EA041632B1 - ANTIBODIES TO C5 AND METHODS FOR THEIR APPLICATION - Google Patents

Description

The field of technology to which the invention belongs

The present invention relates to antibodies to C5 and methods for their use.

Prerequisites for the creation of the invention

The complement system plays a central role in immune complex clearance and immune responses to infectious agents, foreign antigens, virus-infected cells, and tumor cells. Approximately 25-30 complement proteins are known to exist as a complex group of plasma proteins and membrane cofactors. Complement components exercise their immune protective functions by participating in a series of complex enzymatic cleavages and membrane binding events. As a result, the cascades of the complement system lead to the production of products with opsonic, immunoregulatory and lytic functions.

It is now generally accepted that the complement system can be activated in three different ways: the classical pathway, the lectin pathway, and the alternative pathway. There are many components involved in these pathways, and although they differ in their initial stages, they then converge and include the same terminal components of the complement system (C5 to C9), which are responsible for the activation and destruction of target cells.

The classical pathway is usually activated by the formation of antigen-antibody complexes. Regardless, the first step in the activation of the lectin pathway is the binding of specific lectins such as mannan-binding lectin (MBL), H-ficolin, M-ficolin, L-ficolin, and C-type lectin CL-11. In contrast, the alternative pathway undergoes spontaneous activation with low levels of turnover, which can easily rise on foreign or other abnormal surfaces (bacteria, yeast, virus-infected cells, or damaged tissue). These pathways converge at a point where the complement component C3 is cleaved by an active protease to form C3a and C3b.

C3a is an anaphylatoxin. C3b binds to bacterial and other cells, as well as certain viruses and immune complexes, and marks them for removal from the bloodstream (plays the role of an opsonin). C3b also forms a complex with other components to form C5 convertase, which cleaves C5 to form C5a and C5b.

C5 is a protein with a molecular weight of 190 kDa, which is present in the serum of a healthy individual at a concentration of approximately 80 μg/ml (0.4 μm). C5 is glycosylated, with about 1.5-3% of its mass being carbohydrate. Mature C5 is a heterodimer in which the 115 kDa alpha chain is disulfide-linked to the 75 kDa beta chain. C5 is synthesized as a single chain precursor protein (pro-C5 precursor) consisting of 1676 amino acids (see, for example, PTL 1 and PTL 2). The pro-C5 precursor is cleaved to release the beta chain as an amino-terminal fragment and the alpha chain as a carboxy-terminal fragment. Polypeptide fragments, representing the alpha chain and the beta chain, are connected to each other through a disulfide bond to form the mature C5 protein.

Mature C5 is cleaved to form the C5a and C5b fragments during the activation of the pathways of the complement system. As a result of the action of C5 convertase, C5a is cleaved from the C5 alpha chain in the form of an amino-terminal fragment containing the first 74 amino acids of the alpha chain. The remainder of the mature C5 is a C5b fragment which contains an alpha chain residue linked by a disulfide to the beta chain. Approximately 20% of the mass of C5a, which is 11 kDa, is accounted for by carbohydrate.

C5a is another anaphylatoxin. C5b combines with C6, C7, C8 and C9 to form a membrane attack complex (MAC, C5b-9, Terminal Complement Component Complex (TCC)) on the surface of the target cell. When a sufficient amount of MAC is incorporated into the membranes of target cells, the pores formed by the MAC mediate rapid osmotic lysis of the target cells.

As stated above, C3a and C5a are anaphylatoxins. They can trigger mast cell degranulation, which causes the release of histamine and other inflammatory mediators, which lead to smooth muscle contraction, increased vascular permeability, leukocyte activation, and other manifestations of inflammation, including cell proliferation leading to increased parenchymal cell counts. C5a also functions as a chemotactic peptide that serves to recruit granulocytes such as neutrophils, eosinophils, basophils, and monocytes to the site of complement activation.

C5a activity is regulated by the plasma enzyme carboxypeptidase N, which removes the carboxy-terminal arginine from C5a, resulting in the formation of the C5a-des-Arg derivative. C5a-des-Arg has only 1% of the anaphylactic activity and polymorphonuclear chemotactic activity inherent in unmodified C5a.

While a properly functioning complement system provides effective protection against infecting organisms, misregulation or activation of the complement system has been implicated in the pathogenesis of various disorders including, for example, rheumatoid arthritis (RA); lupus nephritis; damage caused by ischemia-reperfusion; paroxysmal nocturnal hemoglobi

- 1 041632 nuria (PNH); atypical hemolytic uremic syndrome (aHUS); dense deposit disease (DDD); macular degeneration (eg age-related macular degeneration (AMD)); syndrome: hemolysis, increased activity of liver enzymes and thrombocytopenia (HELLP); thrombotic thrombocytopenic purpura (TTP); spontaneous loss of the fetus; vasculitis with hypocomplementemia; bullous epidermolysis; repeated loss of the fetus; multiple sclerosis (MS); traumatic brain injury and damage due to myocardial infarction, cardiopulmonary bypass and hemodialysis (see, for example, NPL 1). Thus, inhibition of excessive or uncontrolled activation of the complement cascade may have a beneficial clinical effect on patients with these disorders.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare blood disorder in which red blood cells are defective and therefore destroyed faster than healthy red blood cells. PNH is caused by clonal expansion of hematopoietic stem cells with somatic mutations in the PIG-A (class A phosphatidylinositolglycan) gene located on the X chromosome. Mutations in PIG-A result in early blockade of the synthesis of glycosylphosphatidylinositol (GPI), a molecule that is required to anchor many proteins to cell surfaces. As a result, in PNH, blood cells are deficient in GPI-anchored proteins, which include the complement-regulating proteins CD55 and CD59. Under normal circumstances, these complement-regulating proteins block the formation of MAC on cell surfaces, thereby preventing erythrocyte lysis. The absence of GPI-anchored proteins causes complement-mediated hemolysis in PNH.

PNH is characterized by hemolytic anemia (low red blood cell count), hemoglobinuria (the presence of hemoglobin in the urine, especially after sleep), and hemoglobinemia (the presence of hemoglobin in the bloodstream). Paroxysms are known to occur in PNH-affected individuals, which in this case are instances of dark-colored urine. Hemolytic anemia is caused by intravascular destruction of red blood cells by components of the complement system. Other known symptoms include dysplasia, fatigue, erectile dysfunction, thrombosis, and recurring abdominal pain.

Eculizumab is a humanized anti-complement C5 monoclonal antibody and is the first-line therapeutic agent approved for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS) (see e.g. NPL 2). Eculizumab inhibits the cleavage of C5 by C5 convertase with the formation of C5a and C5b, which prevents the formation of the complex of the terminal components of the C5b-9 complement system. Both C5a and C5b-9 induce terminal complex mediated events characteristic of PNH and aHUS (see also PTL 3, PTL 4, PTL 5 and PTL 6).

Several publications describe antibodies to C5. For example, PTL 7 describes an anti-C5 antibody that binds to the C5 alpha chain but does not bind to C5a and blocks C5 activation, 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 a proteolytic C5 convertase cleavage site on the C5 alpha chain and inhibits the conversion of C5 to C5a and C5b. PTL 10 describes an anti-C5 antibody that has an affinity constant of at least 1x10 ⁷ M' ¹ .

Antibodies (IgG) bind to the neonatal Fc receptor (FcRn) and are characterized by long plasma retention times. IgG binding to FcRn typically occurs under acidic conditions (eg, pH 6.0) and rarely occurs under neutral conditions (eg, pH 7.4). Typically, IgGs are non-specifically incorporated into cells via endocytosis and returned to cell surfaces via binding to endosomal FcRn under acidic conditions in endosomes. The IgGs are then separated from the FcRn by dissociation under neutral conditions in the plasma. IgGs that fail to bind to FcRn are cleaved in lysosomes. If the ability of IgG to bind to FcRn under acidic conditions is eliminated by the introduction of mutations in its Fc region, then IgG does not return from endosomes to plasma, which leads to a marked decrease in plasma IgG retention. Published a method to increase its binding to FcRn under acidic conditions, designed to increase the retention of IgG in plasma. When IgG binding to FcRn under acidic conditions is increased by introducing an amino acid substitution into its Fc region, IgG is more efficiently returned from endosomes to plasma, and as a result, its plasma retention is increased. In addition, data have also been published that IgG with increased ability to bind to FcRn under neutral conditions is not separated by dissociation from FcRn under neutral conditions in plasma, even if it returns to the cell surface due to its binding to FcRn under acidic conditions in endosomes. , and hence its plasma retention does not change or rather worsens (see, for example, NPL 3; NPL 4; NPL 5).

In recent years, antibodies have been described that bind to antigens in a pH dependent manner (see, for example, PTL 11 and PTL 12). These antibodies bind more strongly to antigens under neutral conditions in plasma and are separated by dissociation from antigens under acidic conditions that occur in endosomes. After separation by dissociation from antigens, antibodies regain the ability to bind to antigens when they are returned to plasma via FcRn. Thus, one antibody molecule can re-associate with several antigen molecules.

- 2 041632

In general, the plasma retention time of an antigen is much shorter than that of an antibody that has the above FcRn-mediated recycling mechanism. Thus, when an antigen is bound to an antibody, the antigen normally has a prolonged plasma retention, resulting in an increase in the plasma concentration of the antigen. On the other hand, it has been found that the antibodies described above, which bind to antigens in a pH-dependent manner, allow the elimination of antigens from plasma faster than typical antibodies, since they are separated by dissociation from antigens in endosomes during FcRn-mediated recycling. PTL 13 also describes a computer simulation based assay demonstrating that an anti-C5 antibody having pH dependent binding can enhance antigen knockout.

List of links.

Patent Literature.

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

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

PTL 3: WO 2005/074607,

PTL4: WO 2007/106585,

PTL 5: WO 2008/069889,

PTL6: WO 2010/054403,

PTL 7: WO 95/29697,

PTL 8: WO 02/30985,

PTL9: WO 2004/007553,

PTL 10: WO 2010/015608,

PTL I: WO 2009/125825, PTL 12: WO 2011/122011, PTL 13: WO 2011/111007.

non-patent literature.

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

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

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

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

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

Brief summary of the invention

The technical problem underlying the present invention.

The aim of the present invention was to develop antibodies to C5 and methods for their use.

The solution of the problem.

In some embodiments, an isolated anti-C5 antibody of the present invention binds to an epitope on the C5 beta chain. In some embodiments, an isolated anti-C5 antibody of the present invention binds to an epitope in the MG1-MG2 domain of the C5 beta chain. In some embodiments, an isolated anti-C5 antibody of the present invention binds to an epitope within amino acids 33-124 of the C5 beta chain (SEQ ID NO: 40). In some embodiments, an isolated anti-C5 antibody of the present invention binds to an epitope in the beta chain (SEQ ID NO: 40) of C5 that contains at least one fragment selected from the group consisting of amino acids 47-57, 70-76 and 107-110. In some embodiments, an isolated anti-C5 antibody of the present invention binds to an epitope in the beta chain fragment (SEQ ID NO: 40) of C5 that contains at least one amino acid residue selected from the group consisting of Glu48, Asp51 , His70, His72, Lys109 and His110 SEQ ID NO: 40. In other embodiments, the antibody binds to C5 with higher affinity at neutral pH than at acidic pH. In other embodiments, the antibody binds to C5 with higher affinity at pH 7.4 than at pH 5.8. In another embodiment, the isolated anti-C5 antibody of the present invention binds to the same epitope as the antibody described in Table 1. 2. In other embodiments of the invention, the antibody binds to the same epitope as the antibody described in table. 2 with higher affinity at pH 7.4 than at pH 5.8. In a further embodiment, an anti-C5 antibody of the present invention binds to the same epitope as the antibody described in Table 1. 7 or 8. In other embodiments of the invention, the antibody binds the same epitope as the antibody described in table. 7 or 8, with higher affinity at pH 7.4 than at pH 5.8.

- 3 041632

In some embodiments, an anti-C5 antibody of the present invention competes for binding to C5 with an antibody comprising a VH and VL pair selected from (a) a VH having SEQ ID NO: 1 and a VL having SEQ ID NO : eleven;

(b) VH having SEQ ID NO: 5 and VL having SEQ ID NO: 15;

(c) VH having SEQ ID NO: 4 and VL having SEQ ID NO: 14;

(d) VH having SEQ ID NO: 6 and VL having SEQ ID NO: 16;

(e) VH having SEQ ID NO: 2 and VL having SEQ ID NO: 12;

(e) VH having SEQ ID NO: 3 and VL having SEQ ID NO: 13;

(g) VH having SEQ ID NO: 9 and VL having SEQ ID NO: 19;

(h) VH having SEQ ID NO: 7 and VL having SEQ ID NO: 17;

(i) VH having SEQ ID NO: 8 and VL having SEQ ID NO: 18; and (j) VH having SEQ ID NO: 10 and VL having SEQ ID NO: 20.

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

In some embodiments, an isolated anti-C5 antibody of the present invention has a characteristic selected from the group consisting of the following characteristics:

(a) the antibody contacts amino acids D51 and K109 C5 (SEQ ID NO: 39);

(b) the affinity of the antibody to C5 (SEQ ID NO: 39) is higher than the affinity of the antibody to the mutant C5 containing the E48A substitution in SEQ ID NO: 39; or (c) the antibody binds to the C5 protein having the amino acid sequence of SEQ ID NO: 39 at pH 7.4 but does not bind to the C5 protein having the amino acid sequence of SEQ ID NO: 39 with the H72Y substitution at pH 7.4.

In other embodiments, the antibody binds to C5 with higher affinity at neutral pH than at acidic pH. In 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 present invention inhibits C5 activation. In some embodiments, the isolated anti-C5 antibody of the present invention inhibits the activation of R885H variant C5. In some embodiments, the isolated anti-C5 antibody of the present invention is a monoclonal antibody. In some embodiments, the isolated anti-C5 antibody of the present invention is a human, humanized, or chimeric antibody. In some embodiments, an isolated anti-C5 antibody of the present invention is an antibody fragment that binds to C5. In some embodiments, the isolated anti-C5 antibody of the present invention is a full length IgG1 or IgG4 class antibody.

In some embodiments, an isolated anti-C5 antibody of the present invention comprises (a) an HVR-H3 comprising the amino acid sequence DX1GYX ₂ X ₃ PTHAMX ₄ X ₅ , wherein X1 is G or A, X ₂ is V, Q, or D, X ₃ is T or Y, X ₄ is Y or H, X ₅ is L or Y (SEQ ID NO: 128);

(b) HVR-L3 containing the amino acid sequence QX1TX2VGSSYGNX3, in which X1 is S, C, N or T, X ₂ is F or K, X ₃ is A, T or H (SEQ ID NO: 131); and (c) HVR-H2 containing the amino acid sequence X ₁ IX ₂ TGSGAX ₃ YX ₄ AX ₅ WX ₆ KG, in which X1 is C, A or G, X ₂ is Y or F, X ₃ is T, D or E , X4 is Y, K or Q, X ₅ is S, D or E, X ₆ is A or V (SEQ ID NO: 127).

In some embodiments, an isolated anti-C5 antibody of the present invention comprises (a) an HVR-H1 comprising the amino acid sequence SSYYX1X2, wherein X1 is M or V, X ₂ is C or A (SEQ ID NO: 126);

(b) HVR-H2 containing the amino acid sequence X ₁ IX ₂ TGSGAX ₃ YX ₄ AX ₅ WX ₆ KG, in which X1 is C, A or G, X ₂ is Y or F, X ₃ is T, D or E, X ₄ is Y, K or Q, X ₅ is S, D or E, X ₆ is A or V (SEQ ID NO: 127); and (c) HVR-H3 containing the amino acid sequence DX1GYX ₂ X ₃ PTHAMX4X ₅ in which X1 is G or A, X ₂ is V, Q or D, X ₃ is T or Y, X4 is Y or H, X ₅ denotes L or Y (SEQ ID NO: 128).

In other embodiments, the antibody comprises (a) an HVR-L1 comprising the amino acid sequence X1ASQX2IX3SX4LA, wherein X1 is Q or R, X ₂ is N, Q or G, X ₃ is G or S, X ₄ is D, K or S (SEQ ID NO: 129);

- 4 041632 (b) HVR-L2 contains the amino acid sequence GASX ₁ X2X ₃ S, in which X ₁ denotes

K, E or T, X ₂ is L or T, X ₃ is A, H, E or Q (SEQ ID NO: 130); and (c) an HVR-L3 containing the amino acid sequence QX ₁ TX2VGSSYGNX ₃ in which X ₁ is S, C, N or T, X ₂ is F or K, X ₃ is A, T or H (SEQ ID NO: 131 ).

In some embodiments, an isolated anti-C5 antibody of the present invention comprises (a) an HVR-L1 comprising the amino acid sequence X ₁ ASQX ₂ IX ₃ SX4LA, wherein X ₁ is Q or R, X ₂ is N, Q or G, X ₃ is G or S, X ₄ is D, K or S (SEQ ID NO: 129);

(b) HVR-L2 containing the amino acid sequence GASX ₁ X2X ₃ S, wherein X ₁ is K, E or T, X ₂ is L or T, X ₃ is A, H, E or Q (SEQ ID NO: 130 ); and (c) an HVR-L3 containing the amino acid sequence QX ₁ TX2VGSSYGNX ₃ in which X ₁ is S, C, N or T, X ₂ is F or K, X ₃ is A, T or H (SEQ ID NO: 131 ).

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

In some embodiments, an isolated anti-C5 antibody of the present invention comprises (a) a VH sequence that is at least 95% identical to one of the amino acid sequences of SEQ ID NOs: 10, 106-110;

(b) a VL sequence at least 95% identical to one of the amino acid sequences of SEQ ID NOs: 20, 111-113; or (c) the VH sequence indicated in (a) and the VL sequence indicated in (b).

In other embodiments, the antibody contains a VH having one of the sequences of SEQ ID NOs: 10, 106-110. In other embodiments, the antibody comprises a VL having one of the sequences of SEQ ID NOs: 20, 111-113.

The invention provides an antibody comprising a VH having one of the sequences of SEQ ID NOs: 10, 106-110 and a VL having one of the sequences of SEQ ID NOs: 20, 111-113.

The invention also provides isolated nucleic acids that encode an anti-C5 antibody of the present invention. The invention also provides host cells that contain the nucleic acid of the present invention. The invention also provides a method for producing an antibody, comprising culturing a host cell of the present invention, resulting in an antibody.

The invention also provides a method for producing an anti-C5 antibody. In some embodiments, the method includes immunizing the animal with a polypeptide that contains the MG1MG2 domain (SEQ ID NO: 43) of the C5 beta chain. In some embodiments, the method includes immunizing the animal with a polypeptide that contains a region corresponding to amino acids at positions 33-124 of the C5 beta chain (SEQ ID NO: 40). In some embodiments, the method includes immunizing the animal with a polypeptide that contains at least one fragment selected from amino acids 47-57, 70-76, and 107-110 of the C5 beta chain (SEQ ID NO: 40). In some embodiments, the method comprises immunizing an animal with a polypeptide containing a C5 beta chain fragment (SEQ ID NO: 40) that contains at least one amino acid selected from Glu48, Asp51, His70, His72, Lys109, and His110.

The invention also provides a pharmaceutical composition which comprises an anti-C5 antibody of the present invention and a pharmaceutically acceptable carrier.

Antibodies to C5 proposed in the present invention can be used as a drug. Anti-C5 antibodies of the present invention can be used to treat a complement-mediated disease or condition that involves excessive or uncontrolled C5 activation. The anti-C5 antibodies of the present invention can be used to enhance the clearance of C5 from plasma.

The anti-C5 antibodies of the present invention can be used in the preparation of a medicament. In some embodiments, the drug is for the treatment of a complement-mediated disease or condition that involves excessive or uncontrolled C5 activation. In some embodiments of the invention

- 5 041632 the drug is intended to enhance the clearance of C5 from plasma.

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

Brief description of the drawings

The drawings show the following.

Figure 1 - grouping of antibodies to C5 depending on the epitope, the procedure of which is described in example 2.2. Antibodies grouped into bins for the same epitope are enclosed in thick boxes.

In FIG. 2A - BIACORE® sensograms for antibodies to C5 at pH 7.4 (solid line) and at pH 5.8 (dashed line) to evaluate the dependence on pH, as described in example 3.2. CFA0305, CFA0307, cFa0366, CFA0501, CFA0538 and CFA0599 are antibodies belonging to the epitope group B as described in example 2.2.

In FIG. 2B - BIACORE® sensograms for antibodies to C5 at pH 7.4 (solid line) and at pH 5.8 (dashed line) to assess the dependence on pH, as described in example 3.2. CFA0666, CFA0672 and CFA0675 are antibodies belonging to the epitope group B, and CFA0330 and CFA0341 are antibodies belonging to the epitope group B, as described in example 2.2. 305LO5 is the humanized CFA0305 antibody described in Example 2.3.

In FIG. 3 - Western blot analysis of the binding of C5 beta chain derived fragments (amino acids 19-180, 161-340, 321-500 and 481-660 of SEQ ID NO: 40) fused to a GST tag as described in the example 4.1. CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672, and CFA0675 are epitope group B antibodies. An anti-GST antibody served as a positive control. The positions of C5 fragments (46-49 kDa) fused to GST are indicated by an arrow.

In FIG. 4 are BIACORE® generated sensorgrams characterizing the binding of anti-C5 antibodies to the MG1-MG2 domain of the C5 beta chain as described in Example 4.3. The top panel shows the results for CFA0305 (solid line), CFA0307 (dashed line), CFA0366 (dashed-dashed line), and eculizumab (dashed line). The middle panel shows the results for CFA0501 (solid line), CFA0599 (dashed line), CFA0538 (dashed-dashed line), and eculizumab (dashed line). The bottom panel shows the results for CFA0666 (solid line), CFA0672 (dashed line), CFA0675 (dashed-dashed line), and eculizumab (dashed line). CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672 and CFA0675 are epitope group B antibodies. Eculizumab was used as a control anti-C5 antibody.

In FIG. 5A shows the results of Western blot analysis of binding derived from the MG1-MG2 domain of peptide fragments (amino acids 33-124, 45-124, 52-124, 33-111, 33-108 and 45-111 of SEQ ID NO: 40) fused to GST label as described in example 4.4. An anti-GST antibody was used as the antibody used in the reaction. The positions of C5 fragments fused with GST (35-37 kDa) are indicated by an arrow.

In FIG. 5B - Western blot analysis of binding derived from the MG1-MG2 domain of peptide fragments (amino acids 33-124, 45-124, 52-124, 33-111, 33-108 and 45-111 of SEQ ID NO: 40) fused to GST label as described in example 4.4. The CFA0305 antibody was used as the antibody used in the reaction.

In FIG. 5B is a summary of the results of anti-C5 antibody binding reactions with C5 beta chain-derived fragments as described in Example 4.4. Fragments to which antibodies to C5, included in the epitope group B (CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672 and CFA0675) bind, are highlighted in gray, and fragments to which they did not bind are indicated in white .

In FIG. 6 shows the results of a Western blot binding analysis of point mutant C5 mutants in which E48, D51 and K109 in the beta chain are replaced by alanine (E48A, D51A and K1O9A respectively) as described in Example 4.5. Left panel: eculizumab (anti-C5 antibody, alpha chain binding agent) was used as the antibody used in the reaction, the position of the C5 alpha chain (about 113 kDa) is indicated by an arrow. Right panel: CFA0305 (antibody belonging to the epitope group B, beta chain binding agent) was used as the antibody used in the reaction, the position of the C5 beta chain (about 74 kDa) is indicated by the arrowhead.

In FIG. 7 are BIACORE® generated sensorgrams showing the interaction of eculizumab-P76004 (upper panel) or 305LO5 (lower panel) with C5 mutants as described in Example 4.6. Sensorgrams were obtained by injecting C5-wt (bold solid curve), C5-E48A (short dashed curve), C5-D51A (long dashed curve) and C5-K1O9A (thin solid curve), respectively, onto the sensor surface on which eculizumab was immobilized. -F760G4 or

305LO5. Eculizumab is a control antibody to C5. 305LO5 is a humanized CFA0305 antibody (part of epitope group C) as described in Example 2.3.

In FIG. 8 are BIACORE®-derived sensograms demonstrating interaction of 305LO5 with C5-His mutants to assess pH dependence as described in Example 4.7. Sensorgrams were obtained by injecting C5-wt (bold solid curve), C5-H70Y (long dash curve), C5-H72Y (short dash curve), C5-H110Y (dashed curve) and C5-H70Y+H110Y (thin solid curve). ) respectively on the sensor surface on which 305LO5 is immobilized. The antibody/antigen complexes were allowed to dissociate at pH 7.4 and then further dissociate at pH 5.8 (arrowed) to evaluate pH dependent interactions.

In FIG. 9A is data showing inhibition of complement-activated liposome lysis by anti-C5 antibodies as described in Example 5.1. Results are shown for CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672 and CFA0675, which are members of epitope group B, as described in Example 2.2.

In FIG. 9B is data showing inhibition of complement-activated liposome lysis by anti-C5 antibodies as described in Example 5.1. The results for antibodies CFA0330 and CFA0341 included in the epitope group B are presented, as described in example 2.2.

In FIG. 10A is data showing inhibition of C5a formation by anti-C5 antibodies as described in Example 5.2. C5a concentrations were quantified in supernatants from the liposome lysis assay, the results of which are illustrated in FIG. 9A.

In FIG. 10B is data showing inhibition of C5a production by anti-C5 antibodies as described in Example 5.2. C5a concentrations were quantified in supernatants from the liposome lysis assay, the results of which are illustrated in FIG. 9B.

In FIG. 11 is data showing inhibition of complement-activated hemolysis by anti-C5 antibodies as described in Example 5.3. Complement activation occurred in the classical way.

In FIG. 12 is data showing inhibition of complement-activated hemolysis by anti-C5 antibodies as described in Example 5.4. Complement activation occurred in an alternative way.

In FIG. 13 is a time course of plasma concentration of human C5 after intravenous administration of human C5 alone or human C5 and an anti-human C5 antibody to mice, as determined by assessing C5 clearance using the method described in Example 6.2. CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672 and CFA0675 are epitope group B antibodies, and CFA0330 and CFA0341 are epitope group B antibodies as described in Example 2.2.

In FIG. 14 is a time course of plasma concentrations of anti-human C5 antibody after intravenous administration of human C5 and anti-human C5 antibody to mice, determined by evaluating the pharmacokinetics of the antibody according to the method described in Example 6.3. CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672 and CFA0675 are epitope group B antibodies, and CFA0330 and CFA0341 are epitope group B antibodies as described in Example 2.2.

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

In FIG. 16 Data showing inhibition of complement-activated liposome lysis by anti-C5 antibodies as described in Example 9.1. The results for antibodies 305LO15-SG115 and 305LO23-SG429 are presented.

In FIG. 17 Data showing inhibition of complement-activated liposome lysis by anti-C5 antibodies as described in Example 9.1. The results for antibodies 305LO22-SG115, 305LO22-SG422, 305LO23-SG115 and 305LO23-SG422 are presented.

In FIG. 18 - data showing inhibition of C5a production by anti-C5 antibodies as described in Example 9.2. C5a concentrations were quantified in supernatants from the liposome lysis assay, the results of which are illustrated in FIG. 15.

In FIG. 19 - data showing inhibition of C5a production by anti-C5 antibodies as described in Example 9.2. C5a concentrations were quantified in supernatants from the liposome lysis assay, the results of which are illustrated in FIG. 16.

In FIG. 20 is data showing inhibition of complement activity in monkey plasma by anti-C5 antibodies as described in Example 9.3. Antibodies to C5 were administered to cynomolgus monkeys and complement activity was measured in monkey plasma by haemolysis assay.

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

- 7 041632

In FIG. 22 data showing inhibition of biological activity of C5 wild-type (WT) and C5 variants (V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N and E1437D) by anti-C5 antibody (variant 305) as described in Example 9.4.

In FIG. 23 data showing inhibition of complement-activated liposome lysis by anti-C5 antibodies (BNJ441 and variant 305) as described in Example 9.5.

In FIG. 24 is a time course of plasma concentration of C5 cynomolgus monkeys after intravenous administration of anti-human C5 antibody to cynomolgus monkeys, as determined by assessing C5 clearance using the method described in Example 10.2.

In FIG. 25 is a time course of plasma concentration of anti-human C5 antibody following intravenous administration of anti-human C5 antibody to Cynomolgus monkeys, as determined by evaluating the pharmacokinetics of the antibody using the method described in Example 10.3.

In FIG. 26A and 26B show the crystal structure of the 305 Fab associated with the MG1 domain of human C5 (hC5) as described in Example 11.6. In FIG. 26A is an image of an asymmetric unit cell. MG1 is depicted as a surface and 305 Fab is depicted with stripes (dark grey: heavy chain, light grey: light chain). In FIG. 26B shows an overlay of molecules 1 and 2 (dark gray: molecule 1, light gray: molecule 2).

In FIG. 27A is an illustration of an epitope for the 305 Fab contact region in the MG1 domain described in Example 11.6. In FIG. 27A shows MG1 amino acid sequence mapping data (dark gray: distance less than 3.0 A, light gray: distance less than 4.5 A).

In FIG. 27B is an illustration of an epitope for the 305 Fab contact region in the MG1 domain described in Example 11.6. In FIG. 27B shows mapping data in the crystal structure (dark gray spheres: distance less than 3.0 A, light gray spheres: distance less than 4.5 A).

In FIG. 28A is an enlarged view illustrating the interactions of E48, D51 and K109 (shown as rods) with 305 Fab (shown as surface) as described in Example 11.7.

In FIG. 28B illustrates interactions between E48 and its environment (dark gray dotted line: Fab hydrogen bond, light gray dotted line: water-mediated hydrogen bond) as described in Example 11.7.

In FIG. 28B illustrates interactions between D51 and its environment (dark gray dotted line: hydrogen bonding to Fab) as described in Example 11.7.

In FIG. 28D is an illustration of the interactions between K109 and its environment (dark gray dotted line: hydrogen bonding to Fab, light gray dotted line: salt bridged to H-CDR3_D95) as described in Example 11.7.

In FIG. 29A is an enlarged view illustrating the interactions of H70, H72, and H110 (shown as rods) with 305 Fab (shown as a surface) as described in Example 11.8, in the same orientation as in FIG. 28A.

In FIG. 29B is an illustration of the interactions between H70 and its environment as described in Example 11.8. This histidine residue is depicted with sticks and grid. The hydrogen bond is shown as a dotted line.

In FIG. 29B illustrates the interactions between H72 and its environment as described in Example 11.8. This histidine residue is depicted with sticks and grid. The hydrogen bond is shown as a dotted line.

In FIG. 29D is an illustration of the interactions between an NGO and its environment as described in Example 11.8. This histidine residue is depicted with sticks and grid. The distance between the NGO and the H-CDR3_H100c is indicated by a dotted line.

Description of embodiments of the invention

The techniques and procedures described or referenced herein are generally well known to those skilled in the art and are widely applied using conventional methodologies such as those described in

Sambrook et al., Molecular Cloning: A

Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold

Spring Harbor, N.Y. 2001; Current Protocols in Molecular Biology (ed.R.M.

