Antibodies against human complement factor c2b and use methods

Abstract

To provide antibodies and antigen binding fragment thereof that specifically bind to human complement factor C2 and are capable of inhibiting activation of the classical and lectin pathways of the complement system.SOLUTION: The invention provides a monoclonal antibody or antigen binding fragment thereof that specifically binds to human complement factor C2, and comprises: a VH domain comprising a specific amino acid sequence; and a VL domain comprising a specific amino acid sequence. Also provided is a pharmaceutical composition comprising the monoclonal antibody or antigen binding fragment thereof.SELECTED DRAWING: Figure 14

Description

[Technical field]

[0001] (Related Applications) This application is a continuation of U.S. Provisional Patent Application No. 62 / 779,102, filed December 13, 2018. The benefit of prior art is claimed, the entire contents of which are incorporated herein by reference. .

[0002] (Sequence Listing) This application contains a Sequence Listing which has been submitted electronically in ASCII format, and is hereby incorporated by reference in its entirety. The ASCII copy created on November 22, 2019 is 618634_AGX5-04 It is called 8PC_ST25.txt and is 94,329 bytes in size.

[0003] FIELD OF THEINVENTION The present invention relates to the fields of immunology and molecular biology. More specifically, the present invention relates to a method for the treatment of the complement system. Compositions and methods for inhibiting classical and lectin pathway activation and treatment of human diseases The present invention relates in particular to binding molecules that bind to human complement factor C2 and their use in the treatment of The present invention relates to a method for producing and using the same. [Background technology]

[0004] BACKGROUND OF THEINVENTION The complement system consists of a cascade of plasma enzymes, regulatory proteins, and Prior to activation, various complement factors are converted into inactive precursor proteins. Activation of the system leads to an activation cascade, which The factor(s) specifically proteolyze the complement proteins further downstream in the cascade. This activates the subsequent factors.

[0005] Complement activation occurs via three pathways: the classical (or classical) pathway, the alternative pathway, and the lectin-mediated pathway. The classical pathway involves the interaction of an antigen with an IgM, IgG1, IgG2, or IgG3 antibody. The immune complex is activated by the formation of an immune complex using the complement component C1. The alternative pathway is mediated by IgA-containing immune complexes or by the binding of bacteria and other The lectin pathway is activated by recognition of the activated surface of mannose-binding lectin or Mannan-binding lectin (MBL), also known as mannan-binding protein (MBP), is a Antibody-independent complement activation is initiated by binding to specific carbohydrates on the surface of various pathogens. It is responsible for the sexualization pathway.

[0006] Activation of the classical pathway begins with the sequential activation of C1, C4, and C2; C2 is further cleaved Activation of the alternative pathway involves the sequential activation of complement components D, C3, and B. Each pathway begins with the activation of complement factor C3, a common central component that is involved in the cleavage of the third complement component. This results in activation of the common terminal pathway, which in turn activates the membrane attack complex. (MAC, which contains complement component C5b-9; Muller-Eberhard, Annu Rev Biochem 1988, 57:32) 1) is formed during complement activation. Not only MAC but also the anaphylatoxins C3a and C These activation products also produce several inflammatory peptides, such as 5a. chemotaxis of leukocytes, degranulation of phagocytes, mast cells, and basophils, smooth muscle contraction, and increased vascular permeability. The drug induces multiple biological effects, including oxidative stress, cytotoxicity, and cell lysis (Hugh, 1999; ment 1986, 3:111). The products of complement activation are also involved in the production of toxic oxygen radicals, particularly by phagocytes. It induces the production of dicarbamate and the synthesis and release of arachidonic acid metabolites and cytokines, This further amplifies the inflammatory response.

[0007] Although complement is an important line of defense against pathogenic organisms, its activation is essential for healthy survival. Inhibition of complement activation can also result in damage to host cells that would otherwise be normal. It is believed that the present invention is useful for the treatment and prevention of complement-mediated tissue damage. The present invention relates to novel therapeutic agents that inhibit one or more key components of the complement cascade. There remains a pressing need for Summary of the Invention

[0008] (Summary of the invention) A novel monoclonal anti-human C2b antibody and method for treating the same, which have improved characteristics compared to existing antibodies. The novel antibody is characterized by a heavy chain variable domain (VH) frame. The main feature is the lack of a glycosylation site in framework region 3 (FR3). The antibodies have improved homogeneity and therefore manufacturability compared to existing antibodies; and unexpectedly improved functional properties, such as increased pI, and enhanced potential for so-called antigen sweeping. and antigen-binding fragments thereof find use in human therapy.

[0009] One aspect of the present invention is a monoclonal antibody that specifically binds to human complement factor C2 or an antibody thereof. A native binding fragment comprising: a VH domain comprising the amino acid sequence set forth in SEQ ID NO:1; and comprising a VL domain comprising the amino acid sequence set forth in SEQ ID NO:2; Here, amino acid residues 72-74 (Kabat numbering) of the VH domain each consist of X1X2X3, and X2 is The monoclonal antibody or the above, wherein X1X2X3 is any amino acid and is not NX2S or NX2T. is an antigen-binding fragment thereof.

[0010] One aspect of the present invention is a monoclonal antibody or an antigen-binding fragment thereof according to the present invention and a pharmaceutical The pharmaceutical composition comprises a carrier that is acceptable for administration to the subject pharmaceutical.

[0011] One aspect of the present invention is a method for producing a nucleic acid encoding a monoclonal antibody or an antigen-binding fragment thereof according to the present invention. The nucleic acid molecule or molecules

[0012] One aspect of the invention is a method for producing a nucleic acid molecule comprising one or more vectors comprising one or more nucleic acid molecules according to the invention. be.

[0013] One aspect of the present invention is a method for producing a nucleic acid encoding a monoclonal antibody or an antigen-binding fragment thereof according to the present invention. The host cell comprises one or more nucleic acid molecules that

[0014] One aspect of the invention is a method for producing a nucleic acid molecule comprising the steps of: The host cell is

[0015] One aspect of the present invention is a method for producing the monoclonal antibody or antigen-binding fragment thereof according to the present invention. 1. A method for producing a monoclonal antibody or antigen-binding fragment thereof, comprising the steps of: The method comprises culturing the cell population according to the present invention.

[0016] One aspect of the invention is a method for inhibiting activation of the classical pathway or the lectin pathway in a subject. and administering to a subject in need thereof an effective amount of the monoclonal antibody according to the present invention or an antibody thereof. The method further comprises administering an intact binding fragment.

[0017] The following embodiments apply to all aspects of the present invention.

[0018] In some embodiments, X1X2X3 consists of DX2S.

[0019] In some embodiments, X1X2X3 consists of DKS.

[0020] In one embodiment, the VH domain comprises the amino acid sequence set forth in SEQ ID NO:3.

[0021] In certain embodiments, the VL domain comprises the amino acid sequence set forth in SEQ ID NO:2.

[0022] In one embodiment, the VH domain comprises the amino acid sequence set forth in SEQ ID NO:3, The VL domain comprises the amino acid sequence set forth in SEQ ID NO:2.

[0023] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof is a full-length monoclonal antibody. Includes monoclonal antibodies.

[0024] In certain embodiments, the monoclonal antibody comprises a human IgG heavy chain constant domain.

[0025] In some embodiments, the heavy chain constant domain comprises a human IgG1 heavy chain constant domain. In one embodiment, the human IgG1 heavy chain constant domain has the amino acid sequence set forth in SEQ ID NO:4. include.

[0026] In certain embodiments, the heavy chain constant domain comprises a human IgG4 heavy chain constant domain. In some embodiments, the human IgG4 heavy chain constant domain comprises the amino acid sequence set forth in SEQ ID NO:5. Contains arrays.

[0027] In one embodiment, the monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO:6. and a light chain comprising the amino acid sequence set forth in SEQ ID NO:7.

[0028] In one embodiment, the monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO:8. and a light chain comprising the amino acid sequence set forth in SEQ ID NO:7. [Brief description of the drawings]

[0029] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Polyacrylamide gels loaded with the indicated samples are shown, with the larger molecular weight bands (arrows) for samples in lanes 4, 5, 8, and 9 indicating split and shifted bands for antibodies containing VH3 and VH4. [Diagram 2] Graph showing total levels of the indicated antibodies over the course of 31 days in cynomolgus monkeys. The following antibodies were tested: BRO2-glyc-IgG4 (monkeys 1 and 2, glycosylated VH) and BRO2-IgG4 (monkeys 5 and 6, non-glycosylated VH). [Diagram 3]Figures 3A-3I are graphs showing the levels of free C2 (plotted as OD 450 nm over time) in serum over the course of 31 days following administration of various monoclonal antibodies to cynomolgus monkeys. The following antibodies were tested: BRO2-glyc-IgG4 (Figure 3A; monkeys 1 and 2), negative control (Figure 3B; monkeys 3 and 4), BRO2-IgG4 (Figure 3C; monkeys 5 and 6), BRO2-IgG4-NH (Figure 3D; monkeys 7 and 8), BRO2-IgG1-LALA-NH (Figure 3E; ARGX-117; monkeys 9 and 10), His1-IgG4 (Figure 3F; monkeys 11 and 12), His1-IgG4-NH (Figure 3G; monkeys 13 and 14), His1-IgG1-LALA-NH (Figure 3H; monkeys 15 and 16), and His2-IgG4 (Figure 3I, monkeys 17 and 18). [Figure 4] FIG. 1 is a graph showing mean free C2 levels (plotted as OD 450 nm over time) in serum over a 31 day course from cynomolgus monkeys administered various monoclonal antibodies as indicated. [Diagram 5] Graph showing free C2 levels (plotted as OD 450 nm over time) in serum of cynomolgus monkeys treated with the indicated non-glycosylated antibodies. [Figure 6] Figures 6A-6D are a series of graphs showing free C2 levels (plotted as OD 450 nm) in cynomolgus monkeys determined at the indicated times before or after administration of antibody. Monkeys are the same as in Figures 3A-3I. Figure 6A, pre vs pre + 500 mg / ml BRO-2; Figure 6B, 4 hr vs 1 day; Figure 6C, 4 hr vs 2 days; Figure 6D, day 11 vs day 27. ADA, anti-drug antibodies. [Figure 7]7A-7P are a series of graphs showing the immunogenicity (plotted as OD 450 nm) over a 30 day period of anti-C2 or negative control monoclonal antibodies administered to cynomolgus monkeys. Monkeys are the same as in Figs. 3A-3I. Fig. 7A, monkey 1; Fig. 7B, monkey 2; Fig. 7C, monkey 5; Fig. 7D, monkey 6; Fig. 7E, monkey 7; Fig. 7F, monkey 8; Fig. 7G, monkey 9; Fig. 7H, monkey 10; Fig. 7I, monkey 11; Fig. 7J, monkey 12; Fig. 7K, monkey 13; Fig. 7L, monkey 14; Fig. 7M, monkey 15; Fig. 7N, monkey 16; Fig. 7O, monkey 17; Fig. 7P, monkey 18. [Figure 8] 8A-8F are a series of graphs showing the immunogenicity (plotted as OD 450 nm over time) of anti-C2 monoclonal antibodies administered to cynomolgus monkeys over a 60-day period. Monkeys are the same as in Figs. 3A-3I. Fig. 8A, monkey 5; Fig. 8B, monkey 6; Fig. 8C, monkey 9; Fig. 8D, monkey 10; Fig. 8E, monkey 15; Fig. 8F, monkey 16. ADA, anti-drug antibodies. [Figure 9]Figures 9A-9D show the binding of ARGX-117 to C2, assessed by Western blot analysis and surface plasmon resonance (SPR). Figure 9A shows Western blotting analysis of serum containing ARGX-117 (representative results): lane 1: MW size marker; lane 2: recombinant human C2 control (size approx. 100 kDa); lane 3: serum; lane 4: induction of complement activation by addition of aggregated IgG to serum and incubation at 37°C; lane 5: C2-deficient serum. Figure 9B shows SPR analysis with C2 immobilized on the chip surface and various ARGX-117 Fabs as eluates. Figure 9C shows SPR analysis with biotin-C4b and human C2 immobilized on a streptavidin chip with and without the addition of mAb as eluate; black: no preincubation; grey: anti-FXI; control human IgG4 mAb; turquoise: non-inhibitory anti-C2 clone anti-C2-63, clone 63 recognizing the large subunit of C2 (C2a); red: ARGX-117. All at a 5:1 molar ratio, curves were normalized to the signal immediately before injection of C2 onto the C4b chip surface. Figure 9D shows SPR analysis with biotin-C4b immobilized on a streptavidin chip and subsequent human C2 and mAb as eluate; black: running buffer, grey: anti-FXI; control human IgG4 mAb; turquoise: non-inhibitory anti-C2 clone anti-C2-63; red: ARGX-117; curves were normalized immediately before addition of mAb. [Figure 10]Schematic diagram of domain swap mutants between complement factor C2 (SEQ ID NO:21) and complement factor B (FB) (SEQ ID NO:50). In both proteins, the small fragment (C2b in complement factor C2; SEQ ID NO:44 or FBa in complement factor B; SEQ ID NO:51) consists of three Sushi (or complement control protein (CCP)) domains, while the large fragment is composed of the von Willebrand factor type A (VWFA) domain and the peptidase S1 domain. Note that the sequences between the individual domains were not simultaneously adopted in these mutants, but they may also consist of epitopes. Additional sequences include C2a, SEQ ID NO:43; C2b S1, SEQ ID NO:45; C2b S2, SEQ ID NO:46; C2b S3, SEQ ID NO:47; C2 VWFA, SEQ ID NO:48; C2 peptidase S1, SEQ ID NO:49; FBb, SEQ ID NO:52; FBa S1, SEQ ID NO:53; FBa S2, SEQ ID NO:54; FBa S3, SEQ ID NO:55; FB VWFA, SEQ ID NO:56; and FB peptidase 1, SEQ ID NO:57. [Figure 11] Results are shown using an anti-FLAG ELISA performed on the domain-swap mutants, coated with 5-fold dilutions of supernatant from transfected HEK293 cells and detected with an anti-FLAG mouse monoclonal Ab in combination with an HRP-labeled anti-mouse IgG. [Figure 12] Shown are results obtained by domain swap ELISA performed with anti-C2-5F2.4. Anti-C2-5F2.4 mAb (human IgG4 S241P VH4 / VL3 LC-13 / 03-163A Bioceros) was used to coat, plates were incubated with 20-fold diluted supernatants of HEK293 transfectants, and binding was detected with anti-FLAG Ab. Representative results from two independent experiments with similar results. [Figure 13] Figure 1 shows an alignment of the amino acid sequences of the Sushi 2 (S2) domain of human and mouse C2b. Human S2, SEQ ID NO: 46; mouse S2, SEQ ID NO: 58. Asterisks indicate sequence identity. [Figure 14]Results obtained using anti-FLAG ELISA for fine-mapped mutants are shown, coated with undiluted supernatant from transfected HEK293 cells, and detected with biotin-labeled anti-FLAG mouse monoclonal Ab in combination with HRP-labeled SA conjugate. [Figure 15] Results for fine mapping mutants are shown. Anti-C2-5F2.4 mAb (human IgG4 S241P VH4 / VL3 LC-13 / 03-163A Bioceros) was used to coat, plates were incubated with 20-fold diluted supernatants of HEK293 transfectants, and binding was detected by anti-FLAG Ab. [Figure 16] The cluster mapping mutant concept is shown with three amino acid mutations in each cluster, the positions of which are shown in bold font in the human sequence. Each human sequence was mutated to replace the human amino acids shown in bold with the corresponding mouse amino acids. Human S2, SEQ ID NO: 46; Mouse S2, SEQ ID NO: 58. Asterisks indicate sequence identity. [Figure 17] Figures 17A and 17B show anti-FLAG ELISA for cluster mapping mutants. Figure 17A shows that 5-fold dilutions of supernatants from transfected HEK293 cells were used to coat and anti-FLAG mouse monoclonal Ab was combined with HRP-labeled anti-mouse IgG as detection reagent. GFP, green fluorescent protein. Figure 17B shows binding of anti-C2-5F2.4 to cluster mutants. Anti-C2-5F2.4 mAb (human IgG4 S241P VH4 / VL3, LC-13 / 03-163A, Bioceros) was used to coat and plates were incubated with 20-fold diluted supernatants from HEK293 transfectants and binding was detected by anti-FLAG Ab. GFP, green fluorescent protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] (Detailed Description) (definition) "Antibody" or "Immunoglobulin" - As used herein, the term "immunoglobulin" It is a compound that comprises two heavy chains and two nucleotides, regardless of whether it has any associated specific immunoreactivity. As used herein, the term "antibody" includes any polypeptide having a combination of a light chain and a nucleic acid sequence. , with remarkable specificity for an antigen of interest (e.g., a complement protein complex that includes C2) The term "C2 antibody" as used herein refers to an assembly having immune reactive activity. In particular, the human C2 protein and the domains formed via cleavage of C2, as well as several In one embodiment, the immunological specificity for a complex of complement proteins, including their homologous species, is The term "antibody" is used to refer to an antibody that is an immunoglobulin. Antibodies and immunoglobulins include light and heavy chains, They may or may not have covalent interchain bonds between them. The structure of the cyclic amines is relatively well understood.

