Antibodies against human complement factor C2b and methods of use

Abstract

The present application relates to antibodies against human complement factor C2b and methods of use. Antibodies and antigen-binding fragments thereof that specifically bind to human complement factor C2 and are capable of inhibiting activation of the classical pathway and lectin pathway of the complement system are provided. The antibodies and antigen binding fragments exhibit improved manufacturability, pharmacokinetics, and antigen sweeping. Also provided are pharmaceutical compositions comprising the antibodies and antigen-binding fragments, nucleic acids and vectors encoding the antibodies and antigen-binding fragments, host cells comprising the nucleic acids or vectors, and methods of making and using the antibodies and antigen-binding fragments. The antibodies and antigen-binding fragments can be used to inhibit the classical pathway of complement activation in a subject, such as a human. The antibodies and antigen-binding fragments may also be used to inhibit complement-activated lectin pathways in a subject, such as a human.

Description

[0001] Related Applications

[0002] This application is a divisional application of the Chinese patent application with application number 201980082826.8 and invention name “Antibodies against human complement factor C2b and methods of use”. The original application is the PCT international application PCT / IB2019 / 060802 filed on December 13, 2019, which entered the Chinese national phase on June 11, 2021.

[0003] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 779,102, filed on December 13, 2018, the entire contents of which are incorporated herein by reference.

[0004] Sequence Listing

[0005] 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 was created on November 22, 2019, is named 618634_AGX5-048PC_ST25.txt, and is 94,329 bytes in size. Technical Field

[0006] The present invention relates to the fields of immunology and molecular biology. More specifically, the present invention relates to compositions and methods for inhibiting activation of the classical pathway and lectin pathway of the complement system, and their use in treating human disorders. The present invention particularly relates to binding molecules that bind to human complement factor C2 and methods for their preparation and use. Background Art

[0007] The complement system involves a cascade of plasma enzymes, regulatory proteins, and proteins with cell lysis capabilities. Prior to activation, multiple complement factors circulate as inactive precursor proteins. Activation of the system leads to an activation cascade in which one factor activates a later factor by specific proteolysis of a complement protein further downstream in the cascade.

[0008] Activation of the complement system can occur through three pathways: the classical (or typical) pathway, the alternative pathway, and the lectin pathway. The classical pathway is activated by the interaction of antigen with IgM, IgG1, IgG2, or IgG3 antibodies to form an immune complex that binds C1q (a subunit of complement component C1). The alternative pathway is activated by immune complexes containing IgA or by recognition of bacteria and other activating surfaces. The lectin pathway is responsible for the antibody-independent pathway of complement activation, which is initiated by the binding of mannan-binding lectin (MBL) (also known as mannose-binding lectin or mannan-binding protein (MBP)) to certain carbohydrates on the surface of various pathogens.

[0009] The activation of the classical pathway begins with the sequential activation of C1, C4 and C2; C2 is then cleaved into C2a and C2b. The activation of the alternative pathway begins with the sequential activation of complement components D, C3 and B. Each pathway cleaves and activates the common central component C3 or the third complement factor, which leads to the activation of the common terminal pathway, resulting in the formation of a membrane attack complex (membrane-attack complex, MAC, containing complement components C5b-9; Muller-Eberhard, Annu Rev Biochem 1988, 57: 321). During complement activation, several inflammatory peptides such as anaphylatoxins C3a and C5a, and MAC are produced. These activation products cause pleiotropic biological effects, such as chemotaxis of leukocytes, degranulation of phagocytes, mast cells and basophils, contraction of smooth muscles, increased vascular permeability and cell lysis (Hugh, Complement 1986, 3: 111). Complement activation products also induce the generation of toxic oxygen free radicals and the synthesis and release of arachidonic acid metabolites and cytokines, particularly by phagocytes, which further amplify the inflammatory response.

[0010] Although complement is an important line of defense against pathogenic organisms, its activation can also cause damage to other healthy host cells. Therefore, it is believed that inhibition of complement activation is beneficial in the treatment and prevention of complement-mediated tissue damage. Therefore, there remains an urgent need in the art for new therapeutic agents that inhibit one or more key components of the complement cascade. Summary of the invention

[0011] New monoclonal anti-human C2b antibodies and antigen-binding fragments thereof having improved characteristics relative to existing antibodies are provided. One feature of the new antibodies is the absence of glycosylation sites in the framework region 3 (FR3) of the heavy chain variable domain (VH). Notably, the new antibodies provide improved homogeneity and therefore improved manufacturability, as well as unexpectedly improved functional properties compared to existing antibodies. The improved functional properties include, for example, increased pI and enhanced potential for so-called antigen sweeping. The antibodies and antigen-binding fragments thereof will be used for human therapy.

[0012] One aspect of the present invention is a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to human complement factor C2, wherein the monoclonal antibody or fragment thereof comprises:

[0013] A VH domain comprising the amino acid sequence shown in SEQ ID NO: 1; and

[0014] A VL domain comprising the amino acid sequence shown in SEQ ID NO: 2;

[0015] Wherein amino acid residues 72 to 74 (Kabat numbering) of the VH domain accordingly consist of X1X2X3, wherein X2 is any amino acid and X1X2X3 is not NX2S or NX2T.

[0016] One aspect of the present invention is a pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment thereof according to the present invention, and a pharmaceutically acceptable carrier.

[0017] One aspect of the invention is a nucleic acid molecule or a plurality of nucleic acid molecules encoding a monoclonal antibody or antigen-binding fragment thereof according to the invention.

[0018] One aspect of the invention is a vector or vectors comprising a nucleic acid molecule or nucleic acid molecules according to the invention.

[0019] One aspect of the invention is a host cell comprising a nucleic acid molecule or a plurality of nucleic acid molecules encoding a monoclonal antibody or antigen-binding fragment thereof according to the invention.

[0020] One aspect of the invention is a host cell comprising a vector or vectors comprising a nucleic acid molecule or molecules according to the invention.

[0021] One aspect of the present invention is a method for preparing a monoclonal antibody or an antigen-binding fragment thereof according to the present invention, the method comprising culturing a cell population according to the present invention under conditions allowing expression of the monoclonal antibody or an antigen-binding fragment thereof.

[0022] One aspect of the invention is a method of inhibiting activation of the classical pathway or the lectin pathway in a subject, comprising administering to a subject in need thereof an effective amount of a monoclonal antibody or antigen-binding fragment thereof according to the invention.

[0023] The following embodiments apply to all aspects of the invention.

[0024] In certain embodiments, X1X2X3 consists of DX2S.

[0025] In certain embodiments, X1X2X3 consists of DKS.

[0026] In certain embodiments, the VH domain comprises the amino acid sequence shown in SEQ ID NO:3.

[0027] In certain embodiments, the VL domain comprises the amino acid sequence shown in SEQ ID NO:2.

[0028] In certain embodiments, the VH domain comprises the amino acid sequence shown in SEQ ID NO:3, and the VL domain comprises the amino acid sequence shown in SEQ ID NO:2.

[0029] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a full-length monoclonal antibody.

[0030] In certain embodiments, the monoclonal antibody comprises a human IgG heavy chain constant domain.

[0031] In certain embodiments, the heavy chain constant domain comprises a human IgG1 heavy chain constant domain.In certain embodiments, the human IgG1 heavy chain constant domain comprises the amino acid sequence shown in SEQ ID NO:4.

[0032] 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 shown in SEQ ID NO:5.

[0033] In certain embodiments, the monoclonal antibody comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO:6 and a light chain comprising the amino acid sequence shown in SEQ ID NO:7.

[0034] In certain embodiments, the monoclonal antibody comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO:8 and a light chain comprising the amino acid sequence shown in SEQ ID NO:7. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1Depicted are polyacrylamide gels loaded with the indicated samples. The larger molecular weight bands of the samples in lanes 4, 5, 8 and 9 (arrows) show the band splitting and migration of antibodies with VH3 and VH4.

[0036] Figure 2 is a graph depicting total levels of the indicated antibodies in cynomolgus monkeys over a 31 day period. The following antibodies were tested: BRO2-glyc-IgG4 (monkeys 1 and 2, glycosylated VH) and BRO2-IgG4 (monkeys 5 and 6, non-glycosylated VH).

[0037] Figures 3A to 3I is a graph depicting free C2 levels in serum over a 31-day period from administration of various monoclonal antibodies to cynomolgus monkeys (plotted as OD 450nm over time). The following antibodies were tested: BRO2-glyc-IgG4 ( Figure 3A ; monkeys 1 and 2), negative control ( Figure 3B ; Monkey 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 ; Monkey 11 and 12), His1-IgG4-NH ( Figure 3G ; Monkey 13 and 14), His1-IgG1-LALA-NH ( Figure 3H ; monkeys 15 and 16) and His2-IgG4 ( Fig. 3I ; Monkeys 17 and 18).

[0038] Figure 4 is a graph depicting the mean free C2 levels in serum over a 31-day period from administration of various indicated monoclonal antibodies to cynomolgus monkeys (plotted as OD 450nm over time).

[0039] Figure 5 is a graph depicting free C2 levels (plotted as OD 450nm over time) in sera of cynomolgus monkeys treated with the indicated aglycosylated antibodies.

[0040] Figures 6A to 6D is a series of graphs depicting free C2 levels in cynomolgus monkeys measured at the indicated times before or after administration of the antibody (plotted as OD 450nm). Figures 3A to 3I In. Fig. 6A , pre and pre plus 500mg / ml BRO-2; Figure 6B , 4 hours and 1 day; Figure 6C , 4 hours and 2 days; Fig.6D, day 11 and day 27. ADA, antidrug antibodies.

[0041] Figures 7A to 7P is a series of graphs depicting the immunogenicity of anti-C2 antibody or negative control monoclonal antibody administered to cynomolgus monkeys over a 30-day period (plotted as OD 450nm). Figures 3A to 3I In. Fig. 7A , Monkey 1; Figure 7B , Monkey 2; Figure 7C , Monkey 5; Fig.7D , Monkey 6; Fig. 7E , Monkey 7; Figure 7F , Monkey 8; Figure 7G , Monkey 9; Figure 7H , Monkey 10; Fig.7I , Monkey 11; Figure 7J , Monkey 12; Figure 7K , Monkey 13; Figure 7L , Monkey 14; Figure 7M , Monkey 15; Figure 7N , Monkey 16; Fig.7O , Monkey 17; Figure 7P , Monkey-18.

[0042] Figures 8A to 8F is a series of graphs depicting the immunogenicity of anti-C2 monoclonal antibodies administered to cynomolgus monkeys over 60 days (plotted as OD 450nm over time). Figures 3A to 3I In. Fig. 8A , Monkey 5; Figure 8B , Monkey 6; Figure 8C , Monkey 9; Fig.8D , Monkey 10; Fig. 8E , Monkey 15; Fig.8F , monkey 16. ADA, antidrug antibody.

[0043] Figures 9A to 9D Depicted is the binding of ARGX-117 to C2 assessed by Western blot analysis and surface plasmon resonance (SPR). Fig.9A Western blot analysis of serum with ARGX-117 is depicted (representative results): Lane 1: MW size marker; Lane 2: recombinant human C2 control (size is approximately 100 kDa); Lane 3: serum; Lane 4: complement activation induced by adding aggregated IgG to serum and incubating at 37°C; Lane 5: serum lacking C2.

[0044] Fig. 9B Depicted is the SPR analysis with C2 immobilized on the chip and different ARGX-117 Fabs as eluents.

[0045] Fig. 9C Depicted are SPR analysis with biotin-C4b and human C2 immobilized to a streptavidin chip, and with and without mAb as eluent; black: no pre-incubation; grey: anti-FXI; control human IgG4 mAb; turquoise: non-inhibitory anti-C2 clone anti-C2-63, clone 63 that recognizes the large subunit of C2 (C2a); red: ARGX-117; all at a molar ratio of 5 to 1; curves are normalized to the signal just before injection of C2 onto the C4b chip.

[0046] Fig.9D Depicted is the SPR analysis with biotin-C4b immobilized to a streptavidin chip and successively human C2 and mAb as eluents; black: running buffer; grey: anti-FXI; control human IgG4 mAb; turquoise: non-inhibitory anti-C2 clone anti-C2-63; red: ARGX-117; curves are normalized just before addition of mAb.

