C5. In some specific embodiments, in any of the sequence identification numbers: 11 to 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 a region outside the HVR (ie, in the FR). Optionally, the anti-C5 antibody includes a sequence identification number: the VL sequence of any one of 11 to 20, which contains a post-translational modification of that sequence. In a specific embodiment, the VL includes one, two, or three selected from: (a) HVR-L1 including an amino acid sequence of any of sequence numbers 75 to 84, and (b) including sequence identification No .: HVR-L2 of amino acid sequence of any of 85 to 94, and (c) includes HVR of HVR-L3 of amino acid sequence of any of 95 to 104.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises VH in any of the embodiments provided above, and VL in any of the embodiments provided above. In one embodiment, the antibodies include sequence identification numbers: 1 to VH sequence in any of 10 and sequence identification number: VL sequence in any of 11 to 20, which contains post-translational modifications of those sequences.

In another aspect, the primary anti-C5 antibody includes a heavy chain variable domain (VH) sequence, which has at least 90%, 91%, for an amino acid sequence of any of sequence identification numbers: 10, 106 to 110, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some specific embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity relative to a reference sequence Anti-C5 antibodies that include substitutions (eg, conservative substitutions), insertions or deletions, but that include the sequence retain the ability to bind to C5. In some specific embodiments, in any of the sequence identification numbers: 10, 106 to 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 a region outside the HVR (ie, in the FR). Optionally, the anti-C5 antibody comprises a VH sequence in any of sequence identification numbers: 10, 106 to 110, which contains a post-translational modification of that sequence. In a specific embodiment, VH includes one, two, or three selected from: (a) HVR-H1, (b) including an amino acid sequence of any one of the sequence identification numbers: 45, 54, 117, 126 ) HVR-H2 including the amino acid sequence of any of the sequence identification numbers: 55, 64, 118-120, 127, and (c) includes the amino group of any of the sequence identification numbers: 65, 74, 121, 128 HVR of acid sequence of HVR-H3.

In another aspect, the primary anti-C5 antibody includes a VH sequence having at least 90%, 91%, 92%, 93%, 94% of the amino acid sequence of the sequence identification number: 10, 106 to 110 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some specific embodiments, have a phase of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% A homologous VH sequence, relative to a reference sequence, contains substitutions (eg, conservative substitutions), insertions, or deletions, but anti-C5 antibodies that include that sequence retain the ability to bind to C5. In some specific embodiments, in any of the sequence identification numbers: 10, 106 to 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 a region outside the HVR (ie, in the FR). Optionally, the anti-C5 antibody comprises a VH sequence in any of sequence identification numbers: 10, 106 to 110, which contains a post-translational modification of that sequence. In a specific embodiment, VH includes one, two, or three selected from: (a) HVR-H1, (b) including an amino acid sequence of any one of the sequence identification numbers: 45, 54, 117, 126 ) HVR-H2 including the amino acid sequence of any of the sequence identification numbers: 55, 64, 118-120, 127, and (c) includes the amino group of any of the sequence identification numbers: 65, 74, 121, 128 HVR of acid sequence of HVR-H3.

In another aspect, the primary anti-C5 antibody includes a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96% for the amino acid sequence of sequence identification number: 10 , 97%, 98%, 99%, or 100% sequence identity. In some specific embodiments, the VH sequence is an amino acid sequence of sequence identification number: 10. In some specific embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity relative to a reference sequence Anti-C5 antibodies that include substitutions (eg, conservative substitutions), insertions or deletions, but that include the sequence retain the ability to bind to C5. In some specific embodiments, in the sequence identification number: 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 a region outside the HVR (ie, in the FR). Optionally, the anti-C5 antibody includes the VH sequence in sequence identification number: 10, which contains a transposition of that sequence Post-translation modification. In a specific embodiment, the VH includes one, two, or three selected from: (a) HVR-H1 including the amino acid sequence of the sequence identification number: 54, (b) including the amine of the sequence identification number: 64 HVR-H2 of amino acid sequence, and (c) includes HVR of HVR-H3 of amino acid sequence of sequence identification number: 74.

In another aspect, the primary anti-C5 antibody includes a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96% for the amino acid sequence of sequence identification number: 106 , 97%, 98%, 99%, or 100% sequence identity. In some specific embodiments, the VH sequence is the amino acid sequence of sequence identification number: 106. In some specific embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity relative to a reference sequence Anti-C5 antibodies that include substitutions (eg, conservative substitutions), insertions or deletions, but that include the sequence retain the ability to bind to C5. In some specific embodiments, in sequence identification number: 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 a region outside the HVR (ie, in the FR). Alternatively, the anti-C5 antibody includes the VH sequence in sequence identification number: 106, which contains a post-translational modification of that sequence. In a specific embodiment, VH includes one, two, or three selected from: (a) HVR-H1 including the amino acid sequence of sequence identification number: 117, (b) including the amine of sequence identification number: 118 HVR-H2 of amino acid sequence, and (c) includes HVR of HVR-H3 of amino acid sequence of SEQ ID NO: 121.

In another aspect, the primary anti-C5 antibody includes a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96% for the amino acid sequence of sequence identification number: 107 , 97%, 98%, 99%, or 100% sequence identity. In some specific embodiments, the VH sequence is an amine of sequence identification number: 107 Amino acid sequence. In some specific embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity relative to a reference sequence Anti-C5 antibodies that include substitutions (eg, conservative substitutions), insertions or deletions, but that include the sequence retain the ability to bind to C5. In some specific embodiments, in sequence identification number: 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 a region outside the HVR (ie, in the FR). Optionally, the anti-C5 antibody includes the VH sequence in sequence identification number: 107, which contains a post-translational modification of that sequence. In a specific embodiment, the VH includes one, two, or three selected from: (a) HVR-H1 including the amino acid sequence of the sequence identification number: 117, (b) including the amine of the sequence identification number: 119 HVR-H2 of amino acid sequence, and (c) includes HVR of HVR-H3 of amino acid sequence of SEQ ID NO: 121.

In another aspect, the primary anti-C5 antibody includes a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96% for the amino acid sequence of sequence identification number: 108 , 97%, 98%, 99%, or 100% sequence identity. In some specific embodiments, the VH sequence is an amino acid sequence of sequence identification number: 108. In some specific embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity relative to a reference sequence Anti-C5 antibodies that include substitutions (eg, conservative substitutions), insertions or deletions, but that include the sequence retain the ability to bind to C5. In some specific embodiments, in sequence identification number: 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 a region outside the HVR (ie, in the FR). Optionally, the anti-C5 antibody includes the VH sequence of SEQ ID NO: 108, which contains that sequence Post-translation modification. In a specific embodiment, VH includes one, two, or three selected from: (a) HVR-H1 including the amino acid sequence of sequence identification number: 117, (b) including the amine of sequence identification number: 118 HVR-H2 of amino acid sequence, and (c) includes HVR of HVR-H3 of amino acid sequence of SEQ ID NO: 121.

In another aspect, the primary anti-C5 antibody includes a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96% for the amino acid sequence of sequence identification number: 109 , 97%, 98%, 99%, or 100% sequence identity. In some specific embodiments, the VH sequence is an amino acid sequence of sequence identification number: 109. In some specific embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity relative to a reference sequence Anti-C5 antibodies that include substitutions (eg, conservative substitutions), insertions or deletions, but that include the sequence retain the ability to bind to C5. In some specific embodiments, in sequence identification number: 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 a region outside the HVR (ie, in the FR). Optionally, the anti-C5 antibody includes the VH sequence in sequence identification number: 109, which contains a post-translational modification of that sequence. In a specific embodiment, VH includes one, two, or three selected from: (a) HVR-H1 including the amino acid sequence of sequence identification number: 117, (b) including the amine of sequence identification number: 118 HVR-H2 of amino acid sequence, and (c) includes HVR of HVR-H3 of amino acid sequence of SEQ ID NO: 121.

In another aspect, the primary anti-C5 antibody includes a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96% for the amino acid sequence of sequence identification number: 110 , 97%, 98%, 99%, or 100% sequence identity. In some specific embodiments, the VH sequence is an amine of sequence identification number: 110 Amino acid sequence. In some specific embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity relative to a reference sequence Anti-C5 antibodies that include substitutions (eg, conservative substitutions), insertions or deletions, but that include the sequence retain the ability to bind to C5. In some specific embodiments, in sequence identification number: 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 a region outside the HVR (ie, in the FR). Optionally, the anti-C5 antibody includes the VH sequence in sequence identification number: 110, which contains a post-translational modification of that sequence. In a specific embodiment, the VH includes one, two, or three selected from: (a) HVR-H1 including the amino acid sequence of the sequence identification number: 117, (b) including the amine of the sequence identification number: 120 HVR-H2 of amino acid sequence, and (c) includes HVR of HVR-H3 of amino acid sequence of SEQ ID NO: 121.

In another aspect, an anti-C5 antibody is provided, wherein the antibody includes a light chain variable domain (VL), which has at least 90% of the amino acid sequence of any of sequence identification numbers: 20, 111-113, 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 a reference sequence Anti-C5 antibodies that include substitutions (eg, conservative substitutions), insertions or deletions, but that include the sequence retain the ability to bind to C5. In some specific embodiments, in any of the sequence identification numbers: 20, 111-113, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted. In some specific embodiments, substitutions, insertions, or deletions occur in a region outside the HVR (ie, in the FR). Optionally, the anti-C5 antibody includes a sequence identification number: VL sequence in any of 20, 111-113, which contains a translation of that sequence After modification. In a specific embodiment, the VL includes one, two, or three selected from: (a) HVR-L1, (b) including an amino acid sequence of any of the sequence identification numbers: 75, 84, 122, and 129 ) HVR-L2 including the amino acid sequence of any of the sequence identification numbers: 85, 94, 123-124, 130, and (c) includes the amino group of any of the sequence identification numbers: 95, 104, 125, 131 HVR of acid sequence of HVR-L3.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises VL, which has at least 90%, 91%, 92%, 93%, 94%, 95% for the amino acid sequence of sequence identification number: 20 , 96%, 97%, 98%, 99%, or 100% sequence identity. In some specific embodiments, the VL sequence is an amino acid sequence of sequence identification number: 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 a reference sequence Anti-C5 antibodies that include substitutions (eg, conservative substitutions), insertions or deletions, but that include the sequence retain the ability to bind to C5. In some specific embodiments, in the sequence identification number: 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 a region outside the HVR (ie, in the FR). Optionally, the anti-C5 antibody includes the VL sequence in SEQ ID NO: 20, which contains a post-translational modification of that sequence. In a specific embodiment, the VL includes one, two, or three selected from: (a) HVR-L1 including an amino acid sequence of sequence identification number: 84, (b) including an amine of sequence identification number: 94 HVR-L2 of amino acid sequence, and (c) includes HVR of HVR-L3 of amino acid sequence of sequence identification number: 104.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises VL, which has at least 90%, 91%, 92%, 93%, 94%, 95% for the amino acid sequence of sequence identification number: 111 , 96%, 97%, 98%, 99%, or 100% Sequence identity. In some specific embodiments, the VL sequence is an amino acid sequence of sequence identification number: 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 a reference sequence Anti-C5 antibodies that include substitutions (eg, conservative substitutions), insertions or deletions, but that include the sequence retain the ability to bind to C5. In some specific embodiments, in sequence identification number: 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 a region outside the HVR (ie, in the FR). Optionally, the anti-C5 antibody includes the VL sequence in sequence identification number: 111, which contains a post-translational modification of that sequence. In a specific embodiment, the VL includes one, two, or three selected from: (a) HVR-L1 including the amino acid sequence of the sequence identification number: 122, and (b) amine including the sequence identification number: 123 HVR-L2 of amino acid sequence, and (c) includes HVR of HVR-L3 of amino acid sequence of sequence identification number: 125.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises VL, which has at least 90%, 91%, 92%, 93%, 94%, 95% for the amino acid sequence of sequence identification number: 112 , 96%, 97%, 98%, 99%, or 100% sequence identity. In some specific embodiments, the VL sequence is an amino acid sequence of sequence identification number: 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 a reference sequence Anti-C5 antibodies that include substitutions (eg, conservative substitutions), insertions or deletions, but that include the sequence retain the ability to bind to C5. In some specific embodiments, in sequence identification number: 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 a region outside the HVR (ie, in the FR). can Alternatively, the anti-C5 antibody includes the VL sequence in sequence identification number: 112, which contains a post-translational modification of that sequence. In a specific embodiment, the VL includes one, two, or three selected from: (a) HVR-L1 including the amino acid sequence of the sequence identification number: 122, and (b) amine including the sequence identification number: 123 HVR-L2 of amino acid sequence, and (c) includes HVR of HVR-L3 of amino acid sequence of sequence identification number: 125.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises VL, which has at least 90%, 91%, 92%, 93%, 94%, 95% for the amino acid sequence of sequence identification number: 113 , 96%, 97%, 98%, 99%, or 100% sequence identity. In some specific embodiments, the VL sequence is an amino acid sequence of sequence identification number: 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 a reference sequence Anti-C5 antibodies that include substitutions (eg, conservative substitutions), insertions or deletions, but that include the sequence retain the ability to bind to C5. In some specific embodiments, in sequence identification number: 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 a region outside the HVR (ie, in the FR). Optionally, the anti-C5 antibody includes the VL sequence in SEQ ID NO: 113, which contains a post-translational modification of that sequence. In a specific embodiment, the VL includes one, two, or three selected from: (a) HVR-L1 including the amino acid sequence of the sequence identification number: 122, (b) including amines of the sequence identification number: 124 HVR-L2 of amino acid sequence, and (c) includes HVR of HVR-L3 of amino acid sequence of sequence identification number: 125.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises VH in any of the embodiments provided above, and VL in any of the embodiments provided above. In one embodiment, the antibodies include sequence identification numbers: 10, VH sequence in any one of 106 to 110 and sequence identification number: VL sequence in any one of 20, 111 to 113, which contains post-translational modifications of those sequences. In one embodiment, the antibody includes a VH sequence having a sequence identification number: 10 and a VL sequence having a sequence identification number: 20. In one embodiment, the antibody includes a VH sequence of sequence identification number: 106 and a VL sequence of sequence identification number: 111. In another embodiment, the antibody includes a VH sequence of sequence identification number: 107 and a VL sequence of sequence identification number: 111. In yet another embodiment, the antibody includes a VH sequence of sequence identification number: 108 and a VL sequence of sequence identification number: 111. In another embodiment, the antibody includes a VH sequence of sequence identification number: 109 and a VL sequence of sequence identification number: 111. In another embodiment, the antibody includes a VH sequence of sequence identification number: 109 and a VL sequence of sequence identification number: 112. In another embodiment, the antibody includes a VH sequence of sequence identification number: 109 and a VL sequence of sequence identification number: 113. In another embodiment, the antibody includes a VH sequence of sequence identification number: 110, and a VL sequence of sequence identification number: 113.

In one aspect, an anti-C5 antibody is provided, wherein the antibody comprises a VH sequence comprising (a) HVR-H1 including an amino acid sequence of sequence identification number: 54, (b) including an amine of sequence identification number: 64 HVR-H2 of amino acid sequence, and (c) HVR-H3 including amino acid sequence of sequence identification number: 74, and VL sequence including (a) HVR including amino acid sequence of sequence identification number: 84 -L1; (b) HVR-L2 including the amino acid sequence of sequence identification number: 94; and (c) HVR-L3 including the amino acid sequence of sequence identification number: 104.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises a VH sequence comprising (a) HVR-H1 comprising an amino acid sequence of sequence identification number: 117, (b) comprising a sequence identification number: 118 of HVR-H2 with amino acid sequence And (c) HVR-H3 including the amino acid sequence of the sequence identification number: 121, and VL sequence including (a) HVR-L1 including the amino acid sequence of the sequence identification number: 122; (b) including the sequence HVR-L2 of amino acid sequence of identification number: 123; and (c) HVR-L3 of amino acid sequence of sequence identification number: 125.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises a VH sequence comprising (a) HVR-H1 including an amino acid sequence of sequence identification number: 117, (b) including a sequence identification number: 119 of HVR-H2 of amino acid sequence, and (c) includes HVR-H3 of amino acid sequence of sequence identification number: 121, and VL sequence including (a) the amino acid sequence of amino acid sequence including sequence identification number: 122 HVR-L1; (b) HVR-L2 including the amino acid sequence of sequence identification number: 123; and (c) HVR-L3 including the amino acid sequence of sequence identification number: 125.