[0031] Five different antibody classes (IgG, IgM, IgA, IgD, and IgE) are biochemically distinguishable. All five antibody classes are within the scope of the present invention. Generally, the following discussion focuses on the IgG class. For IgG, the immunoglobulin is typically a molecule Two identical light chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical light chain polypeptides with a molecular weight of 53,000 to 70,000 It contains four identical heavy chains. The four chains are linked in a "Y" configuration by disulfide bonds. In this "Y" configuration, the light chain starts at the mouth of the "Y" and continues through the variable region. Bracket for the heavy chain.

[0032] Antibody light chains are classified as kappa (κ) or lambda (λ). Generally, the light and heavy chains are covalently linked to each other and are used to produce immunoglobulins. The globulin is produced by either hybridomas, B cells, or genetically engineered host cells. When constructed, the "tail" portions of the two heavy chains are bonded by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence is The heavy chain runs from the end to the C-terminus at the bottom of each chain. Classified as alpha, delta, or epsilon (gamma, mu, alpha, delta, or epsilon) and between It will be recognized that there are several subclasses of antibodies (e.g., γ1-γ4). It is the nature of this chain that determines the antibody class as IgG, IgM, IgA, IgD, or IgE, respectively. Immunoglobulin subclass (isotype), e.g. IgG1, IgG2, IgG3, IgG4, IgA 1) are well characterized and are known to confer functional specialization. The class and isotype modifications are readily discernible by one of skill in the art in light of the brief disclosure. and therefore is within the scope of the present invention.

[0033] As described above, the variable region of an antibody allows the antibody to selectively recognize an epitope on an antigen, In other words, the VL domain and the VH domain of the antibody can bind specifically to the VL domain and the VH domain of the antibody. These four chains combine to form the variable regions that define the three dimensional antigen binding site. The structure forms an antigen-binding site at the end of each arm of the Y. More specifically, The original binding site is defined by three complementarity determining regions (CDRs) on each VH and VL chain.

[0034] "Binding Molecule" - As used herein, the term "binding molecule" refers to antibodies and It is a generic term intended to encompass antigen-binding fragments thereof.

[0035] "Binding Site" - As used herein, the term "binding site" refers to a binding site that binds to a target antigen of interest. A binding domain comprises a region of a polypeptide that is responsible for selectively binding to an antigen. Exemplary binding domains include antibody variable domains. The molecule may contain a single binding site or multiple (eg, 2, 3, or 4) binding sites.

[0036] "Variable Region" or "Variable Domain" - The term "variable" refers to the VH and VL domains. The specific portion varies extensively in sequence between antibodies, making each specific antibody specific to its target antigen However, variability refers to the fact that the variable domains of an antibody are used in its binding and specificity. The variability is not uniformly distributed throughout the entire antibody domain. It forms part of the antigen-binding site. In each of the VL and VH domains, there are three segments called "hypervariable loops". The first, second, and third hypervariable loops of the Vλ light chain domain are defined herein as In the VL domain, residues 24-33 (L1(λ)) are designated L1(λ), ​​L2(λ) and L3(λ) in ), 49-53 (L2(λ), consisting of 3 residues), and 9 0-96 (L3(λ), consisting of 5 residues) (Morea et al., Meth ods 20:267-279 (2000). The first, second, and third hypervariable loops of the Vκ light chain domain are These are referred to as L1(κ), L2(κ), and L3(κ) in the VL domain and consist of residues 25-33 (L1 (κ), consisting of 6, 7, 8, 11, 12, or 13 residues), 49-53 (L2(κ), consisting of 3 residues) , and 90-97 (L3(κ), consisting of 6 residues) (Morea et al., (Methods 20:267-279 (2000)). The first, second, and third hypervariable loops of the VH domain are In the specification, these are referred to as H1, H2, and H3, and consist of residues 25-33 (H1, 7, 8, or 9) of the VH domain. ), 52-56 (H2, consisting of 3 or 4 residues), and 91-105 (H3, highly variable in length (Morea et al., Methods 20:267-279 (2000)).

[0037] Unless otherwise indicated, the terms L1, L2, and L3 refer to the first, second, and third VL domains, respectively. 3 hypervariable loops and includes hypervariable loops from both Vκ and Vλ isotypes. The terms H1, H2, and H3 refer to the first, second, and third hypervariable loops of the VH domain, respectively. and hypervariable heavy chains derived from any of the known heavy chain isotypes, including gamma, mu, alpha, delta, or epsilon. Contains a loop.

[0038] The hypervariable loops L1, L2, L3, H1, H2, and H3 are each a "complementarity determining region" as defined below. That is, it may include portions of the "CDRs." The terms "hypervariable loop" and "complementarity determining region" are used interchangeably. The terms are not strictly synonymous because the hypervariable loops (HV) are defined based on structure. In contrast, complementarity determining regions (CDRs) are defined based on sequence variability (Kabat et al. Literature, "Sequences of Proteins of Immunologic Interest" al Interest), 5th edition, Public Health Service, National Institutes of Health, Beth Esda, MD., 1983), and the boundaries of the HV and CDRs may differ in some VH and VL domains. This is because.

[0039] The CDRs of the VL and VH domains may typically be defined as comprising the following amino acids: Residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in the light chain variable domain HCDR3), and residues 31-35 or 31-35b (HCDR1), 50-65 (HCDR2) in the heavy chain variable domain 95-102 (HCDR3) (Kabat et al., "Sequences of Proteins of Interest in Immunology" of Proteins of Immunological Interest), 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Therefore, HVs are and, unless otherwise indicated, the "hypervariable loops" of the VH and VL domains herein are Any reference to a CDR should be construed to encompass the corresponding CDR, and vice versa.

[0040] The more highly conserved portions of the variable domains are generally comprised of framework amino acids, as defined below. The variable domains of native heavy and light chains each have four FRs (respectively It comprises FR1, FR2, FR3, and FR4, and is primarily composed of a β-sheet connected by three hypervariable loops. The hypervariable loops of each chain are held together in close proximity by FRs and are isolated from the other chain. Together with the hypervariable loops, they contribute to the formation of the antigen-binding site of antibodies. Relationship between the sequence and shape of the binding site formed by the complementarity determining region has been elucidated. (Chothia et al., J. Mol Biol. 227:799-817 (1992); Tramontano et al., J. Mol. Biol, 215:175-182 (1990)). Despite their high sequence variability, the six loops Five of them are main-chain conformations with only a small repertoire of so-called "canonical structures." These conformations are primarily determined by the length of the loop. and second, their packing, hydrogen bonds, or unusual main-chain conformations are hypothesized. The ability of the protein to determine conformation through its ability to determine the conformation of the protein is important. These are determined by the presence of key residues at certain positions.

[0041] "Framework Region" - As used herein, "framework region" or "FR region" The term includes amino acid residues that are part of a variable region but are not part of a CDR (e.g. (For example, the Kabat definition of CDRs is used.) Thus, the length of the variable region framework is approximately 100–120 Specific examples of heavy chain variable domains include: For the CDRs defined in the literature by Kabat et al., framework region 1 consists of amino acids 1-30. framework region 1 corresponds to the domain of the variable region encompassing amino acids 36-49; framework region 2 corresponds to the domain of the variable region encompassing amino acids 36-49; framework region 3 corresponds to the domain of the variable region encompassing amino acids 66-94; framework region 4 corresponds to the domain of the variable region from amino acid 103 to the end of the variable region. The light chain framework regions correspond to the domains of the variable regions in each of the light chain variable region CDRs. Similarly, the CDRs according to Chothia et al. or McCallum et al. Using the above definition, the boundaries of the framework regions are defined as In a preferred embodiment, the CDRs are as defined by Kabat.

[0042] In natural antibodies, the six CDRs present on each monomeric antibody help the antibody to organize its three-dimensional structure in an aqueous environment. It is a short, non-contiguous amino acid sequence that takes a specific configuration when synthesized to form an antigen-binding site. The remainder of the heavy and light variable domains are shown in the amino acid sequence. The variability between the children is smaller and is termed the framework region. Generally, the CDRs form loops that form a β-sheet conformation. These frameworks are therefore The cleavage region provides the correct orientation of the six CDRs through interchain non-covalent interactions. The antigen-binding site formed by the arranged CDRs acts as a scaffold for binding the It defines a surface that is complementary to an epitope on an immunoreactive antigen. The location of the CDRs is known to those skilled in the art. If so, it can be easily identified.

[0043] "Aglycosylated" - As used herein, the term "aglycosylated" refers to an antibody or A form of an antibody or antigen-binding fragment thereof that lacks glycosylation at potential glycosylation sites. In certain embodiments, the term "non-glycosylated" refers to an antigen-binding fragment of an antigen. A form that lacks glycosylation at potential N-linked glycosylation sites of the antibody or antigen-binding fragment. In one embodiment, the term "non-glycosylated" refers to an antibody or antigen-binding fragment thereof. The term "antibody" refers to an antibody that lacks glycosylation at potential N-linked glycosylation sites in the variable region of the heavy chain. The term "antibody" refers to an antibody or an antigen-binding fragment thereof in a form that is homozygous for the antibody.

[0044] "Constant Region" - As used herein, the term "constant region" refers to a region that is a variable domain or variable region. The term refers to the portion of an antibody molecule that is outside the constant domain. Immunoglobulin light chains consist of a single domain, the “constant domain.” This domain is typically referred to as the "CL or CL1 domain." This domain is located in the VL domain. The immunoglobulin heavy chains are the chains that bind to the immunoglobulin classes (gamma, mu, alpha, The heavy chains γ, α, and δ have three immunoglobulin domains. The constant region consists of three domains (termed CH1, CH2, and CH3) that separate the CH1 and CH2 domains. The heavy chains μ and ε have four domains (CH1-CH4) that form a constant domain. The heavy chain constant domain is C-terminal to the VH domain.

[0045] The numbering of amino acids in heavy and light immunoglobulin chains is based on the N-terminal The constants of the heavy and light immunoglobulin chains are determined sequentially from the C-terminus at the bottom of each chain. Different numbering systems are used to define the domains. According to the EU numbering system, the IgG domains are The heavy chain constant domains of the molecule are defined as follows: CH1 - amino acid residues 118-215; CH2 - amino acid residues 118-215; CH1 domain - amino acid residues 231-340; CH3 - amino acid residues 341-446. The "hinge region" connects the CH1 domain to the CH The hinge region contains the portion of the heavy chain molecule that connects the two domains. This region contains approximately 25 residues and is The hinge is flexible and allows the two N-terminal antigen-binding regions to move independently. The regions are the upper, middle, and lower hinge domains (Roux KH et al., J. Immunol. 1999). 61:4083-90 1998), which can be subdivided into three distinct domains: the "fully human" hinge region; The antibody of the invention comprising may comprise one of the hinge region sequences shown in Table 1 below.