Ausubel et al., 2003); Methods in Enzymology series (Academic Press, Inc.):

PCR 2: A Practical Approach (ed. M.J. MacPherson, B.D. Hames and G.R. Taylor, 1995), Antibodies, A Laboratory Manual, ed. Harlow and Lane, 1988 and Animal Cell Culture (ed. R.I. Freshney 1987); Oligonucleotide Synthesis (ed. M.J.

Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A

- 8 041632

Laboratory Notebook (ed. J.E. Cellis, 1998) from Academic Press; Animal Cell

Culture (ed. R.I. Freshney, 1987); Introduction to Cell and Tissue Culture (J.P.

Mather and P.E. Roberts, 1998), Plenum Press; Cell and Tissue Culture:

Laboratory Procedures (ed. A. Doyle, J. B. Griffiths and D. G. Newell, 1993-8), J. Wiley and Sons; Handbook of Experimental Immunology (ed. D.M.

Weir and C.C. Blackwell); Gene Transfer Vectors for Mammalian Cells (ed. J.M.

Miller and M.P. Calos, 1987); PCR: The Polymerase Chain Reaction, (ed. Mullis et al., 1994); Current Protocols in Immunology (ed. J.E. Coligan et al., 1991);

Short Protocols in Molecular Biology (Wiley and Sons, 1999);

Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997);

Antibodies: A Practical Approach (ed. D. Catty., IRL Press, 1988-1989);

Monoclonal Antibodies: A Practical Approach (ed. 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 (ed. M. Zanetti and J. D. Capra, Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., J.B. Lippincott Company, 1993).

I. Definitions.

Unless otherwise indicated, the technical and scientific terms used in the present description have the meanings well known to the ordinary specialist in the field in which the present invention relates. In Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd Ed., J. Wiley & Sons, New York, N.Y., 1994; and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th Ed., John Wiley & Sons, New York, N.Y., 1992, generally provides an interpretation intended for those skilled in the art for many of the concepts used in this application. All references cited herein, including patent applications and patent publications, are incorporated herein by reference in their entirety.

For the purposes of interpreting the present specification, the following terms are to be used and, whenever possible, terms used in the singular also include their use in the plural and vice versa. It should be obvious that the terminology used in the present description is presented only for the purpose of describing specific embodiments of the invention and is not intended to limit its scope. In the event that any definition below is inconsistent with that specified in any document included in the description by reference, then the definition below should be used.

For the purposes of this specification, an acceptor human framework region means a framework region that contains the amino acid sequence of a light chain variable domain (VL) framework region or a heavy chain variable domain (VH) framework region derived from a human immunoglobulin framework region or a human consensus framework region as defined below. . An acceptor human framework derived from a human immunoglobulin framework or a human consensus framework may have the same amino acid sequence or may contain alterations in the amino acid sequence. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the sequence of the human VL acceptor framework is identical to the sequence of the human immunoglobulin VL framework or the sequence of the human consensus framework.

The term affinity refers to the total strength of all non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). As used herein, unless otherwise indicated, binding affinity refers to the intrinsic binding affinity of a molecule, reflecting a 1:1 interaction between the components of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can generally be characterized by the dissociation constant (Kd). Affinity can be assessed using conventional methods known in this field, including those presented in the present description. Specific options for measuring binding affinity are provided below by way of illustration.

An affinity matured antibody means an antibody with one or more changes in one or more hypervariable regions (HVRs) compared to the parent antibody,

- 9 041632 which does not contain these changes, these changes lead to an increase 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 has the ability to bind to C5 with sufficient affinity to enable the antibody to be used as a diagnostic and/or therapeutic agent that targets C5. In one embodiment, the level of binding of an anti-C5 antibody to an unrelated non-C5 protein is less than about 10% of the binding of the antibody to C5 as measured by, for example, radioimmunoassay (RIA). In some embodiments, the antibody that binds to C5 has a dissociation constant (Kd) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM, or less than or 0.001 nM or less (for example, 10 ^-8 M or less, for example, from 10 ^-8 to 10 ^-13 M, for example, from 10 ^-9 to 10 ^-13 M). In some embodiments, an anti-C5 antibody binds to a C5 epitope that is conserved for C5 from different species.

In the context of the present description, the term antibody is used in its broadest sense and refers to various antibody structures, including (but not limited to) monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies) and antibody fragments, provided that they have desired antigen-binding activity.

The term antibody fragment refers to a molecule, other than an intact antibody, that contains a portion of the intact antibody that 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 ; dimeric antibodies (diabodi); linear antibodies; single chain antibody molecules (eg scFv); and multispecific antibodies formed from antibody fragments.

The term antibody that binds to the same epitope as the reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen when assessed by a competition assay (competitive assay) and/or vice versa the reference antibody blocks binding. an antibody with its antigen when assessed by a competitive assay. An example of competitive analysis is presented in the present description.

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

The term antibody class refers to the type of constant domain or constant region that/which is included in its heavy chain. Five main classes of antibodies are known: IgA, IgD, IgE, IgG and IgM, and some of them can be further divided into subclasses (isotypes), for example IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains that correspond to the various immunoglobulin classes are referred to as alpha, delta, epsilon, gamma, and mu (α, δ, ε, γ, and μ), respectively.

The concept of a cytotoxic agent in the context of the present description refers to a substance that inhibits or interferes with cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (eg, At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and radioactive isotopes Lu) ; chemotherapeutic agents or drugs (eg, methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins such as low molecular weight toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various anti-tumor or anti-cancer agents listed below.

The concept of effector functions refers to those types of biological activity that are associated with the Fc region of the 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-mediated cell-dependent cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (eg B cell receptor) and activation of B cells.

The concept of an effective amount of an agent, for example, a pharmaceutical composition, means an amount effective in doses and for a period of time necessary to achieve the desired therapeutic or prophylactic result.

The term epitope includes any determinant that is capable of being bound by an antibody. An epitope is a region of an antigen that binds to an antibody whose target is the antigen and includes specific amino acids that are in direct contact with the antibody. Epitopic determinants may include reactive surface groups of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structure characteristics and/or specific charge characteristics. In general, antibodies specific for a particular target antigen should preferentially recognize an epitope on the target antigen in a complex mixture of proteins and/or macromolecules.

The term Fc-region in the context of the present description refers to the C-terminal region of the heavy chain of an immunoglobulin, which contains at least part of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise noted, the numbering of amino acid residues in the Fco region or constant region follows the EU numbering system, which is also referred to as the EU index, as described by Kabat E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

The term framework region or FR refers to variable domain residues other than hypervariable region (HVR) residues. The variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences typically have the following location in the VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms full-length antibody, intact antibody, and whole antibody are used interchangeably herein to refer to an antibody having a structure substantially similar to the native antibody structure, or having heavy chains that contain the Fc region described herein.

The terms host cell, host cell line, and host cell culture are used interchangeably and refer to cells into which an exogenous nucleic acid has been introduced, including progeny of said cells. Host cells include transformants and transformed cells, which include the primary transformed cell and its progeny, regardless of the number of passages. The offspring may not be completely identical in nucleic acid composition to the parent cell, but may contain mutations. Under the scope of the invention falls mutant progeny, which has the same function or biological activity, which is found as a result of screening or selected from the original transformed cell.

A human antibody is an antibody having an amino acid sequence that corresponds to that of an antibody produced in the human body or human cells, or derived from a non-human source that uses a human antibody spectrum or other human antibody-encoding sequences. A humanized antibody containing non-human antigen-binding residues is specifically excluded from this definition of a human antibody.

The human consensus framework region is a framework region that includes the most frequently occurring amino acid residues in selected human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is made from a subset of variable domain sequences. Generally, a subset of sequences is the subset described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., NIH Publication 91-3242, Bethesda MD, vols. 1-3, 1991. In one embodiment, implementation of the invention regarding VL subgroup is a subgroup kappa I according to Kabat and others, above. In one embodiment of the invention regarding VH, the subgroup is subgroup III according to Kabat et al., supra.

The term humanized antibody refers to a chimeric antibody that contains amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, a humanized antibody may comprise substantially all of at least one, and typically two, variable domains in which all or substantially all of the HVRs (e.g., CDRs) correspond to regions of the non-human antibody and all or substantially all of the FRs correspond to regions human antibody. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. The term humanized form of an antibody, such as a non-human antibody, refers to an antibody that has been humanized.

The concept of a hypervariable region or HVR in the context of the present description refers to each of the regions of the variable domain of an antibody, the sequences of which are hypervariable (complementarity determining regions or CDRs), and / or form loops of a certain structure (hypervariable loops), and / or contain antigen-contacting residues (contacts with antigen). Typically, antibodies contain six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). As used herein, examples of HVRs include (a) hypervariable loops comprising amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol., 196, 1987, c. 901-917);

(b) CDRs including 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., Pub.

- 11 041632 lic Health Service, National Institutes of Health, Bethesda, MD, 1991, NIH Publication 91-3242);

(c) areas of contact with the antigen, including 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, 1996, c. 732-745) ; and (d) combinations of residues specified in subparagraphs (a), (b) and/or (c), including amino acid residues HVR 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 specifically stated otherwise, residues in HVR and other residues in the variable domain (eg, residues in FR) are numbered according to Kabat et al., supra.

An immunoconjugate is an antibody conjugated to one or more heterologous molecule(s), including (but not limited to) a cytotoxic agent.

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

An isolated antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic assays (such as, for example, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic assays (such as, for example, ion exchange chromatography or reverse phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chrom. V, 848, 2007, p. 79-87.

An isolated nucleic acid is a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally includes a nucleic acid molecule, but in which the nucleic acid molecule is present outside the chromosome or in which its localization on the chromosome differs from its naturally occurring localization on the chromosome.

The term isolated nucleic acid encoding an anti-C5 antibody refers to one or more nucleic acid molecules encoding heavy and light chains of an antibody (or fragments thereof), including the specified molecule(s) in one vector or in different vectors, and the specified ( s) nucleic acid(s) molecule(s) present(s) at one or more positions in the host cell.

The term monoclonal antibody in the context of the present description refers to an antibody obtained from a population of substantially homogeneous antibodies, i. the individual antibodies in a population are identical and/or bind to the same epitope, with the exception of possible variants of antibodies, for example, containing mutations occurring naturally or arising during the production of a preparation of a monoclonal antibody, these variants are usually present in minor quantities. Unlike polyclonal antibody preparations, which typically include different antibodies to different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single antigen determinant. Thus, the adjective monoclonal defines the specificity of an antibody, characterizing it as derived from a substantially homogeneous population of antibodies, rather than being engineered to meet the requirements for producing an antibody by any particular method. For example, monoclonal antibodies that can be used according to the present invention can be created using various technologies, including (but not limited to) the hybridoma method, recombinant DNA methods, phage display methods, and methods based on the use of transgenic animals that contain all or part of the human immunoglobulin loci, these methods and other exemplary methods for obtaining monoclonal antibodies are presented in the present description.

The term naked antibody refers to an antibody that has not been conjugated to a heterologous moiety (eg, a cytotoxic moiety) or a radioactive label. The naked antibody may be present in the pharmaceutical composition.

The term native antibodies refers to naturally occurring immunoglobulin molecules with different structures. For example, native IgG antibodies are heterotetrameric glycoproteins with a molecular weight of approximately 150,000 Da, consisting of two identical light chains and two identical heavy chains linked by disulfide bridges. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also referred to as a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), which is also called a variable light domain or a light chain variable domain, followed by a light chain constant domain.

- 12 041632 chains (CL). The light chain of an antibody, depending on the amino acid sequence of its constant domain, can belong to one of two types, which are kappa (k) and lambda (λ).

The term package insert refers to the instructions that are usually included in the commercial packaging of therapeutic products, containing information about the indications, use, dose, route of administration, combination therapy, contraindications and / or precautions associated with the use of these therapeutic products. .

Percent (%) amino acid sequence identity relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a reference polypeptide sequence, after sequence alignment and, if necessary, the introduction of gaps to achieve the maximum percent sequence identity without considering any conservative substitutions as a component in the evaluation of sequence identity. Comparison for purposes of determining percent amino acid sequence identity can be performed in a variety of ways known in the art using, for example, publicly available computer programs such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the compared sequences. However, for the purposes of the present invention, the % amino acid sequence identity value is determined using the ALIGN-2 sequence comparison computer program. The sequence comparison computer program ALIGN-2 is copyrighted by Genentech, Inc. and the source code has been provided in the U.S. user documentation. Copyright Office, Washington D.C., 20559, where it is registered as U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, CA. California, or it can be compiled from source code. The ALIGN-2 program can be compiled for use on the UNIX operating system, including Digital UNIX V4.0D. All parameters required for sequence comparison are set by ALIGN-2 and should not be changed. In situations in which ALIGN-2 is used to compare amino acid sequences, the % amino acid sequence identity of a given amino acid sequence A compared to or relative to a given amino acid sequence B (which can alternatively be referred to as a given amino acid sequence A that has or contains a certain % amino acid sequence identity compared to or relative to a given amino acid sequence B) is calculated as follows: 100x fraction X/Y where X is the number of amino acid residues determined to be identical matches as assessed by the ALIGN-2 sequence comparison software, where c using the program, a comparison was made between A and B, and where Y denotes the total number of amino acid residues in B. It should be obvious that if the length of the amino acid sequence A is not equal to the length of the amino acid sequence B, then % id The amino acid sequence identity of A with respect to B cannot be equal to the % amino acid sequence identity of B with respect to A. Unless specifically stated otherwise, all % amino acid sequence identity values specified herein were obtained using the ALIGN-2 computer program described in the previous paragraph.

The term “pharmaceutical composition” refers to a preparation that is in such a form that it provides the biological activity of the active substance included in its composition, which should be effective, and which does not contain additional components that have unacceptable toxicity for the individual to whom the composition should be administered.

A pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical composition, other than the active ingredient, that is non-toxic to the individual. Pharmaceutically acceptable carriers include, but are not limited to, a buffer, excipient, stabilizer, or preservative.

The concept of C5 in the context of the present description can refer to any native C5 from any vertebrate animal, including mammals such as primates (eg, humans and monkeys) and rodents (eg, mice and rats). Unless specifically noted otherwise, the term C5 refers to a human C5 protein having the amino acid sequence shown in SEQ ID NO: 39 and containing the beta chain sequence shown in SEQ ID NO: 40. The term refers to full-length full-length C5, as well as to any form of C5, which is formed as a result of processing in the cell. The term also includes naturally occurring C5 variants, such as splicing variants or allelic variants. The amino acid sequence of exemplary human C5 is shown in SEQ ID NO: 39 (wild-type C5 or WT). The amino acid sequence of exemplary human C5 beta chain is shown in SEQ ID NO: 40. The amino acid sequences of exemplary human C5

Example MG1-, MG2- and MG1-MG2-goMeHoe human C5 beta chains are shown in SEQ ID NOs: 41, and 43, respectively.

The amino acid sequences of exemplary C5 cynomolgus monkeys and mice are shown in SEQ ID NOs: 44 and 105, respectively. Amino acid residues 1-19 of SEQ ID NOs: 39, 40, 43, 44 and 105 correspond to a signal sequence that is removed by cell processing and are therefore absent from the corresponding exemplary amino acid sequences.

As used herein, treatment (and its grammatical variations such as treat or process of treatment) refers to a clinical intervention to alter the natural course of a disease in an individual being treated, and can be done either for prophylaxis or during the development of clinical pathology. The desired actions of treatment are, but are not limited to, prevention of the onset or recurrence of the disease, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastases, reduction in the rate of disease progression, alleviation or temporary relief of the disease state, and remission or improvement in prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or slow the progression of a disease.

The term variable region or variable domain refers to the heavy or light chain domain of an antibody that is involved in the binding of an antibody to an antigen. The heavy chain and light chain variable domains (VH and VL, respectively) of a native antibody generally have similar structures, with each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs) (see, for example, Kindt and et al., Kuby Immunology, 6th ed., W. H. Freeman and Co., 2007, p. 91). A single VH or VL domain may be sufficient to provide antigen binding specificity. In addition, antibodies that bind to a particular antigen can be isolated using the VH or VL domain from an antibody that binds to the antigen to screen for a library of complementary VL or VH domains, respectively (see, e.g., Portolano et al. , J. Immunol., 150, 1993, pp. 880-887; Clarkson et al., Nature, 352, 1991, pp. 624-628).

The concept of a vector in the context of the present description refers to a nucleic acid molecule that has the ability to propagate another nucleic acid with which it is associated. The concept includes a vector in the form of a self-replicating nucleic acid structure, as well as a vector included in the genome of the host cell into which it is introduced. Some vectors have the ability to control the expression of the nucleic acids to which they are operably linked. These vectors in the context of the present description are referred to as expression vectors.

II. Compositions and methods.

One of the objects of the invention are, in particular, antibodies to C5 and their uses. Some embodiments of the invention are antibodies that bind to C5. Antibodies of the invention can be used, for example, to diagnose or treat a complement-mediated disease or condition that involves excessive or uncontrolled C5 activation.

A. Examples of anti-C5 antibodies.

One of the objects of the invention are isolated antibodies that bind to C5. In some embodiments, an anti-C5 antibody of the present invention binds to an epitope in the C5 beta chain. In some embodiments, an anti-C5 antibody binds to an epitope in the MG1-MG2 domain of the C5 beta chain. In some embodiments, an anti-C5 antibody binds to an epitope in a fragment consisting of amino acids 19-180 of the C5 beta chain. In some embodiments, an anti-C5 antibody binds to an epitope in the MG1 domain (amino acids 20-124 of SEQ ID NO: 40 (SEQ ID NO: 41)) of the C5 beta chain. In some embodiments, an anti-C5 antibody binds to an epitope in a fragment consisting of amino acids 33-124 of the C5 beta chain (SEQ ID NO: 40). In another embodiment, the antibody does not bind to a fragment shorter than the fragment consisting of amino acids 33-124 of the C5 beta chain, for example, to a fragment consisting of amino acids 45-124, 52-124, 33-111, 33-108, or 45-111 C5 beta chain (SEQ ID NO: 40).

Another object of the invention are antibodies to C5 with pH-dependent binding characteristics. In the context of the present description, the term pH-dependent binding means that the antibody has a reduced ability to bind to C5 at acidic pH compared to its binding at neutral pH (both terms can be used interchangeably for the purposes of this description). For example, antibodies with pH-dependent binding characteristics include antibodies that bind to C5 with higher affinity at neutral pH than at acidic pH. In some embodiments, the antibodies of the present invention bind to C5 with an affinity of at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10,000 or more times higher at neutral pH than at acidic pH. In some embodiments, antibodies bind to C5 for more than

- 14 041632 higher affinity at pH 7.4 than at pH 5.8. In other embodiments, the antibodies of the present invention bind to C5 with an affinity of at least 2, 3,

5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more times higher at pH 7.4 than at pH 5.8.

For the purposes of this description, the affinity of an antibody for C5 is expressed in terms of the KD of the antibody. The KD of an antibody means the dissociation equilibrium constant of the antibody-antigen interaction. The higher the KD value, which characterizes the binding of an antibody to its antigen, the weaker its binding affinity for the specified specific antigen. Thus, in the context of the present description, the concept of higher affinity at neutral pH than at acidic pH (or the equivalent concept of pH-dependent binding) means that the KD characterizing the binding of an antibody to C5 at acidic pH is higher than the KD characterizing the binding of an antibody to C5 at neutral pH. For example, in the context of the present invention, an antibody is considered to bind to C5 with higher affinity at neutral pH than at acidic pH if the KD characterizing the binding of the antibody to C5 at acidic pH is at least 2 times higher than the KD characterizing binding of the antibody to C5 at neutral pH. Thus, the present invention includes antibodies whose binding to C5 at acidic pH is characterized by a KD value that is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10,000 or more times the KD of C5 binding at neutral pH. In another embodiment, the KD value of the antibody at neutral pH may be 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 , 10 ^-11 , 10 ^-12 M or less. In another embodiment, the KD value of the antibody at acidic pH may be 10 ^-9 , 10 ^-8 , 10 ^-7 , 10 ^-6 M or more.

In other embodiments, an antibody is said to bind to C5 with a higher affinity at neutral pH than at acidic pH if the KD value characterizing the binding of the antibody to C5 at pH 5.8 is at least 2-fold higher than the KD , characterizing the binding of the antibody to C5 at pH 7.4. In some embodiments of the invention, the binding of the proposed antibodies to C5 at pH 5.8 is characterized by a KD value that is at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more times the KD characterizing the binding of the antibody to C5 at pH 7.4. In another embodiment, the KD value of the antibody at pH 7.4 may be 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 , 10 ^-11 , 10 ^-12 M or less. In another embodiment, the KD value of the antibody at pH 5.8 may be 10 ^-9 , 10 ^-8 , 10 ^-7 , 1θ ^-6 M or more.

The binding characteristics of an antibody to a particular antigen can also be expressed as the kd of an antibody. The kd value of an antibody denotes the dissociation rate constant of an antibody relative to a particular antigen and is expressed as reciprocals of a second (ie, s ⁻¹ ). An increased kd value means weaker binding of the antibody to its antigen. Thus, the present invention includes antibodies whose binding to C5 has a higher kd value at acidic pH than at neutral pH. The present invention includes antibodies whose binding to C5 at acidic pH is characterized by a kd value that is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 6θ, 65 , 70, 75, 80, 85, 90, 95, 100, 200, 4θθ, 1000, 10000 or more times the kd characterizing the binding of the antibody to C5 at neutral pH. In another embodiment, the kd value of the antibody at neutral pH may be 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 1/s or less. In another embodiment, the kd value of the antibody at acidic pH may be 10 ^-3 , 10 ^-2 , 10 ^-1 1/s or more. The invention also includes antibodies whose binding to C5 has a higher kd value at pH 5.8 than at pH 7.4. The invention includes antibodies whose binding to C5 at pH 5.8 is characterized by a kd value of at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more times the kd characterizing the binding of the antibody to C5 at pH 7.4. In another embodiment, the kd value of the antibody at pH 7.4 may be 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 1/s or less. In another embodiment, the kd value of the antibody at pH 5.8 may be 10 ^-3 , 10 ^-2 , 10 ^-1 1/s or more.

In some cases, the reduced ability to bind to C5 at acidic pH compared to the ability to bind at neutral pH is expressed as the ratio of the KD value characterizing the binding of the antibody to C5 at acidic pH and the KD value characterizing the binding of the antibody to C5 at neutral pH (or vice versa ). For example, an antibody may be considered to have reduced ability to bind to C5 at acidic pH compared to its ability to bind at neutral pH for the purposes of the present invention if the antibody's acid/neutral pH KD ratio is 2 or more. In some embodiments, the ratio of KD at pH 5.8/pH 7.4 for an antibody of the present invention is 2 or more. In some embodiments, the acid/neutral pH KD ratio for an antibody of the present invention may be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more. In another embodiment, the KD value of the antibody at neutral pH may be 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 , 10 ^-11 , 10 ^-12 M or less. In another embodiment, the KD value of the antibody at acidic pH may be 10 ^-9 ,10 ^-8 , 10 ^-7 , 10 ^-6 M or more. In other cases, an antibody may be considered to have a reduced ability to bind to C5 at acidic pH compared to its ability to bind at neutral pH if the antibody has a KD ratio at pH 5.8/pH 7.4 of 2 or more. In some exemplary embodiments of the invention, the KD ratio at pH 5.8/pH 7.4 for an antibody may be 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more. In another embodiment, the KD value of the antibody at pH 7.4 may be 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 , 10 ^-11 , 10 ^-12 M or less. In another embodiment, the KD value of the antibody at pH 5.8 may be 10 ^-9 , 10 ^-8 , 10 ^-7 , 10 ^-6 M or more.

In some cases, the reduced ability to bind to C5 at acidic pH compared to the ability to bind at neutral pH is expressed as the ratio of the kd value characterizing the binding of the antibody to C5 at acidic pH and the kd value characterizing the binding of the antibody to C5 at neutral pH (or vice versa ). For example, an antibody may be considered to have a reduced ability to bind to C5 at acidic pH relative to its ability to bind at neutral pH for the purposes of the present invention if the antibody's acid/neutral pH kd ratio is 2 or more. In some embodiments, the kd ratio at pH 5.8/7.4 for an anti-C5 antibody of the present invention is 2 or greater. In some embodiments, the acid/neutral pH kd ratio for an antibody of the present invention may be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more. In another embodiment, the kd value of the antibody at neutral pH may be 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 1/s or less. In another embodiment, the kd value of the antibody at acidic pH may be 10 ^-3 , 10 ^-2 , 10 ^-1 1/s or more. In some exemplary embodiments of the invention, the kd ratio at pH 5.8/7.4 for an 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, 10000 or more. In another embodiment, the kd value of the antibody at pH 7.4 may be 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 1/s or less. In another embodiment, the kd value of the antibody at pH 5.8 may be 10 ^-3 , 10 ^-2 , 10 ^-1 1/s or more.

In the context of the present description, the term acidic pH means a pH of 4.0 to 6.5. The concept of acidic pH includes one of the following pH values 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5 .0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2 , 6.3, 6.4 and 6.5. In specific aspects of the invention, the acidic pH is 5.8.

In the context of the present description, the concept of neutral pH means a pH from 6.7 to about 10.0. The concept of neutral pH includes one of the following pH values 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7 .7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 , 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, and 10.0. In specific aspects of the invention, neutral pH is 7.4.

The KD and kd values described herein can be determined using a biosensor based surface plasmon resonance method to characterize antibody-antigen interactions (see, for example, Example 7 herein). The KD values and kd values can be determined at 25 or 37°C.

In some embodiments, an anti-C5 antibody of the present invention binds to an epitope on the C5 beta chain that consists of the MG1 domain (SEQ ID NO: 41). In some embodiments, an anti-C5 antibody of the present invention binds to an epitope in the beta chain (SEQ ID NO: 40) of C5 that contains at least one fragment selected from the group consisting of amino acids 47-57, 70-76 and 107-110. In some embodiments, an anti-C5 antibody of the present invention binds to an epitope in the beta chain fragment (SEQ ID NO: 40) of C5 that contains at least one amino acid selected from the group consisting of Thr47, Glu48, Ala49, Phe50, Asp51, Ala52, Thr53, Lys57, His70, Val71, His72, Ser74, Glu76, Va1107, Ser108, Lys109 and His110. In some embodiments, an anti-C5 antibody of the present invention binds to an epitope in the beta chain fragment (SEQ ID NO: 40) of C5 that contains at least one amino acid selected from the group consisting of Glu48, Asp51, His70, His72, Lys109 and His110. In some embodiments, the binding of an anti-C5 antibody of the present invention to a C5 mutant is reduced compared to its binding to wild-type C5, where the C5 mutant has at least one amino acid substitution at a position selected from the group consisting of Glu48 , Asp51, His72 and Lys109. In another embodiment, the pH-dependent binding of 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 at least one amino acid substitution at a position selected from group that consists of His70, His72 and His110. In a further embodiment of the invention, in the C5 mutant, the amino acid at a position selected from Glu48, Asp51 and Lys109 is changed to alanine and the amino acid at a position selected from His70, His72 and His110,

- 16 041632 replaced by tyrosine.

In some embodiments, an anti-C5 antibody of the present invention competes for binding to C5 with an antibody comprising a VH and VL pair selected from (a) a VH having SEQ ID NO: 1 and a VL having SEQ ID NO : eleven;

(b) VH having SEQ ID NO: 22 and VL having SEQ ID NO: 26;

(c) VH having SEQ ID NO: 21 and VL having SEQ ID NO: 25;

(d) VH having SEQ ID NO: 5 and VL having SEQ ID NO: 15;

(e) VH having SEQ ID NO: 4 and VL having SEQ ID NO: 14;

(e) VH having SEQ ID NO: 6 and VL having SEQ ID NO: 16;

(g) VH having SEQ ID NO: 2 and VL having SEQ ID NO: 12;

(h) VH having SEQ ID NO: 3 and VL having SEQ ID NO: 13;

(i) VH having SEQ ID NO: 9 and VL having SEQ ID NO: 19;

(j) VH having SEQ ID NO: 7 and VL having SEQ ID NO: 17;

(l) VH having SEQ ID NO: 8 and VL having SEQ ID NO: 18;

(m) VH having SEQ ID NO: 23 and VL having SEQ ID NO: 27; and (n) VH having SEQ ID NO: 10 and VL having SEQ ID NO: 20.

In some embodiments, an anti-C5 antibody of the present invention binds to C5 and contacts the amino acid Asp51 (D51) of SEQ ID NO: 39. In additional embodiments, an anti-C5 antibody of the present invention binds to and contacts C5. with the amino acid Lys109 (K109) SEQ ID NO: 39. In a further embodiment, the anti-C5 antibody of the present invention binds to C5 and contacts the amino acid Asp51 (D51) and the amino acid Lys109 (K109) SEQ ID NO: 39.

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

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

Without wishing to be bound by any particular theory, it can be hypothesized that the binding of an anti-C5 antibody to C5 is reduced (or virtually lost) when the amino acid residue at C5 is changed to a different amino acid, which means that the amino acid residue at C5 is critical for the interaction between an antibody to C5 and C5 and that the antibody can recognize an epitope in C5 that contains an amino acid residue.

In the present invention, it has been found that a group of anti-C5 antibodies that compete with each other or bind to the same epitope may have pH-dependent binding characteristics. Among the amino acids, histidine, which has a pKa value of about 6.0-6.5, may have different proton dissociation states at neutral and acidic pH. Therefore, the histidine residue at C5 may contribute to pH dependent interactions between C5 and C5 antibody. Without wishing to be bound by any particular theory, it can be hypothesized that an anti-C5 antibody may recognize a conformational structure around the histidine residue in C5 that varies with pH. This assumption may be consistent with the experimental data described below, indicating that the pH-dependence of the anti-C5 antibody is reduced (or almost lost) when the histidine residue in C5 is replaced with another amino acid (i.e., an anti-C5 antibody with pH-dependent characteristics binding binds to the C5 histidine mutant (C5 His mutant) with similar affinity to wild-type C5 at neutral pH, while the same antibody binds to C5 His mutant with higher affinity than to C5. wild-type, at acidic pH).

In some embodiments, an anti-C5 antibody of the present invention binds to C5 from more than one species. In other embodiments, the anti-C5 antibody binds to human and non-human C5. In other embodiments, the anti-C5 antibody binds to human and monkey C5 (eg, cynomolgus monkeys, rhesus monkeys, marmosets, chimpanzees, or baboons).

One of the objects of the invention are antibodies to C5, which inhibit the activation of C5. In some embodiments, anti-C5 antibodies are provided that prevent C5 cleavage resulting in C5a and C5b, thereby preventing C5a-associated anaphylactic activity, and also prevent assembly of the C5b-9 membrane attack complex (MAC), associated with C5b. In some embodiments, anti-C5 antibodies are provided that block the conversion of C5 to C5a and C5b by C5 convertase. In some embodiments, anti-C5 antibodies are provided that block the access of a C5 convertase to a C5 cleavage site. In some embodiments, anti-C5 antibodies are provided that block hemolytic activity due to C5 activation. In other embodiments, the anti-C5 antibodies of the present invention inhibit C5 activation in a classical and/or alternative manner.

One of the objects of the invention are antibodies to C5, which inhibit the activation of the C5 variant. A C5 variant refers to a C5 genetic variant that is obtained by genetic variation, such as a mutation, polymorphism, or allelic variation. Genetic variation may include deletion, substitution or insertion of one or more nucleotides. The C5 variant may contain one or more genetic variations in C5. In some embodiments, the C5 variant has similar biological activity to wild-type C5. Such a C5 variant may contain at least one variation selected from the group consisting of V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N and E1437D. In the context of the present description, R885H, for example, denotes a genetic variation in which the arginine at position 885 is replaced by histidine. In some embodiments, an anti-C5 antibody of the present invention inhibits the activation of both wild-type C5 and at least one C5 variant selected from the group consisting of V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N and E1437D.

