Anti-OX40L antibody, anti-OX40L and anti-TNFα bispecific antibody, and uses thereof

Abstract

The present invention relates to a novel antibody that specifically binds to OX40L, and a bispecific antibody that specifically binds to OX40L and TNFα. Specifically, the present invention relates to an antibody or bispecific antibody that specifically binds to human OX40L and effectively inhibits the binding of OX40 to an OX40 receptor, nucleic acids encoding the antibodies, expression vectors containing the nucleic acids, transformants containing the expression vectors, methods for producing the antibodies, pharmaceutical compositions for treating autoimmune diseases or inflammatory diseases comprising the antibodies, compositions for diagnosing autoimmune diseases or inflammatory diseases comprising the antibodies, methods for diagnosing autoimmune diseases or inflammatory diseases using the antibodies, and methods for providing information for diagnosing autoimmune diseases or inflammatory diseases using the antibodies, and kits for providing the same.

Description

Technical Field

[0001] The present invention relates to a novel antibody specifically binding to OX40L and a bispecific antibody specifically binding to OX40L and TNFα. Specifically, the present invention relates to an antibody or bispecific antibody that specifically binds to human OX40L and effectively inhibits the binding between OX40 and the OX40 receptor, a nucleic acid encoding the antibody, an expression vector containing the nucleic acid, a transformant containing the expression vector, a method for producing the antibody, a pharmaceutical composition for preventing or treating an autoimmune disease or inflammatory disease containing the antibody, a diagnostic composition for an autoimmune disease or inflammatory disease containing the antibody, a diagnostic method for an autoimmune disease or inflammatory disease using the antibody, a method for providing information for diagnosing an autoimmune disease or inflammatory disease using the antibody, and a kit therefor.

Background Art

[0002] Autoimmune diseases or inflammatory diseases occur due to abnormal activation of the human immune system. Among typical autoimmune diseases, rheumatoid arthritis accounts for 68% of the therapeutic drug market as a TNFα inhibitor.

[0003] Tumor necrosis factor α (TNFα) is a cytokine produced by various cells including monocytes and macrophages, and was originally identified by its ability to induce necrosis of specific mouse tumors [Reference: Old, L. (1985) Science 230: 630-632]. Subsequently, it was revealed that the factor named cachectin, which is related to cachexia, is the same molecule as TNFα. TNFα is involved in the mediation of shock [Reference: Beutler, B. and Cerami, A. (1988) Annu. Rev. Biochem. 57: 505-518; Beutler, B. and Cerami, A. (1989) Annu. Rev. Immunol. 7:625 - 655]. Furthermore, TNFα is involved in the pathophysiology of various human diseases and disorders, including sepsis, infectious diseases, autoimmune diseases, transplant rejection, and graft - versus - host disease [References: Vasili, P. (1992) Annu. Rev. Immunol. 10:411 - 452; Tracey, K. J. and Cerami, A. (1994) Annu. Rev. Med. 45:491 - 503].

[0004] Due to the harmful role of human TNFα (hTNFα) in various diseases, treatment strategies have been planned to inhibit or neutralize hTNFα activity. In particular, antibodies that bind to and neutralize hTNFα have been used as a means to inhibit hTNFα activity. hTNFα - neutralizing antibodies include mouse monoclonal antibodies (mAb) secreted by hybridomas obtained from lymphocytes of mice immunized with hTNFα [References: Hahn T; et a l., (1985) Proc Natl Acad Sci USA 82:3814 - 3818; Liang, C - M., et al. (1986) Biochem. Biophys. Res. Commun. 137:847 - 854; Hirai, M., etal. (1987) J. Immunol. Methods 96:57 - 62; Fendly, B. M., et al. (1987) Hybridoma 6:359 - 370; Muller, A., et alL. (1990) Cytokine 2:162 - 169; U.S. Patent No. 5,231,024 (Moeller et al); European Patent Bulletin No. 186833B1 (Wallach, D.); European Patent Application No. 218868Al (Old et al.); European Patent Bulletin No. 260 610B1 (Moeller, A., et.)] or chimeric antibodies [References: Knight, D. M, et al. (1993) Mol. Immunol. 30:1443 - 1453; PCT Publication WO92 / 16553 (Daddona, P. E., et al.)] or humanized monoclonal antibodies [References : PCT Publication Gazette WO92 / 11383 (Adair, J.R., et al.) or human monoclonal antibodies [Reference: US 10-1142825], etc. There are such anti-hTNFα antibodies that show high affinity for hTNFα (e.g., Kd ≦ 10 -9 M) and can neutralize hTNFα activity. Such anti-hTNFα antibodies have been used as therapeutic agents in various autoimmune diseases, infectious diseases, transplant rejection reactions, and graft-versus-host diseases.

[0005] However, the group of patients who are unresponsive to TNFα inhibitors among these anti-hTNFα antibodies has reached about 50% (Nature Reviews Rheumatology vol.11, 276-289 (2015)). Moreover, the targets of autoimmune diseases developed in recent years are CTLA-4, IL-6, JAK1, JAK2, and CD20, etc., and the pharmaceuticals derived from these do not exhibit the same efficacy as TNFα inhibitors (Nature Reviews Rheumatology vol.11, 276-289 (2015)).

[0006] In particular, autoimmune diseases such as rheumatoid arthritis are caused not by the abnormality of just one type of immune cell but by problems in the entire immune system. Therefore, with the conventional method of developing therapeutic agents that inhibit only one target, there is a limit to improving the effectiveness of therapeutic agents. Thus, in order to overcome this limit of effectiveness, bispecific or multispecific antibodies that control two or more targets with different mechanisms of action at once have been developed. However, conventional bispecific antibodies act only on specific cells among the innate immune system or the adaptive immune system, so they cannot improve the homeostasis of the entire immune system.

Summary of the Invention

Problems to be Solved by the Invention

[0007] An object of the present invention is to provide an anti-OX40L antibody or an antigen-binding fragment thereof that specifically binds to OX40L (OX40 ligand).

[0008] An object of the present invention is to provide an anti - OX40L antibody that specifically binds to OX40L and recognizes a conformational epitope of OX40L containing the amino acid sequences from position 93 to 100 represented by SEQ ID NO:3 and from position 141 to 151 represented by SEQ ID NO:4 in the amino acid sequence of the OX40L protein represented by SEQ ID NO:1.

[0009] Another object of the present invention is to provide a bispecific antibody comprising an anti - OX40L antibody or an antigen - binding fragment thereof that specifically binds to OX40L (OX40 ligand); and an antibody or an antigen - binding fragment thereof that specifically binds to tumor necrosis factor α (TNFα).

[0010] A further object of the present invention is to provide a nucleic acid encoding the anti - OX40L antibody, its binding fragment or the bispecific antibody, an expression vector into which the nucleic acid has been introduced, or a host cell into which the expression vector has been introduced.

[0011] Another object of the present invention is to provide a method for producing an anti - OX40L antibody, its antigen - binding fragment or a bispecific antibody using the above - mentioned host cell. An object of the present invention is to provide a pharmaceutical composition for preventing or treating an autoimmune disease or an inflammatory disease, comprising the anti - OX40L antibody, its antigen - binding fragment or the bispecific antibody.

[0012] An object of the present invention is to provide a diagnostic composition for an autoimmune disease or an inflammatory disease, comprising the anti - OX40L antibody, its antigen - binding fragment or the bispecific antibody.

[0013] An object of the present invention is to provide a composition comprising the anti - OX40L antibody, its antigen - binding fragment or the bispecific antibody for detecting at least one of OXO40L and TNFα.

[0014] An object of the present invention is to provide a method for providing information for diagnosing an autoimmune disease or an inflammatory disease using the anti-OX40L antibody, an antigen-binding fragment thereof, or a bispecific antibody.

[0015] An object of the present invention is to provide a kit for providing information for diagnosing an autoimmune disease or an inflammatory disease, which contains the anti-OX40L antibody, an antigen-binding fragment thereof, or a bispecific antibody.

[0016] An object of the present invention is to provide a method for preventing or treating an autoimmune disease or an inflammatory disease, which includes administering a pharmaceutically effective amount of the anti-OX40L antibody, an antigen-binding fragment thereof, or a bispecific antibody.

[0017] An object of the present invention is to provide the use of the anti-OX40L antibody, an antigen-binding fragment thereof, or a bispecific antibody in the manufacture of a medicament for preventing or treating an autoimmune disease or an inflammatory disease.

[0018] An object of the present invention is to provide the use of the anti-OX40L antibody, an antigen-binding fragment thereof, or a bispecific antibody for preventing or treating an autoimmune disease or an inflammatory disease.

Means for Solving the Problems

[0019] Each description and embodiment disclosed in the present invention can be applied to other descriptions and embodiments respectively. That is, all combinations of various elements disclosed in the present invention belong to the scope of the present invention.

[0020] Also, it cannot be said that the scope of the present invention is limited by the following specific description.

[0021] The present invention provides an anti-OX40L antibody or a binding fragment that specifically binds to OX40L while inhibiting the interaction between OX40L and the OX40 receptor.

[0022] As used herein, the term "antibody" refers to a protein molecule that acts as a receptor to specifically recognize an antigen, including an immunoglobulin molecule that is immunologically reactive with a particular antigen, and includes polyclonal antibodies, monoclonal antibodies, whole antibodies, and binding fragments. The term may also include chimeric antibodies (e.g., humanized murine antibodies), humanized antibodies, human antibodies, and bivalent or bispecific molecules (e.g., bispecific antibodies), diabodies, triabodies, and tetrabodies.

[0023] Typically, immunoglobulins have heavy and light chains, each of which can contain a constant region and a variable region (also known as a domain). The variable regions of the light and heavy chains can contain three hypervariable regions called complementarity-determining regions (hereinafter referred to as "CDRs") and four framework regions. The CDRs mainly play a role in binding to an epitope of an antigen. The CDRs of each chain are typically named CDR1, CDR2, and CDR3, starting from the N-terminus, and can be further identified by the chain in which the particular CDR is located.

[0024] A whole antibody is a structure having two full-length light chains and two full-length heavy chains, each of which is linked to a heavy chain by a disulfide bond. The whole antibody can include IgA, IgD, IgE, IgM, and IgG, with IgG being classified into subtypes IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgG10, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18, IgG19, IgG11, IgG11, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18, IgG19, IgG20, IgG21, IgG22, IgG23, IgG24, IgG25, IgG26, IgG27, 1 , IgG 2 , IgG 3 , and IgG 4It can include. The heavy chain constant region can have gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and can have gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2) as subclasses. The constant region of the light chain can have kappa (κ) and lambda (λ) types.

[0025] As used herein, the terms "fragment", "antibody fragment", "antigen-binding fragment", and "binding fragment" are used interchangeably to mean any fragment of the antibody of the present invention having antigen-binding function, and may include Fab, Fab’, F(ab’)2, and Fv, etc.

[0026] The Fab has a structure having the variable regions of the light chain and the heavy chain, and the "constant region of the light chain and the first constant region (CH1 domain) of the heavy chain", and has one antigen-binding site. Fab’ is different from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. F(ab’) 2 The antibody can be produced by the cysteine residues in the hinge region of Fab’ forming disulfide bonds. Fv (variable fragment) means the smallest antibody fragment having only the variable site of the heavy chain and the variable site of the light chain. The double-stranded Fv (dsFv) has the variable site of the heavy chain and the variable site of the light chain linked by a disulfide bond, and the single-chain Fv (scFv) generally has the variable region of the heavy chain and the variable region of the light chain covalently linked via a peptide linker. These binding fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by limited cleavage of whole antibody with papain, and F(ab’)2 fragment can be obtained by cleavage with pepsin), and can be produced, for example, by genetic recombination technology.

[0027] As used herein, the term "monoclonal antibody" refers to an antibody molecule of single-molecule composition obtained from a substantially identical antibody population, and such a monoclonal antibody exhibits single-binding specificity and affinity for a specific epitope. In one embodiment of the present invention, the anti-OX40L antibody that specifically binds to OX40L of the present invention, or the bispecific antibody that specifically binds to OX40L and TNFα may be a single-molecule antibody. Specifically, the anti-OX40L antibody means an antibody of single-molecule composition that specifically binds to a specific epitope of OX40L, and the bispecific antibody may mean a bispecific antibody of single-molecule composition that simultaneously and specifically binds to specific epitopes of OX40L and TNFα.

[0028] In an embodiment of the present invention, the anti-OX40L antibody of the present invention and the bispecific antibody that specifically binds to OX40L and TNFα may be a chimeric antibody, a humanized antibody, or a human antibody, but are not particularly limited thereto.

[0029] In the present invention, the term "chimeric antibody" refers to an antibody obtained by recombining the variable region of a mouse antibody and the constant region of a human antibody, and is an antibody with a greatly improved immune response compared to a mouse antibody.

[0030] In the present invention, the term "humanized antibody" means an antibody obtained by modifying the protein sequence of a non-human antibody to be similar to an antibody variant naturally produced in humans. For example, the humanized antibody can be prepared by recombining the CDR derived from a mouse with the FR derived from a human antibody to produce a humanized variable region, and then recombining this with the constant region of a desired human antibody to produce a humanized antibody. However, when only CDR grafting is performed, the affinity of the humanized antibody decreases. Therefore, by making some important FR amino acid residues that are thought to affect the three-dimensional structure of the CDR affinity to those of the mouse antibody, the affinity can be increased to the same level as that of the original mouse antibody.

[0031] As used herein, the term "human antibody" refers to a molecule derived from human immunoglobulins, and all amino acid sequences constituting the antibody, including the complementarity-determining regions and framework regions, are composed of the amino acid sequences of human immunoglobulins. Human antibodies are usually used to treat human diseases, which may have three or more potential advantages. First, it can interact better with the human immune system and can more efficiently destroy target cells, for example, by complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). Second, there is an advantage that the human immune system does not recognize the antibody as foreign. Third, there is an advantage that the half-life in the human circulatory system is similar to that of natural antibodies even when the drug is administered in a smaller amount and at a lower frequency. In embodiments of the present invention, the anti-OX40L antibody specifically binding to the OX40L of the present invention and the bispecific antibody specifically binding to OX40L and TNFα may be human antibodies. Therefore, the human anti-OX40L antibody of the present invention and the human bispecific antibody of the present invention not only show strong affinity for OX40L and effectively inhibit the binding of cells expressing OX40L (e.g., monocytes) to the OX40 receptor, but also show low immunogenicity because both the heavy chain and light chain domains are of human origin and are usefully used for the treatment of autoimmune diseases, inflammatory diseases, and the like.

[0032] As used herein, the term "OX40L" refers to a ligand that uses the OX40 protein as a receptor, specifically, a protein that binds to the OX40 receptor. Information on OX40L can be obtained from known databases such as GenBank of the National Institutes of Health, USA. As an example thereof, the Accession Number is Gene ID:54567, NCBI Reference Sequence:NM_003326.5( TNFSF4 ver1), NM_001297562.2 (TNFSF4 ver2) Information on OX40L including the amino acid sequence of SEQ ID NO:1 is provided.

[0033] OX40L is overexpressed in antigen-presenting cells (APCs) and is known to activate several immune cells. Specifically, it has been observed that OX40L is overproduced in patients with autoimmune diseases, similar to TNFα and INFγ (Eur. J. Immunol. 2000. 30: 2815-2823). Different from TNFα that is distributed throughout the body, OX40L is produced only in activated immune cells and is mainly distributed at the lesion sites. OX40L is a multiple immune regulatory protein that can simultaneously participate in antigen-presenting cells (APCs) of the innate immune system and helper T cells of the adaptive immune system to restore immune homeostasis (Nature Reviews Rheumatology vol.12, 74-76 (2016), (Clinic Rev Allerg Immunol, 2016)).

[0034] In the present invention, the term "OX40" means a protein that mediates OX40 / OX40L signal transduction. The OX40 may be included without limitation as long as it is a protein that mediates OX40 / OX40L signal transduction.

[0035] In the present invention, the term "inhibiting the interaction between OX40L and OX40" or "inhibiting the interaction between OX40L and the OX40 receptor" means that an anti-OX40L antibody or a binding fragment thereof that specifically binds to OX40L of the present invention binds to OX40L, and the interaction between OX40L and OX40 It means suppressing or inhibiting the interaction. When an anti-OX40L antibody or its binding fragment binds to OX40L, by suppressing or inhibiting the biological function of OX40L, the binding of OX40L and OX40 is suppressed or inhibited, and the signal transduction of OX40 cannot be brought about. That is, the binding of the anti-OX40L antibody or its binding fragment to OX40L suppresses or inhibits the interaction between OX40L and OX40, and as a result, the signal transduction of OX40 is suppressed or inhibited.

[0036] In the present invention, the "anti-OX40L antibody that specifically binds to OX40L" means an antibody that specifically binds to OX40L and suppresses or inhibits the biological activity of OX40L. The said antibody can suppress or inhibit the biological activity of OX40L and suppress or inhibit the interaction between OX40L and the OX40 receptor. In the present specification, the "anti-OX40L antibody that specifically binds to OX40L" can be used interchangeably with the "antibody that specifically binds to OX40L" or the "anti-OX40L antibody".

