Complement c5-binding antagonist combination

Abstract

The present invention relates to a novel complement C5-binding antagonist combination and a use thereof. The complement C5-binding antagonist combination according to the present invention comprises antagonists specifically binding to different domains of complement C5 and thus can significantly inhibit complement C5 activity, and therefore can be used for preventing or treating various complement-related diseases including myasthenia gravis, age-related macular degeneration, and paroxysmal nocturnal hemoglobinuria.

Description

Complement C5 binding antagonist combination

[0001] The present invention relates to a novel combination of complement C5 binding antagonists that significantly inhibits complement C5 activity by combining complement C5 binding antagonists that target a specific domain of C5, and to the use thereof.

[0002] The complement system is one of the major substances that trigger an innate immune response in our body when activated by foreign pathogens, such as microorganisms, viruses, cancer cells, and antigens.

[0003] The complement system is classified into the classical pathway, which is induced by antibodies, and the alternative pathway, which is activated independently of antibodies. Activation of the complement system's signaling pathways begins with the C1 complex, composed of C1q / r / s (C1qrs), and proceeds through the activation of the C1 complex → C2 and C4 → C3 → C5 to eliminate foreign substances. C5b, generated during the complement activation process, binds to other complement proteins to form the membrane attack complex (MAC), which creates holes in cells to eliminate the target cells or pathogens. If this complement activation occurs unregulated, various autoimmune diseases develop, such as paroxysmal nocturnal hemoglobinuria (PNH), hemolytic uremic syndrome (HUS), myasthenia gravis (gMG), and neuromyelitis optica, in which the body attacks its own cells.

[0004] Meanwhile, existing drugs have limitations in that they cannot completely inhibit C5 activity, requiring additional treatment or leading to LDH levels above the normal range, which causes difficulties in daily life.

[0005] Therefore, in order to solve the aforementioned problems, there is an urgent need to develop improved materials capable of effectively inhibiting C5 activity.

[0006] (Patent Document 1) US 20160108115 A1

[0007] (Patent Document 2) US 20200254092 A1

[0008] This invention is an application supported by a national research and development project of the Republic of Korea, and detailed information is as follows.

[0009] [National R&D projects that supported this invention]

[0010] [Project ID] 171119346

[0011] [Assignment No.] 00258444

[0012] [Buddha Name] Multi-Buddha

[0013] [Name of Project Management (Specialized) Agency] National Drug Development Foundation

[0014] [Research Project Name] National New Drug Development Project (Ministry of Science and ICT, Ministry of Health and Welfare, Ministry of Trade, Industry and Energy)

[0015] [Project Title] US Phase 1 Clinical Study of Complement C5 Inhibitor Antibody for the Development of Treatments for Severe Autoimmune Diseases

[0016] [Name of Project Performing Organization] ImmuneApps

[0017] [Research Period] June 1, 2023 ~ May 31, 2025

[0018] One object of the present invention is to provide a combination comprising: a first antagonist that specifically binds to the MG4 domain of the complement C5 protein beta chain of SEQ ID NO. 11; and a second antagonist that specifically binds to the MG1 domain of the complement C5 protein beta chain.

[0019] Another objective of the present invention is to provide a composition for the prevention or treatment of complement-related diseases comprising the above combination as an active ingredient.

[0020] In one embodiment, it was confirmed that the complement C5 antagonist combination of the present invention significantly inhibits complement activity.

[0021] Accordingly, one aspect of the present invention relates to a combination comprising: a first antagonist that specifically binds to the MG4 domain of the beta chain of the complement C5 protein of SEQ ID NO. 11; and a second antagonist that specifically binds to the MG1 domain of the beta chain of the complement C5 protein.

[0022] In the present invention, the term "antagonist" is a substance that specifically binds to complement C5, and includes antibodies, synthetic peptides, natural peptides, small molecules, etc., and preferably may be antibodies.

[0023] In the present invention, the first antagonist and the second antagonist are substances such as antibodies that bind to complement component 5, act in the final step of the complement cascade reaction, and specifically bind to the beta chain of complement component 5.

[0024] In the present invention, the first antagonist can specifically bind to the MG4 domain of SEQ ID NO. 78, and the second antagonist can specifically bind to the MG1 domain of SEQ ID NO. 75.

[0025] In the present invention, the amino acid sequence of the human C5 protein may be SEQ ID NO. 10, and the amino acid sequence of the beta chain of the human C5 protein may be SEQ ID NO. 11.

[0026] In one embodiment of the present invention, the first antagonist may be a complement-binding protein.

[0027] In the present invention, the term "complement-binding protein" refers to a protein molecule that specifically binds to complement, and preferably may be an antibody.

[0028] In one embodiment of the present invention, the first antagonist may be an anti-C5 antibody comprising heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO. 3; HCDR2 of SEQ ID NO. 4; HCDR3 of SEQ ID NO. 5; light chain complementarity determining region (LCDR) 1 of SEQ ID NO. 6; LCDR2 of amino acid sequence YAS; and LCDR3 of SEQ ID NO. 7.

[0029] In one embodiment of the present invention, the first antagonist comprises: (i) R15-14 comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO. 320, LCDR2 of amino acid sequence SS, LCDR3 of SEQ ID NO. 261, and heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO. 321, HCDR2 of SEQ ID NO. 322, and HCDR3 of SEQ ID NO. 323; (ii) R15-24 comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO. 320, LCDR2 of amino acid sequence SA, LCDR3 of SEQ ID NO. 280, and heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO. 321, HCDR2 of SEQ ID NO. 322, and HCDR3 of SEQ ID NO. 323; (iii) R15-05 comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO. 335, LCDR2 of amino acid sequence SA, LCDR3 of SEQ ID NO. 463, and heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO. 312, HCDR2 of SEQ ID NO. 336, and HCDR3 of SEQ ID NO. 337; (iv) R13-175 comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO. 349, LCDR2 of SEQ ID NO. 350, LCDR3 of SEQ ID NO. 302, and heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO. 351, HCDR2 of SEQ ID NO. 352, and HCDR3 of SEQ ID NO. 353; (v) may be R13-210 comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO 214, LCDR2 of amino acid sequence GA, LCDR3 of SEQ ID NO 306, and heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO 354, HCDR2 of SEQ ID NO 355, and HCDR3 of SEQ ID NO 356.

[0030] In one embodiment of the present invention, the anti-C5 antibody may include a heavy chain variable region of SEQ ID NO. 1; and a light chain variable region of SEQ ID NO. 2.

[0031] In one embodiment of the present invention, the first antagonist may be any one selected from the group consisting of (i) R15-14 comprising a light chain variable region of SEQ ID NO 225 and a heavy chain variable region of SEQ ID NO 387; (ii) R15-24 comprising a light chain variable region of SEQ ID NO 399 and a heavy chain variable region of SEQ ID NO 387; and (iii) R15-05 comprising a light chain variable region of SEQ ID NO 400 and a heavy chain variable region of SEQ ID NO 401; (iv) R13-175 comprising a light chain variable region of SEQ ID NO 408 and a heavy chain variable region of SEQ ID NO 409; and (v) R13-210 comprising a light chain variable region of SEQ ID NO 410 and a heavy chain variable region of SEQ ID NO 411.

[0032] In one embodiment of the present invention, the anti-C5 antibody is a monoclonal antibody that specifically binds to the C5 protein. Additionally, the anti-C5 antibody of the present invention may be an IgG isoform. Preferably, the IgG isoform of the anti-C5 antibody of the present invention may be IgG2 / 4, that is, it may include a fusion protein of the hinge of IgG2 and the Fc of IgG4. By having the IgG2 / 4 hybrid form, the anti-C5 antibody may have excellent production yield and structural stability.

[0033] In one embodiment of the present invention, the first antagonist may be a complement binding protein comprising the amino acid sequence of SEQ ID NO. 80.

[0034] In one embodiment of the present invention, the second antagonist may be an antibody.

[0035] In one embodiment of the present invention, the second antagonist may be any one anti-C5 antibody selected from the group consisting of Ch1-78, Ch2-12, Ch5-13, Ch11-01, Ch11-09, R-C5 / MG1-008-30-#13, R-13-SP#21, R14-16, R14-27, R14-07, R13-04, R14-23, R13-30, R14-19, R14-20, R14-24, R14-26, R15-01, R15-10, R15-21, R13-165, R15-152, R15-126, and Crovalimab, but is not limited thereto.

[0036] In one embodiment of the present invention, the second antagonist comprises: (i) Ch1-78 comprising LCDR (light chain complementarity determining region) 1 of the amino acid sequence SSW, LCDR2 of the amino acid sequence QN, LCDR3 of SEQ ID NO. 150, and HCDR (heavy chain complementarity determining region) 1 of SEQ ID NO. 151, HCDR2 of SEQ ID NO. 152, and HCDR3 of SEQ ID NO. 153; (ii) Ch2-12 comprising LDR1 of the amino acid sequence NSD, LCDR2 of the amino acid sequence DS, LCDR3 of SEQ ID NO. 154, and HCDR1 of SEQ ID NO. 155, HCDR2 of SEQ ID NO. 156, and HCDR3 of SEQ ID NO. 157; (iii) Ch5-13 comprising LCDR1 of SEQ ID NO. 158, LCDR2 of amino acid sequence SN, LCDR3 of SEQ ID NO. 159, and HCDR1 of SEQ ID NO. 160, HCDR2 of SEQ ID NO. 161, and HCDR3 of SEQ ID NO. 162; (iv) Ch11-01 comprising LDR1 of SEQ ID NO. 163, LCDR2 of amino acid sequence DN, LCDR3 of SEQ ID NO. 164, and HCDR1 of SEQ ID NO. 165, HCDR2 of SEQ ID NO. 166, and HCDR3 of SEQ ID NO. 167; (v) Ch11-09 comprising LCDR1 of amino acid sequence SNH, LCDR2 of amino acid sequence DN, LCDR3 of SEQ ID NO. 168, and HCDR1 of SEQ ID NO. 169, HCDR2 of SEQ ID NO. 170, and HCDR3 of SEQ ID NO. 171; (vi) R-C5 / MG1-008-30-#13 comprising LCDR1 of SEQ ID NO. 214, LCDR2 of amino acid sequence GA, LCDR3 of SEQ ID NO. 215, and HCDR1 of SEQ ID NO. 216, HCDR2 of SEQ ID NO. 217, and HCDR3 of SEQ ID NO. 218; (vii) R-13-SP#21 comprising LCDR1 of SEQ ID NO. 219, LCDR2 of amino acid sequence AA, LCDR3 of SEQ ID NO. 220, and HCDR1 of SEQ ID NO. 221, HCDR2 of SEQ ID NO. 222, and HCDR3 of SEQ ID NO. 223;(viii) R14-16 comprising LCDR1 of SEQ ID NO. 310, LCDR2 of SEQ ID NO. 311, LCDR3 of SEQ ID NO. 246, and HCDR1 of SEQ ID NO. 312, HCDR2 of SEQ ID NO. 313, and HCDR3 of SEQ ID NO. 314; (ix) R14-27 comprising LCDR1 of SEQ ID NO. 310, LCDR2 of SEQ ID NO. 311, LCDR3 of SEQ ID NO. 246, and HCDR1 of SEQ ID NO. 315, HCDR2 of SEQ ID NO. 316, and HCDR3 of SEQ ID NO. 314; (x) R14-07 comprising LCDR1 of SEQ ID NO. 310, LCDR2 of SEQ ID NO. 311, LCDR3 of SEQ ID NO. 246, and HCDR1 of SEQ ID NO. 315, HCDR2 of SEQ ID NO. 316, and HCDR3 of SEQ ID NO. 314; (xi) R13-04 comprising LCDR1 of SEQ ID NO. 214, LCDR2 of amino acid sequence GA, LCDR3 of SEQ ID NO. 255, and HCDR1 of SEQ ID NO. 317, HCDR2 of SEQ ID NO. 318, and HCDR3 of SEQ ID NO. 319; (xii) R14-23 comprising LCDR1 of SEQ ID NO. 310, LCDR2 of SEQ ID NO. 311, LCDR3 of SEQ ID NO. 246, and HCDR1 of SEQ ID NO. 315, HCDR2 of SEQ ID NO. 316, and HCDR3 of SEQ ID NO. 314; (xiii) R13-30 comprising LCDR1 of SEQ ID NO. 324, LCDR2 of SEQ ID NO. 311, LCDR3 of SEQ ID NO. 246, and HCDR1 of SEQ ID NO. 315, HCDR2 of SEQ ID NO. 325, and HCDR3 of SEQ ID NO. 314; (xiv) R14-19 comprising LCDR1 of SEQ ID NO. 324, LCDR2 of SEQ ID NO. 311, LCDR3 of SEQ ID NO. 246, and HCDR1 of SEQ ID NO. 315, HCDR2 of SEQ ID NO. 316, and HCDR3 of SEQ ID NO. 314; (xv) R14-20 comprising LCDR1 of SEQ ID NO. 310, LCDR2 of SEQ ID NO. 311, LCDR3 of SEQ ID NO. 246, and HCDR1 of SEQ ID NO. 326, HCDR2 of SEQ ID NO. 327, and HCDR3 of SEQ ID NO. 314;(xvi) R14-24 comprising LCDR1 of SEQ ID NO. 310, LCDR2 of SEQ ID NO. 311, LCDR3 of SEQ ID NO. 246, and HCDR1 of SEQ ID NO. 315, HCDR2 of SEQ ID NO. 328, and HCDR3 of SEQ ID NO. 329; (xvii) R14-26 comprising LCDR1 of SEQ ID NO. 310, LCDR2 of SEQ ID NO. 311, LCDR3 of SEQ ID NO. 246, and HCDR1 of SEQ ID NO. 330, HCDR2 of SEQ ID NO. 331, and HCDR3 of SEQ ID NO. 314; (xviii) R15-01 comprising LCDR1 of SEQ ID NO. 310, LCDR2 of SEQ ID NO. 311, LCDR3 of SEQ ID NO. 246, and HCDR1 of SEQ ID NO. 332, HCDR2 of SEQ ID NO. 333, and HCDR3 of SEQ ID NO. 329; (xix) R15-10 comprising LCDR1 of SEQ ID NO. 310, LCDR2 of SEQ ID NO. 311, LCDR3 of SEQ ID NO. 246, and HCDR1 of SEQ ID NO. 315, HCDR2 of SEQ ID NO. 316, and HCDR3 of SEQ ID NO. 314; (xx) R15-21 comprising LCDR1 of SEQ ID NO. 310, LCDR2 of SEQ ID NO. 311, LCDR3 of SEQ ID NO. 246, and HCDR1 of SEQ ID NO. 334, HCDR2 of SEQ ID NO. 313, and HCDR3 of SEQ ID NO. 314; (xxi) R13-165 comprising LCDR1 of SEQ ID NO. 338, LCDR2 of amino acid sequence RA, LCDR3 of SEQ ID NO. 288, and HCDR1 of SEQ ID NO. 339, HCDR2 of SEQ ID NO. 340, and HCDR3 of SEQ ID NO. 341; (xxii) R15-152 comprising LCDR1 of SEQ ID NO. 342, LCDR2 of SEQ ID NO. 343, LCDR3 of SEQ ID NO. 294, and HCDR1 of SEQ ID NO. 344, HCDR2 of SEQ ID NO. 345, and HCDR3 of SEQ ID NO. 346;and (xxiii) may be any one selected from the group consisting of R15-126 including LCDR1 of SEQ ID NO. 310, LCDR2 of SEQ ID NO. 347, LCDR3 of SEQ ID NO. 246, and HCDR1 of SEQ ID NO. 315, HCDR2 of SEQ ID NO. 328, and HCDR3 of SEQ ID NO. 348. Herein, the sequence constituting the CDR is described according to the IMGT definition.;