In another aspect, an anti-C5 antibody is provided, wherein the antibody comprises a VH sequence comprising (a) HVR-H1 comprising an amino acid sequence of sequence identification number: 117, (b) comprising a sequence identification number: 118 of HVR-H2 of amino acid sequence, and (c) includes HVR-H3 of amino acid sequence of sequence identification number: 121, and VL sequence including (a) the amino acid sequence of amino acid sequence including sequence identification number: 122 HVR-L1; (b) HVR-L2 including the amino acid sequence of sequence identification number: 124; and (c) HVR-L3 including the amino acid sequence of sequence identification number: 125.

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

In some specific embodiments, the anti-C5 antibody of the present invention includes the VH as in any of the embodiments provided above and the amino acid sequence including any of the sequence identification numbers: 33, 34, 35, 114, 115, and 116 Heavy chain constant region. In some specific embodiments, the anti-C5 antibodies of the present invention include VL as in any of the embodiments provided above and a light chain constant region comprising an amino acid sequence of any of sequence identification numbers: 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, the 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 are classified into the same epitope division of C5 and exhibit pH-dependent binding characteristics.

In yet another aspect, the invention provides an antibody that binds to the same epitope as the 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 β (beta) chain (sequence identification number: 40) of C5. In some specific embodiments, an antibody is provided that binds to an epitope within the β chain (sequence identification number: 40) of C5, which includes at least one selected from amino acids 47-57, 70-76, and 107 A fragment of the group of -110. In some specific embodiments, an antibody is provided that binds to an epitope within a fragment of the β chain (sequence identification number: 40) of C5, which includes at least one selected from Thr47, Glu48, Ala49, Phe50, Asp51, Ala52, Thr53, Lys57, Amino acids of the group consisting of 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, comprising a chimeric, humanized, or human antibody. In one embodiment, the anti-C5 antibody is an antibody fragment, such as an Fv, Fab, Fab ', scFv, diabody, or F (ab') ₂ fragment. In another embodiment, the antibody is a full-length antibody, for example, an intact IgG1 or IgG4 antibody or other antibody species or isotype as defined herein.

In yet another aspect, the anti-C5 antibodies according to any of the above embodiments may combine any of the features individually or in combination, as described in the following sections 1-7:

Antibody affinity

In some specific embodiments, the antibodies provided herein have 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 0.001 Dissociation constant (Kd) of nM or less (for example, 10 ^-8 M or less, such as from 10 ^-8 M to 10 ^-13 M, such as from 10 ^-9 M to 10 ^-13 M).

In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, RIA is performed as a Fab of the antibody of interest and its antigen. For example, the solution binding of Fabs to antigens is measured by equilibrating Fabs with a minimum concentration of (125I) -labeled antigens in the presence of a series of titers of unlabeled antigens, and then capturing the bound antigens with anti-Fab antibody-coated discs. Affinity (see, for example, Chen et al., J. Mol. Biol. 293: 865-881 (1999)). In order to establish the conditions for the measurement, a MICROTITER (registered trademark) multiwell disc (Thermo Scientific) was coated with 5 micrograms / ml (μg / ml) of anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) overnight, Blocking was then performed with 2% (w / v) bovine serum albumin in PBS at room temperature (about 23 degrees C (° C)) for 2 to 5 hours. 100 pM or 26 pM [125I] -antigen in a non-adsorbed disk (Nunc # 269620) with serially diluted Fabs of interest (e.g., with Presta et al. Cancer Res. 57: 4593-4599 (1997) Antibodies, Fab-12 were consistently evaluated) mixed. The Fab of interest is then cultured overnight; however, the culture can be continued for a longer time (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to a capture dish and incubated at room temperature (for example, 1 hour). The solution was then removed and the dish was washed 8 times with PBS containing 0.1% polysorbate 20 (TWEEN-20 (registered trademark)). When the plate has dried, add 150 μl / well of scintillation liquid (MICROSCINT-20 ^™ ; Packard), and count the plate on a TOPCOUNT ^™ gamma counter (Packard) for 10 minutes. Each Fab was selected to give a concentration less than or equal to 20% of the maximum binding for a competitive binding assay.

According to another embodiment, Kd is measured using a BIACORE (registered trademark) surface plasma resonance measurement method. For example, an assay using BIACORE (registered trademark) -2000 or BIACORE (registered trademark) -3000 (BIACORE (registered trademark), Inc., Piscataway, NJ) at 25 ° C to immobilize the antigen CM5 wafer in about 10 response units (RU). In one example, N-ethyl-N '-(3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide were prepared according to the supplier's instructions. (NHS) Activated Carboxymethylated Polyglucose Biosensor Chip (CM5, BIACORE (registered trademark), Inc.). Before injecting at a flow rate of 5 μl / min (5 μl / min) to obtain about 10 response units (RU) of the coupled protein, the antigen was diluted to 5 μg / ml (about 0.2 with 10 mM sodium acetate, pH 4.8). μM (micro M)). After injection of the antigen, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serially diluted Fab (0.78nM to 500nM) was injected at 25 ° C at a flow rate of about 25 μl / min into PBS containing 0.05% Polysorbate 20 (TWEEN-20 ^™ ) surfactant. (PBST). A simple one-to-one Langmuir binding model (BIACORE (registered trademark) Evaluation Software version 3.2) was used to calculate the association rate (kon) and dissociation rate (koff) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio koff / kon. See, for example, Chen et al., J. Mol. Biol. 293: 865-881 (1999). If the binding rate exceeds 10 ⁶ M ^-1 s ^-1 according to the above-mentioned surface plasma resonance measurement method, then the binding rate is measured using a fluorescence quenching technique, which is measured at 25 ° C in PBS, pH 7.2, 20nM The increase or decrease in the fluorescence emission intensity (excitation = 295nm; emission = 340nm, 16nm bandpass) of the anti-antigen antibody (Fab form) is measured in a spectrometer, such as a spectrophotometer equipped with a stop-flow device ( (Aviv Instruments) or 8000 series SLM-AMINCO ^™ Spectrophotometer (ThermoSpectronic) with a stirred colorimetric tube, measuring the presence of increased antigen concentration.

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, refer to Hudson et al., Nat. Med. 9: 129-134 (2003). For a review of specific scFv fragments, please refer to, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, edited by Rosenburg and Moore, (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F (ab ') ₂ fragments that include salvage receptor binding epitope residues and have increased in vivo half-life, please refer to US Patent No. 5,869,046.

A diabody (diobody) is an antibody fragment with two antigen-binding sites, which can 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). Tri- and tetra-antibodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).

Single domain antibodies are antibody fragments that include 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, for example, 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 by production of 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, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)). In one example, a chimeric antibody includes a non-human variable region (e.g., derived from a mouse, rat, Rodents, rabbits or non-human primates, such as the variable regions of monkeys) 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 the class or subclass of the parent antibody. Chimeric antibodies contain their antigen-binding fragments.

In some specific embodiments, the chimeric antibody is a humanized antibody. Generally, non-human antibodies are humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibodies. In general, a humanized antibody includes one or more variable domains, where HVRs, for example, CDRs (or portions thereof) are derived from non-human antibodies, and FRs (or portions thereof) are derived from human antibody sequences. Humanized antibodies optionally also include at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are replaced with residues from non-human antibodies (e.g., antibodies from which HVR residues are derived), for example, to restore or improve antibody specificity or affinity Sex.

Humanized antibodies and methods of making them are reviewed in, for example, Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008), and further described in, for example, Riechmann et al., Nature 332: 323-329 (1988) ; Queen et al., Proc. Nat'l Acad. Sci. USA 86: 10029-10033 (1989); US Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36: 25-34 (2005) (Description Specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28: 489-498 (1991) (describes "resurfacing"); Dall'Acqua et al., Methods 36 : 43-60 (2005) (describe "FR shuffling"); and Osbourn et al., Methods 36: 61-68 (2005) and Klimka et al., Br.J. Cancer 83: 252-260 (2000) (Describe the "guided selection" method of FR shuffling).

Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using the "best fit" method (see, for example, Sims et al., J. Immunol. 151: 2296 (1993); derived from specific subtypes Framework regions of the consensus sequence of human antibodies to the variable regions of the light or heavy chain (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA 89: 4285 (1992); and Presta et al., J Immunol. 151: 2623 (1993); human mature (somatic mutant) framework regions or human germline framework regions (see, for example, Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); And framework regions derived from screening FR databases (see, for example, 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 made using a variety of conventional 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 can be prepared by administering immunogens to genetically modified animals that have been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin locus, which replaces the endogenous immunoglobulin locus, or it is present extrachromosomally or randomly integrated within the animal's chromosome. In such genetically transgenic mice, the endogenous immunoglobulin loci are generally not activated. 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 ^TM technology; U.S. Patent No. 5,770,429 which describes HuMab (registered trademark) technology; U.S. Patent No. 7,041,870 which describes KM MOUSE (registered trademark) technology, and U.S. patents Publication number US 2007/0061900, which describes VELOCIMOUSE (registered trademark) technology). Human variable regions can be further modified by intact antibodies produced by such animals, for example, by combining with different human constant regions.

Human antibodies can also be formed by fusion tumor-based methods. Human myeloma and mouse-human fusion myeloma cell lines for producing human monoclonal antibodies have been described. (See, for example, 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 in, for example, U.S. Patent No. 7,189,826 (described in the production of a single human IgM antibody from a fusion tumor cell line) and Ni, Xiandai Mianyixue 26 (4): 265-268 (2006) (described in a human-human fusion tumor 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 generated by isolating variable domain sequences selected from Fv strains derived from a human phage display database. Such variable domain sequences can then be combined with Desired human constant domain combination. Techniques for selecting human antibodies from an antibody database are described below.

5. Antibodies from the database

The antibodies of the present invention can be isolated by screening antibodies in a combinatorial database for 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 in, for example, Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (edited by O'Brien et al., Human Press, Totowa, NJ, 2001), and further in, for example, McCafferty et al., Nature 348: 552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks, Meth. Mol. Biol. 248: 161-575 (Lo, Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338 (2): 299-310 (2004); Lee et al., J. Mol. Biol. 340 (5) : 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101 (34): 12467-12472 (2004); Lee et al., J. Immunol. Methods 284 (1-2): 119-132 (2004).

In a specific phage display method, the repertoire of the VH and VL genes are individually cloned by the polymerase chain reaction (PCR) and randomly recombined into the phage database. Later, for example, by Winter et al., Ann Rev. Immunol. 12: 433-455 (1994), in which antigen-binding phages are screened. The phage usually presents antibody fragments in the form of single-chain Fv (ScFv) fragments or Fab fragments. A database from immune sources can provide high-affinity antibodies to immunogens without the need to construct fusion tumors. Alternatively, natural libraries can be cloned (e.g., from humans) to provide a wide range of non-self without any immunity A single source of antibodies for antigens and autoantigens, as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, it is also possible to synthetically generate a natural database by transposing unrearranged V gene fragments from stem cells, using PCR primers containing random sequences to encode highly variable CDR3 regions, and achieving in vitro rearrangements, As described by Hoogenboom, J. Mol. Biol. 227: 381-388 (1992). Patent publications describing human antibody phage databases include, for example, U.S. Patent No. 5,750,373, and U.S. Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007 / 0292936 and 2009/0002360.

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

6. Multispecific antibodies

In some specific embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different positions. In some specific embodiments, one of the binding specificities is for C5 and the other is for any other antigen. In some specific embodiments, a bispecific antibody can bind to two different epitopes of C5. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing C5. Bispecific antibodies can be made as full-length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-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 transformation (see, for example, US Patent No. 5,731,168). Multispecific antibodies can also be made by the following methods: engineering for electrostatic guidance of antibody Fc-heterodimer molecules (WO 2009 / 089004A1); cross-linking two or more antibodies or fragments ( See, for example, U.S. Patent No. 4,676,980, and Brennan et al., Science 229: 81 (1985); use of a leucine zipper to generate bispecific antibodies (see, for example, Kostelny et al., J. Immunol. 148 (5 ): 1547-1553 (1992)); use "diabody" technology to generate bispecific antibody fragments (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)) ; And the use of single-chain Fv (scFv) dimers (see, for example, Gruber et al., J. Immunol. 152: 5368 (1994)); and the preparation of trispecific antibodies, for example, Tutt et al., J. Immunol. 147: 60 (1991).

Also included herein are engineered antibodies with three or more functional antigen-binding sites, which include "Octopus antibodies" (see, for example, US 2006/0025576).

The antibodies or fragments herein also include a "dual-acting FAb" or "DAF", which includes an antigen-binding site that binds to C5 and another different antigen (see, for example, 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. Variants of the amino acid sequence of an antibody can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Can make any combination of delete, insert and replace reach the final In constructs, the premise is that the final construct has desired characteristics, such as antigen binding.

a. Replace, insert and delete variants

In some specific embodiments, antibody variants having one or more amino acid substitutions are provided. Positions of interest to replace mutagenesis include HVR and FR. Conservative substitutions are shown under the heading "Preferred substitutions" in Table 1. More substantial changes are provided under the heading "Exemplary substitutions" in Table 1 and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into antibodies of interest and screened for products of desired activity, for example, retained / improved antibody binding, reduced immunogenicity, or improved ADCC or CDC.

Amino acids can be classified according to common side chain characteristics: (1) hydrophobic : N-leucine, 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) aromatics: Trp, Tyr, Phe.

Non-conservative substitutions will allow members of one of these categories to swap with the other.

One type of substitution variant involves replacing one or more highly variable region residues of a parent antibody (e.g., a humanized or human antibody). In general, the resulting variants selected for further research will have modifications (e.g., improvements) relative to the parent antibody in specific biological properties (e.g., increased affinity, reduced immunogenicity) and / or will have The specific biological properties of the parent antibody that are substantially retained. Exemplary substitution variants are affinity matured antibodies that 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 specific biological activity (e.g., binding affinity).

Alterations (eg, substitutions) can be made in HVRs, for example, to improve antibody affinity. Such modifications can be formed in HVR "hot spots", that is, residues encoded by codons that undergo mutations at high frequencies during somatic maturation (see, for example, Chowdhury, Methods Mol. Biol. 207: 179- 196 (2008), and / or contact with antigen residues, and test the binding affinity of the variants VH or VL. Affinity maturation by construction and reselection from a second database is already in place, such as Hoogenboom et al. Methods in Molecular Biology 178: 1-37 (O'Brien et al., Ed., Human Press, Totowa, NJ, (2001). In some embodiments of affinity maturity, diversity is introduced to a variable gene selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis) in. A second database is then generated. The database is then screened to identify any antibody variant with the desired affinity. Another method of introducing diversity involves an HVR targeting method, in which several HVR residues are randomized (eg, 4 to 6 residues at a time). HVR residues involved in antigen binding can be specifically identified, such as using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are particularly frequently targeted.

In some specific embodiments, substitutions, insertions, or deletions can 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 (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be formed in the HVR. Such modifications may, for example, be outside of antigen-contacting residues in HVR. In particular embodiments 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 antibodies that can be targeted for mutagenesis is called "alanine scanning mutagenesis", as described in Cunningham, Science 244: 1081-1085 (1989). In this method, a residue or a group of residues (e.g., charged residues such as arg, asp, his, lys, and glu) can be identified and recognized by a neutral or negatively charged amino acid (e.g., alanine Or poly (alanine)) to determine if it affects the antibody-antigen interaction. Additional substitutions can be introduced at amino acid positions that are shown to be functionally sensitive to the initial substitution. Alternatively, or in addition, the crystal structure of the antigen-antibody complex recognizes the point of contact between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as Candidates for the generation. Variants can be screened to determine if they contain the desired properties.

Amino acid sequence inserts include amino- and / or carboxy-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as within a sequence of single or multiple amino acid residues Insert. Examples of the terminal insert include an antibody having an N-terminal methionamine residue. Other insertional variants of the antibody molecule include a fusion of the N or C terminus of the antibody to an enzyme (eg, ADEPT) or a polypeptide, which 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 degree to which the antibody is glycosylated. One or more glycosylation sites can be made or removed by modifying the amino acid sequence to conveniently complete the addition or deletion of the glycosylation site of the antibody.