[0037] The form of the said anti-OX40L antibody can include both the whole antibody and the binding fragment as described above. The anti-OX40L antibody of the present invention specifically binds to human OX40L while inhibiting the interaction between OX40L and the OX40 receptor, and is usefully used for treating autoimmune diseases, inflammatory diseases, etc. By specifically binding to human OX40L that is overexpressed in autoimmune diseases or inflammatory diseases, the therapeutic effect can be maximized while minimizing side effects.

[0038] In an embodiment of the present invention, the anti-OX40L antibody or its antigen-binding fragment that specifically binds to OX40L and inhibits the interaction between OX40L and the OX40 receptor can bind to human OX40L at 3×10 -9 M or less. Specifically, the anti-OX40L antibody or its antigen-binding fragment can bind at 1.5×10 -9 M, 1.3×10 -9 M, particularly at 1×10 -9 M or less of K D .

[0039] In the present invention, the term "binding constant (K on )" means the binding ratio of a specific antibody-antigen interaction, and the term "dissociation constant (K off )" means the dissociation ratio of a specific antibody-antigen interaction. Also, in the present invention, the term "affinity for an antigen (K D )" is the ratio of K off :K on (i.e., K off / K on ) expressed in molar concentration (M). The K D value for an antibody can be measured using methods widely established in the art. For example, as a method for measuring the K D value of an antibody, surface plasmon resonance analysis using a Biacore TM system can be mentioned, but it is not limited thereto.

[0040] The anti-OX40L antibody or antigen-binding fragment thereof of the present invention exhibits a high binding force to OX40L, can suppress or inhibit the activity against OX40L even at low concentrations, and can exhibit an excellent therapeutic effect against autoimmune diseases or inflammatory diseases.

[0041] In an embodiment of the present invention, the anti-OX40L antibody or antigen-binding fragment thereof can recognize a conformational epitope of human OX40L. For example, the anti-OX40L antibody or antigen-binding fragment thereof of the present invention can bind to the amino acid sequences at positions 93 to 100 and 141 to 151 in the amino acid sequence of the human OX40L protein represented by SEQ ID NO: 1 at 3×10 -9 M or less. Specifically, the anti-OX40L antibody or antigen-binding fragment thereof can bind to the amino acid sequences at positions 93 to 100 and 141 to 151 in the amino acid sequence of the human OX40L protein represented by SEQ ID NO: 1 at 1.5×10 -9 M, 1.3×10 -9 M, particularly at 1×10 -9 M or less of K D .

[0042] In an embodiment of the present invention, the anti-OX40L antibody or an antigen-binding fragment thereof can bind to the amino acid sequence of human OX40L protein represented by SEQ ID NO: 3 or SEQ ID NO: 4 with a high binding affinity, and specifically, it can bind at 3×10 -9 M or less. Specifically, the anti-OX40L antibody or an antigen-binding fragment thereof can bind at 1.5×10 -9 M, 1.3×10 -9 M, and particularly, it can bind at K of 1×10 -9 M or less. D

[0043] In an embodiment of the present invention, the K of the anti-OX40L antibody or an antigen-binding fragment thereof for human OX40L D may be measured by surface plasmon resonance (Biacore) analysis.

[0044] Specifically, the anti-OX40L antibody or an antigen-binding fragment thereof may include, but is not limited to, the sequences listed below.

[0045] In an embodiment of the present invention, the anti-OX40L antibody or an antigen-binding fragment thereof has a heavy chain CDR1 as described in one amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, and 14; a heavy chain CDR2 as described in one amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 16, 17, and 18; and a heavy chain CDR3 as described in one amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, 21, and 22, and includes a heavy chain variable region, and has a light chain CDR1 as described in one amino acid sequence selected from the group consisting of SEQ ID NOs: 23 and 24; a light chain CDR2 as described in one amino acid sequence selected from the group consisting of SEQ ID NOs: 25 and 26; and a light chain CDR3 as described in one amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, 29, and 30, and may include a light chain variable region.

[0046] ​In the present invention, the term "heavy chain" may include the full-length heavy chain and its fragments including the variable region domain VH having an amino acid sequence with a variable region sequence sufficient to confer specificity to an antigen and three constant region domains CH1, CH2, and CH3.

[0047] Also, in the present invention, the term "light chain" may include the full-length light chain and its fragments including the variable region domain VL having an amino acid sequence with a variable region sequence sufficient to confer specificity to an antigen and the constant region domain CL.

[0048] In an embodiment of the present invention, the anti-OX40L antibody or its antigen-binding fragment may be an antibody including a heavy chain variable region containing heavy chain CDR1 represented by SEQ ID NO: 12; heavy chain CDR2 represented by SEQ ID NO: 15; and heavy chain CDR3 represented by SEQ ID NO: 19, and a light chain variable region containing light chain CDR1 represented by SEQ ID NO: 23; light chain CDR2 represented by SEQ ID NO: 25; and light chain CDR3 represented by SEQ ID NO: 27, but is not limited thereto. In an embodiment of the present invention, the antibody was named 02C09.

[0049] In an embodiment of the present invention, the anti-OX40L antibody or its antigen-binding fragment may be an antibody including a heavy chain variable region containing heavy chain CDR1 represented by SEQ ID NO: 13; heavy chain CDR2 represented by SEQ ID NO: 16; and heavy chain CDR3 represented by SEQ ID NO: 20, and a light chain variable region containing light chain CDR1 represented by SEQ ID NO: 24; light chain CDR2 represented by SEQ ID NO: 26; and light chain CDR3 represented by SEQ ID NO: 28, but is not limited thereto. In an embodiment of the present invention, the antibody was named hu3F07, I3F07.

[0050] In an embodiment of the present invention, the anti-OX40L antibody or its antigen-binding fragment may be an antibody including a heavy chain variable region containing heavy chain CDR1 represented by SEQ ID NO: 13; heavy chain CDR2 represented by SEQ ID NO: 17; and heavy chain CDR3 represented by SEQ ID NO: 21, and An antibody may include, but is not limited to, a light chain variable region including a light chain CDR1 represented by SEQ ID NO: 24, a light chain CDR2 represented by SEQ ID NO: 26, and a light chain CDR3 represented by SEQ ID NO: 29. In an embodiment of the present invention, the antibody was named 10H07.

[0051] In an embodiment of the present invention, the anti - OX40L antibody or its antigen - binding fragment may include, but is not limited to, a heavy chain variable region including a heavy chain CDR1 represented by SEQ ID NO: 14, a heavy chain CDR2 represented by SEQ ID NO: 18, and a heavy chain CDR3 represented by SEQ ID NO: 22, and a light chain variable region including a light chain CDR1 represented by SEQ ID NO: 24, a light chain CDR2 represented by SEQ ID NO: 26, and a light chain CDR3 represented by SEQ ID NO: 30. In an embodiment of the present invention, the antibody was named 21G07.

[0052] In an embodiment of the present invention, the anti - OX40L antibody or its antigen - binding fragment may include a heavy chain variable region described in one amino acid sequence selected from the group consisting of SEQ ID NOs: 37, 41, 45, 49, and 53, and a light chain variable region described in one amino acid sequence selected from the group consisting of SEQ ID NOs: 38, 42, 46, 50, and 54.

[0053] In an embodiment of the present invention, the anti - OX40L antibody or its antigen - binding fragment (a) a heavy chain variable region described in SEQ ID NO: 37 and a light chain variable region described in SEQ ID NO: 38; (b) a heavy chain variable region described in SEQ ID NO: 41 and a light chain variable region described in SEQ ID NO: 42; (c) a heavy chain variable region described in SEQ ID NO: 45 and a light chain variable region described in SEQ ID NO: 46; (d) a heavy chain variable region described in SEQ ID NO: 49 and a light chain variable region described in SEQ ID NO: 50; or (e) a heavy chain variable region described in SEQ ID NO: 53 and a light chain variable region described in SEQ ID NO: 54.

[0054] In an embodiment of the present invention, the anti - OX40L antibody or its antigen - binding fragment is a heavy - chain constant region described in one amino - acid sequence selected from the group consisting of SEQ ID NO: 5, 6, 7, and 8; and may include a light - chain constant region described in the amino - acid sequence of SEQ ID NO: 10.

[0055] In an embodiment of the present invention, the anti - OX40L antibody or its binding fragment is the (a) heavy - chain variable region described in SEQ ID NO: 37 and the light - chain variable region described in SEQ ID NO: 38; (b) heavy - chain variable region described in SEQ ID NO: 41 and the light - chain variable region described in SEQ ID NO: 42; (c) heavy - chain variable region described in SEQ ID NO: 45 and the light - chain variable region described in SEQ ID NO: 46; (d) heavy - chain variable region described in SEQ ID NO: 49 and the light - chain variable region described in SEQ ID NO: 50; or (e) heavy - chain variable region described in SEQ ID NO: 53 and the light - chain variable region described in SEQ ID NO: 54, and a heavy - chain constant region described in one amino - acid sequence selected from the group consisting of SEQ ID NO: 5, 6, 7, and 8 and a light - chain constant region described in the amino - acid sequence of SEQ ID NO: 10 may be an antibody or its binding fragment containing the same.

[0056] Here, the antibody containing the variable regions of (a) and (b) is a humanized antibody, and the antibody containing the variable regions of (c) - (d) is a chimeric antibody.

[0057] In an embodiment of the present invention, the anti - OX40L antibody or its antigen - binding fragment has physicochemical properties that show sufficient effects in the human body and has excellent thermal stability. For example, the anti - OX40L antibody or its antigen - binding fragment melts at a temperature exceeding 50°C, specifically, at a temperature of 59°C or higher, and can have a half - life of about two weeks or more in the human body.

[0058] In an embodiment of the present invention, when the anti - OX40L antibody of the present invention contains a constant region, it can contain a constant region derived from IgG, IgA, IgD, IgE, IgM or a combination or hybrid thereof.

[0059] As used herein, the term "combination" means that when forming a dimer or multimer, a polypeptide encoding a single-chain immunoglobulin constant region derived from the same species forms a bond with a single-chain polypeptide derived from a different species. As an example, a dimer or multimer can be formed from two or more constant regions selected from the group consisting of the constant regions of IgG, IgA, IgD, IgE, and IgM.

[0060] As used herein, the term "hybrid" means that within a single-chain immunoglobulin heavy-chain constant region, there are sequences corresponding to two or more immunoglobulin heavy-chain constant regions derived from different species. As an example, a hybrid of a domain consisting of 1 to 4 domains selected from the group consisting of CH1, CH2, CH3, and CH4 of IgG, IgA, IgD, IgE, and IgM is possible.

[0061] On the other hand, combinations or hybridizations of the IgG1, IgG2, IgG3, and IgG4 heavy-chain constant regions, which are subtypes of IgG, are also possible. The above combinations and hybridizations are the same as the previous descriptions.

[0062] In an embodiment of the present invention, the IgG1 heavy-chain constant region may be the IgG1 heavy-chain constant region described in SEQ ID NO: 5, the IgG1 N297A heavy-chain constant region may be the heavy-chain constant region described in SEQ ID NO: 6, the IgG4 heavy-chain constant region may be the IgG4 heavy-chain constant region described in SEQ ID NO: 7, and the IgG4 S228P heavy-chain constant region may be the IgG4 heavy-chain constant region described in SEQ ID NO: 8, but is not limited thereto.

[0063] Further, when the anti-OX40L antibody specific to OX40L of the present invention includes a light-chain constant region, the light-chain constant region may be derived from lambda (λ) or kappa (κ) light chain. When the light-chain constant region of the antibody is derived from a kappa light chain, it may be the kappa light-chain constant region described in SEQ ID NO: 10, but is not limited thereto.

[0064] In the present invention, the antibody may include all mouse antibodies produced from mice and mutants in which a part of the amino acid sequence of the parent antibody is substituted, added, and / or deleted in order to improve the affinity, immunity, etc. of the antibody. The mutants may include, but are not limited to, chimeric antibodies, humanized antibodies, affinity-optimized antibodies, etc. In the present invention, the "affinity-optimized antibody" refers to a mutant in which a part of the CDR sequence of a specific antibody is substituted, added, or deleted, and which binds to the same antigen epitope as the specific antibody while having an improved binding affinity for the antigen.

[0065] In the present invention, the mutant comprehensively refers to an antibody in which a part of the CDR amino acid sequence of the parent antibody is mutated (substituted, added, or deleted), provided that it contains the same CDR as the parent antibody or targets the same epitope. Such mutants may be appropriately adjusted by those skilled in the art to improve the affinity, immunity, etc. of the antibody within the range in which the binding ability to the same epitope is maintained.

[0066] The anti-OX40L antibody or antigen-binding fragment thereof of the present invention may include not only the sequence of the anti-OX40L antibody described herein but also its biological equivalents, as long as it can specifically recognize OX40L. For example, further changes may be made to the amino acid sequence of the antibody in order to further improve the binding affinity and / or other biological properties of the antibody. These modifications include, for example, deletion, insertion, and / or substitution of amino acid sequence residues of the antibody. These amino acid mutations are made based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. By analyzing the size, shape, and type of the amino acid side chain substituents, arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; phenylalanine, tryptophan 、and tyrosine are found to have similar shapes. Therefore, based on these, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be said to be biologically functional equivalents. For example, in an embodiment of the present invention, the anti-OX40L antibody or its antigen-binding fragment may contain conservative amino acid changes in one or more residues of the amino acid sequence described in the sequence numbers herein, and the conservative amino acid changes may include the substitutions in Table 1 below.

[0067]

Table 1

[0068] According to an embodiment of the present invention, a novel antibody targeting OX40L was prepared. A library prepared from mice immunized with human OX40L (hOX40L), and antibodies 02C09, hu3F07, 10H07, and 21G07 specific for OX40L were prepared from a human library. The antibodies have an affinity for OX40L at the 0.2 - 0.7 nM level, specifically bind to OX40L with high affinity (Table 32, Figure 4), and the in vitro OX40L inhibitory ability is 0.2 - 0.9 nM, which was confirmed to be significantly superior to the control antibody (Figure 5). Also, in T cells, the result of blocking the immune activity by OX40L was shown (Figure 6). These results indicate that the anti-OX40L antibody specific for OX40L of the present invention can efficiently block the binding to the OX40 receptor, suppress OX40 / OX40L signal transduction, show significantly excellent effects in the treatment of autoimmune diseases and inflammatory diseases, can minimize side effects, and can selectively treat autoimmune diseases and inflammatory diseases while maintaining the homeostasis of the immune system.

[0069] The anti-OX40L antibody or its antigen-binding fragment of the present invention inhibits the function of the said OX40L and is effectively used for the treatment of autoimmune diseases or inflammatory diseases.

[0070] The human immune system consists of two parts: the innate immune system and the adaptive immune system. Autoimmune diseases can occur when the innate immune system and the adaptive immune system are abnormally activated.

[0071] It is known that when OX40L binds to the OX40 receptor, immune cells involved in the innate and adaptive immune systems are overactivated, causing various diseases.

[0072] The above-mentioned OX40L is simultaneously involved in antigen-presenting cells (APCs) of the innate immune system and helper T cells of the adaptive immune system, and can be involved in the homeostasis of the immune system (Nature Reviews Rheumatology vol.12, 74 - 76 (2016), (Clinic Rev Allerg Immunol, 2016)). Also, different from cytokines that are distributed throughout the body, the above-mentioned OX40L is concentratedly distributed at the lesion site. Therefore, the anti-OX40L antibody or its binding fragment of the present invention is likely to bind to OX40L around the lesion.

[0073] Therefore, the anti-OX40L antibody or its antigen-binding fragment can bind to OX40L that is concentratedly distributed around the lesion, maximize the therapeutic effect on autoimmune diseases and inflammatory diseases, reduce side effects, and improve safety.

[0074] In addition, the adaptive immune system has a slower reaction rate but higher persistence than the innate immune system and can be a major factor in autoimmune diseases caused by overactivation of the immune system. The suppression or inhibition of OX40L by the anti-OX40L antibody or its antigen-binding fragment of the present invention has a great advantage in that it can regulate not only innate immune cells but also adaptive immune cells that have not been affected by conventional autoimmune disease therapeutics.

[0075] That is, the present invention that effectively suppresses and inhibits the interaction between OX40L and OX40 can be effectively used for treating autoimmune diseases and inflammatory diseases with an anti-OX40L antibody that specifically binds to OX40L or an antigen-binding fragment thereof.

[0076] The present invention provides a nucleic acid (polynucleotide) encoding the anti-OX40L antibody or an antigen-binding fragment thereof, an expression vector containing the nucleic acid, and a transformant into which the expression vector has been introduced.

[0077] As used herein, the term "nucleic acid" or "polynucleotide" comprehensively includes DNA and RNA molecules, and the nucleotides that are the basic building blocks of the nucleic acid molecules include not only natural nucleotides but also analogues in which the sugar or base moiety is modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, (1990) 90: 543-584).

[0078] The sequences of the nucleic acid molecules encoding the heavy and light chain variable regions of the present invention may be modified, and the modifications include addition, deletion, or non-conservative substitution or conservative substitution of nucleotides.