[0037] In one embodiment of the present invention, the second antagonist comprises: (i) Ch1-78 comprising a light chain variable region of SEQ ID NO. 25 and a heavy chain variable region of SEQ ID NO. 26; (ii) Ch2-12 comprising a light chain variable region of SEQ ID NO. 27 and a heavy chain variable region of SEQ ID NO. 28; (iii) Ch5-13 comprising a light chain variable region of SEQ ID NO. 29 and a heavy chain variable region of SEQ ID NO. 30; (iv) Ch11-01 comprising a light chain variable region of SEQ ID NO. 31 and a heavy chain variable region of SEQ ID NO. 32; (v) Ch11-09 comprising a light chain variable region of SEQ ID NO. 33 and a heavy chain variable region of SEQ ID NO. 34; and (vi) R-C5 / MG1-008-30-#13 comprising a light chain variable region of SEQ ID NO. 236 and a heavy chain variable region of SEQ ID NO. 237; (vii) R-13-SP#21 comprising the light chain variable region of SEQ NO 238 and the heavy chain variable region of SEQ NO 239; (viii) R14-16 comprising the light chain variable region of SEQ NO 381 and the heavy chain variable region of SEQ NO 382; (ix) R14-27 comprising the light chain variable region of SEQ NO 383 and the heavy chain variable region of SEQ NO 384; (x) R14-07 comprising the light chain variable region of SEQ NO 381 and the heavy chain variable region of SEQ NO 224; (xi) R13-04 comprising the light chain variable region of SEQ NO 385 and the heavy chain variable region of SEQ NO 386; (xii) R14-23 comprising the light chain variable region of SEQ NO 381 and the heavy chain variable region of SEQ NO 388; (xiii) R13-30 comprising the light chain variable region of SEQ ID NO 389 and the heavy chain variable region of SEQ ID NO 390; (xiv) R14-19 comprising the light chain variable region of SEQ ID NO 389 and the heavy chain variable region of SEQ ID NO 391; (xv) R14-20 comprising the light chain variable region of SEQ ID NO 392 and the heavy chain variable region of SEQ ID NO 393; (xvi) R14-24 comprising the light chain variable region of SEQ ID NO 381 and the heavy chain variable region of SEQ ID NO 394; (xvii) R14-26 comprising the light chain variable region of SEQ ID NO 381 and the heavy chain variable region of SEQ ID NO 395;(xviii) R15-01 comprising the light chain variable region of SEQ ID NO. 392 and the heavy chain variable region of SEQ ID NO. 396; (xix) R15-10 comprising the light chain variable region of SEQ ID NO. 381 and the heavy chain variable region of SEQ ID NO. 397; (xx) R15-21 comprising the light chain variable region of SEQ ID NO. 381 and the heavy chain variable region of SEQ ID NO. 398; (xxi) R13-165 comprising the light chain variable region of SEQ ID NO. 402 and the heavy chain variable region of SEQ ID NO. 403; (xxii) R15-152 comprising the light chain variable region of SEQ ID NO. 404 and the heavy chain variable region of SEQ ID NO. 405; and (xxiii) R15-126 comprising the light chain variable region of SEQ ID NO. 406 and the heavy chain variable region of SEQ ID NO. 407; and (xxi) any one anti-C5 antibody selected from the group consisting of crovalimab, but not limited thereto.;

[0038] In the present invention, the term "antibody" refers to a glycoprotein that inactivates antigens such as viruses and bacteria and induces extracellular stimulation against microorganisms that have invaded the body, and specifically refers to an immunoglobulin. Since the present invention relates to an anti-C5 antibody, the term "antibody" used without modification, unless otherwise specifically designated, may refer to an anti-C5 antibody that specifically binds to the C5 epitope. The scope of the present invention includes not only the complete antibody form that specifically binds to C5, but also the antigen-binding fragment of said antibody molecule. The complete antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected to the heavy chain by a disulfide bond. The heavy chain invariant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and has subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The light chain invariant region has kappa (κ) and lambda (λ) types.

[0039] In the present invention, the antibody comprises its antigen-binding fragment.

[0040] An antibody antigen-binding fragment or antibody fragment refers to a fragment possessing antigen-binding function and includes Fab, F(ab'), F(ab')2, and Fv. Among antibody fragments, Fab has a structure having variable regions of the light and heavy chains, a constant region of the light chain, and a first constant region (CH1) of the heavy chain, and possesses one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. The F(ab')2 antibody is generated when the cysteine ​​residues in the hinge region of Fab' form disulfide bonds. A recombinant technology for generating the Fv fragment from a minimal antibody fragment possessing only a heavy chain variable region and a light chain variable region is disclosed in PCT international published patent applications WO88 / 10649, WO88 / 106630, WO88 / 07085, WO88 / 07086, and WO88 / 09344. Two-chain Fvs have a variable region of the heavy chain and a variable region of the light chain connected by non-covalent bonds, while single-chain Fvs (scFvs) generally have a variable region of the heavy chain and a variable region of the light chain connected by covalent bonds through a peptide linker or directly connected at the C-terminus, so they can form a dimer-like structure similar to two-chain Fvs. These antibody fragments can be obtained using proteolytic enzymes (for example, restriction cleavage of the whole antibody with papain yields Fab, and cleavage with pepsin yields the F(ab')2 fragment), and can also be produced through genetic recombination technology.

[0041] In the present invention, the antibody may be in the Fv form (e.g., scFv) or in the complete antibody form (IgG). Additionally, the heavy chain constant region may be selected from any one isotype of gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε). For example, the constant region is gamma 1 (IgG1), gamma 3 (IgG3), or gamma 4 (IgG4). The light chain constant region may be in the kappa or lambda form.

[0042] In the present invention, the term "heavy chain (HC or CH)" refers to both a full-length heavy chain and fragments thereof, comprising a variable region domain VH having an amino acid sequence having a sufficient variable region (VR) sequence to confer specificity to an antigen and three constant region domains CH1, CH2, and CH3. Additionally, the term "light chain (LC or CL)" refers to both a full-length light chain and fragments thereof, comprising a variable region domain VL having an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen and a constant region domain CL.

[0043] The above antibodies may include monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, short-chain Fvs (scFv), short-chain antibodies, Fab fragments, F(ab') fragments, disulfide-conjugated Fvs (sdFv) and anti-idiotype (anti-Id) antibodies, or epitope-conjugated fragments of said antibodies. Specifically, said antibodies may be monoclonal antibodies and fully human antibodies. That is, compared to chimeric antibodies, etc., they may have reduced antigenicity when administered to human subjects. The above monoclonal antibodies refer to antibodies obtained from a substantially homogeneous population of antibodies, i.e., identical except for possible naturally occurring mutations that may be present in trace amounts among the individual antibodies comprising the population. Monoclonal antibodies are highly specific and are induced against a single antigenic site.

[0044] The above "humanized" form of the non-human (e.g., murine) antibody is a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. In most cases, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's hypervariable region are replaced with residues from the hypervariable region of a non-human species (donor antibody), e.g., mouse, rat, rabbit, or non-human primate, which possess the desired specificity, affinity, and ability.

[0045] The above "human antibody" refers to a molecule derived from human immunoglobulin, meaning that the entire amino acid sequence constituting the antibody, including the complementarity determining domain and the structural domain, is composed of human immunoglobulin.

[0046] The "chimeric antibody" includes a "chimeric" antibody (immunoglobulin) in which a portion of the heavy chain and / or light chain is derived from a particular species or is identical or homologous to a corresponding sequence within an antibody belonging to a particular class or subclass of antibodies, while the remaining chain(s) are derived from another species or are identical or homologous to a corresponding sequence within an antibody belonging to another class or subclass of antibodies, as well as a fragment of the said antibody exhibiting the desired biological activity.

[0047] In the present invention, the term "antibody variable domain" refers to the light and heavy chain portions of an antibody molecule comprising the amino acid sequences of a Complementary Determining Region (CDR, i.e., CDR1, CDR2, and CDR3) and a Backbone Region (FR). VH refers to the Variable Region of Heavy Chain. VL refers to the Variable Region of Light Chain. Each CDR is defined according to the Kabat definition, the Chothia definition, a combination of the Kabat and Chothia definitions, the IMGT numbering system, the AbM definition, or the contact definition of the CDR, and more preferably, according to the IMGT definition.

[0048] The above "Complementarity Determining Region" (CDR, i.e., CDR1, CDR2, and CDR3) may refer to amino acid residues of the antibody variable domain that are present for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2, and CDR3.

[0049] The term "Framework Region" (FR) in this specification refers to variable domain residues other than CDR residues. Each variable domain typically has four FRs identified as FR1, FR2, FR3, and FR4.

[0050] In the present invention, "antigen-binding fragment" refers to a fragment of an antibody having antigen-binding ability. The "Fv" fragment is an antibody fragment containing a complete antibody recognition and binding site. This region consists of one heavy chain variable domain and one light chain variable domain. The "Fab" fragment contains a light chain variable and constant domain and a heavy chain variable and first constant domain (CH1). The F(ab')2 antibody fragment generally comprises a pair of Fab fragments covalently linked near their carboxyl ends by a hinge cysteine ​​between them. The "single-chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of the antibody, which exist within a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker between the VH domain and the VL domain to enable the scFv to form a structure intended for antigen binding.

[0051] In addition, the antibody or its antigen-binding fragment may have species cross-reactivity. The individual may be a vertebrate, mammal, amphibian, reptile, bird, etc., and the species may be, for example, humans (Homo sapiens), monkeys, rats, mice, etc.

[0052] Additionally, the heavy chain constant region and light chain constant region of the antibody may be antibody constant regions that are IgG, IgM, IgE, IgA, IgD, or a combination thereof. The constant region may be derived, for example, from the IgG2 / 4 (γ2 / 4-chain) antibody constant region.

[0053] The antibody or antibody fragment of the present invention may include not only the sequence of the anti-C5 antibody described herein but also biological equivalents thereof, within a range capable of specifically recognizing C5. For example, additional changes may be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletion, insertion, and / or substitution of amino acid sequence residues of the antibody. Such amino acid modifications are made based on the relative similarity of amino acid side chain substituents, e.g., hydrophobicity, hydrophilicity, charge, size, etc. By analyzing the size, shape, and type of amino acid side chain substituents, it can be seen that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; And phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents.

[0054] In introducing the above mutation, the hydropathic index of the amino acids may be considered. Each amino acid is assigned a hydropathic index according to its hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The hydrophobic amino acid index is very important in conferring interactive biological functions of proteins. It is a known fact that similar biological activity can be achieved by substituting with amino acids having similar hydrophobic indices. When introducing a variation based on the hydrophobic index, the substitution is preferably made between amino acids exhibiting a difference in hydrophobic index within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

[0055] Meanwhile, it is also known that substitution between amino acids having similar hydrophilicity values ​​results in proteins having uniform biological activity. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​are assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); aspalate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); Tryptophan (-3.4). Amino acid exchanges in proteins that do not alter the overall activity of the molecule are known in the art (H. Neurath, RLHill, The Proteins, Academic Press, New York, 1979). The most common exchanges are exchanges between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0056] Considering the variant having the aforementioned biological equivalence activity, the antibody of the present invention or the nucleic acid molecule encoding it may be interpreted to include a sequence that exhibits substantial identity with the sequence described in SEQ ID NO. The above substantial identity refers to a sequence that exhibits at least 61% homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology when any other sequence is aligned to correspond as much as possible with the sequence of the present invention described above and the aligned sequence is analyzed using an algorithm commonly used in the art. Alignment methods for sequence comparison are known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) is accessible from NBCI, etc., and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx on the internet.

[0057] Another aspect of the present invention provides a nucleic acid encoding the antibody or its antigen-binding fragment, an expression vector comprising the nucleic acid, and a cell transformed with the expression vector.

[0058] According to a specific example, nucleic acids encoding the heavy chain of SEQ ID NO. 1 and the light chain of SEQ ID NO. 2 may be provided. For example, the nucleic acid encoding the heavy chain of SEQ ID NO. 1 may be represented by SEQ ID NO. 8, and the nucleic acid encoding the light chain of SEQ ID NO. 2 may be represented by SEQ ID NO. 9.

[0059] According to a specific other example, a nucleic acid encoding the light chain variable region of SEQ ID NO. 25 and the heavy chain variable region of SEQ ID NO. 26; a nucleic acid encoding the light chain variable region of SEQ ID NO. 27 and the heavy chain variable region of SEQ ID NO. 28; a nucleic acid encoding the light chain variable region of SEQ ID NO. 29 and the heavy chain variable region of SEQ ID NO. 30; a nucleic acid encoding the light chain variable region of SEQ ID NO. 31 and the heavy chain variable region of SEQ ID NO. 32; a nucleic acid encoding the light chain variable region of SEQ ID NO. 33 and the heavy chain variable region of SEQ ID NO. 34; a nucleic acid encoding the light chain variable region of SEQ ID NO. 236 and the heavy chain variable region of SEQ ID NO. 237; a nucleic acid encoding the light chain variable region of SEQ ID NO. 238 and the heavy chain variable region of SEQ ID NO. 239; a nucleic acid encoding the light chain variable region of SEQ ID NO. 381 and the heavy chain variable region of SEQ ID NO. 382; Nucleic acid encoding the light chain variable region of SEQ NO 383 and the heavy chain variable region of SEQ NO 384; nucleic acid encoding the light chain variable region of SEQ NO 381 and the heavy chain variable region of SEQ NO 224; nucleic acid encoding the light chain variable region of SEQ NO 385 and the heavy chain variable region of SEQ NO 386; nucleic acid encoding the light chain variable region of SEQ NO 225 and the heavy chain variable region of SEQ NO 387; nucleic acid encoding the light chain variable region of SEQ NO 381 and the heavy chain variable region of SEQ NO 388; nucleic acid encoding the light chain variable region of SEQ NO 389 and the heavy chain variable region of SEQ NO 390; nucleic acid encoding the light chain variable region of SEQ NO 389 and the heavy chain variable region of SEQ NO 391; nucleic acid encoding the light chain variable region of SEQ NO 392 and the heavy chain variable region of SEQ NO 393; Nucleic acid encoding the light chain variable region of SEQ ID NO. 381 and the heavy chain variable region of SEQ ID NO. 394; nucleic acid encoding the light chain variable region of SEQ ID NO. 381 and the heavy chain variable region of SEQ ID NO. 395; nucleic acid encoding the light chain variable region of SEQ ID NO. 392 and the heavy chain variable region of SEQ ID NO. 396; nucleic acid encoding the light chain variable region of SEQ ID NO. 381 and the heavy chain variable region of SEQ ID NO. 397; nucleic acid encoding the light chain variable region of SEQ ID NO. 381 and the heavy chain variable region of SEQ ID NO. 398;Nucleic acid encoding the light chain variable region of SEQ ID NO. 399 and the heavy chain variable region of SEQ ID NO. 387; nucleic acid encoding the light chain variable region of SEQ ID NO. 400 and the heavy chain variable region of SEQ ID NO. 401; nucleic acid encoding the light chain variable region of SEQ ID NO. 402 and the heavy chain variable region of SEQ ID NO. 403; nucleic acid encoding the light chain variable region of SEQ ID NO. 404 and the heavy chain variable region of SEQ ID NO. 405; nucleic acid encoding the light chain variable region of SEQ ID NO. 406 and the heavy chain variable region of SEQ ID NO. 407; nucleic acid encoding the light chain variable region of SEQ ID NO. 408 and the heavy chain variable region of SEQ ID NO. 409; and nucleic acid encoding the light chain variable region of SEQ ID NO. 410 and the heavy chain variable region of SEQ ID NO. 411 may be provided. For example, the nucleic acid encoding the light chain variable region of SEQ ID NO. 25 may be denoted by SEQ ID NO. 35, and the nucleic acid encoding the heavy chain variable region of SEQ ID NO. 26 may be denoted by SEQ ID NO. 36; the nucleic acid encoding the light chain variable region of SEQ ID NO. 27 may be denoted by SEQ ID NO. 37, and the nucleic acid encoding the heavy chain variable region of SEQ ID NO. 28 may be denoted by SEQ ID NO. 38; the nucleic acid encoding the light chain variable region of SEQ ID NO. 29 may be denoted by SEQ ID NO. 39, and the nucleic acid encoding the heavy chain variable region of SEQ ID NO. 30 may be denoted by SEQ ID NO. 40; the nucleic acid encoding the light chain variable region of SEQ ID NO. 31 may be denoted by SEQ ID NO. 41, and the nucleic acid encoding the heavy chain variable region of SEQ ID NO. 32 may be denoted by SEQ ID NO. 42; The nucleic acid encoding the light chain variable region of SEQ ID NO. 33 may be represented by SEQ ID NO. 43, and the nucleic acid encoding the heavy chain variable region of SEQ ID NO. 34 may be represented by SEQ ID NO. 44; the nucleic acid encoding the light chain variable region of SEQ ID NO. 236 may be represented by SEQ ID NO. 240, and the nucleic acid encoding the heavy chain variable region of SEQ ID NO. 237 may be represented by SEQ ID NO. 241;The nucleic acid encoding the light chain variable region of SEQ ID NO. 238 may be represented by SEQ ID NO. 242, and the nucleic acid encoding the heavy chain variable region of SEQ ID NO. 239 may be represented by SEQ ID NO. 243; the nucleic acid encoding the light chain variable region of SEQ ID NO. 381 may be represented by SEQ ID NO. 412, and the nucleic acid encoding the heavy chain variable region of SEQ ID NO. 382 may be represented by SEQ ID NO. 413; the nucleic acid encoding the light chain variable region of SEQ ID NO. 383 may be represented by SEQ ID NO. 414, and the nucleic acid encoding the heavy chain variable region of SEQ ID NO. 384 may be represented by SEQ ID NO. 415; the nucleic acid encoding the light chain variable region of SEQ ID NO. 381 may be represented by SEQ ID NO. 412, and the nucleic acid encoding the heavy chain variable region of SEQ ID NO. 224 may be represented by SEQ ID NO. 416; The nucleic acid encoding the light chain variable region of SEQ ID NO. 385 may be represented by SEQ ID NO. 417, and the nucleic acid encoding the heavy chain variable region of SEQ ID NO. 386 may be represented by SEQ ID NO. 418; the nucleic acid encoding the light chain variable region of SEQ ID NO. 225 may be represented by SEQ ID NO. 419, and the nucleic acid encoding the heavy chain variable region of SEQ ID NO. 387 may be represented by SEQ ID NO. 420; the nucleic acid encoding the light chain variable region of SEQ ID NO. 381 may be represented by SEQ ID NO. 412, and the nucleic acid encoding the heavy chain variable region of SEQ ID NO. 388 may be represented by SEQ ID NO. 421; the nucleic acid encoding the light chain variable region of SEQ ID NO. 389 may be represented by SEQ ID NO. 422, and the nucleic acid encoding the heavy chain variable region of SEQ ID NO. 390 may be represented by SEQ ID NO. 423; The nucleic acid encoding the light chain variable region of SEQ ID NO. 389 can be represented by SEQ ID NO. 424, and the nucleic acid encoding the heavy chain variable region of SEQ ID NO. 391 can be represented by SEQ ID NO. 425;The nucleic acid encoding the light chain variable region of SEQ ID NO. 392 may be represented by SEQ ID NO. 426, and the nucleic acid encoding the heavy chain variable region of SEQ ID NO. 393 may be represented by SEQ ID NO. 427; the nucleic acid encoding the light chain variable region of SEQ ID NO. 381 may be represented by SEQ ID NO. 412, and the nucleic acid encoding the heavy chain variable region of SEQ ID NO. 394 may be represented by SEQ ID NO. 428; the nucleic acid encoding the light chain variable region of SEQ ID NO. 381 may be represented by SEQ ID NO. 429, and the nucleic acid encoding the heavy chain variable region of SEQ ID NO. 395 may be represented by SEQ ID NO. 430; the nucleic acid encoding the light chain variable region of SEQ ID NO. 392 may be represented by SEQ ID NO. 426, and the nucleic acid encoding the heavy chain variable region of SEQ ID NO. 396 may be represented by SEQ ID NO. 431; The nucleic acid encoding the light chain variable region of SEQ ID NO. 381 may be represented by SEQ ID NO. 412, and the nucleic acid encoding the heavy chain variable region of SEQ ID NO. 397 may be represented by SEQ ID NO. 432; the nucleic acid encoding the light chain variable region of SEQ ID NO. 381 may be represented by SEQ ID NO. 412, and the nucleic acid encoding the heavy chain variable region of SEQ ID NO. 398 may be represented by SEQ ID NO. 433; the nucleic acid encoding the light chain variable region of SEQ ID NO. 399 may be represented by SEQ ID NO. 434, and the nucleic acid encoding the heavy chain variable region of SEQ ID NO. 387 may be represented by SEQ ID NO. 420; the nucleic acid encoding the light chain variable region of SEQ ID NO. 400 may be represented by SEQ ID NO. 435, and the nucleic acid encoding the heavy chain variable region of SEQ ID NO. 401 may be represented by SEQ ID NO. 436; The nucleic acid encoding the light chain variable region of SEQ ID NO. 402 may be represented by SEQ ID NO. 437, and the nucleic acid encoding the heavy chain variable region of SEQ ID NO. 403 may be represented by SEQ ID NO. 438;The nucleic acid encoding the light chain variable region of SEQ ID NO. 404 may be represented by SEQ ID NO. 439, and the nucleic acid encoding the heavy chain variable region of SEQ ID NO. 405 may be represented by SEQ ID NO. 440; the nucleic acid encoding the light chain variable region of SEQ ID NO. 406 may be represented by SEQ ID NO. 441, and the nucleic acid encoding the heavy chain variable region of SEQ ID NO. 407 may be represented by SEQ ID NO. 442; the nucleic acid encoding the light chain variable region of SEQ ID NO. 408 may be represented by SEQ ID NO. 443, and the nucleic acid encoding the heavy chain variable region of SEQ ID NO. 409 may be represented by SEQ ID NO. 444; the nucleic acid encoding the light chain variable region of SEQ ID NO. 410 may be represented by SEQ ID NO. 445, and the nucleic acid encoding the heavy chain variable region of SEQ ID NO. 411 may be represented by SEQ ID NO. 446.

