Anti-ox40l antibody, Anti-ox40l / Anti-TNFα bispecific antibody, and uses thereof

Abstract

To provide a novel antibody specifically binding to OX40L and a bispecific antibody specifically binding to OX40L and TNFα.SOLUTION: The present invention discloses an antibody or a bispecific antibody specifically binding to human OX40L to effectively inhibit the binding of OX40 to an OX40 receptor; a nucleic acid encoding the antibody; an expression vector comprising the nucleic acid; a transformant comprising the expression vector; a method for preparing the antibody; a pharmaceutical composition for treating autoimmune diseases or inflammatory diseases comprising the antibody; a composition for diagnosing autoimmune diseases or inflammatory diseases comprising the antibody; a method for diagnosing autoimmune diseases or inflammatory diseases by using the antibody; a method for providing information on diagnosis of autoimmune diseases or inflammatory diseases by using the antibody; and a kit for providing the same.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] 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 the prevention or treatment of autoimmune diseases or inflammatory diseases, which contain the antibodies, compositions for diagnosing autoimmune diseases or inflammatory diseases, which contain 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 such purposes. [Background technology]

[0002] Autoimmune diseases or inflammatory diseases occur when the human immune system becomes abnormally activated. Rheumatoid arthritis is a typical autoimmune disease, and TNFα inhibitors account for 68% of the therapeutic drug market.

[0003] Tumor necrosis factor alpha (TNFα) is a cytokine produced by a variety of cells, including monocytes and macrophages, that was originally identified by its ability to induce necrosis of certain mouse tumors [Reference: Old, L. (1985) Science 230:630-632]. A factor named cachectin, which is associated with cachexia, was subsequently identified as the same molecule as TNFα. TNFα is involved in mediating 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 has been implicated in the pathophysiology of a variety of human diseases and disorders, including sepsis, infections, autoimmune diseases, transplant rejection, and graft-versus-host disease [References: Vasili, P. (1992) Annu. Rev. Immunol. 10:411-452; Tracey, KJ and Cerami, A. (1994) Annu. Rev. Med. 45:491-503].

[0004] Due to the deleterious role of human TNFα (hTNFα) in various diseases, therapeutic strategies have been designed to inhibit or counteract 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 murine monoclonal antibodies (mAbs) secreted by hybridomas derived from lymphocytes of mice immunized with hTNFα [references: Hahn T; et al. l.,(1985)Proc Natl Acad Sci USA 82:3814-3818;Liang,CM.,et al.(1986)Biochem.Biophys.Res.Commun.137:847-854;Hirai,M.,etal.(1987)J.Immunol.Methods 96:57-62;Fendly,BM,et al. (1987) Hybridoma6:359-370; Muller, A., et al. (1990) Cytokine2:162-169; U.S. Patent No. 5,231,024 (Moeller et al); European Patent Publication No. 186833B1 (Wallach, D.); al.);European Patent Publication No. 260 No. 610B1 (Moeller, A., et.)] or chimeric antibodies [references: Knight, DM, et al. (1993) Mol. Immunol. 30:1443-1453; PCT Publication WO92 / 16553 (Daddona, PE, et al.)] or humanized monoclonal antibodies [references These anti-hTNFα antibodies have high affinity for hTNFα (e.g., Kd≦10 -9 M) and can neutralize hTNFα activity. Such anti-hTNFα antibodies are used as therapeutic agents for various autoimmune diseases, infectious diseases, transplant rejection, graft-versus-host disease, and the like.

[0005] However, approximately 50% of patients are refractory to these anti-hTNFα antibody TNFα inhibitors (Nature Reviews Rheumatology vol.11, 276-289(2015)). Furthermore, targets for autoimmune diseases that have been developed in recent years include CTLA-4, IL-6, JAK1, JAK2, and CD20, and pharmaceuticals derived from these targets have not been as effective as TNFα inhibitors (Nature Reviews Rheumatology vol.11, 276-289(2015)).

[0006] In particular, autoimmune diseases such as rheumatoid arthritis are caused not by abnormalities in a single type of immune cell but by problems in the immune system as a whole. Therefore, conventional therapeutic drug development methods that inhibit only one target have limitations in improving the efficacy of therapeutic drugs. Therefore, to overcome this limitation in efficacy, bispecific or multispecific antibodies that simultaneously control two or more targets with different mechanisms of action have been developed. However, conventional bispecific antibodies act only on specific cells of the innate immune system or the adaptive immune system, and therefore cannot improve the homeostasis of the immune system as a whole. Summary of the Invention [Problem 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 comprising the amino acid sequence of positions 93 to 100 shown in SEQ ID NO:3 and positions 141 to 151 shown in SEQ ID NO:4 in the amino acid sequence of the OX40L protein shown in SEQ ID NO:1.

[0009] An 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 alpha (TNFα).

[0010] An object of the present invention is to provide a nucleic acid encoding the anti-OX40L antibody, a binding fragment thereof, 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] An object of the present invention is to provide a method for producing an anti-OX40L antibody, an antigen-binding fragment thereof, or a bispecific antibody using the 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, an antigen-binding fragment thereof, or the bispecific antibody.

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

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

[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, its antigen-binding fragment, or bispecific antibody.

[0015] An object of the present invention is to provide a kit that provides information for diagnosing an autoimmune disease or an inflammatory disease, comprising the anti-OX40L antibody, its antigen-binding fragment, or 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 comprises administering a pharmaceutically effective amount of the anti-OX40L antibody, antigen-binding fragment thereof, or bispecific antibody.

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

[0018] An object of the present invention is to provide 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. [Means for solving the problem]

[0019] Each description and embodiment disclosed in the present invention may be applied to each of the other descriptions and embodiments, i.e., all combinations of the various elements disclosed in the present invention fall within the scope of the present invention.

[0020] Furthermore, the scope of the present invention is not limited to the specific description below.

[0021] The present invention provides anti-OX40L antibodies or binding fragments that specifically bind to OX40L and inhibit 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 immunoglobulin molecules immunologically reactive with a specific 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, 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 primarily play a role in binding to an epitope of an antigen. The CDRs of each chain are typically designated CDR1, CDR2, and CDR3, in order from the N-terminus, and can be further identified by the chain in which the particular CDR is located.