[0079] The nucleic acids of the present invention are also interpreted to include nucleotide sequences that exhibit substantial identity to the nucleotide sequences. In the present invention, substantial identity means that when the nucleotide sequence of the present invention and any other sequence are aligned to maximize correspondence and the aligned sequences are analyzed using an algorithm commonly used in the art, it can mean a nucleotide sequence showing at least 80% homology, specifically, at least 90% homology, more specifically, at least 95% homology.

[0080] As used herein, the term "vector" or "expression vector" is a means for expressing a gene of interest in a host cell and includes plasmid vectors; cosmid vectors; and viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors. Specifically, it may be a plasmid vector, but is not limited thereto.

[0081] In the vector of the present invention, the nucleic acid molecule encoding the light chain variable region and the nucleic acid molecule encoding the heavy chain variable region may be operatively linked to a promoter.

[0082] In the present invention, the term "operatively linked" means a functional binding of a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of binding positions of a transcription regulatory factor) to another nucleic acid sequence, whereby the regulatory sequence regulates the transcription and / or translation of the other nucleic acid sequence.

[0083] The recombinant vector system of the present invention can be constructed by various methods known in the art. For example, the specific method is disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Sp ring Harbor Laboratory Press (2001), which is incorporated herein by reference.

[0084] In the present invention, an expression vector containing a nucleic acid (polynucleotide) encoding an anti-OX40L antibody or an antigen-binding fragment thereof is not particularly limited, but may be a vector capable of replicating and / or expressing the nucleic acid in eukaryotic cells or prokaryotic cells including mammalian cells (for example, human, monkey, rabbit, rat, hamster, mouse cells, etc.), plant cells, yeast cells, insect cells, or bacterial cells (for example, Escherichia coli, etc.). Specifically, it may be a vector operably linked to an appropriate promoter so that the nucleic acid is expressed in a host cell and contains at least one selectable marker. More specifically, it may be in a form in which the nucleic acid is introduced into a phage, plasmid, cosmid, minichromosome, virus, retroviral vector, etc.

[0085] The expression vector containing the nucleic acid (polynucleotide) encoding the anti-OX40L antibody may be an expression vector containing the nucleic acid encoding the heavy chain or the light chain of the anti-OX40L antibody respectively, or an expression vector containing both the nucleic acid encoding the heavy chain and the light chain.

[0086] In the present invention, the transformant into which the expression vector is introduced is not particularly limited, but bacterial cells such as Escherichia coli, Streptomyces, Salmonella typhimurium, etc. transformed by introducing the expression vector; yeast cells; fungal cells such as Pichia pastoris; insect cells such as Drosophila, Spodoptera Sf9 cells; CHO (Chinese hamster ovary cells), SP2 / 0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T, Bowes melanoma cells, HT-1080, BHK (baby hamster kidney cells), HEK (human embryonic kidney cells), PERC.6 (human retinal cells), etc. animal cells; or plant cells. In an embodiment of the present invention, HEK cells or the like are used as host cells.

[0087] As used herein, the term "introduction" means a method of delivering a vector containing a nucleic acid (polynucleotide) encoding an anti-OX40L antibody or an antigen-binding fragment thereof to a host cell. This introduction can be carried out by various methods known in the art, such as calcium phosphate coprecipitation, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofectamine and protoplast fusion methods. In addition, transduction means delivering an object into a cell using virus particles by means of infection. Incidentally, gene bombardment such as a gene gun can be used to introduce the vector into the host cell. In the present invention, introduction may be used interchangeably with transformation.

[0088] The present invention provides a method for producing an anti-OX40L antibody or an antigen-binding fragment thereof.

[0089] The anti-OX40L antibody or an antigen-binding fragment thereof of the present invention can be easily produced by known monoclonal antibody production techniques. For example, the production of monoclonal antibodies can be carried out by producing hybridomas using B lymphocytes obtained from immunized animals (Koeher and Milstein, 1976, Nature, 256:495), or by using phage display technology, but is not limited thereto.

[0090] An antibody library using phage display technology is a method of directly obtaining antibody genes from B lymphocytes and expressing antibodies on the surface of phages (phage) without producing hybridomas. By using phage display technology, many conventional difficulties associated with the production of monoclonal antibodies due to B cell immortalization can be overcome. Generally, phage display technology includes: 1) inserting a random sequence oligonucleotide into a gene site corresponding to the N-terminus of the phage coat protein pIII (or pIV); 2) expressing a fusion protein of a polypeptide encoded by a part of the native coat protein and the random sequence oligonucleotide; 3) treating a receptor substance capable of binding to the polypeptide encoded by the oligonucleotide; 4) eluting the peptide phage particles bound to the receptor using a low pH molecule or a molecule with binding competitiveness; 5) amplifying the phage eluted by panning in a host cell; 6) repeating the method to obtain a desired amount; and 7) determining the active peptide sequence from the DNA sequence of the phage clone selected by panning.

[0091] In an embodiment of the present invention, the method for producing the anti-OX40L antibody or an antigen-binding fragment thereof of the present invention can be carried out using phage display technology. Those skilled in the art are familiar with known phage display technologies, such as Barbas et al. (METHODS: A Companion Referring to the methods known from the papers of Methods in Enzymology 2:119, 1991 and J. Virol. 2001 Jul;75(14):6692 - 9 and Winter et al. (Ann. Rev. Immunol. 12:433, 1994), each step in the production method of the present invention can be easily carried out. The phages used to construct the antibody library include, for example, filamentous phages such as fd, M13, f1, If1, Ike, Zj / Z, Ff, Xf, Pf1 or Pf3 phages, but are not limited thereto. Also, as vectors used to express heterologous genes on the surface of the filamentous phage, for example, phage vectors such as fUSE5, fAFF1, fd - CAT1 or fdtetDOG, or phagemid vectors such as pHEN1, pComb3, pComb8 or pSEX can be mentioned, but are not limited thereto. Also, as helper phages used to provide the wild - type coat protein necessary for reinfection of the recombinant phage for amplification, for example, M13K07 or VSCM13 can be mentioned, but are not limited thereto.

[0092] The polynucleotide encoding the phage display clone of the present invention can be easily isolated and sequenced by ordinary methods. As an example, oligonucleotide primers designed to specifically amplify the heavy - chain and light - chain coding regions from hybridoma or phage template DNA can be used. Once the polynucleotide is isolated, it can be incorporated into an expression vector, and then the expression vector can be introduced into an appropriate host cell to produce the desired monoclonal antibody from the transformed host cell (i.e., transformant). Therefore, the method for producing the human monoclonal antibody of the present invention may be a method for producing a human monoclonal antibody including the step of amplifying an expression vector containing a polynucleotide encoding a human monoclonal antibody, but is not limited thereto.

[0093] The anti - OX40L antibody or antigen - binding fragment thereof of the present invention can be prepared by the aforementioned known recombinant means or biochemical methods, and the antibody can be recovered from the culture broth of a transformant into which an expression vector containing a nucleic acid encoding the antibody has been introduced into an appropriate host cell.

[0094] In an embodiment of the present invention, a method for preparing (producing) an anti - OX40L antibody or antigen - binding fragment thereof that specifically binds to OX40L is (a) culturing the transformant to produce an anti - OX40L antibody or antigen - binding fragment thereof; and (b) recovering the anti - OX40L antibody or antigen - binding fragment thereof produced in step (a). It may be a method for preparing an anti - OX40L antibody or antigen - binding fragment thereof that specifically binds to OX40L.

[0095] In an embodiment of the present invention, the anti - OX40L antibody or antigen - binding fragment thereof can be isolated by known isolation methods. Examples thereof include appropriately isolating from the culture medium by ordinary immunoglobulin purification methods such as protein A sepharose, gel electrophoresis, dialysis, or affinity chromatography, but are not limited thereto.

[0096] The present invention provides a bispecific antibody comprising an anti - OX40L antibody or antigen - binding fragment thereof; and an antibody or antigen - binding fragment thereof that specifically binds to tumor necrosis factor α (TNFα).

[0097] As used herein, the term "bispecific antibody" means an antibody that can bind to two different types of antigens (target proteins). Specifically, it may be a form that does not exist naturally and is produced by genetic engineering or any method.

[0098] The bispecific antibody of the present invention is an antibody that can bind to two different targets, and the bispecific antibody can bind to OX40L and TNFα.

[0099] The "bispecific antibody" of the present invention may be used interchangeably with "dual-target protein", "bispecific antibody" or "bispecific antibody protein".

[0100] The antibody or its binding fragment that specifically binds to OX40L, which is a component of the bispecific antibody of the present invention, includes an antibody or its binding fragment that can specifically bind to OX40L and block the OX40L / OX40L signaling pathway. In an embodiment of the present invention, the antibody or its binding fragment that specifically binds to OX40L, which is a component of the bispecific antibody of the present invention, may be substantially the same anti-OX40L antibody or its binding fragment of the present invention as long as it does not conflict with the foregoing content regarding the anti-OX40L antibody or its binding fragment that specifically binds to OX40L. Further, in an embodiment of the present invention, the antibody or its binding fragment that specifically binds to OX40L, which is a component of the bispecific antibody of the present invention, is an antibody that can specifically bind to OX40L and block the OX40L / OX40L signaling pathway, and may be an antibody or its binding fragment described in WO2018083248, WO2009141239, US2017260279, WO2006029879, or WO2011073180.

[0101] The antibody or its binding fragment that specifically binds to OX40L, which is a component of the bispecific antibody of the present invention, specifically binds to OX40L overexpressed in immune cells, and can not only concentrate the bispecific antibody of the present invention on immune cells expressing TNFα, but may also have the ability to bind to TNFα and reduce immune cell activity by itself.

[0102] In addition, OX40L is overexpressed in APCs, interacts with the OX40 receptor expressed on T cells to induce the proliferation / differentiation / activation of immune cells, and induces the secretion of various inflammatory cytokines, and is one of the upstream signals that simultaneously activate innate immunity and adaptive immunity. The anti-OX40L antibody or its antigen-binding fragment can inhibit the OX40 / OX40L signal and reduce the immune response that is over-activated in patients with autoimmune diseases and inflammatory diseases.

[0103] Furthermore, the anti-TNFα antibody or its binding fragment can also be effective for patients with autoimmune diseases and inflammatory diseases who show resistance to the treatment against TNFα, which is a downstream signal of the inflammatory response.

[0104] As used herein, the term "bispecific antibody comprising an antibody or its antigen-binding fragment that specifically binds to OX40L and an antibody or its antigen-binding fragment that specifically binds to TNFα" may be included without limitation as long as it is a bispecific protein capable of simultaneously inhibiting two signal transduction pathways by OX40L and TNFα. The antibody or its antigen-binding fragment that specifically binds to TNFα and the antibody or its antigen-binding fragment that specifically binds to OX40L constituting the bispecific antibody may each include any of the full-length antibody and antibody fragment forms described for the anti-OX40L antibody or its binding fragment.

[0105] In the present invention, the term "inhibiting the interaction between OX40L and OX40" means that the bispecific antibody that specifically binds to OX40L of the present invention binds to OX40L and inhibits the interaction between OX40L and OX40. Due to the inhibition of the interaction between OX40L and OX40 by the binding of the bispecific antibody, the structural change of OX40 caused by the binding of OX40L to OX40 is not brought about, so it cannot be hydrolyzed and the signal transduction of OX40 is made impossible.

[0106] As used herein, the term "antibody that specifically binds to TNFα" encompasses all antibodies that specifically bind to TNFα as an antigen over a wide range in the body. In one embodiment of the present invention, the antibody that specifically binds to TNFα is a therapeutic antibody targeting TNFα, which may be, but is not limited to, an antibody or its binding fragment described in WO1997029131, WO2003045400, WO2004050683, WO1998011917, EP1097945, WO2001037874, US2006024310, WO2006125229, WO2007056540, WO1994006476, WO2000059530, or WO2001000229. In one embodiment of the present invention, the antibody that specifically binds to TNFα may be adalimumab (trade name Humira, AbbVie), a therapeutic antibody approved by the US FDA, the European EMA, etc. and capable of being stably used, but is not necessarily limited thereto. Such an antibody that specifically binds to TNFα includes any of the aforementioned full-length antibodies or antibody fragments, and may be in the form of an IgG antibody, but is not limited thereto.

[0107] TNFα is a cytokine that regulates immune cells, acts as a pyrogen in the body to generate heat and induce cell death, and produces inflammatory cytokines to cause autoimmune diseases and inflammatory diseases. TNFα is mainly secreted from activated macrophages, but is also secreted from other various immune cells such as nerve cells. The most important role of TNFα is the regulation of immune cells. Inhibiting overexpressed TNFα can suppress autoimmune diseases and inflammatory diseases.

[0108] The bispecific antibody binds to TNFα and binds to human TNFα and the TNFα receptor (TNFα Suppresses or inhibits the interaction with the receptor, TNFα Rc), specifically, a bispecific antibody specific for TNFα, which is a component of the bispecific antibody, binds to TNFα, and may mean suppressing or inhibiting the interaction between TNFα and the TNFα receptor, but is not limited thereto.

[0109] For the purposes of the present invention, the TNFα receptor may be included without limitation as long as it is a protein that binds to mammalian TNFα, and specifically, may mean a protein that binds to human TNFα.

[0110] Inhibition of the interaction between TNFα and the TNFα receptor by the bispecific antibody specific for TNFα of the present invention or its binding fragment will suppress TNFα / TNFα receptor signal transduction by the binding of TNFα and the TNFα receptor. When TNFα and the TNFα receptor bind in the immune system, TNFα / TNFα receptor signal transduction is activated in immune cells, which regulates immune cell differentiation and the like by a mechanism different from the mechanism of action of the OX40L / OX40 signal transduction pathway, and is used as a therapeutic agent for various autoimmune diseases.

[0111] Therefore, the bispecific antibody specific for OX40L and TNFα of the present invention exhibits inhibitory ability against immune cells that are hyperactivated by different mechanisms, and is used as a therapeutic agent having a better therapeutic effect on autoimmune diseases and inflammatory diseases.

[0112] Therefore, the bispecific antibody or its antigen-binding fragment that specifically binds to OX40 and TNFα of the present invention, which effectively inhibits OX40L / OX40 signal transduction and TNFα / TNFα receptor signal transduction, can effectively treat autoimmune diseases and inflammatory diseases, and can maximize the therapeutic effect while minimizing side effects.

[0113] In an embodiment of the present invention, the anti-TNFα antibody or its antigen-binding fragment that specifically binds to TNFα is A heavy chain variable region comprising a heavy chain CDR1 set forth in SEQ ID NO: 89, a heavy chain CDR2 set forth in SEQ ID NO: 90, and a heavy chain CDR3 set forth in SEQ ID NO: 91, and It may also include a light chain variable region comprising a light chain CDR1 set forth in SEQ ID NO: 92, a light chain CDR2 set forth in SEQ ID NO: 93, and a light chain CDR3 set forth in SEQ ID NO: 94.

[0114] In an embodiment of the present invention, the anti-TNFα antibody or antigen-binding fragment thereof that specifically binds to TNFα may include a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 35 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 36, and specifically, it may include the variable region of Humira.

[0115] In an embodiment of the present invention, the form of the bispecific antibody is not particularly limited, but a bispecific antibody in which a binding fragment is linked by a linker to an antibody in IgG form is provided. Specifically, the bispecific antibody of the present invention may be one in which an antibody or antigen-binding fragment thereof that specifically binds to OX40L and an antibody or antigen-binding fragment thereof that specifically binds to TNFα are linked by a linker.

[0116] In an embodiment of the present invention, the linker may be a peptide or non-peptide set forth in the sequence of SEQ ID NO: 31 or SEQ ID NO: 32.

[0117] As used herein, the term "linker" basically refers to two different fusions It refers to a linker that can link partners (such as biopolymers, etc.) using hydrogen bonds, electrostatic interactions, van der Waals forces, disulfide bonds, salt bridges, hydrophobic interactions, covalent bonds, etc. Specifically, it may have at least one cysteine that can participate in at least one disulfide bond under physiological conditions or other standard peptide conditions (such as peptide purification conditions, peptide storage conditions). In addition to simply linking each fusion partner, it can also play a role in providing a certain interval between fusion partners, or serving as a hinge that provides flexibility or rigidity to the fusion body. The linker may be a non-peptide linker or a peptide linker, and may include any that are directly linked by peptide bonds, disulfide bonds, etc.

[0118] In the present invention, the linker is not particularly limited. Specifically, it may be a polypeptide that can link an antibody that specifically binds to OX40L and an antibody that specifically binds to TNFα. More specifically, it may be a peptide linker that can link the C-terminal of the Fc region or the C-terminal of the light chain region of the antibody that specifically binds to OX40L and the antibody that specifically binds to TNFα. Even more specifically, it may be a peptide linker consisting of an amino acid sequence in which the GGGGS motif is repeated. The GGGGS motif may be repeated 1 to 10 times. Most specifically, it may consist of the amino acid sequence of SEQ ID NO: 31 in which the GGGGS motif is repeated 3 times or the amino acid sequence of SEQ ID NO: 32 in which the GGGGS motif is repeated 4 times, but it is not limited thereto, and various linkers may be used within the range that can be easily derived by those with ordinary knowledge in the art.