One of the objects of the invention is an antibody to C5, which contains at least one, two, three, four, five or six HVR selected from (a) HVR-H1, which contains one of the amino acid sequences of SEQ ID NOs: 45-54;

(b) HVR-H2 which contains one of the amino acid sequences of SEQ ID NOs: 55-64;

(c) HVR-H3 which contains one of the amino acid sequences of SEQ ID NOs: 65-74; (d) HVR-L1 which contains one of the amino acid sequences of SEQ ID NOs: 75-84; (e) HVR-L2 which contains one of the amino acid sequences of SEQ ID NOs: 85-94; and (e) HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 95-104. One object of the invention is an antibody that contains at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1, which contains one of the amino acid sequences of SEQ ID NOs: 45-54;

(b) HVR-H2 which contains one of the amino acid sequences of SEQ ID NOs: 55-64; and (c) HVR-H3 which contains one of the amino acid sequences of SEQ ID NOs: 65-74.

In one of the embodiments of the invention, the antibody contains HVR-H3, which contains one of the amino acid sequences of SEQ ID NOs: 65-74. In another embodiment of the invention, the antibody contains HVR-H3, which contains one of the amino acid sequences of SEQ ID NOs: 65-74. and HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 95-104. In a further embodiment, the antibody comprises HVR-H3, which contains one of the amino acid sequences of SEQ ID NOs: 65-74, HVR-L3, which contains one of the amino acid sequences of SEQ ID NOs: 95-104, and HVR-H2, which contains one of the amino acid sequences of SEQ ID NOs: 55-64. In a further embodiment of the invention, the antibody comprises (a) HVR-H1 which contains one of the amino acid sequences of SEQ ID NOs: 45-54;

(b) HVR-H2 which contains one of the amino acid sequences of SEQ ID NOs: 55-64; and (c) HVR-H3 which contains one of the amino acid sequences of SEQ ID NOs: 65-74. Another object of the invention is an antibody containing at least one, at least two or all three HVR VL sequences selected from (a) HVR-L1, which contains one of the amino acid sequences of SEQ ID NOs: 75-84;

(b) HVR-L2 which contains one of the amino acid sequences of SEQ ID NOs: 85-94; and (c) HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 95-104. In one of the embodiments of the invention, the antibody contains (a) HVR-L1, which contains one of the amino acid sequences of SEQ ID NOs: 75-84;

(b) HVR-L2 which contains one of the amino acid sequences of SEQ ID NOs: 85-94; and (c) HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 95-104. According to another aspect of the invention, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three HVR VH sequences selected from (I) HVR-H1, which comprises one of the amino acid sequences SEQ ID NOs: 45-54;

(II) HVR-H2 which contains one of the amino acid sequences of SEQ ID NOs: 55-64; and (III) HVR-H3, which contains one of the amino acid sequences of SEQ ID NOs: 65-74; and (b) a VL domain containing at least one, at least two, or all three sequences

- 18 041632 HVR VL values selected from (I) HVR-L1, which contains one of the amino acid sequences of SEQ ID NOs: 75-84;

(II) HVR-L2 which contains one of the amino acid sequences of SEQ ID NOs: 85-94; and (III) HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 95-104.

Another object of the invention is an antibody containing (a) HVR-H1, which contains one of the amino acid sequences of SEQ ID NOs: 45-54;

(b) HVR-H2 which contains one of the amino acid sequences of SEQ ID NOs: 55-64;

(c) HVR-H3 which contains one of the amino acid sequences of SEQ ID NOs: 65-74;

(d) HVR-L1 which contains one of the amino acid sequences of SEQ ID NOs: 75-84;

(e) HVR-L2 which contains one of the amino acid sequences of SEQ ID NOs: 85-94; and (e) HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 95-104. One of the objects of the invention is an antibody to C5 containing at least one, two, three, four, five or six HVRs selected from (a) HVR-H1, which contains one of the amino acid sequences of SEQ ID NOs: 45, 54, 117 . 126;

(b) HVR-H2 which contains one of the amino acid sequences of SEQ ID NOs: 55, 64, 118-120, 127;

(c) HVR-H3 which contains one of the amino acid sequences of SEQ ID NOs: 65, 74, 121, 128;

(d) HVR-L1 which contains one of the amino acid sequences of SEQ ID NOs: 75, 84, 122, 129;

(e) HVR-L2 which contains one of the amino acid sequences of SEQ ID NOs: 85, 94, 123-124, 130; and (e) HVR-L3 which contains one of the amino acid sequences of SEQ ID Nos: 95, 104, 125, 131.

One of the objects of the invention is an antibody containing at least one, at least two or all three HVR VH sequences selected from (a) HVR-H1, which contains one of the amino acid sequences of SEQ ID NOs: 45, 54, 117, 126 ;

(b) HVR-H2 which contains one of the amino acid sequences of SEQ ID NOs: 55, 64, 118-120, 127; and (c) HVR-H3 which contains one of the amino acid sequences of SEQ ID NOs: 65, 74, 121, 128.

In one embodiment, the antibody contains an HVR-H3 that contains one of the amino acid sequences of SEQ ID NOs: 65, 74, 121, 128. In another embodiment, the antibody contains an HVR-H3 that contains one of the amino acid sequences of SEQ ID NOs : 65, 74, 121, 128, and HVR-L3, which contains one of the amino acid sequences of SEQ ID NOs: 95, 104, 125, 131. In another embodiment, the antibody contains HVR-H3, which contains one of the amino acid sequences of SEQ ID NOs: 65, 74, 121, 128, HVR-L3, which contains one of the amino acid sequences of SEQ ID NOs: 95, 104, 125, 131, and HVR-H2, which contains one of the amino acid sequences of SEQ ID NOs: 55, 64, 118-120, 127.

In the following embodiment, the antibody contains (a) HVR-H1, which contains one of the amino acid sequences of SEQ ID NOs: 45, 54, 117, 126;

(b) HVR-H2 which contains one of the amino acid sequences of SEQ ID NOs: 55, 64, 118-120, 127; and (c) HVR-H3 which contains one of the amino acid sequences of SEQ ID NOs: 65, 74, 121, 128.

Another object of the invention is an antibody containing at least one, at least two or all three HVR VL sequences selected from (a) HVR-L1, which contains one of the amino acid sequences of SEQ ID NOs: 75, 84, 122, 129;

(b) HVR-L2 which contains one of the amino acid sequences of SEQ ID NOs: 85, 94, 123-124, 130; and (c) HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 95, 104, 125, 131.

In one of the embodiments of the invention, the antibody contains (a) HVR-L1, which contains one of the amino acid sequences of SEQ ID NOs: 75, 84, 122, 129;

(b) HVR-L2 which contains one of the amino acid sequences of SEQ ID NOs: 85, 94, 123-124, 130; And

- 19 041632 (c) HVR-L3, which contains one of the amino acid sequences of SEQ ID NOs: 95, 104,

125, 131.

Another object of the invention is an antibody according to the invention, which contains (a) a VH domain containing at least one, at least two or all three HVR VH sequences selected from (I) HVR-H1, which contains one of the amino acids sequences of SEQ ID NOs: 45, 54, 117, 126, (II) HVR-H2, which contains one of the amino acid sequences of SEQ ID NOs: 55, 64, 118-120, 127, and (III) HVR-H3, which contains one of the amino acid sequences of SEQ ID NOs: 65, 74, 121, 128; and (b) a VL domain comprising at least one, at least two, or all three HVR VL sequences selected from (I) HVR-L1, which comprises one of the amino acid sequences of SEQ ID NOs: 75, 84, 122, 129, (II) HVR-L2, which contains one of the amino acid sequences of SEQ ID NOs: 85, 94, 123-124, 130; and (c) HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 95, 104, 125, 131.

Another object of the invention is an antibody containing (a) HVR-H1, which contains one of the amino acid sequences of SEQ ID NOs: 45, 54, 117, 126;

(b) HVR-H2 which contains one of the amino acid sequences of SEQ ID NOs: 55, 64, 118-120, 127;

(c) HVR-H3 which contains one of the amino acid sequences of SEQ ID NOs: 65, 74, 121, 128;

(d) HVR-L1 which contains one of the amino acid sequences of SEQ ID NOs: 75, 84, 122, 129;

(e) HVR-L2 which contains one of the amino acid sequences of SEQ ID NOs: 85, 94, 123-124, 130; and (e) HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 95, 104, 125, 131.

In some embodiments, one or more amino acids of the anti-C5 antibody as described above are substituted at the following positions in the HVR:

(a) in HVR-H1 (SEQ ID NO: 45) at positions 5 and 6;

(b) in HVR-H2 (SEQ ID NO: 55) at positions 1, 3, 9, 11, 13 and 15;

(c) in HVR-H3 (SEQ ID NO: 65) at positions 2, 5, 6, 12 and 13;

(d) in HVR-L1 (SEQ ID NO: 75) at positions 1, 5, 7 and 9;

(e) in HVR-L2 (SEQ ID NO: 85) at positions 4, 5 and 6; and (e) in HVR-L3 (SEQ ID NO: 95) at positions 2, 4 and 12.

In some embodiments, the substitutions are conservative substitutions as described herein.

In some embodiments, one or more of the following substitutions may be made in any combination:

(a) in HVR-H1 (SEQ ID NO: 45) M5V or C6A;

(b) in HVR-H2 (SEQ ID NO: 55) C1A or G, Y3F, T9D or E, Y11K or Q, S13D or E or A15V;

(c) in HVR-H3 (SEQ ID NO: 65) G2A, V5Q or D, T6Y, Y12H or L13Y;

(d) in HVR-L1 (SEQ ID NO: 75) Q1R, N5Q or G, G7S, D9K or S;

(e) in HVR-L2 (SEQ ID NO: 85) K4T or E, L5T or A6H, A6E or A6Q;

(e) in HVR-L3 (SEQ ID NO: 95) C2S, C2N or C2T, F4K; or A12T, or A12H.

All possible combinations of the above substitutions are included in the consensus sequences of SEQ ID NOs: 126, 127, 128, 129, 130 and 131 for HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3, respectively .

In any of the above embodiments, the anti-C5 antibody is humanized. In one embodiment, the anti-C5 antibody comprises the HVRs of any of the above embodiments and also contains an acceptor human framework, eg, a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-C5 antibody comprises an HVR as provided in any of the above embodiments and also contains a VH or VL containing an FR sequence, where the FR sequences are the following sequences. In the case of the heavy chain variable domain, FR1 contains one of the amino acid sequences of SEQ ID NOs: 132-134, FR2 contains

- 20 041632 one of the amino acid sequences of SEQ ID NOs: 135-136, FR3 contains one of the amino acid sequences of SEQ ID NOs: 137-139, FR4 contains one of the amino acid sequences of SEQ ID NOs: 140-141. In the case of the light chain variable domain, FR1 contains one of the amino acid sequences of SEQ ID NOs: 142-143, FR2 contains one of the amino acid sequences of SEQ ID NOs: 144-145, FR3 contains one of the amino acid sequences of SEQ ID NOs: 146-147, FR4 contains one of the amino acid sequences of SEQ ID NO: 148. In another aspect of the invention, the anti-C5 antibody comprises a heavy chain variable domain (VH) sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to one of the amino acid sequences of SEQ ID NO: 1-10. In some embodiments, a VH sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-C5 antibody containing the specified sequence retains the ability to bind to C5. In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in one of SEQ ID NOs: 13, 16-30, and 32-34. In some embodiments, the substitutions, insertions, or deletions affect regions outside of the HVR (ie, FR). Optionally, the anti-C5 antibody contains the VH sequence shown in one of SEQ ID NOs: 1-10, including post-translational modifications of said sequence. In a specific embodiment of the invention, the VH contains one, two or three HVRs selected from (a) HVR-H1, which contains one of the amino acid sequences of SEQ ID NOs: 45-54; (b) HVR-H2 which contains one of the amino acid sequences of SEQ ID NOs: 55-64; and (c) HVR-H3 which contains one of the amino acid sequences of SEQ ID NOs: 65-74. In another aspect of the invention, the anti-C5 antibody contains a VL sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to one of the amino acid sequences of SEQ ID NOs: 11-20 . In some embodiments, a VL sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence , but an anti-C5 antibody containing this sequence retains the ability to bind to C5. In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in one of SEQ ID NOs: 11-20. In some embodiments, the substitutions, insertions, or deletions affect regions outside of the HVR (ie, FR). Optionally, the anti-C5 antibody contains the VL sequence shown in one of SEQ ID NOs: 15, 31, 35-38 and 96-99, including post-translational modifications of said sequence. In a specific embodiment, the VL contains one, two or three HVRs selected from (a) HVR-L1 which contains one of the amino acid sequences of SEQ ID NOs: 75-84;

(b) HVR-L2 which contains one of the amino acid sequences of SEQ ID NOs: 85-94; and (c) HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 95-104. Another object of the invention is an antibody to C5, where the antibody contains VH, presented in one of the above embodiments of the invention, and VL, presented in one of the above embodiments of the invention. In one embodiment, the antibody comprises the VH and VL sequences shown in one of SEQ ID NOs: 1-10 and one of SEQ ID NOs: 11-20, respectively, including post-translational modifications of these sequences.

In another aspect of the invention, the anti-C5 antibody comprises a heavy chain variable domain (VH) sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to one of the 10 amino acid sequences , 106-110. In some embodiments, a VH sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence. , but an anti-C5 antibody containing this sequence retains the ability to bind to C5. In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in one of SEQ ID NOs: 10, 106-110. In some embodiments, the substitutions, insertions, or deletions affect regions outside of the HVR (ie, FR). Optionally, the anti-C5 antibody contains the VH sequence shown in one of SEQ ID NOs: 10, 106-110, including post-translational modifications of said sequence. In a specific embodiment of the invention, the VH contains one, two or three HVRs selected from (a) HVR-H1, which contains one of the amino acid sequences of SEQ ID NOs: 45, 54, 117, 126;

(b) HVR-H2 which contains one of the amino acid sequences of SEQ ID NOs: 55, 64, 118-120, 127; and (c) HVR-H3 which contains one of the amino acid sequences of SEQ ID NOs: 65, 74, 121, 128.

In another aspect of the invention, an anti-C5 antibody contains a VH sequence that is at least

90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of the amino acid sequences of SEQ ID NOs: 10, 106-110. In some embodiments, a VH sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence. , but an anti-C5 antibody containing this sequence retains the ability to bind to C5. In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in one of SEQ ID NOs: 10, 106-110. In some embodiments, the substitutions, insertions, or deletions affect regions outside of the HVR (ie, FR). Optionally, the anti-C5 antibody contains the VH sequence shown in one of SEQ ID NOs: 10, 106-110, including post-translational modifications of said sequence. In a specific embodiment of the invention, the VH contains one, two or three HVRs selected from (a) HVR-H1, which contains one of the amino acid sequences of SEQ ID NOs: 45, 54, 117, 126;

(b) HVR-H2 which contains one of the amino acid sequences of SEQ ID NOs: 55, 64, 118-120, 127; and (c) HVR-H3 which contains one of the amino acid sequences of SEQ ID NOs: 65, 74, 121, 128.

In another aspect of the invention, the anti-C5 antibody contains a VH sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10. In some embodiments, implementation of the invention, the VH sequence is the amino acid sequence of SEQ ID NO: 10. In some embodiments, the VH sequence, which is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical, contains substitutions (eg, conservative substitutions), insertions or deletions relative to the reference sequence, but an anti-C5 antibody containing the indicated sequence retains the ability to bind to C5. In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in SEQ ID NO: 10.

In some embodiments, the substitutions, insertions, or deletions affect regions outside of the HVR (ie, FR). Optionally, the anti-C5 antibody contains the VH sequence shown in SEQ ID NO: 10, including post-translational modifications of said sequence. In a specific embodiment of the invention, the VH contains one, two or three HVRs selected from (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 54;

(b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 64; and (c) HVR-H3 which contains the amino acid sequence of SEQ ID NO: 74.

In another aspect of the invention, the anti-C5 antibody contains a VH sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 106. In some embodiments, carrying out the invention, the VH sequence is the amino acid sequence of SEQ ID NO: 106. In some embodiments, the VH sequence, which is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical, contains substitutions (eg, conservative substitutions), insertions or deletions relative to the reference sequence, but an anti-C5 antibody containing the indicated sequence retains the ability to bind to C5. In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in SEQ ID NO: 106. In some embodiments, the substitutions, insertions, or deletions affect regions outside the HVR (i.e., FR). Optionally, the anti-C5 antibody contains the VH sequence shown in SEQ ID NO: 106, including post-translational modifications of said sequence. In a specific embodiment of the invention, the VH contains one, two or three HVRs selected from (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 117;

(b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 118; and (c) HVR-H3 which contains the amino acid sequence of SEQ ID NO: 121.

In another aspect of the invention, the anti-C5 antibody contains a VH sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 107. In some embodiments, carrying out the invention, the VH sequence is the amino acid sequence of SEQ ID NO: 107. In some embodiments, the VH sequence, which is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical, contains substitutions (eg, conservative substitutions), insertions or deletions relative to the reference sequence, but an anti-C5 antibody containing the indicated sequence retains the ability to bind to C5. In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in SEQ ID NO: 107. In some embodiments, the substitutions, insertions, or deletions affect regions outside the HVR (i.e., FR). Optionally, the anti-C5 antibody contains the VH sequence shown in SEQ ID NO: 107, including post-translational modifications of said sequence. In a specific embodiment of the invention, the VH contains one, two or three HVRs selected from

- 22 041632 (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 117;

(b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 119; and (c) HVR-H3 which contains the amino acid sequence of SEQ ID NO: 121.

In another aspect of the invention, the anti-C5 antibody contains a VH sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 108. In some embodiments, carrying out the invention, the VH sequence is the amino acid sequence of SEQ ID NO: 108. In some embodiments, the VH sequence, which is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical, contains substitutions (eg, conservative substitutions), insertions or deletions relative to the reference sequence, but an anti-C5 antibody containing the indicated sequence retains the ability to bind to C5. In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in SEQ ID NO: 108. In some embodiments, the substitutions, insertions, or deletions affect regions outside the HVR (i.e., FR). Optionally, the anti-C5 antibody contains the VH sequence shown in SEQ ID NO: 108, including post-translational modifications of said sequence. In a specific embodiment of the invention, the VH contains one, two or three HVRs selected from (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 117;

(b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 118; and (c) HVR-H3 which contains the amino acid sequence of SEQ ID NO: 121.

In another aspect, the anti-C5 antibody comprises a VH sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 109. In some embodiments, carrying out the invention, the VH sequence is the amino acid sequence of SEQ ID NO: 109. In some embodiments, the VH sequence, which is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical, contains substitutions (eg, conservative substitutions), insertions or deletions relative to the reference sequence, but an anti-C5 antibody containing the indicated sequence retains the ability to bind to C5. In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in SEQ ID NO: 109. In some embodiments, the substitutions, insertions, or deletions affect regions outside of the HVR (i.e., FR). Optionally, the anti-C5 antibody contains the VH sequence shown in SEQ ID NO: 109, including post-translational modifications of said sequence. In a specific embodiment of the invention, the VH contains one, two or three HVRs selected from (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 117;

(b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 118; and (c) HVR-H3 which contains the amino acid sequence of SEQ ID NO: 121.

In another aspect of the invention, the anti-C5 antibody contains a VH sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 110. In some embodiments, carrying out the invention, the VH sequence is the amino acid sequence of SEQ ID NO: 110. In some embodiments, the VH sequence, which is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical, contains substitutions (eg, conservative substitutions), insertions or deletions relative to the reference sequence, but an anti-C5 antibody containing the indicated sequence retains the ability to bind to C5. In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in SEQ ID NO: 110. In some embodiments, the substitutions, insertions, or deletions affect regions outside the HVR (i.e., FR). Optionally, the anti-C5 antibody contains the VH sequence shown in SEQ ID NO: 110, including post-translational modifications of said sequence. In a specific embodiment of the invention, the VH contains one, two or three HVRs selected from (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 117;

(b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 120; and (c) HVR-H3 which contains the amino acid sequence of SEQ ID NO: 121.

In another aspect of the invention, the anti-C5 antibody comprises a light chain variable domain (VL) sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to one of the amino acid sequences of SEQ ID NOs: 20, 111-113. In some embodiments, a VL sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence , but an anti-C5 antibody containing this sequence retains the ability to bind to C5. In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in one of SEQ ID NOs: 20, 111-113. In some embodiments, the substitutions, insertions, or deletions affect regions outside of the HVR (ie, FR). Optionally, the anti-C5 antibody contains the VL sequence shown in one of SEQ ID NOs: 20, 111-113, including post-translational modifications of said sequence. In a specific embodiment, the VL contains one, two or three HVRs selected from (a) HVR-L1 which contains one of the amino acid sequences of SEQ ID NOs: 75, 84,

122, 129;

(b) HVR-L2 which contains one of the amino acid sequences of SEQ ID NOs: 85, 94, 123-124, 130; and (c) HVR-L3 which contains one of the amino acid sequences of SEQ ID NOs: 95, 104, 125, 131.

In another aspect of the invention, the anti-C5 antibody contains a VL sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 20. In some embodiments, implementation of the invention, the VL sequence is the amino acid sequence of SEQ ID NO: 20. In some embodiments, the VL sequence, which is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical, contains substitutions (eg, conservative substitutions), insertions or deletions relative to the reference sequence, but an anti-C5 antibody containing the indicated sequence retains the ability to bind to C5. In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in SEQ ID NO: 20. In some embodiments, the substitutions, insertions, or deletions affect regions outside the HVR (i.e., FR). Optionally, the anti-C5 antibody contains the VL sequence shown in SEQ ID NO: 20, including post-translational modifications of said sequence. In a specific embodiment of the invention, the VL contains one, two or three HVRs selected from (a) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 84;

(b) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 94; and (c) HVR-L3 which contains the amino acid sequence of SEQ ID NO: 104.

In another aspect of the invention, the anti-C5 antibody comprises a VL sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 111. In some embodiments, implementation of the invention, the VL sequence is the amino acid sequence of SEQ ID NO: 111. In some embodiments, the VL sequence, which is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical, contains substitutions (eg, conservative substitutions), insertions or deletions relative to the reference sequence, but an anti-C5 antibody containing the indicated sequence retains the ability to bind to C5. In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in SEQ ID NO: 111. In some embodiments, the substitutions, insertions, or deletions affect regions outside the HVR (i.e., FR). Optionally, the anti-C5 antibody contains the VL sequence shown in SEQ ID NO: 111, including post-translational modifications of said sequence. In a specific embodiment of the invention, the VL contains one, two or three HVRs selected from (a) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 122;

(b) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 123; and (c) HVR-L3 which contains the amino acid sequence of SEQ ID NO: 125.

In another aspect of the invention, the anti-C5 antibody comprises a VL sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 112. In some embodiments, carrying out the invention, the VL sequence is the amino acid sequence of SEQ ID NO: 112. In some embodiments, the VL sequence, which is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical, contains substitutions (eg, conservative substitutions), insertions or deletions relative to the reference sequence, but an anti-C5 antibody containing the indicated sequence retains the ability to bind to C5. In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in SEQ ID NO: 112. In some embodiments, the substitutions, insertions, or deletions affect regions outside the HVR (i.e., FR). Optionally, the anti-C5 antibody contains the VL sequence shown in SEQ ID NO: 112, including post-translational modifications of said sequence. In a specific embodiment of the invention, the VL contains one, two or three HVRs selected from (a) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 122;

(b) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 123; and (c) HVR-L3 which contains the amino acid sequence of SEQ ID NO: 125.

In another aspect of the invention, the anti-C5 antibody comprises a VL sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 113. In some embodiments, implementation of the invention, the VL sequence is the amino acid sequence of SEQ ID NO: 113. In some embodiments of the invention, the VL sequence, which is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical, contains substitutions (for example, conservative substitutions), insertions or deletions relative to

- 24 041632 reference sequences, but an antibody to C5 containing the specified sequence retains the ability to bind to C5. In some embodiments, a total of 1 to 10 amino acids are changed, inserted, and/or deleted by deletion in SEQ ID NO: 113. In some embodiments, the substitutions, insertions, or deletions affect regions outside of the HVR (i.e., FR). Optionally, the anti-C5 antibody contains the VL sequence shown in SEQ ID NO: 113, including post-translational modifications of said sequence. In a specific embodiment of the invention, the VL contains one, two or three HVRs selected from (a) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 122;

(b) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 124; and (c) HVR-L3 which contains the amino acid sequence of SEQ ID NO: 125.

Another object of the invention is an antibody to C5, where the antibody contains VH, presented in one of the above embodiments of the invention, and VL, presented in one of the above embodiments of the invention. In one embodiment, the antibody comprises the VH and VL sequences shown in one of SEQ ID NOs: 10, 106-110 and one of SEQ ID NOs: 20, 111-113, respectively, including post-translational modifications of these sequences. In one embodiment, the antibody contains the VH sequence shown in SEQ ID NO: 10 and the VL sequence shown in SEQ ID NO: 20. In one embodiment, the antibody contains the VH sequence shown in SEQ ID NO: 106, and a VL sequence as set forth in SEQ ID NO: 111. In another embodiment, the antibody comprises a VH sequence as set forth in SEQ ID NO: 107 and a VL sequence as set forth in SEQ ID NO: 111. In a further embodiment, the antibody comprises the sequence VH shown in SEQ ID NO: 108 and a VL sequence shown in SEQ ID NO: 111. In another embodiment, the antibody comprises a VH sequence shown in SEQ ID NO: 109 and a VL sequence shown in SEQ ID NO: 111. In another embodiment, the antibody contains the VH sequence shown in SEQ ID NO: 109 and a VL sequence as set forth in SEQ ID NO: 112. In another embodiment, the antibody comprises a VH sequence as set forth in SEQ ID NO: 109 and a VL sequence as set forth in SEQ ID NO: 113. In another embodiment, of the invention, the antibody contains the VH sequence shown in SEQ ID NO: 110 and the VL sequence shown in SEQ ID NO: 113.

One of the objects of the invention is an antibody to C5, where the antibody contains a VH sequence containing (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 54;

(b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 64; and (c) HVR-H3 which contains the amino acid sequence of SEQ ID NO: 74 and a VL sequence containing (a) HVR-L1 which contains the amino acid sequence of SEQ ID NO: 84;

(b) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 94; and (c) HVR-L3 which contains the amino acid sequence of SEQ ID NO: 104.

Another object of the invention is an antibody to C5, where the antibody contains a VH sequence containing (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 117;

(b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 118; and (c) HVR-H3 which contains the amino acid sequence of SEQ ID NO: 121 and a VL sequence containing (a) HVR-L1 which contains the amino acid sequence of SEQ ID NO: 122;

(b) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 123; and (c) HVR-L3 which contains the amino acid sequence of SEQ ID NO: 125.

Another object of the invention is an antibody to C5, where the antibody contains a VH sequence containing (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 117;

(b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 119; and (c) HVR-H3 which contains the amino acid sequence of SEQ ID NO: 121 and a VL sequence containing (a) HVR-L1 which contains the amino acid sequence of SEQ ID NO: 122;

(b) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 123; and (c) HVR-L3 which contains the amino acid sequence of SEQ ID NO: 125.

Another object of the invention is an antibody to C5, where the antibody contains a VH sequence containing (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 117;

(b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 118; And

- 25 041632 (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 121, and a VL sequence, containing (a) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 122;

(b) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 124; and (c) HVR-L3 which contains the amino acid sequence of SEQ ID NO: 125.

Another object of the invention is an antibody to C5, where the antibody contains a VH sequence containing (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 117;

(b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 120; and (c) HVR-H3 which contains the amino acid sequence of SEQ ID NO: 121 and a VL sequence containing (a) HVR-L1 which contains the amino acid sequence of SEQ ID NO: 122;

(b) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 124; and (c) HVR-L3 which contains the amino acid sequence of SEQ ID NO: 125.

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

The following object of the invention is an antibody that binds to the same epitope as the antibody to C5 presented in the present description. For example, in some embodiments, the invention provides an antibody that binds to the same epitope as the antibody listed in Table. 2. As demonstrated in the following examples of the invention, all antibodies to C5 listed in table. 2 are grouped into one basket for the indicated C5 epitope and have pH-dependent binding characteristics.

An additional object of the invention is an antibody that binds to the same epitope as the antibody presented in the present description. Another object of the invention is an antibody that binds to the same epitope as the antibody listed in table. 7. In some embodiments, the invention provides an antibody that binds to an epitope present in a fragment that consists of amino acids 33-124 of the C5 beta chain (SEQ ID NO: 40). In some embodiments, an antibody is provided that binds to an epitope on the C5 beta chain (SEQ ID NO: 40) that contains at least one fragment selected from the group consisting of amino acids 47-57, 70-76, and 107 -110. In some embodiments, the invention provides an antibody that binds to an epitope in the C5 beta chain fragment (SEQ ID NO: 40) that contains at least one amino acid selected from the group consisting of Thr47, Glu48, Ala49, Phe50, Asp51 , Ala52, Thr53, Lys57, His70, Val71, His72, Ser74, Glu76, Val107, Ser108, Lys109 and His110. In another embodiment, the epitope for an anti-C5 antibody of the present invention is a conformational epitope.

In another aspect of the invention, the anti-C5 antibody provided in one of the above embodiments of the invention is a monoclonal antibody, including a chimeric, humanized or human antibody. In one embodiment, the anti-C5 antibody is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabodi, or F(ab')2 fragment. In another embodiment, the antibody is a full length antibody, such as an intact IgG1 or IgG4 class antibody, or an antibody of another class or isotype as defined herein.

According to another aspect of the invention, an anti-C5 antibody according to one of the above embodiments of the invention may have any of the characteristics described below in sections 1-7, individually or in combination.

1. Affinity of antibodies.

In some embodiments, an antibody provided herein has a dissociation constant (Kd) value of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0, 01 nM or less, or 0.001 nM or less (eg 10 ^-8 M or less, eg 10 ^-8 to 10 ^-13 M, eg 10 ^-9 to 10 ^-13 M).

In one embodiment, the Kd value is measured using a radiolabeled antigen (RIA) binding assay. In one of the embodiments of the invention, RIA is carried out using the Fab version of the antibody of interest and its antigen. For example, the binding affinity of a Fab to an antigen is measured in solution by equilibrating the Fab with a minimum concentration of ¹²⁵ I-labeled antigen in the presence of titrated dilutions of unlabeled antigen, then capturing the bound antigen on an anti-Fab sensitized plate (see, e.g., Chen et al., J. Mol. Biol., 293, 1999, pp. 865-881). To ensure

MICROTITER® multiwell plates (Thermo Scientific) are sensitized overnight with an anti-Fab capture antibody (Cappel Labs) at a concentration of 5 µg/ml in 50 mM sodium carbonate (pH 9.6) and then blocked with 2% (w/v) bovine serum albumin in PBS for 2-5 hours at room temperature (about 23°C). In a non-absorbent plate (Nunc, no. 269620), 100 or 26 pM ¹²⁵ I-labeled antigen is mixed with serial dilutions of the Fab of interest (e.g., the anti-VEGF antibody for evaluation, Fab-12, see Presta et al. , Cancer Res., 57, 1997, pp. 4593-4599). The Fab of interest is then incubated overnight; however, the incubation can be carried out for a longer period of time (for example, for 65 hours) in order to ensure that equilibrium is reached. The mixtures are then transferred to a prepared capture plate and incubated at room temperature (eg, for 1 hour). The solution is then removed and the plate washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. After the plates are dry, 150 μl/well of scintillation fluid (MICROSCINT-20™; Packard) is added to the plates and the plates are read using a TOPCOUNT™ gamma counter (Packard) for 10 minutes. To perform a competitive binding assay, concentrations of each Fab are chosen to provide a binding level less than or equal to 20% of the maximum.