[0024] A whole antibody has two full-length light chains and two full-length heavy chains, with each light chain linked to a heavy chain by a disulfide bond. The whole antibody can include IgA, IgD, IgE, IgM, and IgG, with IgG subtypes including IgG1, IgG2, IgG3, and IgG4. The heavy chain constant region can have gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, with gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2) subclasses. The light chain constant region 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 an antibody of the present invention that has antigen-binding function, and may include Fab, Fab', F(ab')2, and Fv.

[0026] The Fab has a structure comprising light-chain and heavy-chain variable regions, a light-chain constant region, and the first heavy-chain constant region (CH1 domain) and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy-chain CH1 domain. F(ab')2 antibodies can be produced by disulfide bond formation between cysteine residues in the hinge region of Fab'. Fv (variable fragment) refers to the minimum antibody fragment comprising only the heavy-chain variable region and the light-chain variable region. In a double-chain Fv (dsFv), the heavy-chain variable region and the light-chain variable region are linked by a disulfide bond, while in a single-chain Fv (scFv), the heavy-chain variable region and the light-chain variable region are generally linked by a covalent bond via a peptide linker. These binding fragments can be obtained using proteolytic enzymes (e.g., whole antibodies can be subjected to limited cleavage with papain to give Fab fragments or pepsin to give F(ab')2 fragments) or can be produced, for example, by recombinant DNA techniques.

[0027] As used herein, the term "monoclonal antibody" refers to an antibody molecule of a single molecular 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 of the present invention that specifically binds to OX40L or the bispecific antibody that specifically binds to OX40L and TNFα may be a single molecular antibody. Specifically, the anti-OX40L antibody refers to an antibody of a single molecular composition that specifically binds to a specific epitope of OX40L, and the bispecific antibody may refer to a bispecific antibody of a single molecular composition that specifically binds to specific epitopes of OX40L and TNFα simultaneously.

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

[0029] In the present invention, the term "chimeric antibody" refers to an antibody obtained by combining the variable region of a mouse antibody and the constant region of a human antibody, and which exhibits a significantly improved immune response compared to mouse antibodies.

[0030] In the present invention, the term "humanized antibody" refers to an antibody in which the protein sequence of a non-human antibody has been modified to resemble an antibody variant naturally produced in humans. For example, the humanized antibody can be produced by recombining mouse-derived CDRs with human-derived FRs to produce a humanized variable region, which is then recombined with the constant region of a desired human antibody. However, simply performing CDR grafting alone reduces the affinity of the humanized antibody, and therefore, it is necessary to recombine the CDRs. By altering the affinity of some important FR amino acid residues, which are thought to affect the three-dimensional structure of R, 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 immunoglobulin, in which all amino acid sequences constituting the antibody, including complementary crystalline regions and framework regions, are composed of human immunoglobulin amino acid sequences. Human antibodies are commonly used to treat human diseases and may have three or more potential advantages. First, they interact better with the human immune system, enabling more efficient destruction of target cells, for example, by complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). Second, they have the advantage that the human immune system does not recognize them as foreign. Third, they have the advantage that their half-life in the human circulation is similar to that of natural antibodies, allowing for smaller and less frequent drug administration. In embodiments of the present invention, the anti-OX40L antibodies that specifically bind to OX40L and the bispecific antibodies that specifically bind to OX40L and TNFα of the present invention may be human antibodies. Therefore, the human anti-OX40L antibodies and human bispecific antibodies of the present invention not only exhibit strong affinity for OX40L and effectively inhibit the binding of OX40L-expressing cells (e.g., monocytes) to the OX40 receptor, but also exhibit low immunogenicity because both the heavy chain and light chain domains are human-derived, making them useful for treating autoimmune diseases, inflammatory diseases, and the like.

[0032] As used herein, the term "OX40L" refers to a ligand for the OX40 protein as a receptor, specifically a protein that binds to the OX40 receptor. Information about OX40L can be obtained from publicly known databases, such as GenBank of the National Institutes of Health. For example, GenBank has the Accession Number Gene ID: 54567, and the NCBI Reference Sequence: NM_003326.5 ( TNFSF4 ver1), NM_001297562.2(TNFSF4 ver2) The information on OX40L includes the amino acid sequence of SEQ ID NO:1, which is

[0033] OX40L is overexpressed in antigen-presenting cells (APCs) and is known to activate several immune cells. Specifically, OX40L, like TNFα and INFγ, has been observed to be overproduced in patients with autoimmune diseases (Eur. J. Immunol. 2000. 30:2815-2823). Unlike TNFα, which is distributed throughout the body, OX40L is produced exclusively by activated immune cells and primarily localized at the site of the lesion. OX40L is a multi-immunomodulatory protein that can simultaneously interact with antigen-presenting cells (APCs) of the innate immune system and T helper cells of the adaptive immune system to restore immune system homeostasis (Nature Reviews Rheumatology vol. 12, 74-76 (2016) (Clinic Rev Allerg Immunol, 2016)).

[0034] In the present invention, the term "OX40" refers to a protein that mediates OX40 / OX40L signaling. The OX40 may include, without limitation, any protein that mediates OX40 / OX40L signaling.

[0035] In the present invention, the term "inhibits the interaction between OX40L and OX40" or "inhibits the interaction between OX40L and OX40 receptor" means that the anti-OX40L antibody or binding fragment thereof that specifically binds to OX40L of the present invention binds to OX40L, and the interaction between OX40L and OX40 is inhibited. This means that the antibody or binding fragment thereof inhibits or inhibits the interaction between OX40L and OX40. When an anti-OX40L antibody or a binding fragment thereof binds to OX40L, it inhibits or inhibits the biological function of OX40L, thereby inhibiting or inhibiting the binding of OX40L to OX40, and thereby preventing OX40 signal transduction. In other words, the binding of an anti-OX40L antibody or a binding fragment thereof to OX40L inhibits or inhibits the interaction between OX40L and OX40, resulting in the inhibition or suppression of OX40 signal transduction.

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

[0037] The form of the anti-OX40L antibody may include both a whole antibody and a binding fragment, as described above. The anti-OX40L antibody of the present invention specifically binds to human OX40L and inhibits the interaction between OX40L and the OX40 receptor, and is useful for treating autoimmune diseases, inflammatory diseases, etc., and by specifically binding to human OX40L that is overexpressed in autoimmune diseases or inflammatory diseases, it can maximize therapeutic effects while minimizing side effects.