[0119] In the present invention, the term "non-peptide linker" means a biocompatible linker in which two or more repeating units are bonded, and the repeating units may be linked to each other by any covalent bond rather than a peptide bond.

[0120] The non-peptide linker of the present invention is polyethylene glycol (polyethylene glycol The polymer may be a biodegradable polymer such as a polyethylene glycol (PEG) homopolymer, a polypropylene glycol homopolymer, an ethylene glycol-propylene glycol copolymer, a polyoxyethylated polyol, a polyvinyl alcohol, a polysaccharide, a dextran, or a polyvinyl ethyl ether, a lipid polymer, a chitin, or a hyaluronic acid, or a combination thereof. Specifically, the polymer may be a polyethylene glycol homopolymer, and derivatives thereof known in the art and derivatives that can be easily produced in the art may also be included in the scope of the present invention.

[0121] More specifically, it may be a polyethylene glycol homopolymer having a molecular weight of 1 to 5 kDa, and most specifically, it may be a linker having a molecular weight of about 3.4 kDa at both ends in the form of a bifunctional aldehyde that can link an antibody that specifically binds to OX40L and an antibody that specifically binds to TNFα. In particular, when it has aldehyde-reactive groups at both ends, it is effective in minimizing non-specific reactions.

[0122] The site directly or indirectly linked via the linker is not particularly limited, and may be an Fc portion, Fab', F(ab')2, Fab, Fv, etc. The bispecific antibody may be in a form in which all or a part (fragment) of an antibody that specifically binds to OX40L and all or a part (fragment) of an antibody that specifically binds to TNFα are linked, but is not particularly limited thereto.

[0123] Also, a form in which all or a part of a protein that specifically binds to OX40L and all or a part of a heavy chain of an antibody that specifically binds to TNFα are linked by a peptide linker; a form in which all or a part of a protein that specifically binds to OX40L and all or a part of a light chain of an antibody that specifically binds to TNFα are linked by a peptide linker; or a combination thereof. It may be a combination.

[0124] In an embodiment of the present invention, the bispecific antibody may be in a form in which an antibody specifically binding to OX40L in the form of Immunoglobulin G (IgG), and a full-length antibody, Fab’, F(ab’)2, Fab, Fv, rIgG or scFv-type antibody specifically binding to TNFα are linked by a linker.

[0125] In an embodiment of the present invention, it may be in a form in which an antibody specifically binding to TNFα in the form of Immunoglobulin G (IgG), and a full-length antibody, Fab’, F(ab’)2, Fab, Fv, rIgG or scFv-type antibody specifically binding to OX40L are linked by a linker, but is not limited thereto.

[0126] In the present invention, the term "binding fragment" includes antigen-binding forms of an antibody, including fragments having antigen-binding ability, for example, Fab’, F(ab’)2, Fab, Fv, rIgG and scFv. In particular, the term includes scFv (single-chain variavle fragment), and includes bivalent or Diabody, Triabody, and Tetrabody.

[0127] In the present invention, the term "scFv (single-chain variavle fragment)" means the smallest antibody fragment having a complete antigen recognition site and antigen-binding site, and includes the VH and VL domains of an antibody, where the domains may be present in a single polypeptide chain.

[0128] In an embodiment of the present invention, the bispecific antibody may be one in which an anti-TNFα antibody or its antigen-binding fragment specifically binding to TNFα is linked to at least one end of the light chain and the heavy chain of the anti-OX40L antibody.

[0129] In an embodiment of the present invention, the bispecific antibody may be one in which the anti-TNFα antibody or its antigen-binding fragment is linked to at least one of the C-termini of the light chain and the heavy chain of the anti-OX40L antibody. Specifically, in the bispecific antibody, the anti-TNFα antibody or its antigen-binding fragment may be linked to at least one of the C-termini of the light chain and the heavy chain of the anti-OX40L antibody via a linker. For example, in the bispecific antibody, the anti-TNFα antibody or its antigen-binding fragment may be linked to at least one of the C-termini of the light chain and the heavy chain of the anti-OX40L antibody via a linker having the amino acid sequence set forth in SEQ ID NO: 31 or SEQ ID NO: 32, but is not particularly limited thereto.

[0130] In an embodiment of the present invention, the bispecific antibody may be one in which an anti-OX40L antibody or its antigen-binding fragment that specifically binds to OX40L is linked to at least one of the termini of the light chain and the heavy chain of the anti-TNFα antibody.

[0131] In an embodiment of the present invention, the bispecific antibody may be one in which the anti-OX40L antibody or its antigen-binding fragment is linked to at least one of the C-termini of the light chain and the heavy chain of the anti-TNFα antibody. Specifically, in the bispecific antibody, the anti-OX40L antibody or its antigen-binding fragment may be linked to at least one of the C-termini of the light chain and the heavy chain of the anti-TNFα antibody by a linker. For example, the anti-OX40L antibody or its antigen-binding fragment may be linked to at least one of the C-termini of the light chain and the heavy chain of the anti-TNFα antibody via a linker having the amino acid sequence set forth in SEQ ID NO: 31, but is not particularly limited thereto.

[0132] In an embodiment of the present invention, the bispecific antibody may be one in which a binding fragment of an anti-OX40L antibody that specifically binds to OX40L and a binding fragment of an anti-TNFα antibody are linked via a linker. The linker may be a linker having the amino acid sequence set forth in SEQ ID NO: 31 or SEQ ID NO: 32.

[0133] In an embodiment of the present invention, the bispecific antibody is a) a heavy chain variable region comprising a heavy chain CDR1 selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 12, 13, and 14; a heavy chain CDR2 selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 15, 16, 17, and 18; and a heavy chain CDR3 selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 19, 20, 21, and 22, and a light chain variable region comprising a light chain CDR1 selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 23 and 24; a light chain CDR2 selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 25 and 26; and a light chain CDR3 selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 27, 28, 29, and 30, an anti-OX40L antibody or a binding fragment thereof that specifically binds to OX40L, and b) a heavy chain variable region comprising a heavy chain CDR1 of the amino acid sequence set forth in SEQ ID NO: 89; a heavy chain CDR2 of the amino acid sequence set forth in SEQ ID NO: 90; and a heavy chain CDR3 of the amino acid sequence set forth in SEQ ID NO: 91, and a light chain variable region comprising a light chain CDR1 of the amino acid sequence set forth in SEQ ID NO: 92; a light chain CDR2 of the amino acid sequence set forth in SEQ ID NO: 93; and a light chain CDR3 of the amino acid sequence set forth in SEQ ID NO: 94, an anti-TNFα antibody or a binding fragment thereof that specifically binds to TNFα, which may be linked via a linker.

[0134] The linker may be a linker having the amino acid sequence set forth in SEQ ID NO: 31 or SEQ ID NO: 32.

[0135] In an embodiment of the present invention, the bispecific antibody is: a) (i) a heavy chain variable region comprising a heavy chain CDR1 set forth in SEQ ID NO: 12; a heavy chain CDR2 set forth in SEQ ID NO: 15; and a heavy chain CDR3 set forth in SEQ ID NO: 19, and a light chain variable region comprising a Light chain CDR1 set forth in SEQ ID NO: 23; a Light chain CDR2 set forth in SEQ ID NO: 25; and a light chain CDR3 set forth in SEQ ID NO: 27, an anti-OX40L antibody or a binding fragment thereof; (ii) A heavy chain variable region comprising the heavy chain CDR1 set forth in SEQ ID NO: 13, the heavy chain CDR2 set forth in SEQ ID NO: 16, and the heavy chain CDR3 set forth in SEQ ID NO: 20, and a light chain variable region comprising the light chain CDR1 set forth in SEQ ID NO: 24, the light chain CDR2 set forth in SEQ ID NO: 26, and the light chain CDR3 set forth in SEQ ID NO: 28; an anti-OX40L antibody or an antigen-binding fragment thereof; (iii) A heavy chain variable region comprising the heavy chain CDR1 set forth in SEQ ID NO: 13, the heavy chain CDR2 set forth in SEQ ID NO: 17, and the heavy chain CDR3 set forth in SEQ ID NO: 21, and a light chain variable region comprising the light chain CDR1 set forth in SEQ ID NO: 24, the light chain CDR2 set forth in SEQ ID NO: 26, and the light chain CDR3 set forth in SEQ ID NO: 29; an anti-OX40L antibody or an antigen-binding fragment thereof; or (iv) A heavy chain variable region comprising the heavy chain CDR1 set forth in SEQ ID NO: 14, the heavy chain CDR2 set forth in SEQ ID NO: 18, and the heavy chain CDR3 set forth in SEQ ID NO: 22, and a light chain variable region comprising the light chain CDR1 set forth in SEQ ID NO: 24, the light chain CDR2 set forth in SEQ ID NO: 26, and the light chain CDR3 set forth in SEQ ID NO: 30; an anti-OX40L antibody or an antigen-binding fragment thereof, and b) A heavy chain variable region comprising the heavy chain CDR1 of the amino acid sequence set forth in SEQ ID NO: 89, the heavy chain CDR2 of the amino acid sequence set forth in SEQ ID NO: 90, and the heavy chain CDR3 of the amino acid sequence set forth in SEQ ID NO: 91; and A light chain variable region comprising the light chain CDR1 of the amino acid sequence set forth in SEQ ID NO: 92, the light chain CDR2 of the amino acid sequence set forth in SEQ ID NO: 93, and the light chain CDR3 of the amino acid sequence set forth in SEQ ID NO: 94; an anti-TNFα antibody that specifically binds to TNFα or a binding fragment thereof, and They may be linked via a linker.

[0136] The linker may be a linker having the amino acid sequence set forth in SEQ ID NO: 31 or SEQ ID NO: 32.

[0137] In an embodiment of the present invention, the bispecific antibody is: a) A heavy chain variable region described in one amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 37, 41, 45, 49 and 53; and a light chain variable region described in one amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 38, 42, 46, 50 and 54; an anti-OX40L antibody or a binding fragment thereof, and b) An anti-TNFα antibody or an antigen-binding fragment thereof comprising a heavy chain variable region described in SEQ ID NO: 35 and a light chain variable region described in SEQ ID NO: 36 may be linked via a linker.

[0138] The linker may be a linker having the amino acid sequence described in SEQ ID NO: 31 or SEQ ID NO: 32.

[0139] In an embodiment of the present invention, the bispecific antibody is: a) (a) A heavy chain variable region described in SEQ ID NO: 37 and a light chain variable region described in SEQ ID NO: 38; (b) A heavy chain variable region described in SEQ ID NO: 41 and a light chain variable region described in SEQ ID NO: 42; (c) A heavy chain variable region described in SEQ ID NO: 45 and a light chain variable region described in SEQ ID NO: 46; (d) A heavy chain variable region described in SEQ ID NO: 49 and a light chain variable region described in SEQ ID NO: 50; (e) A heavy chain variable region described in SEQ ID NO: 53 and a light chain variable region described in SEQ ID NO: 54; or (f) A heavy chain variable region having the amino acid sequence described in SEQ ID NO: 33 and a light chain variable region having the amino acid sequence described in SEQ ID NO: 34; an anti-OX40L antibody or a binding fragment thereof, and b) An anti-TNFα antibody or an antigen-binding fragment thereof comprising a heavy chain variable region described in SEQ ID NO: 35 and a light chain variable region described in SEQ ID NO: 36 may be linked via a linker.

[0140] The linker may be a linker having the amino acid sequence described in SEQ ID NO: 31 or SEQ ID NO: 32.

[0141] For example, the structure of the bispecific antibody may have a structure in the form shown in FIG. 1.

[0142] As another example, the bispecific antibody may have a structure in which an antigen-binding fragment of an anti-OX40L antibody and an antigen-binding fragment of an anti-TNFα antibody are linked.

[0143] When the bispecific antibody of the present invention contains a constant region, specifically, the bispecific antibody may include, but is not limited to, a heavy chain constant region described in one amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, and 8; and all or a part of the light chain constant region described in the amino acid sequence of SEQ ID NO: 10. When the bispecific antibody contains a constant region, it means a case where an anti-OX40L antibody or its binding fragment that specifically binds to OX40L constituting the bispecific antibody, or an anti-TNFα antibody or its binding fragment that specifically binds to TNFα contains all or a part of the constant region.

[0144] The bispecific antibody of the present invention has physicochemical properties capable of showing sufficient effects in the human body and excellent thermal stability. For example, the bispecific antibody melts at a temperature exceeding 50°C, specifically, at a temperature of 59°C or higher, and can maintain its binding for a long period, and can have a half-life of about two weeks or more in the human body.

[0145] That is, the bispecific antibody containing an antibody or its antigen-binding fragment that specifically binds to TNFα and an antibody or its antigen-binding fragment that specifically binds to OX40L according to the present invention shows strong affinity for human-derived TNFα and OX40L, not only effectively inhibits cells expressing OX40L (e.g., immune cells) from binding to OX40, but also TNFα binds to the TNF receptor to suppress inflammatory reactions, and can show more significant therapeutic effects in the treatment of autoimmune diseases and the like.

[0146] In the bispecific antibody of the present invention, the antibody or its antigen-binding fragment that specifically binds to TNFα, and the antibody or its antigen-binding fragment that specifically binds to OX40L each retain their specific binding, and in particular, can simultaneously inhibit two targets (antigens) without reducing the affinity for each target. Therefore, two signals are inhibited simultaneously, which can be more effective than binding and inhibiting a single target.

[0147] In an example of the present invention, a nucleic acid (polynucleotide) encoding the bispecific antibody of the present invention was inserted into a vector, which was introduced into animal cells to express and isolate an OX40L-TNFα binding bispecific antibody, and an OX40L-TNFα bispecific antibody that specifically binds to OX40L and TNFα was prepared.

[0148] The bispecific antibody molecule has a structure in which an OX40L IgG antibody molecule and a TNFα-binding scFv are linked by a linker, or a structure in which a TNFα IgG antibody molecule and an OX40L-binding scFv are linked by a linker (Figure 1). The OX40L-TNFα binding bispecific antibody introduced and expressed in the animal cells was isolated, and its expression and purity were confirmed by SDS-PAGE (Figure 2).

[0149] In addition, as a result of analyzing the binding ability assay of the bispecific antibody against OX40L and TNFα by ELISA (enzyme-linked immunosorbent assay), it was confirmed that the OX40L-TNFα binding bispecific antibody specifically binds to OX40L and TNFα, which are the targets of the bispecific antibody (Table 32).

[0150] In an embodiment of the present invention, the bispecific antibody can bind to human OX40L at 3×10 -9 M or less. Specifically, the anti-OX40L antibody or its antigen-binding fragment has a K -9 M, 1.3×10 -9 M, or 1×10 -9 M or less. Dand bind, and for human TNFα, 1×10 -9 K below M D can bind. Specifically, the equilibrium dissociation constant (K D ) values for OX40L and TNFα, which are the antigens of the bispecific antibody, were measured by Biacore analysis. As a result, the bispecific antibody had a K D value of 0.4 - 0.5 nM for human OX40L and a K D value of 0.2 - 0.5 nM for human TNFα (Table 32, Figure 4). Also, the in vitro OX40L inhibitory ability was 0.2 - 1.3 nM, significantly superior to the control antibody, and the in vitro TNFα inhibitory ability was also 0.05 - 0.07 nM, indicating excellent performance (Table 35, Figure 5). Additionally, the results of blocking immune activity in T cells were shown (Figure 6).

[0151] Therefore, the bispecific antibody of the present invention can simultaneously bind to OX40L and TNFα while maintaining the binding ability to each antigen, and can effectively treat diseases related to the target.

[0152] Furthermore, in the in vitro blockade assay experiment where the bispecific antibody of the present invention can simultaneously bind to OX40L and TNFα, the respective signal transduction pathways by the binding of OX40L of immune cells to human OX40 and the binding of TNFα to the TNFα receptor were effectively inhibited by the treatment with the bispecific antibody (Table 35 and Figure 5). From these results, it can be seen that the bispecific antibody specific for OX40L and TNFα of the present invention efficiently blocks the binding of the respective receptors OX40 and TNFα receptor, shows the effect of calming the over-activated immune system, and can simultaneously bind to OX40L and TNFα to effectively treat diseases related to the target.

[0153] The present invention provides a nucleic acid (polynucleotide) encoding a bispecific antibody that specifically binds to the aforementioned OX40L and TNFα, an expression vector containing the nucleic acid (polynucleotide), and a transformant into which the expression vector has been introduced.

[0154] In the present invention, regarding the nucleic acid (polynucleotide), expression vector, transformant, and introduction related to the bispecific antibody that specifically binds to the aforementioned OX40L and TNFα, as long as there is no contradiction, it is the same as the foregoing description.

[0155] The present invention provides a method for producing a bispecific antibody that specifically binds to OX40L and TNFα.