When an antibody includes an Fc region, the sugar attached to it can be modified. Natural antibodies produced by mammalian cells usually include branched, biantennary oligosaccharides, which are generally attached to the As2297 of the CH2 domain of the Fc region by N-links. See, for example, Wright et al., TIBTECH 15: 26-32 (1997). Oligosaccharides can include a variety of sugars, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, and fucose attached to GicNAc in the "backbone" of the diantenna oligosaccharide structure. In some embodiments, oligosaccharides can be modified in the antibodies of the invention to produce antibody variants with specific improved properties.

In one embodiment, provided antibody variants have a sugar structure that lacks fucose attached (directly or indirectly) to the Fc region. For example, in such antibodies The amount of fucose may be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. The amount of fucose is 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 (e.g., complex, Hybrids and high mannose structures) are determined, for example, as described in WO 2008/077546. Asn297 refers to an asparagine residue located at about position 297 (Eu numbering of Fc region residues) in the Fc region; however, due to minor sequence variations in the antibody, Asn297 can also be located at about +/- 3 of position 297 The amino acid is 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 No. 2003/0157108 (Presta, L.); 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of published publications related 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; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742 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 protein fucosylated Lecl3 CHO cells (Ripka et al., Arch. Biochem. Biophys. 249: 533-545 (1986); US 2003/0157108, Presta, L; and WO2004 / 056312, Adams et al., Particularly in Example 11), and knockout cell lines such as the α-1,6-fucose transferase gene, FUT8, Knock out CHO cells (see, for example, Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004); Kanda et al., Biotechnol. Bioeng. 94 (4): 680-688 (2006); and WO2003 / 085107).

Further provided are antibody variants having bisected oligosaccharides, for example, wherein the two antenna oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in 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, for example, in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S); and WO 1999/22764 (Raju, S).

c. Fc region variants

In some specific embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby producing an Fc region variant. Fc region variants can include human Fc region sequences (eg, human IgG1, IgG2, IgG3, or IgG4 Fc regions), which 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, which makes them ideal candidates for applications in which the in vivo half-life of the antibody is important However, certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks Fcγ (gamma) R binding (and therefore may lack ADCC activity), but retains FcRn binding capacity. The major cells that regulate ADCC, NK cells, only express FcyRIII, while 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 the ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, for example, 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 Patent No. 5,821,337 (see, Bruggemann et al., J. Exp. Med. 166: 1351-1361 ( 1987)). Alternatively, non-radioactive assays can be used (see, for example, ACTI ^™ Non-Radioactive Cytotoxicity Assay (CellTechnology, Inc. Mountain View, CA) for flow cytometry; and CytoTox 96 (registered trademark) non-radiocytotoxic Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or in addition, the ADCC activity of the molecule of interest can be assessed in vivo, such as in animal models, such as Clynes et al., Proc. Nat'l Acad. Sci. USA 95: 652-656 (1998) Publicly. A Clq binding assay can also be performed to determine that the antibody cannot bind Clq and therefore lacks CDC activity. Please refer to, for example, Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay can be performed (see, for example, 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, for example, 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 include two or more substituted Fc mutants at amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" with residues 265 and 297 substituted with alanine "Fc mutant (US Patent No. 7,332,581).

Specific antibody variants with improved or reduced binding to FcR have been described. (See, for example, US Patent No. 6,737,056; WO 2004/056312; and Shields et al., J. Biol. Chem. 9 (2): 6591-6604 (2001)).

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

In some embodiments, changes are formed in the Fc region that result in altered (ie, improved or reduced) Clq binding and / or complement-dependent cytotoxicity (CDC), for example, as in U.S. Patent 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 neonatal Fc receptors (FcRn) are described in US2005 / 0014934 (Hinton et al.), Which is responsible for delivering maternal IgG to the fetus (Guyer et al. J. Immunol. 117: 587 (1976) and Kim et al. J. Immunol. 24: 249 (1994)). Those antibodies include having one or more Fc regions substituted therein, which improve the binding of the Fc region to FcRn. Such Fc variants are contained in Fc region residues: 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 substitutions, such as the substitution of residue 434 of the Fc region (US Patent 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. US 5,624,821; and WO 1994/29351.

d) Cysteine engineered antibody variants

In some specific embodiments, it is desirable to produce cysteine engineered antibodies, for example, "thioMAb", in which one or more residues of the antibody are replaced by cysteine residues . In particular embodiments, the substituted residues are present in an easily accessible position of the antibody. By replacing those residues with cysteine, the reactive thiol group is placed in an easily accessible position of the antibody and can be used to couple the antibody to other parts, such as the drug part or link the drug part, to create an immune couple Conjugates, as described further 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, for example, as described in US Patent No. 7,521,541.

e) Antibody derivatives

(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。 In some specific embodiments, the antibodies provided herein can be further modified to include additional non-protein moieties known and readily available in the art. Suitable portions for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, polydextrose, polyvinyl alcohol, polyvinyl pyrrolidone, Poly-1,3-dioxolane, poly-1,3,6-tri

(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, polyoxyethylene polyol (eg, glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde can be advantageous in production due to its stability in water. Polymers can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can be different, 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 based on, including but not limited to, considering the particular nature or function of the antibody to be improved, whether the antibody derivative will be used under defined conditions The treatment is moderate.

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

B. Recombination methods and compositions

Antibodies can be produced using recombinant methods and compositions, for example, as described in US 4,816,567. In one embodiment, an antibody encoding an anti-C5 antibody described herein is provided. Isolated nucleic acid. Such nucleic acids may encode an amino acid sequence that includes a VL and / or an amino acid sequence that includes a VH of an antibody (eg, the light and / or heavy chain of an antibody). In yet another embodiment, one or more vectors (eg, expression vectors) including such nucleic acids are provided. In yet another embodiment, a host cell comprising such a nucleic acid is provided. In this embodiment, the host cell includes (eg, has been transformed with): (1) a vector, including a nucleic acid, which encodes the amino acid sequence of the VL including the antibody and the amino acid sequence of the VH of the antibody, or ) A first vector including a nucleic acid encoding an amino acid sequence including a VL of an antibody, and a second vector including a nucleic acid encoding an amino acid sequence of a VH including an antibody. In one embodiment, the host cell is a eukaryotic organism, such as a Chinese hamster ovary (CHO) cell or a lymphoblast (eg, a Y0, NSO, Sp20 cell). In one embodiment, a method of making an anti-C5 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding an antibody as provided above under conditions suitable for expression of the antibody, and optionally from the host cell (Or host cell culture medium).

For the recombinant production of anti-C5 antibodies, for example, a nucleic acid encoding an antibody as described above is isolated and inserted into one or more vectors for further colonization and / or expression in a host cell. Such nucleic acids can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes, which can specifically bind to genes encoding the heavy and light chains of antibodies).

Suitable host cells for the selection or expression of vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, especially when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, please refer to, for example, US 5,648,237, US 5,789,199, and US 5,840,523. (See also 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, the antibodies can be isolated from the bacterial cell paste of the soluble fraction and can be further purified.

Except for prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeast, are suitable breeding or expression hosts for vectors encoding antibodies, including fungi and yeast strains, whose glycosylation pathways have been "humanized", resulting in partial Production of antibodies to all or all human glycosylation patterns. Please refer to Gerngross, Nat. Biotech. 22: 1409-1414 (2004) and Li et al., Nat. Biotech. 24: 210-215 (2006).

Host cells suitable for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and spinal organisms). Examples of invertebrate biological cells include plant and insect cells. A variety of baculovirus strains have been identified that can be used with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts. Please refer to, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (described in the PLANTIBODIES ^(TM) technology for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, a mammalian cell line adapted to grow in suspension can 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, for example, Graham et al., J. Gen Virol. 36:59 (293 cells described in (1977)); baby hamster kidney cells (BHK); mouse supporter cells (e.g., TM4 cells as 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 (Hep G2); Mouse breast tumors (MMT 060562); For example, as described in Mather et al., Annals NYAcad. Sci. 383: 44-68 (1982); TRI cells; MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO ) Cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. Suitable for use in For a review of specific mammalian host cell lines produced by antibodies, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (edited by BKCLo, Humana Press, Totowa, NJ), pp. 255-268 (2003).

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

By adding, for example, 100 micrograms (μg) or 5 micrograms of protein or conjugate (For rabbits and mice, respectively) in combination with 3 times the volume of Freund's complete adjuvant, and intradermally injected the solution at multiple locations to allow animals (usually non-human mammals) to react with antigens, immunogenic conjugates or Derivative immunity. After 1 month, the animals were boosted by subcutaneous injections in multiple locations with 1/5 to 1/10 of the original amount of peptide or conjugate in Freund's complete adjuvant. On days 7-14 after booster injection, the animals were bled and the antibody titer concentration in the serum was determined. Animals were boosted until titer plateaus were reached. Preferably, the animals are boosted with conjugates of the same antigen, but coupled to different proteins and / or by different cross-linking agents. Conjugates can also be produced in the form of protein fusions in recombinant cell culture. Coagulants, like alum, are also suitable for boosting the immune response.

Monoclonal antibodies can be obtained from a substantially homogeneous antibody population, that is, the individual antibodies comprising the population are the same, except that mutations and / or post-translational modifications that may occur naturally in small amounts (e.g., isomerization, amidation) ). Therefore, the modifier "single strain" indicates the characteristics of the antibody, and it is not a mixture of dispersed antibodies.

For example, monoclonal antibodies can be formed using the fusion tumor method first described by Kohler et al., Nature 256 (5517): 495-497 (1975). In a fusion tumor method, a mouse or other suitable host animal, such as a hamster, is immunized as described herein to induce lymphospheres, which produce or are capable of producing antibodies that will specifically bind to a protein for immunization. Alternatively, lymphocytes can be immunized in vitro.

Immunization reagents will typically contain a protein of the antigen or a fusion variant thereof. In general, if cells of human origin are desired, peripheral blood lymphocytes (PBL) are used, or if cells of non-human mammal origin are desired, spleen cells or lymph node cells are used. Lymphocytes are then fused with an immortal cell line using a suitable fusion agent, such as polyethylene glycol, to form a fusion tumor 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. Generally, a rat or mouse myeloma cell line is used. The fused tumor 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 unfused, parental myeloma cells. For example, if the parental myeloma cells lack hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the medium for the fusion tumor will usually contain hypoxanthine, aminopterin, and thymine (HAT medium), which prevent HGPRT-Growth of Defective Cells.

Preferred immortal bone follow-up tumor cells are those that efficiently fuse, support stable production of antibody support by selected antibody-producing cells, and are sensitive to media such as HAT media. Of these, mouse myeloma lines are preferred, 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 available from American Type Culture Collection, Manassas, Virginia USA, SP-2 cells (and derivatives thereof, such as X-63-Ag8-653). Human myeloma and murine-human hybrid myeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor et al., J Immunol. 133 (6): 3001-3005 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications; Marcel Dekker, Inc., New York (1987), pp. 51-63).

It was determined whether a monoclonal antibody against the antigen was produced in the medium in which the fusion tumor cells were grown. Preferably, the monoclonal antibody produced by fusion tumor cells Specificity is determined by immunoprecipitation or by in vitro binding assays (eg, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). Such techniques and tests are known in the art. Binding avidity can be determined by Scatchard analysis of Munson, Anal Biochem. 107 (1): 220-239 (1980).

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

Using conventional immunoglobulin purification procedures such as, for example, Protein A-Sepharose, Hydroxyl Phosphate Lime Chromatography, Gel Electrophoresis, Dialysis, or Affinity Chromatography, the monoclonal antibodies secreted by the secondary strain are removed from the culture medium, ascites (ascites) fluid) or serum is suitably isolated.

Antibodies can be produced by immunizing a suitable host animal against an antigen. In one embodiment, the antigen is a full-length C5 polypeptide. In one embodiment, the antigen is a polypeptide comprising a β (beta) chain (sequence identification number: 40) of C5. In one embodiment, the antigen is a polypeptide including the MG1-MG2 domain (sequence identification number: 43) of the β chain of C5. In one embodiment, the antigen is a polypeptide including the MG1 domain (sequence identification number: 41) of the β chain of C5. In one embodiment, the antigen is a polypeptide comprising a region of an amino acid corresponding to the β chain position of C5 from 19 to 180. In one embodiment, the antigen is a polypeptide comprising a region of an amino acid corresponding to the β chain positions 33 to 124 of C5. In one embodiment, the antigen is a fragment including amino acids 47-57, 70-76, and 107-110 selected from at least one β chain (sequence identification number: 40) selected from C5. Of peptides. In one embodiment, the antigen is a polypeptide comprising a fragment of the β chain of C5, which includes at least one selected from Thr47, Glu48, Ala49, Phe50, Asp51, Ala52, Thr53, Lys57, His70, Val71, His72, Ser74, Glu76 , Val107, Ser108, Lys109, and His110. In one embodiment, the antigen is a polypeptide including a fragment of the β chain of C5, which includes at least one amino acid selected from the group consisting of Glu48, Asp51, His70, His72, Lys109, and His110. The present invention also includes antibodies produced by immunizing animals against the aforementioned antigens. Antibodies can incorporate any of these features, singly or in combination, as described above for "exemplary anti-C5 antibodies."

C. Determination

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

1. Combined determination and other determinations

In one aspect, for example, the antibody of the present invention is tested for its antigen-binding activity by a conventional method such as ELISA, Western Dot, BIACORE (registered trademark), and the like.

In another aspect, a competition assay 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 antibodies are present in excess, it prevents (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 examples, 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., an antibody as described in Table 2) that is bound by an anti-C5 antibody described herein (e.g., an antibody as described in Table 2) (Eg, linear or conformational epitopes). Detailed exemplary methods for positioning epitopes for antibody binding are provided in Morris, "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ) (1996).

In an exemplary competition assay, fixed C5 is cultured in a solution that includes a first labeled (reference) antibody that binds to C5 and a second antibody that has been tested for its ability to compete with the first antibody for binding to C5 Labeled antibody. The secondary antibody may be present in the fusion tumor supernatant. As a control group, fixed C5 was cultured in a solution including the first-labeled antibody but excluding the second-labeled antibody. After culturing under conditions that allow the primary antibody to bind to C5, excess unbound antibody is removed, and the amount of label that binds to fixed C5 is measured. If the amount of labeling that binds to fixed C5 is substantially reduced in the test sample relative to the control group sample, it means that the second antibody competes with the first antibody for binding to C5. See Harlow and Lane, Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) (1988).

In another exemplary competition assay, BIACORE (registered trademark) analysis is used to determine the ability of the tested anti-C5 antibody to compete for binding to C5 with a second (reference) anti-C5 antibody. In yet another aspect, the BIACORE (registered trademark) equipment (for example, BIACORE (registered trademark) 3000) is operated according to the manufacturer's recommendations, and the C5 protein is captured on a CM5 BIACORE (registered trademark) chip using a conventional standard technique to Manufacture C5 coated surface. Generally, C5 of 200 to 800 resonance units is connected to the chip (this amount gives a simple measurable degree of binding, but can be easily saturated by the concentration of the test antibody used). To be evaluated they compete with each other The two antibodies (ie, the test and reference antibodies) were mixed in a suitable buffer at a molar ratio of 1: 1 to produce a test mixture. When the concentration is calculated based on the binding site, the molecular weight of the test or reference antibody is assumed to be the total molecular weight of the corresponding antibody divided by the number of C5 binding sites on the antibody. The concentration of each antibody (ie, the test and reference antibodies) in the test mixture should be high enough to easily saturate the binding sites of the antibodies on the C5 molecule captured on a BIACORE (registered trademark) wafer. The antibodies in the mixed solution have the same molar concentration (on the basis of binding), usually between 1.00 and 1.5 micromolar (on the basis of binding sites). Different solutions containing separate test antibodies and separate reference antibodies were also prepared. The test and reference antibodies in these solutions should be in the same buffer as the test mix and at the same concentration and conditions. The test mixture containing the test antibody and the reference antibody was passed through a C5 coated BIACORE (registered trademark) wafer, and the total amount of binding was recorded. The wafer is then processed in a manner that removes the bound test or reference antibody without destroying the wafer-bound C5. Typically, this is done by treating the wafer with 30 mM HCl for 60 seconds. A solution of the individual test antibody was then passed over the C5-coated surface and the amount of binding was recorded. The wafer was processed again to remove all bound antibodies without destroying the wafer-bound C5. A solution of the individual reference antibody was then passed over the C5-coated surface and the amount of binding was recorded. The maximum theoretical binding of a mixture of test and reference antibodies is then calculated, which is the sum of the binding of each antibody (i.e., test and reference) when only passing through the C5 surface. If the actual recorded mixture binding is less than this theoretical maximum, then the test antibody and reference antibody compete with each other for C5 binding. Therefore, in general, the competition test anti-C5 antibody binds to C5 in the above-mentioned BIACORE (registered trademark) blocking assay such that during the assay and in the presence of the reference anti-C5 antibody, The recorded binding is between 80% and 0.1% of the maximum theoretical binding (for example, 80% to 4%), specifically between 75% and 0.1% of the maximum theoretical binding (for example, 75% to 4%), More specifically is between 70% and 0.1% (eg 70% to 4%) of the maximum theoretical binding (as defined above) of the combined test and reference antibody.