[0060] In the present invention, the term "epitope" refers to a protein determinant to which an antibody can specifically bind. An epitope typically consists of a group of chemically active surface molecules, such as amino acids or sugar side chains, and generally possesses specific charge characteristics as well as specific three-dimensional structural features. Stereoscopic epitopes and non-stereoscopic epitopes are distinguished in that, in the presence of a denaturing solvent, binding to the former is lost but not to the latter.

[0061] In one embodiment of the present invention, the combination may further include a complement C5 inhibitor.

[0062] In one embodiment of the present invention, the complement C5 inhibitor may be one or more selected from the group consisting of Eculizumab, Pozelimab, Coversin, Tesidolumab, Ravulizumab, Zilucoplan, KP-104, etc., but is not limited thereto.

[0063] In one embodiment of the present invention, complement C1s inhibitors, complement C1q inhibitors, and / or complement C1r inhibitors may be additionally included, but are not limited thereto.

[0064] The antibody or its antigen-binding fragment can be recombinantly produced by isolating the nucleic acid encoding the antibody or its antigen-binding fragment of the present invention. The nucleic acid is isolated and inserted into a replicable vector to further clone (amplify DNA) or further express it. Based on this, the present invention relates, in another aspect, to a vector comprising said nucleic acid.

[0065] The term "nucleic acid" in this specification has a meaning that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acids, include not only natural nucleotides but also analogues in which sugar or base sites are modified. The sequences of nucleic acids encoding the heavy chain and light chain variable regions of the present invention may be modified. Such modifications include the addition, deletion, non-conservative, or conservative substitution of nucleotides.

[0066] The above nucleic acid is interpreted to include a nucleotide sequence that exhibits substantial identity with respect to the above nucleotide sequence. Substantial identity means a nucleotide sequence that exhibits at least 80% homology, more preferably at least 90% homology, and most preferably at least 95% homology when the nucleotide sequence of the present invention is aligned with any other sequence to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art.

[0067] The DNA encoding the antibody is easily isolated or synthesized using conventional processes (e.g., by using an oligonucleotide probe capable of specifically binding to the DNA encoding the heavy and light chains of the antibody). Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, a replication origin, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0068] The term "vector" as used herein includes plasmid vectors; cosmid vectors; and viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors, as means for expressing a target gene in a host cell. In said vector, the nucleic acid encoding the antibody is operatively linked to a promoter.

[0069] "Operational linkage" refers to a functional linkage between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another nucleic acid sequence, thereby allowing the regulatory sequence to regulate the transcription and / or translation of the other nucleic acid sequence.

[0070] In the case of a prokaryotic host, it is common to include a powerful promoter capable of proceeding transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter, etc.), a ribosome binding site for initiating translation, and a transcription / translation termination sequence. Additionally, for example, when a eukaryotic cell is used as a host, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rhoese's sarcoma virus (RSV) promoter) may be used, and generally have a polyadenylation sequence as a transcription termination sequence. In some cases, the vector may be fused with other sequences to facilitate the purification of the antibody expressed therefrom. The sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6xHis (hexahistidine; Quiagen, USA). The vector contains antibiotic resistance genes commonly used in the art as selection markers, such as resistance genes for ampicillin, gentamicin, cabbageillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0071] In another aspect, the present invention relates to cells transformed with the vector mentioned above. The cells used to produce the antibodies of the present invention may be prokaryotic, yeast, or higher eukaryotic cells, but are not limited thereto. Prokaryotic host cells may be used, such as strains of the genus Bacillus, such as Escherichia coli, Bacillus subtilis, and Bacillus churingensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, and Staphylococcus (e.g., Staphylococcus carnosus). However, there is the greatest interest in animal cells, and examples of useful host cell lines may be, but are not limited to, COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080.

[0072] In another aspect, the present invention relates to a method for producing said antibody or said antigen-binding fragment, comprising the steps of: (a) culturing said cells; and (b) recovering said antibody or said antigen-binding fragment from said cultured cells. said cells may be cultured in various media. Any commercially available media may be used as a culture medium without limitation. Any other essential supplement known to those skilled in the art may be included in appropriate concentrations. Culture conditions, such as temperature, pH, etc., are already in use with host cells selected for expression, and this will be obvious to those skilled in the art. The recovery of said antibody or said antigen-binding fragment may remove impurities by, for example, centrifugation or ultrafiltration, and the resulting product may be purified by, for example, affinity chromatography. Additional purification techniques, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, etc., may be used.

[0073] Another aspect of the present invention relates to a composition for the prevention or treatment of complement-related diseases comprising the above-mentioned combination of complement C5 binding antagonists as an active ingredient.

[0074] Another aspect of the present invention provides a method for preventing or treating a complement-related disease, comprising the step of administering a combination of complement C5 binding antagonists according to the present invention to a patient in an effective amount required.

[0075] In one embodiment of the present invention, the complement-related disease may be one or more selected from the group consisting of myasthenia gravis (gMG), age-related macular degeneration (AMD), and paroxysmal nocturnal hemoglobinuria (PNH), but is not limited thereto.

[0076] The specific details regarding the above-mentioned complement C5 binding antagonist combination are as described above. As demonstrated in the following examples, the complement C5 binding antagonist combination according to the present invention can significantly inhibit complement activity and can be effectively used for the prevention and / or treatment of complement-related diseases.

[0077] The term "prevention" above refers to any act of suppressing or delaying the progression of complement-related diseases through the administration of the above composition, and "treatment" refers to suppressing, alleviating, or eliminating the development of complement-related diseases.

[0078] In one embodiment of the present invention, the composition may include a pharmaceutically acceptable carrier that is commonly used in formulations, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, but is not limited thereto. In addition to the above components, the composition of the present invention may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc.

[0079] The composition of the present invention may be administered orally or parenterally. Preferably, it may be administered parenterally, and in the case of parenteral administration, it may be administered via intravenous infusion, subcutaneous infusion, intramuscular infusion, intraperitoneal infusion, endothelial infusion, topical infusion, intranasal infusion, intrapulmonary infusion, and rectal infusion. In one embodiment, it may be administered in the form of an intravenous injection. When administered orally, since proteins or peptides are digested, the oral composition must be formulated to coat the active agent or protect it from degradation in the stomach. Additionally, the pharmaceutical composition may be administered by any device capable of delivering the active substance to target cells.

[0080] The suitable dosage of the composition according to the present invention varies depending on factors such as the formulation method, mode of administration, patient's age, body weight, sex, pathological condition, food, time of administration, route of administration, excretion rate, and response sensitivity, and a physician of ordinary skill can easily determine and prescribe a dosage effective for the desired treatment or prevention. In the present invention, the term "pharmaceutical effective dose" means an amount sufficient to prevent or treat myasthenia gravis (gMG), age-related macular degeneration (AMD), and paroxysmal nocturnal hemoglobinuria (PNH), etc.

[0081] The composition of the present invention may be prepared in a unit volume form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier and / or excipient, according to a method that can be easily carried out by a person skilled in the art to which the invention belongs. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, suppository, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0082] The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

[0083] The complement C5 binding antagonist combination according to the present invention comprises antagonists that specifically bind to different domains of complement C5, thereby significantly inhibiting complement C5 activity; thus, it can be used for the prevention or treatment of various complement-related diseases, including myasthenia gravis, age-related macular degeneration, and paroxysmal nocturnal hemoglobinuria.

[0084] Figure 1 shows the structure of a vector used to manufacture an anti-C5 antibody, which is the first antagonist of a complement C5 binding antagonist combination according to one aspect.

[0085] Figure 2 shows the results of analyzing the binding power of anti-C5 antibodies (Ch2-12, Ch5-13, Ch1-78), which are the second antagonists of the complement C5 binding antagonist combination according to one aspect, to human complement C5.

[0086] Figure 3 shows the results of analyzing the binding power of anti-C5 antibodies (Ch11-01, Ch11-09), which are the second antagonists of the complement C5 binding antagonist combination according to one aspect, to human complement C5.

[0087] Figure 4 shows the results of analyzing the binding power of the anti-C5 antibody (R-C5 / MG1-008-30-#13), which is the second antagonist of the complement C5 binding antagonist combination according to one aspect, to human complement C5.

[0088] Figure 5 shows the results of analyzing the binding power of the anti-C5 antibody (R-13-SP#21), which is the second antagonist of the complement C5 binding antagonist combination according to one aspect, to human complement C5.

[0089] Figure 6 shows the inhibitory effect of Ch1-78, one of the anti-C5 antibodies that is the second antagonist of the complement C5 binding antagonist combination according to one aspect, on complement C5 activity in an alternative pathway-mediated hemolysis assay.

[0090] Figure 7 shows the inhibitory effect of Ch2-12, one of the anti-C5 antibodies that is the second antagonist of the complement C5 binding antagonist combination according to the pattern, on complement C5 activity in an alternative pathway-mediated hemolysis assay.

[0091] Figure 8 shows the inhibitory effect of Ch5-13, one of the anti-C5 antibodies that is the second antagonist of the complement C5 binding antagonist combination according to one aspect, on complement C5 activity in an alternative pathway-mediated hemolysis assay.

[0092] Figure 9 shows the inhibitory effect of Ch11-01, one of the anti-C5 antibodies that is the second antagonist of the complement C5 binding antagonist combination according to one aspect, on complement C5 activity in an alternative pathway-mediated hemolysis assay.

[0093] Figure 10 shows the inhibitory effect of Ch11-09, one of the anti-C5 antibodies that is the second antagonist of the complement C5 binding antagonist combination according to one aspect, on complement C5 activity in an alternative pathway-mediated hemolysis assay.

[0094] Figure 11 shows the inhibitory effect of R-13-SP#21, one of the anti-C5 antibodies that is the second antagonist of the complement C5 binding antagonist combination according to one aspect, on complement C5 activity in an alternative pathway-mediated hemolysis assay.

[0095] Figure 12 shows the inhibitory effect of R-C5 / MG1-008-30-#13, one of the anti-C5 antibodies that is the second antagonist of the complement C5 binding antagonist combination according to one aspect, in an alternative pathway-mediated hemolysis assay of complement C5 activity.

[0096] Figure 13 shows the results of identifying the binding sites on human complement C5 of the anti-C5 antibodies (Ch2-12, Ch5-13, Ch1-78, Ch11-01, Ch11-09), which are the second antagonists of the complement C5 binding antagonist combination according to one aspect.

[0097] Figure 14 shows the results of identifying the binding sites on the beta chain of human complement C5 by the anti-C5 antibody (Ch2-12, Ch5-13, Ch1-78, Ch11-01, Ch11-09), which is the second antagonist of the complement C5 binding antagonist combination according to one aspect.

[0098] Figure 15 shows the results of confirming the binding of the anti-C5 antibody (R-C5 / MG1-008-30-#13, R-13-SP#21), which is the second antagonist of the combination of complement C5 binding antagonists according to one aspect, to the human complement C5 full length, MG1, or MG4.

[0099] Figure 16 shows the results of confirming the binding of anti-C5 antibodies (R14-27, R14-07, R14-24, R15-01), which are the second antagonists of the combination of complement C5 binding antagonists according to one aspect, to the human complement C5 full length, MG1, or MG4.

[0100] Figure 17 shows the results of confirming the binding of anti-C5 antibodies (R14-19, R14-23, R14-20, R14-26), which are the second antagonists of the combination of complement C5 binding antagonists according to one aspect, to the human complement C5 full length, MG1, or MG4.

[0101] Figure 18 shows the results of confirming the binding of anti-C5 antibodies (R15-24, R15-14, R14-16, R15-21, R15-10, R15-05, R13-30, R13-04) to the human complement C5 full length, MG1, or MG4 according to one aspect.

[0102] Figure 19 shows the results of confirming the binding of anti-C5 antibodies (R13-165, R13-175, R13-210, R15-126, R15-152) to the human complement C5 full length, MG1, or MG4 according to one aspect.

[0103] Figure 20 shows the results of analyzing the combined effect of CirpT1 and crovalimab according to the daily pattern.

[0104] The present invention will be described in detail below.

[0105] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention, and the scope of the invention is not limited by the following embodiments.