[0038] In an embodiment of the present invention, the anti-OX40L antibody or antigen-binding fragment thereof that specifically binds to OX40L and inhibits the interaction between OX40L and OX40 receptor is administered to human OX40L at a concentration of 3×10 -9 Specifically, the anti-OX40L antibody or antigen-binding fragment thereof can bind to the antibody at a concentration of 1.5 × 10 -9 M, 1.3 x 10 -9 M, especially, 1×10 -9 K below M D can be combined with

[0039] In the present invention, the term "binding constant (K on ) refers to the binding rate of a particular antibody-antigen interaction, and the term "dissociation constant (K off )" refers to the dissociation rate of a specific antibody-antigen interaction. In the present invention, the term "affinity for an antigen (K D )" is K off :K on The ratio of (i.e., K off / K on ) expressed as molar concentration (M). D The K value can be measured using methods widely established in the art. For example, the K value of an antibody D As a method for measuring values, BioCore TM Examples of such a method include, but are not limited to, surface plasmon resonance analysis using the system.

[0040] The anti-OX40L antibody or antigen-binding fragment thereof of the present invention exhibits high binding affinity to OX40L, can suppress or inhibit activity against OX40L even at low concentrations, and can exhibit excellent therapeutic effects 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 recognizes 3×10 amino acid sequences at positions 93 to 100 and positions 141 to 151 in the amino acid sequence of human OX40L protein shown in SEQ ID NO: 1. -9 Specifically, the anti-OX40L antibody or antigen-binding fragment thereof can bind to the amino acid sequences of positions 93 to 100 and positions 141 to 151 in the amino acid sequence of human OX40L protein shown in SEQ ID NO: 1 at a binding affinity of 1.5 × 10 -9 M, 1.3 x 10 -9 M, especially, 1×10 -9 K below M D can be combined with

[0042] In an embodiment of the present invention, the anti-OX40L antibody or antigen-binding fragment thereof binds with high binding affinity to the amino acid sequence of human OX40L protein shown in SEQ ID NO: 3 or SEQ ID NO: 4. Specifically, 3 × 10 -9 Specifically, the anti-OX40L antibody or antigen-binding fragment thereof can bind to the antibody at a concentration of 1.5 × 10 -9 M, 1.3 x 10 -9 M, especially, 1×10 -9 K below M D can be combined with

[0043] In an embodiment of the present invention, the K D may be measured by surface plasmon resonance (Biacore) analysis.

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

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

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

[0047] Furthermore, in the present invention, the term "light chain" may include all full-length light chains and fragments thereof, including a variable region domain VL and a constant region domain CL, each of which contains an amino acid sequence having a variable region sequence sufficient to confer specificity to an antigen.

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

[0049] In an embodiment of the invention, the anti-OX40L antibody or antigen-binding fragment thereof a heavy chain variable region comprising a heavy chain CDR1 represented by SEQ ID NO: 13; a heavy chain CDR2 represented by SEQ ID NO: 16; and a heavy chain CDR3 represented by SEQ ID NO: 20; and The antibody may be, but is not limited to, an antibody comprising a light chain variable region comprising 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. In an embodiment of the present invention, the antibody is designated as hu3F07 or I3F07.

[0050] In an embodiment of the invention, the anti-OX40L antibody or antigen-binding fragment thereof a heavy chain variable region comprising a heavy chain CDR1 represented by SEQ ID NO: 13; a heavy chain CDR2 represented by SEQ ID NO: 17; and a heavy chain CDR3 represented by SEQ ID NO: 21; and The antibody may be, but is not limited to, an antibody comprising a light chain variable region comprising 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 is designated 10H07.

[0051] In an embodiment of the invention, the anti-OX40L antibody or antigen-binding fragment thereof a heavy chain variable region comprising 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 The antibody may be, but is not limited to, an antibody comprising a light chain variable region comprising 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 is designated 21G07.

[0052] In an embodiment of the invention, the anti-OX40L antibody or antigen-binding fragment thereof a 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 It may comprise 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.

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

[0054] In an embodiment of the invention, the anti-OX40L antibody or antigen-binding fragment thereof a 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 It may comprise the light chain constant region set forth in the amino acid sequence of SEQ ID NO:10.

[0055] In an embodiment of the invention, the anti-OX40L antibody or binding fragment thereof (a) a heavy chain variable region set forth in SEQ ID NO: 37 and a light chain variable region set forth in SEQ ID NO: 38; (b) a heavy chain variable region set forth in SEQ ID NO: 41 and a light chain variable region set forth in SEQ ID NO: 42; (c) a heavy chain variable region set forth in SEQ ID NO: 45 and a light chain variable region set forth in SEQ ID NO: 46; (d) a heavy chain variable region set forth in SEQ ID NO: 49 and a light chain variable region set forth in SEQ ID NO: 50; or (e) a heavy chain variable region set forth in SEQ ID NO: 53 and a light chain variable region set forth in SEQ ID NO: 54, and A 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 a light chain constant region set forth in the amino acid sequence of SEQ ID NO: 10. The antibody may be an antibody or a binding fragment thereof comprising:

[0056] Here, an antibody comprising the variable regions (a) and (b) is a humanized antibody, and an antibody comprising the variable regions (c) to (d) is a chimeric antibody.

[0057] In an embodiment of the present invention, the anti-OX40L antibody or antigen-binding fragment thereof has physicochemical properties that are sufficient for its efficacy in the human body and has excellent thermal stability, for example, the anti-OX40L antibody or antigen-binding fragment thereof melts at temperatures above 50°C, specifically at temperatures above 59°C, and has a half-life of about 2 weeks or more in the human body.

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

[0059] As used herein, the term "combination" refers to the formation of a dimer or multimer in which a polypeptide encoding a single-chain immunoglobulin constant region derived from the same species is bound to a single-chain polypeptide derived from a different species. For example, a dimer or multimer can be formed from two or more constant regions selected from the group consisting of IgG, IgA, IgD, IgE, and IgM constant regions.