[0046] (Table 1. Human hinge sequences) [Table 1]

[0047] "Fragment" - The term "fragment" as used in the context of an antibody of the present invention refers to an intact or a portion of an antibody or antibody chain that contains fewer amino acid residues than the entire antibody or antibody chain. The term "antigen-binding fragment" refers to a portion of an antigen that specifically binds to an antigen or is antigen-specific. Binding (e.g., specific binding to C2 protein or a portion thereof) to an intact antibody of immunoglobulins or antibodies that compete (i.e., with the intact antibody from which they are derived) As used herein, the term "fragment" of an antibody molecule refers to a polypeptide fragment of an antibody. Antigen-binding fragments, such as antibody light chain variable domains (VL), antibody heavy chain variable domains (VH), Single chain antibody (scFv), F(ab')2 fragment, Fab fragment, Fd fragment, Fv fragment, single arm (monovalent) antibody or combinations of antigen-binding fragments thereof. Any antigen-binding molecule formed by combination, assembly, or conjugation. As used herein, the term "antigen-binding fragment" includes unibodies, domain antigens, and the like. The present invention is further intended to encompass antibody fragments selected from the group consisting of antibodies, mAbs, and nanobodies. Fragments include, for example, chemical or non-chemical fragments of intact or complete antibodies or antibody chains. It can be obtained by enzymatic treatment or by recombinant means.

[0048] (Complement component C2) The second component of human complement (C2) is involved in complement activation of the classical and lectin pathways. C2 is a glycoprotein of 0.5 kDa. It is activated by C1s in the classical pathway or activated MASP2 in the lectin pathway. C2 can be activated by (Mg 2+ (in the presence of ) binds to surface-bound C4b to form the C4bC2 complex This complex is subsequently cleaved by activated C1s or MASP2 into two fragments: The larger 70 kDa fragment, traditionally called C2a, remains bound to C4b to form the C3 convertase C4bC2a. The smaller 30 kDa N-terminal fragment, conventionally called C2b, is released into the fluid phase. Recently, some researchers have reversed the names of C2a and C2b, with C2b referring to the larger 70 kDa fragment and C2a the smaller. Some use it to refer to the smaller 30 kDa fragment. As used herein, C2a refers to the larger C2b refers to the 70 kDa fragment of C1b, and C2b refers to the smaller 30 kDa fragment. Upon binding to C4b, C2a is capable of cleaving C3 and C5, respectively, by the C3 and C5 convertases It constitutes the catalytic subunit of

[0049] The amino acid sequence of human C2 is known (GenBank accession number NM_000063) and is set forth as SEQ ID NO: 21. It is shown as follows.

[0050] Amino acid sequence of human C2 (SEQ ID NO:21): [ka]

[0051] Like many other plasma proteins, C2 has a modular structure. Starting from the chromosome 1, three complement control protein modules (CCP1-3, short consensus primers) were identified. also known as sushi-domain repeats (SCRs) or sushi-domain repeats, metal ion-dependent adhesion von Willebrand factor type A (vWFA) domain containing the site, serine protease (SP ) domain (Arlaud et al., Adv Immunol, 1998, 69:249). Electron microscopy studies From these results, it was revealed that C2 consists of three domains. Three CCP modules (CCP1-3 ) together form the N-terminal domain, which corresponds to C2b. The vWFA domain is the second The second and third domains are: Together they make up the larger C2a portion of the molecule.

[0052] The CCP module is a common structural motif found in several proteins. The globular unit of β-amino acids consists of approximately 60 amino acid residues and contains four invariant disulfide-bonded systems. It folds into a compact β-sheet structure of 6–8 strands built around the amino acid residues. The adjacent CCP modules fold (Norman et al., J Mol Biol 1991, 219: 717). , covalently linked by a less conserved linker.

[0053] The initial binding of C2 to surface-bound C4b is mediated by two low affinity sites, one of which One is C2b (Xu and Volanakis, J Immunol 1997, 158:5958), and the other is C2a vWFA domain. The binding of C2b and C2a to the IgG domain (Horiuchi et al., J Immunol 1991, 47: 584) The crystal structure has been determined to a resolution of 1.8 Å (Milder et al., Structure 2006, 14:1587; Krishnan et al., J Mol Biol 2007, 367: 224; Krishnan et al., Acta Crit. stallogr D Biol Crystallogr 2009, D65: 266), which constitutes the contact site with C4 and C3 on C2. The exact topology and structure of the amino acid residues involved in the interaction with C4 are unknown. The amino acid residues of C2 involved remain to be established (Krishnan et al., 2003). ta Cristallogr D Biol. Crystallogr 2009, D65: 266).

[0054] (anti-C2 antibody) One aspect of the present invention is a monoclonal antibody that specifically binds to human complement factor C2 or an antibody thereof. A native binding fragment comprising: a VH domain comprising the amino acid sequence set forth in SEQ ID NO:1; and comprising a VL domain comprising the amino acid sequence set forth in SEQ ID NO:2; Here, amino acid residues 72-74 (Kabat numbering) of the VH domain each consist of X1X2X3, and X2 is The monoclonal antibody or the above, wherein X1X2X3 is any amino acid and is not NX2S or NX2T. is an antigen-binding fragment thereof.

[0055] The VH domain contains the complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3. contains CDRs LCDR1, LCDR2, and LCDR3. The amino acid sequence of DR3 is shown in Table 2.

[0056] (Table 2. CDR) [Table 2]

[0057] In one embodiment, the monoclonal antibody or antigen-binding fragment thereof is In one embodiment, the monoclonal antibody or its antigen-binding The antibody fragment specifically binds to an epitope in the portion of human complement factor C2 that corresponds to human complement factor C2b. To combine.

[0058] In some embodiments, the heavy chain variable domain is aglycosylated. wherein the amino acid sequence of the heavy chain variable domain is the sequence NXS / T (where N represents asparagine, X represents any amino acid, and S / T represents serine or threonine. Thus, in one embodiment, the sequence NXS / T An antibody comprising a VH domain comprising: (wherein X2 is any amino acid and X1X2X3 is not NX2S or NX2T). That is, X1 is any amino acid other than N, and / or X3 is any amino acid other than S or T. In one embodiment, an antibody comprising a VH domain comprising the sequence NXS or NXT is modified. In some other embodiments, each of these three residues may be altered to consist of DXS. Antibodies containing a VH domain containing the sequence NXS or NXT are engineered to modify these three residues accordingly. Each can be from DXT.

[0059] In one embodiment, the heavy chain amino acids at residues 72-74 (Kabat numbering) are each (wherein X2 is any amino acid, and X1X2X3 is not NX2S or NX2T).

[0060] In certain embodiments, the heavy chain amino acids at residues 72-74 (Kabat numbering) consist of DX2S.

[0061] In certain embodiments, the heavy chain amino acids from residues 72 to 74 (Kabat numbering) consist of DKS.

[0062] In one embodiment, the VH domain comprises the amino acid sequence set forth in SEQ ID NO:3.

[0063] In one embodiment, the amino acid sequence of the VH domain consists of the sequence set forth in SEQ ID NO:3. do.

[0064] In certain embodiments, the VL domain comprises the amino acid sequence set forth in SEQ ID NO:2.

[0065] In one embodiment, the amino acid sequence of the VL domain consists of the sequence set forth in SEQ ID NO:2. do.

[0066] In one embodiment, the VH domain comprises the amino acid sequence set forth in SEQ ID NO:3, and the VL The domain comprises the amino acid sequence set forth in SEQ ID NO:2.

[0067] In one embodiment, the amino acid sequence of the VH domain consists of the sequence set forth in SEQ ID NO:3. The amino acid sequence of the VL domain consists of the sequence set forth in SEQ ID NO:2.

[0068] The amino acid sequences of SEQ ID NO:3 and SEQ ID NO:2 are shown in Table 3. SEQ ID NO:2 is the amino acid sequence of Broteio Humanized 5F2.4 (BRO2), as disclosed in U.S. Patent No. 9,944,717 to Pharma BV SEQ ID NO:28 in Table 3 corresponds to the VL (VK3) domain of The VH (V H4) Corresponds to a domain.

[0069] Table 3. VH and VL domains [Table 3]

[0070] In one embodiment, the monoclonal antibodies of the present invention are human antibodies, particularly human IgG1, Ig The CH1 domain, hinge domain, CH2 domain, and CH3 domain of IgG2, IgG3, or IgG4 are include.

[0071] In one embodiment, the antibody comprises the CH1 domain, hinge domain, CH2 domain of human IgG1. , and a CH3 domain and containing substitutions L234A and L235A in the CH2 domain.

[0072] In one embodiment, the antibody comprises the CH1 domain, hinge domain, CH2 domain of human IgG1. , and a CH3 domain, and containing the substitutions H433K and N434F in the CH3 domain.

[0073] In one embodiment, the antibody comprises the CH1 domain, hinge domain, CH2 domain of human IgG1. and a CH3 domain, and has substitutions L234A and L235A in the CH2 domain, and It contains the substitutions H433K and N434F in the amino acid sequence.

[0074] In one embodiment, the antibody comprises the CH1 domain, hinge domain, CH2 domain of human IgG4. In one embodiment, the antibody comprises a CH1 domain of human IgG4, a CH3 domain, and a CH4 domain. The present invention includes a nucleotide sequence for the nucleotide sequence of the nucleotide 1444, 1445, 1446, 1447, 1448, 1449, 1450, 1451, 1452, 1453, and 1454 in the hinge domain. Includes.

[0075] In one embodiment, the antibody comprises the CH1 domain, hinge domain, CH2 domain of human IgG4. , and a CH3 domain and containing the substitution L445P in the CH3 domain.

[0076] In one embodiment, the antibody comprises the CH1 domain, hinge domain, CH2 domain of human IgG4. and a CH3 domain, and having the substitution S228P in the hinge domain, and Contains both substitutions L445P.

[0077] In one embodiment, the antibody comprises the CH1 domain, hinge domain, CH2 domain of human IgG4. , and a CH3 domain, and containing the substitutions H433K and N434F in the CH3 domain.

[0078] In one embodiment, the antibody comprises the CH1 domain, hinge domain, CH2 domain of human IgG4. and a CH3 domain and containing the substitution S228P in the hinge domain and contains the substitutions H433K and N434F.

[0079] In one embodiment, the antibody comprises the CH1 domain, hinge domain, CH2 domain of human IgG4. , and a CH3 domain, and containing the substitutions H433K, N434F and L445P in the CH3 domain.

[0080] In one embodiment, the antibody comprises the CH1 domain, hinge domain, CH2 domain of human IgG4. and a CH3 domain and containing the substitution S228P in the hinge domain and contains the substitutions H433K, N434F, and L445P.

[0081] In some embodiments, the monoclonal antibody comprises a human IgG heavy chain constant domain. In some embodiments, the heavy chain constant domain comprises a human IgG1 heavy chain constant domain. In embodiments, the heavy chain constant domain consists of a human IgG1 heavy chain constant domain.

[0082] In one embodiment, the heavy chain constant domain comprises the amino acid sequence set forth in SEQ ID NO:29. In one embodiment, the heavy chain constant domain comprises a human IgG1 heavy chain constant domain having an amino acid sequence similar to that of the heavy chain constant domain. The acid sequence consists of the sequence set forth in SEQ ID NO:29.

[0083] In one embodiment, the heavy chain constant domain comprises the amino acid sequence set forth in SEQ ID NO:4. In one embodiment, the heavy chain constant domain comprises a human IgG1 heavy chain constant domain having an amino acid sequence similar to that of the heavy chain constant domain. The acid sequence consists of the sequence set forth in SEQ ID NO:4.

[0084] In some embodiments, the heavy chain constant domain comprises a human IgG4 heavy chain constant domain. In an embodiment, the heavy chain constant domain consists of a human IgG4 heavy chain constant domain.

[0085] In one embodiment, the heavy chain constant domain comprises the amino acid sequence set forth in SEQ ID NO:30. In one embodiment, the heavy chain constant domain comprises a human IgG4 heavy chain constant domain comprising the amino acid sequence of The acid sequence consists of the sequence set forth in SEQ ID NO:30.

[0086] In one embodiment, the heavy chain constant domain comprises the amino acid sequence set forth in SEQ ID NO:31. In one embodiment, the heavy chain constant domain comprises a human IgG4 heavy chain constant domain comprising the amino acid sequence of The acid sequence consists of the sequence set forth in SEQ ID NO:31.

[0087] In one embodiment, the heavy chain constant domain comprises the amino acid sequence set forth in SEQ ID NO:5. In one embodiment, the heavy chain constant domain comprises a human IgG4 heavy chain constant domain comprising the amino acid sequence of The acid sequence consists of the sequence set forth in SEQ ID NO:5.

[0088] The amino acid sequences of SEQ ID NOs: 4, 5, and 29 to 31 are shown in Table 4.

[0089] Table 4. Heavy chain constant domains [Table 4]

[0090] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof is a full-length monoclonal antibody. Includes monoclonal antibodies.

[0091] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof is a full-length monoclonal antibody. It consists of a monoclonal antibody.