[0106] [Example]

[0107] Example 1. Preparation of a first antagonist that specifically binds to the MG4 domain

[0108] 1-1. Preparation of the first antagonist (anti-C5 antibody) cell line

[0109] A recombinant anti-C5 antibody with improved C5 inhibitory efficacy and safety was produced. Specifically, the DNA for the heavy and light chains of the anti-C5 antibody, respectively, was obtained from the Antibody Engineering Laboratory of Professor Junho Jeong at Seoul National University and inserted into Vector A and Vector C, respectively, by enzymatic digestion with NheI (NEB, R0131S) and PmeI (NEB, R0560S). Vector A, into which the heavy chain DNA of the anti-C5 antibody was inserted, and Vector C, into which the light chain DNA of the anti-C5 antibody was inserted, were named Vector B and Vector D, respectively (see Fig. 1). The DNA sequences of the expression vectors were confirmed through DNA sequencing analysis.

[0110] CHO-K1 cells (purchased from ATCC, CCL-61) were transfected in 3×10 wells of MEM-α medium supplemented with 10% dFBS (Gibco, 30067-334) (Gibco, 12561) for transformation. 5 Cells were cultured after inoculation, and a total of 2.5 μg of DNA was transfected into the cells using Lipofectamine™ LTX Reagent (Invitrogen, 15338) according to the manufacturer's instructions.

[0111] Three days after transformation, the transformed cells were screened by inoculating them at a concentration of 1,000 cells / well (200 µl) into SFM4CHO medium (HyClone, SH30549.02) free of hypoxanthine and thymidine and supplemented with 500 nM MTX (Yuhan, A193722). These cells were cultured in a 37.0±1 ℃, 5.0±1% CO2 incubator. After about three weeks, when the cells filled more than 70% of the wells, the titer was measured by ELISA. Clones with high titers were sequentially scaled to 24-well plates, 6-well plates, and 125 mL shake flasks.

[0112] Selected clones were inoculated into SFM4CHO medium supplemented with 2% dFBS at a concentration of 0.5 cells per well and cultured in a 37.0±1 ℃, 5.0±1% CO2 incubator. After 3–4 weeks, titers were measured when the cell count exceeded 70% of the wells. Based on the results, cells with high titers were sequentially cultured in 24-well plates, 12-well plates, 6-well plates, and 125 mL shake flasks.

[0113] Subsequently, stability tests were performed on cell lines with high titers for 30 passages. Specifically, 0.3×10 6 The above cell lines were inoculated into a 125 mL shake flask containing 25 mL of SFM4CHO medium at a cell / mL concentration and subcultured every 3 days. The cells were cultured at 37.0±1 ℃ in a 5.0±1% CO2 incubator with stirring (120 rpm). Based on stability tests and batch culture results (production stability, maximum titer, and quality consistency), the final RCB (Research cell bank) of the anti-C5 antibody was selected.

[0114] The sequence of the selected anti-C5 antibody (hereinafter referred to as antibody A) is as shown in Table 1 below. The sequence of antibody A was verified using IMGT / DomainGapAlign (http: / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi).

[0115] Sequence of the first antagonist, anti-C5 antibody (Antibody A) Antibody A Classification Sequence Number Sequence HCDR13GFSFSGRYWHCDR24SGWPGATGDTHCDR35AREPVAWGGGLDLLCDR16QSINNQLCDR2-YASLCDR37QGSYYSGGWDYG Heavy Chain 1EVQLVESGGG LVQPGGSLRL SCAASGFSFS GRYWIQWVRQ APGKGLEWVA SGWPGATGDT NYANWAKGRF TISRDDSKNT LYLQMNSLRA EDTAVYYCAR EPVAWGGGLD LWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSNFGT QTYTCNVDHK PSNTKVDKTV ERKCCVECPP CPAPPVAGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNST YRVVSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGKLight Chain2DIQMTQSPSS LSASVGDRVT ITCQASQSIN NQLSWYQQKP GKAPKLLIYY ASTLASGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQG SYYSGGWDYG FGQGTKVEIKRTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

[0116] (Bold text indicates variable regions, and the rest indicates immutable regions)

[0117] 1-2. Antibody A Culture

[0118] The final cell line selected in Example 1-1 above was cultured in large quantities in a seed bioreactor to produce antibody A. Specifically, the antibody A cell line vial was thawed quickly in a 37.0 °C water bath, inoculated into a 125 mL shake flask supplemented with SFM4CHO medium, and cultured in a 37.0 ± 1 °C, 5.0 ± 1% CO2 incubator. The shake flask culture was expanded through multiple subculturings to inoculate into an N-3 seed bioreactor. During the culture process of antibody A, the inoculated cell concentration at each subculturing was approximately 0.3 x 10⁶. 6 The cells / mL level was maintained, and SFM4CHO was used as the medium.

[0119] After completing inoculation into the N-3 Seed Bioreactor, the dissolved oxygen level was maintained at 10–30%, the culture temperature at 37±1 ℃, and the pH at 6.9±0.2. The culture medium was periodically collected and the cell condition was observed under a microscope. In addition, cell number, cell activity, pH, and osmotic pressure were analyzed, and subculturing was performed once sufficient cell growth was achieved to match the initial inoculation concentration of the N-2 Seed Bioreactor.

[0120] Subsequently, once inoculation into the N-2 Seed Bioreactor was completed, the reactor was maintained under the same conditions as above, and cells were analyzed using the same method as above; further subculturing was performed when sufficient cell growth was achieved to match the initial inoculation concentration of the N-1 Seed Bioreactor.

[0121] Afterward, once inoculation into the N-1 Seed Bioreactor was completed, the reactor was maintained under the same conditions as above, and the cells were analyzed in the same manner as above, except that additional metabolites were analyzed, and subculturing was performed when sufficient cell growth was achieved to the initial inoculation concentration of the 1000L Main Bioreactor.

[0122] Subsequently, once inoculation was completed in the 1000L Main Bioreactor, a cell booster (Hyclone) was periodically added to increase the number of cells during culture, and the reactor was maintained under the same conditions as above, except that the culture temperature was maintained at 35±1 ℃ starting from the 6th day of culture to maintain cell activity. In addition, cells were analyzed using the same method as above, and cell growth was induced by adjusting the supply amounts of glucose and glutamine according to the analysis results.

[0123] After inoculating a 1000L culture vessel, if cell activity decreased rapidly, cell culture was stopped and cells were separated from the culture medium using a Millipore POD filter system. The culture supernatant from which the cells were separated was filtered using a 0.1 µm membrane filter, collected in a container, and stored at ambient temperature.

[0124] 1-3. Antibody A Purification

[0125] To purify antibody A from the culture medium obtained in Examples 1-2 above, two-step chromatography, concentration, and buffer exchange were performed. Specifically, affinity chromatography was performed using MabSelect PrismA (Cytiva) resin to obtain antibody A from the culture recovery medium. The column was equilibrated with an equilibration buffer solution (pH 5.8–6.2) containing 50 mM sodium phosphate, and then the culture recovery medium was loaded. After loading was complete, the column was washed once with the equilibration buffer solution, then washed a second time with a wash buffer solution containing sodium chloride, and then washed a third time with the equilibration buffer solution. After the third wash was completed, the target protein was eluted using an elution buffer solution (pH 3.4–3.6) containing 50 mM sodium citrate.

[0126] Viruses that may originate from host cells or auxiliary materials used in the process were inactivated using low pH conditions. Specifically, 1M Citric acid was added to the eluent obtained from the MabSelect PrismA chromatography step to adjust the pH to 3.5–3.7. After about 1 hour, 1M Tris was added to adjust the pH to 5.6–6.0.

[0127] Once virus inactivation was complete, Mix Mode chromatography was performed using Capto Adhere (Cytiva) resin to remove impurities. The column was equilibrated with an equilibration buffer (pH 5.6–6.0) containing 20 mM histidine and a small amount of sodium chloride, and then the virus-inactivated solution was loaded. After loading was complete, the equilibration buffer was additionally loaded to recover the target protein with high purity (SE-HPLC, 99.0% or higher) and low impurity content (HCP, 100 ng or lower).

[0128] Ultrafine filtration was performed using a virus filter (Virosart HF, Sartorius) to remove viruses that may originate from host cells or auxiliary materials used in the process. After equilibration with an equilibration buffer (pH 5.6–6.0) containing 20 mM histidine and a small amount of sodium chloride, the eluent solution obtained from the Mix mode chromatography step was loaded. After loading was complete, the equilibration buffer was additionally loaded to recover the target protein.

[0129] An ultrafiltration device (Tangential Flow Filtration Membrane System) was used to adjust the protein concentration of the above antibody A and to exchange the purified protein with a formulation buffer. The membrane (Merck, Cut-off: 30 kDa) in the ultrafiltration device was washed with sterile water for injection and equilibrated with an equilibration buffer (pH 5.6–6.0) containing 20 mM histidine and a small amount of sodium chloride. After concentrating the target protein to a concentration of approximately 60 mg / mL or higher, a buffer exchange was performed with a buffer (pH 5.3–5.7) containing 20 mM succinate and 60 mM Arg-HCl. After the buffer exchange was completed, further concentration was performed. After further concentration was completed, sucrose and polysorbate 80 were added to adjust to the target protein concentration and formulation of the above antibody A (Antibody A protein concentration: 108–132 mg / mL. Formulation: 20 mM Succinate, 50 g / L sucrose, 60 mM Arg-HCl, 0.045 % (w / v) PS80).

[0130] Example 2. Preparation of a second antagonist specifically binding to the MG1 domain and analysis of complement C5 binding ability

[0131] 2-1. Preparation of C5 Immunoantibody Library

[0132] 2-1-1. Preparation of C5 Immunotherapy Chicken Antibody Library

[0133] 25 µg of human C5 protein (Acrobiosystems) was mixed with a complete freund adjuvant (Sigma, Cat# F5881) or an incomplete freund adjuvant (Sigma, Cat# F5506) and injected subcutaneously into chickens at 2-week intervals. Spleen, bone marrow, and synovial sacs were obtained from immunized chickens, and total RNA was extracted from them using the Rneasy mini kit (Qiagen, Cat# 74104). First-strand cDNA was synthesized using oligo-dT primers and the SuperScript™ III First-Strand Synthesis System (Invitrogen, Cat# 18080-051). An antigen-binding fragment (Fab) library was constructed using primers specific to the heavy and light chain variable regions of chicken immunoglobulins (Table 2).

[0134] Primer Information for Vλ and VH in Chicken Antigen-Binding Segment Library Type Name Sequence Number Sequence (5' - 3') VH 5' Sense Primers CHybVH(sense) 12GCT GCC CAA CCA GCC ATG GCC GCC GTG ACG TTG GAC GAG TCC VH 3' Reverse Primers CHybIg-B(reverse) 13CGA TGG GCC CTT GGT GGA GGC GGA GGA GAC GAT GAC TTC GGT CCC Vλ 5' Sense Primers CSCVK(sense) 14GTG GCC CAG GCG GCC CTG ACT CAG CCG TCC TCG GTG TC Vλ 3' Reverse Primers CHybL-B(reverse) 15AGA TGG TGC AGC CAC AGT TCG TAG GAC GGT CAG GGT TGT CCC GGC Cloned Human Primers for amplification of the human CHI chain from a cloned human Fab: HIgGCH1-F(sense) 16GCC TCC ACC AAG GGC CCA TCG GTC dpseq(reverse) 17AGA AGC GTA GTC CGG AAC GTC Primers for amplification of the human Ck region and the pelB leader sequence from a cloned human Fab: HKC-F(sense) 18CGA ACT GTG GCT GCA CCA TCT GTC Lead-B(reverse) 19GGC CAT GGC TGG TTG GGC AGC Primers for PCR assembly of chicken VH sequence with the human CHI PCR productproduct) leadVH(sense) 20 GCT GCC CAA CCA GCC ATG GCC dpseq(reverse) 17 AGA AGC GTA GTC CGG AAC GTC Primers for PCR assembly of Chicken VL sequence with the human Ck PCR product CSC-F(sense) 21 GAG GAG GAG GAG GAG GAG GTG GCC CAG GCG GCC CTG ACT CAG Lead-B(reverse) 22 GGC CAT GGC TGG TTG GGC AGC Primers for PCR assembly of chimeric light chain sequences with chimeric heavy chain sequences CSC-F(sense) 23 GAG GAG GAG GAG GAG GAG GTG GCC CAG GCG GCC CTG ACT CAG dp-EX(reverse) 24 GAG GAG GAG GAG GAG GAG AGA AGC GTA GTC CGG AAC GTC

[0135] For the first PCR, to clone the light chain variable region (VL) and heavy chain variable region (VH), 1 µl of cDNA, 25 pmol of each primer, 10 µl of 5X reaction buffer, 1 µl of 10 mM dNTPs, and 1 µl of Taq DNA polymerase were mixed with water to make a final volume of 50 µl. To clone the light chain constant region (CL) and heavy chain first constant region (CHI), 100 ng of pComb3XTT vector, 25 pmol of each primer, 10 µl of 5X reaction buffer, 1 µl of 10 mM dNTPs, and 1 µl of Taq DNA polymerase were mixed with water to make a final volume of 50 µl. The PCR reactions were performed under the following conditions.

[0136] First PCR Condition Step Temperature (°C) Time (sec) Number of Cycles Pre-incubation 98 30 1 Denaturation 98 10 30 Annealing 60 10 Elongation 72 30 Final elongation 72 30 01

[0137] Approximately 350 bp amplified fragments were loaded onto a 1% agarose gel and purified using a Gel & PCR clean-up kit (Cosmojintech, cat# CMA0112). In the second PCR, the first VL and CL, and VH and CHI, were randomly combined by overlap extension PCR. Each PCR reaction was performed with a 50 µl mixture containing 100 ng of each product, 25 pmol of each primer, 10 µl of 5X reaction buffer, 1 µl of 10 mM dNTP, and 1 µl of Taq DNA polymerase. The PCR reactions were performed under the following conditions.

[0138] Second PCR Condition Step Temperature (°C) Time (sec) Number of Cycles Pre-incubation 98 30 1 Denaturation 98 10 15 Annealing 60 10 Elongation 72 30 Final elongation 72 30 01

[0139] Approximately 750 bp amplified fragments were loaded onto a 1% agarose gel and run, after which they were purified using a Gel & PCR clean-up kit (Cosmojintech, cat# CMA0112). In the third PCR, the overlap extension PCR products of VL and CL and the overlap extension PCR products of VH and CHI were randomly combined by overlap extension PCR. Each PCR reaction was performed with a 50 µl mixture containing 100 ng of each product, 25 pmol of each primer, 10 µl of 5X reaction buffer, 1 µl of 10 mM dNTPs, and 1 µl of Taq DNA polymerase. The PCR reactions were performed under the following conditions.

[0140] Third PCR Condition Step Temperature (°C) Time (sec) Number of Cycles Pre-incubation 98 30 1 Denaturation 98 10 10 Annealing 60 10 Elongation 72 45 Final elongation 72 30 01

[0141] Approximately 1500 bp amplified fragments were loaded onto a 1% agarose gel and run, after which they were purified using a Gel & PCR clean-up kit (Cosmogenetec, cat# CMA0112). The Fab fragments and pComb3XSS vector were cleaved with Sfi I restriction enzyme (NEB, Cat# R0123L) at 50°C for 12 hours. 1400 ng of Sfi I-cleaved Fab and 1400 ng of pComb3X vector were mixed and treated with T4 DNA ligase at 16°C for at least 12 hours to induce ligation, followed by ethanol precipitation. The ligated libraries were transformed into E. coli ER2738 by electroporation. Cells were mixed into 3 mL of Super Broth (SB) medium at 37 °C and cultured for 1 hour with stirring at 250 rpm; subsequently, 10 mL of SB medium and 3 µL of 100 mg / mL Cabenicillin were added to the culture medium. The library size was determined by the number of transformed colonies formed after plating an appropriately diluted culture onto Luria broth (LB) plates containing 100 µg / mL Cabenicillin and incubating overnight. After 1 hour of incubation, 4.5 µL of 100 mg / mL Cabenicillin was added, and the cells were cultured for an additional 1 hour. 1 mL of VCSM13 helper phage (> 10 11183 ml of SB medium and 92.5 µl of 100 mg / ml cabbageillin were added, and the mixture was incubated at 37°C for 2 hours with stirring at 250 rpm. Afterward, 280 µl of 50 mg / ml kanamycin was added and the mixture was stirred overnight at 37°C at 250 rpm. The next day, the culture medium was centrifuged at 4°C at 7000 rpm for 10 minutes to extract phagemid DNA from the bacterial pellet. The supernatant was transferred to a clean centrifuge bottle, and then 50 ml of 5XPEG / NaCl solution was added, or 8 g of polyethylene glycol-8000 (PEG-8000, Sigma, Cat# P2139) and 6 g of sodium chloride (Sigma, cat# S9888) were added to dissolve it, and the mixture was stored on ice for 30 minutes to 1 hour. Next, the supernatant was centrifuged at 4°C and 8000 rpm for 30 minutes to 1 hour, and after removing the supernatant, the phage pellet was suspended in PBS containing 1% BSA and 0.02% NaN3 and filtered through a 0.45 µm filter to prepare an antibody library.