[0047] Fig.10 Schematic representation of domain swap mutants between C2 (SEQ ID NO: 21) and complement factor B (FB) (SEQ ID NO: 50) is depicted. In both proteins, the small fragment (C2b in complement 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 von Willebrand Factor type A (von Willebrand Factor type A, VWFA) domains and a peptidase S1 domain. Note that the sequences between the domains are not carried in these mutants, but 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; C2bS3, 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.

[0048] Fig.11Depicted are the results of anti-FLAG ELISA for domain swap mutants. 5-fold dilutions of supernatant from transfected HEK293 cells were used for coating and anti-FLAG mouse monoclonal Ab combined with HRP-labeled anti-mouse IgG was used for detection.

[0049] Fig.12 Depicted are the results of a 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 for coating, plates were incubated with 20-fold diluted supernatants of HEK293 transfectants, and binding was detected by anti-FLAG Ab. Representative results are from two independent experiments with similar outcomes.

[0050] Fig.13 Depicted is an amino acid sequence alignment of human and mouse C2b Sushi 2 (S2) domains. Human S2, SEQ ID NO: 46; mouse S2, SEQ ID NO: 58. Asterisks indicate sequence identity.

[0051] Fig.14 Depicted are the results of anti-FLAG ELISA for fine mapping mutants. Undiluted supernatants from transfected HEK293 cells were used for coating and biotinylated anti-FLAG mouse monoclonal Ab combined with HRP-labeled SA conjugate was used for detection.

[0052] Fig.15 Results of fine mapping mutants are depicted. Anti-C2-5F2.4 mAb (Human IgG4S241P VH4 / VL3 LC-13 / 03-163A Bioceros) was used for coating, plates were incubated with 20-fold diluted supernatants of HEK293 transfectants and binding was detected by anti-FLAG Ab.

[0053] Fig.16 A plan for cluster mapping mutants using three amino acid mutations for each cluster is depicted, the positions of which are indicated in bold in the human sequence. Each human sequence was mutated to replace the human amino acids in bold with the corresponding mouse amino acids. Human S2, SEQ ID NO: 46; Mouse S2, SEQ ID NO: 58. Asterisks indicate sequence identity.

[0054] Fig.17A and 17B Anti-FLAG ELISA performed on cluster localization mutants is depicted. Fig.17ADepicted are five-fold diluted supernatants from transfected HEK293 cells used for coating, and anti-FLAG mouse monoclonal Ab combined with HRP-labeled anti-mouse IgG was used for detection. GFP, green fluorescent protein.

[0055] Fig. 17B Binding of anti-C2-5F2.4 to cluster mutants is depicted. Anti-C2-5F2.4 mAb (human IgG4 S241PVH4 / VL3, LC-13 / 03-163A, Bioceros) was used as coating, plates were incubated with 20-fold diluted supernatants of HEK293 transfectants, and binding was detected by anti-FLAG Ab. GFP, green fluorescent protein. DETAILED DESCRIPTION

[0056] definition

[0057] "Antibody" or "Immunoglobulin" - The term "immunoglobulin" as used herein includes polypeptides having a combination of two heavy chains and two light chains, whether or not they have any associated specific immunoreactivity. The term "antibody" as used herein refers to such a combination that has significant specific immunoreactivity to the target antigen (e.g., a complex of complement proteins including C2). The term "C2 antibody" is used herein to refer to antibodies that exhibit immunological specificity for a complex of complement proteins including C2, particularly human C2 protein, and domains formed by cleavage of C2, and in some cases species homologs thereof. Antibodies and immunoglobulins comprise light and heavy chains, with or without interchain covalent bonds between them. The basic immunoglobulin structure in vertebrate systems is relatively well understood.

[0058] Five different classes of antibodies (IgG, IgM, IgA, IgD and IgE) can be distinguished biochemically. All five classes of antibodies are within the scope of the present invention. The following discussion is generally directed to immunoglobulin molecules of the IgG class. With regard to IgG, immunoglobulins typically comprise two identical light polypeptide chains of about 23,000 daltons and two identical heavy chains of 53,000 to 70,000 daltons. The four chains are connected by disulfide bonds in a "Y" configuration, with the light chain starting from the mouth of the "Y" and continuing through the variable region to bracket the heavy chain.

[0059] The light chains of antibodies are classified as kappa (κ) or lambda (λ). Each heavy chain category can be combined with a κ or λ light chain. Typically, the light chain and heavy chain are covalently bound to each other, and when immunoglobulins are produced by hybridomas, B cells, or genetically modified host cells, the "tails" of the two heavy chains are bound to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus of the forked end of the Y configuration to the C-terminus at the bottom of each chain. It will be appreciated by those skilled in the art that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, or ε), with some subclasses (e.g., γ1-γ4) among them. The properties of the chain determine the "class" of the antibody as IgG, IgM, IgA, IgD, or IgE, respectively. Immunoglobulin subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA1, etc. have been well characterized and are known to confer functional specialization. Modified forms of each of these classes and isotypes are readily discernible to the skilled artisan in view of the instant disclosure and are therefore within the scope of the instant invention.

[0060] As mentioned above, the variable region of an antibody allows the antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the VL domain and VH domain of an antibody combine to form a variable region that defines a three-dimensional antigen binding site. This quaternary antibody structure forms an antigen binding site present at the end of each arm of the Y. More specifically, the antigen binding site is defined by three complementary determining regions (CDRs) on each of the VH and VL chains.

[0061] "Binding molecule" - The term "binding molecule" as used herein is a general term intended to cover antibodies and antigen-binding fragments thereof according to the present disclosure.

[0062] "Binding site" - As used herein, the term "binding site" comprises a region of a polypeptide responsible for selective binding to a target antigen of interest. A binding domain comprises at least one binding site. Exemplary binding domains include antibody variable domains. Antibody molecules of the invention may comprise a single binding site or multiple (e.g., two, three, or four) binding sites.

[0063] "Variable Region" or "Variable Domain" - The term "variable" refers to the fact that certain portions of the variable domains, VH and VL, differ extensively in sequence between antibodies and are used in the binding and specificity of each particular antibody for its target antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called "hypervariable loops" in each of the VL and VH domains that form part of the antigen binding site. λThe first, second and third hypervariable loops of the light chain domain are referred to herein as L1 (λ), L2 (λ) and L3 (λ), and can be defined as comprising residues 24 to 33 (L1 (λ), consisting of 9, 10 or 11 amino acid residues), 49 to 53 (L2 (λ), consisting of 3 residues) and 90 to 96 (L3 (λ), consisting of 5 residues) in the VL domain (Morea et al., Methods 20: 267-279 (2000)). κ The first, second and third hypervariable loops of the light chain domain are referred to herein as L1 (κ), L2 (κ) and L3 (κ), and can be defined as comprising residues 25 to 33 (L1 (κ), consisting of 6, 7, 8, 11, 12 or 13 residues), 49 to 53 (L2 (κ), consisting of 3 residues) and 90 to 97 (L3 (κ), consisting of 6 residues) in the VL domain (Morea et al., Methods 20:267-279 (2000)). The first, second and third hypervariable loops of the VH domain are referred to herein as H1, H2 and H3, and can be defined as comprising residues 25 to 33 (H1, consisting of 7, 8 or 9 residues), 52 to 56 (H2, consisting of 3 or 4 residues) and 91 to 105 (H3, highly variable in length) in the VH domain (Morea et al., Methods 20:267-279 (2000)).

[0064] Unless otherwise indicated, the terms L1, L2, and L3 refer to the first, second, and third hypervariable loops of the VL domain, respectively, and encompass the entire VL domain. κ and V λ Hypervariable loops obtained from both isotypes. The terms H1, H2, and H3 refer to the first, second, and third hypervariable loops of a VH domain, respectively, and encompass hypervariable loops obtained from any known heavy chain isotype, including gamma, mu, alpha, delta, or epsilon.

[0065] Hypervariable loops L1, L2, L3, H1, H2 and H3 may each comprise a portion of a "complementarity determining region" or "CDR", as defined below. The terms "hypervariable loop" and "complementarity determining region" are not strictly synonymous, because hypervariable loops (HV) are defined based on structure, while complementarity determining regions (CDRs) are defined based on sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition. Public Health Service, National Institutes of Health, Bethesda, MD., 1983) and the limits of HV and CDR may be different in some VH and VL domains.

[0066] The CDRs of VL and VH domains can generally be defined as comprising the following amino acids: residues 24 to 34 (LCDR1), 50 to 56 (LCDR2), and 89 to 97 (LCDR3) in the light chain variable domain, and residues 31 to 35 or 31 to 35b (HCDR1), 50 to 65 (HCDR2), and 95 to 102 (HCDR3) in the heavy chain variable domain; (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Therefore, HV can be contained within the corresponding CDRs, and references to the "hypervariable loops" of VH and VL domains herein should be interpreted as also covering the corresponding CDRs, and vice versa, unless otherwise indicated.

[0067] The more highly conserved parts of the variable domains are called framework regions (FRs), as defined below. The variable domains of native heavy and light chains each contain four FRs (FR1, FR2, FR3, and FR4, respectively), mainly in a β-folded configuration, which is connected by three hypervariable loops. The hypervariable loops in each chain are closely held together by FRs, and together with the hypervariable loops from another chain, contribute to the formation of the antigen binding site of the antibody. The structural analysis of antibodies reveals the relationship between the sequence and the shape of the binding site formed by the complementary determining region (Chothia et al., J.Mol.Biol.227:799-817 (1992)); Tramontano et al., J.Mol.Biol, 215:175-182 (1990)). Although it has high sequence variability, five of the six loops only adopt a small part in the main chain conformation, referred to as "canonical structure". These conformations are determined firstly by the length of the loop and secondly by the presence of key residues at certain positions within the loop and in the framework regions that dictate the conformation by their ability to pack, hydrogen bond, or assume unusual main-chain conformations.

[0068] "Framework Region" - The term "framework region" or "FR region" as used herein includes amino acid residues that are part of the variable region but not part of the CDR (e.g., using the Kabat definition of CDR). Thus, the variable region framework is about 100 to 120 amino acids in length, but only includes those amino acids outside the CDR. For the specific example of the heavy chain variable domain and the CDRs defined by Kabat et al., framework region 1 corresponds to the variable region domain covering amino acids 1 to 30; framework region 2 corresponds to the variable region domain covering amino acids 36 to 49; framework region 3 corresponds to the variable region domain covering amino acids 66 to 94; and framework region 4 corresponds to the variable region domain from amino acid 103 to the end of the variable region. The framework region of the light chain is similarly separated by each light chain variable region CDR. Similarly, using the CDR definitions of Chothia et al. or McCallum et al., the framework region boundaries are separated by the ends of the corresponding CDRs as described above. In some preferred embodiments, the CDRs are as defined by Kabat.

[0069] In naturally occurring antibodies, the six CDRs present in each monomer antibody are short, non-continuous amino acid sequences that are specifically positioned to form an antigen binding site when the antibody presents its three-dimensional configuration in an aqueous environment. The rest of the heavy and light chain variable domains show less intermolecular variability in the amino acid sequence and are referred to as framework regions. The framework region mainly adopts a β-folded configuration, and the CDR forms a loop connecting the β-folded structure and forms a part of the β-folded structure in some cases. Therefore, these framework regions play a role in forming a scaffold that positions the six CDRs in the correct direction by non-covalent interactions between chains. The antigen binding site formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. The complementary surface promotes the non-covalent binding of the antibody to the immunoreactive antigen epitope. One of ordinary skill in the art can easily identify the position of the CDR.

[0070] "Aglycosylated" - As used herein, the term "aglycosylated" refers to a form of an antibody or antigen-binding fragment thereof that lacks glycosylation at a potential glycosylation site in the antibody or antigen-binding fragment. In certain embodiments, the term "aglycosylated" refers to a form of an antibody or antigen-binding fragment thereof that lacks glycosylation at a potential N-linked glycosylation site in the antibody or antigen-binding fragment. In certain embodiments, the term "aglycosylated" refers to a form of an antibody or antigen-binding fragment thereof that lacks glycosylation at a potential N-linked glycosylation site in the heavy chain variable region.