[0092] In one embodiment, the present invention relates to a method for identifying a nucleic acid sequence having at least one amino acid sequence similar to that shown in SEQ ID NO:32. at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the sequence In one embodiment, the present invention provides a monoclonal antibody comprising a heavy chain having the same sequence as that of the present invention. As used herein, a heavy chain having 100% sequence identity with the amino acid sequence shown as SEQ ID NO:32 is In one embodiment, the monoclonal antibody provided herein comprises the sequence represented by SEQ ID NO: 7. At least 90%, at least 95%, at least 97%, at least and a light chain having at least 98% or at least 99% sequence identity with the antibody. In one embodiment, the amino acid sequence shown herein as SEQ ID NO:7 is provided. In one embodiment, the monoclonal antibody comprises a light chain having 100% sequence identity to In the present specification, the amino acid sequence shown as SEQ ID NO: 32 has at least 90% At least 95%, at least 97%, at least 98%, or at least 99% sequence identity. and a heavy chain having at least 90%, at least 95%, , at least 97%, at least 98%, or at least 99% sequence identity with a light chain. In one embodiment, the monoclonal antibody provided herein is selected from the group consisting of SEQ ID NO: 32, and a heavy chain having 100% sequence identity with the amino acid sequence shown as SEQ ID NO:7. The present invention provides a monoclonal antibody comprising a light chain having 100% sequence identity to the amino acid sequence shown. do.

[0093] In one embodiment, the present invention relates to a method for identifying a nucleic acid sequence having at least one amino acid sequence similar to that shown in SEQ ID NO:6. At least 90%, at least 95%, at least 97%, at least 98%, or at least 99% In one embodiment, the monoclonal antibody comprises a heavy chain having sequence identity with As used herein, a heavy chain having 100% sequence identity with the amino acid sequence shown as SEQ ID NO:6. In one embodiment, the monoclonal antibody provided herein comprises SEQ ID NO: At least 90%, at least 95%, at least 97%, at least A monoclonal antibody comprising a light chain having at least 98%, or at least 99%, sequence identity with the In one embodiment, the amino acid sequence shown herein as SEQ ID NO:7 is provided. In one embodiment, the monoclonal antibody comprises a light chain having 100% sequence identity with the sequence. In an embodiment, the amino acid sequence shown herein is at least 90% identical to that shown in SEQ ID NO:6, At least 95%, at least 97%, at least 98%, or at least 99% sequence identity. and a heavy chain having at least 90%, at least 95%, , at least 97%, at least 98%, or at least 99% sequence identity with a light chain. In one embodiment, the monoclonal antibody provided herein is selected from the group consisting of SEQ ID NO: 6, and a heavy chain having 100% sequence identity with the amino acid sequence shown as SEQ ID NO: 7. The present invention provides a monoclonal antibody comprising a light chain having 100% sequence identity with an amino acid sequence of do.

[0094] In one embodiment, the present invention relates to a method for identifying a nucleic acid sequence having at least one amino acid sequence similar to that shown in SEQ ID NO:33. At least 90%, at least 95%, at least 97%, at least 98%, or at least 99% In one embodiment, the monoclonal antibody comprises a heavy chain having sequence identity with As used herein, a heavy chain having 100% sequence identity with the amino acid sequence set forth as SEQ ID NO:33. In one embodiment, the monoclonal antibody provided herein comprises SEQ ID NO: At least 90%, at least 95%, at least 97% of the amino acid sequence shown in No. 33; a heavy chain having at least 98%, or at least 99% sequence identity to SEQ ID NO:7; At least 90%, at least 95%, at least 97%, at least 98% with the amino acid sequence shown or a light chain having at least 99% sequence identity to the antibody. In some embodiments, the amino acid sequence shown herein as SEQ ID NO: 33 corresponds to 100% and a heavy chain having 100% sequence identity with the amino acid sequence shown as SEQ ID NO:7. The present invention provides a monoclonal antibody comprising a light chain having the same identity as the antibody.

[0095] In one embodiment, the present invention relates to a method for identifying a nucleic acid sequence having at least one amino acid sequence similar to that shown in SEQ ID NO:34. At least 90%, at least 95%, at least 97%, at least 98%, or at least 99% In one embodiment, the monoclonal antibody comprises a heavy chain having sequence identity with As used herein, a heavy chain having 100% sequence identity with the amino acid sequence shown as SEQ ID NO:34. In one embodiment, the monoclonal antibody provided herein comprises SEQ ID NO: At least 90%, at least 95%, at least 97% of the amino acid sequence shown in No. 34; a heavy chain having at least 98%, or at least 99% sequence identity to SEQ ID NO:7; At least 90%, at least 95%, at least 97%, at least 98% with the amino acid sequence shown or a light chain having at least 99% sequence identity to the antibody. In some embodiments, the amino acid sequence shown herein as SEQ ID NO: 34 corresponds to 100% and a heavy chain having 100% sequence identity with the amino acid sequence shown as SEQ ID NO:7. The present invention provides a monoclonal antibody comprising a light chain having the same identity as the antibody.

[0096] In one embodiment, the present invention relates to a method for identifying a nucleic acid sequence having at least one amino acid sequence similar to that shown in SEQ ID NO:8. At least 90%, at least 95%, at least 97%, at least 98%, or at least 99% In one embodiment, the monoclonal antibody comprises a heavy chain having sequence identity with As used herein, a heavy chain having 100% sequence identity with the amino acid sequence shown as SEQ ID NO:8. In one embodiment, the monoclonal antibody provided herein comprises SEQ ID NO: At least 90%, at least 95%, at least 97%, at least a heavy chain having at least 98%, or at least 99%, sequence identity with the nucleic acid sequence shown as SEQ ID NO:7; At least 90%, at least 95%, at least 97%, at least 98%, Alternatively, a monoclonal antibody is provided, comprising a light chain having at least 99% sequence identity thereto. In one embodiment, 100% identity with the amino acid sequence set forth herein as SEQ ID NO:8. Heavy chain with sequence identity and 100% sequence identity with the amino acid sequence shown as SEQ ID NO:7 The present invention provides a monoclonal antibody comprising a light chain having the same affinity as the antibody of the present invention.

[0097] The amino acid sequences of SEQ ID NOs:6 to 8 and 32 to 34 are shown in Table 5.

[0098] Table 5. Heavy and Light Chains [Table 5]

[0099] The heavy and / or light chains of the antibody have a particular percentage of sequence identity to a reference sequence. For the embodiments defined herein, the heavy and / or light chains may each independently be selected from the C The CDR sequences are kept identical to the DR sequences, so that alterations may only occur outside the CDR regions.

[0100] Unless otherwise stated in this application, percent sequence identity between two amino acid sequences is given in the most appropriate format. (In this alignment, the amino acid sequence to be compared is Contains additions or deletions compared to the reference sequence for optimal alignment between the two sequences. The percentage of identity can be determined by comparing these two sequences. The sequence determines the number of identical positions where the amino acid residues between the two sequences are identical, and the number of identical positions is then calculated. The number of positions in the comparison region is divided by the total number of positions in the comparison region, and the identity between the two sequences is calculated. To get the percentage, multiply the result by 100. For example, BLAST program, "BLAST2 sequences" (Tatusova et al., "BLAST2 sequences" A new tool for comparing protein and nucleotide sequences (Blast 2 sequences - a new tool for comparing protein and nucleotide sequences)” FEMS Microbiol Let 174:247-250) and the parameters used were the default conditions. (In particular, the parameters "Open Gap Penalty": 5 and "Extended Gap Penalty": ":2, the matrix to be selected is, for example, "BLOSUM 62" proposed by the program. The percentage of identity between the two sequences you want to compare (as a matrix) is entered into the program. It can be calculated directly by

[0101] In a non-limiting embodiment, the antibody of the invention comprises a CH1 domain and / or a CL domain (which The amino acid sequences of the human IgG1 and IgG2A polypeptides are fully or substantially human. When the antibody or antigen-binding fragment of the present invention is an antibody intended for use in human therapy, In this case, the entire constant region of the antibody or at least a portion thereof is composed of completely or substantially human amino acids. Thus, the CL domain, the CH1 domain, the hinge region, the CH2 domain, domain, CH3 domain, and CH4 domain (if present), or any combination thereof can be completely or substantially human with respect to its amino acid sequence.

[0102] Advantageously, the CL domain, the CH1 domain, the hinge region, the CH2 domain, the CH3 domain and the CH4 domain The domains, if present, may all have completely or substantially human amino acid sequences. In the context of a constant region of a humanized or chimeric antibody, or an antibody fragment, "substantially The term "human" refers to a polypeptide having at least 90%, at least 92%, at least 95% human constant regions. , at least 97%, or at least 99% amino acid sequence identity. The term "human amino acid sequence" refers to an amino acid sequence encoded by a human immunoglobulin gene. The immunoglobulin genes include germline genes, rearranged genes, and genes that have undergone somatic mutations. The present invention also provides one or more The constant domains of the "human" sequence are altered by the addition, deletion, or substitution of amino acids of Where a polypeptide is intended, but the presence of a "fully human" hinge region is clearly required, The embodiments in which

[0103] The presence of a "fully human" hinge region in the C2-binding antibodies of the present invention minimizes immunogenicity. It may be beneficial to both reduce the amount of cleavage and optimize the stability of the antibody.

[0104] C2-binding antibodies can be modified in the Fc region to increase binding affinity to the neonatal Fc receptor FcRn Increased binding affinity at acidic pH (e.g., from about pH 5.5 to about pH 6.0) is measurable. The increased binding affinity is also due to the fact that the binding affinity at neutral pH (e.g., from about pH 6.9 to about pH 7.4) In this embodiment, an "increase in binding affinity" refers to an increase in the binding affinity of the unmodified Fc region. This means an increase in binding affinity to FcRn relative to the binding affinity of the unmodified Fc region. Typically, the unmodified Fc region , having the wild-type amino acid sequence of human IgG1, IgG2, IgG3, or IgG4. In the present study, the increase in FcRn binding affinity of the antibody molecule having an engineered Fc region was compared with that of the wild-type IgG1 and Binding affinity to IgG2, IgG3, or IgG4 is measured.

[0105] The C2-binding antibody has modifications in the Fc region that increase its binding affinity to the human neonatal Fc receptor FcRn. A measurable increase in binding affinity at acidic pH (e.g., from about pH 5.5 to about pH 6.0) is obtained. The increase in binding affinity is also observed at neutral pH (e.g., from about pH 6.9 to about pH 7.4). In this embodiment, an "increase in binding affinity" refers to an increase in the binding affinity of the unmodified Fc region. This refers to an increase in the binding affinity to human FcRn relative to the binding affinity of the unmodified Fc region. The region has the wild-type amino acid sequence of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the increased binding affinity of the Fc region-modified antibody molecule to human FcRn is determined by measuring the binding affinity of the antibody molecule to human FcRn. The binding affinity of Rn to wild-type IgG1, IgG2, IgG3, or IgG4 is measured.

[0106] Pharmaceutical Composition One aspect of the present invention is a monoclonal antibody that specifically binds to human complement factor C2 or an antibody thereof. A pharmaceutical composition comprising an original binding fragment and a pharma- ceutical acceptable carrier, The antibody or fragment thereof comprises: a VH domain comprising the amino acid sequence set forth in SEQ ID NO:1; and comprising a VL domain comprising the amino acid sequence set forth in SEQ ID NO:2; wherein amino acid residues 72-74 (Kabat numbering) of the VH domain each consist of X1X2X3; X2 is any amino acid, and X1X2X3 is neither NX2S nor NX2T. do.

[0107] The pharmaceutical compositions of the present invention are described in detail in the Remington: The Science and Practice of Pharmaceuticals. "The Practice of Pharmacy" 19th ed., edited by Gennaro, Mack Publishing Co., Easton, In accordance with conventional techniques, such as those disclosed in U.S. Pat. No. 6,239,922, 1995, a pharma- ceutical acceptable carrier or The may be formulated with a diluent, as well as any other known adjuvants and excipients.

[0108] The term "pharmaceutical acceptable carrier" refers to a carrier or excipient that is essentially non-toxic. Examples of such excipients include, but are not limited to, saline, Ringer's solution, Non-aqueous excipients such as fixed oils and ethyl oleate are also suitable. Vehicles may also be used.

[0109] Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition may be in the form of a solution, microemulsion, liposome, or other suitable formulation for increasing drug concentration. The pharmaceutical compositions of the present invention may be formulated as any other suitable ordered structure. Examples of suitable aqueous and non-aqueous carriers that may be used include water, ethanol, polyols (e.g., glycerol, roll, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof. oils, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. For example, the use of coating substances such as lecithin can reduce the amount of dispersing agent required. Proper fluidity is ensured by maintaining the desired particle size and by the use of surfactants. It can be maintained.

[0110] The pharmaceutical compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be achieved by sterilization procedures and the use of various antibacterial and antifungal agents, e.g., parabens, chloramphenicol, chloroquine ... This can be ensured by the inclusion of isobutanol, phenol, sorbic acid, etc. The composition may contain sugars, polyhydric alcohols such as mannitol, sorbitol, glycerol, or It may be desirable to include an isotonicity agent such as sodium chloride. (1) Water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, etc. sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, e.g. Ascorbyl phosphate, butylated hydroxyanisole (BHA), butylated hydroxy Toluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal ions. rate agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, Phosphoric acid and the like may also be included.

[0111] A sterile injectable solution is prepared by mixing the required amount of monoclonal antibody in an appropriate solvent, e.g., as needed. For example, it can be prepared by combining one or a combination of ingredients as listed above, followed by Generally, dispersions are used to dissolve the active compound in a basic dispersion. By combining the carrier with a sterile vehicle containing the required other ingredients, e.g., from those enumerated above, In the case of a sterile powder for preparing a sterile injectable solution, the preferred preparation method is The methods are vacuum drying and freeze drying (lyophilization), and the active ingredient and its pre-sterilized filtration are A powder of any additional desired ingredients is obtained from the resulting solution.