[0142] 2-1-2. Preparation of C5 Immunized Rabbit Fab Library

[0143] 25 µg of human C5 protein (Acrobiosystems) and 50 µg of the MG1 domain of human C5 protein were mixed with a complete freund adjuvant (Sigma, Cat# F5881) or an incomplete freund adjuvant (Sigma, Cat# F5506) and injected subcutaneously into rabbits at 2-week intervals. Spleen and bone marrow were obtained from immunized rabbits, and total RNA was extracted from them using the Rneasy mini kit (Qiagen, Cat# 74104). First-strand cDNA was synthesized using oligo-dT primers and the SuperScript™ III First-Strand Synthesis System (Invitrogen, Cat# 18080-051). An antigen-binding fragment (Fab) library was constructed using primers specific to the heavy and light chain variable regions of rabbit immunoglobulin (Table 6).

[0144] Primer Information for Vκ, Vλ, and VH in Rabbit Antigen-Binding Segment Library Type Name Sequence Number Sequence (5' - 3') VH 5' Sense Primers RHyVH182GCT GCC CAA CCA GCC ATG GCC CAG TCG GTG GAG GAG TCC RGG RHyVH283GCT GCC CAA CCA GCC ATG GCC CAG TCG GTG AAG GAG TCC GAG RHyVH384GCT GCC CAA CCA GCC ATG GCC CAG TCG YTG GAG GAG TCC GGG RHyVH485GCT GCC CAA CCA GCC ATG GCC CAG SAG CAG CTG RTG GAG TCC GG VH 3' Reverse Primers RHyIgGCH1-B(reverse)86CGA TGG GCC CTT GGT GGA GGC TGA RGA GAY GGT GAC CAG GGT GCCVλ 5' Sense Primer RSCλ187GGG CCC AGG CGG CCG AGC TCG TGC TGA CTC AGT CGC CCT CVλ 3' Reverse Primer RHybL-B88AGA TCC TGC AGC CAC AGT TCG GCC TGT GAC GGT CAG CTG GGT CCCVκ 5' Sense Primer RSCVK189GGG CCC AGG CGG CCG AGC TCG TGM TGA CCC AGA CTC CARSCVK290GGG CCC AGG CGG CCG AGC TCG ATM TGA CCC AGA CTC CARSCVK391GGG CCC AGG CGG CCG AGC TCG TGA TGA CCC AGA CTG AAVκ 3' Reverse Primer RHybK1-B92AGA TGG TGC AGC CAC AGT TCG TTT GAT TTC CAC ATT GGT GCCRHybK2-B93AGA TGG TGC AGC CAC AGT TCG TAG GAT CTC CAG CTC GGT CCCRHybK3-B94AGA TGG TGC AGC CAC AGT TCG TTT GAC SAC CAC CTCGGT CCC Primers for amplification of the human CHI chain from a cloned human Fab HIgGCH1-F(sense) 95GCC TCC ACC AAG GGC CCA TCG GTCdpseq(reverse) 96AGA AGC GTA GTC CGG AAC GTC Primers for amplification of the human Ck region and the pelB leader sequence from a cloned human Fab HKC-F(sense) 97CGA ACT GTG GCT GCA CCA TCT GTCLead-B(reverse) 98GGC CAT GGC TGG TTG GGC AGC Primers for PCR assembly of chicken VH sequence with the human CHI PCR product leadVH(sense) 99GCT GCC CAA CCA GCC ATG GCCdpseq(reverse)100AGA AGC GTA GTC CGG AAC GTC Primers for PCR assembly of Chicken VL sequence with the human Ck PCR product RSC-F(sense)101GAG GAG GAG GAG GAG GAG GCG GGG CCC AGG CGG CCG AGC TCLead-B(reverse)102GGC CAT GGC TGG TTG GGC AGC Primers for PCR assembly of chimeric light chain sequences withchimeric heavy chain sequences)RSC-F(sense)103GAG GAG GAG GAG GAG GAG GCG GGG CCC AGG CGG CCG AGC TCdp-EX(reverse)104GAG GAG GAG GAG GAG GAG AGA AGC GTA GTC CGG AAC GTC

[0145] For the first PCR, to clone the light chain variable region (VL) and heavy chain variable region (VH), 1 µl of cDNA, 25 pmol of each primer, 10 µl of 5X reaction buffer, 1 µl of 10 mM dNTPs, and 1 µl of Taq DNA polymerase were mixed with water to make a final volume of 50 µl. To clone the light chain constant region (CL) and heavy chain first constant region (CHI), 100 ng of pComb3XTT vector, 25 pmol of each primer, 10 µl of 5X reaction buffer, 1 µl of 10 mM dNTPs, and 1 µl of Taq DNA polymerase were mixed with water to make a final volume of 50 µl. The PCR reactions were performed under the following conditions.

[0146] First PCR Condition Step Temperature (°C) Time (sec) Number of Cycles Pre-incubation 98 30 1 Denaturation 98 10 30 Annealing 60 10 Elongation 72 30 Final elongation 72 30 01

[0147] Approximately 350 bp amplified fragments were loaded onto a 1% agarose gel and purified using a Gel & PCR clean-up kit (Cosmojintech, cat# CMA0112). In the second PCR, the first VL and CL, and VH and CHI, were randomly combined by overlap extension PCR. Each PCR reaction was performed with a 50 µl mixture containing 100 ng of each product, 25 pmol of each primer, 10 µl of 5X reaction buffer, 1 µl of 10 mM dNTP, and 1 µl of Taq DNA polymerase. The PCR reactions were performed under the following conditions.

[0148] Second PCR Condition Step Temperature (°C) Time (sec) Number of Cycles Pre-incubation 98 30 1 Denaturation 98 10 15 Annealing 60 10 Elongation 72 30 Final elongation 72 30 01

[0149] Approximately 750 bp amplified fragments were loaded onto a 1% agarose gel and run, after which they were purified using a Gel & PCR clean-up kit (Cosmojintech, cat# CMA0112). In the third PCR, the overlap extension PCR products of VL and CL and the overlap extension PCR products of VH and CHI were randomly combined by overlap extension PCR. Each PCR reaction was performed with a 50 µl mixture containing 100 ng of each product, 25 pmol of each primer, 10 µl of 5X reaction buffer, 1 µl of 10 mM dNTPs, and 1 µl of Taq DNA polymerase. The PCR reactions were performed under the following conditions.

[0150] Third PCR Condition Step Temperature (°C) Time (sec) Number of Cycles Pre-incubation 98 30 1 Denaturation 98 10 10 Annealing 60 10 Elongation 72 45 Final elongation 72 30 01

[0151] Approximately 1500 bp amplified fragments were loaded onto a 1% agarose gel and run, after which they were purified using a Gel & PCR clean-up kit (Cosmogenetec, cat# CMA0112). The Fab fragments and pComb3XSS vector were cleaved with Sfi I restriction enzyme (NEB, Cat# R0123L) at 50°C for 12 hours. 1400 ng of Sfi I-cleaved Fab and 1400 ng of pComb3X vector were mixed and treated with T4 DNA ligase at 16°C for at least 12 hours to induce ligation, followed by ethanol precipitation. The ligated libraries were transformed into E. coli ER2738 by electroporation. Cells were mixed into 3 mL of Super Broth (SB) medium at 37 °C and cultured for 1 hour with stirring at 250 rpm; subsequently, 10 mL of SB medium and 3 µL of 100 mg / mL Cabenicillin were added to the culture medium. The library size was determined by the number of transformed colonies formed after plating an appropriately diluted culture onto Luria broth (LB) plates containing 100 µg / mL Cabenicillin and incubating overnight. After 1 hour of incubation, 4.5 µL of 100 mg / mL Cabenicillin was added, and the cells were cultured for an additional 1 hour. 1 mL of VCSM13 helper phage (> 10 11183 ml of SB medium and 92.5 µl of 100 mg / ml cabbageillin were added, and the mixture was incubated at 37°C for 2 hours with stirring at 250 rpm. Afterward, 280 µl of 50 mg / ml kanamycin was added and the mixture was stirred overnight at 30°C at 250 rpm. The next day, the culture medium was centrifuged at 4°C at 7000 rpm for 10 minutes to extract phagemid DNA from the bacterial pellet. The supernatant was transferred to a clean centrifuge bottle, and then 50 ml of 5XPEG / NaCl solution was added, or 8 g of polyethylene glycol-8000 (PEG-8000, Sigma, Cat# P2139) and 6 g of sodium chloride (Sigma, cat# S9888) were added to dissolve it, and the mixture was stored on ice for 30 minutes to 1 hour. Next, the supernatant was centrifuged at 4°C and 8000 rpm for 30 minutes to 1 hour, and after removing the supernatant, the phage pellet was suspended in PBS containing 1% BSA and 0.02% NaN3 and filtered through a 0.45 µm filter to prepare an antibody library.

[0152] 2-1-3. Preparation of a C5 Immunized Rabbit ScFv Library

[0153] 50 µg of human C5 protein (Acrobiosystems) was mixed with a complete freund adjuvant (Sigma, Cat# F5881) or an incomplete freund adjuvant (Sigma, Cat# F5506) and injected subcutaneously into rabbits at 2-week intervals. Spleen and bone marrow were obtained from immunized rabbits, and total RNA was extracted from them using the Rneasy mini kit (Qiagen, Cat# 74104). First-strand cDNA was synthesized using oligo-dT primers and the SuperScript™ III First-Strand Synthesis System (Invitrogen, Cat# 18080-051). A library of single-chain variable fragment (scFv) antibody fragments was constructed using primers specific to the heavy and light chain variable regions of rabbit immunoglobulins (Table 10).

[0154] Primer Information for Vκ, Vλ, and VH in Rabbit Antigen-Binding Segment Library Type Name Sequence Number Sequence (5' - 3') VH 5' Sense Primers RSC VH1105 GGT GGT TCC TCT AGA TCT TCC CAG TCG GTG GAG GAG TCC RGGR SCVH2106 GGT GGT TCC TCT AGA TCT TCC CAG TCG GTG AAG GAG TCC GAGR SCVH3107 GGT GGT TCC TCT AGA TCT TCC CAG TCG YTG GAG GAG TCC GGGR SCVH4108 GGT GGT TCC TCT AGA TCT TCC CAG SAG CAG CTG RTG GAG TCC GGVH 3' Reverse Primers RSCG-B109 CCT GGC CGG CCT GGC CAC TAG TGA CTG AYG GAG CCT TAG GTT GCC CVλ 5' Sense Primer RJλo-B110GGA AGA TCT AGA GGA ACC ACC GCC TGT GAC GGT CAG CTG GGT CCCVλ 3' Reverse Primer RJλo-BL111GGA AGA TCT AGA GGA ACC ACC CCC ACC ACC GCC CGA GCC ACC GCC ACC AGA GGA GCC TGT GAC GGT CAG CTG GGT CCCVκ 5' Sense Primer RSCVK1112GGG CCC AGG CGG CCG AGC TCG TGM TGA CCC AGA CTC CARSCVK2113GGG CCC AGG CGG CCG AGC TCG ATM TGA CCC AGA CTC CARSCVK3114GGG CCC AGG CGG CCG AGC TCG TGA TGA CCC AGA CTG AAVκ 3' Reverse Primer,long linker (linker amino acid sequence : SSGGGGSGGGGGGSSRSS (Sequence No.: 462))RKB9J1o-BL115GGA AGA TCT AGA GGA ACC ACC ACC CCC ACC ACC GCC CGA GCC ACC GCC ACC AGA GGA TTT GAT TTC CAC ATT GGT GCCRKB9Jo-BL116GGA AGA TCT AGA GGA ACC ACC ACC CCC ACC ACC GCC CGA GCC ACC GCC ACC AGA GGA TAG GAT CTC CAG CTC GGT CCCRKB42Jo-BL117GGA AGA TCT AGA GGA ACC ACC CCC ACC ACC GCC CGA GCC ACC GCC ACC AGA GGA TTT GAC SAC CAC CTC GGT CCC Primers for PCR assembly of chimeric light chain sequences with chimeric heavy chain sequences chimeric heavy chain sequences)RSC-F(sense)118GAG GAG GAG GAG GAG GAG GCG GGG CCC AGG CGG CCG AGC TCRSC-B(reverse)119GAG GAG GAG GAG GAG GAG CCT GGC CGG CCT GGC CAC TAG TG,

[0155] To clone the light chain variable region (VL) and heavy chain variable region (VH) in the first PCR, 1 µl of cDNA, 25 pmol of each primer, 10 µl of 5X reaction buffer, 1 µl of 10 mM dNTPs, and 1 µl of Taq DNA polymerase were mixed with water to make a final volume of 50 µl. The PCR reaction was performed under the following conditions.

[0156] First PCR Condition Step Temperature (°C) Time (sec) Number of Cycles Pre-incubation 98 30 1 Denaturation 98 10 30 Annealing 60 10 Elongation 72 30 Final elongation 72 30 01

[0157] Approximately 350 bp amplified fragments were loaded onto a 1% agarose gel and purified using a Gel & PCR clean-up kit (Cosmojintech, cat# CMA0112). In the second PCR, the VL PCR product and the VH PCR product were randomly combined by overlap extension PCR. Each PCR reaction was performed with a 50 µl mixture containing 100 ng of each product, 25 pmol of each primer, 10 µl of 5X reaction buffer, 1 µl of 10 mM dNTP, and 1 µl of Taq DNA polymerase. The PCR reactions were performed under the following conditions.

[0158] Second PCR Condition Step Temperature (°C) Time (sec) Number of Cycles Pre-incubation 98 30 1 Denaturation 98 10 20 Annealing 60 10 Elongation 72 45 Final elongation 72 30 01

[0159] Approximately 750 bp amplified fragments were loaded and run on a 1% agarose gel, after which they were purified using a Gel & PCR clean-up kit (Cosmojin Tech, cat# CMA0112).

[0160] scFv fragments and pComb3XSS vector were cleaved with Sfi I restriction enzyme (NEB, Cat# R0123L) at 50 °C for 12 hours. 1400 ng of Sfi I-cleaved scFv and 1400 ng of pComb3X vector were mixed and treated with T4 DNA ligase at 16 °C for more than 12 hours to induce ligation, followed by ethanol precipitation. The ligated library was transformed into E. coli ER2738 by electroporation. Cells were mixed into 3 mL of Super Broth (SB) medium at 37 °C and cultured for 1 hour with stirring at 250 rpm; subsequently, 10 mL of SB medium and 3 µL of 100 mg / mL cabbageillin were added to the culture medium. Library size was determined by the number of transformed colonies formed after inoculating appropriately diluted cultures onto Luria broth (LB) plates containing 100 µg / ml cabbageillin and incubating overnight. After 1 hour of incubation, 4.5 µl of 100 mg / ml cabbageillin was added, and the culture was incubated for an additional 1 hour. 1 ml of VCSM13 helper phage (> 10 11183 ml of SB medium and 92.5 µl of 100 mg / ml cabbageillin were added, and the mixture was incubated at 37°C for 2 hours with stirring at 250 rpm. Afterward, 280 µl of 50 mg / ml kanamycin was added and the mixture was stirred overnight at 37°C at 250 rpm. The next day, the culture medium was centrifuged at 4°C at 7000 rpm for 10 minutes to extract phagemid DNA from the bacterial pellet. The supernatant was transferred to a clean centrifuge bottle, and then 50 ml of 5XPEG / NaCl solution was added, or 8 g of polyethylene glycol-8000 (PEG-8000, Sigma, Cat# P2139) and 6 g of sodium chloride (Sigma, cat# S9888) were added to dissolve it, and the mixture was stored on ice for 30 minutes to 1 hour. Next, the supernatant was centrifuged at 4°C and 8000 rpm for 30 minutes to 1 hour, and after removing the supernatant, the phage pellet was suspended in PBS containing 1% BSA and 0.02% NaN3 and filtered through a 0.45 µm filter to prepare an antibody library.

[0161] 2-2. Bio-panning

[0162] 1 mg of magnetic bead (Dynabeads M270-Epoxy, Invitrogen, Cat# 14301) was coated with 30 µg of recombinant human complement C5 (Acrobiosystems, Cat# CO5-H52Ha) or human complement C5 extracted from human serum (MERCK, Cat# 204888), suspended in 3% BSA / PBS, and stored at 4 ℃. After washing the human complement C5-coated beads, they were incubated with 1 ml of phage-displayed Fab library at room temperature for 2 hours. The phage treated on the beads was appropriately diluted in SB medium and used to infect E. coli ER2738. The samples were then plated onto LB plates containing 100 µg / ml of cabbageillin and incubated overnight at 37 ℃ to induce colony formation. The number of formed colonies was determined and used to calculate the input titer.