[0071] "Constant region" - As used herein, the term "constant region" refers to the part of the antibody molecule outside the variable domain or variable region. The immunoglobulin light chain has a single domain "constant region", which is usually called the "CL or CL1 domain". This domain is located at the C-terminus of the VL domain. The constant region of the immunoglobulin heavy chain varies according to the immunoglobulin class (γ, μ, α, δ, ε). The heavy chains γ, α and δ have a constant region composed of three immunoglobulin domains (called CH1, CH2 and CH3) and a flexible hinge region that separates the CH1 and CH2 domains. The heavy chains μ and ε have a constant region composed of four domains (CH1 to CH4). The constant domain of the heavy chain is located at the C-terminus of the VH domain.

[0072] The numbering of amino acids in immunoglobulin heavy and light chains extends from the N-terminus of the forked end of the Y configuration to the C-terminus at the bottom of each chain. Different numbering schemes are used to define the constant domains of immunoglobulin heavy and light chains. According to the EU numbering scheme, the heavy chain constant domain of the IgG molecule is identified as follows: CH1-amino acid residues 118 to 215; CH2-amino acid residues 231 to 340; CH3-amino acid residues 341 to 446. The "hinge region" includes the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain. The hinge region contains about 25 residues and is flexible, thereby allowing the two N-terminal antigen binding regions to move independently. The hinge region can be subdivided into three different domains: upstream, middle and downstream hinge domains (Roux KHetal.J.Immunol.161:4083-90 1998). The antibody of the present invention comprising a "full human" hinge region may include one of the hinge region sequences shown in Table 1 below.

[0073] Table 1. Human hinge sequences

[0074]

[0075] "Fragment" - The term "fragment" used in the context of the antibodies of the present invention refers to a segment or portion of an antibody or antibody chain that contains fewer amino acid residues than a complete or full antibody or antibody chain. The term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that specifically binds to an antigen or competes with a complete antibody (i.e., the complete antibody from which it is derived) for specific binding to an antigen (e.g., specifically binds to a C2 protein or a portion thereof). The term "fragment" of an antibody molecule used herein includes an antigen-binding fragment of an antibody, for example, an antibody light chain variable domain (VL), an antibody heavy chain variable domain (VH), a single-chain antibody (scFv), a F(ab')2 fragment, a Fab fragment, a Fd fragment, a Fv fragment, a one-armed (monovalent) antibody, a diabody, a tribody, a tetrabody, or any antigen-binding molecule formed by combining, assembling or conjugating such antigen-binding fragments. The term "antigen-binding fragment" used herein is also intended to encompass antibody fragments selected from monomers (unibody), domain antibodies, and nanobodies (nanobody). Fragments can be obtained, for example, by chemical or enzymatic treatment of a complete or full antibody or antibody chain or by recombinant means.

[0076] Complement component C2

[0077] The second component of human complement (C2) is a 90 to 100 kDa glycoprotein that participates in both the classical and lectin pathways of complement activation. C2 can be activated by C1s of the classical pathway or by MASP2 of the lectin pathway. C2 binds to surface-bound C4b (in the presence of Mg 2+ C4b) binds to form the C4bC2 complex, which is subsequently cleaved by activated C1s or MASP2 into two fragments: a larger 70 kDa fragment, traditionally referred to as C2a, which remains attached to C4b to form the C3 convertase C4bC2a; and a smaller 30 kDa N-terminal fragment, traditionally referred to as C2b, which is released into the fluid phase. Some authors have recently interchanged the names of C2a and C2b, so that C2b refers to the larger 70 kDa fragment and C2a refers to the smaller 30 kDa fragment. As used herein, C2a shall refer to the larger 70 kDa fragment and C2b shall refer to the smaller 30 kDa fragment. Once C2a is activated and binds to C4b, it constitutes the catalytic subunit of the C3 and C5 convertases capable of cleaving C3 and C5, respectively.

[0078] The amino acid sequence of human C2 is known (GenBank Accession No.: NM_000063) and is shown as SEQ ID NO:21.

[0079] Amino acid sequence of human C2 (SEQ ID NO: 21):

[0080]

[0081] Like many other plasma proteins, C2 has a modular structure. Starting from its N-terminus, C2 consists of three complement control protein modules (CCP1-3, also known as short consensus repeats (SCR) or sushi domain repeats), a von Willebrand factor type A (vWFA) domain containing a metal ion-dependent adhesion site, and a serine protease (serine protease, SP) domain (Arlaud et al., Adv Immunol 1998, 69: 249). Electron microscopy studies have shown that C2 consists of three domains. The three CCP modules (CCP1-3) together form the N-terminal domain, which corresponds to C2b. The vWFA domain constitutes the second domain, and the SP domain constitutes the third domain. The second and third domains together constitute the larger C2a portion of the molecule.

[0082] The CCP module is a common structural motif present in many proteins. These globular units are composed of about 60 amino acid residues and folded into a compact six to eight-chain β-sheet structure built around four invariant disulfide-bonded cysteine ​​residues (Norman et al., J Mol Biol 1991, 219: 717). Adjacent CCP modules are covalently linked by poorly conserved joints.

[0083] Initial binding of C2 to surface-bound C4b is mediated by two low-affinity sites: one on C2b (Xu & Volanakis, J Immunol 1997, 158:5958) and the other on the vWFA domain of C2a (Horiuchi et al., J Immunol 1991, 47:584). Although the crystal structures of C2b and C2a have been determined, The C2 amino acid residues involved in the interaction with C4 remain to be determined (Krishnan et al., Acta Cristallogr D Biol. Crystallogr 2009, D65: 266).

[0084] Anti-C2 antibody

[0085] One aspect of the present invention is a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to human complement factor C2, wherein the monoclonal antibody or fragment thereof comprises:

[0086] A VH domain comprising the amino acid sequence shown in SEQ ID NO: 1; and

[0087] A VL domain comprising the amino acid sequence shown in SEQ ID NO: 2;

[0088] Wherein amino acid residues 72 to 74 (Kabat numbering) of the VH domain accordingly consist of X1X2X3, wherein X2 is any amino acid and X1X2X3 is not NX2S or NX2T.

[0089] The VH domain comprises the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3. The VL domain comprises CDRs LCDR1, LCDR2 and LCDR3. The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in Table 2.

[0090] Table 2. CDR

[0091]

[0092] In certain embodiments, the monoclonal antibody or antigen binding fragment thereof specifically binds to human complement factor C2b. In certain embodiments, the monoclonal antibody or antigen binding fragment thereof specifically binds to an epitope in a portion of human complement factor C2 corresponding to human complement factor C2b.

[0093] In certain embodiments, the variable domain of the heavy chain is non-glycosylated. In certain embodiments, the amino acid sequence of the variable domain of the heavy chain does not contain a potential glycosylation site characterized by the sequence NXS / T, wherein N represents asparagine, X represents any amino acid, and S / T represents serine or threonine. Therefore, in certain embodiments, an antibody having a VH domain comprising the sequence NXS / T can be modified so that these residues are composed of X1X2X3, respectively, wherein X2 is any amino acid, and X1X2X3 is not NX2S or NX2T. That is, X1 can be any amino acid except N, and / or X3 can be any amino acid except S or T. In certain embodiments, an antibody having a VH domain comprising the sequence NXS or NXT can be modified so that these three residues are composed of DXS, respectively. In certain other embodiments, an antibody having a VH domain comprising the sequence NXS or NXT can be modified so that these three residues are composed of DXT, respectively.

[0094] In certain embodiments, the heavy chain amino acids at residues 72 to 74 (Kabat numbering) consist of X1X2X3, respectively, wherein X2 is any amino acid and X1X2X3 is not NX2S or NX2T.

[0095] In certain embodiments, the heavy chain amino acids at residues 72 to 74 (Kabat numbering) consist of DX2S.

[0096] In certain embodiments, the heavy chain amino acids at residues 72 to 74 (Kabat numbering) consist of DKS.

[0097] In certain embodiments, the VH domain comprises the amino acid sequence shown in SEQ ID NO:3.

[0098] In certain embodiments, the amino acid sequence of the VH domain consists of the sequence shown in SEQ ID NO:3.

[0099] In certain embodiments, the VL domain comprises the amino acid sequence shown in SEQ ID NO:2.

[0100] In certain embodiments, the amino acid sequence of the VL domain consists of the sequence shown in SEQ ID NO:2.

[0101] In certain embodiments, the VH domain comprises the amino acid sequence shown in SEQ ID NO:3, and the VL domain comprises the amino acid sequence shown in SEQ ID NO:2.

[0102] In certain embodiments, the amino acid sequence of the VH domain consists of the sequence shown in SEQ ID NO:3, and the amino acid sequence of the VL domain consists of the sequence shown in SEQ ID NO:2.

[0103] The amino acid sequences of SEQ ID NO:3 and SEQ ID NO:2 are shown in Table 3. SEQ ID NO:2 corresponds to the VL (VK3) domain of humanized 5F2.4 (BRO2) disclosed in U.S. Patent No. 9,944,717 to Broteio Pharma BV. Also shown in Table 3, SEQ ID NO:28 corresponds to the VH (VH4) domain of humanized 5F2.4 (BRO2) disclosed in U.S. Patent No. 9,944,717, which is incorporated herein by reference.

[0104] Table 3. VH and VL domains

[0105]

[0106] In certain embodiments, the monoclonal antibodies of the invention comprise the CH1 domain, hinge domain, CH2 domain, and CH3 domain of a human antibody, particularly human IgG1, IgG2, IgG3, or IgG4.

[0107] In certain embodiments, the antibody comprises a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain of human IgG1, and comprises the substitutions L234A and L235A in the CH2 domain.

[0108] In certain embodiments, the antibody comprises a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain of human IgG1, and comprises the substitutions H433K and N434F in the CH3 domain.

[0109] In certain embodiments, the antibody comprises a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain of human IgG1, and comprises substitutions L234A and L235A in the CH2 domain and substitutions H433K and N434F in the CH3 domain.

[0110] In certain embodiments, the antibody comprises a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain of human IgG4. In certain embodiments, the antibody comprises a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain of human IgG4, and comprises the substitution S228P in the hinge domain.

[0111] In certain embodiments, the antibody comprises a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain of human IgG4, and comprises the substitution L445P in the CH3 domain.

[0112] In certain embodiments, the antibody comprises a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain of human IgG4, and comprises both the substitution S228P in the hinge domain and the substitution L445P in the CH3 domain.

[0113] In certain embodiments, the antibody comprises a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain of human IgG4, and comprises the substitutions H433K and N434F in the CH3 domain.

[0114] In certain embodiments, the antibody comprises a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain of human IgG4, and comprises the substitution S228P in the hinge domain and the substitutions H433K and N434F in the CH3 domain.

[0115] In certain embodiments, the antibody comprises a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain of human IgG4, and comprises the substitutions H433K, N434F, and L445P in the CH3 domain.

[0116] In certain embodiments, the antibody comprises a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain of human IgG4, and comprises the substitution S228P in the hinge domain and the substitutions H433K, N434F, and L445P in the CH3 domain.

[0117] In certain embodiments, the monoclonal antibody comprises a human IgG heavy chain constant domain. In certain embodiments, the heavy chain constant domain comprises a human IgG1 heavy chain constant domain. In certain embodiments, the heavy chain constant domain consists of a human IgG1 heavy chain constant domain.

[0118] In certain embodiments, the heavy chain constant domain comprises a human IgG1 heavy chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 29. In certain embodiments, the amino acid sequence of the heavy chain constant domain consists of the sequence shown in SEQ ID NO: 29.

[0119] In certain embodiments, the heavy chain constant domain comprises a human IgG1 heavy chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 4. In certain embodiments, the amino acid sequence of the heavy chain constant domain consists of the sequence shown in SEQ ID NO: 4.

[0120] In certain embodiments, the heavy chain constant domain comprises a human IgG4 heavy chain constant domain. In certain embodiments, the heavy chain constant domain consists of a human IgG4 heavy chain constant domain.