[0156] Specifically, the production method includes: (a) culturing a transformant into which an expression vector containing a nucleic acid (polynucleotide) encoding a bispecific antibody that specifically binds to the aforementioned OX40L and TNFα has been introduced to produce a bispecific antibody; and (b) recovering the bispecific antibody produced in step (a). The antibody may contain an antibody that binds bispecifically to OX40L and TNFα.

[0157] As long as there is no contradiction, the method for producing the bispecific antibody that specifically binds to the aforementioned OX40L and TNFα of the present invention may be applied substantially in the same manner as the production method described for the anti-OX40L antibody or its antigen-binding fragment.

[0158] The present invention provides a pharmaceutical composition for preventing or treating an autoimmune disease or an inflammatory disease, which contains an anti-OX40L antibody or its antigen-binding fragment, or a bispecific antibody that specifically binds to the aforementioned OX40L and TNFα.

[0159] The pharmaceutical composition may further contain a pharmaceutically acceptable carrier.

[0160] In the present invention, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate the living body and does not inhibit the biological activity and properties of the administered compound. Pharmaceutically acceptable carriers that are acceptable in compositions formulated as liquid solutions are sterile and suitable for living organisms, such as physiological saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed and used. If necessary, other ordinary additives such as antioxidants, buffers, and bacteriostatic agents may be added. Further, a diluent, a dispersant, a surfactant, a binder, and a lubricant may be added, and it may be formulated into injectable preparations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets.

[0161] The anti-OX40L antibody or its antigen-binding fragment, or the bispecific antibody that specifically binds to OX40L and TNFα can be involved in the inhibition of over-activated immune cells by binding to OX40L and inhibiting the binding to the OX40 receptor. The OX40L / OX40 receptor is the same as the above description.

[0162] Furthermore, the bispecific antibody binds to TNFα in addition to OX40L and inhibits the interaction between TNFα and the TNFα receptor, thereby regulating the differentiation of immune cells and the like by a mechanism different from the mechanism of action of the OX40L / OX40 signaling pathway, and can be involved in the suppression of various autoimmune diseases. That is, it can regulate the differentiation of immune cells and the like by a mechanism different from the mechanism of action of the OX40L / OX40 signaling pathway, and can be involved in the suppression of various autoimmune diseases.

[0163] Therefore, the pharmaceutical composition of the present invention can significantly and effectively prevent or treat autoimmune diseases or inflammatory diseases, minimize side effects, and enhance safety.

[0164] In the present invention, the autoimmune disease or inflammatory disease may include rheumatoid arthritis. Rheumatoid arthritis is a disease that is both an inflammatory disease and classified as an autoimmune disease. In the present invention, the terms "autoimmune disease", "inflammatory disease" or "autoimmune disease and inflammatory disease" include rheumatoid arthritis.

[0165] As used herein, the term "prevention" means suppressing or delaying the onset of a disease, and suppressing or delaying the recurrence of the disease in a subject in which the disease has been treated.

[0166] In the present invention, the term "treatment" can mean any act of improving or favorably changing the symptoms of an autoimmune disease by administration of a composition.

[0167] The present invention provides a method for preventing or treating an autoimmune disease or inflammatory disease using the anti-OX40L antibody or an antigen-binding fragment thereof, or the bispecific antibody that specifically binds to OX40L and TNFα.

[0168] The present invention provides a method for preventing or treating an autoimmune disease or inflammatory disease using a pharmaceutical composition comprising the anti-OX40L antibody or an antigen-binding fragment thereof, or the bispecific antibody that specifically binds to OX40L and TNFα.

[0169] The method for preventing or treating the autoimmune disease or inflammatory disease includes the step of administering to an individual the anti-OX40L antibody or an antigen-binding fragment thereof, or the bispecific antibody that specifically binds to OX40L and TNFα.

[0170] The method for preventing or treating the autoimmune disease or inflammatory disease includes the step of administering to an individual a pharmaceutical composition comprising the anti-OX40L antibody or an antigen-binding fragment thereof, or the bispecific antibody that specifically binds to OX40L and TNFα.

[0171] The individual may be an individual with an autoimmune disease or an inflammatory disease that has developed or is suspected of developing. Specifically, it may include mammals and birds such as cows, pigs, sheep, chickens, dogs, humans, etc., but is not particularly limited thereto.

[0172] The pharmaceutical composition may be in various dosage forms, oral or parenteral. When formulating, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with one or more compounds. Further, in addition to mere excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as wetting agents, sweeteners, flavoring agents, preservatives, etc., may also be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. are used. As the base of suppositories, witepsol, macrogol, tween 61, cocoa butter, laurin fat, glycerogelatin, etc. are used.

[0173] The pharmaceutical composition may have any dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories.

[0174] The anti-OX40L antibody or antigen-binding fragment thereof of the present invention, or the bispecific antibody that specifically binds to the OX40L and TNFα, or the pharmaceutical composition is administered in a pharmaceutically effective amount. In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level can be determined according to factors including the type and severity of the individual, age, gender, type of cancer, activity of the drug, sensitivity to the drug, administration time, administration route and excretion rate, treatment period, factors including drugs used simultaneously, and other factors well-known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it may be administered once or multiple times. Considering all the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which may be determined by those skilled in the art.

[0175] Here, the composition may be administered once or multiple times in a pharmaceutically effective amount. In this case, the composition may be administered in the form of a solution, powder, aerosol, capsule, enteric-coated tablet or capsule, or suppository. Administration routes include, but are not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, endothelial administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, etc. However, in the case of oral administration, since the peptide is digested, the oral composition should be formulated to coat the active drug or be protected from degradation in the stomach. Furthermore, the pharmaceutical composition may be administered by any device capable of moving the active substance to the target cells.

[0176] The present invention also provides the use of the anti-OX40L antibody or antigen-binding fragment thereof or bispecific antibody in the manufacture of a medicament for preventing or treating an autoimmune disease or inflammatory disease.

[0177] The present invention provides the use of a pharmaceutical composition comprising the anti-OX40L antibody or antigen-binding fragment thereof or bispecific antibody in the manufacture of a medicament for preventing or treating an autoimmune disease or inflammatory disease.

[0178] The present invention provides the use of the anti-OX40L antibody or its antigen-binding fragment or bispecific antibody for the prevention or treatment of autoimmune diseases or inflammatory diseases.

[0179] The present invention provides the use of a pharmaceutical composition comprising the anti-OX40L antibody or its antigen-binding fragment or bispecific antibody for the prevention or treatment of autoimmune diseases or inflammatory diseases.

[0180] Regarding the anti-OX40L antibody or its antigen-binding fragment, or the bispecific antibody that specifically binds to OX40L and TNFα, the pharmaceutical composition, autoimmune diseases, inflammatory diseases, prevention, or treatment, unless otherwise inconsistent, is the same as the foregoing description.

[0181] The present invention comprises the anti-OX40L antibody or its antigen-binding fragment or the bispecific antibody that specifically binds to OX40L and TNFα, and detects the OX40L protein in an isolated biological sample of an individual suspected of having an autoimmune disease or inflammatory disease by an antigen-antibody reaction. A diagnostic composition is provided.

[0182] In an embodiment of the present invention, the diagnostic composition can diagnose the presence or absence of an autoimmune disease or inflammatory disease.

[0183] Regarding the anti-OX40L antibody or its antigen-binding fragment, or the bispecific antibody that specifically binds to OX40L and TNFα, autoimmune diseases and inflammatory diseases, unless otherwise inconsistent, is the same as the foregoing description.

[0184] In the present invention, the term "diagnosis" means to confirm the presence or characteristics of a pathological condition. For the purposes of the present invention, diagnosis is to confirm the presence or absence of an autoimmune disease or inflammatory disease.

[0185] This invention ClearlyIn this case, the anti-OX40L antibody or its antigen-binding fragment or the diagnostic composition may be used to measure the level of OX40L protein in an isolated sample of an individual suspected of having an autoimmune disease or an inflammatory disease using the anti-OX40L antibody or its antigen-binding fragment of the present invention, or a bispecific antibody that specifically binds to the OX40L and TNFα, and the measured level of OX40L protein is compared with the samples of healthy individuals and / or patient control groups to determine an autoimmune disease or an inflammatory disease.

[0186] Examples of methods for measuring the level of the protein for this purpose include Western blot, ELISA (Enzyme Linked Immunosorbent Assay), radioimmunoassay, radioimmunodiffusion, Ouchterlony method, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, and protein chip, etc., but are not limited thereto. Using the above analysis methods, the levels of OX40L protein in a healthy control group and an individual suspected of having an autoimmune disease can be compared, thereby enabling the diagnosis of the presence or absence of an autoimmune disease in a patient suspected of having the disease.

[0187] The diagnostic composition for an autoimmune disease or an inflammatory disease of the present invention may further include, without limitation, those known in the art as necessary for carrying out the method for measuring the level of the protein in addition to the antibody of the present invention.

[0188] When the diagnostic composition of the present invention contains a bispecific antibody that specifically binds to the OX40L and TNFα, the diagnostic composition may be a composition for detecting TNFα protein in an isolated biological sample of an individual suspected of having an autoimmune disease or an inflammatory disease by an antigen-antibody reaction. Specifically, when containing a bispecific antibody that specifically binds to the OX40L and TNFα, the diagnostic composition may be a composition for detecting at least one of the OX40L protein and the TNFα protein in an isolated biological sample of an individual suspected of having an autoimmune disease or an inflammatory disease by an antigen-antibody reaction.

[0189] The anti-OX40L antibody or its antigen-binding fragment, or the bispecific antibody that specifically binds to the OX40L and TNFα, or the diagnostic composition may be used to measure the level of at least one of the OX40L protein and the TNFα protein in an isolated sample of an individual suspected of having an autoimmune disease or an inflammatory disease, and to determine whether it is an autoimmune disease or an inflammatory disease.

[0190] The method for measuring the level of the protein is the same as the above description as long as there is no contradiction.

[0191] The present invention relates to an anti-OX40L antibody or its antigen-binding fragment, or a bispecific antibody that specifically binds to the OX40L and TNFα 、 or Including them diagnostic composition By used By doing To provide a method for diagnosing an autoimmune disease or an inflammatory disease or a method for providing information for diagnosis. Specifically, the present invention provides (a) a step of measuring the level of the OX40L protein in an isolated sample of an individual suspected of having an autoimmune disease or an inflammatory disease using the anti-OX40L antibody or its antigen-binding fragment, or the bispecific antibody that specifically binds to the OX40L and TNFα; and (b) a step of determining an autoimmune disease or an inflammatory disease using the level of the OX40L protein measured in the step (a), to provide a method for diagnosing an autoimmune disease or an inflammatory disease, or a method for providing information for diagnosing an autoimmune disease or an inflammatory disease.

[0192] For example, the step of determining an autoimmune disease or an inflammatory disease using the measured level of the OX40L protein may be performed by comparing the measured level of the OX40L protein with the protein level of a general healthy individual and / or a patient.

[0193] The present invention provides a method for diagnosing an autoimmune disease or an inflammatory disease, or a method for providing information for diagnosing an autoimmune disease or an inflammatory disease, including: (a) measuring the level of at least one of the OX40L protein and the TNFα protein in an isolated sample of an individual suspected of having an autoimmune disease using a bispecific antibody that specifically binds to the OX40L and TNFα; and (b) determining an autoimmune disease or an inflammatory disease using the level of at least one of the OX40L protein and the TNFα protein measured in step (a).

[0194] For example, the step of determining an autoimmune disease or an inflammatory disease using the level of at least one of the measured OX40L protein and the TNFα protein may be performed by comparing the level of at least one of the measured OX40L protein and the TNFα protein with the level of at least one of the OX40L protein and the TNFα protein of a general healthy individual and / or a patient.

[0195] The present invention provides a kit for providing information for diagnosing an autoimmune disease or an inflammatory disease, including the anti-OX40L antibody or an antigen-binding fragment thereof, or the bispecific antibody that specifically binds to the OX40L and TNFα.

[0196] Regarding the anti-OX40L antibody or an antigen-binding fragment thereof, or the bispecific antibody that specifically binds to the OX40L and TNFα, autoimmune disease, inflammatory disease, individual, diagnosis, and the step (method) of measuring the protein level, unless there is a contradiction, it is the same as the above description.

[0197] In the present invention, the term "sample" includes, but is not limited to, samples such as whole blood, serum, blood, plasma, saliva, urine, sputum, lymph, cerebrospinal fluid, and interstitial fluid in which the expression level of OX40L is different in patients with autoimmune diseases.

Advantages of the Invention

[0198] The anti-OX40L antibody or its antigen-binding fragment of the present invention not only specifically binds to OX40L and effectively inhibits the binding between receptors, but also has excellent immunosuppressive ability and can show significantly excellent effects in the fields of treatment and diagnosis of autoimmune diseases and inflammatory diseases.

[0199] Furthermore, the bispecific antibody that specifically binds to the above-mentioned OX40L and TNFα shows strong affinity not only for OX40L but also for TNFα, and has excellent immunosuppressive ability because it retains binding in the human body for a long time, and can show significantly excellent effects in the fields of treatment and diagnosis of autoimmune diseases and inflammatory diseases.

Brief Description of the Drawings

[0200]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Mode for Carrying Out the Invention

[0201] Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are for illustrative purposes only, and the present invention is not limited by the following examples.

[0202] Example 1: Selection of OX40L-Specific Antibody Clones

[0203] Example 1-1: Preparation of OX40L Antigen

[0204] The antigen of human OX40L utilized the extracellular domain and was the human OX40L amino acid sequence (SEQ ID NO: 1) of Accession No. NP_003317 from the 51st to the 183rd amino acid sequence (Q51~L183) with a histidine tag fused to the N-terminus, and the human OX40L protein (Cat# OXL-H52Q8) provided by Acrobiosystems was obtained and used.

[0205] The amino acid sequence of the human OX04L protein (antigen) of Acrobiosystems is shown in SEQ ID NO: 2. The provided antigen was produced in HEK293 cells and has a size of 16.9 kDa.

[0206]

Table 2

[0207] Example 1-2: Preparation of Human Library Phage

[0208] 7.5×10 human-derived scFv library cells with diversity 10 individuals were cultured in 2×YT-glucose-Mgcl2-chloramphenicol (CM) medium at 37°C until the absorbance of the culture solution reached OD 600 = 0.5 - 0.7.

[0209] The cells were infected with helper phage and cultured at 37°C for about 1 hour. After centrifuging the cultured cells (5000 rpm, 4°C, 10 minutes), 2×YT-IPTG-Mgcl 2 -kanamycine (KM)-CM medium was added to remix the cells, and the cells were cultured in a shaking incubator at 30°C for 16 hours. The cultured cells were centrifuged (5000 rpm, 4°C, 10 minutes), 4% PEG (Sigma, 81253) and 3% NaCl (Junsei, 1905-0350) were added to the supernatant and dissolved well, and then reacted on ice for about 1 hour. Centrifuged again (7500 rpm, 4°C, 30 minutes), DPBS (Wellgene, LB001-02) was added to the pellet and dissolved, and then centrifuged (10000 rpm, 4°C, 10 minutes) to obtain a supernatant containing library phage, which was placed in a new tube and stored at 5 ± 3°C.

[0210] Example 1-3: Preparation of Immune Library Phage

[0211] Using the human OX40L antigen (SEQ ID NO: 2) of Example 1-1, immune library phage was prepared using Balb / C mouse and SD RAT.

[0212] After purifying 10 7-week-old Balb / C mice and 4 8-week-old male SD rats, they were immunized with human OX40L antigen on days 0, 21, 42, and 63, and the spleen was excised on day 84 to elute RNA. Using the eluted RNA, cDNA was synthesized, and the heavy chain variable region and the light chain variable region were amplified. The heavy chain variable region and the light chain variable region were mixed, and the DNA amplified in the scFv form was inserted into the phage vector (pYG100) to prepare RAT-derived immune scFv library cells. The prepared cells were cultured in 2×YT-glucose-Mgcl2-chloramphenicol (CM) medium at 37°C until the absorbance of the culture solution reached OD 600 = 0.5 - 0.7.

[0213] The cultured cells were infected with helper phage and cultured at 37°C for about 1 hour. After centrifuging the cultured cells (5000 rpm, 4°C, 10 minutes), 2×YT-IPTG-Mgcl 2 -kanamycine (KM)-CM medium was added to remix the cells, and the cells were cultured in a shaking incubator at 30°C for 16 hours. The cultured cells were centrifuged (5000 rpm, 4°C, 10 minutes), 4% PEG (Sigma, 81253) and 3% NaCl (Junsei, 1905-0350) were added to the supernatant and dissolved well, and then reacted on ice for about 1 hour. Centrifuged again (7500 rpm, 4°C, 30 minutes), DPBS (Wellgene, LB001-02) was added to the pellet and dissolved, and then centrifuged (10000 rpm, 4°C, 10 minutes) to obtain the supernatant containing library phage, which was put into a new tube and stored at 5 ± 3°C.