In another embodiment of the invention, Kd is measured by surface plasmon resonance using a BIACORE® device. For example, the assay is performed with a BIACORE®-2000 or BIACORE®-3000 device (GE Healthcare Inc., Piscataway, NJ) at 25° C. using antigen immobilized on CM5 chips with an immobilization level corresponding to ~ 10 response units (RU). In one embodiment, carboxymethylated dextran (CM5, GE Healthcare Inc.) biosensor chips are activated with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) hydrochloride according to the supplier's instructions. . The antigen is diluted with 10 mM sodium acetate, pH 4.8 to a concentration of 5 μg/ml (~ 0.2 μM) before injection at a flow rate of 5 μl/min to achieve an immobilization level corresponding to about 10 response units (RU) of the cross-linked protein. After injection of the antigen, 1 M ethanolamine is injected to block the unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (from 0.78 to 500 nM) in PBS supplemented with 0.05% polysorbate 20 (TWEEN-20™) as a surfactant (PBS) are injected at 25°C with a flow rate of approximately 25 μl /min The rate of association reaction (k _on ) and dissociation reaction (ko _f f) is calculated using a simple 1:1 Langmuir binding model (Evaluation software version 3.2, BIACORE®) by simultaneously fitting association and dissociation sensorgrams. The equilibrium constant of the dissociation reaction (Kd) is calculated as the ratio k _off /k _on (see, for example, Chen et al., J. Mol. Biol., 293, 1999, pp. 865-881). If the association reaction rate exceeds 106 M ^-1 s ^-1 as measured by the surface plasmon resonance method described above, then the association reaction rate can be determined using the fluorescence quenching method, which measures the increase or decrease in the intensity of fluorescence emission (excitation wavelength 295 nm ; emission wavelength 340 nm, bandwidth 16 nm) at 25°C applied at a concentration of 20 nM anti-antigen antibody (Fab-format) in PBS, pH 7.2, in the presence of increasing concentrations of antigen, measuring with a spectrometer such as as a spectrophotometer equipped with a flow stop unit (Aviv Instruments) or an SLM-AMINCO™ 8000 series spectrophotometer (ThermoSpectronic) while stirring the contents of the cuvette.

2. Fragments of antibodies.

In some embodiments, the antibody of the present invention is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') ₂ , Fv, and scFv fragments, and others described below. For a review of some antibody fragments, see Hudson et al., Nat. Med., 9, 2003, p. 129-134. For a review of scFv fragments, see, for example, Plueckthun in The Pharmacology of Monoclonal Antibodies, ed. Rosenburg and Moore, Springer, New York, vol. 113, 1994, p. 269-315; see also WO 93/16185; and US Pat _.

Dimeric antibodies are antibody fragments with two antigen-binding sites, which can be bivalent or bispecific (see, for example, EP 0404097; WO 1993/01161; Hudson et al., Nat. Med., 9, 2003, c. 129-134 and Holliger et al., Proc. Natl. Acad. Sci., USA, 90, 1993, pp. 6444-6448). Trimeric and tetrameric antibodies are also described in Hudson et al., Nat. Med., 9, 2003, p. 129-134).

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

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Antibody fragments can be created by a variety of techniques, including (but not limited to) proteolytic cleavage of intact antibodies, as well as obtained using recombinant host cells (eg, E. coli or phage) described in the present description.

3. Chimeric and humanized antibodies.

In some embodiments of the invention, the antibody specified in the present description is a chimeric antibody. Some chimeric antibodies are described, for example, in US No. 4816567; and Morrison et al., Proc. Natl. Acad. Sci., USA, 81, 1984, p. 6851-6855. In one example, a chimeric antibody comprises a non-human variable region (eg, a variable region from an antibody from mice, rats, hamsters, rabbits, or non-human primates such as monkeys) and a human constant region. In another example, the chimeric antibody is a class-switched antibody whose class or subclass has been changed from the parent antibody. Chimeric antibodies include their antigen-binding fragments.

In some embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parent non-human antibody. Typically, a humanized antibody contains one or more variable domains in which the HVRs, eg CDRs (or portions thereof), are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. The humanized antibody may optionally contain at least a portion of a human constant region. In some embodiments, certain FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (eg, an antibody from which HVR residues are derived), for example, to maintain or improve the specificity or affinity of the antibody.

Information about humanized antibodies and methods for their creation are summarized in, for example, Almagro, Front. Biosci., 13, 2008, p. 1619-1633, and also described in Riechmann et al., Nature, 332, 1988, p. 323-329; Queen et al., Proc. Nail. Acad. Sci., USA, 86, 1989, p. 10029-10033; US No. 5821337, 7527791, 6982321 and 7087409; Kashmiri et al., Methods, 36, 2005, p. 25-34 (description of transplantation of SDR (a-CDR)); Padlan, Mol. Immunol., 28, 1991, p. 489-498 (description of applying a new coating); Dall'Acqua et al., Methods, 36, 2005, p. 43-60 (description of permutation FR); and Osbourn et al., Methods, 36, 2005, p. 61-68; and Klimka et al., Br. J. Cancer, 83, 2000, p. 252-260 (describing a directed selection approach for FR permutation).

Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using best fit methods (see, for example, Sims et al., J. Immunol., 151, 1993, c. 2296-2308, framework regions derived from the consensus sequence of a specific subgroup of human antibody light and heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci., USA, 89, 1992, c. 4285 -4289 and Presta et al., J. Immunol., 151, 1993, pp. 2623-2632), human mature (with somatic mutations) or human germline framework regions (see, e.g., Almagro and Fransson, Front Biosci., 13, 2008, pp. 1619-1633), and framework regions obtained by screening FR libraries (see, e.g., Vasa et al., J. Biol. Chem., 272, 1997, p. 10678-10684 and Rosok et al., J. Biol Chem., 271, 1996, pp. 22611-22618).

4. Human antibodies.

In some embodiments, the antibody of the present invention is a human antibody. Human antibodies can be generated using a variety of techniques known in the art. Human antibodies are described in general by van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5, 2001, p. 368-374; and Lonberg, Curr. Opin. Immunol., 20, 2008, p. 450-459.

Human antibodies can be generated by administering an immunogen to a transgenic animal modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. These animals typically contain all or part of the human immunoglobulin loci instead of endogenous immunoglobulin loci that are either present outside the chromosomes or integrated arbitrarily into the chromosomes of the animal. In such transgenic mice, endogenous immunoglobulin loci are usually inactivated. For a review of methods for generating human antibodies in transgenic animals, see Lonberg, Nat. Biotech., 23, 2005, p. 1117-1125 (see, for example, also US No. 6075181 and US No. 6150584 for the XENOMOUSE™ Technology; US No. 5770429 for the HUMAB® Technology; US No. 7041870 for the K-M MOUSE® Technology and US Patent Application Publication 2007/0061900 description of VELOCIMOUSE® technology). Human variable regions from intact antibodies derived from these animals can be further modified, for example by combining with another human constant region.

Human antibodies can be generated using hybridoma-based methods. Human myeloma and murine-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described (see, for example, Kozbor, J. Immunol., 133, 1984, c. 3001-3005; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, published by Marcel Dekker, Inc.,

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New York, 1987, p. 51-63; and Boerner et al., J. Immunol., 147, 1991, p. 86-95). Human antibodies generated using human B-cell hybridoma technology are also described in Li et al., Proc. Nail. Acad. Sci., USA, 103, 2006, p. 3557-3562. Additional methods include those described, for example, in US No. 7189826 (description of obtaining monoclonal human antibodies of the IgM type from hybridoma cell lines) and in Ni, Xiandai Mianyixue, 26, 2006, c. 265-268 (description of human-human hybridomas). Human hybridoma technology (Trioma technology) is also described by Vollmers and Brandlein, Histology and Histopathology, 20, 2005, p. 927-937; and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27, 2005, p. 185-191.

Human antibodies can also be generated by isolating the variable domain sequences of Fv clones selected from phage display libraries that have been generated using human antibodies. These variable domain sequences can then be combined with the desired human constant domain. Methods for selecting human antibodies from antibody libraries are described below.

5. Antibodies obtained from libraries.

Antibodies of the invention can be isolated by screening combinatorial libraries of antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening said libraries for antibodies having the desired binding characteristics. These methods are summarized in, for example, Hoogenboom et al., Methods in Molecular Biology, 178, 2001, c. 1-37 and are also described, for example, in McCafferty et al., Nature, 348, 1990, c. 552-554; Clackson et al., Nature, 352, 1991, p. 624-628; Marks et al., J. Mol. Biol., 222, 1992, p. 581-597; Marks and Bradbury, Methods in Molecular Biology, 248, 2003, p. 161-175; Sidhu et al., J. Mol. Biol., 338, 2004, p. 299-310; Lee et al., J. Mol. Biol., 340, 2004, p. 1073-1093; Fellouse, Proc. Natl. Acad. Sc., USA, 101, 2004, p. 12467-12472 and Lee et al., J. Immunol. Methods, 284, 2004, p. 119-132.

In some phage display techniques, populations of VH and VL genes are cloned individually by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which are then screened for antigen-binding phage according to the method described by Winter et al., Ann . Rev. Immunol., 12, 1994, p. 433-455. The phage typically displays antibody fragments either as single chain Fv fragments (scFv) or as Fab fragments. Libraries derived from immunized sources include antibodies with high affinity for the immunogen without the need for hybridomas. Alternatively, an untreated (naive) population (e.g. from a human body) can be cloned, producing a single source of antibodies to a wide range of foreign as well as self antigens without any immunization, as described in Griffiths et al., EMBO J., 12, 1993, p. 725-734. Finally, naïve libraries can also be generated synthetically by cloning non-rearranged V-gene segments from stem cells and using random sequence PCR primers to encode CDR3 hypervariable regions and to perform in vitro rearrangement according to the method described by Hoogenboom. and Winter, J. Mol. Biol., 227, 1992, p. 381-388. Human antibody phage libraries are described, for example, in the following patent publications: US 5750373 and US Patent Application Publications 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936 and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody libraries are considered to be human antibodies or human antibody fragments.

6. Multispecific antibodies.

In some embodiments, the antibody described herein is a multispecific antibody, such as a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have specificities that bind to at least two different sites. In some embodiments, one of the binding specificities allows binding to C5 and the other to any other antigen. In some embodiments, the bispecific antibodies can bind to two different C5 epitopes. Bispecific antibodies can also be used to localize cytotoxic agents in cells that express C5. Bispecific antibodies can be obtained as full length antibodies or antibody fragments.

Techniques for generating multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature, 305, 1983, c. 537-540, WO 93/08829 and Traunecker et al., EMBO J., 10, 1991, pp. 3655-3659), and knob-in-hole technology (providing boss-to-bottom interaction, see, for example, US No. 5731168). Multispecific antibodies can also be obtained by creating controlled electrostatic influences to obtain antibody molecules with heterodimeric Fc (WO 2009/089004); cross-linking two or more antibodies or fragments (see, for example, US No. 4676980 and Brennan and others, Science, 229, 1985, c. 81-83); the use of leucine zippers to produce bispecific antibodies (see, for example, 29 041632, Kostelny et al., J. Immunol. 148, 1992, pp. 1547-1553); the use of dimeric antibody technology (diabodies) to create bispecific antibody fragments (see, for example, Holliger et al., Proc. Natl.

Acad. Sci., USA, 90, 1993, p. 6444-6448); and the use of single chain Fv (sFv) dimers (see, for example,

Gruber et al., J. Immunol., 152, 1994, p. 5368-5374); and obtaining trispecific antibodies according to the method described, for example, Tutt and others, J. Immunol., 147, 1991, c. 60-69).

Also within the scope of the present invention are engineered antibodies with three or more functional antigen-binding sites, including octopus antibodies (see, for example, US 2006/0025576 A1).

The antibody or fragment thereof may also include a dual action Fab or DAF containing an antigen-binding site that binds to C5 as well as to another antigen other than it (see, for example, US 2008/0069820).

7. Antibody variants.

Some embodiments of the invention relate to variants of the amino acid sequences of the antibodies presented in the present description. For example, it may be desirable to increase the binding affinity and/or other biological properties of an antibody. Variants of the amino acid sequence of an antibody can be obtained by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by synthesizing peptides. These modifications include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequences of the antibody. Any combination of deletions, insertions, and substitutions can be used to obtain the final construct, provided that the final construct has the desired characteristics, such as the ability to bind to an antigen.

a) Variants obtained by substitution, insertion and deletion.

Some embodiments of the invention are antibody variants that have one or more amino acid substitutions. Sites of interest for displacement mutagenesis include HVR and FR. Conservative substitutions are presented below in table. 1 under the heading preferred substitutions and are described below with reference to the classes of amino acid side chains. More important substitutions are presented below in Table. 1 under the heading exemplified substitutions and are described below with reference to the amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the products screened for desired activity, eg, retention/increase in antigen binding, reduced immunogenicity, or increased ADCC or CDC.

Table 1

Original balance Exemplary substitutions Preferred substitutions

Ala (A) Val; Leu; He Val Arg(R) Lys; gin; Asn Lys Asn(N) gin; His; Asp, Lys; Arg gin Asp(D) Glu; Asn Glu Cys(C) Ser; Ala Ser Gin (Q) asn; Glu Asn Glu(G) asp; gin asp Gly (G) Ala Ala His(H) asn; gin; Lys; Arg Arg He(I) Leu; Val; met; Ala; Ph; norleucine Leu Leu(L) norleucine; He; Val; met; Ala; Phe lie Lys (K) Arg; gin; Asn Arg Met(M) Leu; Ph; He Leu Phe(F) trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser(S) Thr Thr Thr(T) Val; Ser Ser TRP(W) Tyr; Phe Tyr Tyr (Y) trp; Ph; Thr; Ser Phe Val(V) lie; Leu; met; Ph; Ala; norleucine Leu

Amino acids can be grouped based on common side chain properties as follows:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) main: His, Lys, Arg;

(5) residues that affect chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions mean the replacement of a representative of one of the specified classes with a representative from another class.

One type of resulting replacement option involves replacing one or more

- 30 041632 residues of the hypervariable region of the parent antibody (for example, humanized or human antibodies). Typically, the resulting variant(s) selected for further study should have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/ or must(s) have substantially retained certain biological properties of the parent antibody. An example of a substitution variant is an affinity matured antibody, which can be generated, for example, using phage display-based affinity maturation technologies, such as those described herein. In general, the method is as follows: one or more HVR residues are mutated and antibody variants are displayed on phage and screened for a particular biological activity (eg, binding affinity).

Changes (eg substitutions) can be made in the HVR, eg to increase the affinity of the antibody. These changes can be made in hot (active) spots in HVR, i.e. residues encoded by codons that mutate at a high rate during somatic maturation (see, for example, Chowdhury, Methods Mol. Biol., 207, 2008, c. 179-196), and/or in residues that come into contact with antigen , followed by testing the VH or VL variant for binding affinity. Affinity maturation by building and reselecting from secondary libraries is described, for example, in Hoogenboom et al., Methods in Molecular Biology, 178, 2002, c. 1-37. In some embodiments, the affinity maturation process introduces diversity into the variable region genes selected for maturation using any of a wide variety of methods (eg, reduced precision PCR, strand shuffling, or site directed mutagenesis using oligonucleotides). Then a secondary library is created. The library is then screened to identify any antibody variants with the desired affinity. Another method used to introduce diversity includes HVR-targeting approaches that randomize multiple HVR residues (eg, 4-6 residues at a time). HVR residues involved in antigen binding can be specifically identified, for example, using alanine-scanning mutagenesis or modeling. Often the targets are primarily CDR-H3 and CDRL3.

In some embodiments, the substitutions, insertions, or deletions may affect one or more HVRs, as long as the changes do not significantly reduce the ability of the antibody to bind to the antigen. For example, conservative changes can be made to the HVR (eg conservative substitutions as described herein) that do not significantly reduce binding affinity. These changes, for example, may be outside of the HVR residues involved in contacting the antigen. In some embodiments, in the VH and VL variant sequences above, each HVR is either unchanged or contains no more than one, two, or three amino acid substitutions.

A valuable method for identifying antibody residues or regions that can be mutated is the so-called alanine-scanning mutagenesis described in Cunningham and Wells, Science, 244, 1989, c. 1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to decide whether whether this will affect the interaction of the antibody with the antigen. Additional substitutions can be introduced at amino acid positions that have been shown to be functionally sensitive to the initial substitutions. Alternatively or additionally, the crystal structure of the antigen-antibody complex is examined to identify points of contact between the antibody and antigen. These contacting residues and neighboring residues can be considered as targets or excluded from consideration as candidates for replacement. Variants can be screened to determine if they have the desired properties.

Insertions into amino acid sequences include amino- and/or carboxy-terminal fusions ranging in length from a single residue to polypeptides containing one hundred or more residues, as well as insertions of one or more amino acid residues into the sequence. An example of the result of end insertions is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion of the N- or C-terminus of the antibody with an enzyme (eg, for ADEPT (antibody-directed enzyme prodrug therapy) or a polypeptide that prolongs the serum half-life of the antibody.

b) Variants of glycosylation.

In some embodiments of the invention, the antibody of the invention is modified to increase or decrease the degree of glycosylation of the antibody. The addition or deletion of glycosylation sites in an antibody can be easily accomplished by altering the amino acid sequence in such a way that one or more glycosylation sites are created or removed.

If the antibody contains an Fc region, then you can change the carbohydrate attached to it. native

- 31 041632 antibodies that are produced by mammalian cells, as a rule, contain a branched biantenna oligosaccharide, which, as a rule, is attached via an N-linkage to the Asn297 CH2 domain of the Fc region (see, for example, Wright et al., TIBTECH, 15, 1997, pp. 26-32). The oligosaccharide may include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose attached to the GlcNAc in the stem of the biantenna oligosaccharide structure. In some embodiments, modifications to the oligosaccharide in an antibody of the invention can be made to create antibody variants with certain improved properties.

One of the embodiments of the invention are variants of the antibody having a carbohydrate structure in which the content of fucose attached (directly or indirectly) to the Fc region is reduced. For example, the fucose content in said Fc region may be 1 to 80%, 1 to 65%, 5 to 65%, or 20 to 40%. The fucose content is determined by calculating the average sugar chain fucose content per Asn297 relative to the sum of all glycostructures attached to Asn297 (e.g. complex, hybrid and high mannose structures), which is measured by MALDI-TOF mass spectrometry according to the method described, for example, in WO 2008/077546. Asn297 denotes an asparagine residue present at approximately position 297 in the Fc region (EU numbering of Fc region residues); however, due to minor sequence variations, the Asn297 antibody can also be located at a position about ±3 amino acids upstream or downstream from position 297, i.e. between positions 294 and 300. These fucosylated variants may have improved ADCC function (see, for example, US 2003/0157108 (Presta L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd.). defucosylated or fucose-deficient antibody variants are US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140/0; US 2004/0132140/0; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; Jazakiol et al. ., 336, 2004, pp. 1239-1249; Yamane-Ohnuki et al., Biotech. Bioeng., 87, 2004, pp. 614-622). Examples of cell lines that have the ability to produce defucosylated antibodies are protein fucosylation deficient Lec 13 CHO cells (Ripka et al., Arch. Biochem. Biophys., 249, 1986, c. 533-545; US 2003/0157108 (Presta L.) and WO 2004/056312 (Adams et al., see especially Example 11), and gene knockout cell lines, e.g. alpha-1,6-fucosyltransferase knockout CHO cells, FUT8 (see ., for example, Yamane-Ohnuki et al., Biotech Bioeng., 87, 2004, pp. 614-622), Kanda et al., Biotechnol. and WO 2003/085107).

In addition, variants of antibodies with bisected oligosaccharides are described, for example, in which the bisected oligosaccharide attached to the Fc region of the antibody is bisected with GlcNAc. These antibody variants may have reduced fucosylation and/or increased ADCC function. Examples of these antibody variants are described, for example, in WO 2003/011878 (Jean-Mairet et al.); US No. 6602684 (Umana et al.) and US 2005/0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also proposed. These antibody variants may have increased CDC function. These 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.

According to specific embodiments of the invention, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby creating a variant Fc region. A variant Fc region may comprise a human Fc region sequence (eg, human IgG1, IgG2, IgG3, or IgG4 Fc regions) comprising an amino acid modification (eg, substitution) at one or more amino acid positions.

Some embodiments of the invention relate to an antibody variant that has some but not all effector functions, making it a promising candidate for applications where the in vivo half-life of the antibody is important, but some effector functions (such as CDC and ADCC) are not are necessary or harmful. In vitro and/or in vivo cytotoxicity assays can be performed to confirm the reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks the ability to bind to Fc-gamma R (and therefore likely lacks ADCC activity), but retains the ability to bind to FcRn. Primary cells mediating ADCC, ie. NK cells express only Fc-gamma RIII while monocytes express Fc-gamma RI, Fc-gamma RII and Fc-gamma RIII. Data on FcR expression on hematopoietic cells are summarized in Table 1. 3 on p. 464 by Ravetch and Kinet, Annu. Rev. Immunol., 9, 1991, p. 457-492. Examples of in vitro assays for ADCC activity of a molecule of interest are, but not limited to, those described in US No. 5500362 (see, for example, Hellstrom et al., Proc. Nail. Acad. Sci., USA, 83, 1986 , pp. 7059-7063; and Hellstrom et al., Proc. Nat'l Acad. Sci., USA, 82, 1985, pp. 1499-1502); US No. 5821337 (see Bruggemann et al., J. Exp. Med., 166, 1987, c. 1351-1361). In al

Alternatively, non-radioactive assays can be used (see, for example, non-radioactive flow cytometry-based ACTI™ cytotoxicity assay (CellTechnology, Inc. Mountain View, Calif.); and non-radioactive CytoTox 96® cytotoxicity assay (Promega , Madison, Wisconsin. Suitable effector cells for such assays are peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or in addition, the ADCC activity of a molecule of interest can be assessed in vivo, for example, by animal model, for example, as described by Clynes et al., Proc. Natl. Acad. Sci., USA, 95, 1998, pp. 652-656 Clq binding assays can also be performed to confirm that the antibody cannot bind Clq and therefore lacks CDC activity (see e.g. WO 2006/029879 and WO 2005/100402 ELISA for assessing Clq and C3c binding). perform CDC analysis (see, for example, Gazzano-Santoro et al., J. Immunol. Methods, 202, 1996, p. 163-171; Cragg et al., Blood, 101, 2003, pp. 1045-1052; and Cragg, Blood, 103, 2004, p. 2738-2743). Determination of FcRn binding and in vivo clearance/half-life can also be performed using methods known in the art (see, for example, Petkova et al., Int'1. Immunol., 18(12), 2006, c. 1759 -1769).

Antibodies with reduced effector function include antibodies with the replacement of one or more of the following residues of the Fc region, such as 238, 265, 269, 270, 297, 327 and 329 (see, for example, US No. 6737056). These Fc mutants include Fc mutants with substitutions in two or more of the following amino acid positions: 265, 269, 270, 297 and 327, including the so-called DANA Fc region mutant carrying the replacement of residues 265 and 297 with alanine (US No. 7332581 ).

Some variants of antibodies with increased or decreased ability to bind to FcR have been described (see, for example, US No. 6737056; WO 2004/056312; and Shields et al., J. Biol. Chem., 276, 2001, c. 6591-6604) .

In specific embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that increase ADCC, such as substitutions at positions 298, 333, and/or 334 of the Fc region (EU residue numbering).

In some embodiments, changes are made in the Fc region that result in an altered (i.e., either increased or decreased) Clq binding ability and/or complement dependent cytotoxicity (CDC), as described, for example, in US No. 6,194,551, WO 1999/51642 and Idusogie et al., J. Immunol., 164, 2000, p. 4178-4184.

Antibodies with extended half-life and increased ability to bind to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the embryo (Guyer et al., J. Immunol., 117, 1976, c. 587-593; and Kim and et al., J. Immunol., 24, 1994, pp. 2429-2434), described in US 2005/0014934 A1 (Hinton et al.). These antibodies contain an Fc region with one or more substitutions that increase the binding of the Fc region to FcRn. These Fc region variants include variants with substitutions of one or more of the following Fc region residues: 378, 380, 382, 413, 424 or 434, for example, a substitution of residue 434 in the Fc region (US No. 7371826).

Additional examples regarding other variants of the Fc region are also described in Duncan, Nature, 322, 1988, c. 738-740; in US No. 5648260 and 5624821 and in WO 1994/29351.

d) Cysteine engineered antibody variants.

In some embodiments of the invention, it may be desirable to create cysteine-engineered antibodies, such as thioMAbs, in which one or more residues in the antibody are replaced with cysteine residues. In specific embodiments of the invention, the substituted residues are located at accessible sites on the antibody. By replacing these residues with cysteine, reactive thiol groups are placed at accessible sites on the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or drug linker moieties, to generate an immunoconjugate as indicated below in this description. In some embodiments, any one or more of the following residues may be replaced by a cysteine: V205 (Cabot numbering) light chain; A118 (EU number) of the heavy chain; and S400 (EU number) of the heavy chain Fc region. Antibodies constructed with cysteine can be generated according to the method, for example, described in US No. 7521541.

e) Derivatives of antibodies.

In some embodiments, an antibody provided herein can be further modified to contain additional non-protein fragments known in the art and readily available. Fragments suitable for antibody derivatization include, but are not limited to, water-soluble polymers. Examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, copolymer ethylene/maleic anhydride, polyamino acids (either homopolymers or random copolymers) and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene copolymers

- 33 041632 side/ethylene oxide, polyoxyethylene polyols (eg glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde may have a manufacturing advantage due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. In general, the amount and/or type of polymers used for derivatization can be determined in terms of, but not limited to, specific properties or functions of the antibody to be improved, whether the antibody derivative is intended to be used in therapy under certain conditions, etc. .d.

Another embodiment of the invention are conjugates of an antibody and a non-protein fragment, which can be selectively heated by exposing it to radiation. In one embodiment, the non-protein fragment is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci., USA, 102, 2005, pp. 11600-11605). The radiation can be of any wavelength and includes, but is not limited to, wavelengths that do not damage normal cells, but that heat the non-protein fragment to a temperature that kills cells closest to the conjugate: antibody-non-protein fragment.

B. Methods of recombination and composition.

Antibodies can be obtained using recombination and composition techniques such as those described in US No. 4,816,567. One embodiment of the invention is an isolated nucleic acid encoding an anti-C5 antibody as described herein. Another embodiment of the invention is an isolated nucleic acid that encodes a polypeptide containing a variant Fc region or parental Fc region as defined herein. Said nucleic acid may encode an amino acid sequence containing the VL and/or an amino acid sequence containing the VH of an antibody (eg, antibody light and/or heavy chains). The following embodiment of the invention is(are) one or more vectors (for example, expression vectors), which(e) contains(at) the specified nucleic acid. Another embodiment of the invention is a host cell that contains the specified nucleic acid. In one of these embodiments of the invention, the host cell contains (for example, as a result of transformation with these vectors):

(1) a vector containing a nucleic acid that encodes an amino acid sequence containing the VL of the antibody and an amino acid sequence containing the VH of the antibody; or (2) a first vector containing a nucleic acid that encodes an amino acid sequence containing the VL of an antibody and a second vector containing a nucleic acid that encodes an amino acid sequence containing the VH of an antibody.

In one embodiment, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (eg, Y0, NS0, Sp20 cell). One embodiment of the invention is a method for producing an anti-C5 antibody comprising culturing a host cell containing a nucleic acid that encodes an antibody as described above under conditions suitable for expressing the antibody, and optionally isolating the antibody from the host cell (or culture medium). host cells). Another embodiment of the invention is a method for producing a polypeptide that contains a variant Fc region or a parent Fc region, comprising culturing a host cell that contains nucleic acid(s) encoding(s) a polypeptide, such as an antibody, Fc- an Fc region or variant as defined above under conditions suitable for expression of the polypeptide, and optionally isolating the polypeptide from the host cell (or host cell culture medium).

For recombinant production of an anti-C5 antibody, antibody-encoding nucleic acids, such as those described above, are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. For recombinant production of an Fc region, a nucleic acid encoding an Fc region is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. The specified nucleic acid can be easily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that have the ability to specifically bind to the genes encoding the heavy and light chains of antibodies).

Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be made in bacteria, particularly when glycosylation and Fc-related effector function are not required. For information on the expression of antibody fragments and polypeptides in bacteria, see, for example, US No. 5648237, 5789199 and 5840523 (see also Charlton in: Methods in Molecular Biology, edited by Lo B.K.C, ed. Humana Press , Totowa, NJ, 248, 2003, pp. 245-254 for a description of the expression of antibody fragments in E. coli). After expression, the antibody can be isolated from the bacterial cell paste as a soluble fraction and can be further purified.

In addition to prokaryotic organisms, eukaryotic microorganisms such as filamentous fungi or yeasts, including strains of fungi and yeasts whose glycosylation pathways have been humanized to produce an antibody, can be used as hosts suitable for cloning or expression of plasmids that encode antibodies. with partially or fully human glycosylation scheme (see Gemgross, Nat. Biotech., 22, 2004, pp. 1409-1414 and Li et al., Nat. Biotech., 24,

2006, p. 210-215).

Host cells that can be used to express the glycosylated antibody are also obtained from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells are insect cells, and plant cells can also be used. Numerous strains of baculoviruses and corresponding suitable insect cells as hosts have been identified, primarily for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts (see, for example, US Pat.

Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to growth in suspension can be used. Other examples of suitable mammalian host cell lines are the CV1 monkey kidney cell line transformed with OB40 (COS-7); a human embryonic kidney cell line (HEK 293 cells or 293 cell line as described, for example, in Graham et al., J. Gen. Virol., 36, 1977, pp. 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells described, for example, in Mather, Biol. Reprod., 23, 1980, c. 243-251); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma (HELA) cells; dog kidney cells (MDCK); bovine rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor cells (MMT 060562); TRI cells as described, for example, in Mather et al., Annals N.Y. Acad. Sci. 383, 1982, p. 44-68); MRC 5 cells and FS4 cells. Other valuable mammalian host cell lines are Chinese Hamster Ovary (CHO) cells, including DHFRCHO cells (Urlaub et al., Proc. Natl. Acad. Sci., USA, 77, 1980, c. 4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of specific mammalian host cell lines that can be used to produce antibodies, see, for example, Yazaki and Wu, in: Methods in Molecular Biology, ed. V.K.S. Lo, Humana Press, Totowa, NJ, vol. 248, 2003, p. 255-268.