[0060] As used herein, the term "hybrid" means that a single-chain immunoglobulin heavy chain constant region contains sequences corresponding to immunoglobulin heavy chain constant regions derived from two or more different species. An example of such a hybrid may be a domain hybrid consisting of one to four domains selected from the group consisting of CH1, CH2, CH3, and CH4 of IgG, IgA, IgD, IgE, and IgM.

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

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

[0063] Furthermore, when the anti-OX40L antibody of the present invention specific for OX40L comprises a light chain constant region, the light chain constant region may be derived from a 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, but is not limited to, the kappa light chain constant region set forth in SEQ ID NO: 10.

[0064] In the present invention, the antibody may include all of mouse antibodies produced from mice and mutants thereof in which a portion of the amino acid sequence of the parent antibody is substituted, added, and / or deleted 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, an "affinity-optimized antibody" refers to a mutant in which a portion 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 improved binding affinity to the antigen.

[0065] In the present invention, the mutants comprehensively refer to antibodies in which a portion of the parent antibody CDR amino acid sequence has been mutated (substitution, addition, or deletion) provided that the mutants contain the same CDRs as the parent antibody or target the same epitope. Such mutants may be appropriately adjusted by those skilled in the art to improve the affinity, immunoactivity, etc. of the antibody, as long as the binding ability to the same epitope is maintained.

[0066] The anti-OX40L antibodies or antigen-binding fragments thereof of the present invention may include not only the sequences of the anti-OX40L antibodies described herein but also biological equivalents thereof, as long as they can specifically recognize OX40L. For example, to further improve the binding affinity and / or other biological properties of the antibody, additional changes may be made to the amino acid sequence of the antibody. These modifications include, for example, deletion, insertion, and / or substitution of residues in the amino acid sequence of the antibody. These amino acid mutations are made based on the relative similarity of the amino acid side chain substitutes, such as hydrophobicity, hydrophilicity, charge, size, etc. By analyzing the size, shape, and type of amino acid side chain substitutes, it has been found that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine and tryptophan are similarly charged residues. It can be seen that amino acids such as arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents. For example, in embodiments of the present invention, the anti-OX40L antibody or antigen-binding fragment thereof may contain conservative amino acid changes at one or more residues of the amino acid sequence set forth in SEQ ID NO: herein, and the conservative amino acid changes may include substitutions in Table 1 below.

[0067] [Table 1]

[0068] According to an embodiment of the present invention, novel antibodies targeting OX40L were generated. OX40L-specific antibodies 02C09, hu3F07, 10H07, and 21G07 were generated from a library generated from mice immunized with human OX40L (hOX40L) and the human library. These antibodies specifically bind to OX40L with high affinity, ranging from 0.2 to 0.7 nM (Table 32, Figure 4). Their in vitro OX40L inhibitory potency ranged from 0.2 to 0.9 nM, significantly superior to that of control antibodies (Figure 5). Furthermore, these antibodies were shown to block OX40L-mediated immune activation in T cells (Figure 6). These results indicate that the anti-OX40L antibody specific to OX40L of the present invention can efficiently block binding to the OX40 receptor, suppress OX40 / OX40L signaling, and exhibit significantly superior effects in the treatment of autoimmune diseases and inflammatory diseases, minimize side effects, and selectively treat autoimmune diseases and inflammatory diseases while maintaining the homeostasis of the immune system.

[0069] The anti-OX40L antibody or antigen-binding fragment thereof of the present invention inhibits the function of OX40L and is effectively used in 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 either the innate or adaptive immune system is abnormally activated.

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

[0072] OX40L is involved in both antigen-presenting cells (APCs) of the innate immune system and T helper cells of the acquired immune system, and can be involved in the homeostasis of the immune system (Nature Review). Newz Rheumatology vol. 12, 74-76 (2016), (Clinic Rev Allerg Immunol, 2016). Furthermore, unlike cytokines that are distributed throughout the body, OX40L is distributed intensively at lesion sites. Therefore, the anti-OX40L antibody or binding fragment thereof of the present invention is highly likely to bind to OX40L around lesions.

[0073] Therefore, anti-OX40L antibodies or their antigen-binding fragments can bind to OX40L, which is concentrated around the lesion, thereby maximizing the therapeutic effect against autoimmune diseases and inflammatory diseases, while reducing side effects and improving safety.

[0074] Furthermore, the acquired immune system has a slower response rate than the innate immune system but is more persistent, and may be a major cause of autoimmune diseases caused by excessive activity of the immune system. Suppression or inhibition of OX40L by the anti-OX40L antibody or antigen-binding fragment thereof of the present invention has a significant advantage in that it can regulate not only innate immune cells but also acquired immune cells, which are not affected by conventional therapeutic agents for autoimmune diseases.

[0075] In other words, the present invention effectively suppresses and inhibits the interaction between OX40L and OX40, and anti-OX40L antibodies or antigen-binding fragments thereof that specifically bind to OX40L can be effectively used in the treatment of autoimmune diseases and inflammatory diseases.

[0076] The present invention provides nucleic acids (polynucleotides) encoding the anti-OX40L antibodies or antigen-binding fragments thereof, expression vectors containing the nucleic acids, and transformants incorporating the expression vectors.

[0077] The terms "nucleic acid" or "polynucleotide" as used herein comprehensively include DNA and RNA molecules, and nucleotides, which are the basic building blocks of 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 said modifications include the addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0079] The nucleic acid of the present invention is also understood to include a nucleotide sequence that shows substantial identity to the nucleotide sequence. In the present invention, substantial identity may mean a nucleotide sequence that shows at least 80% homology, specifically at least 90% homology, more specifically at least 95% homology, when the nucleotide sequence of the present invention is aligned with any other sequence to maximize correspondence and the aligned sequences are analyzed using an algorithm commonly used in the art.

[0080] As used herein, the term "vector" or "expression vector" refers to 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, and specifically may be, but is not limited to, a plasmid vector.

[0081] In the vectors 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 "operably linked" refers to the functional association of a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of binding sites for a transcriptional regulator) with another nucleic acid sequence, whereby said control sequence controls the transcription and / or translation of said 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, specific methods are described in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999. Ring Harbor Laboratory Press (2001), which is incorporated herein by reference.