[0214] Examples 1-4: Panning of phage display

[0215] To select an OX40L antibody that binds to human OX40L, a solution containing the human OX40L protein of Example 1-1 was added to an immunotube at a concentration of 1 to 10 μg / mL, and the OX40L protein was adsorbed onto the surface of the immunotube overnight at 5 ± 3 °C. After that, a 1% solution of bovine serum albumin was added to the tube to protect the surface where OX40L was not adsorbed. After emptying the tube, 10 12 CFU of a human antibody phage library (Example 1-2) or an immune phage library (Example 1-3) was placed in the tube and bound to the antigen. Phages that bound non-specifically were washed 5 to 20 times with a PBS-T (Phosphate buffered saline - 0.05% Tween20) solution and further washed 1 to 5 times with DPBS. Then, the remaining antigen-specific phage antibodies were recovered using a 0.1M TAE solution.

[0216] The recovered phages were neutralized with 1M Tris buffer (pH 7.5), then infected into XL1Blue Escherichia coli at 37 °C for 1 hour. The infected Escherichia coli was spread on an SOBCG plate using glass beads and cultured in an incubator at 37 °C for about 16 hours. The next day, the cultured Escherichia coli was suspended in 4 ml of an SB (superbroth) carbenicillin culture solution, 15% glycerol was added, and a part was stored at -80 °C. Of the remaining, 50 μl was added to 20 ml of an SB carbenicillin culture solution with a 2% glucose solution and cultured at 37 °C. When the absorbance of the culture solution reached 0.6 at 600 nm, it was centrifuged to remove the culture solution, and this was suspended again in 20 ml of an SB carbenicillin culture solution. Then, 10 12 PFU of M13 helper phage was added and cultured at 37 °C with slow stirring. The next day, the culture solution was centrifuged to take only the culture solution, polyethylene glycol and sodium chloride (NaCl) were added, and it was precipitated at 4 °C for 30 minutes and then centrifuged. The supernatant was removed, and the precipitated phages were suspended in 1 ml of PBS and used as a library to repeat the panning process 3 to 5 times to amplify / concentrate antigen-specific clones.

[0217] Example 1-5: Selection of Specific Clones after Phage Panning

[0218] To select antibodies (scFv) that bind to human OX40L protein, antigen-specific clones were selected using one of the following two methods.

[0219] First, after panning, it was spread and cultured on an agar medium to obtain single colonies, which were inoculated into 1 - 1.5 mL of culture solution, cultured, then induced with IPTG, and the scFv-type protein was expressed in E. coli. The E. coli culture was centrifuged to obtain the supernatant, which was used to confirm the binding between recombinant human OX40L antigen and scFv by ELISA method (Steinberger. Rader and BarbasIII. 2000. Phage display vectors. In: Phage Display Laboratory Manual. 1st ed. ColdSpringHarborLaboratoryPress. NY, USA. pp. 11.9 - 11.12). The bound scFv was detected using HRP (Horseradish peroxidase)-anti-His antibody and tetramethylbenzidine (TMB) substrate.

[0220] Second, after panning, single phages were obtained from SOBCG plates, which were inoculated into deep well plates with 1 mL of medium dispensed each, and cultured with shaking at 37 °C for 16 hours. The amplified cells were diluted 10-fold and inoculated into the deep well plates again, and cultured in a shaking incubator at 37 °C until the OD 600 reached 0.5. When the OD 600 reached 0.5, M13 helper phage was added at a level of 10 9 CFU, and the cells were infected in a static incubator at 37 °C for 30 minutes and then in a shaking incubator at 37 °C for 30 minutes. After the infection was completed, the cells were centrifuged to precipitate and the supernatant was obtained, and scFv that binds to human OX40L antigen was confirmed by ELISA method. The bound scFv was detected using HRP (Horseradish It was detected using peroxidase-anti-M13 antibody and tetramethylbenzidine (TMB) substrate. The antigen-specific antibody (scFv) clones confirmed from these were analyzed by nucleotide sequencing analysis.

[0221] Example 2: Preparation of anti-OX40L antibody

[0222] Based on the sequences for the antibody variable regions (scFv) obtained in Examples 1-5 above, the heavy chain variable region was ligated to the heavy chain constant region (SEQ ID NO: 8), and the light chain variable region was ligated to the light chain constant region (SEQ ID NO: 10) to prepare the antibody. The antibodies were named 02C09, Hu3F07, 10H07, 21G07, and I3F07.

[0223] [Table 3-1] [Table 3-2]

[0224] (1) Anti-OX40L antibody 02C09

[0225] The 02C09 antibody contains heavy chain CDR1 shown in SEQ ID NO: 12; heavy chain CDR2 shown in SEQ ID NO: 15; heavy chain CDR3 shown in SEQ ID NO: 19; light chain CDR1 shown in SEQ ID NO: 23; light chain CDR2 shown in SEQ ID NO: 25; and light chain CDR3 shown in SEQ ID NO: 27.

[0226] The anti-OX40L antibody 02C09 was prepared using the pcDNA3.1 expression vector (Invitrogen) and the animal cell line FreeStyle™ 293-F (Invitrogen).

[0227] Using PEI, a polymer that enhances the intracellular delivery efficiency of genes, suspension FreeStyle™ 293-F animal cells transfected with an expression vector containing the gene encoding the anti-OX40L antibody 02C09 were cultured at 200 mL per flask in 500-mL Erlenmeyer flasks (Corning) and, if necessary, cultured in large quantities.

[0228] The transfected FreeStyle™ 293-F cells were cultured in suspension at 37 °C under 8% CO 2 conditions, and the medium used for culturing was FreeStyle™ 293 Expression Medium AGT™ (Invitrogen, AG1000D9P1). When overexpressing, 500 μg of PEI (Polysciences, 23966-2) and 125 μg of the DNA to be overexpressed were mixed in 5 mL of the culture medium. Approximately 24 hours after adding the DNA-PEI, 10 mL of 10% soytone (BD, 212488) was added, and after further culturing for approximately 5 days, only the supernatant was obtained and used for antibody purification.

[0229] To purify the antibody, the antibody was first purified from the culture using a recombinant protein-A sepharose column. When further purification was required to improve purity, secondary purification was performed using the primary purification product. In this case, purification was carried out using Superdex200 gel filtration chromatography or hydroxyapatite chromatography was performed.

[0230]

Table 4

[0231]

Table 5

[0232] (2) Anti-OX40L antibody Hu3F07

[0233] The Hu3F07 antibody contains a heavy-chain CDR1 represented by SEQ ID NO: 13; a heavy-chain CDR2 represented by SEQ ID NO: 16; a heavy-chain CDR3 represented by SEQ ID NO: 20; a light-chain CDR1 represented by SEQ ID NO: 24; a light-chain CDR2 represented by SEQ ID NO: 26; and a light-chain CDR3 represented by SEQ ID NO: 28. Including.

[0234] The anti-OX40L antibody Hu3F07 was prepared using the expression vector pcDNA3.1 (Invitrogen) and the animal cell line FreeStyle™ 293-F (Invitrogen). The specific culture conditions, culture method, and purification method were carried out in substantially the same manner as those described for the anti-OX40L antibody 02C09 in (1) above.

[0235]

Table 6

[0236]

Table 7

[0237] (3) Anti-OX40L antibody 10H07

[0238] The 10H07 antibody contains a heavy-chain CDR1 represented by SEQ ID NO: 13; a heavy-chain CDR2 represented by SEQ ID NO: 17; a heavy-chain CDR3 represented by SEQ ID NO: 21; a light-chain CDR1 represented by SEQ ID NO: 24; a light-chain CDR2 represented by SEQ ID NO: 26; and a light-chain CDR3 represented by SEQ ID NO: 29.

[0239] The anti-OX40L antibody 10H07 was prepared using the expression vector pcDNA3.1 (Invitrogen) and the animal cell line of FreeStyle™ 293-F (Invitrogen). The specific culture conditions, culture method, and purification method were carried out in substantially the same manner as those described for the anti-OX40L antibody 02C09 in (1) above.

[0240]

Table 8

[0241]

Table 9

[0242] (4) Anti-OX40L antibody 21G07

[0243] The 21G07 antibody contains a heavy chain CDR1 represented by SEQ ID NO: 14; a heavy chain CDR2 represented by SEQ ID NO: 18; a heavy chain CDR3 represented by SEQ ID NO: 22; a light chain CDR1 represented by SEQ ID NO: 24; a light chain CDR2 represented by SEQ ID NO: 26; and a light chain CDR3 represented by SEQ ID NO: 30. 。

[0244] The anti - OX40L antibody 21G07 was prepared using the expression vector pcDNA3.1 (Invitrogen) and the animal cell line FreeStyle™ 293 - F (Invitrogen). The specific culture conditions, culture methods, and purification methods were carried out in substantially the same manner as those described for the above - mentioned (1) anti - OX40L antibody 02C09.

[0245]

Table 10

[0246]

Table 11

[0247] (5) Anti-OX40L antibody I3F07

[0248] The I3F07 antibody comprises a heavy chain CDR1 represented by SEQ ID NO: 13; a heavy chain CDR2 represented by SEQ ID NO: 16; a heavy chain CDR3 represented by SEQ ID NO: 20; a light chain CDR1 represented by SEQ ID NO: 24; a light chain CDR2 represented by SEQ ID NO: 26; and a light chain CDR3 represented by SEQ ID NO: 28.

[0249] The anti-OX40L antibody I3F07 was prepared using the expression vector pcDNA3.1 (Invitrogen) and the animal cell line FreeStyle™ 293-F (Invitrogen). Specific culture conditions, culture methods, and purification methods were carried out in substantially the same manner as those described for the anti-OX40L antibody 02C09 in (1) above.

[0250] [Table 12]

[0251] [Table 13]

[0252] Example 3: Preparation of Bispecific Antibody Targeting OX40L and TNFα

[0253] An antibody (02C09, Hu3F07, I3FO7) or a fragment (scFv) thereof that binds to human OX40L prepared in Example 2 above was linked to an anti-TNFα antibody (TNFαi antibody (Humira)) or a fragment (scFv) thereof using a linker to prepare a bispecific antibody that can also bind to human TNFα (see Figure 1).

[0254] Specifically, the bispecific antibody is in the form of (i) linking the heavy chain variable region and the light chain variable region of the anti-TNFα antibody to the C-terminus of the heavy chain constant region of the anti-OX40L antibody with a linker; and (ii) A form in which the variable heavy chain region and the variable light chain region of an anti-TNFα antibody are linked by a linker to the C-terminus of the constant light chain region of an anti-OX40L antibody; (iii) A form in which the variable heavy chain region and the variable light chain region of an anti-OX40L antibody are linked by a linker to the C-terminus of the constant heavy chain region of an anti-TNFα antibody; (iv) A form in which the variable heavy chain region and the variable light chain region of an anti-OX40L antibody are linked by a linker to the C-terminus of the constant light chain region of an anti-TNFα antibody, and they were prepared in the order of IgG-scFv-binding positions as 02C09-TNFαi HC, 02C09-TNFαi LC, hu3F07-TNFαi HC, hu3F07-TNFαi LC, TNFαi-02C09 HC, TNFαi-02C09 LC, TNFαi-hu3F07 HC, TNFαi-hu3F07 LC, I3F07-TNFαi HC, I3F07-TNFαi LC, TNFαi-I3F07 HC, TNFαi-I3F07 LC, respectively.

[0255] The linker used was GGGGSGGGGSGGGGS represented by SEQ ID NO: 31.

[0256] [Table 14]

[0257] [Table 15]

[0258] The anti-TNFαi antibody was prepared by a method substantially the same as the method for producing (preparing) the anti-OX40L antibody of Example 2, in which the variable heavy chain region of the anti-TNFα antibody was linked to the constant heavy chain region (SEQ ID NO: 8), and the variable light chain region was linked to the constant light chain region (SEQ ID NO: 10).

[0259] (1) Bispecific antibody 02C09-TNFαi HC

[0260] The bispecific antibody 02C09-TNFαi HC has a binding fragment (ScFv) of a TNFαi antibody, in which the heavy chain variable region (SEQ ID NO: 35) and the light chain variable region (SEQ ID NO: 36) of the TNFαi antibody (Humira) are linked by a linker (SEQ ID NO: 32), linked to the C-terminus of the constant region of the heavy chain of the anti-OX40L antibody 02C09 by a linker (SEQ ID NO: 31).

[0261] The bispecific antibody 02C09-TNFαi HC was prepared using the expression vector pcDNA3.1 (Invitrogen) and the animal cell line FreeStyle™ 293-F (Invitrogen). The specific culture conditions, culture method, and purification method were carried out in substantially the same manner as those described for the anti-OX40L antibody 02C09 in (1) above.

[0262]

Table 16-1

Table 16-2

[0263] (2) Bispecific antibody 02C09-TNFαi LC

[0264] 02C09-TNFαi LC has a binding fragment (ScFv) of a TNFαi antibody, in which the heavy chain variable region (SEQ ID NO: 35) and the light chain variable region (SEQ ID NO: 36) of the TNFαi antibody (Humira) are linked by a linker (SEQ ID NO: 32), linked to the C-terminus of the constant region of the light chain of the anti-OX40L antibody 02C09 by a linker (SEQ ID NO: 31).

[0265] The bispecific antibody 02C09-TNFαi LC was prepared using the expression vector pcDNA3.1 (Invitrogen) and the animal cell line FreeStyle™ 293-F (Invitrogen). The specific culture conditions, culture method, and purification method were carried out in substantially the same manner as those described for the anti-OX40L antibody 02C09 in (1) above.

[0266]

Table 17-1

Table 17-2

[0267] (3) Bispecific antibody Hu3F07-TNFαi HC The heavy chain constant region C-terminus of the anti-OX40L antibody hu3F07 is linked by a linker (SEQ ID NO: 31) to a binding fragment (ScFv) of a TNFαi antibody, in which the heavy chain variable region (SEQ ID NO: 35) and the light chain variable region (SEQ ID NO: 36) of the TNFαi antibody are linked by a linker (SEQ ID NO: 32).

[0268] The bispecific antibody Hu3F07-TNFαi HC was prepared using the expression vector pcDNA3.1 (Invitrogen) and the animal cell line FreeStyle™ 293-F (Invitrogen). The specific culture conditions, culture methods, and purification methods were carried out in substantially the same manner as those described for the above-mentioned (1) anti-OX40L antibody 02C09.

[0269]

Table 18-1

Table 18-2

[0270] (4) Bispecific antibody Hu3F07-TNFαi LC

[0271] Hu3F07-TNFαi LC is a binding fragment (ScFv) of a TNFαi antibody, in which the heavy chain variable region (SEQ ID NO: 35) and the light chain variable region (SEQ ID NO: 36) of the TNFαi antibody are linked by a linker (SEQ ID NO: 32) to the C-terminus of the constant region of the light chain of the anti-OX40L antibody hu3F07, and are linked by a linker (SEQ ID NO: 31).

[0272] The bispecific antibody Hu3F07-TNFαi LC was prepared using the expression vector pcDNA3.1 (Invitrogen) and the animal cell line FreeStyle™ 293-F. The specific culture conditions, culture method, and purification method were carried out in substantially the same manner as those described for the above (1) anti-OX40L antibody 02C09.

[0273]

Table 19-1

Table 19-2

[0274] (5) Bispecific antibody TNFαi-02C09 HC

[0275] TNFαi-02C09 HC is a binding fragment (ScFv) of the anti-OX40L antibody 02C09, in which the heavy chain variable region (SEQ ID NO: 37) and the light chain variable region (SEQ ID NO: 38) of the anti-OX40L antibody 02C09 are linked by a linker (SEQ ID NO: 32) to the C-terminus of the constant region of the heavy chain of the TNFαi antibody (Humira), and are linked by a linker (SEQ ID NO: 31).

[0276] The bispecific antibody TNFαi-02C09 HC was prepared using the expression vector pcDNA3.1 (Invitrogen) and the animal cell line FreeStyle™ 293-F. The specific culture conditions, culture method, and purification method were carried out in substantially the same manner as those described for the above (1) anti-OX40L antibody 02C09.

[0277]

Table 20-1

Table 20-2

[0278] (6) Bispecific antibody TNFαi-02C09 LC

[0279] In TNFαi-02C09 LC, a binding fragment (ScFv) of anti-OX40L antibody 02C09, in which the heavy-chain variable region (SEQ ID NO: 37) and the light-chain variable region (SEQ ID NO: 38) of anti-OX40L antibody 02C09 are linked by a linker (SEQ ID NO: 32), is linked by a linker (SEQ ID NO: 31) to the C-terminus of the constant region of the light chain of the TNFαi antibody.

[0280] The bispecific antibody TNFαi-02C09 LC was prepared using the expression vector pcDNA3.1 (Invitrogen) and the animal cell line FreeStyle™ 293-F. The specific culture conditions, culture method, and purification method were carried out in substantially the same manner as those described for the above (1) anti-OX40L antibody 02C09.

[0281]

Table 21-1

Table 21-2

[0282] (7) Bispecific antibody TNFαi-hu3F07 HC

[0283] TNFαi-hu3F07 HC is an anti-OX40L antibody in which the heavy-chain variable region (SEQ ID NO: 41) and the light-chain variable region (SEQ ID NO: 42) of the anti-OX40L antibody hu3F07 are linked by a linker (SEQ ID NO: 32) to the C-terminus of the heavy-chain constant region of the TNFαi antibody. hu3F07 The binding fragment (ScFv) of the antibody is linked by a linker (SEQ ID NO: 31).