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

In some specific embodiments, the anti-C5 antibodies of the invention compete with antibodies that include VH and VL pairs of antibodies CFA0305 or 305LO5 to bind C5.

In yet other embodiments, the anti-C5 antibody binds to C5 with a higher affinity at a neutral pH compared to an acidic pH. In some specific embodiments, the anti-C5 antibodies of the invention compete with antibodies comprising VH and VL pairs selected from: CFA0538, CFA0501, CFA0599, CFA0307, CFA0366, CFA0675, and CFA0672 to bind C5. 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 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 a neutral pH compared to an acidic pH. In some specific embodiments, the anti-C5 antibodies of the invention compete with antibodies that include VH and VL pairs of antibodies CFA0305 or 305LO5 to bind C5. In still other embodiments, compared to At pH 5.8, the anti-C5 antibody binds to C5 with higher affinity at pH 7.4.

In some specific embodiments, the anti-C5 antibody of the present invention includes a VH selected from a sequence identification number: 22 and a VL selected from a sequence identification number: 26, or a VH selected from a sequence identification number: 21 and a sequence identification number: 25. Antibodies to VH of VL and VL pairs compete for binding to C5. In some embodiments, the anti-C5 antibody is selected from: (a) a VH with a sequence identification number: 5 and a VL with a sequence identification number: 15; (b) a VH with a sequence identification number: 4 and a sequence identification number: 14 (C) sequence identification number: VH of 6 and sequence identification number: VL of 16; (d) sequence identification number: 2 of VH and sequence identification number: VL of 12; (e) sequence identification number: 3 VH and sequence identification number: 13 VL; (f) sequence identification number: 1 VH and sequence identification number: 11 VL; (g) sequence identification number: 9 VH and sequence identification number: 19 VL; (h) ) VH of sequence identification number: 7 and VL of sequence identification number: 17; and (i) VH of sequence identification number: 8 and sequence identification number number of 18: VH and VL pair of antibodies compete for binding to C5. In some embodiments, the anti-C5 antibody competes for binding to C5 with an antibody comprising a VH of sequence identification number: 23 and a VL of sequence identification number: 27. In some embodiments, the anti-C5 antibody competes with antibodies that include VH of sequence identification number: 7 and VL of sequence identification number: 17 to bind C5.

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

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

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

In yet other embodiments, the anti-C5 antibody binds to C5 with a higher affinity at a neutral pH compared to an acidic pH. In some specific embodiments, The anti-C5 antibody binds to C5 with higher affinity at neutral pH than at acidic pH, and interacts with VH selected from: (a) a sequence identification number: 1 and a VL sequence number: 11; (b) a sequence Identification number: VH of 5 and sequence identification number: 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: 16 (E) VH of sequence identification number: 2 and VL of sequence identification: 12; VL of sequence identification number: 3; and VL of sequence identification number: 13; VL of sequence identification number: 9 VH and sequence identification number: 19 VL; (h) sequence identification number: 7 VH and sequence identification number: 17 VL; (i) sequence identification number: VH of 8 and sequence identification number: 18 VL; and ( j) VH and VL pairs of sequence identification number: 10 and sequence identification number: 20 compete with antibodies against C5. In yet other embodiments, the anti-C5 antibody binds to C5 with higher affinity at pH 7.4 than at pH 5.8.

In some embodiments, the anti-C5 antibody binds to C5 with a higher affinity at neutral pH than at acidic pH, and interacts with VH selected from: (a) a sequence identification number: 5 and a sequence identification number: 15 VL; (b) sequence identification number: 4 VH and sequence identification number: 14 VL; (c) sequence identification number: 6 VH and sequence identification number: 16 VL; (d) sequence identification number: 2 VH And sequence identification number: 12 VL; (e) sequence identification number: 3 VH and sequence identification number: 13 VL; (f) sequence identification number: VH of 1 and sequence identification number: 11 VL; (g) Sequence identification number: VH of 9 and sequence identification number: VL of 19; (h) Sequence identification number: VH of 7 and sequence identification number: VL of 17; and (i) Sequence identification number: VH and sequence identification number of 8 : 18 VL VH and VL pair antibodies compete for binding to C5. In yet other embodiments, the anti-C5 antibody binds to C5 with higher affinity at pH 7.4 than at pH 5.8.

In some embodiments, the anti-C5 antibody binds to C5 with a higher affinity at neutral pH than at acidic pH, and interacts with a VL comprising: VH selected from: sequence identification number: 1 and sequence identification number: 11 or The VH of the sequence identification number: 10 and the sequence identification number: 20 of the VL and VL pairs compete for binding to C5. In yet other embodiments, the anti-C5 antibody binds to C5 with higher affinity at pH 7.4 than at pH 5.8.

In some specific embodiments, whether the anti-C5 antibody of the present invention binds to a specific epitope can be determined as follows: C5 point mutation in which the amino acid (except alanine) on C5 is replaced by alanine is expressed in 293 cells And the binding of anti-C5 antibodies to C5 mutants was tested by ELISA, Western blot, or BIACORE (registered trademark); wherein the anti-C5 antibodies had substantially reduced C5 mutants compared to their binding to wild-type C5. Or disappeared binding represents the binding of an 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 (sequence identification number: 40). In still other embodiments, the amino acid on C5 to be substituted with alanine is Asp51 or Lys109 of the β chain of C5 (sequence identification number: 40).

In another embodiment, whether an anti-C5 antibody with pH-dependent binding properties binds to a specific epitope can be determined as follows: In 293 cells, the expression of a histidine residue on C5 by another amino acid (eg, , 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); which compared to C5 mutants at acidic pH Binding, the substantially reduced binding of anti-C5 antibodies to wild-type C5 at acidic pH represents the binding of anti-C5 antibodies to C5 The epitope of that histidine residue. 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 C5 mutants at neutral pH. In some specific embodiments, the histidine residue on C5 to be replaced by another amino acid is selected from the group consisting of His70, His72, and His110 of the β chain of C5 (sequence identification number: 40). In yet another embodiment, the histidine residue His70 is replaced with tyrosine.

2. Activity determination

In the same state, an assay is provided to identify the biological activity that an anti-C5 antibody has. Biological activities may include, for example, inhibiting the activation of C5, preventing the cleavage of C5 to form C5a and C5b, preventing the C5 convertase from approaching the cleavage site on C5, preventing 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, such biological activities of the antibodies of the invention are tested.

In some specific examples, testing whether the antibody inhibits C5 cleavage to C5a and C5b is determined by, for example, a method described in, for example, 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 proteins, such as ELISA or Western blotting. In the presence of a test antibody (or after contact with the test antibody) and a decrease in the amount of C5 cleavage products (C5a and / or C5b) is detected, the test antibody is identified as an antibody that inhibits C5 cleavage. In some specific embodiments, the concentration and / or physiological activity of C5a can be measured by, for example, chemotaxis assay, RIA, or ELISA (see, for example, Ward and Zvaifler J. Clin. Invest. 50 (3 ): 606-616 (1971)).

In some specific embodiments, testing whether the antibody prevents the C5 converting enzyme from approaching C5 is determined by a method for detecting a protein interaction between the C5 converting enzyme and C5, for example, ELISA or BIACORE registered trademark. In the case where the test antibody is present (or after contact with the test antibody) and the interaction is reduced, the test antibody is identified as an antibody that prevents the C5 convertase from approaching C5.

In some specific embodiments, C5 activity can be measured as a function of its ability to lyse cells in a subject's body fluids. Methods known in the art, such as conventional hemolysis 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 conventional variation of the assay, such as the chicken red blood cell hemolysis method described in Hillmen et al. (2004), N. Engl. J. Med. 350 (6): 552-559 (2004), measures The cytolytic capacity of C5 or its decline. In some specific embodiments, the C50eq assay is used to quantify C5 activity or its inhibition. The CH50eq assay is a method for measuring total classical complement activity in serum. This test is a lytic assay that uses antibody-sensitized red blood cells as an activator of the classical complement pathway, and uses various dilutions of the test serum to determine the amount required to obtain 50% cytolysis (CH50). The percentage of hemolysis can be determined, for example, using a spectrophotometer. The CH50eq assay provides an indirect measurement 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 methods exemplified and exemplified in the operating examples. Using these or other types of assays, candidate antibodies capable of inhibiting C5 activation can be screened. In some specific embodiments, compared to the effect of the negative control group under similar conditions, the inhibition of C5 activation is included in the assay by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40%, or more 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. Immunoconjugate

The invention also provides immunoconjugates comprising an anti-C5 antibody herein conjugated to one or more cytotoxic agents, for example, a chemotherapeutic agent or drug, a growth inhibitor, a toxin (e.g., bacterial, fungal, plant or animal origin) Protein toxins, enzymatically active toxins, or fragments thereof) or radioisotopes.

In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC), wherein the antibody is conjugated to one or more drugs, including but not limited to maytansinoid (see, U.S. Patent) No. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); auristatin such as monomethyl auristatin drug part DE and DF (MMAE and MMAF) (please refer to US Patent Nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin ); Calicheamicin or its derivatives (see, U.S. Pat. 1993); and Lode et al. Cancer Res. 58: 2925-2928 (1998)); anthracycline antibiotics such as daunycin (daunomycin) or doxorubicin (see Kratz et al., Current Med Chem. 13: 477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16: 358-362 (2006); Torgov et al., Bioconj. Chem. 16: 717-721 (2005); Nagy Et al. Proc. Natl. Acad. Sci. USA 97: 829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12: 1529-1532 (2002); King et al., J. Med. Chem. 45: 4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, Iarotaxel, tesetaxel, and ortataxe; Trichothecene; and CC1065.

In another embodiment, an immunoconjugate includes an antibody as described herein coupled to an enzyme-active toxin or a fragment thereof, including but not limited to diphtheria A chain, a non-binding active fragment of diphtheria toxin, an exotoxin A chain ( (From Pseudomonas aeruginosa), ricin A chain, acacia A chain, modeccin A chain, alpha-sarcin, sarcin, Aleutites fordii, dianthin, Phytolaca americana (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin ), Crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, ectomycin Economycin and trichothecene.

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

A variety of bifunctional protein coupling agents can be used to form conjugates of antibodies and cytotoxic agents, such as N-succinoimino 3- (2-pyridyldithio) propionate (SPDP), succinimino-4 -(N-maleimidoiminomethyl) cyclohexane-I-carboxylate (SMCC), iminosulfane (IT), imidate (e.g. dimethyl adipamide hydrochloride dimethyl ester) Bifunctional derivatives, active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis (p-azidobenzoyl) hexane) Amines), double nitrogen derivatives (e.g. bis (p-diazobenzyl) -ethylenediamine), diisocyanates (e.g. toluene 2,6-diisocyanate), and direactive fluorine compounds (e.g. 1,5- Difluoro-2,4-dinitrobenzene). For example, a bacterin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14 labeled benzyl-1-isothiocyanate-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelator used to couple radionucleotides to antibodies. Please refer to WO94 / 11026. A linker may be a "cleavable linker" that facilitates the release of a cytotoxic drug in a cell. For example, acid labile linkers, peptidase sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al. Cancer Res. 52: 127-131 (1992 ); US Patent No. 5,208,020).

The immunoconjugates or ADCs herein explicitly contemplate, but are not limited to, such conjugates prepared with a cross-linking agent 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 (succinizone-imino- (4-vinylfluorene) benzoate), which are 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 is useful for detecting the presence of C5 in a biological sample. The term "detection", as used herein, encompasses quantitative or qualitative detection. In some specific embodiments, the biological sample includes 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 fluid, joint fluid, abdominal fluid, intraocular fluid and mucus.

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

In another embodiment, a method of selecting an individual with a complement-regulated disease or condition involving excessive or uncontrolled C5 activation is provided as a therapy suitable to include an anti-C5 antibody of the invention. In some specific embodiments, the method comprises (a) detecting a genetic variation derived from an individual's C5, and (b) when a genetic variation derived from an individual's C5 is detected, selecting to be suitable for inclusion in the present invention Of Individuals with anti-C5 antibody therapy. In another embodiment, a method is provided for selecting a therapy for an individual with an over- or uncontrolled C5-activated complement-modulating disease or condition. In some specific embodiments, the method comprises (a) detecting a genetic variation derived from an individual's C5 and (b) when a genetic variation derived from an individual's C5 is detected, selecting the individual for inclusion in the invention 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 derived from an individual's C5, and (b) when detecting a genetic variation derived from an individual's C5, selecting as suitable for including the present invention Individuals with anti-C5 antibody therapy and (c) administering an anti-C5 antibody of the invention to the individual.

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

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

It has been reported that some patients with a genetic variation in C5 show adverse reactions to therapies comprising pre-existing anti-C5 antibodies (Nishimura et al., N. Engl. J. Med. 370: 632-639 (2014)). Such patients are recommended to be treated with a therapy comprising the present anti-C5 antibody, since this antibody has inhibitory activity on the activation of C5 variants and the activation of wild-type C5, as shown in the following examples.

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, but not limited to, southern blot analysis or northern blot analysis. The C5 variant may include at least one genetic variation. The genetic variation can be selected from the group consisting of V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N and E1437D. In the text, R885H, for example, represents a genetic variation in which the arginine at position 885 was replaced with a histidine. In some specific embodiments, the C5 variant has a biological activity similar to that of wild-type C5.

Exemplary diseases that can be diagnosed using the antibodies of the present invention include rheumatoid arthritis (RA); systemic lupus erythematosus (SLE); lupus nephritis; ischemia reperfusion injury (IRI); asthma ; Paroxysmal nocturnal hemoglobinuria (PNH); hemolytic uremic syndrome (HUS) (e.g., atypical hemolytic uremic syndrome (aHUS)); dense deposit disease (DDD) ; Neuromyelitis optica (NMO); multifocal motor neuropathy (MMN); multiple sclerosis (MS); systemic sclerosis; Macular lesions (e.g., senile macular degeneration (AMD)); hemolysis, elevated liver enzymes, and low platelet (HELLP) syndrome; thrombotic thrombocytopenic purpura (TTP); spontaneous fetal loss; Epidermolysis bullosa; recurrent fetal loss; pre-eclampsia; traumatic brain injury; myasthenia gravis; cold agglutinin syndrome; Sjogren's syndrome; dermatomyositis; large Bullous pemphigoid; phototoxicity; Shiga toxin E. coli-related hemolytic uremic syndrome; typical or infectious hemolytic uremic syndrome (tHUS); C3 glomerulonephritis; anti-neutrophil cytoplasmic antibody (ANCA) -associated vasculitis; humoral and vascular graft rejection; acute antibody-mediated rejection (AMR); graft dysfunction; myocardial infarction; Allograft; sepsis; coronary artery disease; hereditary angioedema; dermatomyositis; Graves' disease; atherosclerosis; Alzheimer's disease (AD); Huntington's disease; Creutzfeld-Jacob; Parkinson's disease; cancer; wound; septic shock; spinal cord injury; uveitis; diabetic eye disease; retinopathy of prematurity; glomerulonephritis; membranous nephritis; immunoglobulin A Nephropathy; adult respiratory distress syndrome (ARDS); chronic obstructive pulmonary disease (COPD); cystic fibrosis; hemolytic anemia; paroxysmal cold hemoglobinuria; Anaphylactic shock (anaphylactic shock); allergies; osteoporosis; osteoarthritis; Hashimoto's thyroiditis; type I diabetes; psoriasis; pemphigus; autoimmune Autoimmune hemolytic anemia (AIHA); idiopathic thrombocytopenic purpura (ITP); Goodpasture syndrome; Degos disease; antiphospholipid Antiphospholipid syndrome (APS); Catastrophic APS (CAPS); Cardiovascular disease; Myocarditis; Cerebrovascular disorder (Cerebrovascular disorder); Peripheral vascular disease; Renal vascular disease; Mesenteric / intestinal 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); after stent placement Restenosis following stent placement; rotation atherectomy; membranous nephropathy; Guillain-Barresyndrome (GBS); Fisher syndrome; antigen-induced arthritis Synovial inflammation; viral infection; bacterial infection; fungal infection; and damage caused by myocardial infarction, cardiopulmonary bypass, and hemodialysis.