[0121] In certain embodiments, the heavy chain constant domain comprises a human IgG4 heavy chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 30. In certain embodiments, the amino acid sequence of the heavy chain constant domain consists of the sequence shown in SEQ ID NO: 30.

[0122] In certain embodiments, the heavy chain constant domain comprises a human IgG4 heavy chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 31. In certain embodiments, the amino acid sequence of the heavy chain constant domain consists of the sequence shown in SEQ ID NO: 31.

[0123] In certain embodiments, the heavy chain constant domain comprises a human IgG4 heavy chain constant domain comprising the amino acid sequence shown in SEQ ID NO: 5. In certain embodiments, the amino acid sequence of the heavy chain constant domain consists of the sequence shown in SEQ ID NO: 5.

[0124] The amino acid sequences of SEQ ID NOs: 4, 5, and 29 to 31 are shown in Table 4.

[0125] Table 4. Heavy chain constant domain

[0126]

[0127] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a full-length monoclonal antibody.

[0128] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof consists of a full-length monoclonal antibody.

[0129] In certain embodiments, provided herein are monoclonal antibodies comprising a heavy chain having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 32. In certain embodiments, provided herein are monoclonal antibodies comprising a heavy chain having 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 32. In certain embodiments, provided herein are monoclonal antibodies comprising a light chain having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, provided herein are monoclonal antibodies comprising a light chain having 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, provided herein are monoclonal antibodies comprising a heavy chain having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 32, and a light chain having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, provided herein are monoclonal antibodies comprising a heavy chain having 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 32, and a light chain having 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7.

[0130] In certain embodiments, provided herein are monoclonal antibodies comprising a heavy chain having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, provided herein are monoclonal antibodies comprising a heavy chain having 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, provided herein are monoclonal antibodies comprising a light chain having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, provided herein are monoclonal antibodies comprising a light chain having 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, provided herein are monoclonal antibodies comprising a heavy chain having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6 and a light chain having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, provided herein are monoclonal antibodies comprising a heavy chain having 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6 and a light chain having 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7.

[0131] In certain embodiments, provided herein are monoclonal antibodies comprising a heavy chain having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 33. In certain embodiments, provided herein are monoclonal antibodies comprising a heavy chain having 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 33. In certain embodiments, provided herein are monoclonal antibodies comprising a heavy chain having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 33 and a light chain having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, provided herein are monoclonal antibodies comprising a heavy chain having 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 33 and a light chain having 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7.

[0132] In certain embodiments, provided herein are monoclonal antibodies comprising a heavy chain having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, provided herein are monoclonal antibodies comprising a heavy chain having 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, provided herein are monoclonal antibodies comprising a heavy chain having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 34 and a light chain having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, provided herein are monoclonal antibodies comprising a heavy chain having 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 34 and a light chain having 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7.

[0133] In certain embodiments, provided herein are monoclonal antibodies comprising a heavy chain having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, provided herein are monoclonal antibodies comprising a heavy chain having 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, provided herein are monoclonal antibodies comprising a heavy chain having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8 and a light chain having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, provided herein are monoclonal antibodies comprising a heavy chain having 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8 and a light chain having 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7.

[0134] The amino acid sequences of SEQ ID NOs: 6 to 8 and 32 to 34 are shown in Table 5.

[0135] Table 5. Heavy and light chains

[0136]

[0137]

[0138] For some embodiments in which the heavy and / or light chains of an antibody are defined by a particular percentage sequence identity to a reference sequence, the heavy and / or light chains may retain CDR sequences that are identical to those present in the reference sequence such that the variations exist only outside of the CDR regions.

[0139] Unless otherwise specified in the present application, the % sequence identity between two amino acid sequences can be determined by comparing the two sequences arranged in an optimal manner, and wherein the amino acid sequence to be compared may comprise additions or deletions relative to the reference sequence for optimal alignment between the two sequences. The identity percentage is calculated as follows: the number of identical positions where the amino acid residues are identical between the two sequences is determined, the number of identical positions is divided by the total number of positions in the comparison window and the result is multiplied by 100 to obtain the identity percentage between the two sequences. For example, the BLAST program "BLAST 2sequences" (Tatusova et al, "Blast 2sequences-anew tool forcomparing protein and nucleotide sequences", FEMS Microbiol Lett. 174: 247-250) can be used, the parameters used are those given by default (particularly the parameters "open gap penalty": 5, and "extended gap penalty": 2; the selected matrix is, for example, the matrix "BLOSUM 62" proposed by the program), and the identity percentage between the two sequences to be compared is directly calculated by the program.

[0140] In some non-limiting embodiments, the antibodies of the invention may comprise a CH1 domain and / or a CL domain (from the heavy chain and the light chain, respectively) whose amino acid sequence is completely or substantially human. In the case where the antibody or antigen-binding fragment of the invention is an antibody intended for human therapeutic use, the entire constant region of the antibody or at least a portion thereof typically has a completely or substantially human amino acid sequence. Thus, one or more or any combination of the CL domain, CH1 domain, hinge region, CH2 domain, CH3 domain and CH4 domain (if present) may be completely or substantially human in terms of their amino acid sequence.

[0141] Advantageously, the CL domain, CH1 domain, hinge region, CH2 domain, CH3 domain and CH4 domain (if present) may all have completely or substantially human amino acid sequences. In the context of the constant region of a humanized or chimeric antibody or antibody fragment, the term "substantially human" refers to an amino acid sequence identity of at least 90%, or at least 92%, or at least 95%, or at least 97%, or at least 99% with a human constant region. In this context, the term "human amino acid sequence" refers to an amino acid sequence encoded by a human immunoglobulin gene, including germline, rearranged and somatically mutated genes. The present invention also contemplates polypeptides comprising "human" sequence constant domains that have been altered relative to a human sequence by making one or more amino acid additions, deletions or substitutions, except for those embodiments that explicitly require the presence of a "fully human" hinge region.

[0142] The presence of a "fully human" hinge region in the C2-binding antibodies of the invention may be beneficial both for minimizing immunogenicity and for optimizing antibody stability.

[0143] C2 binding antibodies can be modified in the Fc region to improve the binding affinity to neonatal Fc receptor FcRn. The improved binding affinity can be measurable at acidic pH (e.g., from about pH 5.5 to about pH 6.0). The improved binding affinity can also be measurable at neutral pH (e.g., from about pH 6.9 to about pH 7.4). In this embodiment, "improved binding affinity" means to improve the binding affinity to FcRn relative to the binding affinity of the unmodified Fc region. Typically, the unmodified Fc region will have the wild-type amino acid sequence of human IgG1, IgG2, IgG3 or IgG4. In some such embodiments, the antibody molecule with a modified Fc region will measure the improved binding affinity to FcRn relative to the binding affinity of wild-type IgG1, IgG2, IgG3 or IgG4 to FcRn.

[0144] C2 binding antibodies can be modified in the Fc region to improve the binding affinity to human neonatal Fc receptor FcRn. The improved binding affinity can be measurable at acidic pH (e.g., from about pH 5.5 to about pH 6.0). The improved binding affinity can also be measurable at neutral pH (e.g., from about pH 6.9 to about pH 7.4). In this embodiment, "improved binding affinity" means to improve the binding affinity to human FcRn relative to the binding affinity of the unmodified Fc region. Typically, the unmodified Fc region will have the wild-type amino acid sequence of human IgG1, IgG2, IgG3 or IgG4. In some such embodiments, the antibody molecule with a modified Fc region will measure the improved binding affinity to human FcRn relative to the binding affinity of wild-type IgG1, IgG2, IgG3 or IgG4 to human FcRn.

[0145] Pharmaceutical composition

[0146] One aspect of the present invention is a pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to human complement factor C2, and a pharmaceutically acceptable carrier, wherein the monoclonal antibody or fragment thereof comprises:

[0147] A VH domain comprising the amino acid sequence shown in SEQ ID NO: 1; and

[0148] A VL domain comprising the amino acid sequence shown in SEQ ID NO: 2;

[0149] Wherein amino acid residues 72 to 74 (Kabat numbering) of the VH domain accordingly consist of X1X2X3, wherein X2 is any amino acid and X1X2X3 is not NX2S or NX2T.

[0150] The pharmaceutical composition of the present invention can be formulated together with a pharmaceutically acceptable carrier or diluent and any other known adjuvants and excipients according to conventional techniques such as those disclosed in (Remington: The Science and Practice of Pharmacy, 19th edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995).

[0151] The term "pharmaceutically acceptable carrier" refers to an inherently non-toxic carrier or excipient. Some examples of such excipients include, but are not limited to, saline, Ringer's solution, dextrose solution, and Hanks' solution. Non-aqueous excipients such as fixed oils and ethyl oleate may also be used.

[0152] Pharmaceutical compositions must generally be sterile and stable under manufacturing and storage conditions. The composition may be formulated into solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. Some examples of suitable aqueous and non-aqueous carriers that may be used for pharmaceutical compositions of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof; vegetable oils (e.g., olive oil); and injectable organic esters (e.g., ethyl oleate). Appropriate fluidity may be maintained, for example, by using a coating material (e.g., lecithin), by maintaining a desired particle size in the case of a dispersion, and by using a surfactant to maintain appropriate fluidity.

[0153] The pharmaceutical composition may also include excipients such as preservatives, wetting agents, emulsifiers and dispersants. The presence of microorganisms can be prevented by sterilization procedures and by including a variety of antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. It is also desirable to include isotonic agents in the composition, such as sugars, polyols such as mannitol, sorbitol, glycerol, or sodium chloride. Pharmaceutically acceptable antioxidants may also be included, such as: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0154] Sterile injectable solutions can be prepared by incorporating the desired amount of monoclonal antibody into a suitable solvent together with, for example, one or a combination of the ingredients listed above (as required), followed by sterilization microfiltration. Typically, dispersions are prepared by incorporating the active compound into a sterile carrier containing a basic dispersion medium and desired other ingredients, such as those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze drying (lyophilization), which produce a powder of the active ingredient plus any additional desired ingredients from its previously sterile filtered solution.

[0155] The pharmaceutical composition is preferably administered parenterally, preferably by intravenous (iv) or subcutaneous (sc) injection or infusion.

[0156] As used herein, the phrases "parenteral administration" and "administered parenterally" mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intraperitoneal, subcutaneous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0157] Prolonged absorption of the injectable anti-C2 mAb or fragment thereof can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.

[0158] The mAb or its fragment can be prepared with a carrier that will protect the compound from rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations are generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0159] The pharmaceutical compositions can be administered using medical devices known in the art.

[0160] The dosage regimen is adjusted to provide the optimum desired response (e.g., a therapeutic response).For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0161] Actual dosage levels of the mAb or fragments thereof in the pharmaceutical compositions of the invention may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response in a particular patient without being toxic to the patient.

[0162] In one embodiment, the binding molecules, in particular antibodies, according to the invention can be administered at 10 to 500 mg / m 2 , for example 200 to 400 mg / m 2 The weekly dose of 1% oxadiazine is administered by infusion. Such administration may be repeated, for example 1 to 8 times, for example 3 to 5 times. Administration may be performed by continuous infusion over a period of 1 to 24 hours, for example 2 to 12 hours. In some embodiments, administration may be performed by one or more bolus injections.

[0163] In one embodiment, the binding molecules according to the present invention, particularly antibodies, can be administered by infusion at a weekly dose of 1 to 50 mg / kg body weight (mg / kg), for example 5 to 25 mg / kg. Such administration can be repeated, for example 1 to 8 times, for example 3 to 5 times. Administration can be performed by continuous infusion over a period of 1 to 24 hours, for example 2 to 12 hours. In some embodiments, administration can be performed by one or more bolus injections.

[0164] In another embodiment, the mAb or fragment thereof or any other binding molecule disclosed in the present invention can be administered as a maintenance therapy, for example, once a week for 6 months or longer.