[0284] The bispecific antibody TNFαi-hu3F07 HC was prepared using the expression vector pcDNA3.1 (Invitrogen) and the animal cell line FreeStyle™ 293-F. The specific culture conditions, culture method, and purification method were carried out in substantially the same manner as those described for the anti-OX40L antibody 02C09 in (1) above.

[0285]

Table 22-1

Table 22-2

[0286] (8) Bispecific antibody TNFαi-hu3F07 LC

[0287] TNFαi-hu3F07 LC is an anti-OX40L antibody in which the heavy-chain variable region (SEQ ID NO: 41) and the light-chain variable region (SEQ ID NO: 42) of the anti-OX40L antibody hu3F07 are linked by a linker (SEQ ID NO: 32) to the C-terminus of the light-chain constant region of the TNFαi antibody. The binding fragment (ScFv) of the anti-OX40L antibody hu3F07 is linked by a linker (SEQ ID NO: 31).

[0288] The bispecific antibody TNFαi-02C09 LC was prepared using the expression vector pcDNA3.1 (Invitrogen) and the animal cell line FreeStyle™ 293-F. The specific culture conditions, culture method, and purification method were carried out in substantially the same manner as those described for the anti-OX40L antibody 02C09 in (1) above.

[0289]

Table 23-1

Table 23-2

[0290] (9) Bispecific antibody I3F07-TNFαi HC

[0291] The bispecific antibody I3F07-TNFαi HC has a binding fragment (ScFv) of a TNFαi antibody, in which the variable region of the heavy chain (SEQ ID NO: 35) and the variable region of the light chain (SEQ ID NO: 36) of the TNFαi antibody (Humira) are linked by a linker (SEQ ID NO: 32), linked to the C-terminus of the constant region of the heavy chain of the anti-OX40L antibody I3F07 by a linker (SEQ ID NO: 31).

[0292] The bispecific antibody I3F07-TNFαi HC was prepared using the expression vector pcDNA3.1 (Invitrogen) and the animal cell line FreeStyle (trademark) 293-F (Invitorgen). The specific culture conditions, culture methods, and purification methods were carried out substantially in the same manner as those described for the above (1) anti-OX40L antibody 02C09.

[0293]

Table 24-1

Table 24-2

[0294] (10) Bispecific antibody I3F07-TNFαi LC

[0295] The binding fragment (ScFv) of the TNFαi antibody, in which the heavy chain variable region (SEQ ID NO: 35) and the light chain variable region (SEQ ID NO: 36) of Humira are linked by a linker (SEQ ID NO: 32) to the C-terminus of the light chain constant region of the anti-OX40L antibody I3F07, is linked by a linker (SEQ ID NO: 31).

[0296] The bispecific antibody I3F07-TNFαi LC was prepared using the expression vector pcDNA3.1 (Invitrogen) and the animal cell line FreeStyle™ 293-F (Invitrogen). The specific culture conditions, culture method, and purification method were carried out in substantially the same manner as those described for the anti-OX40L antibody 02C09 in (1) above.

[0297]

Table 25-1

Table 25-2

[0298] (11) Bispecific antibody TNFαi-I3F07 HC

[0299] TNFαi-I3F07 HC is an anti-OX40L antibody in which the heavy chain variable region (SEQ ID NO: 53) and the light chain variable region (SEQ ID NO: 54) of the anti-OX40L antibody I3F07 are linked by a linker (SEQ ID NO: 32) to the C-terminus of the heavy chain constant region of the TNFαi antibody I3F07 of the binding fragment (ScFv) is linked by a linker (SEQ ID NO: 31).

[0300] The bispecific antibody TNFαi-I3F07 HC was prepared using the expression vector pcDNA3.1 (Invitrogen) and the animal cell line FreeStyle™ 293-F. The specific culture conditions, culture method, and purification method were carried out in substantially the same manner as those described for the anti-OX40L antibody 02C09 in (1) above.

[0301]

Table 26-1

Table 26-2

[0302] (12) Bispecific antibody TNFαi-I3F07 LC

[0303] In TNFαi-I3F07 LC, a binding fragment (ScFv) of anti-OX40L antibody I3F07, in which the heavy chain variable region (SEQ ID NO: 53) and the light chain variable region (SEQ ID NO: 54) of anti-OX40L antibody I3F07 are linked by a linker (SEQ ID NO: 32), is linked by a linker (SEQ ID NO: 31) to the C-terminus of the constant region of the light chain of the TNFαi antibody.

[0304] The bispecific antibody TNFαi-I3F07 LC was prepared using the expression vector pcDNA3.1 (Invitrogen) and the animal cell line FreeStyle™ 293-F. The specific culture conditions, culture method, and purification method were carried out in substantially the same manner as those described for the above (1) anti-OX40L antibody 02C09.

[0305]

Table 27-1

Table 27-2

[0306] Also, in a method substantially the same as the method for producing the anti-OX40L antibody of Example 2, an anti-OX40L antibody as a reference antibody (hereinafter, Ref.Ab or O4L) was prepared using the VH and VL sequences (SEQ ID NOs: 33 and 34) of oxelumab, which is an anti-OX40L antibody, and an anti-TNFα antibody as a reference antibody (hereinafter, Ref.TNFαi or Humira) was prepared using the VH and VL sequences (SEQ ID NOs: 35 and 36) of Humira, which is an anti-TNFα antibody.

[0307]

Table 28

[0308]

Table 29

[0309] Using the prepared antibody O4L and the Ref.TNFαi, anti-OX40L-TNFαi HC and LC antibodies (hereinafter, O4L-TNFαi HC and O4L-TNFαi LC, respectively) were prepared in a method substantially the same as the method for producing the bispecific antibody of Example 3.

[0310]

Table 30-1

Table 30-2

[0311]

Table 31-1

Table 31-2

[0312] Experimental Example 1: Analysis of Antibodies

[0313] The anti-OX40L antibody obtained in Example 2 and the bispecific antibody obtained in Example 3 were analyzed by SDS-PAGE, and the results are shown in Figure 2.

[0314] Experimental Example 2: Epitope of OX40L Antigen for Anti-OX40L Antibody

[0315] The epitope of the OX40L antigen for the anti-OX40L antibody was analyzed by HDX-MS. Using 0.12 - 2.00 M Urea, 0.12 - 1.00 M TCEP (pH 2.6) quench buffer and pepsin column, D 2 labeling and analysis were performed with O-based buffer solution. The data were processed using PLGS and DynamX, and the results as shown in Figure 3 were obtained.

[0316] Experimental Example 3: Characteristics of Antibodies

[0317] Experimental Example 3-1. Analysis of Equilibrium Dissociation Constant (K D ) for Antigens of Anti-OX40L Antibody and Bispecific Antibody

[0318] The affinity of the anti-OX40L antibody and the bispecific antibody that simultaneously controls OX40L and TNFα, which were isolated and purified in Examples 2 and 3 above, for antigens was analyzed as follows.

[0319] Among the above antibodies, the anti-OX40L antibody was confirmed for its binding ability to human OX40L (SEQ ID NO: 2), and the OX40L, TNFα bispecific antibody was confirmed for its binding ability to human OX40L (SEQ ID NO: 2) and human TNFα (TNF-H5228 from Acrobiosystems), respectively (Table 32).

[0320] SPR (Surface Plasmon Resonance) analysis was performed using a Bicore T200, and the running buffer used was HBS-EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.15% surfactant P20). Human antibody capture ki t (anti-hFc antibody) was immobilized on the surface of a CM5 chip by the amine coupling method. Human OX40L or human TNFα was diluted to 10 nM in the running buffer and then serially diluted 1 / 2 and analyzed in 5 concentration ranges. The antibody concentration was confirmed by measuring the absorbance (A280) after sterile filtration of the antibody through a 0.2 μm filter. The analysis samples were prepared at high purity / high concentration so that the minimum dilution factor was 100 or more, minimizing the effect of buffer changes (Buffer effect). A regeneration step was provided during all analyses to keep the experimental baseline constant. The Biacore analysis results are as shown in Table 32 and Figure 4.

[0321] In Figure 4, Ag1 indicates human OX40L and Ag2 indicates human TNFα.

[0322]

Table 32

[0323] As can be confirmed from Table 32 above, it was confirmed that the anti-OX40L antibody of Example 2 strongly binds to OX40L, and the bispecific antibody of Example 3 strongly binds to both OX40L and TNFα. Specifically, it was confirmed that both the anti-OX40L antibody and the bispecific antibody have a binding affinity for OX40L at the nM level, and in particular, the bispecific antibody also has a binding affinity for TNFα at the nM level. The above results suggest that the bispecific antibody retains a high level without being hindered in its binding ability to each antigen.

[0324] Experimental Example 3-2. Thermal Stability Test of Anti-OX40L Antibody and Bispecific Antibody

[0325] A test was conducted to confirm the characteristics regarding the thermal stability of the bispecific antibody of Example 3 and the anti-OX40L antibody of Example 2 (Table 33).

[0326] After diluting the antibody in DPBS to prepare 3 μM / 45 μL, it was mixed with 5 μL of 200×sypro orange dye (#S6650, Thermo), and 50 μL each was dispensed into qPCR Tubes (#B77009, B57651, bioplastics). qPCR was performed using a Biorad CFX96 real-time PCR instrument. The conditions for qPCR were: after reacting at 25°C for 30 seconds, the temperature was raised to 99°C at a rate of 1°C per minute and reacted at each temperature for 1 minute, and finally reacted at 25°C for 10 seconds before ending. As the rate constant for the dissociation of the antibody structure, Tm (Melting temperature) was used, and the results are as shown in Table 33 below.

[0327]

Table 33

[0328] As can be confirmed from the above table, the melting temperatures of the anti-OX40L antibody and the bispecific antibody are between 59 and 65°C, indicating that the bispecific antibody has the same thermal stability as the anti-OX40L antibody.

[0329] Experimental Example 3-3. PK (Pharmacokinetics analysis) of Anti-OX40L Antibody and Bispecific Antibody

[0330] Analysis was conducted to confirm the pharmacokinetics when the bispecific antibody of Example 3 and the anti-OX40L antibody of Example 2 were administered (Table 34).

[0331] Since it has a stable drug reaction and a stable supply system, male Sprague-Dawley rats, which are widely used in pharmacokinetic tests, were used. The test was conducted without fasting. Three 7-week-old male Sprague-Dawley (SD) rats were used and administered a single intravenous bolus at 5 mpk (2.5 - 2.4 mg / ml). After administration, a total of 10 times of blood sampling was performed from the jugular vein at 3 min, 3, 8, 24, 48, 72, 96, 120, 144, 168 - hr Approximately 150 μl of blood was collected from the jugular vein at each time point, isolated using Sodium heparin as an anticoagulant, and the sample was centrifuged (12,000 rpm, 3 min) to obtain approximately 70 μ l of plasma, which was immediately stored in an ultra-low temperature freezer. General symptoms were observed more than once a day during the experiment. After the last blood sampling, the experimental animals were 2 euthanized by CO inhalation.

[0332] The plasma obtained from the PK test was analyzed using the Gyrolab xPlore (registered trademark) (Cat.#P0020300, GYROS PROTEIN) and Gyrolab PK kit (Cat.#P0020499, GYROS PROTEIN) of the analyzer. Parameter values such as AUC(last), AUC(inf), Cmax, Tmax, and Half life were obtained using the BA Calc 2007 1.0.0 or PK Solver 2.0 program from the results of Gyrolab. The results are as shown in Table 34.

[0333]

Table 34

[0334] The half-lives of the anti-OX40L antibodies in Example 2 were 3.6 days and 4.0 days respectively, and the bispecific antibodies in Example 3 were observed to be 3.6 - 6.0 days. Therefore, it can be inferred that in humans, it can also show a half-life of about 2 weeks or more.

[0335] Experimental Example 4: Efficacy of Antibodies

[0336] Experimental Example 4-1. Evaluation of the Signal Inhibition Ability of Antibodies (1)

[0337] The activity evaluation experiments of the anti-OX40L antibody of Example 2 and the bispecific antibodies of Example 3 (Hu3F07-TNFαi HC, 02C09-TNFαi HC, O4L-TNFαi HC) were carried out using an OX40L / OX40 blockade bioassay kit (blockade bioassay kit: Promega CS197706) and a TNFα / TNFα Rc blockade bioassay kit (blockade bioassay kit: Promega CS177503) (Table 35, Figure 5).

[0338] (1) Using the OX40L / OX40 blockade bioassay kit, the activities of the anti-OX40L antibody of Example 2 and the bispecific antibodies of Example 3 were evaluated.

[0339] NFκB-luc2 / OX40 Jurkat cells were immersed in RPMI1640 (10% FBS) culture medium and statically cultured overnight at 37°C and 5% CO 2 in an incubator. The next day, antigens and antibodies to react with the cells were prepared. The OX40L antigen was prepared in RPMI1640 (10% FBS) culture medium so that the final concentration when incorporated into the cells was 15 ng / mL. The anti-OX40L antibody, bispecific antibody, and control antibody were diluted so that the final concentration at the start concentration was 33 μg / mL when incorporated into the cells, and then serially diluted by one-third to prepare nine steps. The tenth concentration was replaced with 0 and prepared to have a total concentration gradient of ten steps. 25 μL each of the prepared antigen dilution and antibody dilution were dispensed into the cells. Dispensing was done so that each sample had three replicates. After dispensing, the cells, antigen dilution, and antibody dilution were made to total 100 μL. CO 2 The reaction was carried out in an incubator for about 5 hours and then.

[0340] Bio-Glo was dispensed in 75 μL aliquots and allowed to react for about 10 minutes. After analyzing the samples by luminescence using a microplate reader, they were analyzed with 4-parameter (X-axis log(concentration)).

[0341] IC in μg / mL, the concentration unit of the antibody 50 The analysis results were converted to nM to obtain the final IC 50 This was because the anti-OX40L antibody is 150 kDa and the bispecific antibody is 200 kDa, so that they could be viewed under the same conditions.

[0342] (2) The activity of the bispecific antibody was evaluated using a TNFα / TNFα Rc blocking bioassay kit.

[0343] NFκB-RE HEK293 cells were placed in DMEM (10% FBS) culture medium and statically cultured in an incubator at 37 °C and 5% CO 2 Antigens and antibodies to react with the cells were prepared. When diluting the TNFα antigen in DMEM (10% FBS) culture medium to react with the cells, the final concentration was set to 3 ng / mL. The starting concentrations of the bispecific antibody and the control antibody Humira (Ref. TNFαi) were diluted in DMEM (10% FBS) culture medium at a concentration of 10 nM and then serially diluted by half to prepare eight steps. When reacting with the cells, the final concentration was set so that the starting concentration was 0.8 nM.

[0344] The prepared antigen diluent and antibody diluent were mixed at a ratio of 3:2 and dispensed in 20 μL aliquots to the cells. Dispensing was done so that each sample had two replicates. After dispensing, they were reacted in a CO 2 incubator for about 4 hours

[0345] Bio-Glo was dispensed in 100 μL aliquots and reacted. After analyzing the samples by luminescence using a microplate reader, they were analyzed with 4-parameter (X-axis log(concentration)) to obtain the IC 50 ​​

[0346] The said IC 50 The values are as shown in Table 35 and Figure 5 below.

[0347]

Table 35

[0348] As can be seen from Table 35 and Figure 5, the OX40L blocking ability of the bispecific antibodies and anti-OX40L antibodies according to Examples 2 and 3 is at a level of 1.3×10 -9 M (nM) or less. In particular, the OX40L blocking ability of the anti-OX40L antibody is at a level of 0.9×10 -9 M (nM) or less. The bispecific antibody also has a TNFα blocking ability at a level of 1×10 -9 M (nM) or less, and it was found to exhibit significantly excellent blocking ability. That is, it can be seen that the anti-OX40L antibody of the present invention exhibits excellent OX40L blocking ability, and the bispecific antibody prepared using the said antibody exhibits excellent blocking ability not only against OX40L but also against TNFα.

[0349] Experimental Example 4-2. Evaluation of the signal suppression ability of antibodies (2)

[0350] The activity of the bispecific antibody of Example 3 (02C09-TNFαi LC, TNFαi-02C09 HC, TNFαi 02C09 LC) was evaluated using a TNFα / TNFα Rc blockade bioassay kit (Promega CS1775036) (Table 36). The specific evaluation conditions and methods are substantially the same as those described in the said Experimental Example 4-1. Evaluation of the signal suppression ability of antibodies (1).

[0351]

Table 36

[0352] As can be seen from Table 36 above, the TNFα blocking ability of the bispecific antibodies 02C09-TNFαi LC, TNFαi-02C09 HC, and TNFαi 02C09 LC of Example 3 was found to be at a level of 1×10 -9 M (nM) or less, indicating excellent blocking ability.