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

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

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

[0087] As used herein, the term "transduction" refers to a method of delivering a vector containing a nucleic acid (polynucleotide) encoding an anti-OX40L antibody or an antigen-binding fragment thereof into a host cell. This transduction can be performed 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. Furthermore, transduction refers to the delivery of a target substance into a cell by infection using viral particles. Incidentally, a vector can also be introduced into a host cell by gene bombardment or the like. In the present invention, "transduction" may be used interchangeably with "transformation."

[0088] The present invention provides methods for producing anti-OX40L antibodies or antigen-binding fragments thereof.

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

[0090] Phage display antibody libraries are developed by directly extracting antibody genes from B lymphocytes and expressing them on the surface of phages, without the need for hybridoma production. Phage display overcomes many of the traditional challenges associated with monoclonal antibody production through B cell immortalization. Generally, phage display involves the following steps: 1) inserting random-sequence oligonucleotides into the 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 portion of the native coat protein and the random-sequence oligonucleotide; 3) treating the phage with a receptor substance capable of binding to the polypeptide encoded by the oligonucleotide; 4) eluting the receptor-bound peptide phage particles with a low pH molecule or a molecule that competes with binding; 5) amplifying the phage eluted by panning in host cells; 6) repeating the above process to obtain the desired amount; and 7) determining active peptide sequences from the DNA sequences of the phage clones selected by panning.

[0091] In an embodiment of the present invention, the method for producing the anti-OX40L antibody or antigen-binding fragment thereof of the present invention can be carried out using phage display technology. Those skilled in the art can use known phage display techniques, for example, those described in Barbas et al. (METHODS: A Companion Each step of the production method of the present invention can be easily performed by referring to known methods described in the papers by (J. Virol. 2001 Jul;75(14):6692-9) and (Winter et al. (Ann. Rev. Immunol. 12:433, 1994). Phages used to construct antibody libraries include, but are not limited to, fd, M13, f1, If1, Ike, Zj / Z, Ff, Xf, Pf1, and Pf3 filamentous phages. Vectors used to express heterologous genes on the surface of the filamentous phages include, but are not limited to, phage vectors such as fUSE5, fAFF1, fd-CAT1, and fdtetDOG, and phagemid vectors such as pHEN1, pComb3, pComb8, and pSEX. In addition, helper phages used to provide wild-type coat proteins necessary for reinfection of recombinant phages for amplification include, but are not limited to, M13K07 or VSCM13.

[0092] Polynucleotides encoding the phage display clones of the present invention can be easily isolated and sequenced using conventional methods. For example, oligonucleotide primers designed to specifically amplify the heavy and light chain coding regions from hybridoma or phage template DNA can be used. Once the polynucleotides are isolated, they can be incorporated into an expression vector, which can then be introduced into appropriate host cells to produce the desired monoclonal antibody from the transformed host cells (i.e., transformants). Therefore, the method for producing the human monoclonal antibody of the present invention may be, but is not limited to, a method for producing a human monoclonal antibody, which includes a step of amplifying an expression vector containing a polynucleotide encoding the human monoclonal antibody.

[0093] The anti-OX40L antibody or antigen-binding fragment thereof of the present invention can be produced by the above-mentioned known recombinant or biochemical methods, and the antibody can be recovered from the culture medium of a transformant obtained by introducing an expression vector containing a nucleic acid encoding the antibody into a suitable host cell.

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

[0095] In an embodiment of the present invention, the anti-OX40L antibody or antigen-binding fragment thereof can be isolated by known isolation methods, including, but not limited to, conventional immunoglobulin purification methods such as protein A sepharose, gel electrophoresis, dialysis, or affinity chromatography, and can be suitably isolated from the culture medium.

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

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

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

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

[0100] Antibodies or binding fragments thereof that specifically bind to OX40L and are components of the bispecific antibodies of the present invention include antibodies or binding fragments thereof that can specifically bind to OX40L and block the OX40L / OX40L signaling pathway. In an embodiment of the present invention, antibodies or binding fragments thereof that specifically bind to OX40L and are components of the bispecific antibodies of the present invention may be substantially similar anti-OX40L antibodies or binding fragments thereof of the present invention, as long as they are not inconsistent with the above-described anti-OX40L antibodies or binding fragments thereof that specifically bind to OX40L. Furthermore, in an embodiment of the present invention, antibodies or binding fragments thereof that specifically bind to OX40L and are components of the bispecific antibodies of the present invention may be antibodies or binding fragments thereof that can specifically bind to OX40L and block the OX40L / OX40L signaling pathway, and may be antibodies or binding fragments thereof described in WO2018083248, WO2009141239, US2017260279, WO2006029879, or WO2011073180.

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

[0102] OX40L is also overexpressed in APCs and interacts with the OX40 receptor expressed on T cells. OX40L is one of the upstream signals that simultaneously activate both innate and adaptive immunity, inducing the proliferation / differentiation / activation of immune cells and secreting various inflammatory cytokines. Anti-OX40L antibodies or their antigen-binding fragments can inhibit OX40 / OX40L signaling and reduce overactivated immune responses in patients with autoimmune and inflammatory diseases.

[0103] Furthermore, anti-TNFα antibodies or binding fragments thereof can also be effective for patients with autoimmune diseases and inflammatory diseases who are resistant to treatment against TNFα, a downstream signal of inflammatory responses.

[0104] As used herein, the term "bispecific antibody comprising 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" may include, without limitation, any bispecific protein that can simultaneously inhibit two signaling pathways mediated by OX40L and TNFα. The antibody or antigen-binding fragment thereof that specifically binds to TNFα and the antibody or antigen-binding fragment thereof that constitute the bispecific antibody may take the form of either the full-length antibody or antibody fragment described above for the anti-OX40L antibody or binding fragment thereof.

[0105] In the present invention, the term "inhibiting the interaction between OX40L and OX40" means that the bispecific antibody of the present invention that specifically binds to OX40L binds to OX40L and inhibits the interaction between OX40L and OX40; the binding of the bispecific antibody inhibits the interaction between OX40L and OX40, and does not bring about a structural change in OX40 due to the binding of OX40L to OX40, making it unable to be hydrolyzed and preventing OX40 signal transduction.