In some specific embodiments, a labeled anti-C5 antibody is provided. Labels include, but are not limited to, labels or portions that are directly detected (e.g., fluorescent, chromogenic, electron-intensive, chemiluminescent, and radioactive labels), and indirect (e.g., through enzymatic reactions or molecular interactions) Parts, like enzymes or ligands. Exemplary labels include, but are not limited to, radioisotopes ³² P, ¹⁴ C, ¹²⁵ I, ³ H, and ¹³¹ I, fluorophores such as rare earth chelates or fluorescein and their derivatives, rhodamine and its Derivatives, dansyl, umbellifeixme, luciferiferase, for example, firefly luciferase and bacterial luciferase (US Patent No. 4,737,456), luciferin , 2,3-dihydrophthalazine dione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lyase, carbohydrate oxidase, for example, glucose oxidase , Galactose oxidase and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as urase and xanthine oxidase, combined with enzymes that use hydrogen peroxide to oxidize dye precursors such as HRP, lactoperoxidase, or micro Peroxidase (microperoxidase), biotin / avidin, spin labels, phage labels, stable free radicals, etc.

F. Pharmaceutical formula

The pharmaceutical formulation of an anti-C5 antibody as described herein is obtained by combining this antibody with a desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. 1980)) mixed and prepared as a lyophilized formulation or as an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations used, and include but are not limited to: buffering agents such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine Preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexahydroxy quaternary ammonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butanol or benzyl alcohol; alkyl parabens, such as Methyl parahydroxybenzoate or propyl parahydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; Proteins such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine Glycine, glutamine, asparagine, histamine, arginine, or lysine; monosaccharides, disaccharides, and other sugars, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar , Such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counter ions, such as sodium; metal complexes (such as Zn-protein complexes); and / or non-ionic surfactants, such as polyethylene glycol (PEG) . Exemplary pharmaceutically acceptable carriers herein also include pharmaceutical 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.)). Specific 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 glucosaminoglycanases, such as chondroitinase.

An exemplary lyophilized antibody formulation is 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 containing a histidine-acetate buffer.

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

The active ingredient can be trapped in, for example, microcapsules prepared by coacervation technology or by interfacial polymerization, such as hydroxymethyl cellulose or gelatin microcapsules and poly- (methyl methacrylate) microcapsules, respectively, In colloidal drug delivery systems (e.g. liposomes, albumin microspheres, microemulsions, nanoparticle and nanocapsules) Medium or in coarse drops. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

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

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

G. Methods of treatment and compositions

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

In one aspect, an anti-C5 antibody is provided for use as a medicament. In yet another aspect, an anti-C5 antibody is provided for treating a complement-regulated disease or condition involving excessive or uncontrolled C5 activation. In some specific embodiments, anti-C5 antibodies are provided for use in a method of treatment. In some specific embodiments, the invention provides anti-C5 antibodies for use in a method of treating an individual having a complement-modulated 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.

When the antibody is a soluble protein, the binding of the antibody to its antigen can cause an increase in the half-life of the antigen in the plasma (ie, reduce the elimination of the antigen from the plasma), since the antibody itself has a longer half-life in the plasma and acts as an antigen Carrier. This is due to the recovery of the antigen-antibody complex through the endosomal pathway in the cell by FcRn (Roopenian, Nat. Rev. Immunol. 7 (9): 715-725 (2007)). However, antibodies with pH-dependent binding properties Binding to its antigen in the external environment, and then releasing it into the acidic intracellular compartment after entering the cell, is expected to have superior properties in antigen neutralization and clearance, compared to its pH-independent form Binding counterparts (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 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 the elimination of C5 from plasma in an individual, said method comprising administering an effective amount of an anti-C5 antibody to the individual to enhance the elimination of C5 from plasma. In one embodiment, the anti-C5 antibody enhances the elimination of C5 from plasma compared to traditional anti-C5 antibodies that do not have pH-dependent binding properties. According to any of the above embodiments, the "individual" is preferably a human.

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

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

In yet another aspect, the invention provides the use of an anti-C5 antibody in the production or manufacture of a medicament. In one embodiment, the medicament is for the treatment of a disease or condition involving complement or excessive C5 activation. In yet another embodiment, a medicament is for use in a method of treating a complement-regulated disease or condition involving excessive or uncontrolled C5 activation, which comprises modulating a disease or condition to a complement with excessive or uncontrolled C5 activation The individual 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.

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

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

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 the activation of C5 in an individual, the method comprising administering an effective amount of the medicament to the individual to inhibit the activation of C5. In one embodiment, C5-regulated cytotoxicity can be inhibited by inhibiting C5 activation. According to any of the above embodiments, the "individual" may be a human.

In yet another aspect, the invention provides a method for treating a complement-regulated disease or condition involving excessive or uncontrolled C5 activation. In one embodiment, the method comprises administering an effective amount of an anti-C5 antibody to an individual having said complement-regulated disease or condition involving excessive or uncontrolled C5 activation. 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 may be a human.

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

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

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

In yet another aspect, the invention provides a pharmaceutical formulation comprising any of the anti-C5 antibodies provided herein, for example, for use in any one or more of the methods of treatment. In one embodiment, the pharmaceutical formulation includes any of the anti-C5 antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, the 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 disease or condition involving complement or excessive control of C5 activation. In yet another embodiment, the pharmaceutical formula is used to enhance the elimination of C5 from plasma. In one embodiment, the anti-C5 antibody enhances the elimination of C5 from plasma compared to traditional anti-C5 antibodies that do not have pH-dependent binding properties. In yet another embodiment, the pharmaceutical formula is used to inhibit the accumulation of C5 in plasma. In one embodiment, the accumulation of C5 in plasma is the result of the formation of an antigen-antibody complex. In another embodiment, the anti-C5 antibody inhibits the accumulation of C5 in plasma compared to traditional 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 another embodiment, the pharmaceutical formula is used to inhibit the activation of C5. In one embodiment, C5-regulated cytotoxicity is inhibited by inhibiting C5 activation. In one embodiment, the pharmaceutical formulation is administered to an individual with a complement-regulated disease or condition involving excessive or uncontrolled C5 activation. The "individual" according to any of the above embodiments is preferably a human.

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

In yet another aspect, the present invention provides a method for preparing a medicament or a pharmaceutical formulation, comprising mixing any of the anti-C5 antibodies 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 pharmaceutical or pharmaceutical formulation further includes adding at least one additional therapeutic agent to the pharmaceutical or pharmaceutical formulation.

In some specific embodiments, complement-regulated diseases or conditions involving excessive or uncontrolled C5 activation are selected from the group consisting of: rheumatoid arthritis (RA); systemic lupus erythematosus (SLE); lupus Nephritis (lupus nephritis); ischemia reperfusion injury (IRI); asthma; paroxysmal nocturnal hemoglobinuria (PNH); hemolytic uremic syndrome (HUS) (for example, 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 (for example, senile macular degeneration (AMD)); hemolysis, elevated liver enzymes and low platelet (HELLP) syndrome; thrombocytopenic purpura (thrombotic thrombocytopenic) purpura, TTP); spontaneous fetal loss; epidermolysis bullosa; recurrent fetal loss; pre-eclampsia; traumatic brain injury; myasthenia gravis; cold agglutinin syndrome; Sjogren syndrome 'ssyndrome); dermatomyositis; bullous pemphigoid; phototoxic response; Shiga toxin E. coli-related hemolytic uremic syndrome; typical or infection Hemolytic uremic syndrome (tHUS); C3 glomerulonephritis; antineutrophil cytoplasmic antibody (ANCA) -associated vasculitis; humoral and vascular graft rejection; acute antibody-mediated rejection (AMR) Graft dysfunction myocardial infarction allograft septicemia 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; glomerulonephritis; membranous nephritis nephritis); immunoglobulin A nephropathy; adult respiratory distress syndrome (ARDS); chronic obstructive pulmonary disease (COPD); cystic fibrosis; hemolytic anemia; paroxysmal cold hemoglobin Urine (paroxysmal cold hemoglobinuria); anaphylactic shock; allergies; osteoporosis; osteoarthritis; Hashimoto's thyroiditis; Type I diabetes; psoriasis; pemphigus; autoimmune hemolytic anemia (AIHA); idiopathic thrombocytopenic purpura (ITP); guppa Goodpasture syndrome; Degos disease; Antiphospholipid syndrome (APS); Catastrophic APS (CAPS); Cardiovascular disease; Myocarditis; Cerebrovascular disease (cerebrovascular disorder); peripheral vascular disease; renal vascular disease; mesenteric / enteric vascular disorder (mesentic / 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 (restenosis following stent placement ); Rotational atherectomy; membranous nephropathy; Granbali syndrome (G uillain-Barre syndrome (GBS); Fisher syndrome; antigen-induced arthritis; synovial inflammation; viral infection; bacterial infection; fungal infection; and damage caused by myocardial infarction, cardiopulmonary bypass and hemodialysis.

In some specific embodiments, the complement-regulated disease or condition is an ophthalmic condition. In still other embodiments, the eye condition is a macular lesion. In yet other embodiments, the macular lesion is senile macular degeneration (AMD). In yet other embodiments, AMD is a dry AMD.

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

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

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

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

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

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

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

Combination therapies as described above include combined administration (where two or more therapeutic agents are contained in the same or separate formulations), and separate administration, in which case administration of the antibodies of the invention may occur in additional Before, at the same time 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 about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other occur.

The antibodies (and any additional therapeutic agents) of the invention can be administered by any suitable method, including parenteral, intrapulmonary, and intranasal administration, and, if required by local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. To some extent, depending on whether the medication is short-term or long-term, it can be by any suitable route, such as by injection, For example, intravenous or subcutaneous injection. Various dosing schedules are covered herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

The 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 specific condition to be treated, the specific mammal to be treated, the clinical status of the individual patient, the cause of the condition, the location of the delivered agent, the method of administration, the timing of administration, and what is known to the medical practitioner Other factors. The antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat problematic conditions. The effective amount of other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used at the same dose, and using the route of administration as described herein, or at about 1 to 99% of the dose described herein, or at any dose and route that is empirically / clinically determined appropriate.

For the prevention or treatment of a disease, the appropriate dosage of the antibodies 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 progress of the disease The antibody is administered for preventive or therapeutic purposes, previous treatment, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitably administered to the patient in a single treatment or after a series of treatments. Depending on the type and severity of the disease, antibodies from about 1 microgram / kg (μg / kg) to 15 milligrams per kilogram (mg / kg) (e.g., 0.1 mg / kg-10 mg / kg) can be used for administration to patients The initial candidate dose, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dose may range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated application for several days or longer, the root Depending on the condition, treatment will usually be continued until the desired suppression of disease symptoms occurs. An exemplary dose of the antibody should 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) can be administered to a patient. Such doses may be administered at intervals, such as weekly or every three weeks (eg, so that the patient receives about 2 to about 20, or, for example, about 6 doses of the antibody). An initially higher loading dose may be administered, followed by one or more lower doses. The progress of this therapy can be easily monitored by conventional methods and assays.

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

F. Products

In another aspect of the present invention, there is provided an article of manufacture comprising a material useful for treating, preventing, and / or diagnosing the aforementioned conditions. Articles of manufacture include containers and labels or package inserts on or in combination with the containers. Suitable containers include, for example, bottles, vials, syringes, IV solution packs, and the like. The container may be made of various materials such as glass or plastic. A container holds a composition which is alone or in combination with another composition which is effective for treating, preventing and / or diagnosing a condition, a composition which is effective for the treatment of symptoms, and which can have sterile access Mouth (e.g., a container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an antibody of the invention. A label or pharmaceutical imitation indicates that the composition is used to treat a particular condition. In addition, 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. In this embodiment of the invention The article of manufacture may further include a pharmaceutical copy indicating that the composition can be used to treat specific symptoms. Alternatively or additionally, the article of manufacture may further include a second (or third) container that includes a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and glucose solution. From a commercial and user standpoint, it can also contain other materials needed, including other buffers, diluents, filters, needles, and syringes.

It should be understood that any of the above preparations may include an immunoconjugate of the invention to replace or supplement the anti-C5 antibody.

[Example]

The following are examples of the methods and compositions of the present invention. It should be understood that various other embodiments may be implemented in accordance with the general description provided above.

[Example 1]

Preparation of C5

1.1. Expression and purification of recombinant human and rhesus C5

The FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA) was used to transiently express recombinant human C5 (NCBI GenBank accession number: NP_001726.2, SEQ ID NO: 39). The conditioned medium expressing human C5 was diluted with an equal volume of milliQ water, and then used on a Q-sepharose FF or Q-sepharose HP anion exchange column (GE healthcare, Uppsala, Sweden), and then eluted with a NaCl gradient. The fractions containing human C5 were collected, and then the salt concentration and pH were adjusted to 80 mM NaCl and pH 6.4, respectively. The obtained sample was applied to a SP-sepharose HP cation exchange column (GE healthcare, Uppsala, Sweden) and eluted with a NaCl gradient. Fractions containing human C5 were collected and applied to a CHT ceramic hydroxyphosphate lime column (Bio-Rad Laboratories, Hercules, CA, USA). The human C5 eluent 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 C5 (NCBI GenBank Accession No .: XP_005580972, SEQ ID NO: 44) was performed in the same manner as the human part.

1.2. Purification of Rhesus 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. The fraction containing cynoC5 was immediately neutralized and applied to protein A HP columns (GE healthcare, Uppsala, Sweden) and peptide M agarose (Invivogen, San Diego, CA, USA) one after the other. The flow-through fraction was then applied to a Superdex 200 colloidal filtration column (GE healthcare, Uppsala, Sweden). Fractions containing cynoC5 were collected and stored at -80 degrees C (° C).

[Example 2]

Production of anti-C5 antibodies

2.1. Antibody screening

Anti-C5 antibodies are prepared, selected, and measured in the following ways: NZW rabbits 12 to 16 weeks old were immunized intradermally with human C5 and / or monkey C5 (50-100 micrograms (μg) / dose / rabbit). This dose is repeated 4-5 times over a 2 month period. One week after the last immunization, the spleen and blood of the immunized rabbits were collected. Antigen-specific B cells were stained with labeled antigen and classified by the FCM cell classifier (FACS aria III, BD). The cells were plated in 96-well plates at a density of 1 cell / well together with 25,000 cells / well of EL4 cells. (European Collection of Cell Cultures), and the activated rabbit T cell conditioned medium was diluted 20-fold and cultured for 7-12 days. EL4 cells were previously treated with mitomycin C (Sigma, Cat No. M4287) for 2 hours and washed 3 times. Rabbit thymus cells were cultured in a plant containing hemagglutinin-M (Roche, Cat No. 1 1082132-001), phorbol 12-myristate 13-acetate (Sigma, Cat No. P1585), and 2 % FBS in RPMI-1640 to prepare rabbit T cell conditioned medium. 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 the antibodies in the B cell culture supernatant. Biotin (GeneScript, Cat No. Z02043) was covered with 50 nM PBS on 384-well MAXISorp (Nunc, Cat No. 164688), and left at room temperature for 1 hour. The discs were then blocked with a 5-fold dilution of 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 the blocked ELISA plate, cultured for 1 hour and washed. The B cell culture supernatant was added to the ELISA plate, cultured for 1 hour and washed. Binding was detected by goat anti-rabbit IgG-horseradish peroxidase (BETHYL, Cat No. A120-111P) followed by the addition of ABTS (KPL, Cat No. 50-66-06).