[0165] Nucleic acid molecules and vectors

[0166] One aspect of the present invention is a nucleic acid molecule or multiple nucleic acid molecules encoding a monoclonal antibody or its antigen-binding fragment according to the present invention. In certain embodiments, a single nucleic acid molecule encodes both the VH and VL domains of a monoclonal antibody or its antigen-binding fragment according to the present invention. In certain embodiments, a single nucleic acid molecule encodes both the heavy chain (HC) and the light chain (LC) of a monoclonal antibody or its antigen-binding fragment according to the present invention. In certain embodiments, a first nucleic acid molecule encodes the VH domain of a monoclonal antibody or its antigen-binding fragment according to the present invention, and a second nucleic acid molecule encodes the VL domain. In certain embodiments, a first nucleic acid molecule encodes the heavy chain (HC) of a monoclonal antibody or its antigen-binding fragment according to the present invention, and a second nucleic acid molecule encodes the light chain (LC).

[0167] In certain embodiments, the nucleic acid molecule encoding the VH domain comprises the nucleic acid sequence shown in SEQ ID NO:35.

[0168] In certain embodiments, the nucleic acid molecule encoding the VL domain comprises the nucleic acid sequence shown in SEQ ID NO:36.

[0169] In certain embodiments, the nucleic acid molecule encoding HC comprises the nucleic acid sequence shown in SEQ ID NO:37.

[0170] In certain embodiments, the nucleic acid molecule encoding HC comprises the nucleic acid sequence shown in SEQ ID NO:38.

[0171] In certain embodiments, the nucleic acid molecule encoding HC comprises the nucleic acid sequence shown in SEQ ID NO:39.

[0172] In certain embodiments, the nucleic acid molecule encoding HC comprises the nucleic acid sequence shown in SEQ ID NO:40.

[0173] In certain embodiments, the nucleic acid molecule encoding HC comprises the nucleic acid sequence shown in SEQ ID NO:41.

[0174] In certain embodiments, the nucleic acid molecule encoding the LC domain comprises the nucleic acid sequence shown in SEQ ID NO:42.

[0175] In certain embodiments, the nucleic acid sequence of the nucleic acid molecule encoding the VH domain consists of the sequence shown in SEQ ID NO:35.

[0176] In certain embodiments, the nucleic acid sequence of the nucleic acid molecule encoding the VL domain consists of the sequence shown in SEQ ID NO:36.

[0177] In certain embodiments, the nucleic acid sequence of the nucleic acid molecule encoding HC consists of the sequence shown in SEQ ID NO:37.

[0178] In certain embodiments, the nucleic acid sequence of the nucleic acid molecule encoding HC consists of the sequence shown in SEQ ID NO:38.

[0179] In certain embodiments, the nucleic acid sequence of the nucleic acid molecule encoding HC consists of the sequence shown in SEQ ID NO:39.

[0180] In certain embodiments, the nucleic acid sequence of the nucleic acid molecule encoding HC consists of the sequence shown in SEQ ID NO:40.

[0181] In certain embodiments, the nucleic acid sequence of the nucleic acid molecule encoding HC consists of the sequence shown in SEQ ID NO:41.

[0182] In certain embodiments, the nucleic acid sequence of the nucleic acid molecule encoding the LC domain consists of the sequence shown in SEQ ID NO:42.

[0183] The nucleic acid sequences corresponding to SEQ ID NOs: 35 to 42 are shown in Table 6.

[0184] Table 6. Nucleic acid sequences of VH, VL, HC and LC

[0185]

[0186]

[0187]

[0188]

[0189] For SEQ ID NO: 35 and 39, a217g generates the N72D mutation

[0190] The present invention also provides gene delivery vehicles or vectors comprising nucleic acid molecules according to the present invention. The gene delivery vehicle or vector can be a plasmid or other nucleic acid replicated by bacteria. Such a gene delivery vehicle or vector can be easily transferred to, for example, production cells. The gene delivery vehicle can also be a viral vector. Some preferred viral vectors are adenoviral vectors, lentiviral vectors, adeno-associated viral vectors and retroviral vectors.

[0191] The present invention also provides a vector comprising a nucleic acid molecule or multiple nucleic acid molecules according to the present invention. In certain embodiments, a single vector comprises a single nucleic acid molecule encoding both the VH and VL domains of a monoclonal antibody or its antigen-binding fragment according to the present invention. In certain embodiments, a single vector comprises a single nucleic acid molecule encoding both the heavy chain (HC) and light chain (LC) of a monoclonal antibody or its antigen-binding fragment according to the present invention.

[0192] In certain embodiments, the first vector comprises a first nucleic acid molecule encoding a VH domain of a monoclonal antibody or antigen-binding fragment thereof according to the present invention, and the second vector comprises a second nucleic acid molecule encoding a VL domain. In certain embodiments, the first vector comprises a nucleic acid molecule encoding a heavy chain (HC) of a monoclonal antibody or antigen-binding fragment thereof according to the present invention, and the second vector comprises a second nucleic acid molecule encoding a light chain (LC).

[0193] The vectors according to the present invention include expression vectors suitable for expressing monoclonal antibodies or antigen-binding fragments thereof by host cells. The host cells can be eukaryotic cells or prokaryotic cells.

[0194] The present invention provides host cells comprising nucleic acid molecules or multiple nucleic acid molecules encoding antibodies or antigen-binding fragments thereof according to the present invention. As an alternative or in addition, the present invention provides host cells comprising vectors or multiple vectors encoding antibodies or antigen-binding fragments thereof according to the present invention. Any suitable technique can be used, including, for example, but not limited to, transduction, transformation, transfection and injection, to introduce one or more nucleic acid molecules or similarly one or more vectors into host cells. Various forms of these methods are well known in the art, including, for example, electroporation, calcium phosphate transfection, lipid transfection (lipofection), cell squeezing (cell squeezing), sonoporation, optical transfection and gene gun.

[0195] In certain embodiments, the host cell is a eukaryotic cell. In certain embodiments, the host cell is a yeast cell. In certain embodiments, the host cell is an insect cell. In certain embodiments, the host cell is a mammalian cell. In certain embodiments, the host cell is a human cell. In certain embodiments, the host cell is selected from hybridoma cells, Chinese hamster ovary (CHO) cells, NSO cells, human embryonic kidney (HEK293) cells and PER.C6 TM The present invention also contemplates other host cells than those mentioned above. Host cells also include cell lines developed for commercial production of antibodies and antigen-binding fragments thereof according to the present invention.

[0196] The cell line provided with the nucleic acid can produce binding molecules / antibodies in a laboratory or a production plant. Alternatively, the nucleic acid is transferred to cells in an animal body in need thereof, and the binding molecules / antibodies are produced in vivo by transformed cells. The nucleic acid molecules of the present invention are generally provided with regulatory sequences to express binding molecules in cells. However, today's homologous recombination techniques have become much more efficient. These techniques involve, for example, double-strand break-assisted homologous recombination using site-specific double-strand break-inducing nucleases such as TALEN. Such or similar homologous recombination systems can insert nucleic acid molecules into regions that provide one or more cis-required regulatory sequences.

[0197] The present invention also provides an isolated or recombinant cell or in vitro cell culture cell comprising a nucleic acid molecule or vector according to the present invention. The present invention also provides an isolated or recombinant cell or in vitro cell culture cell comprising a binding molecule according to the present invention. Preferably, the cell produces the binding molecule. In certain embodiments, the cell secretes the binding molecule. In a preferred embodiment, the cell is a hybridoma cell, a CHO cell, a NS0 cell, a HEK293 cell or a PER-C6 TM Cells. In a particularly preferred embodiment, the cells are CHO cells. Cell cultures comprising cells according to the invention are also provided. A number of institutions and companies have developed cell lines for large-scale production of antibodies, e.g., for clinical use. Some non-limiting examples of such cell lines are CHO cells, NS0 cells, or PER.C6 TMCells. These cells are also used for other purposes, such as the production of proteins. Cell lines developed for industrial-scale production of proteins and antibodies are also referred to herein as industrial cell lines. The present invention provides industrial cell lines comprising nucleic acid molecules, binding molecules and / or antibodies according to the present invention. The present invention also provides cell lines comprising binding molecules or antibodies of the present invention developed for large-scale production of proteins and / or antibodies. The present invention also provides uses of cell lines developed for large-scale production of binding molecules and / or antibodies of the present invention.

[0198] Methods for preparing antibodies

[0199] The present invention also provides a method for preparing a monoclonal antibody or an antigen-binding fragment thereof according to the present invention, comprising culturing a host cell population according to the present invention under conditions that allow expression of the monoclonal antibody or its antigen-binding fragment. In certain embodiments, the method further comprises harvesting the monoclonal antibody or its antigen-binding fragment from the culture. Preferably, the cells are cultured in a serum-free medium. Preferably, the cells are suitable for suspension growth. Also provided is an antibody obtainable by a method for producing an antibody according to the present invention. The antibody is preferably purified from the culture medium of the culture. Preferably, the antibody is affinity purified.

[0200] How to use

[0201] One aspect of the invention is a method of inhibiting activation of the classical pathway or the lectin pathway in a subject, comprising administering to a subject in need thereof an effective amount of a monoclonal antibody or antigen-binding fragment thereof according to the invention. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a mouse, rat, hamster, guinea pig, rabbit, goat, sheep, pig, cat, dog, horse or cattle. In certain embodiments, the subject is a non-human primate, such as a monkey. In certain embodiments, the subject is a human.

[0202] The inhibitory effect of the antibody or antigen-binding fragment can be assessed using any suitable method, including, for example, measuring total complement activity, i.e., a hemolytic activity test based on the ability of a serum sample to lyse sheep erythrocytes coated with anti-sheep antibodies. A reduction in hemolysis compared to an untreated control sample indicates an inhibitory effect of the antibody or antigen-binding fragment. In one embodiment, the untreated control sample can be a historical sample obtained before the start of treatment with the antibody or antigen-binding fragment. Typically, a reduction in total complement activity of at least 5% compared to a control indicates effectiveness. In certain embodiments, a reduction in total complement activity of at least 10% compared to a control indicates effectiveness.

[0203] Diseases that can be treated or prevented by the methods or monoclonal antibodies or antigen-binding fragments thereof according to the present invention are: autoimmune diseases, such as experimental allergic neuritis, type II collagen-induced arthritis, myasthenia gravis, hemolytic anemia, glomerulonephritis, idiopathic membranous nephropathy, rheumatoid arthritis, systemic lupus erythematosus, immune complex-induced vasculitis, adult respiratory distress syndrome, stroke, xenotransplantation, allotransplantation, multiple sclerosis, burns, extracorporeal dialysis and blood oxygenation, inflammatory diseases, including sepsis and septic shock, toxicity induced by in vivo administration of cytokines or mAbs, antibody-mediated rejection of allogeneic transplants (e.g., renal allografts), multiple trauma, ischemia-reperfusion injury and myocardial infarction.

[0204] Individuals suffering from diseases involving complement-mediated damage or at risk of such complement-mediated damage can be treated by administering an effective amount of a monoclonal antibody or antigen-binding fragment thereof according to the present invention to an individual in need thereof. Thus, biologically active complement-derived peptides are reduced in the individual and the lysis and other destructive effects of complement on cells and tissues are weakened or prevented. An "effective amount" means an amount sufficient to achieve a desired biological response. In one embodiment, an "effective amount" means an amount of a monoclonal antibody or antigen-binding fragment thereof according to the present invention that is capable of inhibiting complement activation in an individual.

[0205] Treatment (prophylactic or therapeutic) usually consists of parenteral administration of a monoclonal antibody or its antigen-binding fragment according to the present invention, such as intravenous, subcutaneous or topical administration, together with a pharmaceutical carrier. Administration can usually be completed by injection or infusion. The dosage and administration regimen of the monoclonal antibody or its antigen-binding fragment according to the present invention will depend on the degree of complement activation intended to be inhibited. Usually, for the monoclonal antibody of the present invention, the amount is 2 to 20 mg / kg body weight. For parenteral administration, the monoclonal antibody or its antigen-binding fragment according to the present invention will be formulated into an injectable form combined with a pharmaceutically acceptable parenteral carrier. Such carriers are well known in the art, and some examples include saline, dextrose solution, Ringer's solution, and a solution containing a small amount of human serum albumin.