[0163] Phages that were not bound to the beads were removed by washing with 0.05% tween-20 / PBS, and phages bound to the beads were eluted with 500 µl of 0.1 M glycine-HCl (pH 2.2) and neutralized by mixing with 50 µl of 1 M Tris (pH 9.0). The eluted phages were used to infect E. coli ER2738, and a portion was appropriately diluted in SB medium and plated onto LB plates containing 100 µg / ml of cabbageillin. Colony formation was induced by incubating overnight at 37°C, and the number of formed colonies was counted and used to calculate the output titer. The remaining infected E. coli ER2738 were treated with SB medium, cabbageillin, VCSM13 helper phage (Agilent technologies, Cat# 200251), and kanamycin, and then cultured overnight at 37°C or 30°C at 250 rpm. The next day, 100 ml of the culture medium was centrifuged at 4°C at 4000 rpm for 15 minutes, and the supernatant was transferred to a clean centrifuge bottle. Then, 25 ml of 5XPEG / NaCl solution was added, or 4 g of polyethylene glycol-8000 (PEG-8000, Sigma, Cat# P2139) and 3 g of sodium chloride (Sigma, Cat# S9888) were added and dissolved, and the mixture was stored on ice for 30 minutes. Next, the supernatant was centrifuged at 15,000 g at 4 ℃ for 15 minutes, and after removing the supernatant, the phage pellet was suspended in TBS (Tris-Buffered Saline) containing 1% BSA and 0.02% NaN3 and filtered through a 0.45 µm filter to prepare an antibody library to be used for the next panning cycle.

[0164] 2-3. Selection of Anti-C5 Binder by ELISA

[0165] 2-3-1. ELISA for screening anti-C5 binders from chicken or rabbit Fab libraries

[0166] Enzyme immunoassay was performed to select clones that bind to human complement C5 from biopanning. Recombinant human complement C5 was diluted to 1 μg / ml in PBS, placed in a 96-well plate, coated overnight at 4°C, and blocked with Superblock blocking buffer in PBS (Thermo, Cat# 37515) or 3% BSA / PBS. To measure the output phage titer, E. coli ER2738 colonies infected with the prepared phage were cultured overnight at 37°C or 30°C at 250 rpm, and the supernatant separated by centrifugation was mixed in equal parts with 6% BSA / PBS and placed in a 96-well plate coated with human complement C5, and cultured at room temperature for 1 hour. After culture, the colonies were washed with 0.05% tween-20 / PBS, and then HRP-conjugated anti-human Fab antibody (Sigma, Cat# A0293) was diluted in 3% BSA / PBS and cultured at room temperature for 1 hour. After the culture was finished, washing with 0.05% tween-20 / PBS was performed, TMB (3,3,5,5-Tetramethylbenzidine liquid substrate, Sigma, Cat# T0440) was added and incubated at room temperature for up to 30 minutes, then reaction stop solution (Invitrogen, Cat# SS04) was added and absorbance was measured at 450 nm. Based on the measurement results, clones showing an absorbance 2.5 times higher than that of wells not coated with human complement C5 were selected.

[0167] Phage DNA was extracted from E. coli ER2738 cells that had been cultured overnight after being infected with phages from selected clones using the NICSROprep plasmid DNA miniprep kit (Bionics, Cat# BNROP-0068), and genetic analysis was performed. Through this, sequence information of anti-human complement C5 clones was obtained, and the CDR sequences of each clone were derived through analysis.

[0168] The CDR sequence (Table 13), amino acid sequences of the heavy chain and light chain variable regions (Table 14), and DNA sequences (Table 15) of the anti-human complement C5 clone are summarized in the table below.

[0169]

[0170]

[0171]

[0172] 2-3-2. ELISA for screening anti-C5 binders from rabbit scFv library

[0173] Enzyme immunoassay was performed to select clones binding to human complement C5 from biopanning. Recombinant human complement C5 was diluted to 1 μg / ml in PBS, placed in a 96-well plate, coated overnight at 4°C, and blocked with 3% BSA / PBS. To measure the output phage titer, E. coli ER2738 colonies infected with the prepared phage were cultured overnight at 30°C and 250 rpm. The supernatant separated by centrifugation was mixed in equal parts with 6% BSA / PBS, placed in a 96-well plate coated with human complement C5, and incubated at room temperature for 1 hour. After incubation, the colonies were washed with 0.05% tween-20 / PBS, and an HRP-conjugated anti-HA antibody (Roche, 12013819001) was diluted in 3% BSA / PBS and incubated at room temperature for 1 hour. After the culture was finished, washing with 0.05% tween-20 / PBS was performed, TMB (3,3,5,5-Tetramethylbenzidine liquid substrate, Sigma, Cat# T0440) was added and incubated at room temperature for up to 30 minutes, then reaction stop solution (Invitrogen, Cat# SS04) was added and absorbance was measured at 450 nm. For clones showing an absorbance more than 10 times higher than that of wells not coated with human complement C5, phage DNA was extracted from E. coli ER2738 cells infected with phages and cultured overnight using the NICSROprep plasmid DNA miniprep kit (Bionics, Cat# BNROP-0068), and genetic information of the heavy and light chain variable regions of the anti-human complement C5 clones was decoded through genetic analysis.

[0174]

[0175]

[0176]

[0177] 2-4. Production of IgG2 / 4 form anti-C5 antibody

[0178] 2-4-1. Cloning of Full-Length IgG Vectors

[0179] 2-4-1-1. Cloning of the full-length IgG vector of the C5 binder found in the chicken Fab library

[0180] To convert Fab-type clones present in phage DNA that specifically bind to human complement C5 into IgG forms, the light chain variable region (VL) and heavy chain variable region (VH) were cloned from the pComb3X vector. To clone VL and VH, 2 µl of phage DNA, 25 pmol of each primer, 10 µl of 5X reaction buffer, 1 µl of 10 mM dNTP, and 1 µl of Taq DNA polymerase were mixed with water to make a final volume of 50 µl.

[0181] Primer sequence for cloning VL Cloning sequence number sequence (5'-3') Forward primer, CVL-F45CTACACGGGTGCTTAGCCTGACTCAGCCGTCCTCGGT Reverse primer, CVL-R46GAGGGCGCGGCCTTAGGTTGTCCTAGGACGGTCAGGGTTGTCCCVH Cloning sequence number sequence (5'-3') Forward primer, CVH-F47CTACACGGGTGCTTAGCGCCGTGACGTTGGACGAGTC Reverse primer, CVH-R48AGGGGAAATACACTGGGCCCTTTGGTGCTAGCGGAGGAGACGATGACTTCGGTC

[0182] The PCR reaction was performed under the following conditions.

[0183] PCR Condition Step Temperature (°C) Time (sec) Number of Cycles Pre-incubation 98 30 1 Denaturation 98 10 30 Annealing 60 10 Elongation 72 30 Final elongation 72 30 01

[0184] Approximately 350 bp amplified fragments were loaded and run on a 1% agarose gel, after which they were purified using a Gel & PCR clean-up kit (Cosmojintech, cat# CMA0112). For the construction of light chain expression vectors, VL PCR products were cloned into a pcDNA3.4 animal cell expression vector containing the human light chain constant region (CL), and for the construction of heavy chain expression vectors, VH PCR products were cloned into a pcDNA3.4 animal cell expression vector containing the human heavy chain constant first region (CHI), the IgG2 hinge, the heavy chain constant second region (CH2), and the heavy chain constant third region (CH3) of IgG4 using an in-fusion HD cloning kit (Clontech, cat# 639648).

[0185] 제2 길항물질, 항-C5 IgG 항체의 경쇄 및 중쇄 아미노산 서열클론명서열번호서열Ch1-78 LC49LTQPSSVSANPGETVKITCSGDSSWYGWYQQKSPGSAPVTVIYQNTKRPSDIPSRFSGSKSGSTATLTITGVRAEDEAVYFCGGYDSSSNAGTFGAGTTLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSCh1-78 HC50AVTLDESGGGLQTPGGGLSLVCKASGFDFSSYDMAWVRQAPGKGLEWVAGISSSGRYTYYGAAVKGRATISRDNGQSIVRLQLNNLRAEDTGTYYCAKSADTACGSTAAGCIDAWGHGTEVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKCh2-12 LC51LTQPSSVSANPGETVKITCSGGNSDYGWFQQKAPGSTPVTLIYDSTNRPSDIPSRFSGSKSGSTHTLTITGVQVDDEAVYYCASYDSSTDSFMFGAGTTLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSCh2-12HC52AVTLDESGGGLQTPGGALSLVCKGSGFTLSSYGMNWVRQAPGKGLEWVAAINAAGSSTRYGAAVKGRATISRDNGQSTLRLQLNNLRAEDTATYYCAKSSYECVYCWYGDTGDIDAWGHGTEVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKCh5-13 LC53LTQPSSVSANPGETVKITCSGGSSWYGYGWYQQKSPGSAPVTLIYSNDKRPSNIPSRFSGSTSGSTGTLTITGVQAEDEAVYYCGSEDSTNSNPGIFGAGTTLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSCh5-13HC54AVTLDESGGGLQTPGGALSLVCKASGFTFRTYGMYWVRQAPGKGLEFVAGIIDDGSDTFYGPAVKGRATISRDNGQSTVRLQLNNLRAEDTGTYYCAKGGYGGIWSGDIDAWGHGTEVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKCh11-01 LC55LTQPSSVSANLGGTVEITCSGGSGKYYGWYQQKSPGSAPVTLIYDNNKRPSDIPSRFSGALSGSTATLTITGVRAEDEAVYFCGSWDSDTDAGIFGAGTTLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSCh11-01HC56AVTLDESGGGLQTPGGALSLVCKGSGFTFSSHGMFWVRQTPGKGLEWVAGIEDTGSDPHYGAAVKGRATISRDNGQSTLRLQLNNLRAEDTGTYYCARSGYGGWHVGHIDAWGHGTEVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKCh11-09 LC57LTQPSSVSANLGGTVKITCSGDSNHYGWYQQKAPASAPVTLIYDNNKRPSDIPSRFSGSKSDSTHTLTITGVQAEDEAVYFCGAWDSSTDAGIFGAGTTLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSCh11-09HC58AVTLDESGGGLQTPGGALSLVCKASGFSFSSHGMFWVRQTPGKGLEWVAGIEDTGSDPHYGAAVKGRATISRDNGQSTVRLQLNNLRAEDTGTYYCAKSAYGGWHVGHI DAWGHGTEVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVER KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0186] (Bold text indicates variable regions (heavy chain variable regions and light chain variable regions), and the rest indicate invariant regions)

[0187]

[0188] (Bold text indicates variable regions (heavy chain variable regions and light chain variable regions), and the rest indicate invariant regions)

[0189] 2-4-1-2. Cloning of the full-length IgG vector of the C5 binder found in the rabbit Fab library

[0190] To convert Fab-type clones present in phage DNA that specifically bind to human complement C5 into IgG forms, the light chain variable region (VL) and heavy chain variable region (VH) were cloned from the pComb3X vector. To clone VL and VH, 2 µl of phage DNA, 25 pmol of each primer, 10 µl of 5X reaction buffer, 1 µl of 10 mM dNTP, and 1 µl of Taq DNA polymerase were mixed with water to make a final volume of 50 µl.

[0191] Primer sequence for cloning VL Cloning sequence number sequence (5'-3') Forward primer, RVK1A-F447CTACACGGGTGCTTAGCGAGCTCGTGATGACCCAGACTCCA Reverse primer, RVK1-R448GAGCGGCCACCGTACGTTTGATTTCCACATTGGTGCC Reverse primer, RVK2-R449GAGCGGCCACCGTACGTAGGATCTCCAGCTCGGTCCCVH Cloning sequence number sequence (5'-3') Forward primer, RVH4GA-F450CTACACGGGTGCTTAGCCAGGAGCAGCTGATGGAGTCCGG Reverse primer, RVH1A-R451AGGGGAAATACACTGGGCCCTTTGGTGCTAGCTGAAGAGACGGTGACCAGGGTGCC Forward primer, RVH4CA-F452CTACACGGGTGCTTAGCCAGCAGCAGCTGATGGAGTCCGGReverse Primer, RVH2A-R453AGGGGAAATACACTGGGCCCTTTGGTGCTAGCTGAAGAGATGGTGACCAGGGTGCC

[0192] The PCR reaction was performed under the following conditions.

[0193] PCR Condition Step Temperature (°C) Time (sec) Number of Cycles Pre-incubation 98 30 1 Denaturation 98 10 30 Annealing 60 10 Elongation 72 30 Final elongation 72 30 01

[0194] Approximately 350 bp amplified fragments were loaded and run on a 1% agarose gel, after which they were purified using a Gel & PCR clean-up kit (Cosmojintech, cat# CMA0112). For the construction of light chain expression vectors, VL PCR products were cloned into a pcDNA3.4 animal cell expression vector containing the human light chain constant region (CL), and for the construction of heavy chain expression vectors, VH PCR products were cloned into a pcDNA3.4 animal cell expression vector containing the human heavy chain constant first region (CHI), the IgG2 hinge, the heavy chain constant second region (CH2), and the heavy chain constant third region (CH3) of IgG4 using an in-fusion HD cloning kit (Clontech, cat# 639648).

[0195]

[0196] (Bold text indicates variable regions (heavy chain variable regions and light chain variable regions), and the rest indicate invariant regions)

[0197]

[0198] (Bold text indicates variable regions (heavy chain variable regions and light chain variable regions), and the rest indicate invariant regions)

[0199] 2-4-2. Transfection and Protein Purification

[0200] A transient overexpression system utilizing the transfection of an antibody expression vector into the Expi293F cell line was used to produce IgG2 / 4-type anti-human complement C5 binding antibodies. On the day before transfection, Expi293F (Gibco, Cat# A14527) cells were placed in fresh Expi293 medium (Gibco, Cat# A1435102) at a rate of 3 x 10⁶ 6 Prepared at 1 / mL and cultured overnight at 8% CO2, 37°C, and 120 rpm. The next day, checked whether the ratio of living cells to the total number of cells was 95% or higher, and whether the number of living cells was 4.5 x 10⁶ 6 Check if it is at least / mL, add fresh Expi293 medium, and 3X10 6 It was prepared at a concentration of / mL. For IgG expression, a light chain expression vector and a heavy chain expression vector were prepared at a ratio of 2:1 at 1.25 μg / mL per 1 mL of Expi293F and incubated at room temperature for 5 minutes. Meanwhile, PEI (polyethylenimine, Polysciences, Cat# 24765) was prepared in Expi293 medium at 3.75 μg / mL per 1 mL of Expi293F and incubated at room temperature for 5 minutes. Next, the prepared expression vector solution and PEI solution were mixed and incubated at room temperature for 5 minutes; afterward, the mixture was added to the prepared Expi293F and incubated for 5 days under 8% CO2 at 37°C and 120 rpm. After the incubation was completed, the supernatant was collected, and the IgG antibody protein was isolated and purified using protein A affinity chromatography.

[0201] 2-5. Measurement of Complement C5 Binding Ability of Second Antagonist, Anti-C5 Antibody

[0202] Enzyme immunoassay was performed to analyze the binding affinity of anti-human complement C5 antibodies, which are second antagonists prepared in IgG form. Recombinant human complement C5 was diluted to 1 µg / ml in PBS, placed in a 96-well plate, coated overnight at 4°C, and blocked with Superblock blocking buffer in PBS (Thermo, Cat# 37515). Next, anti-human complement C5 antibodies were diluted in 3% BSA / PBS in increments of 1 / 3 or 1 / 4 starting from 1 µg / ml, placed in the 96-well plate coated with human complement C5, and incubated at room temperature for 1 hour. After incubation, the plates were washed with 0.05% tween-20 / PBS, and then HRP-conjugated anti-human IgG antibodies (abcam, Cat# 98624) were diluted in 3% BSA / PBS and incubated at room temperature for 1 hour. After the culture was finished, washing with 0.05% tween-20 / PBS was performed, TMB (3,3,5,5-Tetramethylbenzidine liquid substrate, Sigma, Cat# T0440) was added and the mixture was incubated at room temperature for up to 30 minutes, followed by the addition of a reaction stop solution (Invitrogen, Cat# SS04) and the measurement of absorbance at 450 nm. As a result, it was confirmed that all of the second antagonists, the anti-C5 antibodies, exhibited binding affinity to human complement C5 (Figs. 2 to 5).