An ELISA assay was used to assess the pH-dependent binding of antibodies to C5. Goat anti-rabbit IgG-Fc (BETHYL, Cat No. A120-111A) diluted to 1 μg / ml (μg / ml) with PBS (-) was added to MAXISorp (Nunc, Cat No. 164688) in 384 wells. Incubate at room temperature for 1 hour and block with a 5-fold dilution of Blocking One (Nacalai Tesque, Cat No. 03953-95). After incubation, the dishes were washed and B cell culture supernatant was added. The plates were incubated for 1 hour, washed, and 500 pM biotinylated human or monkey C5 was added and incubated for 1 hour. After incubation, wash the dishes and incubate for 1 hour at room temperature with pH 7.4 MES buffer (20 mM MES, 150 mM NaCl, and 1.2 mM CaCl ₂ ) or pH 5.8 MES buffer (20 mM MES, 150 mM NaCl, and 1 mM EDTA). . After incubation, the biotinylated C5 binding 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 the affinity and pH-dependent binding of antibodies to C5. The antibody secreted by the B cell culture supernatant was loaded on a protein A biosensing probe (Pall Life Sciences) and immersed in 50 nM human or monkey C5 in pH 7.4 MES buffer to analyze binding kinetics . Dissociation kinetics was analyzed in both pH 7.4 MES buffer and pH 5.8 MES buffer.

A total of 41,439 B cell lines were screened for affinity and pH-dependent binding to human or monkey C5, and 677 lines were selected and designated CFA0001-0677. The ZR-96 Quick-RNA kit (ZYMO RESEARCH, Cat No. R1053) was used to purify the RNA of the selected cell line from the cryopreservation pellet. The DNA encoding the variable region of the antibody heavy chain in the selected cell line was amplified by reverse transcription PCR and recombined with the DNA encoding the constant region of the heavy chain of F760G4 (sequence identification number: 33) or F939G4 (sequence identification number: 34). The DNA encoding the variable region of the antibody light chain was amplified by reverse transcription PCR and recombined with the DNA encoding the constant region (sequence identification number: 36) of the k0MTC light chain. The heavy and light chain genes of existing humanized anti-C5 antibodies, eculizumab (EcuH-G2G4, sequence identification number: 29 and EcuL-k0, sequence identification number: 30), were synthesized separately. A DNA encoding VH (EcuH, sequence identification number: 31) was fused in-frame to a DNA encoding a modified human IgG4 CH (F760G4, sequence identification number: 33), and a DNA encoding VL (EcuL , Sequence identification number: 32) fused in-frame to the DNA encoding the constant region of the k0 light chain (sequence identification number: 37). Each fused coding sequence was transfected into an expression vector. Antibodies were expressed in FreeStyle ^(TM) 293-F cells (Invitrogen) and purified from culture supernatants to assess functional activity. Using the liposomal cell lysis assay described in Example 5.1, the neutralizing activity of the antibodies was evaluated by testing the inhibition of complement activity.

2.2. Epitope binning by sandwich ELISA

Anti-C5 antibodies with high affinity, pH-dependent or neutralizing activity were selected for further analysis. Sandwich ELISA assays are used to classify selected antibodies into different epitope bins that bind to the same or overlapping epitopes of the C5 protein. Unlabeled capture antibody was diluted to 1 microgram / ml (μg / ml) with PBS (-) and added to a 384-well MAXISorp plate (Nunc, Cat No. 164688). The plates were incubated at room temperature for 1 hour and blocked with a 5-fold dilution of Blocking One (Nacalai Tesque, Cat No. 03953-95). The plates were incubated for 1 hour, washed, and 2 nM of human C5 was added and incubated for 1 hour. After incubation, the dishes were washed and labeled detection antibodies (1 μg / ml (μg / mL), biotinylated by NHS-PEG4-Biotin) were added. After 1 hour incubation, ABTS (KPL, Cat No. 50-66-06) was added via biotin-horseradish peroxidase conjugate (Thermo Scientific, Cat No. 21132) to detect biotinylated antibodies Combination.

All anti-C5 antibodies are used as capture and detection antibodies and are fully paired. As shown in Figure 1, competing antibodies are classified into 7 epitope bins: CFA0668, CFA0334, and CFA0319 are classified To epitope A, CFA0647, CFA0589, CFA0341, CFA0639, CFA0635, CFA0330, and CFA0318 are classified into epitope B, CFA0538, CFA0501, CFA0599, CFA0307, CFA0366, CFA0305, CFA0675, CFA0666, and CFA0672 are classified to antigen In determinant C, eculizumab and CFA0322 are assigned to epitope D, CFA0329 is assigned to epitope E, CFA0359 and CFA0217 are assigned to epitope F, and CFA0579, CFA0328 and CFA0272 are assigned to epitope In base G. Figure 1 shows some epitope bins of the anti-C5 chimeric antibody. Table 2 lists the sequences of VH and VL of anti-C5 antibodies classified to epitope C.

2.3. Humanization and optimization

To reduce the potential immunogenicity of the antibodies, humanization of some variable regions of anti-C5 antibodies was performed. The complementarity-determining region (CDR) of the anti-C5 rabbit antibody was transplanted onto the homologous human antibody framework (FR) by a conventional CDR grafting method (Nature 321: 522-525 (1986)). Genes encoding humanized VH and VL were synthesized and recombined with modified human IgG4 CH (SG402, sequence identification number: 35) and human CL (SK1, sequence identification number: 38), and each recombination sequence The columns are transgenic in expression vectors.

Detection of some mutations and combinations of mutations to identify mutations and combinations of mutations that improve the binding characteristics of some lead antibodies. Multiple mutations are then introduced into the humanized variable region to enhance the binding affinity for C5 at neutral pH or to reduce the binding affinity for C5 at acidic pH. One of the optimized variants, 305LO5 (VH, sequence identification number: 10; VL, sequence identification number: 20; HVR-H1, sequence identification number: 54; HVR-H2, sequence identification number: 64; HVR-H3 , Sequence identification number: 74; HVR-L1, sequence identification number: 84; HVR-L2, sequence identification number: 94; and HVR-L3, sequence identification number: 104), thus generated from CFA0305.

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

[Example 3]

Anti-C5 antibody binding characteristics

3.1. Expression and purification of recombinant antibodies

The FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA) was used to express recombinant antibodies transiently. Purification was performed using protein A from conditioned medium expressing antibodies using conventional methods. Further colloidal filtration is performed if necessary.

3.2. Evaluation of pH dependence

The kinetic parameters of anti-C5 antibody to recombinant human C5 were evaluated using a BIACORE (registered trademark) T200 instrument (GE Healthcare) at 37 ° C (° C) at pH 7.4 and pH 5.8. An amine coupling kit (GE Healthcare) was used to fix ProA / G (Pierce) on the CM4 sensor wafer according to the settings recommended by GE Healthcare. The antibodies and analytes were diluted in their respective running buffers, ACES pH 7.4 and pH 5.8 (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl ₂ , 0.05% Tween 20, 0.005% NaN ₃ ). Each antibody is captured on the sensing surface by ProA / G. The degree of antibody capture is typically 60-90 resonance units (RU). Then, recombinant human C5 was injected at a concentration of 10 and 20 nM or 20 and 40 nM, followed by dissociation. The surface was regenerated with 25 mM NaOH. Using BIACORE (registered trademark) T200 evaluation software, version 2.0 (GE Healthcare) fitted the sensor map with a 1: 1 binding model to determine the kinetic parameters under two pH conditions. Panels 2A and 2B show sensory images of all antibodies. Table 3 lists the antibody's binding rate (ka), dissociation rate (kd), and binding affinity (KD). All antibodies except CFA0330 (VH, sequence identification numbers: 21 and VL, sequence identification number: 25) and A0341 (VH, sequence identification numbers: 22 and VL, sequence identification number: 26), compared to pH 7.4 , Showing a relatively fast off-rate at pH 5.8.

3.3. Cross-reactivity check

In order to observe the cross-reactivity of anti-C5 antibodies to human C5 (hc5) and rhesus C5 (cynoC5), BIACORE (registered trademark) kinetic assay was performed. Measured The setup was the same as described in Example 3.2, with recombinant cynoC5 injected at a concentration of 2, 10 and 50 nM. The kinetic parameters were determined by fitting the same data as described in Example 3.2. Table 4 lists the binding kinetics and affinity at pH 7.4. Table 4 shows the kinetic parameters for hC5 as a result of Example 3.2. All anti-C5 antibodies except CFA0672 showed similar KD for hC5 and cynoC5. Compared to hC5, the KD of CFA0672 to cynoC5 is 8 times weaker.

[Example 4]

Epitope mapping of anti-C5 antibodies

4.1. Binding of anti-C5 MAb to C5 β-chain derived peptides

The binding of the anti-C5 monoclonal antibody (MAb) to the β-chain derived peptide of C5 was tested in Western blot analysis. C5 peptides fused to GST markers (pGEX-4T-1, GE Healthcare Life Sciences, 28-9545-49) were expressed in E.coli (DH5α (DH5alpha), TOYOBO, DNA-903): 19-180, 161- 340, 321-550 and 481-660. E.coli samples were collected after incubation with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 5 hours at 37 ° C (° C) and centrifugation at 20,000 xg for 1 minute to obtain a precipitate. . With sample buffer solution (2ME +) (Wako, 191-13272) The precipitate was suspended and used for western blot analysis. The expression of each peptide was confirmed with an anti-GST antibody (Abcam, ab9085) (Figure 3). The arrow indicates the GST-fused C5 peptide (46-49 kDa). Anti-C5 MAb: CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672 and CFA0675, combined with 19-180 of C5 (Figure 3).

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

The FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA) was used to transiently express the recombinant MG1-MG2 domain of the β chain of human C5 (sequence identification number: 43). Conditioned medium expressing the MG1-MG2 domain was diluted with 1/2 volume of milliQ water, and then used in a Q-sepharose FF anion exchange column (GE healthcare, Uppsala, Sweden). The fractions flowing through the anion exchange column were adjusted to pH 5.0 and used on an SP-Sepharose HP cation exchange column (GE healthcare, Uppsala, Sweden) and eluted with a NaCl gradient. Fractions containing the MG1-MG2 domain were collected from the eluate and then applied to a Superdex 75 colloidal filtration column (GE healthcare, Uppsala, Sweden) equilibrated with 1x PBS. Fractions containing the MG1-MG2 domain were then pooled and stored at -80 ° C.

4.3. Binding ability to MG1-MG2 domain

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

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

The binding of anti-C5 Mab to the MG1-MG2 domain-derived peptide was tested in Western blot analysis. C5 peptides fused to GST markers were expressed in E. coli: 33-124, 45-124, 52-124, 33-111, 33-108, and 45-111 (sequence identification number: 40). After incubating with 1 mM IPTG at 37 ° C for 5 hours and centrifuging at 20,000 x g for 1 minute to obtain a precipitate, E. coli samples were collected. The pellet was suspended in a sample buffer solution (2ME +) and used for Western blot analysis. The expression of C5-derived peptide was confirmed with an anti-GST antibody (Figure 5A). CFA0305 only binds to 33-124 peptides (Figure 5B). CFA0305 binds to the beta chain of recombinant human C5 (rhC5) (approximately 70 kDa), which serves as a control group. Figure 5C summarizes the response of anti-C5 MAb to C5-derived peptides.

4.5. Anti-C5 MAb binding to C5 mutants

The three amino acid residues in the β (beta) chain of C5: E48, D51, and K109 were predicted by crystal structure analysis, and involved the binding between C5 and anti-C5-MAb. Analysis of C5 MAb binding to human C5 point mutants. A C5 point mutant was expressed in FS293 cells by lipid transfection (lipofection), in which any one of E48, D51, and K109 was replaced with alanine. The culture medium was collected 5 days after lipid transfection, and then used for western blotting. SDS-PAGE was performed under reducing conditions, and the results are shown in FIG. 6. Eculizumab binds to the alpha (alpha) chain of wild-type (WT) C5 and three C5 point mutants, while CFA0305 strongly binds to the beta chain of WT C5, weakly to the beta chain of E48A C5 mutant, and does not bind to The β chain of the D51A and K109A C5 mutants, which represents that these 3 amino acid residues are involved in antibody / antigen interactions. Table 5 shows the western blot analysis of anti-C5 MAb (CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675). to sum up. Anti-C5 MAb is classified into the same epitope C, but the binding patterns between antibodies are slightly different, suggesting that the binding regions of C5 anti-C5 MAb are close to each other but not the same.

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

To test that residues E48, G51, and K109 did indeed involve antibody / antigen interactions, a BIACORE (registered trademark) binding analysis was performed. Three C5 mutants were prepared: E48A, G51A and K109A, as described in Example 4.5. A sample of culture supernatant containing mutant C5 overexpressed in FS293 cells was prepared at 40 micrograms per milliliter (μg / ml) of mutant C5. For BIACORE (registered trademark) binding analysis, the samples were diluted 10-fold with BIACORE (registered trademark) running buffer (ACES pH7.4, 10 mg / ml BSA, 1 mg / ml carboxymethyldextran) to the mutant C5. The final sample concentration was 4 μg / ml (μg / ml).

Using the measurement conditions described in Example 3.2, the interaction of the three C5 mutants with the anti-C5 antibody was evaluated with a BIACORE (registered trademark) T200 instrument (GE Healthcare) at 37 ° C. As an electrophoresis buffer, an ACES pH 7.4 buffer containing 10 mg / ml BSA and 1 mg / ml carboxymethyldextran was used. Eculizumab-F760G4 and 305LO5 were captured by different mouse anti-human IgG and Fc fragment-specific antibodies (GE Healthcare) on different flow cells. Flow path 1 is used as a reference surface. Wild-type and mutant C5 proteins were injected onto 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 ₂ . Results were analyzed with Bia Evaluation software, version 2.0 (GE Healthcare). The blank injection curve of the reference flow path (flow path 1) and the running buffer was subtracted from the curve of the flow path with the capture antibody.

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

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

Crystal structure analysis showed that three histidine residues on human C5 were 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)). In order to investigate which His residue at the antibody / antigen interface contributes to the pH-dependent interaction between the anti-C5 antibody and C5, a BIACORE (registered trademark) binding assay was performed. Three human C5 mutants with a single His mutation (H70Y, H72Y, and H110Y) and mutants with a double His mutation (H70Y + H110Y) were prepared as follows: H70, H72, and H110 were expressed in FS293 cells by lipid transfection Tyrosine Single His mutants replaced, and double His mutants in which both H70 and H110 were replaced with tyrosine. The antigen-binding properties of the C5 His mutant to 305LO5, a pH-dependent anti-C5 antibody were determined by a modified BIACORE (registered trademark) assay as described in Example 4.6. In short, an additional dissociation phase at pH 5.8 was added to the BIACORE (registered trademark) measurement immediately after the pH 7.4 dissociation phase to assess the pH dependence between the antibody and the anti-source from the complex formed at pH 7.4 Sexual dissociation. The data was processed and fitted using Scrubber 2.0 (BioLogic Software) curve fitting software to determine the off-rate at pH 5.8.

As shown in Figure 8, C5 single His mutants and double His mutants (H70 + H110) at H70 or H110 did not affect the binding of C5 to 305LO5 at neutral pH. At the same time, a single His mutant in H72 showed a significant reduction in C5 binding to 305LO5. The dissociation rates of C5 His mutant and C5-wt protein at pH 5.8 are shown in Table 6. As shown in Table 6, among the C5 antigens tested, C5-wt showed the fastest dissociation from 305LO5 at pH 5.8. Compared to C5-wt, the single His mutant in H70 showed an almost twice slower off-rate at pH 5.8, and the single His mutant in H110 caused a slightly slower off-rate at pH 5.8. The double His mutants in H70 and H110 had a greater effect on pH-dependent binding, and had a dissociation rate almost three times slower than C5-wt at pH 5.8.