Polyclonal antibodies are preferably produced in animals after several subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antibody and adjuvant. The relevant antigen can be conjugated to a protein immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctional or derivatizing agent, e.g., maleimidobenzoyl sulfosuccinimide ester (conjugation via cysteine residues), N -hydroxysuccinimide (conjugation through lysine residues), glutaraldehyde, succinic anhydride, SOCl ₂ or R1N=C=NR, where R and R ¹ represent different alkyl groups.

Animals (generally non-human mammals) are immunized with the antigen, immunogenic conjugates or derivatives by combining, for example, 100 or 5 μg of the protein or conjugate (for rabbits and mice, respectively) with 3 volumes of complete Freund's adjuvant and administering the solution intradermally into several places. After 1 month, the animals are booster vaccinated (revaccinated) using from 1/5 to 1/10 of the original amount of the peptide or conjugate in complete Freund's adjuvant by subcutaneous injection at several sites. After 7-14 days, blood samples of the animals are obtained and the serum antibody titer is assessed. Animals are subjected to revaccination until the titer reaches a plateau. Preferably, the animals are revaccinated using a conjugate containing the same antigen but conjugated to a different protein and/or via a different cross-linking agent. Conjugates can also be obtained in recombinant cell culture as protein fusions. In addition, aggregating agents such as alum can be used to enhance the immune response.

Monoclonal antibodies are derived from a population of substantially homogeneous antibodies, ie. the individual antibodies in a population are identical except for possible naturally occurring mutations and/or post-translational modifications (eg, isomerization, amidation), which may be present in minor amounts. The adjective monoclonal indicates that the antibody is not present in a mixture of different antibodies.

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

The immunizing agent will typically include an antigenic protein or a fusion variant thereof. Typically, either peripheral blood lymphocytes (PBL) are used if cells of human origin are required, or splenic or lymph node cells are used if non-human mammalian sources are used. The lymphocytes are then fused to an immortalized cell line using a suitable fusion agent such as polyethylene glycol to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic

Press, 1986, p. 59-103).

Immortalized cell lines are generally transformed mammalian cells, primarily rodent, bovine and human myeloma cells. Typically, rat or mouse myeloma cell lines are used. The hybridoma cells thus obtained are seeded and grown in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT) is absent in parental myeloma cells, the culture medium for hybridomas typically includes hypoxanthine, aminopterine, and thymidine (HAT medium), i.e. substances that interfere with the growth of HGPRT-deficient cells.

Preferred immortalized myeloma cells are cells that can be efficiently fused, that maintain a stable high level of antibody production by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, murine myeloma lines such as those derived from MOPC-21 and MPC-11 murine tumors, which are available from the Salk Institute Cell Distribution Center, San Diego, PA, are preferred. California, USA, and SP-2 cells (and their derivatives, eg X63-Ag8653), which can be obtained from the American Type Culture Collection, Manassas, pc. Virginia, USA. Also described are human myeloma and mouse-human heteromyeloma cell lines that can be used to produce human monoclonal antibodies (Kozbor et al., J. Immunol., 133(6), 1984, c. 3001-3005; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, 1987, pp. 51-63).

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

Once hybridoma cells are found to produce antibodies of the desired specificity, affinity and/or activity, clones can be subcloned using serial dilution procedures and grown using standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as tumors in mammals.

Monoclonal antibodies secreted by subclones can be separated from the culture medium, ascitic fluid or serum using conventional immunoglobulin purification procedures, such as chromatography for protein A-sepharose, hydroxyapatite, gel electrophoresis, dialysis or affinity chromatography.

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

- 36 041632

B. Analyzes.

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

1. Binding assays and other assays.

In one aspect of the invention, an antibody of the invention is tested for antigen-binding activity using known methods such as ELISA, Western blotting, BIACORE®, etc. In one aspect of the invention, a polypeptide containing a variant Fc region of the invention is tested for binding activity to its Fc receptor using known methods such as BIACORE®, etc.

According to another aspect of the invention, competition assays can be used to identify an antibody that competes for C5 binding with any of the anti-C5 antibodies described herein. In some embodiments of the invention, when the specified competitive antibody is present in excess, it blocks (for example, 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 cases, binding is inhibited by at least 80%, 85%, 90%, 95% or more. In some embodiments, said competing antibody binds to the same epitope (eg, a linear or conformational epitope) that an anti-C5 antibody provided herein binds (eg, an anti-C5 antibody described in Table 2). A detailed description of exemplary methods for mapping an epitope to which an antibody binds is provided by Morris, Epitope Mapping Protocols, in: Methods in Molecular Biology, vol. 66, Humana Press, Totowa, NJ, 1996.

As an example of a competition assay, immobilized C5 is incubated in a solution containing a first labeled antibody that binds to C5 and a second unlabeled antibody to be tested for its ability to compete with the first antibody for binding to C5. The second antibody may be present in the hybridoma supernatant. As a control, immobilized C5 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions suitable for binding of the first antibody to C5, the excess of unbound antibody is removed and the amount of label associated with immobilized C5 is measured. If the amount of label associated with immobilized C5 is significantly reduced in the test sample compared to the control sample, then this indicates that the second antibody competes with the first antibody for binding to C5 (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988).

Another example of a competitive assay is the BIACORE® assay, which evaluates the ability of a test anti-C5 antibody to compete for binding to C5 with a second (reference) anti-C5 antibody. In a further embodiment using a BIACORE® device (eg, BIACORE®3000) as recommended by the manufacturer, the C5 protein is captured on the BIACORE® CM5 chip using a standard method known in the art to create a C5 coated surface. Typically, the amount immobilized on the C5 chip should be between 200-800 resonant units (an amount that provides easily measurable levels of binding, but which can easily be saturated with the test antibody at the concentrations used). The two antibodies (ie, test and reference antibody) for which the ability to compete with each other is to be assessed are mixed in a 1:1 molar ratio of binding sites in a suitable buffer to form a test mixture. When calculating concentrations based on binding sites, the molecular weight of the test or reference antibody is taken as the total mass of the corresponding antibody divided by the number of C5 binding sites on the antibody. The concentration of each antibody (ie, test and reference antibody) in the test mixture should be high enough to easily saturate the binding sites for that antibody on the C5 molecules captured on the BIACORE® chip. The test antibody and the reference antibody in the mixture are present at the same molar concentration (based on binding sites), typically between 1.00 and 1.5 μM (based on binding sites). Separate solutions are also prepared containing only the test antibody and only the reference antibody. The antibody to be tested and the reference antibody in these solutions must be in the same buffer and at the same concentrations and conditions as in the test mixture. The test mixture containing the test antibody and the reference antibody is passed over the surface of the C5 coated BIACORE® chip and the total binding level is recorded. The chip is then processed in such a way as to remove the bound test or reference antibody without damaging the C5 bound to the chip. Typically, this is done by treating the chip with 30 mM HCl for 60 seconds. A solution containing only the test antibody is then passed over the C5 coated surface and the level of binding recorded. The chip is processed again to completely remove the bound antibody without damaging the C5 bound to the chip. The solution containing only the reference antibody is then passed over the C5-coated surface and the level of binding is recorded. After that, they calculate

- 37 041632 theoretical maximum possible binding level for a mixture of test antibody and reference antibody, which is the sum of the binding levels for each antibody (i.e. test antibody and reference antibody) when passing them over a C5 coated surface individually. If the actual measured binding level for the mixture is less than the specified theoretical maximum, then the test antibody and the reference antibody compete with each other for binding to C5. Thus, as a rule, a competitive test antibody to C5 is an antibody that should bind to C5 according to the BIACORE® blocking assay described above in such a way that when the assay is carried out and in the presence of a reference antibody to C5, the measured level of binding is from 80 to 0.1% (e.g., less than 80 to 4%) of the theoretical maximum binding level, specifically 75 to 0.1% (e.g., 75 to 4%) of the theoretical maximum binding level, and more specifically, from 70 to 0.1% (eg, 70 to 4%) of the theoretical maximum binding level (as defined above) for the combination of test antibody and reference antibody.

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

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

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

In other embodiments, the anti-C5 antibody binds to C5 with higher affinity at neutral pH than at acidic pH. In some embodiments, an anti-C5 antibody of the present invention competes for binding to C5 with an antibody that contains a VH and VL pair from a CFA0305 or 305LO5 antibody. In other embodiments, the anti-C5 antibody binds to C5 with higher affinity at pH 7.4 than at pH 5.8.

In some embodiments, an anti-C5 antibody of the present invention competes for binding to a C5 antibody that contains a VH and VL pair selected from a VH having SEQ ID NO: 22 and a VL having SEQ ID NO: 26, or VH having SEQ ID NO: 21 and VL having SEQ ID NO: 25. In some embodiments, an anti-C5 antibody competes for binding to C5 with an antibody that contains a VH and VL pair selected from (a) a VH having SEQ ID NO: 5, and VL having SEQ ID NO: 15;

(b) VH having SEQ ID NO: 4 and VL having SEQ ID NO: 14;

(c) VH having SEQ ID NO: 6 and VL having SEQ ID NO: 16;

(d) VH having SEQ ID NO: 2 and VL having SEQ ID NO: 12;

(e) VH having SEQ ID NO: 3 and VL having SEQ ID NO: 13;

(e) VH having SEQ ID NO: 1 and VL having SEQ ID NO: 11;

(g) VH having SEQ ID NO: 9 and VL having SEQ ID NO: 19;

(h) VH having SEQ ID NO: 7 and VL having SEQ ID NO: 17; and (i) VH having SEQ ID NO: 8 and VL having SEQ ID NO: 18.

In some embodiments, an anti-C5 antibody competes for binding to C5 with an antibody that contains a VH having SEQ ID NO: 23 and a VL having SEQ ID NO: 27. In some embodiments, an anti-C5 antibody competes for binding to C5 with an antibody that contains VH having SEQ ID NO: 7 and VL having SEQ ID NO: 17.

In some embodiments, an anti-C5 antibody of the present invention competes for binding to C5 with an antibody that contains a VH and VL pair selected from (a) VH having SEQ ID NO: 1, VL having SEQ ID NO: eleven;

(b) VH having SEQ ID NO: 22, VL having SEQ ID NO: 26;

(c) VH having SEQ ID NO: 21 and VL having SEQ ID NO: 25;

(d) VH having SEQ ID NO: 5 and VL having SEQ ID NO: 15;

(e) VH having SEQ ID NO: 4, VL having SEQ ID NO: 14;

(e) VH having SEQ ID NO: 6 and VL having SEQ ID NO: 16;

(g) VH having SEQ ID NO: 2, VL having SEQ ID NO: 12;

- 38 041632 (h) VH having SEQ ID NO: 3, and VL having SEQ ID NO: 13;

(i) VH having SEQ ID NO: 9 and VL having SEQ ID NO: 19;

(j) VH having SEQ ID NO: 7 and VL having SEQ ID NO: 17;

(l) VH having SEQ ID NO: 8 and VL having SEQ ID NO: 18;

(m) VH having SEQ ID NO: 23 and VL having SEQ ID NO: 27; and (n) VH having SEQ ID NO: 10 and VL having SEQ ID NO: 20.

In some embodiments, an anti-C5 antibody of the present invention competes for binding to C5 with an antibody that contains a VH and VL pair selected from (a) a VH having SEQ ID NO: 22 and a VL having SEQ ID NO : 26;

(b) VH having SEQ ID NO: 21 and VL having SEQ ID NO: 25;

(c) VH having SEQ ID NO: 5 and VL having SEQ ID NO: 15;

(d) VH having SEQ ID NO: 4 and VL having SEQ ID NO: 14;

(e) VH having SEQ ID NO: 6 and VL having SEQ ID NO: 16;

(e) VH having SEQ ID NO: 2 and VL having SEQ ID NO: 12;

(g) VH having SEQ ID NO: 3 and VL having SEQ ID NO: 13;

(h) VH having SEQ ID NO: 9 and VL having SEQ ID NO: 19;

(i) VH having SEQ ID NO: 7 and VL having SEQ ID NO: 17;

(j) VH having SEQ ID NO: 8 and VL having SEQ ID NO: 18;

(l) VH having SEQ ID NO: 23 and VL having SEQ ID NO: 27.

In some embodiments, an anti-C5 antibody of the present invention competes for binding to C5 with an antibody that contains a VH and VL pair selected from VH having SEQ ID NO: 1 and VL having SEQ ID NO: 11, or VH having SEQ ID NO: 10 and VL having SEQ ID NO: 20.

In other embodiments, the anti-C5 antibody binds to C5 with higher affinity at neutral pH than at acidic pH. In some embodiments, an anti-C5 antibody binds to C5 with higher affinity at neutral pH than at acidic pH and competes for binding to C5 with an antibody that contains a VH and VL pair selected from (a) a VH having SEQ ID NO : 1, and VL having SEQ ID NO: 11;

(b) VH having SEQ ID NO: 5 and VL having SEQ ID NO: 15;

(c) VH having SEQ ID NO: 4 and VL having SEQ ID NO: 14;

(d) VH having SEQ ID NO: 6 and VL having SEQ ID NO: 16;

(e) VH having SEQ ID NO: 2 and VL having SEQ ID NO: 12;

(e) VH having SEQ ID NO: 3 and VL having SEQ ID NO: 13;

(g) VH having SEQ ID NO: 9 and VL having SEQ ID NO: 19;

(h) VH having SEQ ID NO: 7 and VL having SEQ ID NO: 17;

(m) VH having SEQ ID NO: 8 and VL having SEQ ID NO: 18; and (j) VH having SEQ ID NO: 10 and VL having SEQ ID NO: 20.

In other embodiments, the anti-C5 antibody binds to C5 with higher affinity at pH 7.4 than at pH 5.8.

In some embodiments, an anti-C5 antibody binds to C5 with higher affinity at neutral pH than at acidic pH and competes for binding to C5 with an antibody that contains a VH and VL pair selected from (a) a VH having SEQ ID NO: 5, and VL having SEQ ID NO: 15;

(b) VH having SEQ ID NO: 4 and VL having SEQ ID NO: 14;

(c) VH having SEQ ID NO: 6 and VL having SEQ ID NO: 16;

(d) VH having SEQ ID NO: 2 and VL having SEQ ID NO: 12;

(e) VH having SEQ ID NO: 3 and VL having SEQ ID NO: 13;

(e) VH having SEQ ID NO: 1 and VL having SEQ ID NO: 11;

(g) VH having SEQ ID NO: 9 and VL having SEQ ID NO: 19;

(h) VH having SEQ ID NO: 7 and VL having SEQ ID NO: 17; and (i) VH having SEQ ID NO: 8 and VL having SEQ ID NO: 18.

In other embodiments, the anti-C5 antibody binds to C5 with higher affinity at pH 7.4 than at pH 5.8.

In some embodiments, an anti-C5 antibody binds to C5 with higher affinity at neutral pH than at acidic pH and competes for binding to C5 with an antibody that contains a VH and VL pair selected from a VH having SEQ ID NO: 1 , and VL having SEQ ID NO: 11, or VH having SEQ ID NO: 10, and VL having SEQ ID NO: 20. In other embodiments, the anti-C5 antibody binds to C5 with higher affinity at pH 7, 4 than at pH 5.8.

In some embodiments, it can be determined whether an anti-C5 antibody of the present invention binds to a specific epitope as follows: expression of C5 point mutants in which the amino acid (except for

- 39 041632 alanine) in C5 is replaced by alanine, in cell line 293 and evaluate the binding of antibodies to C5 with C5 mutants by ELISA, Western blot or BIACORE®; however, a significant reduction or elimination of the binding of the C5 antibody to the C5 mutant compared to binding to wild-type C5 indicates that the C5 antibody binds to an epitope containing the specified amino acid in C5. In some embodiments, the amino acid at C5 to be replaced by alanine is selected from the group consisting of Glu48, Asp51, His70, His72, Lys109, and His110 of the C5 beta chain (SEQ ID NO: 40). In other embodiments, the amino acid at C5 to be replaced by alanine is Asp51 or Lys109 of the C5 beta chain (SEQ ID NO: 40).

In another embodiment of the invention, it can be determined whether a pH dependent anti-C5 antibody binds to a specific epitope as follows: C5 point mutant mutants in which the histidine residue in C5 is replaced by another amino acid (e.g. tyrosine) are expressed , in 293 cell line and evaluate the binding of anti-C5 antibody to C5 mutants by ELISA, Western blotting or BIACORE®; however, a significant decrease in the binding of antibodies to C5 wild-type C5 at acidic pH compared with its binding to the C5 mutant at acidic pH, indicates that the antibody to C5 binds to an epitope containing the indicated histidine residue on C5. In other embodiments, the binding of an anti-C5 antibody to wild-type C5 at neutral pH is not significantly reduced compared to its binding to a C5 mutant at neutral pH. In some embodiments, the C5 histidine residue to be changed to another amino acid is selected from the group consisting of His70, His72, and His110 of the C5 beta chain (SEQ ID NO: 40). In a further embodiment of the invention, the His70 histidine residue is replaced with a tyrosine.

2. Activity assays.

One of the objects of the invention are assays designed to identify antibodies to C5 that have biological activity. Biological activity may include, for example, inhibition of C5 activation, prevention of C5 cleavage to form C5a and C5b, blocking C5 convertase access to a C5 cleavage site, blocking hemolytic activity caused by C5 activation, and so on. Also provided are antibodies having said biological activity in vivo and/or in vitro.

In some embodiments, an antibody of the invention is tested for a specified biological activity.

In some embodiments, the determination of whether the test antibody inhibits the cleavage of C5 with the formation of C5a and C5b, is carried out using the methods described, for example, Isenman and others, J. Immunol., 124(1), 1980, c. 326-331. In another embodiment of the invention, this is determined using methods that allow specific detection of cleaved C5a and/or C5b proteins, for example, using ELISA variants or Western blotting. If a reduced amount of C5 cleavage product (i.e., C5a and/or C5b) is detected in the presence of the test antibody (or after contact with it), then the test antibody is identified as an antibody that can inhibit C5 cleavage. In some embodiments of the invention, the concentration and/or physiological activity of C5a can be measured using methods such as, for example, chemotactic analysis, RIA, or ELISA (see, for example, Ward and Zvaifler J. Clin. Invest., 50(3 ), 1971, pp. 606-616).

In some embodiments of the invention, the determination of whether the test antibody blocks the access of C5 convertase to C5 is carried out using methods that allow detection of protein interactions between C5 convertase and C5, for example, ELISA or BIACORE®. If there is a decrease in interactions in the presence of the test antibody (or after contact with it), then the test antibody is identified as an antibody that can block the access of C5 convertase to C5.

In some embodiments of the invention, the activity of C5 can be assessed by its ability to carry out cell lysis in the total water of the individual. The ability of C5 to carry out cell lysis or its reduction can be assessed using methods well known in this field, for example, using a standard hemolytic assay, such as the hemolytic assay described in Experimental Immunochemistry, ed. Kabat and Mayer, 2nd ed., 135-240, CC Thomas, Springfield, IL, 1961, p. 135-139, or a standard variant of this assay, such as the chicken erythrocyte hemolysis method described, for example, in Hillman et al., N. Engl. J. Med., 350(6), 2004, p. 552-559. In some embodiments of the invention, the activity of C5 or its inhibition is quantified using CH50eq analysis. The CH50eq assay is a method for measuring the total activity of the classical complement pathway in serum. This test is a lytic activity assay that uses antibody-sensitized erythrocytes as an activator of the classical complement pathway and uses various dilutions of test serum to determine the amount needed to achieve 50% lysis (CH50). The percentage of hemolysis can be determined, for example, using a spectrophotometer. CH50eq analysis is a means of indirect

- 40 041632 measurements of the formation of the complex of terminal complement components (TSC), since the TCCs themselves are directly responsible for the measured hemolysis. Inhibition of C5 activation can be detected and/or measured using the methods set forth and illustrated in the Experimental Examples. Using assays of these or other suitable types, candidate antibodies having the ability to inhibit C5 activation can be screened. In some embodiments, the inhibition of C5 activation comprises an assayed decrease in C5 activation of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40%, or more, compared to exposure to a negative control under similar conditions. In some embodiments, it refers to inhibition of C5 activation by at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more.

D. Immunoconjugates.

The invention also provides immunoconjugates comprising the C5 antibody described herein conjugated to one or more cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active bacterial, fungal, of plant or animal origin or their fragments) or radioactive isotopes.

In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including (but not limited to) maytansinoid (see US No. 5208020, 5416064 and EP 0425235 B1 ); auristatin, for example, fragments DE and DF of monomethylauristatin (MMAE and MMAF) (see US No. 5635483, 5780588 and 7498298); dolastatin; calicheamicin or its derivatives (see US No. 5712374, 5714586, 5739116, 5767285, 5770701, 5770710, 5773001 and 5877296; Hinman and others, Cancer Res., 53, 1993, pp. 3336-3342; and Lode and others. Cancer Res., 58, 1998, pp. 2925-2928); anthracycline such as daunomycin or doxorubicin (see Kratz et al., Curr. Med. Chem., 13, 2006, pp. 477-523; Jeffrey et al., Bioorg. Med. Chem. Lett., 16, 2006, pp. 358-362; Torgov et al., Bioconjug. Chem., 16, 2005, pp. 717-721; Nagy et al., Proc. Natl. Acad. Sci., USA, 97, 2000, pp. 829- 834; Dubowchik et al., Bioorg. & Med. Chem. Letters, 12, 2002, pp. 1529-1532; King et al., J. Med. Chem., 45, 2002, pp. 4336-4343; and US No. 6630579); methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel and orthotaxel; trichothecene and CC1065.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including (but not limited to) diphtheria toxin A chain, non-binding diphtheria toxin active fragments, exotoxin A chain (from Pseudomonas aeruginosa), ricin A-chain, abrin A-chain, modecin A-chain, alpha-sarcin, Aleurites fordii proteins, dianthine proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-5), inhibitor from Momordica charantia, curcin, crotin, an inhibitor from Sapaonaria officinalis, gelonin, mitogellin, restrictocin, fenomycin, enomycin and trichothecenes.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A wide variety of radioactive isotopes can be used to prepare radioconjugates. Examples include ²¹¹ At, ¹³¹ I, ¹²⁵ I, ⁹⁰ Y, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹² Bi, ³² P, ²¹² Pb and radioactive isotopes of Lu. When the radioconjugate is used for detection, it may contain a radioactive atom for scintigraphy, such as TC ^99m or I ¹²³ , or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine -123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Antibody-cytotoxic agent conjugates can be prepared using a wide variety of bifunctional protein-binding agents such as N-succinimidyl-3-(2pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional imidoester derivatives (such as dimethyl adipimidateχHCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bisazido derivatives (such as bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (such as bis(para-diazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene-2,6diisocyanate), and dual activity fluorine compounds (such as 1,5-difluoro2,4-dinitrobenzene). For example, the ricin immunotoxin can be prepared according to the method described in Vitetta et al., Science, 238, 1987, c. 1098-1104. C ¹⁴ labeled 1-isothiocyanatebenzyl-3methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an example of a chelating agent used to conjugate a radionucleotide to an antibody (see WO 94/11026). The linker may be a cleavable linker that facilitates the release of the cytotoxic drug. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Res., 52, 1992, c. 127-131; US No. 5208020).

In the context of the present description, immunoconjugates or ADCs are meant (but not limited to

- 41 041632 limited only to them) specified conjugates obtained using cross-linking reagents, which include (but are not limited to) BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SLAB, SMCC, SMPB , SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SLAB, sulfo-SMCC and sulfo-SMPB, as well as SVSB (succinimidyl(4vinylsulfone)benzoate), which are commercially available (for example, from Pierce Biotechnology, Inc., Rockford, Illinois, USA).

E. Methods and compositions for diagnosis and detection.

In some embodiments, any of the anti-C5 antibodies provided herein can be used to detect the presence of C5 in a biological sample. The concept of detection (detection) in the context of the present description provides for quantitative or qualitative detection. In one embodiment, the biological sample comprises a cell or tissue, e.g., serum, whole blood, plasma, biopsy sample, tissue sample, cell suspension, saliva, sputum, oral fluid, cerebrospinal fluid, amniotic fluid, ascitic fluid, milk , colostrum, mammary secretion, lymph, urine, sweat, lacrimal fluid, gastric juice, synovial fluid, peritoneal fluid, lens fluid or mucus.

One embodiment of the invention is an anti-C5 antibody provided herein for use in a diagnostic or detection method. Another object of the invention relates to a method for detecting the presence of C5 in a biological sample. In another embodiment, the method comprises contacting a biological sample with an anti-C5 antibody provided herein under conditions that allow the anti-C5 antibody to bind to C5 and detecting the formation of a complex between the anti-C5 antibody and C5. Said method may be an in vitro or in vivo method. In one embodiment, an anti-C5 antibody is used to select individuals who can be treated with an anti-C5 antibody, eg, where C5 is a biomarker for patient selection.

Another embodiment of the invention is a method of selecting an individual having a complement-mediated disease or condition that involves excessive or uncontrolled C5 activation as a candidate for therapy comprising an anti-C5 antibody of the present invention. In some embodiments, the method includes (a) detecting a genetic variation in C5 obtained from an individual; and (b) selecting the individual as suitable for therapy comprising an anti-C5 antibody of the present invention when a genetic variation is found in C5 obtained from the individual.

Another embodiment of the invention is a method of selecting therapy for an individual having a complement-mediated disease or condition that involves excessive or uncontrolled C5 activation. In some embodiments, the method includes (a) detecting a genetic variation in C5 obtained from an individual; and (b) selecting a therapy comprising an anti-C5 antibody of the present invention for an individual when a genetic variation is found in C5 derived from the individual.

Another embodiment of the invention is a method of treating an individual having a complement-mediated disease or condition that involves excessive or uncontrolled C5 activation. In some embodiments, the method includes (a) detecting a genetic variation in C5 obtained from an individual;

(b) selecting an individual as suitable for therapy comprising an anti-C5 antibody of the present invention when a genetic variation is found in C5 obtained from the individual; and (c) administering to the subject an anti-C5 antibody of the present invention.

Another embodiment of the invention is an anti-C5 antibody of the present invention for use in the treatment of an individual having a complement-mediated disease or condition that involves excessive or uncontrolled C5 activation. In some embodiments, the subject is treated with an anti-C5 antibody of the present invention when a genetic variation is found in the subject's C5.

Another embodiment of the invention is the use of an in vitro genetic variation in C5 to select an individual having a complement-mediated disease or condition that involves excessive or uncontrolled C5 activation as a candidate for therapy comprising an anti-C5 antibody of the present invention. In some embodiments, an individual is selected for therapy when a genetic variation is found in C5 derived from the individual. Another embodiment of the invention is the use of an in vitro genetic variation in C5 to select therapy for an individual having a complement-mediated disease or condition that involves excessive or uncontrolled C5 activation. In some embodiments, a therapy comprising an anti-C5 antibody of the present invention is selected for an individual when a genetic variation is found in C5 derived from the individual.

Some patients with a genetic variation in C5 have been found to respond poorly to therapy involving existing anti-C5 antibodies (Nishimura et al., N. Engl. J. Med., 370, 2014, pp. 632-639). It is recommended that this patient be treated with a therapy comprising an anti-C5 antibody of the present invention, since such an antibody has inhibitory activity on the activation of C5 variants as well as wild-type C5, as demonstrated in the experimental examples below.

Detection of genetic variation in C5 can be performed using a method known in the art. This method includes, but is not limited to, sequencing, PCR, RT-PCR, and a hybridization-based method such as Southern blot or Northern blot. C5 variants may contain at least one genetic variation. The genetic variation can be selected from the group consisting of VM5I, R449G, V802I, R885H, R928Q, D966Y, S1310N and E1437D. In the present description, for example, R885H denotes a genetic variation in which the arginine at position 885 is replaced by histidine. In some embodiments, the C5 variant has similar biological activity to wild-type C5.

Examples of disorders that can be diagnosed using an antibody of the invention include rheumatoid arthritis (RA); systemic lupus erythematosus (SLE); lupus nephritis; damage caused by ischemia-reperfusion; asthma, paroxysmal nocturnal hemoglobinuria (PNH); hemolytic uremic syndrome (HUS) (eg, atypical hemolytic uremic syndrome (aHUS); dense deposit disease (DDD); optic neuromyelitis (NMO); multifocal motor neuropathy (MMN); multiple sclerosis (MS); systemic sclerosis; degeneration macular degeneration (eg, age-related macular degeneration (AMD)); syndrome: hemolysis, elevated liver enzymes, and thrombocytopenia (HELLP); thrombotic thrombocytopenic purpura (TTP); spontaneous fetal loss; epidermolysis bullosa; recurrent fetal loss; preeclampsia; = traumatic brain damage, severe pseudoparalytic myasthenia gravis, cold agglutinin disease, Sjögren's syndrome, dermatomyositis, bullous pemphigoid, phototoxic reactions, hemolytic uremic syndrome associated with E. coI toxin produced Shiga, typical or infectious hemolytic uremic syndrome (tHUS), C3-glomerulonephritis ; antineutrophil cytoplasmic antibody (ANCA) associated vasculitis; humo ral and vascular graft rejection; acute antibody-mediated rejection (AMR); transplant dysfunction; myocardial infarction; allogeneic transplant; sepsis; coronary artery disease; hereditary angioedema; dermatomyositis; Graves' disease; atherosclerosis; Alzheimer's disease (AD); Huntington's disease; Creutzfeldt-Jakob disease; Parkinson's disease; crayfish; wounds; septic shock; spinal nerve injury uveitis; diabetes-related eye diseases; retinopathy of prematurity; glomerulonephritis; membranous nephritis; associated with immunoglobulin A nephropathy; adult respiratory dyspress syndrome (ARDS); chronic obstructive pulmonary disease (COPD); cystic fibrosis; hemolytic anemia; paroxysmal cold hemoglobinuria; anaphylactic shock; allergy; osteoporosis; osteoarthritis; Hashimoto's thyroiditis; type I diabetes; psoriasis; pemphigus; autoimmune hemolytic anemia (AIHA); idiopathic thrombocytopenic purpura (ITP); Goodpasture's syndrome; Degos disease; antiphospholipid syndrome (APS); catastrophic APS (CAPS); cardiovascular disorder; myocarditis; violation of cerebral circulation; peripheral vascular disease; renovascular disorder; mesenteric/enteric vascular disease; vasculitis; nephritis with Henoch-Schönlein purpura; Takayashi's disease; dilating cardiomyopathy; diabetic angiopathy; Kawasaki disease (arteritis); venous air embolism (VGE), restenosis after stenting; rotational atherectomy; membranous nephropathy; Guillain-Barré syndrome (GBS); Fisher's syndrome; antigen-induced arthritis; synovial inflammation; viral infections; bacterial infections; fungal infections; and damage due to myocardial infarction, cardiopulmonary bypass, and hemodialysis.

Some embodiments of the invention are labeled anti-C5 antibodies. Labels include, but are not limited to, labels or moieties that can be detected directly (such as fluorescent, chromophoric, electron dense, chemiluminescent, and radioactive labels) as well as moieties such as enzymes or ligands that are indirectly detectable, such as through an enzymatic reaction or molecular interaction. Examples of labels include, but are not limited to, ³² P, ¹⁴ C, ¹²⁵ I, ³ H, and ¹³¹ I radioisotopes, fluorophores such as rare earth metal chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases , e.g., firefly luciferase and bacterial luciferase (US No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose -6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase linked to an enzyme that uses hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase or microperoxidase, biotin/avidin, spin labels, bacteriophage labels , stable free radicals, etc.