[0106] As used herein, the term "antibody that specifically binds to TNFα" includes 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 that targets TNFα, and may be, but is not limited to, an antibody or a binding fragment thereof 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, but is not limited to, adalimumab (trade name Humira, Abbvie), a therapeutic antibody that has been approved by the US FDA, the European EMA, etc. and can be used stably. Such an antibody that specifically binds to TNFα may be in the form of either the full-length antibody or an antibody fragment as described above, and may be in the form of an IgG antibody, but is not limited thereto.

[0107] TNFα is a cytokine that regulates immune cells. It acts as a heat source in the body, generating fever and inducing cell death, and produces inflammatory cytokines, causing autoimmune and inflammatory diseases. TNFα is primarily secreted by activated macrophages, but is also secreted by various other immune cells, including neurons. The most important role of TNFα is to regulate immune cells. Inhibiting overexpressed TNFα can suppress autoimmune and inflammatory diseases.

[0108] The bispecific antibody binds to TNFα and binds to human TNFα and the TNFα receptor (TNFα Specifically, it can mean that a bispecific antibody specific to TNFα, which is a component of the bispecific antibody, binds to TNFα and suppresses or inhibits the interaction between TNFα and the TNFα receptor; This is not limited to this.

[0109] For the purposes of the present invention, the TNFα receptor may include, without limitation, any protein that binds to mammalian TNFα, and specifically, may refer to a protein that binds to human TNFα.

[0110] Inhibition of the interaction between TNFα and the TNFα receptor by the TNFα-specific bispecific antibody or binding fragment thereof of the present invention suppresses TNFα / TNFα receptor signaling induced by the binding of TNFα to the TNFα receptor. When TNFα binds to the TNFα receptor in the immune system, TNFα / TNFα receptor signaling is activated in immune cells, which regulates immune cell differentiation and other processes via a mechanism different from that of the OX40L / OX40 signaling pathway, and is used as a therapeutic agent for various autoimmune diseases.

[0111] Therefore, the bispecific antibody specific to OX40L and TNFα of the present invention exhibits inhibitory effects on overactivated immune cells through different mechanisms, and can be used as a therapeutic agent with superior therapeutic effects for autoimmune diseases and inflammatory diseases.

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

[0113] In an embodiment of the present invention, the anti-TNFα antibody or antigen-binding fragment thereof 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 comprise 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 comprise 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 may comprise the variable regions of Humira.

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

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

[0117] As used herein, the term "linker" basically refers to a linker that can link two different fusion partners (e.g., biopolymers, etc.) using hydrogen bonds, electrostatic interactions, van der Waals forces, disulfide bonds, salt bridges, hydrophobic interactions, covalent bonds, etc. Specifically, the term "linker" may have at least one cysteine that can participate in at least one disulfide bond under physiological conditions or other standard peptide conditions (e.g., peptide purification conditions, peptide storage conditions), and in addition to simply linking the respective fusion partners, it may also play a role in providing a certain distance between the fusion partners or providing flexibility to the fusion. The linker may be a non-peptide linker or a peptide linker, and may include those directly linked by a peptide bond, a disulfide bond, or the like.

[0118] In the present invention, the linker is not particularly limited, and may be, for example, a polypeptide capable of linking an antibody that specifically binds to OX40L and an antibody that specifically binds to TNFα, or, more specifically, a peptide linker capable of linking the C-terminus of the Fc region or the C-terminus of the light chain region of the antibody that specifically binds to OX40L to the antibody that specifically binds to TNFα, or, even more specifically, a peptide linker consisting of an amino acid sequence in the form of a repeated GGGGS motif. The GGGGS motif may be repeated 1 to 10 times, and most specifically, may be 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. However, the linker is not limited thereto, and various linkers may be used within the scope that can be easily deduced by a person skilled in the art.

[0119] In the present invention, the term "non-peptide linker" refers to a biocompatible linker consisting of two or more repeating units, which may be linked to each other by any covalent bond other than a peptide bond.

[0120] The non-peptide linker of the present invention is polyethylene glycol (PEG). 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 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 are also included within 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 and a bifunctional aldehyde at both ends that can link an antibody that specifically binds to OX40L and an antibody that specifically binds to TNFα. In particular, when the linker has aldehyde-reactive groups at both ends, it is effective in minimizing nonspecific 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 the antibody that specifically binds to OX40L and all or a part (fragment) of the antibody that specifically binds to TNFα are linked, but is not particularly limited thereto.

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

[0124] In an embodiment of the present invention, the bispecific antibody may be in a form in which an antibody that specifically binds 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 that specifically binds to TNFα are linked by a linker.

[0125] In an embodiment of the present invention, an antibody that specifically binds 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 that specifically binds to OX40L may be in a form linked by a linker, but is not limited to this.

[0126] In the present invention, the term "binding fragment" includes fragments having antigen-binding ability, such as antigen-binding forms of antibodies, including Fab', F(ab')2, Fab, Fv, rIgG, and scFv. In particular, the term includes scFv (single-chain variable fragment), including bivalent or diabodies, triabodies, and tetrabodies.

[0127] In the present invention, the term "scFv (single-chain variable fragment)" means the minimum antibody fragment that has a complete antigen-recognition site and antigen-binding site, and includes the VH and VL domains of an antibody, wherein said 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 an antigen-binding fragment thereof that specifically binds to TNFα is linked to at least one terminus of the light chain and the heavy chain of an anti-OX40L antibody.

[0129] In an embodiment of the present invention, the bispecific antibody may be one in which the anti-TNFα antibody or an antigen-binding fragment thereof is linked to the C-terminus of at least one of the light chain and heavy chain of the anti-OX40L antibody. Specifically, the bispecific antibody may be one in which the anti-TNFα antibody or an antigen-binding fragment thereof is linked to the C-terminus of at least one of the light chain and heavy chain of the anti-OX40L antibody via a linker. For example, the bispecific antibody may be one in which the anti-TNFα antibody or an antigen-binding fragment thereof is linked to the C-terminus of at least one of the light chain and heavy chain of the anti-OX40L antibody via a linker having the amino acid sequence of 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 an anti-TNFα antibody in which an anti-OX40L antibody or an antigen-binding fragment thereof that specifically binds to OX40L is linked to at least one terminus of the light chain and the heavy chain.