[0112] The pharmaceutical compositions may be administered parenterally, preferably by intravenous (iv) or subcutaneous (sc) injection or It is administered by injection.

[0113] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to administration that is typically administered intravenously. Means a mode of administration other than enteral and topical administration, usually by injection, including but not limited to intravenous , intraperitoneal, subcutaneous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardial, intradermal, transtracheal, subcutaneous , including intra-articular, subcapsular, subarachnoid, intrathecal, epidural and intrasternal injections and infusions.

[0114] Prolonged absorption of the injectable anti-C2 mAb or fragment thereof can be achieved by incorporating in the composition an agent which delays absorption, This can be achieved, for example, by including monostearate and gelatin.

[0115] The mAb or fragment thereof can be prepared with a carrier that protects the compound against rapid release; For example, controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycols, Colic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations are generally known to those skilled in the art. "Sustained and Controlled Release Drug Delivery Systems," JR Ro binson, ed., Marcel Dekker, Inc., New York, 1978.

[0116] Pharmaceutical compositions can be administered with medical devices known in the art.

[0117] Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). A single bolus may be administered, or several divided doses may be administered over time, depending on the urgency of the treatment situation. As indicated, dosages can be proportionally reduced or increased accordingly.

[0118] The actual dosage level of the mAb or fragment thereof in the pharmaceutical composition of the present invention may be varied to suit a particular patient. The amount of active ingredient effective to achieve a desired therapeutic response in a given patient without causing toxicity to the patient. get.

[0119] In one embodiment, a binding molecule, in particular an antibody, according to the invention is administered once a week at a dose of 10 to 500 mg / m 2 ,example For example, 200 to 400 mg / m 2 Such administration can be by injection at a dosage of 1 The administration may be repeated for 1 to 24 hours, for example 2 to 12 hours. This can be accomplished by continuous infusion over a period of time. In this regard, administration may be performed by one or more bolus injections.

[0120] In one embodiment, a binding molecule, in particular an antibody, according to the invention is administered once a week at a dose of 1 to 50 mg / kg body weight (m g / kg), for example, at a dosage of 5 to 25 mg / kg, by injection. The administration may be repeated, for example, 1 to 8 times, for example, 3 to 5 times. For example, the procedure may be performed by continuous infusion over a period of 2 to 12 hours. In an embodiment, administration can be performed by one or more bolus injections.

[0121] In yet another embodiment, the mAb or fragment thereof disclosed herein, or any other The binding molecule may be administered as a maintenance therapy, e.g., once a week for a period of six months or more. This can be done.

[0122] (Nucleic Acid Molecules and Vectors) One aspect of the present invention is a method for producing a nucleic acid encoding a monoclonal antibody or an antigen-binding fragment thereof according to the present invention. In one embodiment, the single nucleic acid molecule is one or more nucleic acid molecules that Both the VH and VL domains of a monoclonal antibody or an antigen-binding fragment thereof are encoded by In one embodiment, the single nucleic acid molecule comprises a monoclonal antibody or a nucleic acid sequence thereof according to the invention. In one embodiment, the antigen-binding fragment encodes both the heavy chain (HC) and the light chain (LC). The first nucleic acid molecule comprises the VH domain of the monoclonal antibody or antigen-binding fragment thereof according to the present invention. and the second nucleic acid molecule encodes a monoclonal antibody or an antigen-binding fragment thereof according to the invention. In one embodiment, the first nucleic acid molecule encodes a VL domain of the fragment. a second nucleic acid molecule encoding the heavy chain (HC) of the monoclonal antibody or antigen-binding fragment thereof; , which encodes the light chain (LC) of the monoclonal antibody or antigen-binding fragment thereof according to the invention.

[0123] In one embodiment, the nucleic acid molecule encoding the VH domain is the nucleic acid molecule set forth in SEQ ID NO:35. Contains the acid sequence.

[0124] In one embodiment, the nucleic acid molecule encoding the VL domain is the nucleic acid molecule set forth in SEQ ID NO:36. Contains the acid sequence.

[0125] In one embodiment, the nucleic acid molecule encoding HC comprises the nucleic acid sequence set forth in SEQ ID NO:37. include.

[0126] In one embodiment, the nucleic acid molecule encoding HC comprises the nucleic acid sequence set forth in SEQ ID NO:38. include.

[0127] In one embodiment, the nucleic acid molecule encoding HC comprises the nucleic acid sequence set forth in SEQ ID NO:39. include.

[0128] In one embodiment, the nucleic acid molecule encoding HC has the nucleic acid sequence set forth in SEQ ID NO:40. include.

[0129] In one embodiment, the nucleic acid molecule encoding HC has the nucleic acid sequence set forth in SEQ ID NO:41. include.

[0130] In one embodiment, the nucleic acid molecule encoding the LC domain is the nucleic acid molecule set forth in SEQ ID NO:42. Contains the acid sequence.

[0131] In one embodiment, the nucleic acid sequence of the nucleic acid molecule encoding the VH domain is SEQ ID NO:35. It consists of the sequence described in

[0132] In one embodiment, the nucleic acid sequence of the nucleic acid molecule encoding the VL domain is SEQ ID NO:36. It consists of the sequence described in

[0133] In one embodiment, the nucleic acid sequence of the nucleic acid molecule encoding HC is set forth in SEQ ID NO:37. It consists of an array.

[0134] In one embodiment, the nucleic acid sequence of the nucleic acid molecule encoding HC is set forth in SEQ ID NO:38. It consists of an array.

[0135] In one embodiment, the nucleic acid sequence of the nucleic acid molecule encoding HC is set forth in SEQ ID NO:39. It consists of an array.

[0136] In one embodiment, the nucleic acid sequence of the nucleic acid molecule encoding HC is set forth in SEQ ID NO:40. It consists of an array.

[0137] In one embodiment, the nucleic acid sequence of the nucleic acid molecule encoding HC is set forth in SEQ ID NO:41. It consists of an array.

[0138] In one embodiment, the nucleic acid sequence of the nucleic acid molecule encoding the LC domain is SEQ ID NO:42. It consists of the sequence described in

[0139] The nucleic acid sequences corresponding to SEQ ID NOs:35 to 42 are shown in Table 6.

[0140] Table 6. Nucleic acid sequences of VH, VL, HC, and LC [Table 6] TIFF2024160274000009.tif245170TIFF2024160274000010.tif240170TIFF2024160274000011.tif194170For SEQ ID NOs:35 and 39, a217g results in an N72D mutation.

[0141] The present invention further provides a gene delivery vehicle or vector comprising a nucleic acid molecule according to the present invention. The gene delivery vehicle or vector can be a plasmid or other bacterially replicating nucleic acid. Such gene delivery vehicles or vectors can be easily introduced into producer cells, e.g. The gene delivery vehicle can also be a viral vector. The virus vectors are adenovirus vectors, lentivirus vectors, and adeno-associated virus vectors. vectors, and retroviral vectors.

[0142] The present invention further provides a vector comprising one or more nucleic acid molecules according to the invention. In one embodiment, a single vector contains a monoclonal antibody or its antigen-binding domain according to the invention. In one embodiment, the nucleic acid molecule includes a single nucleic acid molecule encoding both the VH and VL domains of the combined fragment. In the present invention, a single vector contains It contains a single nucleic acid molecule encoding both the heavy chain (HC) and the light chain (LC).

[0143] In one embodiment, the first vector comprises a monoclonal antibody according to the present invention or an antibody thereof. The first nucleic acid molecule encodes the VH domain of the original binding fragment, and the second vector is A second nucleic acid sequence encoding the VL domain of the monoclonal antibody or antigen-binding fragment thereof In one embodiment, the first vector comprises a monoclonal antibody according to the present invention. or an antigen-binding fragment thereof, and the second vector comprises a nucleic acid molecule encoding the heavy chain (HC) of A second nucleic acid encoding the light chain (LC) of the monoclonal antibody or antigen-binding fragment thereof according to the present invention. The nucleic acid molecule includes:

[0144] The vector according to the present invention is capable of inducing the expression of a monoclonal antibody or an antigen-binding fragment thereof by a host cell. Host cells include expression vectors suitable for use in expressing the could be.

[0145] The present invention relates to one or more nucleic acid molecules encoding an antibody or an antigen-binding fragment thereof according to the present invention. Alternatively, the present invention provides a host cell comprising the antibody of the present invention or an antibody thereof. A host cell is provided that contains one or more vectors encoding the original binding fragment. The nucleic acid molecule, or likewise one or more vectors, can be used in a variety of applications, including, but not limited to, transfection, transformation, and expression. Using any suitable technique, including transformation, transfection, and injection. The vector can be introduced into a host cell by a method such as the steps of: For example, electroporation, calcium phosphate transfection, and Pofection, cell squeezing, sonoporation, optical transfection, and gene guns.

[0146] In some embodiments, the host cell is a eukaryotic cell. The cell is a yeast cell. In some embodiments, the host cell is an insect cell. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a hybridoma cell, a Chinese hamster ovarian tumor cell, or a human ovarian tumor cell. CHO cells, NS0 cells, human embryonic kidney (HEK293) cells, and PER.C6™ cells. In addition to the above cells, the present invention also relates to mammalian cells selected from the group consisting of: Other host cells are further contemplated. Host cells can be used to express the antibodies and antigen-binding fragments thereof according to the invention. Further included are cell lines developed for commercial production.

[0147] The cell line provided with the nucleic acid can then produce the binding molecule / antibody in the laboratory or in a production facility. Alternatively, the nucleic acid can be introduced into cells in the body of an animal in need thereof, thereby transforming the cells. The binding molecules / antibodies are produced in vivo by cells. The nucleic acid molecules of the invention are typically However, the present homology Recombinant techniques have become much more efficient. These techniques use nucleases, e.g. Double-strand break-assisted interleukin-1 (IL-1) expression of TALEN-induced site-specific double-strand breaks Such analogous homologous recombination systems require the transfer of nucleic acid molecules into one or more cis The nucleic acid sequence can be inserted into a region that provides the desired regulatory sequences.

[0148] The present invention further relates to an isolated or recombinant cell comprising a nucleic acid molecule or a vector according to the invention, or an in vitro cell culture. The present invention further provides a binding molecule according to the present invention. An isolated or recombinant cell, or an in vitro cell culture, is provided. Preferably, said cell In one embodiment, the cells secrete the binding molecule. In a preferred embodiment, the cell is a hybridoma cell, a CHO cell, an NS0 cell, a HEK2 cell, or a . 93 cells, or PER-C6™ cells. In a particularly preferred embodiment, the cells are CH Further provided are cell cultures comprising the cells of the invention. The industry is developing cell lines for the large-scale production of antibodies, e.g., for clinical use. Non-limiting examples of such cell lines are CHO cells, NS0 cells, or PER.C6™ cells. These cells are also used for other purposes, such as for the production of proteins. Cell lines developed for industrial-scale production of proteins and antibodies are called industrial cell lines. The invention provides industrial cell lines comprising the nucleic acid molecules, binding molecules and / or antibodies according to the invention. The present invention also relates to a large scale synthesis of proteins and / or antibodies, including the binding molecules or antibodies of the present invention. The present invention also provides cell lines developed for the production of the binding molecules and / or antibodies of the present invention. The present invention provides for the use of cell lines developed for large-scale production of the antibody.

[0149] (Method of producing antibodies) The present invention relates to a method for producing a monoclonal antibody or an antigen-binding fragment thereof according to the present invention. and producing a monoclonal antibody or antigen-binding fragment thereof according to the present invention under conditions that allow for expression of the monoclonal antibody or antigen-binding fragment thereof. The method further comprises culturing the host cell population according to the invention. The method further comprises recovering the monoclonal antibody or antigen-binding fragment thereof from the culture. Preferably, the cells are cultured in a serum-free medium. The present invention further provides an antibody obtained by the method for producing an antibody according to the present invention. The antibody is preferably purified from the culture medium. Preferably, the antibody is purified by affinity purification. To manufacture.

[0150] (How to use) One aspect of the invention is a method for inhibiting activation of the classical pathway or the lectin pathway in a subject. and administering to a subject in need thereof an effective amount of the monoclonal antibody according to the present invention or an antibody thereof. In one embodiment, the subject is administered In some embodiments, the subject is a mouse, a rat, a hamster, a guinea pig, or a mammal. In some embodiments, the animal is a dog, rabbit, goat, sheep, pig, cat, dog, horse, or cow. In some embodiments, the subject is a non-human primate, e.g., a monkey. It is.

[0151] The inhibitory effect of the antibody or antigen-binding fragment can be determined, for example, by measuring total complement activity, by detecting serum samples with anti-human IgG antibodies, This involves testing for hemolytic activity based on the ability to lyse sheep red blood cells coated with a diantibody. The level of hemolysis compared to untreated control samples can be assessed using any suitable method. A decrease indicates an inhibitory effect of the antibody or antigen-binding fragment. The control sample was a historical sample obtained before treatment with the antibody or antigen-binding fragment was started. Typically, there is at least a 5% decrease in total complement activity compared to a control. In one embodiment, a decrease in total complement activity compared to a control indicates efficacy. A reduction of at least 10% indicates efficacy.

[0152] Treatment or administration of the method or monoclonal antibody or antigen-binding fragment thereof according to the present invention The diseases that can be prevented are experimental allergic neuritis, type II collagen-induced joint inflammation, inflammation, myasthenia gravis, hemolytic anemia, glomerulonephritis, idiopathic membranous nephropathy, rheumatoid arthritis, systemic erythritis Autoimmune diseases such as erythematosus, immune complex vasculitis, adult respiratory distress syndrome, stroke , xenotransplantation, allotransplantation, multiple sclerosis, burns, extracorporeal dialysis and blood oxygenation, sepsis and septicemia Inflammatory disorders, including hemolytic shock, induced by in vivo administration of cytokines or mAbs toxicity, antibody-mediated rejection of allografts such as kidney transplants, multiple trauma, ischemia-reperfusion injury, and myocardial infarction.