[0353] Experimental Example 4-3. Evaluation of the signal suppression ability of antibodies (3)

[0354] The activities of the bispecific antibodies (I3F07-TNFαi HC, I3F07-TNFαi LC, TNFαi-I3F07 HC, TNFαi-I3F07 LC) of Example 3 were evaluated using an OX40L / OX40 blockade bioassay kit (blockade bioassay kit: Promega CS197706) and a TNFα / TNFα Rc blockade bioassay kit (blockade bioassay kit: Promega CS177503) (Table 37). The specific evaluation conditions and methods are substantially the same as those described in the above Experimental Example 4-1. Evaluation of the signal suppression ability of antibodies (1).

[0355]

Table 37

[0356] As can be seen from Table 37 above, the OX40L blocking ability of the bispecific antibodies of Example 3 was at a level of 1×10 -9 M (nM) or less, and the blocking ability of TNFα was also at a level of 1×10 -9 M (nM) or less, indicating excellent blocking ability.

[0357] Experimental Example 4-4. Evaluation of the ability of antibodies to inhibit immune cell activity

[0358] To evaluate the efficacy of the anti-OX40L antibody of Example 2 and the bispecific antibody of Example 3, the ability to inhibit the activity of immune cells was confirmed using PBMC (peripheral blood mononuclear ce ll) from healthy individuals and PBMC from rheumatoid arthritis (RA) patients (Figure 6).

[0359] T cells were isolated from PBMC of healthy individuals and RA patients by the above method, and the reduction level of the secretion of IL-2, a cytokine secreted when T cells are activated, was analyzed.

[0360] To fractionate T cells from PBMC of healthy individuals and patients, anti-CD3 (R&D systems, MAB100) was diluted to 100 ng / well and adhered to 96 wells at 5 ± 3 for about 16 hours. After removing the adhesion solution, it was washed with PBS. After diluting DNase I to 20 U / mL in LGM-3 (10% FBS, 1% P / S), PBMC of healthy individuals were added to prepare a mixed solution, centrifuged (200 g, 15 minutes), the supernatant was removed, and PBMC of healthy individuals were diluted with LGM-3 (10% FBS, 1% P / S) to 1 × 10 6 cell / mL and then dispensed. Human OX40L (ACROBIOSYSTEMS, OXL-H52Q8) and human TNFα (SINO BIOLOGICAL, 10602-HNA) were diluted in LGM-3 (10% FBS, 1% P / S) and 50 μL each was added to the dispensed PBMC of healthy individuals. After preparing the anti-OX40L antibody and the bispecific antibody at 800 ng / mL in LGM-3 (10% FBS, 1% P / S), they were diluted to 1 nM. 50 μL each of the T cells isolated from PBMC, human OX40L, and human TNFα were dispensed into a plate and mixed. After about 24 to 72 hours, only the supernatant was collected to measure IL-2, and the results are shown in Figure 6.

[0361] In Figure 6, A shows the results of the T cell assay from PBMC of healthy individuals, B shows the results of the T cell assay from PBMC of patients.

[0362] As can be confirmed from Fig. 6, it was confirmed that administration of the bispecific antibody of the present invention tended to decrease IL-2. In particular, looking at the T cell results derived from PBMC of RA patients, it was confirmed that the bispecific antibody of the present invention significantly decreased IL-2 expression in T cells derived from PBMC of RA patients compared to Humira (Ref. TNFαi). This means that the bispecific antibody of the present invention can act as an effective therapeutic antibody for RA, and in particular, can effectively act even in RA patients refractory to Humira.

[0363] Therefore, it can be seen that the anti-OX40L antibody and the bispecific antibody of the present invention can suppress the over-activated immune system and exhibit excellent therapeutic effects on autoimmune diseases.

Claims

1. An anti-OX40L antibody or an antigen-binding fragment thereof that specifically binds to OX40L (OX40 ligand) and inhibits the interaction between OX40L and the OX40 receptor, wherein the anti-OX40L antibody or an antigen-binding fragment thereof is: (i) an antibody or an antigen-binding fragment thereof comprising a heavy-chain variable region comprising heavy-chain CDR1 set forth in SEQ ID NO: 12; heavy-chain CDR2 set forth in SEQ ID NO: 15; and heavy-chain CDR3 set forth in SEQ ID NO: 19, and a light-chain variable region comprising light-chain CDR1 set forth in SEQ ID NO: 23; light-chain CDR2 set forth in SEQ ID NO: 25; and light-chain CDR3 set forth in SEQ ID NO: 27; (ii) an antibody or an antigen-binding fragment thereof comprising a heavy-chain variable region comprising heavy-chain CDR1 set forth in SEQ ID NO: 13; heavy-chain CDR2 set forth in SEQ ID NO: 16; and heavy-chain CDR3 set forth in SEQ ID NO: 20, and a light-chain variable region comprising light-chain CDR1 set forth in SEQ ID NO: 24; light-chain CDR2 set forth in SEQ ID NO: 26; and light-chain CDR3 set forth in SEQ ID NO: 28; (iii) an antibody or an antigen-binding fragment thereof comprising a heavy-chain variable region comprising heavy-chain CDR1 set forth in SEQ ID NO: 13; heavy-chain CDR2 set forth in SEQ ID NO: 17; and heavy-chain CDR3 set forth in SEQ ID NO: 21, and a light-chain variable region comprising light-chain CDR1 set forth in SEQ ID NO: 24; light-chain CDR2 set forth in SEQ ID NO: 26; and light-chain CDR3 set forth in SEQ ID NO: 29; or (iv) an antibody or an antigen-binding fragment thereof comprising a heavy-chain variable region comprising heavy-chain CDR1 set forth in SEQ ID NO: 14; heavy-chain CDR2 set forth in SEQ ID NO: 18; and heavy-chain CDR3 set forth in SEQ ID NO: 22, and a light-chain variable region comprising light-chain CDR1 set forth in SEQ ID NO: 24; light-chain CDR2 set forth in SEQ ID NO: 26; and light-chain CDR3 set forth in SEQ ID NO: 30, the anti-OX40L antibody or an antigen-binding fragment thereof.

2. The anti-OX40L antibody or an antigen-binding fragment thereof according to claim 1, wherein the anti-OX40L antibody or an antigen-binding fragment thereof binds to one or more epitopes described in the amino acid sequences selected from the group consisting of SEQ ID NOs: 3 and 4.

3. The anti-OX40L antibody or antigen-binding fragment thereof was administered to human OX40L at a concentration of 1×10 －９ K below M Ｄ wherein said K Ｄ The anti-OX40L antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody is determined by surface plasmon resonance analysis.

4. The anti-OX40L antibody or an antigen-binding fragment thereof is: The anti-OX40L antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3, which comprises a heavy-chain variable region comprising heavy-chain CDR1 described in the amino acid sequence of SEQ ID NO: 13; heavy-chain CDR2 described in the amino acid sequence of SEQ ID NO: 16; and heavy-chain CDR3 described in the amino acid sequence of SEQ ID NO:

20.

5. The anti-OX40L antibody or an antigen-binding fragment thereof is: The heavy-chain variable region comprising the heavy-chain CDR1 set forth in SEQ ID NO: 13; the heavy-chain CDR2 set forth in SEQ ID NO: 16; and the heavy-chain CDR3 set forth in SEQ ID NO: 20, and the light-chain CDR1 set forth in SEQ ID NO: 24; the light-chain CDR2 set forth in SEQ ID NO: 26; and the anti-OX40L antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which is a light-chain variable region comprising the light-chain CDR3 set forth in SEQ ID NO:

28.

6. The anti-OX40L antibody or antigen-binding fragment thereof is: The heavy-chain variable region set forth in one amino acid sequence selected from the group consisting of SEQ ID NOs: 37, 41, 45, 49 and 53; and The anti-OX40L antibody or antigen-binding fragment thereof according to claim 1 or 2, which comprises a light-chain variable region set forth in one amino acid sequence selected from the group consisting of SEQ ID NOs: 38, 42, 46, 50 and 54.

7. The anti-OX40L antibody or antigen-binding fragment thereof is: (a) The heavy-chain variable region set forth in SEQ ID NO: 37 and the light-chain variable region set forth in SEQ ID NO: 38; (b) The heavy-chain variable region set forth in SEQ ID NO: 41 and the light-chain variable region set forth in SEQ ID NO: 42; (c) The heavy-chain variable region set forth in SEQ ID NO: 45 and the light-chain variable region set forth in SEQ ID NO: 46; (d) The heavy-chain variable region set forth in SEQ ID NO: 49 and the light-chain variable region set forth in SEQ ID NO: 50; or (e) The heavy-chain variable region set forth in SEQ ID NO: 53 and the light-chain variable region set forth in SEQ ID NO: 54; The anti-OX40L antibody or antigen-binding fragment thereof according to claim 1 or 2, which comprises the same.

8. The anti-OX40L antibody or antigen-binding fragment thereof is: The heavy-chain constant region set forth in one amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7 and 8; and The anti-OX40L antibody or antigen-binding fragment thereof according to claim 7, which comprises a light-chain constant region set forth in the amino acid sequence of SEQ ID NO:

10.

9. A bispecific antibody comprising an anti-OX40L antibody or antigen-binding fragment thereof that specifically binds to OX40L; and an anti-TNFα antibody or antigen-binding fragment thereof that specifically binds to TNFα, Wherein the anti-OX40L antibody or antigen-binding fragment thereof is: (i) A heavy chain variable region comprising a heavy chain CDR1 set forth in SEQ ID NO: 12, a heavy chain CDR2 set forth in SEQ ID NO: 15, and a heavy chain CDR3 set forth in SEQ ID NO: 19, and a light chain variable region comprising a light chain CDR1 set forth in SEQ ID NO: 23, a light chain CDR2 set forth in SEQ ID NO: 25, and a light chain CDR3 set forth in SEQ ID NO: 27; an antibody or an antigen-binding fragment thereof; (ii) A heavy chain variable region comprising a heavy chain CDR1 set forth in SEQ ID NO: 13, a heavy chain CDR2 set forth in SEQ ID NO: 16, and a heavy chain CDR3 set forth in SEQ ID NO: 20, and a light chain variable region comprising a light chain CDR1 set forth in SEQ ID NO: 24, a light chain CDR2 set forth in SEQ ID NO: 26, and a light chain CDR3 set forth in SEQ ID NO: 28; an antibody or an antigen-binding fragment thereof; (iii) A heavy chain variable region comprising a heavy chain CDR1 set forth in SEQ ID NO: 13, a heavy chain CDR2 set forth in SEQ ID NO: 17, and a heavy chain CDR3 set forth in SEQ ID NO: 21, and a light chain variable region comprising a light chain CDR1 set forth in SEQ ID NO: 24, a light chain CDR2 set forth in SEQ ID NO: 26, and a light chain CDR3 set forth in SEQ ID NO: 29; an antibody or an antigen-binding fragment thereof; or (iv) A heavy chain variable region comprising a heavy chain CDR1 set forth in SEQ ID NO: 14, a heavy chain CDR2 set forth in SEQ ID NO: 18, and a heavy chain CDR3 set forth in SEQ ID NO: 22, and a light chain variable region comprising a light chain CDR1 set forth in SEQ ID NO: 24, a light chain CDR2 set forth in SEQ ID NO: 26, and a light chain CDR3 set forth in SEQ ID NO: 30; an antibody or an antigen-binding fragment thereof, A bispecific antibody.

10. The bispecific antibody according to claim 9, wherein the bispecific antibody is one in which an anti-OX40L antibody or an antigen-binding fragment thereof is linked to an anti-TNFα antibody or an antigen-binding fragment thereof.

11. The bispecific antibody according to claim 10, wherein the anti-OX40L antibody or an antigen-binding fragment thereof binds to one or more epitopes set forth in the amino acid sequences selected from the group consisting of SEQ ID NOs: 3 and 4.

12. The anti-OX40L antibody or an antigen-binding fragment thereof is: The bispecific antibody according to claim 10, which comprises a heavy chain variable region comprising a heavy chain CDR1 set forth in the amino acid sequence of SEQ ID NO: 13, a heavy chain CDR2 set forth in the amino acid sequence of SEQ ID NO: 16, and a heavy chain CDR3 set forth in SEQ ID NO:

20.

13. The anti-OX40L antibody or an antigen-binding fragment thereof is: A heavy chain variable region comprising a heavy chain CDR1 set forth in SEQ ID NO: 13, a heavy chain CDR2 set forth in SEQ ID NO: 16, and a heavy chain CDR3 set forth in SEQ ID NO: 20, and a light chain CDR1 set forth in SEQ ID NO: 24, a light chain CDR2 set forth in SEQ ID NO: 26, and a light chain CDR3 set forth in SEQ ID NO: 28; The bispecific antibody according to claim 10, which is an antibody or an antigen-binding fragment thereof comprising a light chain variable region.

14. The anti-OX40L antibody or an antigen-binding fragment thereof is: A heavy chain variable region described in one amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 37, 41, 45, 49 and 53; and A light chain variable region described in one amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 38, 42, 46, 50 and 54; The bispecific antibody according to claim 10, which comprises.

15. The anti-OX40L antibody or an antigen-binding fragment thereof is: (a) a heavy chain variable region described in SEQ ID NO: 37 and a light chain variable region described in SEQ ID NO: 38; (b) a heavy chain variable region described in SEQ ID NO: 41 and a light chain variable region described in SEQ ID NO: 42; (c) a heavy chain variable region described in SEQ ID NO: 45 and a light chain variable region described in SEQ ID NO: 46; (d) a heavy chain variable region described in SEQ ID NO: 49 and a light chain variable region described in SEQ ID NO: 50; (e) a heavy chain variable region described in SEQ ID NO: 53 and a light chain variable region described in SEQ ID NO: 54; or (f) a heavy chain variable region described in SEQ ID NO: 33 and a light chain variable region described in SEQ ID NO: 34; The bispecific antibody according to claim 10, which comprises.

16. The anti-TNFα antibody or an antigen-binding fragment thereof is: A heavy chain variable region comprising a heavy chain CDR1 set forth in SEQ ID NO: 89, a heavy chain CDR2 set forth in SEQ ID NO: 90, and a heavy chain CDR3 set forth in SEQ ID NO: 91, and A light chain variable region comprising a light chain CDR1 set forth in SEQ ID NO: 92, a light chain CDR2 set forth in SEQ ID NO: 93, and a light chain CDR3 set forth in SEQ ID NO: 94; The bispecific antibody according to any one of claims 10 to 15, which comprises.

17. The anti-TNFα antibody or an antigen-binding fragment thereof comprises a heavy chain variable region described in SEQ ID NO: 35 and a light chain variable region described in SEQ ID NO: 36; The bispecific antibody according to claim 16.

18. The bispecific antibody binds to human OX40L with a K of 1.5×10 －９ M or less, and binds to human TNFα with a K of 1×10 Ｄ M or less, where the K －９ is measured by surface plasmon resonance (Biacore) analysis. The bispecific antibody according to claim 16. Ｄ The bispecific antibody binds to human OX40L with a K of 1.5×10 Ｄ M or less, and binds to human TNFα with a K of 1×10

19. The bispecific antibody according to claim 16, wherein an anti-TNFα antibody or an antigen-binding fragment thereof that specifically binds to TNFα is linked to at least one end of the light chain and the heavy chain of the anti-OX40L antibody.

20. The bispecific antibody according to claim 19, wherein the anti-TNFα antibody or an antigen-binding fragment thereof is linked to at least one C-terminal end of the light chain and the heavy chain of the anti-OX40L antibody.

21. The bispecific antibody according to claim 16, wherein an anti-OX40L antibody or an antigen-binding fragment thereof that specifically binds to OX40L is linked to at least one end of the light chain and the heavy chain of the anti-TNFα antibody.

22. The bispecific antibody according to claim 21, wherein the anti-OX40L antibody or an antigen-binding fragment thereof is linked to at least one C-terminal end of the light chain and the heavy chain of the anti-TNFα antibody.

23. The bispecific antibody according to claim 16, wherein the anti-OX40L antibody or an antigen-binding fragment thereof and the anti-TNFα antibody or an antigen-binding fragment thereof are linked by a linker.

24. The bispecific antibody according to claim 23, wherein the linker is as described in the sequence of SEQ ID NO: 31 or SEQ ID NO:

32.

25. A nucleic acid encoding the antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8, or the bispecific antibody according to any one of claims 9 to 24.

26. An expression vector containing the nucleic acid according to claim 25.

27. A transformant into which the expression vector according to claim 26 has been introduced.

28. A method for producing an antibody or an antigen-binding fragment thereof, or a bispecific antibody using the transformant according to claim 27.

29. A pharmaceutical composition comprising the antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8, or the bispecific antibody according to any one of claims 9 to 24, and a pharmaceutically acceptable carrier, excipient and / or diluent.

30. The pharmaceutical composition according to claim 29 for preventing or treating an autoimmune disease or an inflammatory disease.

31. Use of the antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8, the bispecific antibody according to any one of claims 9 to 24, or the pharmaceutical composition according to claim 29 in the manufacture of a medicament for preventing or treating an autoimmune disease or an inflammatory disease.

Images