[0206] Pharmaceutical compositions must generally be sterile and stable under manufacturing and storage conditions. The composition may be formulated into solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. Some examples of suitable aqueous and non-aqueous carriers that may be used for pharmaceutical compositions of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof; vegetable oils (e.g., olive oil); and injectable organic esters (e.g., ethyl oleate). Appropriate fluidity may be maintained, for example, by using a coating material (e.g., lecithin), by maintaining a desired particle size in the case of a dispersion, and by using a surfactant to maintain appropriate fluidity.

[0207] The pharmaceutical composition may also include excipients such as preservatives, wetting agents, emulsifiers and dispersants. The presence of microorganisms can be prevented by sterilization procedures and by including a variety of antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. It is also desirable to include isotonic agents in the composition, such as sugars, polyols such as mannitol, sorbitol, glycerol, or sodium chloride. Pharmaceutically acceptable antioxidants may also be included, such as: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0208] Sterile injectable solutions can be prepared by incorporating the desired amount of mAb or its fragments into a suitable solvent together with one of the ingredients listed above or in combination (as required), followed by sterilization microfiltration. Typically, dispersions are prepared by incorporating the active compound into a sterile carrier containing a basic dispersion medium and desired other ingredients, such as those from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying (lyophilization), which produce a powder of the active ingredient plus any additional desired ingredients from its previous sterile filtered solution.

[0209] Prolonged absorption of the injectable anti-C2 mAb or fragment thereof can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.

[0210] The mAb or its fragment can be prepared with a carrier that will protect the compound from rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations are generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0211] The pharmaceutical compositions can be administered using medical devices known in the art.

[0212] The dosage regimen is adjusted to provide the optimum desired response (e.g., a therapeutic response).For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0213] Actual dosage levels of the mAb or fragments thereof in the pharmaceutical compositions of the invention may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response in a particular patient without being toxic to the patient.

[0214] In one embodiment, the monoclonal antibodies according to the present invention can be administered at 10 to 500 mg / m 2 , for example 200 to 400 mg / m 2 The weekly dose is administered by infusion. Such administration can be repeated, for example 1 to 8 times, for example 3 to 5 times. Administration can be carried out by continuous infusion over a period of 2 to 24 hours, for example 2 to 12 hours.

[0215] In another embodiment, the mAb or fragment thereof or any other binding molecule disclosed in the present invention can be administered by maintenance therapy, for example, once a week for 6 months or longer.

[0216] The invention will now be illustrated with reference to the following examples, which illustrate some particularly advantageous embodiments. However, it should be noted that these embodiments are merely illustrative and should not be construed as limiting the invention in any way.

[0217] Example

[0218] Example 1: Removal of glycosylation sites from anti-C2b monoclonal antibodies

[0219] BRO2-glyc-IgG4

[0220] U.S. Patent No. 9,944,717 discloses a mouse inhibitory anti-C2b monoclonal antibody (mAb). From this lead, four humanized variants were produced using the composite human antibody technology of Antitope Ltd (Cambridge, UK), which comprised different heavy chain variable domains (VH1, VH2, VH3 or VH4) and kappa light chain variable domains (VK1, VK2, VK3 or VK4). Based on computer simulation analysis, the immunogenicity risk of each humanized VH and VK sequence was predicted. As shown in Table 7, when VH4 was paired with VK3 or VK4, the lowest risk of immunogenicity was predicted, as well as the highest percent identity with the closest human germline variant. This observation was based on the lowest number of promiscuous binding peptides for human MHC class II. VH4 is preferred because it has a higher percent identity with the closest human germline. Furthermore, based on binding and potency, VH4 / VK3 was selected as the anti-human C2b humanized lead antibody and is referred to herein as BRO2-glyc-IgG4.

[0221] Table 7. Immunogenicity risk ranked from 1 (=lowest) to 5 (=highest) (high affinity preferred over medium affinity) and sequence identity to the closest human germline

[0222]

[0223] SDS-PAGE analysis of the BRO2-glyc-IgG4 variants revealed double bands and band migration in the VH3 and VH4 variants. It is assumed that this migration is caused by a potential glycosylation site (motif NXS) at residues 72 to 74 (Kabat numbering) in the framework region 3 (FR3) of VH3 and VH4. Since this potential glycosylation site will not only lead to heterogeneity of antibody products expressed by mammalian cell lines, but also to heterogeneity of antibody function, the potential glycosylation site was removed. The glycosylation site was removed by site-directed mutagenesis to produce the N72D variant of VH, which is referred to herein as VH3.2 or VH4.2. The N72D mutation eliminates the altered banding pattern observed in VH3 and VH4 ( Figure 1 ), confirming that the double bands and band migration were caused by glycosylation and heterogeneity in the heavy chain.

[0224] To further determine whether variant VH4.2, a VH identical to BRO2-glyc-IgG4 but without the glycosylation site in FR3, exhibited improved properties compared to the heterogeneously glycosylated parent mAb BRO2-glyc-IgG4, the thermal tolerance of each antibody was determined.

[0225] In order to test heat tolerance, the temperature was raised from 55°C to 75°C by Thermocycler (Biometra) to treat the humanized variants. The remaining binding capacity was analyzed on a CM5 chip directly coated with human C2 purified from serum (3500RU, Complement Technologies Cat#A112, batch #20) on Biacore 3000. The data were analyzed using BIAevaluation software. The slope of the specific binding of each variant was determined using BIAevaluation software, generally from the linear phase fitting of the sensorgram (starting 5 seconds after the injection started and stopping after 11 seconds). The mean value of the slope obtained for 59°C, 56.9°C, 55°C and 4°C temperatures was then used as 100% activity to calculate the percentage activity. Finally, the percentage activity (Log (agonist) relative to response, variable slope (4 parameters)) was drawn in GraphPad Prism. The temperature (TM50) when the antibody lost its 50% binding capacity is shown in Table 8 below.

[0226] BRO2-IgG4

[0227] Both variants without the glycosylation site present in BRO2-glyc-IgG4 showed improved thermal tolerance (Table 8). BRO2-glyc-IgG4 showed a TM50 of 64.0°C. VH4.2 / VK3 (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.

[0228] Table 8. Percent Identity and Thermal Tolerance of Anti-C2b Monoclonal Antibodies to the Closest Human Germline Sequence

[0229]

[0230] Example 2: Preparation of non-glycosylated IgG4 and non-glycosylated IgG1 variants

[0231] BRO2-IgG4-NH

[0232] Antibodies with pH-dependent antigen binding dissociate bound antigens in acidic endosomes after internalization into cells. Thus, the released antigens are transported to lysosomes and degraded, while the dissociated antibodies freed from antigens are recycled back to the plasma via FcRn. The recycled free antibodies can bind to another target antigen. By repeating this cycle, pH-dependent antigen-binding antibodies can bind to target antigens more than once, and thus improve the neutralizing ability of the antibodies. This process is very important in the use of antibodies. (NH) technology (argenx, Belgium) can be further improved during assembly, (NH) technology enhances antibody binding to FcRn at acidic endosomal pH (pH 6.0) but not at neutral pH (pH 7.4). Therefore, the amino acids in the Fc region of BRO2-IgG4 were mutated to alter the pH-dependent binding to FcRn (H433K, N434F). The resulting antibody is referred to herein as BRO2-IgG4-NH.

[0233] BRO2-IgG1-NH and

[0234] BRO2-IgG1-LALA-NH(ARGX-117)

[0235] The effect of immunoglobulin subclass on potency was also examined. Additional Variants (BRO2-IgG1-NH). Antibody effector function can be further attenuated by mutations in the Fc region that alter antibody binding to Fcγ receptors. Therefore, amino acid substitutions L234A and L235A ("LALA") were incorporated into BRO2-IgG1-NH to generate BRO2-IgG1-LALA-NH, also referred to herein as ARGX-117.

[0236] His1-IgG1-LALA-NH

[0237] To determine whether the pH dependence of BRO2-IgG1-LALA-NH can be improved to prolong its in vivo pharmacokinetic and pharmacodynamic (PK / PD) effects, one amino acid in the VK of the BRO2-IgG1-LALA-NH antibody was mutated to histidine (G29H, the mutant VK is referred to as Vk3m3 herein). The resulting antibody is referred to as His1-IgG1-LALA-NH herein.

[0238] His1-IgG4

[0239] Similarly, to determine whether the pH dependence of BRO2-IgG4 can be improved to prolong its in vivo PK / PD effect, one amino acid in the VK of the BRO2-IgG4 antibody was mutated to histidine (G29H, the mutant VK is referred to as Vk3m3 herein). The resulting antibody (VH4.2 / Vk3m3) is referred to as His1-IgG4 herein.

[0240] His1-IgG4-NH

[0241] To examine the effect of recycling on antibody potency, The mutations were incorporated into the His1-IgG4 (VH4.2 / Vk3m3) antibody. The resulting antibody is referred to herein as His1-IgG4-NH.

[0242] His2-IgG4-NH

[0243] In order to determine whether the pH dependence of BRO2-IgG4-NH can be improved to prolong its in vivo PK / PD effect, one amino acid of VH4 of BRO2-IgG4-NH antibody was mutated to histidine (K26H, VH mutant is referred to as VH4.2m12 herein). In addition, the VK3 light chain of BRO2-IgG4-NH antibody was replaced with the above-mentioned VK4 light chain, and the second amino acid was mutated to histidine (G29H, VK4 mutant is referred to as VK4m3 herein). The resulting antibody (VH4.2m12 / VK4m3) is referred to as His2-IgG4-NH herein.

[0244] Example 3: Improved potency of non-glycosylated BRO2 variants

[0245] Overall pharmacokinetics (PK)

[0246] Cynomolgus monkeys (n=2, 1 male and 1 female per group) were randomly assigned to treatment groups according to Table 9 below.

[0247] Table 9. Treatment group assignment

[0248]

[0249] A serum sample was obtained from each monkey one day prior to receiving the test antibody (day -1, or "PRE"). Then 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 then obtained continuously from each monkey for up to 60 days (to d60).

[0250] For the PK of total antibody (total PK), microtiter plates were coated with 100 μL 5 μg / mL goat anti-human IgG (Bethyl; A80-319A) at 4°C overnight. The plates were washed 3 times with at least 200 μL PBS-0.05% Tween20 and then blocked with 200 μL PBS-2% BSA for 2 hours at room temperature (RT). After the plates were washed 3 times with at least 200 μL PBS-0.05% Tween20, serum samples, standards and QC samples (prepared in mixed untreated cynomolgus monkey serum) diluted in 100 μL PBS-0.2% BSA-1% mixed untreated cynomolgus monkey serum were applied in duplicate. For each antibody, its own frozen standards and QC samples were applied in duplicate (using the same antibody batch as the batch injected into the monkey). The negative control antibody is an antibody that binds to non-C2 complement components. Incubation was carried out at room temperature for 2 hours while shaking the plate. After the plate was washed 5 times with at least 200 μL PBS-0.05% Tween20, 100 μL horseradish peroxidase (HRP)-labeled mouse anti-human IgGκ (Southern Biotech, 9230-05) was diluted 260,000 times in PBS 0.2% BSA and applied to the wells for 1 hour at room temperature. The plate was washed 5 times with at least 200 μL PBS-0.05% Tween20, stained with 100 μL 3,3',5,5'-tetramethylbenzidine (TMB), and stopped with 100 μL 0.5M H2SO4 (CHEM LAB, Cat# CL05-2615-1000) after 10 minutes. OD was measured at 450nm, and the concentration of the sample was calculated back using GraphPad Prism (each using its own standard).

[0251] The results are shown in Table 10, and a comparison of glycosylated BRO2-glyc-IgG4 with non-glycosylated BRO2-IgG4 is shown in Figure 2 In the overall PK assay, the concentration of non-glycosylated BRO2-IgG4 was generally higher than that of glycosylated BRO2-glyc-IgG4. This improvement in overall PK was completely unexpected and represents an important additional advantage of non-glycosylated antibodies.

[0252] Table 10. Overall PK

[0253]

[0254] Free C2

[0255] Cynomolgus monkeys (n=2, 1 male and 1 female per group) received a single intravenous injection of 5 mg / kg of the test antibody as described above.