[0153] Individuals suffering from a disease involving complement-mediated injury or at risk of developing such complement-mediated injury An individual who is exposed to the vaccine may administer an effective amount of the monoclonal antibody of the present invention to an individual in need thereof. The treatment can be achieved by administering a monoclonal antibody or an antigen-binding fragment thereof to the patient. The amount of biologically active complement-derived peptides in the body is reduced, and the lysis of complement to cells and tissues is reduced. The adverse effects of the compound or other compounds on the body are reduced or prevented. In one embodiment, "effective amount" refers to an amount sufficient to achieve a desired response in an individual. The monoclonal antibody or its antibody according to the present invention capable of inhibiting complement activation in It means the amount of intact binding fragment.

[0154] Treatment (prophylactic or therapeutic) generally involves the use of a monoclonal antibody according to the invention or an antigen-binding site thereof. The synthetic fragments can be administered parenterally, for example, intravenously, subcutaneously, or topically, in combination with a pharmaceutical carrier. The administration can typically be accomplished by injection or infusion. The dose and administration regimen of the monoclonal antibody or antigen-binding fragment thereof will depend on the intended complement binding. Typically, for the monoclonal antibodies of the present invention, The amount ranges from 2 to 20 mg per kg of body weight. For parenteral administration, the monoclonal antibody according to the invention is The monoclonal antibody or antigen-binding fragment thereof is combined with a pharma- ceutical acceptable parenteral vehicle. Such vehicles are well known in the art and are suitable for use in the preparation of injectable forms. Examples include saline, glucose solution, Ringer's solution, and a small amount of human serum albumin. There is a solution containing

[0155] Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition may be in the form of a solution, microemulsion, liposome, or other suitable formulation for increasing drug concentration. The pharmaceutical compositions of the present invention may be formulated as any other suitable ordered structure. Examples of suitable aqueous and non-aqueous carriers that may be used include water, ethanol, polyols (e.g., glycerol, glycol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof. vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. For example, the use of a coating material such as lecithin can reduce the amount of water that is required for dispersions. Proper fluidity can be maintained by maintaining the proper particle size and by the use of surfactants. It can be held.

[0156] The pharmaceutical compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be achieved by sterilization procedures and the use of various antibacterial and antifungal agents, e.g., parabens, chloramphenicol, chloroquine ... This can be ensured by the inclusion of isobutanol, phenol, sorbic acid, etc. The composition may contain sugars, polyhydric alcohols such as mannitol, sorbitol, glycerol, or It may be desirable to include an isotonicity agent such as sodium chloride. (1) Water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, etc. sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, e.g. Ascorbyl phosphate, butylated hydroxyanisole (BHA), butylated hydroxy Toluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metals Chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid , phosphoric acid, etc.

[0157] A sterile injectable solution is prepared by dissolving the mAb or fragment thereof in the required amount in an appropriate solvent, e.g. For example, by combining with one or a combination of ingredients as listed above, followed by sterile microfiltration. Generally, the dispersant can be used to disperse the active compound in a basic dispersion. By combining the carrier with a sterile vehicle containing the required other ingredients, e.g., from those enumerated above, In the case of a sterile powder for preparing a sterile injectable solution, the preferred preparation method is The methods used are vacuum drying and freeze drying (lyophilization), and the active ingredient and its previously sterile filtration are Obtain a powder of any additional desired ingredients from the filtered solution.

[0158] Prolonged absorption of the injectable anti-C2 mAb or fragment thereof can be achieved by incorporating in the composition an agent which delays absorption, This can be achieved, for example, by including monostearate and gelatin.

[0159] The mAb or fragment thereof can be prepared with a carrier that protects the compound against rapid release; Controlled release formulations include, for example, implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycols, Colic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations are generally known to those skilled in the art. "Sustained and Controlled Release Drug Delivery Systems," JR Ro binson, ed., Marcel Dekker, Inc., New York, 1978.

[0160] Pharmaceutical compositions can be administered with medical devices known in the art.

[0161] Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). A single bolus may be administered, or several divided doses may be administered over time, depending on the urgency of the treatment situation. As indicated, dosages can be proportionally reduced or increased accordingly.

[0162] The actual dosage level of the mAb or fragment thereof in the pharmaceutical composition of the present invention may be varied to suit a particular patient. The amount of active ingredient effective to achieve a desired therapeutic response in a given patient without causing toxicity to the patient. get.

[0163] In one embodiment, the monoclonal antibody according to the present invention is administered at a dose of 10 to 500 mg / m once a week. 2 ,example For example, 200-400 mg / m 2 Such administration can be by injection at a dosage of, for example The administration may be repeated, for example, 1 to 8 times, for example, 3 to 5 times. The administration may be repeated for 2 to 24 hours, for example, 2 to 12 hours. This can be accomplished by continuous infusion over a period of time.

[0164] In yet another embodiment, the mAb or fragment thereof disclosed herein, or any other The binding molecule may be administered as a maintenance therapy, e.g., once a week for a period of six months or more. This can be done.

[0165] The present invention will now be illustrated with reference to the following examples which set forth particularly advantageous embodiments. However, these embodiments are merely illustrative and are not intended to limit the invention in any way. It should be noted that this is not to be construed as a EXAMPLES

[0166] (Example) Example 1: Removal of glycosylation sites from an anti-C2b monoclonal antibody (BRO2-glyc-IgG4) U.S. Patent No. 9,944,717 discloses a murine inhibitory anti-C2b monoclonal antibody (mAb). From this lead, different heavy chain variable domains (VH1, VH2, VH3, or VH4) and kappa Four humanized variants containing the light chain variable domain (VK1, VK2, VK3, or VK4) were Using Composite Human Antibody technology from e Ltd (Cambridge, UK) Based on in silico analysis, the immunogenicity risk for each humanized VH and VK sequence was As shown in Table 7, pairing VH4 with VK3 or VK4 was associated with the lowest risk of immunogenicity. Low, along with the percentage of identity to the closest related human germline variant This observation is consistent with the promiscuous nature of human MHC class II. Based on the smallest number of binding peptides. VH4 is preferred because it has a relatively high percentage of identity with VH5. Based on this, VH4 / VK3 was selected as the anti-human C2b humanized lead antibody. It is called O2-glyc-IgG4.

[0167] (Table 7.1 Immunogenicity risk ranked from 1 (= lowest) to 5 (= highest) (medium affinity) (higher affinity than 100) and sequence identity to the closest human germline relative) [Table 7]

[0168] SDS-PAGE analysis of the BRO2-glyc-IgG4 variants revealed double binding to the VH3 and VH4 variants. It was found that there was a band and band shift in the VH3 and VH4 frames. A potential glycosylation site (motif) at residues 72-74 (Kabat numbering) in framework region 3 (FR3) We hypothesized that this potential glycosylation site may be responsible for the increased expression of ribosomal glycosylation in mammalian cell lines. Not only will the antibody products expressed from these antibodies be heterogeneous, but the antibody functions may also be heterogeneous. Therefore, potential glycosylation sites were removed. Glycosylation sites were eliminated by site-directed mutagenesis. Induction was used to generate the N72D variant of VH (referred to herein as VH3.2 or VH4.2). The N72D mutation altered the banding patterns observed in VH3 and VH4. This resulted in the doublet band and band shift due to glycosylation of the heavy chain (Figure 1). It was confirmed that this was caused by heterogeneity.

[0169] A variant VH identical to BRO2-glyc-IgG4 but lacking the glycosylation site in FR3 H4.2 exhibits improved properties compared to the heterogeneously glycosylated parent mAb BRO2-glyc-IgG4 To further determine whether this was the case, the heat tolerance of each antibody was determined.

[0170] For the thermal resistance test, the temperature was changed from 55°C to a maximum of 75°C using a thermocycler (Biometra). The humanized variants were treated with increasing concentrations of 100 mM NaCl. Residual binding capacity was analyzed on a Biacore 3000 from serum to Purified human C2 (3500 RU, Complement Technologies Cat#A112, lot#20) was directly coated. The data were analyzed on a CM5 chip surface coated with 100% ethanol. The data were analyzed using the BIAevaluation software. The slope of specific binding for each variant was calculated using the BIAevaluation software's Sensor General fitting from the linear phase of the gram (starting 5 seconds after the start of injection and ending 11 seconds after injection) The activity at 4°C was set as 100%, and the activity was then stopped at 59°C, 56.9°C, and The average value of the slope obtained at a temperature of 55° C. was used to calculate the percentage of activity. , and percentage of activity was plotted in GraphPad Prism (log(agonist) vs. response, Variable slope (4 parameters). The temperature at which the antibody loses 50% of its binding capacity (TM50) is As shown in Table 8.

[0171] (BRO2-IgG4) Both variants lack the glycosylation site present in BRO2-glyc-IgG4 and have improved heat resistance. The results showed that the TM50 of BRO2-glyc-IgG4 was 64.0°C (Table 8). The antibody (also referred to herein as BRO2-IgG4) showed a TM50 of 65.0 or 65.1°C in two independent experiments. VH4.2 / VK4 showed a TM50 of 65.2 or 65.4°C in two independent experiments.

[0172] (Table 8. Percentage of identity of anti-C2b monoclonal antibodies to the closest human germline sequence) (compound and heat resistance) [Table 8]

[0173] Example 2: Preparation of non-glycosylated IgG4 and non-glycosylated IgG1 variants (BRO2-IgG4-NH) Antibodies with pH-dependent antigen binding bind to the antigen in acidic endosomes after internalization into cells. As a result, the released antigen is transported to the lysosome and degraded. On the other hand, dissociated antibodies that are not bound to antigens are recycled back to the plasma by FcRn. The released antibody can then bind to another target antigen. This cycle can then be repeated. Thus, pH-dependent antigen-binding antibodies bind to the target antigen multiple times, thus improving the neutralizing ability of the antibodies. The antibody binds to FcRn at acidic endosomal pH (pH 6.0) and at neutral pH (NH) technology (argenx, Belgium) without fortification (pH 7.4). This process could be further improved if the Fc region of BRO2-IgG4 was The amino acids were mutated (H433K, N434F) to alter the pH-dependent binding to FcRn. The resulting antibody is referred to as BRO2-IgG4-NH herein.

[0174] (BRO2-IgG1-NH and BRO2-IgG1-LALA-NH (ARGX-117)) The effect of immunoglobulin subclass on efficacy was also investigated. The (BRO2) variant was prepared in a human IgG1 background (BRO2-IgG1-NH). Antibody effector functions are mediated by mutations in the Fc region that alter the binding of antibodies to Fcγ receptors. Thus, the amino acid substitutions L234A and L235A ("LALA") can be used to further reduce the ) was incorporated into BRO2-IgG1-NH to give BRO2-IgG1-LALA-NH (also referred to herein as ARGX-117). ) was obtained.

[0175] (His1-IgG1-LALA-NH) Improve the pH dependency of BRO2-IgG1-LALA-NH to improve its pharmacokinetics and pharmacodynamics (PK / To determine whether the PD effect of the BRO2-IgG1-LALA-NH antibody could be augmented, we The acid was mutated to a histidine (G29H mutant VK, referred to herein as Vk3m3). The resulting antibody is referred to herein as His1-IgG1-LALA-NH.

[0176] (His1-IgG4) Similarly, can we improve the pH dependency of BRO2-IgG4 to extend its PK / PD effects in vivo? To determine whether the amino acid in the VK of the BRO2-IgG4 antibody was mutated to histidine, (G29H mutant VK, referred to herein as Vk3m3). The resulting antibody (VH4.2 / V k3m3) is referred to herein as His1-IgG4.

[0177] (His1-IgG4-NH) To examine the effect of recycling on antibody potency, NHance® mutations were The resulting antibody is referred to herein as This is referred to as His1-IgG4-NH.

[0178] (His2-IgG4-NH) To determine whether the pH dependency of BRO2-IgG4-NH can be improved to extend its PK / PD effects in vivo To determine whether the amino acid sequence of VH4 of the BRO2-IgG4-NH antibody was mutated to histidine ( K26H VH mutant, referred to herein as VH4.2m12). The light chain was replaced with the VK4 light chain described above with the second amino acid mutated to a histidine (G29H , VK4 mutant, referred to herein as VK4m3). K4m3) is referred to herein as His2-IgG4-NH.

[0179] Example 3: Improved potency in non-glycosylated BRO2 variants (Overall Pharmacokinetics (PK)) Cynomolgus monkeys (n=2, 1 male and 1 female per group) were randomly assigned to the following groups: 9 and were divided into separate treatment groups.

[0180] Table 9. Treatment group assignment [Table 9]

[0181] Serum samples were obtained from each monkey one day prior to administration of the test antibody (day -1 or "pre"). On day 1 (d1), each monkey received a single intravenous injection of 5 mg / kg of the test antibody according to Table 9. Serum samples were obtained from each monkey consecutively for up to 60 days (d60).

[0182] For PK of all antibodies (total PK), microtiter plates were filled with 100 μL of 5 μg / mL goat anti- The plate was coated with human IgG (Bethyl; A80-319A) overnight at 4°C. Wash three times with 200 μL PBS-0.05% Tween 20, followed by 200 μL PBS-2% BSA at room temperature (RT) for 2 h. The plate was washed three times with at least 200 μL of PBS-0.05% Tween 20. Afterwards, serum samples, standards, and QC samples (prepared in pooled untreated cynomolgus serum) were Duplicate applications were made at 100-fold dilutions and were combined with 100 μL PBS-0.2% BSA-1% pooled untreated For each antibody, their own frozen standards and QC samples were prepared in duplicate. (the same batch of antibody was used as the one injected into the monkeys). The negative control antibody was Antibodies that bind to non-C2 complement components. Incubation was continued at RT with shaking of the plate. The plate was washed five times with at least 200 μL of PBS-0.05% Tween 20 and then incubated for 10 0 μL of Horseradish Peroxidase (HRP)-conjugated mouse anti-human IgGκ (Southern Bio tech, 9230-05) was diluted 260,000-fold in PBS 0.2% BSA, applied to the wells, and incubated at RT for 1 hour. Wash the plate at least five times with 200 μL PBS-0.05% Tween 20 and then add 100 μL 3,3',5,5' - Staining was performed using tetramethylbenzidine (TMB) and 100 μL 0.5 M H2SO4 (CHEM LAB, C The OD at 450 nm was measured and analyzed using GraphPad Prism. The sample concentrations were back-calculated using each one's own standard.