[Example 5]

Inhibitory activity of anti-C5 antibodies on C5 activation

5.1. Inhibition of anti-C5 MAb on lysis of complement-activated liposomes

Inhibition of complement activity against C5 MAb was tested by a liposomal cell lysis assay. 30 microliters of normal human serum (6.7%) (Biopredic, SER018) was mixed with 20 microliters ([mu] L) of diluted MAb in a 96-well plate and incubated on a shaker at 25 ° C for 30 minutes. Liposomes sensitized with antibodies against dinitrophenyl (Autokit CH50, Wako, 995-40801) were transferred to each well, and the plates were placed on a shaker at 25 ° 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 mixed solution was incubated at 37 ° C. for 40 minutes, and then the OD of the mixed solution at 340 nm was measured. The percentage of liposomal cell lysis is defined as 100x [(OD _MAb- OD _{serum and liposome background} )] / [(OD _{does not have MAb-} OD _{serum and liposome background} )]. Figure 9A shows anti-C5 Mabs: CFA 0305, 0307, 0366, 0501, 0538, 0599, 0666, 0672, and 0675, inhibiting liposome cell lysis. Two pH-independent antibodies, CFA0330 and 0341, also inhibited cell lysis (Figure 9B).

5.2. Inhibition of C5a production by anti-C5 MAb

During liposome cell lysis, C5a production was tested against C5 MAb to confirm that anti-C5 MAb inhibited C5 cleavage into C5a and C5b. The C5a ELISA kit (R & D system, DY2037) was used to quantify the degree of C5a in the supernatant from the liposomal cell lysis assay. All MAbs dose-dependently inhibit C5a production in the supernatant (Figures 10A and 10B).

5.3. Inhibition of anti-C5 MAb on complement activation hemolytic response

The inhibition of classical complement activity by anti-C5 MAb was tested in a hemolytic assay. Wash chicken red blood cells (cRBCs) with gelatin / veronal buffered saline containing 0.5 mM MgCl ₂ and 0.15 mM CaCl ₂ (GVB ++) (Boston BioProducts, IBB-300X) (Innovative research, IC05-0810), It was then sensitized with 1 microgram / ml ([mu] g / ml) anti-chicken RBC antibody (Rockland 103-4139) at 4 degrees C for 15 minutes. The cells were then washed with GVB ++ and suspended in the same buffer at 5x10 ⁷ cells / ml. In a separate round-bottomed 96-well microtest plate, 50 microliters (μl) of normal human serum (20%) (Biopredic, SER019) was mixed with 50 microliters of diluted Mab and cultured on a shaker at 37 ° C 30 minutes. 60 microliters of sensitized cRBC supernatant was then added to the wells containing serum, and the antibody mixture was incubated at 37 ° C for 30 minutes. After incubation, the discs were centrifuged at 1000 xg for 2 minutes at 4 ° C. The supernatant (100 microliters) was transferred to a well on a flat-bottomed 96-well microtest plate to measure the OD at 415 nm, which uses 630 nm as the reference wavelength. The percentage of hemolytic response is defined as 100x × [(OD _MAb- OD _{serum and cRBC} )] / [(OD _{has no MAb-} OD _{serum and cRBC background} )]. Figure 11 shows that anti-C5 Mab: CFA0305 and 305LO5 inhibit cRBC hemolytic response.

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

The hemolytic response assay of the alternative pathway is performed in a similar manner to the classical pathway hemolytic response assay. Blood collected from a New Zealand white rabbit (InVivos) was mixed with the same volume of Alsever's solution (Sigma, A3551), and the mixed solution was used as rabbit RBC (rRBC). RRBC was washed with 2 mM MgCl ₂ and 10 mM EGTA supplemented GVB, and suspended in the same buffer at ⁷ × 10 ⁸ cells / ml. In a round-bottomed 96-well microtest disc, mix 40 microliters (μl) of normal human serum (25%) (Biopredic, SER019) with 40 microliters of diluted Mab and incubate on a shaker at 37 ° C for 30 minutes . Then 20 microliters of rRBC supernatant was added to the wells containing serum, and the antibody mixture was incubated at 37 ° C for 60 minutes. After incubation, the discs were centrifuged at 1000 xg for 2 minutes at 4 ° C. The supernatant (70 microliters) was transferred to a well on a flat-bottomed 96-well microtest plate to measure the OD at 415 nm, which uses 630 nm as the reference wavelength. Figure 12 shows that anti-C5 Mab: CFA0305 and CFA0672 inhibit the hemolytic response of rRBC, which represents that these antibodies inhibit the alternative complement pathway.

[Example 6]

Pharmacokinetics of anti-C5 monoclonal antibodies and human C5 in mice

6.1. In vivo testing with C57BL / 6 mice

After administration of human C5 or human C5 and anti-human C5 antibodies to C57BL / 6 mice (In Vivos or Biological Resource Centre, Singapore) alone, the in vivo kinetics of human C5 (Calbiochem) and anti-human C5 antibodies were evaluated. Human C5 solution (0.01mg / ml) or solution containing human C5 and anti-human C5 antibodies (0.01mg / ml and 2mg / 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 to the tail vein at a dose of 10 ml / kg. In this case, anti-human C5 antibodies are present in excess compared to human C5, so it can be assumed that almost every human C5 binds 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 degrees C for 10 minutes to separate the plasma. Separated plasma is stored before measurement In a refrigerator at -80 degrees C. The anti-human C5 antibodies used are the above-mentioned CFA0305-F760G4, CFA0307-F760G4, CFA0330-F760G4, CFA0341-F760G4, CFA0366-F760G4, CFA0501-F760G4, CFA0538-F760G4, CFA0599-F760G4, CFA0666-F760G0, CFA0660 -F760G4.

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

Measurement of total human C5 concentration in mouse plasma by ECL

In the case where CFA0330-F760G4, CFA0341-F760G4 or human C5 alone was present in the plasma sample, the following method was used. The anti-human C5 antibody (Santa Cruz) was dispensed on a MULTI-ARRAY 96-well bare disk (Meso Scale Discovery) and left to stand overnight at 4 ° C to prepare a disk immobilized with anti-human C5. An antibody (CFA0330-F760G4 or CFA0341-F760G4) injected at 1 microgram / ml ([mu] g / ml) injection was prepared by diluting a calibration curve sample and mouse plasma sample 100 times or more, and cultured at 37 ° C for 30 minutes. Next, the sample was dispensed onto a plate to which anti-human C5 was fixed, and left to stand at room temperature for 1 hour. Next, a SULFO-TAG-labeled anti-human IgG antibody (Meso Scale Discovery) was added and reacted at room temperature for 1 hour, followed by washing. Immediately thereafter, Read Buffer T (x4) (Meso Scale Discovery) was allocated and measured using Sector Imager 2400 (Meso Scale Discovery).

In the case where CFA0305-F760G4, CFA0307-F760G4, CFA0366-F760G4, CFA0501-F760G4, CFA0538-F760G4, CFA0599-F760G4, CFA0666-F760G4, CFA0672-F760G4, or CFA0675-F760G4 are present in plasma samples, the following method is used. Will be anti-human C5 antibody (CFA0329-F939G4; VH, SEQ ID NO: 23 and VL, SEQ ID NO: 27) was assigned to MULTI-ARRAY 96-well bare disk (Meso Scale Discovery) and left at 4 ° C overnight to prepare fixed antibodies Human C5 disk. A calibration curve sample and a mouse plasma sample diluted 100 times or more with an acidic solution (pH 5.5) were prepared and incubated at 37 ° C for 30 minutes. Next, the sample was dispensed onto a plate to which anti-human C5 was fixed, and left to stand at room temperature for 1 hour. Next, a 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).

Based on the response of the calibration curve, the human C5 concentration was calculated using the analysis software SOFTmax PRO (Molecular Devices). The time course of plasma human C5 concentration after intravenous administration by this method is shown in FIG. 13. Data are plotted as percentage 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. Distribute anti-human IgG (gamma) -chain specific) F (ab ') 2 antibody fragments (Sigma) or anti-human IgG kappa chain antibodies (Antibody Solutions) to MULTI-ARRAY 96-well bare plates (Meso Scale Discovery) and allowed to stand overnight at 4 ° C to prepare anti-human IgG-immobilized dishes. Prepare calibration curve samples and mouse plasma samples diluted 100-fold or more. Next, the sample was dispensed onto an anti-human IgG-immobilized plate, and allowed to stand at room temperature for 1 hour. Next, add biotinylated anti-human IgG antibody (Southernbiotech) or SULFO-TAG The recorded anti-human IgG Fc antibody (Southernbiotech) was reacted at room temperature for 1 hour and washed. Next, only when a biotinylated anti-human IgG antibody is used, SULFO-TAG-labeled streptavidin is added and reacted 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 anti-human C5 concentration was calculated from the response of the calibration curve using the analysis software SOFTmax PRO (Molecular Devices). The time course of the plasma anti-human C5 antibody concentration after intravenous administration by this method is shown in FIG. 14. Data are plotted as percentage remaining compared to anti-human C5 antibody concentration at 5 minutes.

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

Test pH-dependent anti-human C5 antibodies in vivo (CFA0305-F760G4, CFA0307-F760G4, CFA0366-F760G4, CFA0501-F760G4, CFA0538-F760G4, CFA0599-F760G4, CFA0666-F760G4, CFA0672-F760G4 and CFA0675-F7604) Dependent anti-human C5 antibodies (CFA0330-F760G4 and CFA0341-F760G4), and the plasma anti-human C5 antibody concentration and plasma human C5 concentration were compared. As shown in Figure 14, antibody exposures are comparable. In the meantime, the elimination of human C5 with the simultaneous administration of pH-dependent anti-human C5 antibodies was accelerated compared to pH-free anti-human C5 antibodies (Figure 13).

[Example 7]

Optimization of anti-C5 monoclonal antibodies (305 variants)

Introduction of some mutations to the 305LO5 optimized variable region of the anti-C5 antibody In order to further improve its characteristics and generate optimized variable regions 305LO15, 305LO16, 305LO18, 305LO19, 305LO20, 305LO22, and 305LO23. The amino acid sequences of VH and VL of the 305 variant are listed in Tables 7 and 8, respectively. . Gene encoding humanized VH and modified human IgG1 CH variant SG115 (sequence identification number: 114) and modified human IgG4 CH variant SG422 (sequence identification number: 115) or SG429 (sequence identification number: 116) Combined. The gene encoding the humanized VL binds to human CL (SK1, sequence identification number: 38). The heavy and light chain genes encoding humanized anti-C5 antibodies, BNJ441 (BNJ441H, sequence identification number: 149; BNJ441L, sequence identification number: 150), were synthesized separately, and each was transfected into an expression vector.

Antibodies were expressed in HEK239 cells and purified by protein A, which was co-transfected with a combination of heavy and light chain expression vectors.

[Example 8]

Binding properties of anti-C5 antibody (305 variant)

Using BIACORE (registered trademark) T200 equipment (GE Healthcare) at 37 degrees C to evaluate the kinetic parameters of anti-C5 antibodies against recombinant human C5 under three different conditions; (1) both binding and dissociation are at pH 7.4, (2) Binding and dissociation were both at pH 5.8 and (3) Binding was at pH 7.4, but dissociation was at pH 5.8. An amine coupling kit (GE Healthcare) was used to fix ProA / G (Pierce) on the CM1 sensor chip according to the settings recommended by GE Healthcare. Dilute the antibodies and analytes of conditions (1) and (3) in ACES pH7.4 buffer (20mM ACES, 150mM NaCl, 1.2mM 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 ). Each antibody is captured on the sensing surface by ProA / G. The degree of antibody capture is typically 60-90 resonance units (RU). Then, recombinant human C5 was injected at 3 to 27 nM or 13.3 to 120 nM, which was prepared by three-fold serial dilution, followed by dissociation. The surface was regenerated with 25 mM NaOH. Using BIACORE (registered trademark) T200 evaluation software, version 2.0 (GE Healthcare) fitted the sensing map with a 1: 1 combination model to determine the kinetic parameters under conditions (1) and (2), and MCK A 1: 1 dissociation fit model of the model was used to determine the dissociation rate under condition (3). The pH dependence of all antibodies is expressed as the ratio of the dissociation rate under conditions (2) and (1).

Binding rate (ka), dissociation rate (kd), binding affinity (KD), and pH dependence are listed in Table 9. Compared to pH 7.4, all antibodies showed a faster off-rate at pH 5.8, and their pH dependence was about 20 times.

The binding affinity of anti-C5 antibodies (BNJ441, eculizumab, and 305 variants) to recombinant human C5 at pH 7.4 and pH 5.8 was measured at 37 degrees C using BIACORE (registered trademark) T200 equipment (GE Healthcare) to evaluate Effect of pH on antigen binding. Goat's anti-human IgG (Fc) using an amine coupling kit (GE Healthcare) according to the supplier's recommended settings Multiple antibodies (KPL # 01-10-20) were immobilized on the CM4 sensor wafer. The antibodies and analytes were diluted in ACES pH7.4 buffer or ACES pH5.8 buffer containing 20mM ACES, 150mM NaCl, 1.2mM CaCl2, 0.05% Tween 20, and 0.005% NaN3. The anti-Fc method is used to capture the antibody on the surface of the sensing wafer, and the capture degree is usually 50-80 resonance units (RU). Recombinant human C5 was prepared by three-fold serial dilution starting from 27 nM under the measurement conditions of pH 7.4, or by three-fold serial dilution starting from 135 nM under the measurement conditions of pH 5.8. The surface was regenerated with 20 mM HCl, 0.01% Tween 20. Data were processed and fitted using a BiaEvaluation 2.0 software (GE Healthcare) in a 1: 1 combination model.

The binding affinity (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 that the ratio of (KD at pH 5.8) / (KD at pH 7.4) was about 800, which was 8 times higher than BNJ441, and the ratio of (KD at pH 5.8) / (KD at pH 7.4) was only 93.

[Example 9]

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

9.1. Inhibition of anti-C5 MAb on lysis of complement-activated liposomes

Inhibition of complement activity against C5 MAb was tested by a liposomal cell lysis assay. 30 microliters of normal human serum (6.7%) (Biopredic, SER019) was mixed with 20 microliters of diluted MAb in a 96-well plate and incubated on a shaker for 30 minutes at room temperature. A liposome solution 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 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, after which the OD at 340 nm was measured. The percentage of liposomal cell lysis is defined as 100x [(OD _MAb- OD _{serum and liposome background} )] / [(OD _{does not have MAb-} OD _{serum and liposome background} )]. Figure 15 shows anti-C5 Mabs: 305LO15-SG422, 305LO16-SG422, 305LO18-SG422, 305LO19-SG422, 305LO20-SG422, and 305LO20-SG115, inhibiting liposomal cell lysis. Two antibodies with Fc variants: 305LO15-SG115 and 305LO23-SG429 also inhibited liposomal cell lysis (Figure 16).

The anti-C5 MAb was tested for inhibition of recombinant human C5 (sequence identification number: 39). In a 96-well plate, 10 μl of human serum lacking C5 (Sigma, C1163) was mixed with 20 μl of diluted MAb and 20 μl of recombinant C5 (0.1 μg / mL (μg / mL)) and Incubate at 37 ° C for 1 hour 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, after which the OD at 340 nm was measured. The percentage of liposomal cell lysis is as defined above. Figure 17 shows anti-C5 Mabs: 305LO22-SG115, 305LO22-SG422, 305LO23-SG115, and 305LO23-SG422, inhibiting liposomal cell lysis.

9.2. Inhibition of C5a production by anti-C5 MAb

During liposomal cell lysis; C5a production was tested against C5 MAb to confirm that anti-C5 MAb inhibited C5 cleavage into C5a and C5b. The C5a ELISA kit (R & D system, DY2037) was used to quantify the degree of C5a in the supernatant from the liposomal cell lysis assay. All MAbs inhibited C5a production in the supernatant in a dose-dependent manner (Figures 18 and 19).