E. Pharmaceutical compositions.

Pharmaceutical compositions of the anti-C5 antibody provided herein are prepared by mixing said antibody, having the desired purity, with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th ed. ed. Osol A., 1980) in the form of lyophilized compositions or aqueous solutions. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed and include (but are not limited to): buffers such as phosphate, citrate, and other organic acid buffers; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and meta-cresol); low molecular weight polypeptides (containing less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as poly(vinylpyrrolidone); amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal-containing complexes (eg Zn-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG). As used herein, examples of pharmaceutically acceptable carriers also include interstitial drug dispersing agents such as soluble neutrally active hyaluronidase glycoproteins (sHASEGP), e.g. human soluble hyaluronidase PH-20 glycoproteins such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Some examples of sHASEGP and methods of using them, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. According to one aspect of the invention, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

Examples of lyophilized antibody compositions are described in US No. 6267958. Aqueous antibody compositions include those described in US No. 6171586 and WO 2006/044908, the latter compositions include histidine acetate buffer.

The composition according to the invention may also contain more than one active substance, if necessary for the specific indication to be treated, preferably substances with complementary activities that do not adversely affect each other. Such active substances must be present in combination in amounts effective to achieve the intended purpose.

The active substances can also be included in microcapsules, for example, obtained by coacervation or interfacial polymerization methods, for example, in hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, in liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsion. Such methods are described in Remington's Pharmaceutical Sciences, 16th ed., ed. Osol A., 1980.

Sustained release formulations can be prepared. Suitable examples of sustained release formulations are semi-permeable matrices of solid hydrophobic polymers containing an antibody or conjugate, such matrices are shaped articles such as films or microcapsules.

Compositions intended for use in vivo, as a rule, must be sterile. Sterility can easily be achieved, for example, by filtration through sterile filter membranes.

G. Therapeutic Methods and Compositions.

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

One of the objects of the invention is an antibody to C5 intended for use as a drug. Other aspects of the invention relate to an antibody to C5 intended for use in the treatment of a complement-mediated disease or condition that involves excessive or uncontrolled activation of C5. Some embodiments of the invention provide an anti-C5 antibody for use in a method of treatment. Some embodiments of the invention provide an anti-C5 antibody for use in a method of treating a subject who has a complement-mediated disease or condition that involves excessive or uncontrolled C5 activation, comprising administering to the subject an effective amount of the C5 antibody. In one of these embodiments of the invention, the method includes the introduction to the individual in an effective amount of at least one additional therapeutic agent. An individual in the context of any of the above options

- 44 041632 embodiment of the invention is preferably a human.

When the antigen is a soluble protein, binding of the antibody to the antigen can result in an extended plasma half-life of the antigen (i.e., reduced plasma clearance of the antigen) because the antibody itself has a longer half-life and serves as a carrier for the antigen. This is a consequence of FcRn-mediated recycling of the antigen-antibody complex via the endosomal pathway in the cell (Roopenian, Nat. Rev. Immunol., 7(9), 2007, c. 715-725). However, an antibody with pH-dependent binding characteristics that binds to its antigen in a neutral extracellular environment, while being released into the acidic conditions of endosomal compartments after cell entry, is expected to have improved properties in terms of neutralization and clearance of the antigen relative to its counterpart that binds pH-independent manner (Igawa et al., Nat. Biotech., 28(11), 2010, pp. 1203-1207; Devanaboyina et al., mAbs, 5(6), 2013, pp. 851-859; WO 2009 /125825).

A further embodiment of the invention is an anti-C5 antibody for use in enhancing clearance of C5 from plasma. In some embodiments, an anti-C5 antibody is provided for use in a method of enhancing plasma clearance of C5 in a subject, comprising administering to the subject an effective amount of an anti-C5 antibody to enhance plasma clearance of C5. In one embodiment, an anti-C5 antibody increases the clearance of C5 from plasma compared to a canonical anti-C5 antibody that does not have pH-dependent binding characteristics. The individual in the context of any of the above embodiments of the invention is preferably a human.

A further embodiment of the invention is an anti-C5 antibody for use in suppressing plasma accumulation of C5. In some embodiments, an anti-C5 antibody for use in a method for inhibiting plasma C5 accumulation in a subject, comprising administering to the subject an effective amount of an anti-C5 antibody to inhibit plasma C5 accumulation. In one of the embodiments of the invention, 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 plasma C5 accumulation compared to a canonical anti-C5 antibody that does not have pH-dependent binding characteristics. The individual in the context of any of the above embodiments of the invention is preferably a human.

An anti-C5 antibody of the present invention can inhibit C5 activation. In other embodiments, the invention is an anti-C5 antibody for use in inhibiting C5 activation. In some embodiments, an anti-C5 antibody is provided for use in a method for inhibiting C5 activation in a subject, comprising administering to the subject an effective amount of an anti-C5 antibody to inhibit C5 activation. In one embodiment, C5 mediated cytotoxicity is suppressed by inhibiting C5 activation. The individual in the context of any of the above embodiments of the invention is preferably a human.

The next object of the invention is the use of antibodies to C5 for the preparation or production of drugs. In one embodiment, the drug is for the treatment of a complement-mediated disease or condition that involves excessive or uncontrolled C5 activation. In another embodiment, the drug is used in a method of treating a complement-mediated disease or condition that involves excessive or uncontrolled C5 activation, comprising administering to an individual having a complement-mediated disease or condition that includes excessive or uncontrolled C5 activation, in an effective amount of the drug. In one of these embodiments of the invention, the method also includes the introduction to the individual in an effective amount of at least one additional therapeutic agent. The individual in the context of any of the above embodiments of the invention is preferably a human.

In a further embodiment of the invention, the drug is intended to enhance the clearance of C5 from plasma. In another embodiment, the drug is for use in a method of increasing plasma C5 clearance in an individual, comprising administering to the individual an effective amount of the drug to increase plasma C5 clearance. In one embodiment, an anti-C5 antibody increases the clearance of C5 from plasma compared to a canonical anti-C5 antibody that does not have pH-dependent binding characteristics. An individual in the context of any of the above embodiments of the invention may be a human.

In a further embodiment of the invention, the drug is for suppressing plasma C5 accumulation. In a further embodiment of the invention, the drug is for use in a method for inhibiting plasma C5 accumulation in a subject, comprising administering to the subject an effective amount of the drug to inhibit plasma C5 accumulation. In one of the embodiments of the invention, 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 plasma accumulation of C5 compared to the canonical anti-C5 antibody.

C5, which does not have pH-dependent binding characteristics. An individual in the context of any of the above embodiments of the invention may be a human.

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

Another aspect of the invention is a method of treating a complement-mediated disease or condition that involves excessive or uncontrolled C5 activation. In one embodiment, the method comprises administering to an individual having said complement-mediated disease or condition that involves excessive or uncontrolled C5 activation an effective amount of an anti-C5 antibody provided herein. In one of the embodiments of the invention, the method also includes the introduction to the individual in an effective amount of at least one additional therapeutic agent. An individual in the context of any of the above embodiments of the invention may be a human.

The next object of the invention is a method of increasing the clearance of C5 from plasma in an individual. In one embodiment, the method comprises administering to the subject an effective amount of an anti-C5 antibody to enhance clearance of C5 from plasma. In one embodiment, an anti-C5 antibody increases the clearance of C5 from plasma compared to a canonical anti-C5 antibody that does not have pH-dependent binding characteristics. In one embodiment, the subject is a human.

The next object of the invention is a method of suppressing the accumulation of C5 in plasma in an individual. In one embodiment, the method comprises administering to the subject an effective amount of an anti-C5 antibody to inhibit plasma accumulation of C5. In one of the embodiments of the invention, 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 plasma C5 accumulation compared to a canonical anti-C5 antibody that does not have pH-dependent binding characteristics. In one embodiment, the subject is a human.

An anti-C5 antibody of the present invention can inhibit C5 activation. Another object of the invention is a method of inhibiting C5 activation in an individual. In one embodiment, the method comprises administering to the subject an effective amount of an anti-C5 antibody to inhibit C5 activation. In one embodiment, C5 mediated cytotoxicity is suppressed by inhibiting C5 activation. In one embodiment, the subject is a human.

Another object of the invention are pharmaceutical compositions containing any of the antibodies to C5 presented in the present description, which are intended, for example, for use in any of the above therapeutic methods. In one of the embodiments of the invention, the pharmaceutical composition contains any of the antibodies to C5 presented in the present description, and a pharmaceutically acceptable carrier. In another embodiment of the invention, the pharmaceutical composition contains any of the antibodies to C5 presented in the present description, and at least one additional therapeutic agent.

A further aspect of the invention is a pharmaceutical composition for the treatment of a complement-mediated disease or condition that involves excessive or uncontrolled C5 activation.

In a further embodiment of the invention, the pharmaceutical composition is intended to enhance the clearance of C5 from plasma. In one embodiment, an anti-C5 antibody increases the clearance of C5 from plasma compared to a canonical anti-C5 antibody that does not have pH-dependent binding characteristics. In a further embodiment of the invention, the pharmaceutical composition is for suppressing plasma C5 accumulation. In one of the embodiments of the invention, 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 plasma C5 accumulation compared to a canonical anti-C5 antibody that does not have pH-dependent binding characteristics. An anti-C5 antibody of the present invention can inhibit C5 activation. In a further embodiment of the invention, the pharmaceutical composition is for inhibiting C5 activation. In one embodiment, C5 mediated cytotoxicity is suppressed by inhibiting C5 activation. In one embodiment, the pharmaceutical composition is administered to an individual having a complement-mediated disease or condition that involves excessive or uncontrolled C5 activation. The individual in the context of any of the above embodiments of the invention is preferably a human.

In one aspect of the invention, the individual has wild-type C5. In another aspect of the invention, the individual has the C5 variant. In some embodiments, the C5 variant has similar biological activity to wild-type C5. Said C5 variant may contain at least one variation selected from the group consisting of V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N and E1437D. In the present description, for example, R885H denotes a genetic variation in which the arginine at position 885 is replaced by histidine.

A further aspect of the invention are methods for preparing a medicament or pharmaceutical composition comprising mixing any of the anti-C5 antibodies provided herein with a pharmaceutically acceptable carrier, for example, for use in any of the above therapeutic methods. In one of the embodiments of the invention, methods for preparing a drug or pharmaceutical composition further include adding at least one additional therapeutic agent to the drug or pharmaceutical composition.

In some embodiments, the complement-mediated disease or condition that includes excessive or uncontrolled C5 activation is selected from the group consisting of rheumatoid arthritis (RA); systemic lupus erythematosus (SLE); lupus nephritis; damage caused by ischemia-reperfusion; asthma, paroxysmal nocturnal hemoglobinuria (PNH); hemolytic uremic syndrome (HUS) (eg, atypical hemolytic uremic syndrome (aHUS); dense deposit disease (DDD); optic neuromyelitis (NMO); multifocal motor neuropathy (MMN); multiple sclerosis (MS); systemic sclerosis; degeneration macular degeneration (eg, age-related macular degeneration (AMD)); syndrome: hemolysis, elevated liver enzymes, and thrombocytopenia (HELLP); thrombotic thrombocytopenic purpura (TTP); spontaneous fetal loss; epidermolysis bullosa; recurrent fetal loss; preeclampsia; traumatic injury brain, severe pseudoparalytic myasthenia gravis, cold agglutinin disease, Sjögren's syndrome, dermatomyositis, bullous pemphigoid, phototoxic reactions, hemolytic uremic syndrome associated with E.coU toxin produced by Shiga toxin, typical or infectious hemolytic uremic syndrome (tHUS), C3-glomerulonephritis; antineutrophil cytoplasmic antibody associated (ANCA) vasculitis; ral and vascular graft rejection; acute antibody-mediated rejection (AMR); transplant dysfunction; myocardial infarction; allogeneic transplant; sepsis; coronary artery disease; hereditary angioedema; dermatomyositis; Graves' disease; atherosclerosis; Alzheimer's disease (AD); Huntington's disease; Creutzfeldt-Jakob disease; Parkinson's disease; crayfish; wounds; septic shock; spinal nerve injury uveitis; diabetes-related eye diseases; retinopathy of premature babies; glomerulonephritis; membranous nephritis; associated with immunoglobulin A nephropathy; adult respiratory dyspress syndrome (ARDS); chronic obstructive pulmonary disease (COPD); cystic fibrosis; hemolytic anemia; paroxysmal cold hemoglobinuria; anaphylactic shock; allergy; osteoporosis; osteoarthritis; Hashimoto's thyroiditis; type I diabetes; psoriasis; pemphigus; autoimmune hemolytic anemia (AIHA); idiopathic thrombocytopenic purpura (ITP); Goodpasture's syndrome; Degos disease; antiphospholipid syndrome (APS); catastrophic APS (CAPS); cardiovascular disorder; myocarditis; violation of cerebral circulation; peripheral vascular disease; renovascular disorder; mesenteric/enteric vascular disease; vasculitis; nephritis with Henoch-Schönlein purpura; Takayashi's disease; dilating cardiomyopathy; diabetic angiopathy; Kawasaki disease (arteritis); venous air embolism (VGE), restenosis after stenting; rotational atherectomy; membranous nephropathy; Guillain-Barré syndrome (GBS); Fisher's syndrome; antigen-induced arthritis; synovial inflammation; viral infections; bacterial infections; fungal infections; and damage due to myocardial infarction, cardiopulmonary bypass, and hemodialysis.

In some embodiments, the complement-mediated disease or condition is an ocular disease-associated condition. In other embodiments of the invention, the eye disease is macular degeneration. In other embodiments, the macular degeneration is AMD. In other embodiments of the invention, AMD is a dry form of AMD.

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

In some embodiments, a complement-mediated disease or

- 47 041632 the condition is a myocardial infarction.

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

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

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

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

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

Such combination therapies as mentioned above include co-administration (when two or more therapeutic agents are included in the same or different compositions) and separate administration, in each case administration of an antibody or polypeptide containing an Fc region variant of the invention, may be performed before, concurrently and/or after administration of the additional therapeutic agent or agents. In one embodiment, administration of the anti-C5 antibody and administration of an additional therapeutic agent may be within about a month, or within about 1, 2, or 3 weeks, or within about 1, 2, 3, 4, 5, or 6 days of each other. friend.

The antibody of the invention (and any additional therapeutic agent) can be administered by any suitable route, including parenteral, intrapulmonary and intranasal, and, if necessary, used for topical treatment, injection into lesions. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal or subcutaneous administration. Dosing may be by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending, inter alia, on whether the treatment is short-term or chronic. Various dosing regimens may be used, including (but not limited to) single administration or multiple administrations at different time points, bolus administration, and pulsatile infusion.

The antibodies of the invention may be formulated, dosed and administered in accordance with good clinical practice. Factors considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of administration of the agent, the method of administration, the schedule of administration, and other factors known to medical practitioners. The antibody may, but is not required, be included in the composition in combination with one or more other agents currently used to prevent and treat the disorder in question. The effective amount of these other agents depends on the amount of antibody present in the composition, the type of disorder or treatment, and other factors mentioned above. They are generally used in the same doses and using the same routes of administration as specified in the present description, or in an amount ranging from 1 to 99% of the dose indicated in the present description, or in any dose and with any pathways that are empirically/clinically judged to be suitable.

For the prevention or treatment of a disease, the acceptable dose of an antibody of the invention (when used alone or in combination with one or more other additional therapeutic agents) should depend on the type of disease being treated, the type of antibody, the type of polypeptide containing the variant Fc region , severity and course of disease, whether the antibody or polypeptide containing the Fc region variant is used for preventive or therapeutic purposes, prior therapy, the patient's medical history and response to the antibody or polypeptide containing the Fc region variant, as well as the prescription of the attending physician. An antibody or polypeptide containing a variant Fc region can be administered to a patient at a single time or as a series of treatments. Depending on the type and severity of the disease, from about 1 μg/kg to 15 mg/kg (e.g., 0.5-10 mg/kg) of the antibody may represent the initial possible dose to be administered to the patient, e.g., via one or more separate treatments. or by continuous infusion. One example of a typical daily dose may be from about 1 μg/kg to 100 mg/kg or more, depending on the above factors. When repeated administrations are used over a period of several days or a longer period of time depending on the condition, treatment should generally be continued until suppression of the symptoms of the disease is achieved. One example of doses of an antibody or polypeptide containing a variant Fc region is from about 0.05 to about 10 mg/kg. Thus, one or more of the following doses may be administered to a patient: about 0.5, 2.0, 4.0, or 10 mg/kg (or any combination thereof). These doses can be administered in fractional, for example, every week or every three weeks (for example, so that the patient receives from about two to about twenty, for example, about six doses of an antibody or polypeptide containing

- 48 041632 Fc region variant). An initial increased loading dose may be used followed by one or more lower doses. Using conventional methods and assays, the effect of said therapy can be easily monitored.

Obviously, any of the above compositions or therapeutic methods can be used using the immunoconjugate of the invention, instead of or in addition to the C5 antibody.

3. Products.

Another object of the invention is a product that contains products used for the treatment, prevention and/or diagnosis of the above disorders. The product is a container and a label or package insert placed on or inside the container. Acceptable containers are, for example, jars, vials, syringes, intravenous solution bags, etc. Containers can be made from various materials such as glass or plastic. The container contains a composition that alone or in combination with another composition is effective in treating, preventing and/or diagnosing a condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial provided with a stopper that can be pierced with using a hypodermic needle). At least one active ingredient in the composition is an antibody of the invention. The label or package insert indicates that the composition is used to treat the selected condition. In addition, the product may include (a) a first container containing the composition, where the composition contains the antibody proposed in the present invention; and (b) a second container containing the composition, wherein the composition contains an additional cytotoxic or other therapeutic agent.

According to this embodiment of the invention, the product may contain a package insert leaflet that contains information that the compositions can be used to treat a particular condition. In an alternative or additional embodiment, the product may further include a second (or third) container with a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BSVI), phosphate buffered saline, Ringer's solution and dextrose solution. In addition, it may include other products required from a commercial and consumer point of view, in particular other buffers, diluents, filters, needles and syringes.

As should be obvious, any of the above products may include the immunoconjugate of the invention, instead of or in addition to the antibody to C5.

Examples

The following are examples of methods and compositions proposed in the invention. As should be apparent, various other embodiments of the invention may be practiced based on the general description of the invention provided above.

Example 1. Obtaining C5.

1.1. Expression and purification of recombinant human C5 and cynomolgus monkeys.

Recombinant human C5 (NCBI GenBank, access code: NP_001726.2, SEQ ID NO: 39) was transiently expressed using the FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA). Conditioned media expressing human C5 were diluted with an equal volume of milliQ water, then applied to a Q-sepharose FF or Q-sepharose HP anion exchange column (GE healthcare, Uppsala, Sweden), and then eluted using a NaCl gradient. Fractions containing human C5 were pooled, after which the salt concentration and pH were adjusted to 80 mM NaCl and pH 6.4, respectively. The resulting sample was applied to a HP SP-sepharose cation exchange column (GE healthcare, Uppsala, Sweden) and eluted using a NaCl gradient. Fractions containing human C5 were pooled and applied to a ceramic hydroxyapatite CNT column (Bio-Rad Laboratories, Hercules, CA, USA). The human C5 containing eluate was then applied to a Superdex 200 gel filtration column (GE healthcare, Uppsala, Sweden). Fractions containing human C5 were pooled and stored at -150°C.

Expression and purification of recombinant C5 cynomolgus monkeys (NCBI GenBank, access code: XP_005580972, SEQ ID NO: 44) was carried out in the same manner as that used for the human copy.

1.2. Purification of Cynomolgus monkey C5 (cynoC5) from plasma.

Cynomolgus monkey plasma sample was loaded onto SSL7-agarose (Invivogen, San Diego, CA, USA) and then eluted using 100 mM sodium acetate, pH 3.5. Fractions containing cynoC5 were immediately neutralized and loaded onto a protein A HP column (GE healthcare, Uppsala, Sweden) in tandem with a peptide M-agarose column (Invivogen, San Diego, CA, USA). The run-through fraction was then applied to a Superdex 200 gel filtration column (GE healthcare, Uppsala, Sweden). Fractions containing cynoC5 were pooled and

- 49 041632 stored at -80°C.

Example 2. Creation of antibodies to C5.

2.1. Antibody screening.

Antibodies to C5 were obtained, selected and analyzed as follows.

New Zealand White Rabbits (NZW) aged 12-16 weeks were immunized intradermally with human C5 and/or monkey C5 (50-100 μg/dose/rabbit). Treatment with the indicated dose was repeated 3-5 times over a 2-month period. 1 week after the last immunization, spleen and blood samples from the immunized rabbit were collected. Antigen-specific B cells were stained with labeled antigen, sorted using an FCM cell sorter (FACS aria III, BD) and plated in 96-well plates at a density of 1 cell/well along with 25,000 EL4 cells/well (from the European Cell Culture Collection). ) and conditioned medium for activated rabbit T cells, diluted 20 times, and cultured for 7-12 days. EL4 cells were pretreated with mitomycin C (Sigma, catalog number M4287) for 2 hours and washed 3 times. Conditioned medium for activated rabbit T cells was prepared by culturing rabbit thymocytes in RPMI-1640 medium containing phytohemagglutinin-M (Roche, cat. no. 11082132-001), phorbol-12-myristate-13-acetate (Sigma, cat. no. P1585). ) and 2% FBS. After cultivation, the supernatants of B-cell cultures were collected for additional analysis and the debris was cryopreserved.

An ELISA assay was used to evaluate the specificity of antibodies in the B cell culture supernatant. MAXISorp 384-well plates (Nunc, cat. no. 164688) were sensitized with streptavidin (GeneScript, cat. no. Z02043) at 50 nM in PBS for 1 hour at room temperature. The plates were then blocked with Blocking One reagent (Nacalai Tesque, cat. no. 03953-95) diluted 5 times. Human or simian C5 was labeled with NHS-PEG4-biotin (PIERCE, catalog number 21329) and added to blocked ELISA plates, incubated for 1 hour and washed. B cell culture supernatants were loaded onto ELISA plates, incubated for 1 hour and washed. Binding was detected with goat anti-rabbit IgG conjugated to horseradish peroxidase (BETHYL, cat. no. A120-111P), to which ABTS (KPL, cat. no. 50-66-06) was added.

ELISA analysis 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 with PBS(-) was added to a 384-well MAXISorp plate (Nunc, cat. no. 164688), incubated for 1 hour at room temperature and blocked with Blocking One reagent (Nacalai Tesque, catalog number 03953-95) diluted 5 times. After incubation, the plates were washed and B cell culture supernatants were added. Plates were incubated for 1 hour, washed, 500 pM biotinylated human or monkey C5 was added and incubated for 1 hour. After incubation, plates were washed and incubated with either MES buffer, pH 7.4 (20 mM MES, 150 mM NaCl and 2 mm CaCl ₂ ), or with MES buffer, pH 5.8 (20 mm MES, 150 mm NaCl and 1 mm EDTA) for 1 h at room temperature. After incubation, binding of biotinylated C5 was detected with a streptavidin-horseradish peroxidase conjugate (Thermo Scientific, cat. no. 21132) by adding ABTS (KPL, cat. no. 50-66-06).

The Octet RED384 system (Pall Life Sciences) was used to evaluate the affinity and pH-dependent binding of antibodies to C5. Antibodies secreted into the B cell culture supernatant were applied to the tip of a protein A coated biosensor (Pall Life Sciences) and immersed in 50 nM human or simian C5 MES buffer, pH 7.4 for analysis of association kinetics. Dissociation kinetics were analyzed in both MES buffer pH 7.4 and MES buffer pH 5.8.

A total of 41439 B cell lines were screened for affinity and pH dependent binding to human or simian C5 and 677 lines were selected and designated as CFA0001-0677. RNA of selected lines was purified from cryopreserved cell debris using ZR-96 Quick-RNA kits (ZYMO RESEARCH, catalog number R1053). The DNA encoding the heavy chain variable regions of the antibodies of the selected lines were amplified by reverse transcription PCR and recombined with the DNA encoding the heavy chain constant region of F760G4 (SEQ ID NO: 33) or F939G4 (SEQ ID NO: 34). The DNA encoding the antibody light chain variable regions was amplified by reverse transcription PCR and recombined with the DNA encoding the kOMTC light chain constant region (SEQ ID NO: 36). The heavy and light chain genes of the existing humanized anti-C5 antibody, eculizumab (EcuH-G2G4, SEQ ID NO: 29 and EcuL-k0, SEQ ID NO: 30) were synthesized separately. DNA encoding VH (EcuH, SEQ ID NO: 31) was fused in frame with DNA encoding modified human IgG4 CH (F760G4, SEQ ID NO: 33), and DNA encoding VL (EcuL, SEQ ID NO: 32), was fused in frame with DNA encoding the light chain constant region k0 (SEQ ID NO: 37). Each of the fused coding sequences was also cloned into an expression vector. Antibodies were expressed in FreeStyle™ 293-P cells (Invitrogen) and purified from culture supernatants for evaluation.

- 50 041632 functional activity. Antibody neutralizing activities were assessed by the complement activity inhibition assay using the liposome lysis assay as described in Example 5.1.

2.2. Grouping depending on the epitope using a sandwich ELISA.

Antibodies to C5 with high affinity, pH-dependence or neutralizing activity were selected for further analysis. Sandwich ELISA analysis was used to group selected antibodies binding to the same or overlapping epitopes of the C5 protein into different epitope baskets. Unlabeled capture antibodies were diluted to 1 μg/ml with PBS(-) and added to MAXISorp 384-well plates (Nunc, cat. no. 164688). Plates were incubated for 1 hour at room temperature and blocked with Blocking One reagent (Nacalai Tesque, cat. no. 03953-95) diluted 5 times. The plates were incubated for 1 h, washed, 2 nM human C5 was added and incubated for 1 h. After incubation, the plates were washed and labeled identifying antibodies (1 μg/ml, biotinylated with NHS-PEG4-biotin) were added. After a 1 hour incubation, binding of the biotinylated antibody was detected with a streptavidin-horseradish peroxidase conjugate (Thermo Scientific, cat. no. 21132) by adding ABTS (KPL, cat. no. 50-66-06).

All anti-C5 antibodies were used both as a capture antibody and as an identifying antibody, and a comprehensive pairwise analysis was performed. As shown in FIG. 1, mutually competing antibodies were grouped into 7 epitope bins: CFA0668, CFA0334 and CFA0319 assigned to epitope group A, CFA0647, CFA0589, CFA0341, CfAo639, CFA0635, CFA0330 and CFA0318 assigned to epitope group B, CFA0538, CFA0397, CFA0501, CFA0501 CFA0366, CFA0305, CFA0675, CFA0666 and CFA0672 are classified as epitope B, eculizumab and CFA0322 are classified as epitope D, CFA0329 are assigned as epitope E, CFA0359 and CFA0217 are assigned as epitope E, and CFA0579, CFA0328 and CFA0272 are assigned as epitope G. FIG. 1 shows the grouping depending on the epitope of some chimeric antibodies to C5. The VH and VL sequences of anti-C5 antibodies included in epitope group B are listed in Table 1. 2.

table 2

Antibodies belonging to the epitope group B

SEQIDNO:

Antibody vh VL HVR-H1 HVR-H2 HVR-H3 HVR-L1 HVR-L2 HVR-L3 CFA0305 1 eleven 45 55 65 75 85 95 CFA0307 2 12 46 56 66 76 86 96 CFA0366 3 13 47 57 67 77 87 97 CFA0501 4 14 48 58 68 78 88 98 CFA0538 5 15 49 59 69 79 89 99 CFA0599 6 16 50 60 70 80 90 100 CFA0666 7 17 51 61 71 81 91 101 CFA0672 8 18 52 62 72 82 92 102 CFA0675 9 19 53 63 73 83 93 103

2.3. Humanization and optimization.

The variable region of some anti-C5 antibodies has been humanized to reduce the potential immunogenicity of the antibodies. The hypervariable regions (CDRs) of a rabbit anti-C5 antibody were transplanted into homologous human antibody frameworks (FRs) using a standard CDR grafting method (Nature, 321, 1986, c. 522-525). Genes encoding humanized VH and VL were synthesized and combined with modified human IgG4 CH (SG402, SEQ ID NO: 35) and human CL (SK1, SEQ ID NO: 38), respectively, and each of the combined sequences was cloned into an expression vector.

A large number of mutations and combinations of mutations were analyzed to identify mutations and combinations of mutations that improve the binding properties of some promising antibodies. Then introduced a variety of mutations in humanized variable regions to increase the binding affinity for C5 at neutral pH or to reduce the binding affinity for C5 at acidic pH. In this way, based on CFA0305, one of the optimized variants, 305LO5 (VH, SEQ ID NO: 10; VL, SEQ ID NO: 20; HVR-H1, SEQ ID NO: 54; HVR-H2, SEQ ID NO: 64; HVR-H3, SEQ ID NO: 74; HVR-L1, SEQ ID NO: 84; HVR-L2, SEQ ID NO: 94 and HVR-L3, SEQ ID NO: 104).

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

Example 3. Characterization of the binding of antibodies to C5.

3.1. Expression and purification of recombinant antibodies.

Recombinant antibodies were transiently expressed using FreeStyle293-F (FS293-F cells) (Thermo Fisher, Carlsbad, CA, USA). Purification from conditioned media in which antibodies were expressed was carried out using a standard method using protein A. If necessary, gel filtration was then performed.

3.2. Assessment of pH dependence.

- 51 041632

The kinetic parameters of anti-C5 antibodies against recombinant human C5 were evaluated at pH 7.4 and pH 5.8 at 37° C. using a BIACORE® T200 device (GE Healthcare). ProA/G protein (Pierce) was immobilized on a CM4 sensor chip using an amine coupling kit (GE Healthcare) under conditions recommended by GE Healthcare. Antibodies and analytes were diluted with appropriate running buffers, ACES pH 7.4 and pH 5.8 (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl ₂ , 0.05% Tween 20, 0.005% NaN ₃ ). Each antibody was captured on the sensor surface using ProA/G. Antibody capture levels typically corresponded to 60-90 resonance units (RU). Recombinant human C5 was then injected at concentrations of 10 and 20 nM, or 20 and 40 nM, and then allowed to dissociate. The surface was regenerated using 25 mm NaOH. Kinetic parameters at both pH values were determined by sensogram fit based on a 1:1 binding model using BIACORE® T200 Evaluation software version 2.0 (GE Healthcare). Sensorgrams for all antibodies are presented in Fig. 2A and 2B. Association rate (ka), dissociation rate (kd) and binding affinity (KD) of antibodies are presented in table. 3. All antibodies except CFA0330 (VH, SEQ ID NO: 21 and VL, SEQ ID NO: 25) and CFA0341 (VH, SEQ ID NO: 22 and VL, SEQ ID NO: 26) had a relatively faster rate of dissociation at pH 5.8 than at pH 7.4.