[0183] The results are shown in Table 10. Comparison of glycosylated BRO2-glyc-IgG4 with non-glycosylated BRO2-IgG4 The results are shown in Figure 2. In all PK assays, the concentration of nonglycosylated BRO2-IgG4 was generally The overall PK improvement was completely unexpected. This represents a further important advantage of non-glycosylated antibodies.

[0184] (Table 10. Total PK) [Table 10]

[0185] (Free C2) Cynomolgus monkeys (n=2, 1 male and 1 female per group) were administered 5 mg / kg of the test antigen as described above. The body was given a single intravenous injection.

[0186] In this assay, 100 μL of 2.5 μg / mL mouse anti-human C2 monoclonal antibody mAb32 (Anti-C2 #32 m-IgG@3.31 mg / mL, 0.2 μm PBS, LC-12 / 05-166, 12-apr-13) was used. Microtiter plates were coated overnight at 4°C. This antibody is different from BRO2 of C2. Wash the plate three times with at least 200 μL of PBS-0.05% Tween 20. Then, the plate was blocked with 200 μL of PBS-2% BSA (pH 7.4) for 2 hours at room temperature (RT). , samples, frozen standards (specific for each antibody, prepared in pooled untreated cynomolgus monkey serum) , and frozen QC samples (prepared in pooled untreated cynomolgus serum) were thawed and 80 μL Dilute 6.7-fold in PBS-0.2% BSA. Add 40 μL of biotinylated anti-C2 VH4 / VK3 at 0.6 μg / mL. Each sample was prepared in duplicate. 100 μL of the mixture was diluted with 100 μL of PBS immediately after adding the biotinylated antibody. The plates were incubated at RT for 2 h and then transferred to a washed coated plate. Wash five times with at least 200 μL of PBS-0.02% Tween 20 and add 10 0 μL strep-HRP (Jackson, 016-030-084) was added. After 1 h incubation at RT, Wash the plate at least five times with 200 μL PBS-0.05% Tween 20 and then add 100 μL TMB (Calbioche Staining was performed using 100 μL 0.5 M H2SO4 (CHEM LAB, Cat#CL05-2615-1000) The incubation was stopped after 10 min at RT. The OD at 450 nm was measured and used to determine the levels of C2.

[0187] Sera from the following monkeys were initially tested together using the free C2 assay on different days: Monkeys 1 and 2; monkeys 3 and 4; monkeys 5 and 6; monkeys 7, 8, 9, and 10; monkeys 11, 12, 15, and 16; and monkeys 13, 14, 17, and 18.

[0188] Free C2 levels in all monkeys are shown in Figures 3A-3I and Table 11.

[0189] As expected, monkeys 3 and 4 received the negative control antibody, and therefore There was no decrease in free C2. All monkeys treated with the BRO2 variant had Until the day, the levels of free C2 were very low.

[0190] Regarding monkeys given BRO2-glyc-IgG4 (monkeys 1 and 2) and BRO2-IgG4 (monkeys 5 and 6) C2 levels began to recover from day 4 and returned to baseline levels by day 31. Monkeys 5 and 6 treated with nonglycosylated antibodies were consistently better than those treated with BRO2-glyc-IgG4. The results showed lower free C2 levels than those of the control (Figure 3C, Table 11).

[0191] Except for the monkey with anti-drug antibody (ADA, marked with * in Figures 3D-3I), all other monkeys C2 levels increased much more slowly, and even by day 31, C2 levels were still near baseline. did not return.

[0192] Figure 4 shows the average rise (logarithmic scale) in free C2 levels (OD 450 nm) for two monkeys in each group. The free C2 levels for the BRO2 variant were significantly higher than for the His1 variant. It became lower.

[0193] Monkey 10 injected with BRO2-IgG1-LALA-NH (ARGX-117) had the lowest C2 at all time points tested. The 60-day free C2 levels from monkeys 5 and 6, 9 and 10, and 15 and 16 were A comparison of the levels can be seen in Figure 5. Monkey 10 also had the best overall PK (see above). The raw data are shown in Table 11. Glycosylated and non-glycosylated variants were The compared mean data are shown in Table 12.

[0194] Table 11. Free C2 (OD450nm) for all antibodies [Table 11]

[0195] Table 12. Mean free C2 of glycosylated and non-glycosylated antibodies [Table 12]

[0196] These different monkey assays were performed on different days, so only a selected number of time points (pre Sera from all monkeys were analyzed at a single platelet concentration (4 hours, 1, 2, 4, 11, and 27 days). The "before" sample was also replicated by adding excess BRO2 (500 μg / mL). Tests were conducted with and without the addition of

[0197] The OD of the "before" samples was comparable for all monkeys, indicating that the free C2 levels in the different monkeys The results showed that the β-amyloides expression was comparable (Figure 6A). The “before” sample was preincubated with 500 μg / mL of BRO2. When baptized, all signals were reduced to ODs between 0.013 and 0.015 (Figures 6A and 6B). Such low OD values ​​were not obtained for any of the PK samples, and no free C2 was observed at any time point. The lowest levels were obtained at 4 hours, and these were The lowest was for the variant (OD of 0.02–0.03) (Monkeys 5, 6, 7, 8, 9, and 10, Fig. 6 C) Interpretation of the results on days 11 and 27 was hindered by ADA (anti-drug antibodies), which was observed in several monkeys ( Fig. 6D ).

[0198] (Immunogenicity) Cynomolgus monkeys (n=2, 1 male and 1 female per group) were administered 5 mg / kg of the test antigen as described above. Serum samples from all monkeys were collected at baseline (pre-exposure) and ADA (anti-drug antibodies) was tested for up to 31 days (Figures 7A-7P). Monkeys 5 and 6, 9 and 10 Serum samples from 15 and 16 were further tested up to day 59 (Figures 8A-8F).

[0199] Immunogenicity was determined by incubating the microtiter plate with 100 μL of each antibody at 1 μg / mL at 4°C. Plates were coated overnight with at least 200 μL of PBS-0.05% Wash three times with Tween 20, followed by blocking with 200 μL PBS-1% casein for 2 h at RT. After washing the plate three times with at least 200 μL of PBS-0.05% Tween 20, serum samples were 1 μL PBS-0.1% casein and in the coated wells for 2 h at room temperature (RT). Plates were washed at least five times with 200 μL of PBS-0.05% Tween 20. Then, 100 μL of 8000-fold diluted anti-monkey IgG-HRP (Southern Biotech #4700-05) was added to the wells. The plate was washed at least five times with 200 μL of PBS-0.05% Tween 20. 100 μL TMB was used for staining, and 100 μL 0.5 M H2SO4 (CHEM LAB, Cat#CL05-2615-10 The incubation was stopped after 10 min at 0°C (OD at 450 nm). Representative results are shown in Figures 7A-7P.

[0200] Monkeys 8 (BRO2-IgG4-NH), 11 (His1-IgG4), 13 and 14 (His1-IgG4-NH), and 16 (Hi s1-IgG1-LALA-NH), a clear ADA response was observed (Figures 7F, 7I, 7K, and 7L, respectively). , as well as 7N). Indeed, the signals obtained in the ELISA after antibody injection were significantly higher than the baseline ("pre" ) signal (before antibody injection) was at least two-fold higher.

[0201] on day 11 for monkeys 11, 13, and 16 (Fig. 7I, 7K, and 7N, respectively); ) on day 15; and monkey 14 (FIG. 7L) on day 19. ADA was observed in monkey 15 (FIG. 7C) on day 15; and monkey 16 (FIG. 7D) on day 19.

[0202] For monkey 9 (BRO2-IgG1-LALA-NH) (Figure 7G), no signal was detected in any samples after injection of the antibody. An increase in the signal was observed, but the signal was already high in the baseline sample and the increase over time was small. It was about 1.5 times smaller.

[0203] For monkey 5 (FIG. 7C), abnormally high levels of α-glutamyltransferase were observed in the baseline sample even before the antibody injection. A short signal was observed, which was also higher than the signal at later time points. This could be explained by interference of antibodies (present in serum) with the assay. Therefore, it was not possible to determine whether there was an ADA response in this monkey.

[0204] Example 4: Isoelectric point (pI) The paper by Igawa et al. (Protein Eng Des) investigated VH mutants of a certain IgG1 monoclonal antibody. Sel 2010, 23(5):385-392) has been used to evaluate the pI and clearance of monoclonal antibodies. There is a strong positive correlation between pI and monoclonal antibody half-life, and a negative correlation between pI and monoclonal antibody half-life. In this example, the pI of various forms of anti-human C2b was determined. Shown in Table 13.

[0205] Table 13. Apparent pI of anti-human C2b monoclonal antibodies [Table 13]

[0206] All three antibodies tested have no glycosylation in the VH. As shown in Table 13, ARGX- The pI of 117 was found to be significantly higher than the pI of a closely related IgG4 antibody. The observed pI for 7 indicates an enhanced potential for so-called antigen sweeping. It is expected that this is the case.

[0207] Example 5: Domain Analysis by Western Blotting and Surface Plasmon Resonance (SPR) Analysis mapping) As shown in Figure 1, the binding properties of ARGX-117 were analyzed by Western blotting and surface plasma analysis. The results were evaluated by spin resonance (SPR) analysis. As shown in the figure, ARGX-117 was found to bind to C2 and C2b. The affinity of the C2 (SEQ ID NO: 21) to the solid phase was confirmed by SPR using Biacore 300 as shown in FIG. was further investigated by coating the antibody with various concentrations of ARGX-117 Fab as eluent. The affinity was calculated assuming 1:1 binding between ab and C2, giving a Kd of approximately 0.3 nM. To investigate the mechanism of action of RGX-117, we coated the cells with streptavidin as shown in Figure 9C. Using biotinylated C4 immobilized on a fused chip, the formation of C3 convertase (C4bC2a) was simulated. A similar SPR analysis was performed. C2 was preincubated alone or after preincubation with a control mAb. When added to the flow buffer, binding of C2 was observed on the chip. Preincubation with anti-C2-63 (antibody) enhanced the signal. This is probably because this mAb form forms a complex with C2 and the C2:mAb complex binds together. This is probably because the combination of C2 with ARGX-117 resulted in an increase in molecular weight and an increase in the SPR signal. Preincubation with C2 significantly reduced the binding of C2 to C4b. The interaction is believed to be initiated by the C2b domain (SEQ ID NO: 44). This interaction is mediated by the large C2a domain (SEQ ID NO: 43) of the C3 convertase complex. The results of this experiment show that ARGX-117 inhibits the binding of C4b. It is suggested that this inhibits C2.

[0208] To further understand the mechanism of action of ARGX-117-mediated inhibition of C2, we first isolated C2 from streptavidin-induced After binding to C4b immobilized on a DNA chip and stabilizing with flow buffer alone, The samples were run as shown in Figure 9D. Running buffer or irrelevant soluble antigen targeting antibodies were used. A control human IgG4 mAb (i.e., anti-factor XI (anti-FXI)) caused some signal reduction. The injection of anti-C2-63 increased the signal, which was normalized after the injection was completed. This suggests that the mAb can bind to the C3 convertase (C4bC2a). This is consistent with the predicted binding model, suggesting that after binding to C2, the C2a domain remains largely Interestingly, ARGX-117 has a strong binding to C3 convertase. These results suggest that ARGX-117 can bind to C2, but this result is not consistent with previous results. This suggests that C2 is highly unstable and likely influences C2 in a way that promotes its activation. These results also suggest that ARGX-117 is released together with C2b from the C2 molecule. This suggests that...

[0209] Example 6: Domain Mapping Using Domain Swap Mutants of C2 and Factor B To map the epitope of anti-C2-5F2.4, anti-C2-5F2.4 was cloned into factor B (FB, SEQ ID NO: 50), and C2 and FB are highly homologous proteins with similar domain structures. Both of these proteins contain small and large fragments. The small fragment of complement C2 is called C2b (SEQ ID NO: 44), and the small fragment of factor B is called FBa (SEQ ID NO: 51). Each small fragment contains three Sushis (CCP domains). As shown in Figure 10, each The large fragments contain the von Willebrand factor type A domain (VWFA) and the peptidase S1 domain. The domain swap mutants contained a C-terminal FLAG tag.

[0210] To generate various swap mutants, C2, FB, and 10 domain swapped constructs were used. The DNA constructs of the mutants were obtained from GenScript. The DNA was transformed into competent Escherichia coli (E. The cells were striped onto agar plates. The bacteria were incubated at 37°C for 16 hours. Thirteen bottles of 200 mL LB (Luria Broth) medium were prepared. (MP Bio), autoclaved. Add 300 μL of ampicillin (100 mg / mL) to each bottle. For each construct, preculture was started using 3 mL of LB medium. After 6 hours, Cultures were transferred to bottles and grown with agitation at 37°C for 16 hours. DNA was purified according to the manufacturer's instructions. Plasmid DNA purification kit (MaxiPrep, NucleoBond PC 500, Macherey-Nagel) according to the manufacturer's instructions. Plasmid DNA was purified from bacterial pellets by NanoDro and reconstituted in TE buffer. The plasmid integrity was verified by restriction analysis. For each construct, 1 μL plasmid DNA and 9 μL restriction enzyme mix (PstI and Pv uII) and incubated at 37°C for 2 hours. The DNA was purified using a Bio-Rad ChemiDoc MP system. The gels were analyzed after running on a 1% agarose gel at 100 V for 1 hour using FineMapper. The DNA constructs for sequencing (see below) were treated similarly, but their integrity was verified by sequencing. This was confirmed by standard methods.