9.3. Measurement of complement activity in rhesus monkey plasma

Inhibition of complement activity by anti-C5 MAb was measured in rhesus monkey plasma. Anti-C5 Mab was administered internally to monkeys (20 mg / kg), and plasma samples were collected periodically until day 56. Wash chicken red blood cells (cRBCs) with gelatin / veronal buffered saline containing 0.5 mM MgCl ₂ and 0.15 mM CaCl ₂ (GVB ++) (Boston BioProducts, IBB-300X) (Innovative research, IC05-0810), It was then sensitized with 1 microgram / ml (μg / ml) anti-chicken RBC antibody (Rockland 103-4139) at 4 ° C for 15 minutes. The cells were then washed with GVB ++ and suspended in the same buffer at 1 × 10 ⁸ cells / ml. Monkey plasma was incubated with sensitized cRBC at 37 ° C for 20 minutes in a separate round-bottomed 96-well microtest plate. After incubation, the discs were centrifuged at 1000 xg for 2 minutes at 4 ° C. The supernatant was transferred to a well on a flat-bottomed 96-well micro-test disc to measure the OD at 415 nm, which uses 630 nm as the reference wavelength. The percentage of hemolytic response is defined as 100x [( _post- OD _plasma- OD _{plasma and cRBC background} )] / [( _pre- OD _plasma- OD _{plasma and cRBC background} )]]. Figure 20 shows that anti-C5 Mab: 305LO15-SG422, 305LO15-SG115, 305LO16-SG422, 305LO18-SG422, 305LO19-SG422, 305LO20-SG422, 305LO20-SG115, and 305LO23-SG115 inhibit complement activity in plasma.

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

The anti-C5 MAb was tested for inhibition of recombinant human C5 variants: V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N, and E1437D. PNH patients with R885H mutations in C5 have been reported to respond poorly to eculizumab (see, for example, Nishimura et al., New Engl. J. Med. 370: 632-639 (2014)). Individual human C5 variants were expressed in FS293 cells, and the supernatant was used for subsequent studies. In a 96-well plate, 10 μl of human serum lacking C5 (Sigma, C1163) with 20 μl of diluted Mab and 20 μl of recombinant C5 variants (2-3 μg / ml (μg / mL)) The cell culture medium was mixed and cultured 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, after which the OD at 340 nm was measured. The percentage of liposomal cell lysis is as defined above. Figure 21 shows that the anti-C5 MAb (eculizumab) did not inhibit the R885H C5 variant, but suppressed other tested variants. Figure 22 shows all C5 variants tested against C5 Mab (305 variant) inhibition.

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

The inhibition of complement activity by anti-C5 Mab was tested by a liposomal cell lysis assay. 30 microliters of normal human serum (6.7%) (Biopredic, SER019) was mixed with 20 microliters of diluted MAb in a 96-well plate and incubated on a shaker at room temperature for 30 minutes. A liposome solution 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, after which the OD at 340 nm was measured. The percentage of liposomal cell lysis inhibition was defined as 100x [(OD _MAb- OD _{serum and liposome background} )] / [(OD _{has no MAb-} OD _{serum and liposome background} )]. Figure 13 shows anti-C5 Mabs: 305LO15-SG422, 305LO16-SG422, 305LO18-SG422, 305LO19-SG422, 305LO20-SG422, and 305LO20-SG115, inhibiting liposomal cell lysis. Figure 23 shows that the anti-C5 MAb, BNJ441 and 305 variants inhibit liposomal cell lysis, and the 305 variant has a stronger inhibitory activity than BNJ441.

[Example 10]

Pharmacokinetics of anti-C5 monoclonal antibody (305 variant) in rhesus monkeys

10.1. In vivo testing with rhesus monkeys

After administration of an anti-human C5 antibody to a rhesus monkey (Shin Nippon Biomedical Laboratories, Ltd., Japan), the in vivo kinetics of the anti-human C5 antibody was evaluated. An anti-human C5 antibody solution (2.5 mg / ml) was administered to the cephalic vein of the forearm at a dose of about 8 ml / kg at a time by 30 minutes infusion. Before administration (pre-administration) and after 5 minutes, 7 hours, 1 day, 2 days, 3 days, 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, and 56 days of administration Collect blood. The collected blood was immediately centrifuged at 1,700xg and 4 ° C for 10 minutes to separate the plasma. The separated plasma was stored in a refrigerator at -70 ° C or below before the measurement was performed. The anti-human C5 antibody was prepared as described in Example 7.

10.2. Measurement of Total Rhesus C5 Plasma Concentration by ELISA Analysis

The concentration of total rhesus C5 in rhesus monkey plasma was measured by ELISA. The anti-human C5 antibody (in-house antibody produced by the method described in Example 2) was distributed on Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International), and left to stand at 4 ° C overnight to prepare an immobilized antibody. Plate of river monkey C5. A calibration curve sample and a rhesus monkey plasma sample diluted at 20,000 times with 0.4 μg / ml (μg / ml) injected antibody were prepared and cultured at 37 ° C for 60 minutes. Next, the sample was dispensed onto a plate to which anti-rhesus C5 was immobilized, and allowed to stand at room temperature for 1 hour. Next, an HRP-labeled anti-human IgG antibody (Southernbiotech) was added and reacted at room temperature for 30 minutes, followed by washing. Next, ABTS ELISA HRP Substrate (KPL) was added. The signal was measured at a wavelength of 405 nm by a plate reader. Based on the response of the calibration curve, the anti-rhesus C5 concentration was calculated using the analysis software SOFTmax PRO (Molecular Devices). The time course of plasma rhesus C5 concentration after intravenous administration was measured by this method, as shown in FIG. 24. Data are plotted as percentage remaining compared to plasma rhesus C5 concentration before administration. Compared to pH-independent anti-human C5 antibodies, pH-dependent anti-human C5 antibodies (305LO15-SG422, 305LO15-SG115, 305LO16-SG422, 305LO18-SG422, 305LO19-SG422, 305LO20-SG422, 305LO20-SG115, 305LO22- SG422, 305LO23-SG422, and 305LO23-SG115) show 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 rhesus plasma was measured by ELISA. The anti-human IgG kappa chain antibody (Antibody Solutions) was dispensed on unc-ImmunoPlate MaxiSorp (Nalge Nunc International) and left to stand overnight at 4 ° C to prepare an anti-human IgG-immobilized plate. Preparation diluted 100 times or More calibration curve samples and rhesus monkey plasma samples. Next, the sample was dispensed onto an anti-human IgG-immobilized plate, and allowed to stand at room temperature for 1 hour. Next, an HRP-labeled anti-human IgG antibody (Southernbiotech) was added and reacted at room temperature for 30 minutes, followed by washing. Next, ABTS ELISA HRP Substrate (KPL) was added. The signal was measured by a disk reader at a wavelength of 405 nm. Based on the response of the calibration curve, the analysis software SOFTmax PRO (Molecular Devices) was used to calculate the anti-human C5 antibody concentration. The time course of the plasma anti-human C5 antibody concentration after intravenous administration by this method is shown in FIG. 25. Compared to pH-independent anti-human C5 antibodies, pH-dependent anti-human C5 antibodies (305LO15-SG422, 305LO15-SG115, 305LO16-SG422, 305LO18-SG422, 305LO19-SG422, 305LO20-SG422, 305LO20-SG115, 305LO22- SG422, 305LO23-SG422, and 305LO23-SG115) show longer half-life.

[Example 11]

X-Ray Crystal Structure Analysis of 305 Mutant 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), expression in E.coli strain BL21 DE3 pLysS (Promega) was fused to a GST-tagged MG1 domain (those sequence identification number: 39 amino acid residues 20-124). Protein expression was induced with 0.1 Mm of isopropyl β (beta) -D-1-thiogalactopyranoside (IPTG) at 25 ° C for 5 hours. Bacterial cell pellets were lysed with Bugbuster (Merck) supplemented with a mixture of lysonase (Merck) and a complete protease inhibitor, followed by purification of GST-MG1 from the soluble fraction using a GSTrap column (GE healthcare) according to the supplier's instructions. Yi Ning The GST tag was cleaved by blood enzyme (Sigma), and the resulting MG1 domain was further purified on a Superdex 75 colloidal filtration column (GE healthcare). The fractions containing the MG1 domain were collected and stored at -80 ° C.

11.2. Preparation of 305 variant Fab fragments

Fab fragments prepared from one of the 305 optimized variants were prepared by conventional methods using papain (Roche Diagnostics, Cat No. 1047825), and subsequently loaded on a protein A column (MabSlect SuRe, GE Healthcare) to remove Fc Fragments, cation exchange columns (HiTrap SP HP, GE Healthcare) and colloidal filtration columns (Superdex 200 16/60, GE Healthcare). Fractions containing Fab fragments were collected and stored at -80 ° C.

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

The purified recombinant human C5-MG1 domain was mixed with the purified 305 mutant Fab fragment in a 1: 1 molar ratio. The complex was purified by colloidal filtration chromatography (Superdex 200 10/300 increase, GE Healthcare) using a column equilibrated with 25 mM HEPES pH 7.5 and 100 mM NaCl.

11.4. Crystallization

The purified complex was concentrated to about 10 mg / Ml and crystallized at 4 ° C by a sitting drop vapor diffusion method in combination with a seeding method. The storage solution consisted of 0.2M dehydrated magnesium formate and 15.0% w / v polyethylene glycol 3350. This successfully produced disc-like crystals within a few days. The crystals were immersed in a solution of 0.2M dehydrated magnesium formate, 25.0% w / v polyethylene glycol 3350, and 20% glycerol.

11.5. Data Collection and Structure Determination

X-ray diffraction data were measured by BL32XU at SPring-8. During the measurement, the crystals were continuously placed in a nitrogen flow at -178 degrees C to maintain the frozen state, and the MX-225HS CCD detector (RAYONIX) attached to the rays was used to rotate the crystals by 1.0 degrees each time to collect A total of 180 X-ray diffraction images. Use 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) ) Judgment of cell parameters, indexing of diffraction points, and processing of diffraction data from the obtained diffraction images, finally obtain diffraction intensity data with a resolution of 2.11 Angstroms (Å). The crystal data statistics are shown in Table 11.

a; R _merge = Σ hkl Σ j | Ij ( hkl )-< I ( hkl )> | / Σ hkl Σ j | I ( hkl ) |, where Ij ( hkl ) and < I ( hkl ) are measured respectively Intensity j and the average intensity reflected by the hkl index.

b; R factor = Σ hkl | F _calc ( hkl ) |-| F _obs ( hkl ) | / Σ hkl | F _obs ( hkl ) |, where F _obs and F _calc are observed and calculated structural factor amplitudes, respectively.

c; R _free is calculated by 5% random reserved reflection.

The structure was determined by molecular substitution using 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 derived from the published human C5 crystal structure (PDB code: 3CU7, Nat. Immunol. 9: 753-760 ( 2008)). The Coot program (Acta Cryst.D66: 486-501 (2010)) was used to build the model, and the program Refmac5 (Acta Cryst.D67: 355-367 (2011)) was used to improve the model. The diffraction intensity data has a crystal confidence factor (R) of 20.42% at 25-2.11 Angstroms (Å), with a Free R value of 26.44%. Structure improvement statistics are shown in Table 11.

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

The Fab fragment ("305 Fab") from the 305 optimized variant binds to the human C5-MG1 domain ("MG1") in a 1: 1 ratio, and the asymmetric unit of the crystal structure contains 2 complexes, molecules 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 diagram discussed below was prepared using molecule 1.

In Figures 27A and 27B, the epitope of the 305 Fab contact region is located in the MG1 amino acid sequence and crystal structure, respectively. The epitope comprises the amino acid residue of MG1, which contains one or more atoms within 4.5 Angstroms (Å) from 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

As described in Examples 4.5 and 4.6, Western blot and BIACORE (registered trademark) binding analysis were used to test anti-C5 Mab containing 305 antibody series against three Binding of human C5 point mutants E48, D51 and K109. Although the 305 mutants strongly bound 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 within 3.0 angstroms (Å) from the 305 Fab, and they form several hydrogen bonds with the Fab, as shown in Figure 28A. Show. In more detailed examination, the K109 residue of MG1 is buried in the groove formed at the interface of the heavy chain of the Fab, and through three hydrogen bonds H-CDR3_G97, H-CDR3_Y100, and H-CDR3_T100b and through the salt bridge H-CDR3_D95 interacts closely with Fab (Figure 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 (Figure 28C). These K109 and D51, which represent 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, which suggests that it contributes less to antibody binding than K109 and E51 (Figure 28B). These relationships are consistent with the results of the Western blot and the BIACORE (registered trademark) combination analysis of human C5 mutants (Examples 4.5 and 4.6). Supplementary note: The numbering of Fab amino acids is based on the Kabat numbering scheme (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

11.8. Interactions of H70, H72 and H110 of the human C5 and 305 antibody series

Crystal structure analysis revealed that the three histidine residues on human C5, namely H70, H72, and H110, are contained in the epitope of the 305 mutant Fab, as shown in Figures 27A and 29A. Implementation of BIACORE (registered trademark) analysis To study the contribution of these histidine residues to the pH-dependent protein-protein interactions between human C5 and 305 mutant Fabs using human C5 mutants H70Y, H72Y, H110Y, and H70Y + H110Y (Example 4.7). H72Y caused a complete loss of C5 binding to the 305 mutant Fab. This residue of C5 is located in a pocket formed by the CDR2 loop of the 305 Fab heavy chain 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 tolerable, and because there is not enough space to accommodate the larger side chain of tyrosine, the hydrogen bond with H-CDR2_Y58 cannot be maintained. Regarding the contribution of H70 and H110 to pH dependence, the H70Y and H110Y mutations resulted in a slower dissociation of the 305 mutant Fab from C5 at pH 5.8. H70 forms an intramolecular hydrogen bond with T53 of MG1, which is thought to be disrupted at pH 5.8, when the protonation of H70 of C5 results in a conformational change in the corresponding part of the MG1 interaction interface (Figure 29B). Regarding H110, the protonation of this C5 residue is expected to cause a mutually exclusive charge to the 305 Fab, which can be enhanced by the protonation of the adjacent histidine residue H-CDR3_H100c (Figure 29D).

Although the foregoing invention has been described in detail by way of illustration and examples for the purpose of clear understanding, this description and examples should not be construed as limiting the scope of the invention. The contents of all patents and scientific literature cited herein are expressly incorporated herein by reference in their entirety.

<110> Sino-Foreign Pharmaceutical Co., Ltd.

<120> Anti-C5 antibodies and methods of using them

<130> C1-A1606-TW

<150> JP 2016-120325

<151> 2016-06-17

<160> 150

<170> PatentIn version 3.5

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<210> 137

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400> 137

<210> 138

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400> 138

<210> 139

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400> 139

<210> 140

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400> 140

<210> 141

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400> 141

<210> 142

<211> 23

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400> 142

<210> 143

<211> 23

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400> 143

<210> 144

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400> 144

<210> 145

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400> 145

<210> 146

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400> 146

<210> 147

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400> 147

<210> 148

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400> 148

<210> 149

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400> 149

<210> 150

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400> 150

Claims (4)

An application of an anti-C5 antibody, which is used to manufacture a pharmaceutical formula for treating a disease or condition involving excessive or uncontrolled C5 activation, wherein the antibody includes: (a) a sequence identification number: the VH sequence of 106 And sequence identification number: 111 VL sequence; (b) sequence identification number: 107 VH sequence and sequence identification number: 111 VL sequence; (c) sequence identification number: 108 VH sequence and sequence identification number: 111 VL (D) sequence identification number: VH sequence of 109 and sequence identification number: 111 VL sequence; (e) sequence identification number: 109 of VH sequence and sequence identification number: 112 VL sequence; (f) sequence identification number : VH sequence of 109 and sequence identification number: 113 VL sequence; or (g) sequence identification number: 110 of VH sequence and sequence identification number: 113 VL sequence. An application of an anti-C5 antibody, which is used for manufacturing a pharmaceutical formula for enhancing C5 elimination from plasma, wherein the antibody includes: (a) a sequence identification number: 106 of the VH sequence and a sequence identification number: 111 of the VL sequence; ( b) sequence identification number: 107 VH sequence and sequence identification number: 111 VL sequence; (c) Sequence identification number: VH sequence of 108 and sequence identification number: VL sequence of 111; (d) Sequence identification number: VH sequence of 109 and sequence identification number: VL sequence of 111; (e) Sequence identification number: 109 VH sequence and sequence identification number: 112 VL sequence; (f) sequence identification number: 109 VH sequence and sequence identification number: 113 VL sequence; or (g) sequence identification number: 110 VH sequence and sequence identification number : VL sequence of 113. The use as described in claim 1 or 2, wherein the antibody includes a heavy chain constant region comprising an amino acid sequence of sequence identification number: 114, and a light chain constant region comprising a sequence identification number: 38 Amino acid sequence. The use according to item 1 or 2 of the scope of the patent application, wherein the antibody is a full-length IgG1 antibody or a full-length IgG4 antibody.