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

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

[0133] In an embodiment of the invention, the bispecific antibody comprises: a) an anti-OX40L antibody or a binding fragment thereof that specifically binds to OX40L, comprising a heavy chain variable region comprising a heavy chain CDR1 set forth in an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, and 14; a heavy chain CDR2 set forth in an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 16, 17, and 18; and a heavy chain CDR3 set forth in an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, 21, and 22; and a light chain variable region comprising a light chain CDR1 set forth in an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 and 24; a light chain CDR2 set forth in an amino acid sequence selected from the group consisting of SEQ ID NOs: 25 and 26; and a light chain CDR3 set forth in an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, 29, and 30; b) an anti-TNFα antibody or binding fragment thereof that specifically binds to TNFα, comprising a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 89; a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 90; and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 91; and a light chain variable region comprising a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 92; a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 93; and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 94; They 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 invention, the bispecific antibody comprises: a) (i) an anti-OX40L antibody or binding fragment thereof comprising 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; (ii) an anti-OX40L antibody or antigen-binding fragment thereof comprising 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; (iii) an anti-OX40L antibody or an antigen-binding fragment thereof comprising 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; or (iv) an anti-OX40L antibody or an antigen-binding fragment thereof, comprising 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; b) an anti-TNFα antibody or binding fragment thereof that specifically binds to TNFα, comprising a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 89; a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 90; and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 91; and a light chain variable region comprising a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 92; a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 93; and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 94; 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 invention, the bispecific antibody comprises: a) one selected from the group consisting of SEQ ID NOs: 33, 37, 41, 45, 49 and 53 an anti-OX40L antibody or a binding fragment thereof, comprising a heavy chain variable region set forth in the amino acid sequence set forth in SEQ ID NO: 34, 38, 42, 46, 50, and 54; and a light chain variable region set forth in one amino acid sequence selected from the group consisting of SEQ ID NO: 34, 38, 42, 46, 50, and 54; b) an anti-TNFα antibody or antigen-binding fragment thereof comprising a heavy chain variable region set forth in SEQ ID NO: 35 and a light chain variable region set forth in SEQ ID NO: 36; They may be linked via a linker.

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

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

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

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

[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 together.

[0143] When a bispecific antibody of the present invention comprises a constant region, the bispecific antibody may specifically comprise, but is not limited to, a 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 all or a part of the light chain constant region set forth in the amino acid sequence of SEQ ID NO: 10. When the bispecific antibody comprises a constant region, it means that an anti-OX40L antibody or a binding fragment thereof that specifically binds to OX40L, or an anti-TNFα antibody or a binding fragment thereof that specifically binds to TNFα, which constitutes the bispecific antibody, comprises all or a part of the constant region.

[0144] The bispecific antibodies of the present invention have physicochemical properties that enable them to exert sufficient effects in the human body and have excellent thermal stability: for example, they melt at temperatures above 50°C, specifically at temperatures of 59°C or higher, can retain their binding for long periods, and have a half-life of about two weeks or more in the human body.

[0145] In other words, the bispecific antibody comprising an antibody or antigen-binding fragment thereof that specifically binds to TNFα and an antibody or antigen-binding fragment thereof that specifically binds to OX40L according to the present invention exhibits strong affinity for human-derived TNFα and OX40L, and not only effectively inhibits the binding of OX40L-expressing cells (e.g., immune cells) to OX40, but also suppresses inflammatory responses via the binding of TNFα to TNF receptors, thereby demonstrating more significant therapeutic effects in the treatment of autoimmune diseases and the like.

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

[0147] In the Examples of the present invention, nucleic acids (polynucleotides) encoding bispecific antibodies of the present invention were inserted into vectors, which were then introduced into animal cells to express and isolate the OX40L-TNFα-binding bispecific antibodies, thereby producing OX40L-TNFα bispecific antibodies that specifically bind to OX40L and TNFα.

[0148] The bispecific antibody molecule has a structure in which an OX40L IgG antibody molecule and a TNFα-binding scFv are linked via a linker, or a structure in which a TNFα IgG antibody molecule and an OX40L-binding scFv are linked via 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] Furthermore, the binding assay of the bispecific antibody against OX40L and TNFα was performed using enzyme-linked immunosorbent assay (ELISA), and the results confirmed that the OX40L-TNFα-binding bispecific antibody specifically bound to OX40L and TNFα, the targets of the bispecific antibody (Table 32).

[0150] In an embodiment of the invention, the bispecific antibody binds human OX40L at a concentration of 3×10 -9 Specifically, the anti-OX40L antibody or antigen-binding fragment thereof can bind to the antibody at a concentration of 1.5 × 10 -9 M, 1.3 x 10 -9 M, or 1 x 10 -9 K below M Dand for human TNFα, 1 × 10 -9 K below M D Specifically, the equilibrium dissociation constant (K) of the bispecific antibody for the antigens OX40L and TNFα is D ) values were measured by Biacore analysis, and the bispecific antibody exhibited a K of 0.4 to 0.5 nM for human OX40L. D values of 0.2–0.5 nM for human TNFα. D It was confirmed that the antibody exhibited excellent in vitro OX40L inhibitory activity at 0.2-1.3 nM, significantly superior to the control antibody, and that it also exhibited excellent in vitro TNFα inhibitory activity at 0.05-0.07 nM (Table 35, Figure 5). It also demonstrated blocking of immune activity in T cells (Figure 6).

[0151] Therefore, the bispecific antibody of the present invention can simultaneously bind to OX40L and TNFα while retaining its binding ability to each antigen, and can effectively treat diseases associated with these targets.

[0152] Furthermore, the bispecific antibodies of the present invention can simultaneously bind to OX40L and TNFα. In an in vitro blockade assay, it was confirmed that the signaling pathways mediated by the binding of OX40L to human OX40 in immune cells and the binding of TNFα to the TNFα receptor were effectively inhibited by bispecific antibody treatment (Table 35 and Figure 5). These results demonstrate that the bispecific antibodies specific for OX40L and TNFα of the present invention efficiently block the binding of their respective receptors, OX40 and TNFα receptor, thereby calming an overly activated immune system and simultaneously binding to OX40L and TNFα, thereby effectively treating diseases associated with these targets.

[0153] The present invention provides nucleic acids (polynucleotides) encoding the bispecific antibodies that specifically bind to OX40L and TNFα, expression vectors containing the nucleic acids (polynucleotides), and transformants into which the expression vectors have been introduced.