[0256] In this assay, microtiter plates were coated overnight at 4°C with 100 μL of 2.5 μg / mL mouse anti-human C2 monoclonal antibody mAb32 (anti-C2 #32m-IgG @ 3.31 mg / mL, 0.2 μm PBS, LC-12 / 05-166, April 12, 13). This antibody binds to an epitope on C2 that is different from that of BRO2. The plate was washed 3 times with at least 200 μL PBS-0.05% Tween20 and then blocked with 200 μL PBS-2% BSA (pH 7.4) at room temperature for 2 hours. At the same time, samples, frozen standards (specific for each antibody, prepared in mixed untreated cynomolgus monkey serum) and frozen QC samples (prepared in mixed untreated cynomolgus monkey serum) were thawed and diluted 6.7 times in 80 μL PBS-0.2% BSA. 40 μL of biotinylated anti-C2 VH4 / VK3 was added at 0.6 μg / mL. Each sample was prepared in duplicate. After adding the biotinylated antibody, 100 μL of the mixture was immediately transferred to the washed coated plate. The plate was incubated at room temperature for 2 hours, washed 5 times with at least 200 μL PBS-0.02% Tween20, and 100 μL strep-HRP (Jackson, 016-030-084) diluted 300,000 times in PBS-0.2% BSA was added. After incubation at room temperature for 1 hour, the plate was washed 5 times with at least 200 μL PBS-0.05% Tween20, stained with 100 μL TMB (Calbiochem, CL07), and stopped with 100 μL 0.5M H2SO4 (CHEM LAB, Cat# CL05-2615-1000) after 10 minutes. OD was measured at 450 nm and used to determine C2 levels.

[0257] Sera from the following monkeys were first tested together using the free C2 assay performed 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.

[0258] The free C2 levels of all monkeys are shown in Figures 3A to 3I and Table 11.

[0259] As expected, there was no decrease in free C2 for monkeys 3 and 4, since these monkeys were dosed with the negative control antibody. For all monkeys treated with the BRO2 variants, free C2 levels were very low until after day 2.

[0260] For monkeys receiving BRO2-glyc-IgG4 (monkeys 1 and 2) and BRO2-IgG4 (monkeys 5 and 6), C2 levels began to rise on day 4 and returned to baseline levels by day 31. Monkeys 5 and 6 treated with the aglycosylated antibody consistently showed lower free C2 levels than those treated with BRO2-glyc-IgG4 ( Figure 3C , Table 11).

[0261] For those except in the case of antidrug antibodies (ADA) Figures 3D to 3I In all other monkeys except for those marked with * in the figure, the increase in C2 levels was much slower and the C2 levels did not return to the baseline even by day 31.

[0262] Figure 4 Magnification (logarithmic scale) of the mean free C2 levels (OD 450nm) of 2 monkeys per group is shown. The free C2 levels of the BRO2 variant were lower than those of the His1 variant.

[0263] Monkey 10 injected with BRO2-IgG1-LALA-NH (ARGX-117) had the lowest C2 levels at all time points tested. Figure 5 Comparison of free C2 levels up to 60 days from monkeys 5 and 6, 9 and 10, and 15 and 16 can be seen in Table 10. Monkey 10 also had the best overall PK (see above). The raw data are shown in Table 11, and the mean data comparing the glycosylated and non-glycosylated variants are shown in Table 12.

[0264]

[0265] Table 12. Average free C2 of glycosylated and non-glycosylated antibodies

[0266]

[0267] Since the assays just described were performed on different days for different monkeys, the analysis was repeated for a selected number of time points (pre, 4 hours, days 1, 2, 4, 11, and 27) with sera from all monkeys placed on a single plate ( Figures 6A to 6D The pre-samples were also tested with and without the addition of excess BRO2 (500 μg / mL).

[0268] The OD of the pre-samples from all monkeys was comparable, indicating that the free C2 levels in different monkeys were comparable ( Fig. 6A When the pre-samples were pre-incubated with 500 μg / mL BRO2, all signals dropped to an OD of 0.013 to 0.015 ( Fig. 6A and 6BSuch low OD values ​​were not obtained for any PK sample, indicating that free C2 was not completely depleted at any time point. The lowest level was obtained at 4 hours and it was not significantly different for the BRO2 variants (monkeys 5, 6, 7, 8, 9 and 10, Figure 6C ) was the lowest (OD 0.02 to 0.03). Interpretation of the results on days 11 and 27 was hampered by ADA (anti-drug antibodies), which were observed in several monkeys ( Fig.6D ).

[0269] Immunogenicity

[0270] As described above, cynomolgus monkeys (n=2, 1 male and 1 female per group) received a single intravenous injection of 5 mg / kg of the test antibody. Serum samples obtained from all monkeys from baseline (before exposure) until day 31 were tested for ADA (anti-drug antibodies) ( Figures 7A to 7P ), and serum samples obtained from monkeys 5 and 6, 9 and 10, and 15 and 16 were further tested until day 59 ( Figures 8A to 8F ).

[0271] Immunogenicity was determined by coating the microtiter plates with 100 μL of 1 μg / mL of the corresponding antibodies at 4°C overnight. The plates were washed 3 times with at least 200 μL of PBS-0.05% Tween20 and subsequently blocked with 200 μL of PBS-1% casein at room temperature for 2 hours. After the plates were washed 3 times with at least 200 μL of PBS-0.05% Tween20, serum samples were diluted 20 times or more in 100 μL of PBS-0.1% casein and incubated for 2 hours in the coated wells at room temperature (RT). After the plates were washed 5 times with at least 200 μL of PBS-0.05% Tween20, 100 μL of anti-monkey IgG-HRP (SouthernBiotech #4700-05) was added to the wells for 1 hour at room temperature with 8000-fold dilution. The plate was washed 5 times with at least 200 μL PBS-0.05% Tween 20, stained with 100 μL TMB, and stopped with 100 μL 0.5 M H2SO4 (CHEM LAB, Cat# CL05-2615-1000) after 10 minutes. OD was measured at 450 nm. Representative results are shown in Figures 7A to 7P middle.

[0272] For monkeys 8 (BRO2-IgG4-NH), 11 (His1-IgG4), 13 and 14 (His1-IgG4-NH), and 16 (His1-IgG1-LALA-NH) (respectively Figure 7F , 7I, 7K and 7L and 7N), a clear ADA response was observed. In fact, the signal obtained in ELISA after injection of the antibody was increased by at least 2-fold compared to the baseline ("PRE") signal (before injection of the antibody).

[0273] For monkeys 11, 13, and 16 (respectively Fig.7I , 7K and 7N), ADA was observed from day 11; for monkey 8 ( Figure 7F ), starting from day 15; and for monkey 14 ( Figure 7L ), starting from the 19th day.

[0274] For Monkey 9 (BRO2-IgG1-LALA-NH) Figure 7G ), an increase in signal was observed for all samples after antibody injection, but the signal in the baseline sample was already high and the increase over time was lower (about 1.5-fold).

[0275] For Monkey 5 ( Figure 7C ), an abnormally high signal was observed in the baseline sample before the injection of the antibody. This signal was also higher than the signal at the later time points. This can be explained by interference of the antibody (present in the serum) with the assay. Therefore, it is not possible to determine whether there was an ADA response in this monkey.

[0276] Example 4: Isoelectric point (pI)

[0277] Igawa et al. (Protein Eng Des Sel 2010, 23(5):385-392), studying VH mutants of certain IgG1 monoclonal antibodies, reported a strong positive correlation between isoelectric point (pI) and monoclonal antibody clearance and a negative correlation between pI and monoclonal antibody half-life. In this example, the pIs of various forms of anti-human C2b were determined. The results are shown in Table 13.

[0278] Table 13. Apparent pI of anti-human C2b monoclonal antibodies

[0279]

[0280] All three antibodies tested were not glycosylated in the VH. As shown in Table 13, the pi of ARGX-117 was found to be significantly greater than the pi of the closely related IgG4 antibodies. It was expected that the observed pi of ARGX-117 would manifest as an enhanced potential for so-called antigenic sweeping.

[0281] Example 5: Domain localization by Western blotting and surface plasmon resonance (SPR) analysis

[0282] The binding properties of ARGX-117 were evaluated by Western blotting and surface plasmon resonance (SPR) analysis. Figure 1 Western blot results showed that ARGX-117 bound to C2 and C2b. Fig.9A The binding properties of ARGX-117 were further investigated by SPR using Biacore 300 with different concentrations of ARGX-117 Fab as eluent by coating C2 (SEQ ID NO: 21) on a solid phase, as shown in Fig. 9B As shown in . Affinity was calculated assuming 1:1 binding between Fab and C2 and resulted in a Kd of approximately 0.3 nM. To investigate the mechanism of action of ARGX-117, an SPR analysis was performed that simulated the formation of C3 convertase (C4bC2a) in the presence of biotinylated C4 immobilized to a streptavidin-coated chip, as shown in Fig. 9C As shown in . When C2 in the flow buffer was added alone or pre-incubated with a control mAb, C2 binding was observed on the chip. Pre-incubation with anti-C2 clone 63 (i.e., anti-C2-63) produced a higher signal, presumably because the mAb form complexed with C2 and the C2:mAb complex were combined, resulting in a higher molecular weight and a higher SPR signal. When C2 was pre-incubated with ARGX-117, the binding of C2 to C4b was greatly reduced. The initial interaction of C2 with C4b is believed to be initiated by the C2b domain (SEQ ID NO: 44). Thereafter, the large C2a domain (SEQ ID NO: 43) takes over, and this interaction is essential for the formation of the C3 convertase complex. The results from this experiment indicate that ARGX-117 inhibits C2 by inhibiting binding to C4b.

[0283] To further understand the mechanism of action of ARGX-117 on C2 inhibition, C2 was first allowed to bind to C4b immobilized to a streptavidin chip, and after stabilization by flow buffer alone, the sample was flowed through, e.g. Fig.9DAs shown in . Running buffer or a control human IgG4 mAb targeting an unrelated soluble antigen (i.e., anti-factor XI (anti-FXI)) resulted in some signal reduction, which is normal after stopping the injection. Injection of anti-C2-63 resulted in an increase in signal, indicating that this mAb is able to bind to the C3 convertase (C4bC2a). This is consistent with the predicted binding model of C2 to C4b, which suggests that after binding to C2, the C2a domain is still largely available. Interestingly, ARGX-117 exhibited strong binding to the C3 convertase, which then dissociated rapidly. These results indicate that ARGX-117 is able to bind to C2, but this binding is very unstable and may affect C2 in a way that promotes activation. These results also indicate that ARGX-117 will be released from the C2 molecule along with C2b.

[0284] Example 6: Domain Mapping Using Domain Swap Mutants of C2 and Factor B

[0285] To locate the epitope of anti-C2-5F2.4, the following facts were utilized: Anti-C2-5F2.4 does not cross-react with factor B (FB; SEQ ID NO: 50), and C2 and FB are highly homologous proteins with similar domain structures. Both proteins contain a small fragment and a large fragment. The small fragment in complement C2 is called C2b (SEQ ID NO: 44), and the small fragment in factor B is called FBa (SEQ ID NO: 51). The small fragment in each protein contains three Sushi domains (CCP domains). The large fragment in each protein contains a von Willebrand factor type A domain (VWFA) and a peptidase S1 domain, such as Fig.10 As shown in . The domain swap mutants contained a C-terminal FLAG tag.

[0286] In order to generate a variety of exchange mutants, the DNA constructs of C2, FB and ten domain exchange mutants were obtained from GenScript. DNA heat shock transformation was performed in competent Escherichia coli (E.coli) cells (ThermoFisher). Cells were streaked on agar plates and grown for 16 hours at 37°C. Preparation (MP Bio) 13 bottles of 200mL LB (Luria broth (LuriaBroth)) culture medium were autoclaved. 300 μL of ampicillin (100 mg / mL) were added to each bottle. For each construct, pre-culture was started with 3mL LB culture medium. After 6 hours, the pre-culture was transferred to the bottle and grown for 16 hours at 37°C under agitation. DNA was purified from bacterial precipitation according to the manufacturer's instructions (MaxiPrep, NucleoBondPC 500, Macherey-Nagel) by plasmid DNA purification kit, and reconstructed in TE buffer. Plasmid DNA concentration was measured by NanoDrop, and was set to 1 μg / μL. The integrity of the plasmid was verified by restriction analysis. For each construct, 1 μL of plasmid DNA and 9 μL of restriction enzyme mixture (PstI and PvuII) were mixed and incubated at 37°C for 2 hours. Using the Bio-Rad ChemiDoc MP system, after running for 1 hour at 100V, DNA was analyzed on 1% agarose gel. The DNA constructs for fine positioning (see below) were handled in the same manner, but their integrity was checked by sequencing.