Table 3

Kinetic parameters of antibodies to C5 at pH 7.4 and pH 5.8

Antibody designation ka pH7.4kd KD ka pH5.8kd KD CFA0305 3.82E+04 5.89E-04 1.54Е-08 4.27E+04 1.83E-02 4.30E-07 CFA0307 3.24E+05 2.63E-03 8.1 SE-09 2.04E+05 3.34Е-02 1.64E-07 CFA0366 1.04E+06 9.34Е-03 8.99Е-09 9.35E+05 7.03Е-02 7.52E-08 CFA0501 4.74E+05 1.69E-03 3.56E-09 1.50E+05 2.62E-02 1.74E-07 CFA0538 4.73E+05 1.85E-03 3.91 E-09 1.22E+05 3.01 E-02 2.46E-07 CFA0599 4.74E+05 2.81 E-03 5.93E-09 4.54E+05 3.73E-02 8.21 E-08 CFA0666 3.65E+05 6.26E-04 1.71 E-09 2.82E+05 9.39Е-03 Z.ZZE-08 CFA0672 5.23E+05 1.83E-04 3.51E-10 7.11E+04 9.78Е-03 1.38E-07 CFA0675 3.83E+05 4.12E-04 1.08E-09 3.89E+05 6.61 E-03 1.70E-08 305-LO5 4.48E+05 2.11E-04 4.71E-10 2.03E+06 2.85E-02 1.40E-08 CFA0330 1.66E+06 2.02Е-04 1.22E-10 1.22E+06 2.24Е-04 1.84Е-10 CFA0341 6.28E+05 9.77Е-05 1.55E-10 1.24E+06 7.39Е-05 5.95E-11

3.3. Cross-reactivity assessment.

To detect cross-reactivity of anti-C5 antibodies, namely anti-human C5 (hC5) and anti-C5 cynomolgus monkey (cynoC5) antibodies, a BIACORE® kinetic assay was performed. The analysis was carried out under the same conditions as described in example 3.2. Recombinant cynoC5 was injected at concentrations of 2, 10 and 50 nM. Kinetic parameters were determined using the same data fit as described in Example 3.2. The results of determining the kinetic characteristics of binding and affinity at pH 7.4 are presented in table. 4. Kinetic parameters in relation to hC5 indicated in table. 4 are the results obtained in Example 3.2. All anti-C5 antibodies except CFA0672 had comparable KD values for hC5 and cynoC5. The KD for CFA0672 for cynoC5 was 8 times higher (weaker affinity) than for hC5.

Table 4

Binding kinetics and affinity of antibodies to C5, namely antibodies to hC5 and cynoC5, at pH 7.4

Antibody designation Affinity for hC5 Affinity for supoC5 ka kd KD ka kd KD CFA0305 3.82E+04 5.89E-04 1.54Е-08 1.21E+04 6.70E-04 5.54Е-09 CFA0307 3.24E+05 2.63E-03 8.1 SE-09 2.90E+05 2.23E-03 7.68E-09 CFA0366 1.04E+06 9.34Е-03 8.99Е-09 5.04E+05 9.04Е-03 1.79E-08 CFA0501 4.74E+05 1.69E-03 3.56E-09 2.66E+05 1.56E-03 5.88E-09 CFA0538 4.73E+05 1.85E-03 3.91E-09 3.05E+05 1.66E-03 5.44Е-09 CFA0599 4.74E+05 2.81 E-03 5.93E-09 5.42E+05 2.35E-03 4.33E-09 CFA0666 3.65E+05 6.26E-04 1.71 E-09 3.14E+05 4.93E-04 1.57E-09 CFA0672 5.23E+05 1.83E-04 3.51E-10 6.41E+05 1.85E-03 2.88E-09 CFA0675 3.83E+05 4.12E-04 1.08E-09 2.94E+05 3.78E-04 1.29E-09

Example 4 Epitope mapping for anti-C5 antibodies.

4.1. Binding of MAbs to C5 with peptides derived from the C5 beta chain.

Anti-C5 monoclonal antibodies (MAbs) were tested for binding to C5 beta chain-derived peptides by Western blot analysis. C5 peptides: 19-180, 161-340, 321-500 and 481-660 fused to GST tag (pGEX-4T-1, GE Healthcare Life Sciences, 28-9545-49) expressed in E. coli (DH5alpha, TOYOBO, DNA-903). E. coli containing samples were collected after incubation with 1 mM isopropyl-beta-D-1-thiogalactopyranoside (IPTG) for 5 h at 37° C. and centrifuged at 20,000xg for 1 min to obtain pellets. Debris was suspended in sample buffer solution (2ME+) (Wako, 191-13272) and used for Western blot analysis.

Expression of each peptide was confirmed using an anti-GST antibody (Abcam, ab9085) (FIG. 3). The arrow indicates GST-fused C5 peptides (46-49 kDa). Mat to C5: CFA0305, CFA0307,

CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672 and CFA0675 bind to amino acids

19-180 C5 (Fig. 3).

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

The recombinant MG1-MG2 domain (SEQ ID NO: 43) of the human C5 beta chain was transiently expressed using FreeStyle293-F (FS293-F cells) (Thermo Fisher, Carlsbad, CA, USA). Conditioned media in which MG1-MG2 domain expression occurred were diluted 1/2 volume with milliQ water and then loaded onto a Q-sepharose FF anion exchange column (GE healthcare, Uppsala, Sweden). The pH of the anion exchange column fraction was adjusted to pH 5.0 and loaded onto a HP SP Sepharose cation exchange column (GE healthcare, Uppsala, Sweden) and then eluted using a NaCl gradient. Fractions containing the MG1-MG2 domain were collected from the eluent and then gel-filtered on a Superdex 75 column (GE healthcare, Uppsala, Sweden) equilibrated with 1×PBS. Fractions containing the MG1-MG2 domain were then pooled and stored at -80°C.

4.3. The ability to bind to the MG1-MG2 domain.

The ability of the anti-C5 antibody to bind to the MG1-MG2 domain was evaluated under the same assay conditions as described in Example 3.2, except that measurements were only made under pH 7.4 conditions. The MG1-MG2 domain was injected at concentrations of 20 and 40 nM. As shown in FIG. 4, all antibodies except eculizumab-F760G4 showed an increased binding response, indicating that these antibodies were MG1-MG2 binding agents. Eculizumab-F760G4, which is known to be an alpha chain binding agent, was not found to bind to the MG1-MG2 domain.

4.4. Binding of Mab to C5 with peptides derived from the MG1-MG2 domain of C5.

The anti-C5 mAb was tested for binding to MG1-MP2 domain-derived peptides by Western blot analysis. C5 peptides: 33-124, 45-124, 52-124, 33-111, 33-108 and 45-111 (SEQ ID NO: 40) fused to the GST tag were expressed in E. coli. E. coli containing samples were collected after incubation with 1 mM IPTG for 5 hours at 37° C. and centrifuged at 20,000xg for 1 minute to obtain debris. Debris was suspended in sample buffer solution (2ME+) and used for Western blot analysis. Expression of C5-derived peptides was confirmed using an anti-GST antibody (FIG. 5A). CFA0305 only bound peptide 33-124 (FIG. 5B). CFA0305 bound to the beta chain of recombinant human C5 (rhC5) (approximately 70 kDa), which was used as a control. In FIG. 5B summarizes the interaction of anti-C5 mAbs with C5-derived peptides.

4.5. Mab binding to C5 with C5 mutants.

Since three amino acid residues in the C5 beta chain, namely E48, D51 and K109, were predicted based on crystal structure analysis to be involved in C5 binding to C5 Mab, C5 Mab was tested for binding to human point mutants. C5 by Western blot analysis. Containing point mutations C5 mutants, in which one of the residues E48, D51 and K109 was replaced by alanine, were expressed in FS293 cells using lipofection. Culture media were harvested 5 days after lipofection and used for Western blotting thereafter. SDS-PAGE was carried out under reducing conditions. The results are shown in FIG. 6. Eculizumab bound to wild-type (WT) C5 alpha chain and three C5 point mutants, while CFA0305 antibody was characterized by strong binding to WT C5 beta chain, weak binding to C5 E48A mutant and no binding to beta - chain D51A mutant and K109A mutant C5; this indicates that these 3 amino acid residues are involved in antibody/antigen interactions. In table. 5 is a summary of the results of Western blot analysis of anti-C5 mAbs (CFA0305, CFA0307, CFA0366, CFA0501, CFA0538, CFA0599, CFA0666, CFA0672 and CFA0675). MAbs to C5 belong to the group for the same epitope B, however, the binding patterns of antibodies are somewhat different; this suggests that the regions binding C5 to MAb to C5 are close to each other, but are not identical.

- 53 041632

Table 5 Summary of data on the interaction of Mab to

C5 with C5 mutants

wt E48A D51A K109A eculizumab + + + + CFA0305 + + - - CFA0307 + - - - CFA0366 + - - - CFA0501 + - - - CFA0538 + - - - CFA0599 + - - + CFA0666 + - - + CFA0672 + - - + CFA0675 + + - +

4.6. Analysis of the binding of anti-C5 antibodies to C5 mutants by the BIACORE® method.

To check whether residues E48, G51 and K109 are indeed involved in antibody/antigen interactions, a BIACORE® binding assay was performed. Received three mutants C5, namely containing mutations E48A, G51A and K1O9A, as described in example 4.5. Received samples of culture supernatants containing FS293 cells overexpressing the C5 mutant with a concentration of C5 mutant 40 μg/ml. For BIACORE® binding assay, the sample was diluted 10-fold with BIACORE® running buffer (ACES pH 7.4, 10 mg/kg BSA, 1 mg/mL carboxymethyl dextran) to a final concentration of C5 mutant in the sample of 4 μg/mL.

The interactions of the three C5 mutants with C5 antibodies were evaluated at 37° C. using a BIACORE® T200 device (GE Healthcare) using the assay conditions described in Example 3.2. ACES buffer pH 7.4 containing 10 mg/ml BSA, 1 mg/ml carboxymethyl dextran was used as running buffer. Eculizumab-P760G4 and 305LO5 were captured on various flow cells with an anti-human IgG monoclonal mouse antibody, Fc fragment specific (GE Healthcare). Flow cell 1 was used as a reference surface. Wild-type and C5 mutant C5 proteins were injected onto the sensor surface at a concentration of 4 μg/ml to interact with captured antibodies. After the end of each analysis cycle, the sensor surface was regenerated with 3 M MgCl ₂ . The results were analyzed using Bia Evaluation software version 2.0 (GE Healthcare). The curves obtained for the reference flow cell (flow cell 1) and the curves obtained using empty running buffer injections were subtracted from the curves obtained for the captured antibody flow cells.

As shown in FIG. 7, all three C5 mutants were able to bind to eculizumab with a binding profile comparable to wild-type C5. In the case of 305LO5, all three mutants showed a lower binding response to 305LO5 compared to wild-type C5. Mutations D51A and K1O9A reduced C5 binding to 305LO5 to the background level.

4.7. Identification of His residues in C5 that are involved in pH-dependent interactions between anti-C5 and C5 antibodies.

Crystal structure analysis revealed 3 histidine residues in human C5 located at the antibody/antigen interface. A histidine residue with a typical pKa of about 6.0 is known to be involved in pH-dependent protein-protein interactions (Igawa et al., Biochim Biophys Acta 1844(11), 2014, c. 1943-1950). To investigate whether His residues at the antibody/antigen interface are involved in pH-dependent interactions between C5 and C5 antibody, a BIACORE® binding assay was performed. Three mutants of human C5 with one His mutation (H70Y, H72Y and H110Y) and a double His mutation mutant (H70Y+H110Y) were prepared as follows: single His mutation mutants in which one of H70, H72 and H110 is replaced by tyrosine, and a double His mutation mutant in which both H70 and H110 are replaced by tyrosine, were expressed in FS293 cells using lipofection. The antigen-binding properties of 305LO5, a pH dependent anti-C5 antibody, against C5 His mutants were determined using a modified BIACORE® assay as described in Example 4.6. In general, the method was as follows: in the BIACCGE® assay, an additional dissociation phase at pH 5.8 was included immediately after the dissociation phase at pH 7.4 to assess the pH-dependent dissociation of antibody and antigen from the complexes formed at pH 7.4. The dissociation rate at pH 5.8 was determined by data processing and fitting using Scrubber 2.0 curve fitting software (BioLogic Software).

As shown in FIG. 8, a single His mutation of H70 or NPO and a double His mutation (H70+NPO) of C5 had no effect on C5 binding to 305LO5 at neutral pH. At the same time, one H72 His-mutation led to a significant decrease in C5 binding to 305LO5. Dissociation rates at pH 5.8 for His-mutants of the C5 protein and C5-wt protein are presented in Table 1. 6. As shown in Table. 6, C5-wt was characterized by the fastest dissociation from 305LO5 at pH 5.8 among the tested C5 antigens. One His-mutation H70 led to an almost two-fold slowdown

- 54 041632 dissociation rate at pH 5.8, and one His-mutation of NPO resulted in a slight slowdown in the dissociation rate at pH 5.8 compared to C5-wt. The His double mutation of both H70 and NPO resulted in a more pronounced effect on pH-dependent binding, with the dissociation rate at pH 5.8 being almost three times slower than in the case of C5-wt.

Table 6

Dissociation rate value at pH 5.8 for C5 His mutants binding to 305LO5

Antigens kd (1/s) C5-wt 1.1 E-2 C5-H70Y 5.3E-3 C5-H110Y 9.3E-3 C5-H70Y, H110Y 3.9E-3

Example 5 Activity of C5 Antibodies to Inhibit C5 Activation.

5.1. Inhibition of complement-activated liposome lysis of Mab to C5.

The anti-C5 mAb was tested for inhibition of complement activity using a liposome lysis assay. 30 μl of healthy human serum (6.7%) (Biopredic, SER018) was mixed with 20 μl of diluted Mab in a 96-well plate and incubated on a shaker for 30 min at 25°C. Liposomes sensitized with anti-dinitrophenyl antibodies (Autokit CH50, Wako, 995-40801) were transferred to each well and the plate was placed for 2 minutes on a shaker at 25°C. 50 µl of substrate solution (Autokit CH50) was added to each well and mixed by shaking for 2 min at 25°C. The final mixture was incubated at 37° C. for 40 min and then the OD of the mixture was measured at 340 nm. The percentage of liposome lysis was determined by the formula

100x[(OPmat- ^OP background due to serum and liposomes ^)]/[OD without MAT ^-OP background due to serum and liposomes)]

In FIG. 9A demonstrates that anti-C5 mAbs CFA0305, 0307, 0366, 0501, 0538, 0599, 0666, 0672 and 0675 inhibit liposome lysis. The two non-pH dependent antibodies CFA0330 and 0341 also inhibited lysis (FIG. 9B).

5.2. Inhibition of C5a formation with anti-C5 mAb.

Anti-C5 mAbs were tested for C5a production during liposome lysis to confirm that C5 mAbs inhibit C5 cleavage to form C5a and C5b. The C5a level in the supernatants obtained from the liposome lysis assay was quantified using a C5a ELISA kit (R&D systems, DY2037). All mAbs inhibited the formation of C5a in supernatants in a dose dependent manner (FIGS. 10A and 10B).

5.3. Inhibition of Anti-C5 Mab Complement-Activated Hemolysis Anti-C5 Mab was tested for inhibition of classical complement activity by a hemolytic assay. Chicken red blood cells (cRBC) (Innovative research, IC05-0810) were washed with gelatin/veronal buffered saline containing 0.5 mM MgCl ₂ and 0.15 mM CaCl ₂ (GVB++) (Boston BioProducts, IBB-300X), and then sensitized with an antibody to chicken RBC (Rockland 103-4139) at a concentration of 1 μg/ml for 15 min at 4°C. The cells were then washed with GVB++ and suspended in the same buffer at 5 x 10 ⁷ cells/ml. In a separate round-bottom 96-well plate, 50 µl of healthy human serum (20%) (Biopredic, SER019) was mixed with 50 µl of diluted Mab and incubated on a shaker at 37°C for 30 min. Then, 60 µl of the sensitized cRBC suspension was added to the wells containing serum, and the mixture containing the antibody was incubated at 37°C for 30 min. After incubation, the plate was centrifuged at 1000xg for 2 min at 4°C. Supernatants (100 μl) were transferred to the wells of a flat-bottomed 96-well microassay plate for OD measurements at 415 nm, the reference wavelength was 630 nm. The percentage of hemolysis was determined by the formula ^{100x[( Op} MAb ^{- OP} serum and ^cRBC)]/[(0P without MAT ^{- OP} background due to serum and cRBC ^)]

In FIG. 11 demonstrates that anti-C5 mAbs CFA0305 and 305LO5 inhibit cRBC hemolysis.

5.4. Inhibition of mAb to C5 alternative complement pathway.

Hemolytic analysis in the case of the alternative route was carried out similarly to the implementation of the hemolytic analysis in the case of the classical route. Blood collected from New Zealand white rabbits (InVivos) was mixed with an equal volume of Olsver's solution (Sigma, A3551) and the mixture was used as rabbit RBCs (rRBCs). rRBC was washed with GVB supplemented with 2 mM MgCl ₂ and 10 mM EGTA and suspended in the same buffer at 7x108 cells/ml. In a round bottom 96-well microassay plate, 40 µl of healthy human serum (25%) (Biopredic, SER019) was mixed with 40 µl of diluted Mab and incubated on a shaker at 37°C for 30 min. Then, 20 µl of rRBC suspension was added to the wells containing serum, and the mixture containing the antibody was incubated at 37°C for 60 min. After incubation, the plate was centrifuged at 1000xg for 2 min at 4°C. Supernatants (70 μl) were transferred to the wells of a flat-bottomed 96-well microassay plate to measure OD at 415 nm, the reference wavelength was 630 nm. In FIG. 12 demonstrates that anti-C5 mAbs CFA0305 and CFA0672 inhibited hemolysis.

- 55 041632 rRBC, this indicates that these antibodies inhibit alternative complement pathways.

Example 6 Pharmacokinetic study of anti-C5 monoclonal antibodies using human C5 in mice.

6.1. An in vivo experiment using C57BL/6 mice.

In vivo kinetic analysis of human C5 (Calbiochem) and anti-human C5 antibody was performed after administration of human C5 alone or human C5 and anti-human C5 antibody C57BL/6 mice (In Vivos or Biological Resource Centre, Singapore). Human C5 solution (0.01 mg/ml) or mixture solution containing human C5 and anti-human C5 antibody (0.01 and 2 mg/ml , CFA0599-F760G4, CFA0666-F760G4, CFA0672-F760G4 and CFA0675-F760G4) or 0.2 mg/mL (CFA0330-F760G4 and CFA0341-F760G4), respectively), was administered as a single dose of 10 ml/kg into the caudal vein. In this case, the anti-human C5 antibody was present in excess relative to human C5, and therefore it can be assumed that each human C5 bound to the antibody. Blood was collected at 5 minutes, 7 hours, 1 day, 2 days, 3 days and 7 days after administration. The collected blood was immediately centrifuged at 14,000 rpm and 4° C. for 10 minutes to separate the plasma. The separated plasma was stored in a freezer at -80° C. until analysis. Применяли описанные выше антитела к человеческому С5: CFA0305-F760G4, CFA0307-F760G4, CFA0330-F760G4, CFA0341-F760G4, CFA0366-F760G4, CFA0501-F760G4, CFA0538-F760G4, CFA0599-F760G4, CFA0666-F760G4, CFA0672-F760G4 и CFA0675F760G4.

6.2. Measurement of total plasma concentration of human C5 using electrochemiluminescent (ECL) analysis.

The total plasma concentration of human C5 in mice was measured using ECL.

In the presence of CFA0330-F760G4, CFA0341-F760G4 or human C5 alone in the plasma sample, the following method was used. Anti-human C5 antibody (Santa Cruz) was loaded into an empty 96-well MULTI-ARRAY plate (Meso Scale Discovery) and allowed to stand overnight at 4°C to prepare anti-human C5 antibody immobilized plates. Calibration curve samples and mouse plasma samples diluted 100 times or more were prepared and injected with 1 μg/ml antibody (CFA0330-F760G4 or CFA0341F760G4) and incubated for 30 min at 37°C. The samples were then plated with immobilized anti-human C5 antibody and allowed to stand for 1 h at room temperature. Then, a SULFO-labeled anti-human IgG antibody (Meso Scale Discovery) was added and allowed to react for 1 hour at room temperature, and washing was performed. Thereafter, Read Buffer T (x4) (Meso Scale Discovery) was immediately added and measurements were taken using a Sector Imager 2400 (Meso Scale Discovery).

In the presence of CFA0305-F760G4, CFA0307-F760G4, CFA0366-F760G4, CFA0501F760G4, CFA0538-F760G4, CFA0599-F760G4, CFA0666-F760G4, CFA0672-F760G4, or CFA060G4, CFA0672-F760G4 or CFA060G4, CFA0672-F760G4 or CFA060G4 in the plasma sample, the following method was used. Anti-human C5 antibody (CFA0329-F939G4; VH, SEQ ID NO: 23 and VL, SEQ ID NO: 27) was added to an empty 96-well MULTI-ARRAY plate (Meso Scale Discovery) and allowed to stand overnight at 4° C to obtain plates with immobilized antibodies to human C5. Prepared samples for the construction of the calibration curve and samples of mouse plasma, diluted 100 or more times with an acidic solution (pH 5.5), and incubated for 30 min at 37°C. The samples were then plated with immobilized anti-human C5 antibody and allowed to stand for 1 h at room temperature. Then SULFO-labeled anti-human C5 antibody (CFA0300-F939G4; VH, SEQ ID NO: 24 and VL, SEQ ID NO: 28) was added and allowed to react for 1 hour at room temperature, followed by washing. Thereafter, Reading Buffer T (x4) (Meso Scale Discovery) was immediately added and measurements were taken using a Sector Imager 2400 (Meso Scale Discovery).

Human C5 concentration was calculated from the response obtained from the calibration curve using SOFTmax PRO software (Molecular Devices). A time plot of plasma concentration of human C5 after intravenous administration obtained by this method is shown in FIG. 13. Data plotted as percentage of C5 remaining compared to plasma concentration of human C5 after 5 minutes.

6.3. Plasma measurement of anti-human C5 antibody by ECL assay.

The concentration of antibodies to human C5 in the plasma of mice was measured using ECL. Anti-human IgG (gamma chain specific) F(ab')2 fragment (Sigma) or anti-human IgG kappa chain (Antibody Solutions) were added to an empty 96-well MULTI-ARRAY plate (Meso Scale). Discovery) and allowed to stand overnight at 4°C to obtain tablets with immobilized anti-human IgG antibody. Prepared samples for the construction of the calibration curve and samples of mouse plasma, diluted 100 times or more. The samples were then plated with immobilized anti-human IgG and allowed to stand for

- 56 041632 for 1 hour at room temperature. Then biotinylated anti-human IgG (Southernbiotech) or SULFO-labeled anti-human IgG, Fc (Southernbiotech) was added and allowed to react for 1 hour at room temperature, followed by washing. Then, only when a biotinylated anti-human IgG antibody was used, SULFO-labeled streptavidin (Meso Scale Discovery) was added and allowed to react for 1 hour at room temperature, and washing was also performed. Thereafter, Reading Buffer T (x4) (Meso Scale Discovery) was immediately added and measurements were taken using a Sector Imager 2400 (Meso Scale Discovery). Human C5 concentration was calculated from the response obtained from the calibration curve using SOFTmax PRO software (Molecular Devices). A time plot of plasma concentration of human C5 after intravenous administration obtained by this method is shown in FIG. 14. Data plotted as percentage of C5 remaining compared to plasma concentration of human C5 after 5 minutes.

6.4. Effect of pH-Dependent Anti-Human C5 Antibody Binding on Human C5 Clearance in Vivo.

рН-зависимые антитела к человеческому С5 (CFA0305-F760G4, CFA0307-F760G4, CFA0366F760G4, CFA0501-F760G4, CFA0538-F760G4, CFA0599-F760G4, CFA0666-F760G4, CFA0672-F760G4 и CFA0675-F760G4) и не обладающие рН-зависимостью антитела к human C5 (CFA0330-F760G4 and CFA0341-F760G4) were tested in vivo and the plasma concentration of anti-human C5 antibody was compared with the plasma concentration of human C5. As shown in FIG. 14, antibody exposure was comparable. At the same time, the clearance of human C5, which was administered simultaneously with pH-dependent antibodies to human C5, increased compared to the clearance in the presence of non-pH-dependent antibodies to human C5 (Fig. 13).

Example 7 Optimization of anti-C5 monoclonal antibodies (antibody 305 variants).

A large number of mutations were introduced into the optimized variable region of the anti-C5 antibody 305LO5 to further improve its properties and optimized variable regions 305LO15, 305LO16, 305LO18, 305LO19, 305LO20, 305LO22 and 305LO23 were created. Amino acid sequences of VH and VL variants 305 are presented in table. 7 and 8, respectively. Genes encoding humanized VH were combined with modified human IgG1 CH variant SG1 15 (SEQ ID NO: 114) and modified human IgG4 CH variants SG422 (SEQ ID NO: 115) or SG429 (SEQ ID NO: 116). Genes encoding humanized VL were combined with human CL (SK1, SEQ ID NO: 38). The heavy and light chain genes encoding the humanized anti-C5 antibody BNJ441 (BNJ441H, SEQ ID NO: 149; BNJ441L, SEQ ID NO: 150) were synthesized separately and each cloned into an expression vector.

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

Table 7

Amino acid sequences of VH variants 305

Antibody vh HVR-H1 HVR-H2 HVR-H3 305LO5 SEQID NO: 10 SEQ ID NO: 54 SSYYVA SEQID NO: 64 AIYTGSGATYKASWAKG SEQ ID NO: 74 DGGYDYPTHAMHY 305LO15 SEQ ID NO: 106 SEQID NO: 117 SSYYMA SEQ ID NO: 118 AIFTGSGAEYKAEVAKG SEQ ID NO: 121 DAGYDYPTHAMHY 305LO16 SEQID NO: 107 SEQID NO: 117 SSYYMA SEQ ID NO: 119 AIFTGSGAEYKAEWVKG SEQ ID NO: 121 DAGYDYPTHAMHY 305LO18 SEQID NO: 103 SEQID NO: 117 SSYYMA SEQ ID NO: 118 AIFTGSGAEYKAEVAKG SEQ ID NO: 121 DAGYDYPTHAMHY 305LO19 SEQID NO: 109 SEQID NO: 117 SSYYMA SEQ ID NO: 118 AIFTGSGAEYKAEVAKG SEQ ID NO: 121 DAGYDYPTHAMHY 3Q5LO2D SEQID NO: 109 SEQID NO: 117 SSYYMA SEQ ID NO: 118 AIFTGSGAEYKAEVAKG SEQ ID NO: 121 DAGYDYPTHAMHY 305LO22 SEQID NO: 109 SEQ ID NO: 117 SSYYMA SEQ ID NO: 118 AIFTGSGAEYKAEVAKG SEQ ID NO: 121 DAGYDYPTHAMHY 3Q5LO23 SEQID NO: 110 SEQID NO: 117 SSYYMA SEQ ID NO: 120 GIFTGSGATYKAEWAKG SEQ ID NO: 121 DAGYDYPTHAMHY

- 57 041632

Table 8

Amino acid sequences of VL variants 305

Antibody VL HVR-L1 HVR-L2 HVR-L3 305LO5 SEQIDNO:20 SEQ ID NO: 84 QA5QNIGSSIA SEQ ID NO:94 GASKTHS SEQ ID NO: 104 QSTKVGSSYGNH 305LO15 SEQ ID NO: 111 SEQ ID NO: 122 RASQGISSSLA SEQ ID NO: 123 GASETES SEQ ID NO: 125 QNTKVGSSYGNT 305LO16 SEQ ID NO: 111 SEQ ID NO: 122 RASQGISSSLA SEQ ID NO: 123 GASETES SEQ ID NO; 125 QNTKVGSSYGNT 305LO18 SEQ ID NO: 111 SEQ ID NO: 122 RASQGISSSLA SEQ ID NO: 123 GA5ETES SEQ ID NO: 125 QNTKVGSSYGNT 305LO19 SEQ ID NO: 111 5EQIDNO: 122 RASQGISSSLA SEQ ID NO: 123 GASETES SEQ ID NO: 125 QNTKVGSSYGNT 3O5L02O SEQ ID NO: 112 SEQ ID NO: 122 RASQGISSSLA SEQ ID NO: 123 GASETES SEQ ID NO: 125 QNTKVGSSYGNT 305LO22 SEQ ID NO: 113 SEQ ID NO: 122 RASQGISSSLA SEQ ID NO: 124 GASTTQS SEQ ID NO: 125 QNTKVGSSYGNT 305LO23 SEQ ID NO: 113 SEQ ID NO: 122 RASQGISSSLA SEQ ID NO: 124 GASTTQS SEQ ID NO: 125 QNTKVGSSYGNT

Example 8. Characterization of the binding of antibodies to C5 (options 305).

Kinetic parameters of anti-C5 antibodies, namely binding parameters to recombinant human C5, were evaluated at 37°C using a BIACORE® T200 device (GE Healthcare) under three different conditions:

(1) both association and dissociation took place at pH 7.4, (2) both association and dissociation took place at pH 5.8, and (3) association took place at pH 7.4, but dissociation was place at pH 5.8.

ProA/G (Pierce) was immobilized on a CM1 sensor chip using an amine coupling kit (GE Healthcare) using the conditions recommended by GE Healthcare. Antibodies and analytes for conditions (1) and (3) were diluted in ACES buffer pH 7.4 (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl ₂ , 0.05% Tween 20, 0.005% NaN ₃ ), for conditions (2) they were diluted in ACES buffer pH 5.8 (20 mm ACES, 150 mm NaCl, 1.2 mm CaCl ₂ , 0.05% Tween 20, 0.005% NaN ₃ ). Each antibody was captured on the sensor surface using ProA/G. Antibody capture levels typically corresponded to 60-90 resonance units (RU). Then, recombinant human C5 was injected at concentrations of 3 to 27 nM or 13.3 to 120 nM, obtained by three-fold serial dilution, after which dissociation was carried out. The surface was regenerated using 25 mm NaOH. Kinetic parameters for conditions (1) and (2) were determined by sensorgram fit based on a 1:1 binding model, and dissociation rate for condition (3) was determined by sensorgram fit based on a 1:1 dissociation model for MSCs using BIACORE® software T200 Evaluation Version 2.0 (GE Healthcare). The pH dependence for all antibodies was presented as the ratio of dissociation rates for conditions (2) and (1).

Association rate (ka), dissociation rate (kd), binding affinity (KD), and pH dependence are presented in Table 1. 9. All antibodies were characterized by a higher rate of dissociation at pH 5.8 than at pH 7.4, and their dependence on pH (change from pH 5.8 to pH 7.4) was approximately 20-fold.