[0211] Mutant proteins were produced by transient transfection in HEK293T cells. HEK cells were cultured in complete DMEM (10% fetal calf serum (FCS) and 1% penicillin / streptomycin). The cells were cultured in DMEM (Gibco) supplemented with 100 mM sodium phosphate sulphate (P / S). One day before transfection, 10 cm flask of cells 2 The cells were seeded onto 15 culture dishes (Greiner Bio-One). Prior to transfection, 21 mL of empty DMEM medium was diluted with 630 μL of polyethylenimine (P-Pei, P As controls, the empty plasmid PF45 pcDNA3.1 and PF146 H2B GFP was transfected. 15 μg of plasmid DNA was placed in an Eppendorf tube. The cells were incubated in 1500 μL of empty medium-P-Pei mix in a 20-well tube for 20 min. Carefully add the injection mix to the cells and pipette the medium up and down to remove the After 8 hours, the medium was replaced with 15 mL of empty medium. After 3 days, the cells were examined for GFP expression under a fluorescent microscope. The expression was confirmed. On the fourth day, the supernatant was collected and filtered through a 0.22 μm filter (Sartorius). The mixture was sterile filtered and concentrated on a Vivaspin column (Sartorius) to approximately 1 / 3 of the original volume. The main swap mutants were concentrated on a 30,000 MWCO column and analyzed using C2b fragments for fine mapping. The mutants were concentrated using a 10,000 MWCO column. All supernatants were stored at -20°C and analyzed by SDS-PAGE and Also analyzed by anti-FLAG Western blot.

[0212] To confirm the expression of the various constructs, an anti-FLAG tag ELISA assay was performed. The microplate (Maxisorp, NUNC, Cat#439454) was filled with 5-fold diluted or 100 μL of untreated HEK293T supernatant (domain swap mutants and fine markers, respectively) The plates were coated overnight with PBS (for PBS-treated mutants) and washed four times with 0.05% Tween-20. After washing, 100 μL / well of 1 μg / mL anti-FLAG Ab (Clone) in PBS and 0.1% Tween-20 (PBST) was added. Add 1 mL of 100 mM Trisodium phosphate (LPS; Sigma-Aldrich) and incubate for 1 h at room temperature (RT) with stirring. As detection Ab, 100 μL / well of horseradish peroxidase (HRP)-conjugated yam was used. Add anti-mouse IgG (Santa Cruz Biotechnology, Cat# sc-2005, 1:1000 dilution) to PBST. After the final washing step, 100 μL / well of TMB (Invitrogen) was added to the wells and incubated for 1 h at RT. ogen, Cat#SB02) as substrate and 100 μL / well of HCl (Fischer, Cat#J / 4320 / 15) The reaction was stopped after a few minutes by 50°C and the absorbance was read at 450 nm (BioRad, iMark Micro plate reader).

[0213] As shown in Figure 11, the supernatants of all mutants except C2-(FB-Pep1) were positive by anti-FLAG ELISA. The variation among mutants may be due to differential production or the presence of This can be explained by the different detection efficiencies with anti-FLAG mAb after immunofluorescence.

[0214] Next, we investigated the recognition of the swap mutants by the anti-C2-5F2.4 antibody. 100 μL PBS containing 2 μg / mL anti-C2-5F2.4. The plate was washed four times with PBS containing 0.05% Tween-20 and then added 200 μL The cells were incubated in PBS containing 0.1% Tween-20 and 1% bovine serum albumin (BSA) (PBST-BSA) for 1 h at RT. After washing, 100 μL of culture containing mutants diluted 20-fold in PBST-BSA was added to the wells. The supernatant was added and incubated for 2 h at RT with stirring. After washing, PBST- Add 1 μg / mL biotin-labeled anti-FLAG (clone M2, Sigma-Aldrich) in BSA for 1 h at RT. The plate was washed and incubated with 1 μg / mL streptavidin-POD conjugate (Roche, C At# 11089153001) was added and incubated in the dark for 30 minutes. Add 100 μL / well of TMB (Invitrogen, Cat#SB02) as substrate and 100 μL / well of HCl (Fi The reaction was stopped after a few minutes using a 50-μL antibody (Sigma, Cat# J / 4320 / 15). The absorbance at 450 nm was measured using a microprobe. The measurements were made using a plate reader (BioRad, iMark microplate reader).

[0215] Wild-type C2 showed clear binding, and the complement C2 S2 domain (SEQ ID NO:46) bound to the factor B S2 domain. Loss of binding was observed only for C2-(FB-S2) in which the 5'-amino acid residue was replaced with the 5'-amino acid residue (SEQ ID NO: 54). In contrast, as shown in FIG. 12, no binding was observed with wild-type FB, but the S2 domain of factor B Mutant F in which the complement C2 S2 domain (SEQ ID NO: 54) was replaced with the complement C2 S2 domain (SEQ ID NO: 46). Strong binding was detected for B-(C2-S2). These results suggest that anti-C2-5F2.4 binds to S2 (Su These results clearly show that C2-(FB-P) recognizes an epitope on the shi domain 2. ep1) was produced in sufficient amounts. The same was true when mouse IgG2a anti-C2-5F2.4 was used. Furthermore, similar results were obtained when binding was examined in the presence of 1.25 mM Ca++ in the buffer. The results were obtained using a domain swap mutant between human C2 and mouse C2. Epitope mapping by Bioceros BV has also reached a similar conclusion. The amino acid sequence of Sushi domain 2 of monkey C2 is completely identical to that of Sushi domain 2 of human C2 .

[0216] Example 7: Fine mapping of the epitope of anti-C2-5F2.4 in Sushi domain 2 Anti-C2-5F2.4 did not cross-react with mouse C2, and the mouse S2 domain (SEQ ID NO: 58) was not cross-reactive with mouse C2. As shown, they differ from the human S2 domain (SEQ ID NO: 46) at 10 amino acid positions. To investigate which of the 10 amino acids contributes to the binding with mAb, fine-grained Fine mapping mutants were generated. The fine mapping constructs included the human C2b fragment (huC2b), huC2b with mouse S2 (huC2b-mS2), and mouse to human sequences, respectively. Ten mutants were included, including a single amino acid backmutation of A mutant C2b protein similar to the C2b protein was transiently transfected into HEK293 cells. It was produced by.

[0217] As shown in Figure 14, all mutants were produced and detected by anti-FLAG ELISA. As shown above, anti-C2-5F2.4 bound to huC2b, but not to huC2b bearing mouse S2 (huC2b-mS2). As shown in Figure 15, neither of the reversion point mutations significantly inhibited binding to anti-C2-5F2.4. This suggests that the epitope of this mAb is located on at least two sites in the S2 domain. It is suggested that the protein is composed of amino acids. It binds in the presence of 1.25 mM Ca++ in the buffer. Similar results were obtained when investigating

[0218] As shown in Figure 16, by using publicly available structural data of human C2b, anti-C Ten amino acid positions that may contribute to the epitope of 2-5F2.4 were analyzed. Analysis revealed three possible sequences, each consisting of three amino acids that could contribute to the epitope. The DNA constructs of these cluster mutants were designed. The cluster mutants were designed and obtained by replacing the amino acids of human C2b S2 with the corresponding amino acids of mouse C2b S3. The mutants were created by mutating three amino acids into S2. If these three amino acids contribute to the epitope of anti-C2-5F2.4, binding is lost. Figure 17A shows that the cluster 1 mutants are fully expressed and do not affect binding. Based on the anti-FLAG ELISA, As a result, expression of mutants in cluster 2 was reduced, which led to loss of binding by anti-C2-5F2.4. The mutants in cluster 3 were also poorly expressed, as shown in Figure 17B. This would provide the most plausible explanation for the loss of binding by anti-C2-5F2.4 as shown in Figure 1. Similar results were obtained when binding was examined in the presence of 25 mM Ca++. The amino acid in cluster 1 can be excluded, and the four possible amino acids in cluster 2 and cluster 3 can be excluded. It is narrowed down to acid.

[0219] The domain swap mutants have the epitope on C2 recognized by anti-C2-5F2.4 but not on C2b. These experiments further provided convincing evidence that the sushi domain 2 of These results suggest that the presence of the domain on FB is sufficient for recognition by anti-C2-5F2.4. Considering that anti-C2-5F2.4 does not react with mouse C2, it is possible that human and mouse Sushi 2 Of the 10 amino acids that differ between the domains, at least one must be essential for the epitope. By using single amino acid backmutations, we have identified a single amino acid in Sushi 2. We show that the binding cannot be restored by amino acids. The experiments performed concluded that the amino acids in cluster 1 do not contribute to the epitope of anti-C2-5F2.4. It was suggested that the amino acids in cluster 2 may contribute to the epitope of anti-C2-5F2.4. However, the expression of this mutant is lower than that of the mutants in cluster 1, so the mutants in cluster 2 It cannot be excluded that the folding of the mutants was not optimized. Cluster 3 The mutant was poorly expressed, indicating that the mutation affected folding. likely, and the role of amino acids in cluster 3 remains unclear. do not.

[0220] (Incorporated by reference) All publications and patent documents cited herein are hereby incorporated by reference in their entirety. It is embedded in the The present application provides the following aspects of the invention. (Aspect 1) A monoclonal antibody or an antigen-binding fragment thereof that specifically binds to human complement factor C2. hand: A VH domain comprising the amino acid sequence set forth in SEQ ID NO:3; and The monoclonal antibody comprises a VL domain comprising the amino acid sequence set forth in SEQ ID NO:2. or an antigen-binding fragment thereof. (Aspect 2) The monoclonal antibody according to embodiment 1 or its antigen-binding activity, including a full-length monoclonal antibody. piece. (Aspect 3) 3. The monoclonal antibody of claim 1 or 2, wherein the monoclonal antibody comprises a human IgG heavy chain constant domain. A clonal antibody or an antigen-binding fragment thereof. (Aspect 4) The antibody of embodiment 3, wherein the heavy chain constant domain comprises a human IgG1 heavy chain constant domain. A cloned antibody or an antigen-binding fragment thereof. (Aspect 5)

[0023] According to embodiment 4, the human IgG1 heavy chain constant domain comprises the amino acid sequence set forth in SEQ ID NO:4. A monoclonal antibody or an antigen-binding fragment thereof. (Aspect 6) The antibody of embodiment 3, wherein the heavy chain constant domain comprises a human IgG4 heavy chain constant domain. A cloned antibody or an antigen-binding fragment thereof. (Aspect 7)

[0023] According to embodiment 6, the human IgG4 heavy chain constant domain comprises the amino acid sequence set forth in SEQ ID NO:5. A monoclonal antibody or an antigen-binding fragment thereof. (Aspect 8) The monoclonal antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:6 and a The monoclonal antibody or its derivatives according to embodiment 3, comprising a light chain comprising an amino acid sequence according to embodiment 7. Antigen-binding fragment. (Aspect 9) The monoclonal antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:8 and a The monoclonal antibody or its derivatives according to embodiment 3, comprising a light chain comprising an amino acid sequence according to embodiment 7. Antigen-binding fragment. (Aspect 10) Monoclonal antibody or antigen-binding fragment thereof that specifically binds to human complement factor C2 and a pharmaceutical composition thereof A pharmaceutical composition comprising a pharma- ceutical acceptable carrier, the monoclonal antibody or fragment thereof. but: A VH domain comprising the amino acid sequence set forth in SEQ ID NO:3; and The pharmaceutical composition comprising a VL domain comprising the amino acid sequence set forth in SEQ ID NO:2. (Aspect 11) The monoclonal antibody or antigen-binding fragment thereof comprises a full-length monoclonal antibody. 11. The pharmaceutical composition according to embodiment 10. (Aspect 12) 12. The method of claim 10 or 11, wherein the monoclonal antibody comprises a human IgG heavy chain constant domain. Pharmaceutical composition. (Aspect 13) A nucleic acid encoding the monoclonal antibody or antigen-binding fragment according to any one of aspects 1 to 9. is a plurality of nucleic acid molecules. (Aspect 14) 14. One or more vectors comprising one or more nucleic acid molecules according to embodiment 13. (Aspect 15) 14. A host cell comprising one or more nucleic acid molecules according to embodiment 13. (Aspect 16) 15. A host cell comprising one or more vectors according to embodiment 14. (Aspect 17) 17. The host cell according to any one of aspects 14 to 16, wherein the host cell is a mammalian cell. (Aspect 18) 1. A method for making a monoclonal antibody or antigen-binding fragment thereof, comprising: Under conditions suitable for the expression of the monoclonal antibody or antigen-binding fragment thereof, Cultivating a population of host cells according to any one of claims 1 to 4; and and isolating said monoclonal antibody or antigen-binding fragment from said cell. Law. (Aspect 19) A method for inhibiting the classical pathway of complement activation in a subject, comprising administering to a subject in need thereof an effective amount of the monoclonal antibody or antigen-binding fragment thereof according to any one of aspects 1 to 9, The method comprising administering. (Aspect 20) A method for inhibiting the lectin pathway of complement activation in a subject, comprising administering to said subject a therapeutically effective amount of and administering to the subject an effective amount of the monoclonal antibody or antigen-binding fragment thereof according to any one of aspects 1 to 9. The method comprises administering a piece. (Aspect 21) 21. The method of embodiment 19 or 20, wherein the subject is a human.

Claims

[Claim 1] The novel products, methods and processes substantially as herein described.

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