[0287] The mutant proteins were produced by transient transfection in HEK293T cells. HEK cells were cultured in complete DMEM (DMEM (Gibco) supplemented with 10% fetal calf serum (FCS) and 1% penicillin / streptomycin (P / S)). One day before transfection, cells from two flasks were seeded into 15 10 cm 2Culture dish (Greiner Bio-One).Before transfection, 21mL empty DMEM culture medium is mixed with 630 μL polyethyleneimine (P-Pei, Polysciences, Inc.).As a control, empty plasmid PF45pcDNA3.1 and PF146H2B GFP are transfected.15 μg plasmid DNA is hatched 20 minutes in 1500 μL empty culture medium-P-Pei mixture in Eppendorf tube.Transfection mixture is carefully added to cells, and by pipetting up and down mixed culture medium.After 8 hours, culture medium is replaced with 15mL empty culture medium.After 3 days, the GFP expression of cells is checked by fluorescence microscopy.Supernatant is collected at the 4th day, it is filtered sterilized by 0.22 μm filter (Sartorius), and is concentrated to about 1 / 3rd of original volume with Vivaspin column (Sartorius). The domain swap mutants were concentrated using a 30,000 MWCO column and the C2b mutants used for fine mapping were concentrated using a 10,000 MWCO column. All supernatants were stored at -20°C and also analyzed by SDS-PAGE and anti-FLAG Western blotting.

[0288] In order to verify the expression of different constructs, anti-FLAG tag ELISA was performed. Microplates (Maxisorp, NUNC, Cat#439454) were coated overnight with 100 μL HEK293T supernatants diluted 5 times in PBS or undiluted (for domain exchange mutants and fine positioning mutants, respectively). After washing 4 times with PBS and 0.05% Tween-20, 100 μL / well 1 μg / mL anti-FLAG Ab (clone M2, Sigma-Aldrich) in PBS and 0.1% Tween-20 (PBST) was added and incubated for 1 hour at room temperature (RT) under stirring. As detection Ab, 100 μL / well horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (Santa Cruz Biotechnology, Cat#sc-2005, 1000x dil.) was added in PBST and incubated for 1 hour at room temperature. After the final washing step, 100 μL / well TMB (Invitrogen, Cat#SB02) was added as substrate, the reaction was stopped after a few minutes with 100 μL / well HCl (Fischer, Cat#J / 4320 / 15) and the absorbance was read at 450 nm (BioRad, iMark Microplate reader).

[0289] With the exception of C2-(FB-Pep1), anti-FLAG ELISA detected proteins in the supernatants of all mutants, e.g. Fig.11 The variation between mutants could be explained by different production of anti-FLAG mAb after coating or different detection efficiencies.

[0290] Next, the recognition of exchange mutants by anti-C2-5F2.4 antibodies was studied. To this end, microplates (Maxisorp, NUNC, Cat#439454) were coated overnight with 2 μg / mL anti-C2-5F2.4 in 100 μL PBS. The plates were washed 4 times with PBS with 0.05% Tween-20 and blocked with 200 μL PBS (PBST-BSA) with 0.1% Tween-20 and 1% bovine serum albumin (BSA) for 1 hour at room temperature. After washing, 100 μL culture supernatant containing mutants was added at a 20-fold dilution in PBST-BSA and incubated for 2 hours at room temperature under stirring. After washing, 1 μg / mL biotinylated anti-FLAG (clone M2, Sigma-Aldrich) was added in PBST-BSA at room temperature for 1 hour as a detection antibody. The plate was washed and 1 μg / mL streptavidin-POD conjugate (Roche, Cat#11089153001) was added and incubated in the dark for 30 minutes. The plate was washed and 100 μL / well TMB (Invitrogen, Cat#SB02) was added as substrate, and the reaction was stopped after a few minutes with 100 μL / well HCl (Fischer, Cat#J / 4320 / 15). The absorbance was measured at 450 nm on a microplate reader (BioRad, iMark microplate reader).

[0291] Wild-type C2 showed significant binding, and loss of binding was only observed for C2-(FB-S2), in which the complement C2 S2 domain (SEQ ID NO:46) was replaced with the Factor B S2 domain (SEQ ID NO:54). Fig.12 As shown in , no binding to wild-type FB was observed, but strong binding to the mutant FB-(C2-S2) was detected, in which the factor BS2 domain (SEQ ID NO: 54) was replaced by the complement C2 S2 domain (SEQ ID NO: 46). These results clearly show that anti-C2-5F2.4 recognizes an epitope on S2 (Sushi domain 2) on C2b. The results also show that C2-(FB-Pep1) is produced in sufficient quantities. Similar results were obtained when mouse IgG2a anti-C2-5F2.4 was used. In addition, when 1.25mM Ca was present in the buffer, ++Similar results were obtained when binding was studied in the absence of . Similar conclusions were reached by epitope mapping performed by Bioceros BV using domain swap mutants between human C2 and mouse C2. In addition, the amino acid sequence of Sushi domain 2 of cynomolgus monkey C2 is exactly the same as that of Sushi domain 2 of human C2.

[0292] Example 7: Fine Mapping of the Anti-C2-5F2.4 Epitope within Sushi Domain 2

[0293] Anti-C2-5F2.4 does not cross-react with mouse C2, and the mouse S2 domain (SEQ ID NO:58) differs from the human S2 domain (SEQ ID NO:46) at 10 amino acid positions, such as Fig.13 As shown in . To investigate which of these 10 amino acids is responsible for mAb binding, fine mapping mutants were generated. Fine mapping constructs included human C2b fragments (huC2b), huC2b with mouse S2 (huC2b-mS2), and ten mutants, each containing a single amino acid back mutation from the mouse sequence to the human sequence. Mutant C2b proteins similar to the domain swap mutants were generated by transient transfection into HEK293 cells.

[0294] like Fig.14 All mutants were generated and tested by anti-FLAG ELISA as shown in Figure 1. As expected, anti-C2-5F2.4 bound to huC2b but not to huC2b with mouse S2 (huC2b-mS2). None of the reverse point mutations restored binding of anti-C2-5F2.4, indicating that the epitope of this mAb is composed of at least two amino acids on the S2 domain, such as Fig.15 When 1.25 mM Ca was present in the buffer ++ Similar results were obtained when the combination was studied in the case of

[0295] By using publicly available structural data for human C2b, the positions of ten possible amino acids that could contribute to the anti-C2-5F2.4 epitope were analyzed, such as Fig.16 This analysis revealed three possible clusters, each consisting of three amino acids that could contribute to the epitope. DNA constructs for these cluster mutants were designed and obtained. Cluster mutants were generated by mutating human C2b S2 amino acids to the corresponding mouse C2b S2 amino acids. Three amino acids were changed in each mutant. If these three amino acids contribute to the epitope against C2-5F2.4, loss of binding is expected. Fig.17ACluster 1 mutants were shown to be well expressed and binding was not affected, and thus these amino acids did not contribute to binding. Cluster 2 mutants had lower expression based on anti-FLAG ELISA, and this led to the lack of anti-C2-5F2.4 binding. Cluster 3 mutants were also not well expressed, and this most likely explains the lack of anti-C2-5F2.4 binding, as Fig. 17B When 1.25 mM Ca was present in the buffer ++ Similar results were obtained when studying binding in the case of . From this analysis, the amino acids in cluster 1 could be excluded, leaving four possible amino acids in clusters 2 and 3.

[0296] The domain swap mutants provide strong evidence that the epitope on C2 recognized by anti-C2-5F2.4 is located on Sushi domain 2 on C2b. Furthermore, these experiments show that the presence of this domain on FB is sufficient for recognition by anti-C2-5F2.4. Given that anti-C2-5F2.4 does not react with mouse C2, one or more of the 10 amino acids that differ between the human and mouse Sushi 2 domains should be required for the epitope. By using single amino acid back mutations, we show that a single amino acid in Sushi 2 cannot restore binding. From experiments with cluster mutants, the following conclusions were drawn: Amino acids in cluster 1 do not contribute to the epitope for anti-C2-5F2.4. Amino acids in cluster 2 may contribute to the epitope for anti-C2-5F2.4, but since expression of this mutant was lower than that of cluster 1 mutants, it cannot be ruled out that the folding of cluster 2 mutants is not optimal. Since cluster 3 mutants were not expressed well, it seems likely that the mutations affected folding, so the role of amino acids in cluster 3 remains elusive.

[0297] Incorporated by Reference

[0298] All publications and patent documents cited herein are incorporated by reference in their entirety.

Claims

1. A monoclonal antibody or an antigen-binding fragment thereof that specifically binds to human complement factor C2, wherein the monoclonal antibody or the fragment thereof comprises: A VH domain comprising the amino acid sequence shown in SEQ ID NO: 3; and A VL domain comprising the amino acid sequence shown in SEQ ID NO:

2.

2. The monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a full-length monoclonal antibody.

3. The monoclonal antibody or antigen-binding fragment thereof of claim 1 or claim 2, wherein the monoclonal antibody comprises a human IgG heavy chain constant domain. 4 . The monoclonal antibody or antigen-binding fragment thereof of claim 3 , wherein the heavy chain constant domain comprises a human IgG1 heavy chain constant domain. 5 . The monoclonal antibody or antigen-binding fragment thereof of claim 4 , wherein the human IgG1 heavy chain constant domain comprises the amino acid sequence shown in SEQ ID NO:

4.

6. The monoclonal antibody or antigen-binding fragment thereof of claim 3, wherein the heavy chain constant domain comprises a human IgG4 heavy chain constant domain.

7. The monoclonal antibody or antigen-binding fragment thereof of claim 6, wherein the human IgG4 heavy chain constant domain comprises the amino acid sequence shown in SEQ ID NO:

5.

8. The monoclonal antibody or antigen-binding fragment thereof of claim 3, wherein the monoclonal antibody comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 6 and a light chain comprising the amino acid sequence shown in SEQ ID NO:

7.

9. The monoclonal antibody or antigen-binding fragment thereof of claim 3, wherein the monoclonal antibody comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 8 and a light chain comprising the amino acid sequence shown in SEQ ID NO:

7.

10. A pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to human complement factor C2, and a pharmaceutically acceptable carrier, wherein the monoclonal antibody or the fragment thereof comprises: A VH domain comprising the amino acid sequence shown in SEQ ID NO: 3; and A VL domain comprising the amino acid sequence shown in SEQ ID NO:

2.

11. The pharmaceutical composition of claim 10, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a full-length monoclonal antibody.

12. The pharmaceutical composition of claim 10 or claim 11, wherein the monoclonal antibody comprises a human IgG heavy chain constant domain.

13. A nucleic acid molecule or a plurality of nucleic acid molecules encoding the monoclonal antibody or antigen-binding fragment of any one of claims 1 to 9.

14. A vector or a plurality of vectors comprising the nucleic acid molecule or a plurality of nucleic acid molecules according to claim 13.

15. A host cell comprising the nucleic acid molecule or multiple nucleic acid molecules according to claim 13.

16. A host cell comprising the vector or multiple vectors of claim 14.

17. The host cell of any one of claims 14 to 16, wherein the host cell is a mammalian cell.

18. A method for preparing a monoclonal antibody or an antigen-binding fragment thereof, comprising: Cultivating the host cell population of any one of claims 15 to 17 under conditions suitable for expressing the monoclonal antibody or antigen-binding fragment thereof; as well as The monoclonal antibody or antigen-binding fragment is isolated from the cell.

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 of any one of claims 1 to 9.

20. A method for inhibiting the lectin 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 of any one of claims 1 to 9.

21. The method of claim 19 or claim 20, wherein the subject is a human.

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