Table 9

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

ka (1/Mc) 7.4_7.4 kd (1/c) KD(M) ka (1/Mc) 5.8_5.8 kd (1/c) KD(M) 7.4_5.8 kd (1/c) pH dependency LO15-SG422 1.40E+06 4.19E-04 3.00E-10 1.34E+05 8.79E-03 6.57E-08 1.61 E-02 21.0 LO15-SG115 1.31E+06 3.54E-04 2.70E-10 9.49E+04 8.27E-03 8.72E-08 1.67E-02 23.4 LO16-SG422 1.28E+06 4.12E-04 3.21E-10 1.09E+05 8.69E-03 7.95E-08 1.61 E-02 21.1 LO18-SG422 1.36E+06 4.26E-04 3.14E-10 1.39E+05 8.65E-03 6.24E-08 1.69E-02 20.3 LO19-SG422 1.37E+06 4.76E-04 3.46E-10 1.38E+05 8.30E-03 6.00E-08 1.61 E-02 17.4 LO20-SG115 1.44E+06 4.67E-04 3.24E-10 1.41E+05 8.18E-03 5.81 E-08 1.61 E-02 17.5 LO20-SG422 1.35E+06 4.70E-04 3.49E-10 1.36E+05 8.15E-03 5.99E-08 1.55E-02 17.3 LO22-SG115 1.46E+06 3.82E-04 2.62E-10 1.71E+05 9.30E-03 5.45E-08 1.46E-02 24.3 LO23-SG115 1.53E+06 4.23E-04 2.77E-10 1.33E+05 8.55E-03 6.41 E-08 1.73E-02 20.2

To assess the effect of pH on antigen binding, the binding affinity of anti-C5 antibodies (BNJ441, eculizumab, and variant 305) to recombinant human C5 at pH 7.4 and pH 5.8 at 37°C was determined using a BIACORE® T200 device (GE Healthcare) . Goat polyclonal anti-human IgG (Fc) (KPL, No. 01-10-20) was immobilized on a CM4 sensor chip using an amine coupling kit (GE Healthcare) under conditions recommended by the manufacturer. Antibodies and analytes were diluted either in ACES buffer pH 7.4 or in ACES buffer pH 5.8 containing 20 mM ACES, 150 mM NaCl, 1.2 mM CaCl ₂ , 0.05% Tween 20 and 0.005% NaN ₃ . Antibodies were captured on the surface of the sensor according to the method based on the use of antibodies to Fc, capture levels, as a rule, corresponded to 50-80 resonance units (RU). Samples of recombinant human C5 were prepared by 3-fold serial dilution starting at 27 nM for analysis at pH 7.4 or from 135 nM for analysis at pH 5.8. The surface was regenerated using 20 mM HCl, 0.01% Tween 20. Data processing and fitting based on a 1:1 binding model was performed using BiaEvaluation 2.0 software (GE

healthcare).

The binding affinities (KD) of BNJ441, eculizumab, and variant 305 to recombinant human C5 at pH 7.4 and pH 5.8 are shown in Table 1. 10. For option 305, the ratio (KD at pH 5.8)/(KD at pH 7.4) was almost 800, i.e. 8 times greater than for BNJ441, for which the ratio (KD at pH 5.8)/(KD at pH 7.4) was only 93.

Table 10

Antibody _______KD (M) . KD ratio at pH 7.4 pH 5.8 pH 5.8/pH 7.4 variant 305LO5 1.66E-10 1.32E-07 795 eculizumab 1.42E-10 2.64Е-09 19 BNJ441 1.38E-09 1.28E-07 93

Example 9 Activity of anti-C5 antibodies (variants 305) in relation to C5 activation.

9.1. Inhibition of Mab to C5 complement-activated liposome lysis.

The anti-C5 mAb was tested for inhibition of complement activity using a liposome lysis assay. 30 µl of healthy human serum (6.7%) (Biopredic, SER019) was mixed with 20 µl of diluted Mab in a 96-well plate and incubated on a shaker for 30 minutes at room temperature. Liposomes sensitized with anti-dinitrophenyl antibodies (Autokit CH50, Wako, 995-40801) were transferred to each well and the plate was placed on a shaker at 37°C for 2 minutes. 50 µl of substrate solution (Autokit CH50) was added to each well and mixed by shaking for 2 minutes at 37°C. The final mixture was incubated at 37° C. for 40 min and then the OD of the mixture was measured at 340 nm. The percentage of liposome lysis was determined by the formula

100x[(OPmat-OP background due to serum and liposomes ^)]/[(0P without MAT ^-0P background due to serum and liposomes)]

In FIG. 15 demonstrates that 305LO15-SG422, 305LO16-SG422, 305LO18-SG422, 305LO19-SG422, 305LO20-SG422, and 305LO20-SG115 anti-C5 mAbs inhibit liposome lysis. Two antibodies with Fc variants 305LO15-SG115 and 305LO23-SG429 also inhibited liposome lysis (FIG. 16).

The anti-C5 mAb was tested for inhibition of recombinant human C5 (SEQ ID NO: 39). 10 μl C5 deficient human serum (Sigma, C1163) was mixed with 20 μl diluted Mab and 20 μl recombinant C5 (0.1 μg/ml) in a 96-well plate and incubated on a shaker for 1 hour at 37°C. Liposomes (Autokit CH50) were transferred to each well and placed on a shaker for 2 min at 37°C. 50 µl of substrate solution (Autokit CH50) was added to each well and mixed by shaking for 2 minutes at 37°C. The final mixture was incubated at 37° C. for 180 min and then the OD was measured at 340 nm. The percentage of liposome lysis was determined according to the above procedure. In FIG. 17 demonstrates that the anti-C5 mAbs 305LO22-SG115, 305LO22SG422, 305LO23-SG115, and 305LO23-SG422 inhibit liposome lysis.

9.2. Inhibition of C5a formation with anti-C5 mAbs.

Anti-C5 mAbs were tested for their effect on C5a formation during liposome lysis to confirm that C5 mAbs inhibit the cleavage of C5 to form C5a and C5b. C5a levels in supernatants from the liposome lysis assay were quantified using a C5a ELISA kit (R&D systems, DY2037). All mAbs inhibited the formation of C5a in supernatants in a dose dependent manner (FIGS. 18 and 19).

9.3. Measurement of complement activity in the plasma of cynomolgus monkeys.

The anti-C5 mAb was tested for inhibition of complement activity in the plasma of Cynomolgus monkeys. Anti-C5 mAb was administered to monkeys (20 mg/kg) and plasma samples were collected periodically up to day 56. Chicken red blood cells (cRBC) (Innovative research, IC05-0810) were washed with gelatin/veronal buffered saline containing 0.5 mM MgCl ₂ and 0.15 mM CaCl ₂ (GVB++) (Boston BioProducts, IBB-300X), and then sensitized with an antibody to chicken RBC (Rockland 103-4139) at a concentration of 1 μg/ml for 15 min at 4°C. The cells were then washed with GVB++ and suspended in the same buffer at 1x10 cells/ml. In a separate round-bottom 96-well microassay plate, monkey plasma was incubated with sensitized cRBCs at 37° C. for 20 minutes. After incubation, the plate was centrifuged at 1000xg for 2 min at 4°C. The supernatants were transferred to the wells of flat-bottomed 96-well microassay plates for OD measurements at 415 nm, the reference wavelength was 630 nm. The percentage of hemolysis was determined by the formula ^100x[(0P after administration ^{- OD} background due to plasma and cRBc) ^]/[ ( ^OD before injection ^{- OD} background due to plasma and cRBC ^)]

In FIG. 20 demonstrates that anti-C5 mAbs 305LO15-SG422, 305LO15-SG115, 305LO16-SG422, 305LO18-SG422, 305LO19-SG422, 305LO20-SG422, 305LO20-SG115, and 305LO23-SG115 inhibited plasma complement activity.

9.4. Inhibition of the biological activity of C5 variants by Mab to C5.

Mat to C5 was tested for inhibition of the following recombinant human C5 variants: V145I, R449G, V802I, R885H, R928Q, D966Y, S1310N and E1437D. It is known that patients with

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PNHs that have the R885H mutation at C5 elicit a poor response to eculizumab (see, e.g., Nishimura et al., New Engl. J. Med., 370, 2014, pp. 632-639). Each of the human C5 variants was expressed in FS293 cells and the supernatants were used in the study described below. 10 µl of C5 deficient human serum (Sigma, C1163) was mixed with 20 µl of diluted Mab and 20 µl of cell culture medium containing the recombinant C5 variant (2-3 µg/ml) in a 96-well plate and incubated on a shaker in for 0.5 h at 37°C. Liposomes (Autokit CH50) were transferred to each well and placed on a shaker for 2 min at 37°C. 50 µl of substrate solution (Autokit CH50) was added to each well and mixed by shaking for 2 minutes at 37°C. The final mixture was incubated at 37° C. for 90 min and then the OD was measured at 340 nm. The percentage of liposome lysis was determined according to the procedure described above. In FIG. 21 demonstrates that the anti-C5 mAb (eculizumab) did not inhibit the R885H variant of C5, but did inhibit the other variants tested. In FIG. 22 demonstrates that anti-C5 mAb (variant 305) inhibited all C5 variants tested.

9.5. Inhibition of Complement-Activated Liposomal Lysis by Anti-C5 Mabs.

The anti-C5 mAb was tested for inhibition of complement activity using a liposome lysis assay. 30 µl of healthy human serum (6.7%) (Biopredic, SER019) was mixed with 20 µl of diluted Mab in a 96-well plate on a shaker for 30 min at room temperature. A solution of liposomes sensitized with antibodies to dinitrophenyl (Autokit CH50, Wako, 995-40801) was transferred to each well and placed on a shaker for 2 min at 25°C. 50 µl of substrate solution (Autokit CH50) was added to each well and mixed by shaking for 2 min at 25°C. The final mixture was incubated at 37° C. for 45 min and then the OD was measured at 340 nm. The percentage of inhibition of liposome lysis was determined by the formula

100x[(OPmat-OP background due to serum and liposomes ⁾ ] ^/[(OP without MAT ^-OP background due to serum and liposomes)]

In FIG. 23 demonstrates that the anti-C5 mAb BNJ441 and variant 305 inhibited liposome lysis and that variant 305 had stronger inhibitory activity than BN441.

Example 10 Pharmacokinetic study of monoclonal antibodies to C5 (options 305) using cynomolgus monkeys.

10.1. In vivo evaluation using cynomolgus monkeys.

In vivo kinetic assays for anti-human C5 antibody were performed after administration of anti-human C5 antibody to cynomolgus monkeys (Shin Nippon Biomedical Laboratories, Ltd., Japan). An anti-human C5 antibody solution (2.5 mg/mL) was injected once at a dose of about 8 mL/kg into the cephalic vein of the forearm by a 30-minute infusion. Blood was collected before administration and 5 minutes, 7 hours, 1, 2, 3, 7, 14, 21, 28, 35, 42, 49 and 50 days after administration. The collected blood was immediately centrifuged at 1700xg and 4°C for 10 min to separate the plasma. The separated plasma was stored in a freezer at -70°C or below until analysis. Antibodies to human C5 were obtained according to the method described in example 7.

10.2. Measurement of total plasma C5 concentration in cynomolgus monkeys by ELISA assay.

The total C5 concentration of cynomolgus monkeys in the plasma of cynomolgus monkeys was measured by ELISA. An anti-human C5 antibody (an antibody generated in Applicant's laboratory using the method described in Example 2) was added to a Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and allowed to stand overnight at 4°C to prepare monkey immobilized C5 plates. cynomolgus. Prepared samples for the construction of the calibration curve and plasma samples of cynomolgus, diluted 2000 times, which were injected with antibody at a concentration of 0.4 μg/ml, and incubated for 60 min at 37°C. The samples were then plated with immobilized C5 cynomolgus monkeys and allowed to stand for 1 hour at room temperature. Then, an HRP-labeled anti-human IgG antibody (SouthernBiotech) was added and allowed to react for 30 minutes at room temperature, followed by washing. Then ABTS ELISA HRP substrate (KPL) was added. The signal was measured using a plate reader at a wavelength of 405 nm. The C5 concentration of cynomolgus monkeys was calculated from the response obtained from the calibration curve using SOFTmax PRO analytical software (Molecular Devices). A plot of C5 concentration in plasma of Cynomolgus monkeys after intravenous administration as a function of time, obtained by this method, is presented in Fig. 24. The data in the graph are presented as the percentage of C5 remaining compared to the plasma concentration of C5 in cynomolgus monkeys prior to administration. При применении рН-зависимых антител к человеческому С5 (305LO15SG422, 305LO15-SG115, 305LO16-SG422, 305LO18-SG422, 305LO19-SG422, 305LO20-SG422, 305LO20SG115, 305LO22-SG422, 305LO23-SG422 и 305LO23-SG115) обнаружено меньшее накопление С5 in plasma compared to non-pH dependent antibodies to human C5.

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

The concentration of antibodies to human C5 in the plasma of cynomolgus monkeys was measured using ELISA. An anti-human IgG kappa chain antibody (Antibody Solutions) was added to a Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and allowed to stand overnight at

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4°C to obtain tablets with immobilized anti-human IgG. Prepared samples for the construction of the calibration curve and plasma samples of cynomolgus, diluted 100 times or more. Samples were then plated with immobilized anti-human IgG and allowed to stand for 1 hour at room temperature. Then, an HRP-labeled anti-human IgG antibody (SouthernBiotech) was added and allowed to react for 30 minutes at room temperature, and washing was performed. Then ABTS ELISA HRP substrate (KPL) was added. The signal was measured using a plate reader at a wavelength of 405 nm. Anti-human C5 antibody concentration was calculated from the response obtained from the calibration curve using SOFTmax PRO analytical software (Molecular Devices). A time plot of plasma concentration of anti-human C5 antibody after intravenous administration obtained by this method is shown in FIG. 25. pH dependent antibodies to human C5 (305LO15-SG422, 305LO15-SG115, 305LO16-SG422, 305LO18-SG422, 305LO19-SG422, 305LO20-SG422, 305LO20-SG115, 305LO22-SG422, and 305LO23-SG123-SG123) were characterized by a longer half-life compared to non-pH-dependent antibodies to human C5.

Example 11 X-ray analysis of the crystal structure of a complex containing Fab variant 305 and the MG1 domain of human C5.

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

The MG1 domain (amino acid residues 20-124 of SEQ ID NO: 39) fused to the GST tag via a thrombin-cleavable linker (GST-MG1) was expressed in E. coli strain BL21 DE3 pLysS (Promega) using the vector pGEX-4T- 1 (GE healthcare). Protein expression was induced using 0.1 mM isopropyl-beta-O-1-thiogalactopyranoside (IPTG) at 25°C for 5 h. complete cocktail of protease inhibitors (Roche), followed by purification of GST-MG1 from the soluble fraction using a GSTrap column (GE healthcare) according to the manufacturer's instructions. The GST tag was cleaved with thrombin (Sigma) and the resulting MG1 domain was further purified on a Superdex 75 gel filtration column (GE healthcare). The fractions containing the MG1 domain were pooled and stored at -80°C.

11.2. Obtaining a Fab fragment of variant 305.

Fab fragments of one of the optimized 305 variants were prepared using a standard method using limited papain digestion (Roche Diagnostics, cat. no. 1047825) and then applied to a protein A column (MabSlect SuRe, GE Healthcare) to remove Fc fragments, a cation exchange column (HiTrap SP HP, GE Healthcare); and a gel filtration column (Superdex 200 16/60, GE Healthcare). The fractions containing the Fab fragment were pooled and stored at -80°C.

11.3. Obtaining a complex containing Fab variant 305 and the MG1 domain of human C5.

The purified MG1 domain of recombinant human C5 was mixed with the purified Fab fragment of variant 305 in a 1:1 molar ratio. The complex was purified by gel filtration (Superdex 200 Increase 10/300, GE Healthcare) using a column equilibrated with 25 mM HEPES pH7.5, 100 mM NaCl.

11.4. Crystallization.

Purified complexes were concentrated to about 10 mg/ml and crystallized by vapor diffusion in the sessile drop variant in combination with seeding at 4°C. The solution in the reservoir consisted of 0.2 M magnesium formate, 15.0% (w/v) polyethylene glycol 3350. In this way, lamellar crystals were obtained after a few days. The crystals were impregnated with a solution containing 0.2 M magnesium formate dehydrate, 25.0% (w/v) polyethylene glycol 3350 and 20% glycerol.

11.5. Data collection and structure definition.

X-ray diffraction data were obtained using the BL32XU device at the SPring-8 synchrotron. During measurements, the crystal was constantly in a stream of nitrogen at -178°C to maintain it in a frozen state, and using an MX-225HS CCD detector (RAYONIX) placed on the beam axis, we obtained by rotating the crystal by 1.0° each time , a total of 180 x-ray diffraction patterns. Determination of unit cell parameters, indexing of diffraction spots and processing of diffraction data obtained from diffraction patterns were carried out using the Xia2 program (J. Appl. Cryst., 43, 2010, p. 186-190), the XDS software package (Acta. Cryst . D66, 2010, pp. 125-132) and Scala (Acta. Cryst. D62, 2006, pp. 72-82) and finally obtained data on diffraction intensity with a resolution up to 2.11 A. Results of statistical processing of crystallographic data are presented in table. eleven.

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Table 11

Collected radiographic data and updated statistical data Collected data

space group P1 elementary cell a, b, c (A) 39.79, 55.10, 127.76 α, β, γ (°) 89.18, 86.24, 78.20 resolution (A) 49.49-2.11 total reflections 112.102 unique reflections 56.154 recall (shell maxims, permissions) (%) 92.1 (95.8) Rmerg _e ^a (maximum shell, permissions) (%) 7.2 (31.7) Clarification resolution (A) 25.00-2.11 reflections 53.398 Rfactor ⁶ (Rfree ) (%) 20.42 (26.44) S.K.O. - deviation from the ideal bond lengths (A) 0.0088 bond angles (°) 1.3441

a: R _merge =ZhklZj\Ij(hkl) - <I(hkl)>\/ZhklZj\Ij(hkl)\, rjielj(hkl) and <I(hkl)> denote the intensity of the measured parameter j and the average reflection intensity with index hkl, respectively.

b: factor R=Zhkl\F _ca i _c (hkl)\ - \F _obs (hkl)\/Zhkl\F _obs (hkl)\, where F _abs nF _cak denote the measured and calculated amplitudes of the structure factor, respectively.

c: R _free was calculated using 5% randomly selected reflections.

The structure was determined by the molecular displacement method using the Phaser program (J. Appl. Cryst., 40, 2007, pp. 658-674). A search (starter) model of the Fab domain was derived from the published crystal structure of IgG4 Fab (PDB code: 1BB1), and a starter model of the MG1 domain was derived from the published crystal structure of human C5 (PDB code: 3CU7, Nat. Immunol., 9, 2008, pp. 753-760). The model was built using the Coot program (Acta Cryst. D66, 2010, pp. 486-501) and refined using the Refmac5 program (Acta Cryst. D67, 2011, pp. 355-367). The crystallographic confidence factor (R) for diffraction intensity data at a resolution of 25-2.11 A was 20.42%, while the Free R value was 26.44%. Refined statistical data on the structure are presented in table. eleven.

11.6. General structure of the complex containing Fab variant 305 and MG1 domain C5.

A Fab fragment of an optimized variant 305 (305 Fab) bound to the MG1 domain of human C5 (MG1) in a 1:1 ratio and an asymmetric unit cell of the crystal structure containing two complexes, namely molecules 1 and 2, are shown in FIG. 26A. Molecules 1 and 2 were able to match each other well with a standard deviation (S.D.D.) of 0.717 A at the alpha C position in all residues as shown in FIG. 26B. The drawings described below were made using molecule 1.

In FIG. 27A and 27B map the epitope for the 305 Fab contact region in the amino acid sequence and crystal structure of MG1, respectively. An epitope includes an amino acid residue in MG1 that contains one or more atoms up to 4.5 A away from any part of the 305 Fab in the crystal structure. In addition, in FIG. 27A highlights an epitope within 3.0 A.

11.7. Interactions with E48, D51 and K109.

As described in Examples 4.5 and 4.6, anti-C5 mAbs that included 305 series antibodies were tested for binding to three human C5 point mutants E48A, D51A and K109A using Western blot and BIACORE® binding assays. While the 305 variants exhibited strong binding to WT C5, they showed only weak binding to the C5 E48A mutant and no binding to the D51A and K109A mutants. Analysis of the crystal structure of the complex containing 305 Fab and MG1 revealed that all three amino acids E48, D51 and K109 are up to 3.0 Å away from 305 Fab, forming numerous hydrogen bonds with the Fab as shown in FIG. 28A. In a more detailed study, it was found that the K109 residue of MG1 is located in the groove formed at the interface between MG1 and the heavy chain of Fab and is closely connected to Fab by three hydrogen bonds with H-CDR3_G97, H-CDR3_Y100 and H-CDR3_T100b, and a salt bridge with H-CDR3_D95 (Fig. 28D). D51 is between MG1 and the 305 Fab heavy chain and is double hydrogen bonded to H-CDR1_Ser32 and H-CDR2_Ser54, thus filling the space (FIG. 28B). This indicates that both residues, and K109 and D51, C5 are residues that are crucial for binding antibodies of the 305 series.

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Claims (39)

On the other hand, E48 is located closer to the surface and is bound by only one hydrogen bond with Fab, which suggests that its contribution to antibody binding should be less than the contribution of K109 and E51 (Fig. 28B). These relationships are consistent with Western blot and BIACORE® binding assays for human C5 mutants (Examples 4.5 and 4.6). Additional note: Fab amino acid residue numbering is based on Cabot's 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 between H70, H72 and H110 human C5 and 305 series antibodies. Crystal structure analysis revealed that three histidine residues in human C5, namely H70, H72 and H110, are included in the epitope for Fab variant 305, as illustrated in FIG. 27A and 29A. A BIACORE® binding assay was performed to examine the contribution of these histidine residues to the pH-dependent protein-protein interaction between human C5 and Fab variant 305 using human C5 H70Y-, H72Y-, H110Y- and H70Y+H110Y mutants of human C5 (Example 4.7). The H72Y mutation resulted in complete elimination of Fab variant 305 binding to C5. This residue at C5 resides in a pocket formed by the CDR2 loop in the 305 Fab heavy chain and the loop in MG1 (L73, S74 and E76) densely filling this space, as illustrated in FIG. 29V. In addition, the H72 residue in C5 is hydrogen bonded to H-CDR2_Y58. The H72Y mutation should not be expected to be valid because there is not enough space to include the larger tyrosine side chain. The hydrogen bond with H-CDR2_Y58 also cannot be maintained. Regarding the contribution of H70 and H110 to pH dependence, the H70Y and H110Y mutations result in slower separation by dissociating Fab variant 305 from C5 at pH 5.8. H70 forms an intramolecular hydrogen bond with T53 MG1, which is expected to break at pH 5.8 when protonation of H70 at C5 induces a conformational change in the corresponding region of the MG1 interface involved in the interaction (Fig. 29B). For H110, protonation of this residue at C5 would be expected to cause charge repulsion from 305 Fab, which could be enhanced by protonation of the adjacent histidine residue, H-CDR3_H100c (FIG. 29D). Although the invention has been described above for a better understanding with some details for the purpose of illustration and examples, the description and examples should not be construed as limiting the scope of the invention. The description of all patent and scientific literature cited in the present description is expressly incorporated herein by reference in its entirety. CLAIM 1. The use of a drug containing an anti-C5 antibody for the treatment of a complement-mediated disease or condition that involves excessive or uncontrolled activation of C5, wherein the antibody comprises a VH sequence, any of SEQ ID NOs: 106-110, and a VL sequence, any of SEQ ID NOs: 111-113. 2. The use of a drug containing an anti-C5 antibody to enhance the clearance of C5 from plasma, wherein the antibody contains a VH sequence of any of SEQ ID NOs: 106-110 and a VL sequence of any of SEQ ID NOs: 111-113. 3. Use according to claim 1 or 2, where the antibody contains (a) VH sequence of SEQ ID NO: 106 and VL sequence of SEQ ID NO: 111; (b) VH sequence of SEQ ID NO: 107 and VL sequence of SEQ ID NO: 111; (c) VH sequence of SEQ ID NO: 108 and VL sequence of SEQ ID NO: 111; (d) VH sequence of SEQ ID NO: 109 and VL sequence of SEQ ID NO: 111; (e) VH sequence of SEQ ID NO: 109 and VL sequence of SEQ ID NO: 112; (f) VH sequence of SEQ ID NO: 109 and VL sequence of SEQ ID NO: 113; or (g) VH sequence of SEQ ID NO: 110 and VL sequence of SEQ ID NO: 113. 4. The use of a drug containing an anti-C5 antibody for the treatment of a complement-mediated disease or condition that involves excessive or uncontrolled activation of C5, where the antibody contains (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117; (b) HVR-H2 comprising the amino acid sequence of any of SEQ ID NOs: 118-120; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123 or 124; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125. 5. Use according to claim 4, where the antibody contains (i) (a) HVR-H1, including the amino acid sequence of SEQ ID NO: 117; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123; And - 63 041632 (f) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 125; (ii) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 119; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125; (iii) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 124; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125; or (iv) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 120; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 124; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125. 6. Use according to one of claims 1 to 5, where the antibody further comprises (I) a constant region heavy chain comprising an amino acid sequence, any of SEQ ID NOs: 33, 34, 35, 114, 115 and 116; and (ii) a constant region light chain comprising the amino acid sequence of any of SEQ ID NOs: 36, 37 and 38. 7. Use according to claim 6, wherein the antibody further comprises a constant region heavy chain comprising the amino acid sequence of SEQ ID NO: 114 and a constant region light chain comprising the amino acid sequence of SEQ ID NO: 38. 8. Use according to one of claims 1 to 7, wherein the antibody inhibits C5 activation. 9. Use according to one of claims 1 to 8, wherein the antibody inhibits the activation of C5, variant R885H. 10. Use according to one of claims 1 to 9, wherein the antibody binds to C5 with higher affinity at pH 7.4 than at pH 5.8. 11. Use according to one of claims 1 to 10, wherein the antibody is a monoclonal antibody. 12. Use according to one of claims 1-11, wherein the antibody is a human, humanized or chimeric antibody. 13. Use according to one of claims 1 to 12, wherein the antibody is a full-length IgG1-class or IgG4-class antibody. 14. The use of an anti-C5 antibody for the manufacture of a medicament for the treatment of a complement-mediated disease or condition that involves excessive or uncontrolled activation of C5, wherein the antibody comprises a VH sequence, any of SEQ ID NOs: 106-110, and a VL sequence, any of SEQ ID NOs: 111-113. 15. Use of an anti-C5 antibody for the manufacture of a medicament for enhancing plasma C5 clearance, wherein the antibody comprises a VH sequence of any of SEQ ID NOs: 106-110 and a VL sequence of any of SEQ ID NOs: 111-113. 16. Use according to claim 14 or 15, where the antibody contains (a) VH sequence of SEQ ID NO: 106 and VL sequence of SEQ ID NO: 111; (b) VH sequence of SEQ ID NO: 107 and VL sequence of SEQ ID NO: 111; (c) VH sequence of SEQ ID NO: 108 and VL sequence of SEQ ID NO: 111; (d) VH sequence of SEQ ID NO: 109 and VL sequence of SEQ ID NO: 111; (e) VH sequence of SEQ ID NO: 109 and VL sequence of SEQ ID NO: 112; (f) VH sequence of SEQ ID NO: 109 and VL sequence of SEQ ID NO: 113; or (g) VH sequence of SEQ ID NO: 110 and VL sequence of SEQ ID NO: 113. 17. The use of an anti-C5 antibody for the manufacture of a medicament for the treatment of a complement-mediated disease or condition that involves excessive or uncontrolled C5 activation, wherein the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117; (b) HVR-H2 comprising the amino acid sequence of any of SEQ ID NOs: 118-120; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123 or 124; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125. 18. Use according to claim 17, where the antibody contains (i) (a) HVR-H1, including the amino acid sequence of SEQ ID NO: 117; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123; And - 64 041632 (f) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 125; (ii) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 119; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125; (iii) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 124; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125; or (iv) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 120; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 124; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125. 19. Use according to one of claims 14-18, where the antibody further comprises (I) a heavy chain constant region comprising an amino acid sequence, any of SEQ ID NOs: 33, 34, 35, 114, 115 and 116; and (ii) a constant region light chain comprising the amino acid sequence of any of SEQ ID NOs: 36, 37 and 38. 20. Use according to claim 19, wherein the antibody further comprises a constant region heavy chain comprising the amino acid sequence of SEQ ID NO: 114 and a constant region light chain comprising the amino acid sequence of SEQ ID NO: 38. 21. Use according to one of claims 14-20, wherein the antibody inhibits C5 activation. 22. Use according to one of claims 14-21, wherein the antibody inhibits C5 activation, variant R885H. 23. Use according to one of claims 14-22, wherein the antibody binds to C5 with higher affinity at pH 7.4 than at pH 5.8. 24. Use according to one of claims 14-23, wherein the antibody is a monoclonal antibody. 25. Use according to one of claims 14-24, wherein the antibody is a human, humanized, or chimeric antibody. 26. Use according to one of claims 14-25, wherein the antibody is a full length IgG1 or IgG4 antibody. 27. A method of treating an individual with a complement-mediated disease or condition that involves excessive or uncontrolled C5 activation, comprising administering to the individual an effective amount of an anti-C5 antibody, wherein the antibody comprises a VH sequence, any of SEQ ID NOs: 106-110, and a VL sequence, any of SEQ ID NOs: 111-113. 28. A method of enhancing plasma C5 clearance in an individual, comprising administering to the individual an effective amount of an anti-C5 antibody to enhance plasma C5 clearance, wherein the antibody comprises a VH sequence, any of SEQ ID NOs: 106-110, and a VL sequence, any of SEQ ID NOs: 111-113. 29. The method according to item 27 or 28, where the antibody contains (a) VH sequence of SEQ ID NO: 106 and VL sequence of SEQ ID NO: 111; (b) VH sequence of SEQ ID NO: 107 and VL sequence of SEQ ID NO: 111; (c) VH sequence of SEQ ID NO: 108 and VL sequence of SEQ ID NO: 111; (d) VH sequence of SEQ ID NO: 109 and VL sequence of SEQ ID NO: 111; (e) VH sequence of SEQ ID NO: 109 and VL sequence of SEQ ID NO: 112; (f) VH sequence of SEQ ID NO: 109 and VL sequence of SEQ ID NO: 113; or (g) VH sequence of SEQ ID NO: 110 and VL sequence of SEQ ID NO: 113. 30. A method of treating an individual with a complement-mediated disease or condition that includes excessive or uncontrolled C5 activation, comprising administering to the individual an effective amount of an anti-C5 antibody, wherein the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117; (b) HVR-H2 comprising the amino acid sequence of any of SEQ ID NOs: 118-120; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123 or 124; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125. 31. The method according to item 30, where the antibody contains (i) (a) HVR-H1, including the amino acid sequence of SEQ ID NO: 117; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) HVR-L1, including the amino acid sequence - - 65 041632 SEQ ID NO: 122; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125; (ii) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 119; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125; (iii) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 124; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125; or (iv) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 120; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 124; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125. 32. The method according to one of claims 27-31, where the antibody contains (I) a heavy chain of a constant region comprising an amino acid sequence, any of SEQ ID NOs: 33, 34, 35, 114, 115 and 116; and (ii) a constant region light chain comprising the amino acid sequence of any of SEQ ID NOs: 36, 37 and 38. 33. The method of claim 32, wherein the antibody further comprises a constant region heavy chain comprising the amino acid sequence of SEQ ID NO: 114 and a constant region light chain comprising the amino acid sequence of SEQ ID NO: 38. 34. The method of one of claims 27-33, wherein the antibody inhibits C5 activation. 35. The method according to one of claims 27-34, wherein the antibody inhibits the activation of C5, variant R885H. 36. The method according to one of claims 27-35, wherein the antibody binds to C5 with higher affinity at pH 7.4 than at pH 5.8. 37. The method according to one of claims 27-36, wherein the antibody is a monoclonal antibody. 38. The method according to one of claims 27-37, wherein the antibody is a human, humanized, or chimeric antibody. 39. The method according to one of claims 27-38, wherein the antibody is a full length IgG1 or IgG4 antibody. -