[0203] Example 3. Analysis of the inhibitory effect of a complement C5 binding antagonist combination on complement C5 activity

[0204] 3-1. Analysis of Hemolytic Inhibitory Activity of Complement C5 Antagonist Combinations Using Alternative Pathway-Mediated Hemolytic Analysis

[0205] 3-1-1 Analysis of Hemolytic Inhibitory Effect of C5 Binder Combinations Found in Chicken Fab Library

[0206] Rabbit red blood cells (Innovative research, Cat# IRBRBC10ML) were 5 x 10⁶ in GVBMG (gelatin veronal buffer with Mg, EGTA, Novatein biosciences, Cat# NIBB-320X) solution. 8 The sample was prepared by suspension at 1 / mL, and human serum with live complement activity (Innovative research, Cat# ICSER100ML) was prepared at a 75% concentration in GVBMG solution. The anti-human complement C5 antibody was prepared alone to a final concentration of 1,125 µg / mL (7,500 nM), and when used in combination with antibody A, it was mixed in a 1:1 molar ratio in GVBMG solution to achieve a maximum concentration of 1,125 µg / mL (7,500 nM). A total of six concentrations were prepared by diluting each maximum concentration by 1 / 3 or 1 / 2 increments. Next, 50 µl of each sample was mixed into a 96-well plate and incubated at 37°C at 100 rpm for 2 hours. As a positive control, rabbit erythrocytes were placed in GVBMG solution at a concentration of 5 x 10⁶ 8 Samples were prepared consisting of a suspension at 1 / mL, human serum with live complement activity prepared at a 75% concentration in GVBMG solution, and 50 µl of GVBMG solution mixed with each; as a negative control, rabbit erythrocytes were placed in GVBMG solution at a concentration of 5 x 10⁶ 8 Samples were prepared by suspending human serum at 75% concentration in GVBMG solution after removing complement activity via heat treatment, and mixing 50 µl of GVBMG solution with each sample, and incubated at 37°C and 100 rpm for 2 hours. After the reaction was complete, the samples were centrifuged at 4°C and 4000 rpm for 10 minutes, the supernatant was collected, transferred to a new 96-well plate, and the absorbance was measured at 414 nm. The degree of hemolysis (%) was calculated using Equation 1 below.

[0207] [Equation 1]

[0208]

[0209] As a result, it was confirmed that Ch1-78, Ch2-12, Ch5-13, Ch11-01, and Ch11-09 clones alone had lower hemolytic inhibition ability than antibody A, but when treated in combination with antibody A, their hemolytic inhibition ability was higher than when treated with antibody A alone (Figs. 6 to 10).

[0210] 3-1-2. Analysis of Hemolytic Inhibitory Activity of C5 Binder Combinations Found in Rabbit Fab Library

[0211] Rabbit red blood cells (Innovative research, Cat# IRBRBC 10ML) were 6.67 x 10⁶ in GVBMG (gelatin veronal buffer with Mg, EGTA, Novatein biosciences, Cat# NIBB-320X) solution. 8 The samples were prepared by suspension at µg / mL, and human serum with live complement activity (Innovative research, Cat# ICSER100ML) was prepared. The anti-human complement C5 antibody was prepared alone to a final concentration of 750 µg / mL (5000 nM); when used in combination with antibody A, it was mixed in a 1:1 molar ratio in GVBMG solution to achieve a maximum concentration of 750 µg / mL (5000 nM); and samples were prepared at 1 / 3 of each maximum concentration or a total of 5 concentrations. Next, for each sample, 50 µl of sample, 100 µl of human serum, and rabbit erythrocytes were placed in a 96-well plate at a concentration of 6.67 x 10⁶ in GVBMG solution. 8 50 µl of the suspension at / mL was mixed in a 1:2:1 ratio to achieve 50% human serum conditions, and then incubated at 37°C at 100 rpm for 2 hours. As a positive control, rabbit erythrocytes were placed in GVBMG solution at a concentration of 6.67 x 10⁶ 8 Samples were prepared by suspending the erythrocytes at 1 / mL, mixing 100 µL of human serum with live complement activity with 50 µL of GVBMG solution, and as a negative control, rabbit erythrocytes were placed in GVBMG solution at a concentration of 6.67 x 10⁶ 8Samples were prepared by mixing 100 µl of human serum suspended at 1 / mL, heat-treated to remove complement activity, and 50 µl of GVBMG solution, respectively, and incubated at 37°C at 100 rpm for 2 hours. After the reaction was complete, the supernatant was collected after centrifugation at 4°C at 4000 rpm for 10 minutes, transferred to a new 96-well plate, and the absorbance was measured at 414 nm. The degree of hemolysis (%) was calculated using Equation 1 below.

[0212] [Equation 1]

[0213]

[0214] As a result, it was confirmed that the R-C5 / MG1-008-30-#13 and R-13-SP#21 clones alone had lower hemolytic inhibition ability than antibody A, but when combined with antibody A, the hemolytic inhibition ability was higher than when antibody A was treated alone (Figs. 11 and 12).

[0215] 3-2. Preparation of a Competitor C5 Inhibitor for Hemolytic Inhibition Analysis

[0216] Eculilzumab from Alexion and Zilucoplan from UCB were purchased, while Crovalimab from Roche, Pozelimab from Regeneron, and Tesidolumab from Novartis were produced directly by obtaining amino acid sequence information.

[0217]

[0218] (Bold text indicates variable regions (heavy chain variable regions and light chain variable regions), and the rest indicate invariant regions)

[0219] 3-3. Analysis of Hemolytic Inhibitory Activity of Antibody A and Competitor C5 Inhibitor under Co-treatment Conditions Using Alternative Route-Mediated Hemolytic Assay

[0220] Rabbit red blood cells 6.67 x 10⁶ in GVBMG solution 8 The solution was prepared by suspension at 1 / mL, and human serum with active complement was thawed and stored on ice. The C5 inhibitor was prepared alone to a final concentration of 20 μM, and when used in combination with antibody A, it was mixed in a 1:1 molar ratio in GVBMG solution to achieve a total concentration of 20 μM. Next, 50 μL of rabbit erythrocytes, 100 μL of human serum, and 50 μL of C5 inhibitor were mixed in a 96-well plate and incubated at 37°C at 100 rpm for 2 hours. As a positive control, rabbit erythrocytes were placed in GVBMG solution at a concentration of 6.67 x 10⁶ 8 A sample was prepared by mixing 50 µl of a suspension at 1 / mL with 100 µl of human serum with live complement activity and 50 µl of GVBMG solution, and as a negative control, rabbit erythrocytes were placed in GVBMG solution at a concentration of 6.67 x 10⁶ 8 A sample was prepared by mixing 100 µl of human serum suspended at 1 / mL, from which complement activity had been removed by heat treatment, with 50 µl of GVBMG solution, and incubated at 37°C and 100 rpm for 2 hours. After the reaction was complete, the supernatant was collected after centrifugation at 4°C and 4000 rpm for 10 minutes, transferred to a new 96-well plate, and the absorbance was measured at 414 nm. The degree of hemolysis (%) was calculated using Equation 1 above.

[0221] As a result, it was confirmed that the inhibitory effect on complement activity increased when antibody A was combined with an inhibitor having a different binding site for human complement C5 compared to when antibody A was treated alone, and among them, the inhibitory effect on complement activity was best when antibody A was combined with an antibody that binds to MG1 (Table 28).

[0222] Analysis of hemolytic inhibition of antibodies AP(%) under combination therapy conditions with antibody A and competitor C5 inhibitors Antibody A (MG4)Crovalimab (MG1)Eculizumab (MG7)Pozelimab (MG6)Tesidolumab (α-chain)Zilucoplan (C5d)(-) IgG ((-) control) Antibody A 16.89±1.06 -0.25±0.06 2.07±0.55 2.78±0.52 4.61±0.56 01±0.49 24.47±1.44 Crovalimab 92.58±2.18 2.56±0.12 1.87±0.11 2.69±0.19 66±5.56 95.89±0.38 Eculizumab 83.92±0.054. 62±06.88±0.8667.89±9.3988.69±2.6Pozelimab68.42±1.945.99±0.6354.62±7.4781.04± 1.11Tesidolumab84.7±1.5661.64±3.882.63±1.67Zilucoplan101.04±0.87109.92±0.2(-) IgG112.38±0.92

[0223] 3-4. Epitope Mapping 3-4-1. Confirmation of antibody binding to C5 alpha and beta chains

[0224] Reduced (R, reduced form) and non-reduced (NR, non-reduced form) human complement C5 were each subjected to SDS-PAGE, and the proteins were transferred to a PVDF membrane (Invitrogen, Cat# IB24001). The membrane was blocked for 1 hour with 5% skim milk / 0.05% tween 20 / PBS, washed with 0.05% tween 20 / PBS, and then an anti-human complement C5 antibody was prepared at a concentration of 10 µg / ml in 5% skim milk / 0.05% tween 20 / PBS and incubated on the PVDF membrane for 1 hour. After washing with 0.05% tween 20 / PBS, HRP-conjugated anti-human IgG Fc antibody (abcam, Cat# ab98624) was mixed with 5% skim milk / 0.05% tween 20 / PBS and treated on a PVDF membrane for 1 hour. After washing with 0.05% tween 20 / PBS, development was performed using an ECL western blot substrate kit (Bio-rad, Cat# 1705061). Human complement C5 has a molecular weight of approximately 190 kDa under non-reducing conditions and splits into a beta chain of approximately 74 kDa and an alpha chain of approximately 114 kDa under reducing conditions.

[0225] As a result, as shown in Figure 13, it was confirmed that Ch1-78, Ch2-12, Ch5-13, Ch11-01, and Ch11-09 clones bind to the beta chain of human complement C5.

[0226] 3-4-2. Production of C5 Beta Chain MG1, MG2, MG3, MG4, MG5

[0227] Based on the 2008 paper (Nature Immunology, 2008, vol 9(7), p753-760), five domains constituting the human complement C5 beta chain were constructed. MG1, MG2, MG3, and MG5 were cloned into human Fc fusion protein expression vectors, expressed in Expi293F, and purified using a Protein A column, while MG4 was fused with a histidine tag and a C-tag (EPEA) at the carboxyl terminus, expressed in Expi293F, and purified using a C-tag affinity column. The amino acid sequences of each MG domain are shown in Table 29 below.

[0228] C5 beta chain MG1, MG2, MG3, MG4, and MG5 domain sequences Domain Sequence Number Amino Acid Sequence

[0229] 3-4-3. Identification of the binding site of the second antagonist, anti-C5 antibody, on the C5 beta chain

[0230] 3-4-3-1. Confirmation of epitopes using Western blotting

[0231] Each MG domain was SDS-PAGEd separately, and the proteins were transferred to a PVDF membrane (Invitrogen, Cat# IB24001). The proteins were then leached to 5% skim milk / PBS for 1 hour or NICSRO-Block. TM Block for 5 minutes with Fast 5-minute blocking solution (Bionics, cat# BNROW-0016), wash with 0.05% tween 20 / PBS, and then prepare anti-human complement C5 antibody at a concentration of 10 µg / ml in 5% skim milk / 0.05% tween 20 / PBS or 1% BSA / 0.05% tween 20 / PBS and incubate on a PVDF membrane for 1 hour. After washing with 0.05% tween 20 / PBS, HRP-conjugated anti-human Fab-specific antibody (Sigma, cat# A0293) was mixed with 5% skim milk / 0.05% tween 20 / PBS or 1% BSA / 0.05% tween 20 / PBS and treated on a PVDF membrane for 1 hour. After washing with 0.05% tween 20 / PBS, development was performed using an ECL western blot substrate kit (Bio-rad, Cat# 1705061).

[0232] As a result, as shown in Figure 14, it was confirmed that Ch1-78, Ch2-12, Ch5-13, Ch11-01, and Ch11-09 clones bind to the MG1 region of human complement C5.

[0233] 3-4-3-2. Epitope identification using ELISA

[0234] Epitope confirmation ELISA using a 3-4-3-2-1 IgG-type C5 binder

[0235] Enzyme-linked immunosorbent assay (ELISA) was performed to select antibodies binding to the MG1 and MG4 domains of human complement C5 from among clones binding to human complement C5. The MG1 and MG4 domains of recombinant human complement C5 were diluted to a concentration of 10 μg / mL in PBS, and human complement C5 isolated from human serum was diluted to a concentration of 1 μg / mL in PBS and added to 96-well microplates. The plates were incubated overnight at 4°C to coat the antigens. After coating, the plates were blocked at room temperature with a PBS solution containing 3% (w / v) bovine serum albumin (BSA). The anti-human complement C5 antibody was diluted to 1 μg / ml in 3% BSA / PBS and placed in a 96-well plate coated with human complement C5, and incubated at room temperature for 1 hour. After incubation, the plate was washed with 0.05% tween-20 / PBS, and then an HRP-conjugated anti-human IgG antibody (abcam, Cat# 98624) was diluted in 3% BSA / PBS and incubated at room temperature for 1 hour. After incubation, the plate was washed with 0.05% tween-20 / PBS, TMB (3,3,5,5-Tetramethylbenzidine liquid substrate, Sigma, Cat# T0440) was added and incubated at room temperature for up to 30 minutes. Then, a reaction stop solution (Invitrogen, Cat# SS04) was added, and the absorbance was measured at 450 nm.

[0236] As a result, it was confirmed that the above antibodies bind to human complement C5 and its MG1 domain, respectively (Fig. 15). As a negative control, an irrelevant antibody was used that does not specifically bind to either the MG1 or MG4 domain of human complement C5.

[0237] Epitope confirmation ELISA using a 3-4-3-2-2 scFv C5 binder

[0238] Enzyme-linked immunosorbent assay (ELISA) was performed to select antibody fragments (scFv) that bind to the MG1 and MG4 domains of human complement C5 from among clones that bind to C5. The MG1 and MG4 domains of recombinant human complement C5 were diluted to a concentration of 10 μg / mL in PBS, and human complement C5 isolated from human serum was diluted to a concentration of 1 μg / mL in PBS; these were then added to 96-well microplates. The plates were incubated overnight at 4°C to coat the antigens. After coating, the plates were blocked at room temperature with a PBS solution containing 3% (w / v) bovine serum albumin (BSA). The cell culture supernatant of scFv previously confirmed to bind to human complement C5 was mixed with a 6% BSA / PBS solution in a 1:1 ratio and added to the wells coated with human complement C5 and its MG1 and MG4 domains, respectively. The plates were then incubated at room temperature for 1 hour. After the reaction was complete, the wells were washed with PBS containing 0.05% Tween-20, and an anti-HA antibody labeled with HRP (Peroxidase) (Roche, Cat. No. 12013819001) was diluted with 3% BSA / PBS solution and added to the wells, and the reaction was carried out at room temperature for 1 hour. After performing the same washing process, 3,3', 5.5'-Tetramethylbenzidine (TMB) solution (Sigma, Cat. No. T0440) was added as a substrate and the reaction was carried out at room temperature for up to 30 minutes. After stopping the reaction by adding a reaction stop solution (Invitrogen, Cat. No. SS04), the absorbance was measured at a wavelength of 450 nm.

[0239] As a result, it was confirmed that the scFv binds to human complement C5 and its MG1 and MG4 domains, respectively (Figs. 16 to 19). As a positive control, antibody A, known to bind to MG4 of the C5 domain, was used, and as a negative control, an unrelated antibody that does not specifically bind to either the MG1 or MG4 domain of human complement C5 was used.

[0240] Example 4. Preparation of additional combinations and analysis of their complement C5 activity inhibitory effect

[0241] 4-1. Production of MG4-Binding Protein

[0242] According to a paper published in 2020 (An inhibitor of complement C5 provides structural insights into activation, Proceedings of the National Academy of Sciences of the United States of America, 2020, Jan 7; 117(1), p362-370), a protein named CirpT1, present in the salivary glands of mites (Rhipicephalus pulchellus), is known to inhibit complement activity by binding to MG4 of human complement C5. To produce the CirpT1 protein, a DNA sequence formed by fusing the gene encoding the CirpT1 protein with the gene encoding the human kappa light chain constant region was cloned into a pcDNA3.4 animal cell expression vector. The cells were cultured using a transient overexpression system with the Expi293F cell line, and the protein was purified using a kappa-select affinity column (Cytiva, Cat# 17545811).

[0243] Human Ck Fusion CirpT1 Protein Sequence Classification Sequence Number Sequence Amino Acid Sequence of Human Ck Fusion CirpT1 Protein 80DVQERGHTYVTKNVTVEDGACVYLRNVIPNGETKALNNPCVLSTCYAADRKVNSTLCPNIGVDEGCHVEWTPDGVYPNCCPKHVCPSATASSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC DNA Sequence of Human Ck Fusion CirpT1 Protein

[0244] (Bold text represents CirpT1, the rest represent human Ck sequences)

[0245] 4-2. Measurement of Complement Activation Inhibitory Effect of Combined Administration of MG1-Binding Antibody and MG4-Binding Protein via Alternative Pathway-Mediated Hemolysis Assay

[0246] Rabbit red blood cells 6.67 x 10⁶ in GVBMG solution 8 The samples were prepared by suspension at 1 / mL, and human serum with active complement was thawed and stored on ice. Human complement C5 inhibitors were prepared alone to a peak concentration of 20 μM; when different types of human complement C5 inhibitors were used in combination, they were mixed in a 1:1 molar ratio in GVBMG solution to achieve a peak concentration of 20 μM, and each peak concentration was diluted by 1 / 3 to prepare a total of 6 concentrations. Next, 50 μL of rabbit erythrocytes, 100 μL of human serum, and 50 μL of human complement C5 inhibitor were mixed in a 96-well plate and incubated at 37°C at 100 rpm for 2 hours. As a positive control, rabbit erythrocytes were placed in GVBMG solution at a concentration of 6.67 x 10⁶ 8 A sample was prepared by mixing 50 µl of a suspension at 1 / mL with 100 µl of human serum with live complement activity and 50 µl of GVBMG solution, and as a negative control, rabbit erythrocytes were placed in GVBMG solution at a concentration of 6.67 x 10⁶ 8 A sample was prepared by mixing 100 µl of human serum suspended at 1 / mL, from which complement activity had been removed by heat treatment, with 50 µl of GVBMG solution, and incubated at 37°C and 100 rpm for 2 hours. After the reaction was complete, the supernatant was collected after centrifugation at 4°C and 4000 rpm for 10 minutes, transferred to a new 96-well plate, and the absorbance was measured at 414 nm. The degree of hemolysis (%) was calculated using Equation 1 above.

[0247] As a result, as shown in Figure 20, it was confirmed that the inhibitory effect on complement activity was increased when a combination of clones binding to MG1 and MG4—namely, antibody A and crovalimab or CirpT1 and crovalimab—was used in combination, compared to when crovalimab binding to MG1 of human complement C5, antibody A binding to MG4, and CirpT1 were treated alone.

[0248] The complement C5 binding antagonist combination according to the present invention includes antagonists that specifically bind to different domains of complement C5, and since it has been confirmed that it significantly inhibits complement C5 activity, it can be applied to the prevention and treatment of various diseases such as myasthenia gravis, age-related macular degeneration, and paroxysmal nocturnal hemoglobinuria that are caused or exacerbated by abnormal activation of the complement system.

Claims

1. A combination comprising: a first antagonist that specifically binds to the MG4 domain of the beta chain of the complement C5 protein of SEQ ID NO. 11; and a second antagonist that specifically binds to the MG1 domain of the beta chain of the complement C5 protein.

2. A combination according to claim 1, wherein the first antagonist is a complement-binding protein.

3. A combination according to claim 1, wherein the first antagonist comprises any one selected from the group consisting of the following: (i) an antibody comprising the heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO. 3, the HCDR2 of SEQ ID NO. 4, the HCDR3 of SEQ ID NO. 5, the light chain complementarity determining region (LCDR) 1 of SEQ ID NO. 6, the LCDR2 of the amino acid sequence YAS, and the LCDR3 of SEQ ID NO. 7; (ii) R15-14 comprising the light chain complementarity determining region (LCDR) 1 of SEQ ID NO. 320, the LCDR2 of amino acid sequence SS, the LCDR3 of SEQ ID NO. 261, and the heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO. 321, the HCDR2 of SEQ ID NO. 322, and the HCDR3 of SEQ ID NO. 323; (iii) R15-24 comprising the light chain complementarity determining region (LCDR) 1 of SEQ ID NO. 320, the LCDR2 of amino acid sequence SA, the LCDR3 of SEQ ID NO. 280, and the heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO. 321, the HCDR2 of SEQ ID NO. 322, and the HCDR3 of SEQ ID NO. 323; (iv) R15-05 comprising the light chain complementarity determining region (LCDR) 1 of SEQ ID NO. 335, the LCDR2 of amino acid sequence SA, the LCDR3 of SEQ ID NO. 463, and the heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO. 312, the HCDR2 of SEQ ID NO. 336, and the HCDR3 of SEQ ID NO. 337; (v) R13-175 comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO. 349, LCDR2 of SEQ ID NO. 350, LCDR3 of SEQ ID NO. 302, and heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO. 351, HCDR2 of SEQ ID NO. 352, and HCDR3 of SEQ ID NO. 353; and (vi) R13-210 comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO 214, LCDR2 of amino acid sequence GA, LCDR3 of SEQ ID NO 306, and heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO 354, HCDR2 of SEQ ID NO 355, and HCDR3 of SEQ ID NO 356.

4. In paragraph 3, the combination wherein the first antagonist comprises any one selected from the group consisting of the following: (i) heavy chain variable region of SEQ ID NO. 1 and light chain variable region of SEQ ID NO. 2; (ii) light chain variable region of SEQ ID NO. 225 and heavy chain variable region of SEQ ID NO. 387; (iii) light chain variable region of SEQ ID NO. 399 and heavy chain variable region of SEQ ID NO. 387; (iv) light chain variable region of SEQ ID NO 400 and heavy chain variable region of SEQ ID NO 401; (v) light chain variable region of SEQ ID NO. 408 and heavy chain variable region of SEQ ID NO. 409; and (vi) Light chain variable region of sequence number 410 and heavy chain variable region of sequence number 411.

5. A combination according to claim 1, wherein the first antagonist is a complement binding protein comprising the amino acid sequence of SEQ ID NO.

80.

6. A combination according to paragraph 1, wherein the second antagonist is an antibody.

7. A combination according to claim 1, wherein the second antagonist is any one anti-C5 antibody selected from the group consisting of Ch1-78, Ch2-12, Ch5-13, Ch11-01, Ch11-09, R-C5 / MG1-008-30-#13, R-13-SP#21, R14-16, R14-27, R14-07, R13-04, R14-23, R13-30, R14-19, R14-20, R14-24, R14-26, R15-01, R15-10, R15-21, R13-165, R15-152, R15-126, and Crovalimab.

8. A combination according to claim 7, wherein the second antagonist comprises any one selected from the group consisting of the following: (i) Ch1-78 comprising LCDR (light chain complementarity determining region) 1 of amino acid sequence SSW, LCDR2 of amino acid sequence QN, LCDR3 of SEQ NO. 150, and HCDR (heavy chain complementarity determining region) 1 of SEQ NO. 151, HCDR2 of SEQ NO. 152, and HCDR3 of SEQ NO. 153; (ii) Ch2-12 comprising LDR1 of amino acid sequence NSD, LCDR2 of amino acid sequence DS, LCDR3 of SEQ ID NO. 154, and HCDR1 of SEQ ID NO. 155, HCDR2 of SEQ ID NO. 156, and HCDR3 of SEQ ID NO. 157; (iii) Ch5-13 comprising LCDR1 of SEQ ID NO. 158, LCDR2 of amino acid sequence SN, LCDR3 of SEQ ID NO. 159, and HCDR1 of SEQ ID NO. 160, HCDR2 of SEQ ID NO. 161, and HCDR3 of SEQ ID NO. 162; (iv) Ch11-01 comprising LDR1 of SEQ ID NO. 163, LCDR2 of amino acid sequence DN, LCDR3 of SEQ ID NO. 164, HCDR1 of SEQ ID NO. 165, HCDR2 of SEQ ID NO. 166, and HCDR3 of SEQ ID NO. 167; (v) Ch11-09 comprising LCDR1 of amino acid sequence SNH, LCDR2 of amino acid sequence DN, LCDR3 of SEQ ID NO. 168, and HCDR1 of SEQ ID NO. 169, HCDR2 of SEQ ID NO. 170, and HCDR3 of SEQ ID NO. 171; (vi) R-C5 / MG1-008-30-#13 comprising LCDR1 of SEQ ID NO. 214, LCDR2 of amino acid sequence GA, LCDR3 of SEQ ID NO. 215, and HCDR1 of SEQ ID NO. 216, HCDR2 of SEQ ID NO. 217, and HCDR3 of SEQ ID NO. 218; (vii) R-13-SP#21 comprising LCDR1 of SEQ ID NO. 219, LCDR2 of amino acid sequence AA, LCDR3 of SEQ ID NO. 220, and HCDR1 of SEQ ID NO. 221, HCDR2 of SEQ ID NO. 222, and HCDR3 of SEQ ID NO. 223; (viii) R14-16 comprising LCDR1 of SEQ ID NO. 310, LCDR2 of SEQ ID NO. 311, LCDR3 of SEQ ID NO. 246, and HCDR1 of SEQ ID NO. 312, HCDR2 of SEQ ID NO. 313, and HCDR3 of SEQ ID NO. 314; (ix) R14-27 comprising LCDR1 of SEQ ID NO. 310, LCDR2 of SEQ ID NO. 311, LCDR3 of SEQ ID NO. 246, and HCDR1 of SEQ ID NO. 315, HCDR2 of SEQ ID NO. 316, and HCDR3 of SEQ ID NO. 314; (x) R14-07 comprising LCDR1 of SEQ ID NO. 310, LCDR2 of SEQ ID NO. 311, LCDR3 of SEQ ID NO. 246, and HCDR1 of SEQ ID NO. 315, HCDR2 of SEQ ID NO. 316, and HCDR3 of SEQ ID NO. 314; (xi) R13-04 comprising LCDR1 of SEQ ID NO. 214, LCDR2 of amino acid sequence GA, LCDR3 of SEQ ID NO. 255, and HCDR1 of SEQ ID NO. 317, HCDR2 of SEQ ID NO. 318, and HCDR3 of SEQ ID NO. 319; (xii) R14-23 comprising LCDR1 of SEQ ID NO. 310, LCDR2 of SEQ ID NO. 311, LCDR3 of SEQ ID NO. 246, and HCDR1 of SEQ ID NO. 315, HCDR2 of SEQ ID NO. 316, and HCDR3 of SEQ ID NO. 314; (xiii) R13-30 comprising LCDR1 of SEQ ID NO. 324, LCDR2 of SEQ ID NO. 311, LCDR3 of SEQ ID NO. 246, and HCDR1 of SEQ ID NO. 315, HCDR2 of SEQ ID NO. 325, and HCDR3 of SEQ ID NO. 314; (xiv) R14-19 comprising LCDR1 of SEQ ID NO. 324, LCDR2 of SEQ ID NO. 311, LCDR3 of SEQ ID NO. 246, and HCDR1 of SEQ ID NO. 315, HCDR2 of SEQ ID NO. 316, and HCDR3 of SEQ ID NO. 314; (xv) R14-20 comprising LCDR1 of SEQ ID NO. 310, LCDR2 of SEQ ID NO. 311, LCDR3 of SEQ ID NO. 246, and HCDR1 of SEQ ID NO. 326, HCDR2 of SEQ ID NO. 327, and HCDR3 of SEQ ID NO. 314; (xvi) R14-24 comprising LCDR1 of SEQ ID NO. 310, LCDR2 of SEQ ID NO. 311, LCDR3 of SEQ ID NO. 246, and HCDR1 of SEQ ID NO. 315, HCDR2 of SEQ ID NO. 328, and HCDR3 of SEQ ID NO. 329; (xvii) R14-26 comprising LCDR1 of SEQ ID NO. 310, LCDR2 of SEQ ID NO. 311, LCDR3 of SEQ ID NO. 246, and HCDR1 of SEQ ID NO. 330, HCDR2 of SEQ ID NO. 331, and HCDR3 of SEQ ID NO. 314; (xviii) R15-01 comprising LCDR1 of SEQ ID NO. 310, LCDR2 of SEQ ID NO. 311, LCDR3 of SEQ ID NO. 246, and HCDR1 of SEQ ID NO. 332, HCDR2 of SEQ ID NO. 333, and HCDR3 of SEQ ID NO. 329; (xix) R15-10 comprising LCDR1 of SEQ ID NO. 310, LCDR2 of SEQ ID NO. 311, LCDR3 of SEQ ID NO. 246, and HCDR1 of SEQ ID NO. 315, HCDR2 of SEQ ID NO. 316, and HCDR3 of SEQ ID NO. 314; (xx) R15-21 comprising LCDR1 of SEQ ID NO. 310, LCDR2 of SEQ ID NO. 311, LCDR3 of SEQ ID NO. 246, and HCDR1 of SEQ ID NO. 334, HCDR2 of SEQ ID NO. 313, and HCDR3 of SEQ ID NO. 314; (xxi) R13-165 comprising LCDR1 of SEQ ID NO. 338, LCDR2 of amino acid sequence RA, LCDR3 of SEQ ID NO. 288, and HCDR1 of SEQ ID NO. 339, HCDR2 of SEQ ID NO. 340, and HCDR3 of SEQ ID NO. 341; (xxii) R15-152 comprising LCDR1 of SEQ ID NO. 342, LCDR2 of SEQ ID NO. 343, LCDR3 of SEQ ID NO. 294, and HCDR1 of SEQ ID NO. 344, HCDR2 of SEQ ID NO. 345, and HCDR3 of SEQ ID NO. 346; (xxiii) R15-126 comprising LCDR1 of SEQ ID NO. 310, LCDR2 of SEQ ID NO. 347, LCDR3 of SEQ ID NO. 246, and HCDR1 of SEQ ID NO. 315, HCDR2 of SEQ ID NO. 328, and HCDR3 of SEQ ID NO. 348; and (xxiv) Crovalimab.

9. In paragraph 7, the second antagonist is a combination selected from the group consisting of: (i) Ch1-78 comprising the light chain variable region of SEQ ID NO. 25 and the heavy chain variable region of SEQ ID NO. 26; (ii) Ch2-12 comprising the light chain variable region of SEQ ID NO. 27 and the heavy chain variable region of SEQ ID NO. 28; (iii) Ch5-13 comprising the light chain variable region of SEQ ID NO. 29 and the heavy chain variable region of SEQ ID NO. 30; (iv) Ch11-01 comprising the light chain variable region of SEQ ID NO. 31 and the heavy chain variable region of SEQ ID NO. 32; (v) Ch11-09 comprising the light chain variable region of SEQ ID NO. 33 and the heavy chain variable region of SEQ ID NO. 34; (vi) R-C5 / MG1-008-30-#13 comprising the light chain variable region of SEQ ID NO. 236 and the heavy chain variable region of SEQ ID NO. 237; (vii) R-13-SP#21 comprising the light chain variable region of SEQ ID NO. 238 and the heavy chain variable region of SEQ ID NO. 239; (viii) R14-16 comprising the light chain variable region of SEQ ID NO. 381 and the heavy chain variable region of SEQ ID NO. 382; (ix) R14-27 comprising the light chain variable region of SEQ ID NO. 383 and the heavy chain variable region of SEQ ID NO. 384; (x) R14-07 comprising the light chain variable region of SEQ ID NO. 381 and the heavy chain variable region of SEQ ID NO. 224; (xi) R13-04 comprising the light chain variable region of SEQ ID NO. 385 and the heavy chain variable region of SEQ ID NO. 386; (xii) R14-23 comprising the light chain variable region of SEQ ID NO. 381 and the heavy chain variable region of SEQ ID NO. 388; (xiii) R13-30 comprising the light chain variable region of SEQ ID NO. 389 and the heavy chain variable region of SEQ ID NO. 390; (xiv) R14-19 comprising the light chain variable region of SEQ ID NO. 389 and the heavy chain variable region of SEQ ID NO. 391; (xv) R14-20 comprising the light chain variable region of SEQ ID NO. 392 and the heavy chain variable region of SEQ ID NO. 393; (xvi) R14-24 comprising the light chain variable region of SEQ ID NO. 381 and the heavy chain variable region of SEQ ID NO. 394; (xvii) R14-26 comprising the light chain variable region of SEQ ID NO. 381 and the heavy chain variable region of SEQ ID NO. 395; (xviii) R15-01 comprising the light chain variable region of SEQ ID NO. 392 and the heavy chain variable region of SEQ ID NO. 396; (xix) R15-10 comprising the light chain variable region of SEQ ID NO. 381 and the heavy chain variable region of SEQ ID NO. 397; (xx) R15-21 comprising the light chain variable region of SEQ ID NO. 381 and the heavy chain variable region of SEQ ID NO. 398; (xxi) R13-165 comprising the light chain variable region of SEQ ID NO. 402 and the heavy chain variable region of SEQ ID NO. 403; (xxii) R15-152 comprising the light chain variable region of SEQ ID NO. 404 and the heavy chain variable region of SEQ ID NO. 405; (xxiii) R15-126 comprising the light chain variable region of SEQ ID NO. 406 and the heavy chain variable region of SEQ ID NO. 407; and (xxiv) Crovalimab.

10. A combination according to claim 1, further comprising a complement C5 inhibitor.

11. In paragraph 10, the above complement C5 inhibitor is a combination of one or more selected from the group consisting of eculizumab, ravulizumab, pozelimab, zilucoplan, and tesidolumab.

12. A combination according to claim 1, wherein the first antagonist is an anti-C5 antibody comprising a fusion protein of the hinge of IgG2 and the Fc of IgG4.

13. A composition for the prevention or treatment of complement-related diseases comprising, as an active ingredient, a combination of any one of claims 1 to 12.

14. A composition according to claim 13, wherein the complement-related disease is one or more selected from the group consisting of myasthenia gravis (gMG), age-related macular degeneration (AMD), and paroxysmal nocturnal hemoglobinuria (PNH).

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