[0154] In the present invention, the nucleic acids (polynucleotides), expression vectors, transformants, and introductions related to the bispecific antibodies that specifically bind to OX40L and TNFα are the same as those described above, unless inconsistent.

[0155] The present invention provides methods for producing bispecific antibodies that specifically bind to OX40L and TNFα.

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

[0157] The bispecific antibody of the present invention that specifically binds to OX40L and TNFα may be produced by substantially the same production method as described for the anti-OX40L antibody or antigen-binding fragment thereof, as long as there is no contradiction.

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

[0159] The pharmaceutical composition may further comprise 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 body and does not inhibit the biological activity and properties of the administered compound. Pharmaceutical carriers acceptable for compositions formulated as liquid solutions are sterile and biocompatible, and include physiological saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components. Other common additives, such as antioxidants, buffers, and bacteriostatic agents, may also be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be added to formulate the compositions into injectable solutions, such as aqueous solutions, suspensions, and emulsions, as well as 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 inhibiting overactivated immune cells by binding to OX40L and inhibiting binding to the OX40 receptor. The OX40L / OX40 receptor is as described above.

[0162] Furthermore, the bispecific antibody binds to TNFα in addition to OX40L and inhibits the interaction between TNFα and the TNFα receptor, thereby regulating immune cell differentiation and other functions through a mechanism different from that of the OX40L / OX40 signaling pathway, and may be involved in the suppression of various autoimmune diseases.

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

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

[0165] As used herein, the term "prevention" means inhibiting or delaying the onset of a disease, and also means inhibiting or delaying the recurrence of said disease in a subject who has been treated for said disease.

[0166] In the present invention, the term "treatment" may refer to any action that improves or favorably alters the symptoms of an autoimmune disease by administering a composition.

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

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

[0171] The individual may be an individual suffering from or suspected of suffering from an autoimmune disease or an inflammatory disease, and specifically may include mammals and birds, including cows, pigs, sheep, chickens, dogs, and humans, but is not limited to these.

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

[0173] The pharmaceutical composition may be in the form of a tablet, pill, powder, granule, capsule, suspension, oral liquid, milk, or the like. The pharmaceutical composition may have any dosage form selected from the group consisting of a pharmaceutical preparation, a syrup, a sterile aqueous solution, a non-aqueous solution, a suspension, an emulsion, a freeze-dried preparation, and a suppository.

[0174] The anti-OX40L antibody or antigen-binding fragment thereof, the bispecific antibody specifically binding to OX40L and TNFα, or the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment. The effective dose level can be determined based on factors including the type and severity of the individual, age, sex, type of cancer, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, concurrently used drugs, and other factors well known in the medical field. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. It can also be administered in single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that will achieve maximum efficacy at the minimum dose without side effects, which can be determined by those skilled in the art.

[0175] The composition may be administered in a single or multiple doses in a pharmaceutically effective amount. The composition may be administered in the form of a liquid, powder, aerosol, capsule, enteric-coated tablet or capsule, or suppository. Routes of administration include, but are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, pulmonary, and rectal administration. However, because peptides are digested during oral administration, oral compositions should be formulated to coat or protect the active agent from digestion in the stomach. Furthermore, pharmaceutical compositions may be administered by any device capable of delivering the active agent to target cells.

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

[0177] The present invention provides 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 the prevention or treatment of an autoimmune disease or inflammatory disease.

[0178] The present invention provides 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 use of the anti-OX40L antibody or an antigen-binding fragment thereof, or a pharmaceutical composition comprising the bispecific antibody for the prevention or treatment of an autoimmune disease or an inflammatory disease.

[0180] The anti-OX40L antibody or antigen-binding fragment thereof, or the bispecific antibody that specifically binds to OX40L and TNFα, pharmaceutical composition, autoimmune disease, inflammatory disease, prevention, or treatment are the same as those described above, unless inconsistent.

[0181] The present invention provides a diagnostic composition comprising the anti-OX40L antibody or its antigen-binding fragment, or the bispecific antibody that specifically binds to OX40L and TNFα, and used to detect OX40L protein in an isolated biological sample from an individual suspected of having an autoimmune disease or an inflammatory disease through an antigen-antibody reaction.

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

[0183] The anti-OX40L antibody or antigen-binding fragment thereof, or the antibody specific for OX40L and TNFα The bispecific antibodies that specifically bind to the inflammatory disease and autoimmune disease are as described above unless otherwise contradictory.

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

[0185] In the present invention, the anti-OX40L antibody or antigen-binding fragment thereof or the diagnostic composition may be used to determine the presence of an autoimmune disease or inflammatory disease by measuring the level of OX40L protein in an isolated sample from an individual suspected of having an autoimmune disease or inflammatory disease using the anti-OX40L antibody or antigen-binding fragment thereof of the present invention, or the bispecific antibody that specifically binds to OX40L and TNFα, and comparing the measured OX40L protein level with samples from healthy individuals and / or patient control groups.

[0186] Methods for measuring protein levels include, but are not limited to, Western blot, ELISA (Enzyme-Linked Immunosorbent Assay), radioimmunoassay (otA), radioimmunodiffusion, Ouchterlony immunoassay, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, and protein chip. Using these analytical methods, OX40L protein levels can be compared between healthy controls and individuals suspected of having an autoimmune disease, thereby enabling the diagnosis of the onset of an autoimmune disease in patients suspected of having the disease.

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

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

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

[0190] The method for measuring the level of said protein is the same as described above unless contradicted.

[0191] The present invention provides a method for diagnosing an autoimmune disease or an inflammatory disease or a method for providing information for diagnosis, by using an anti-OX40L antibody or an antigen-binding fragment thereof, or a bispecific antibody that specifically binds to OX40L and TNFα, or a diagnostic composition containing them. Specifically, 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, comprising the steps of: (a) measuring the level of OX40L protein in an isolated sample from an individual suspected of having an autoimmune disease or an inflammatory disease using the anti-OX40L antibody or an antigen-binding fragment thereof, or the bispecific antibody that specifically binds to OX40L and TNFα; and (b) determining the presence of an autoimmune disease or an inflammatory disease using the level of OX40L protein measured in step (a).

[0192] For example, the step of determining an autoimmune disease or an inflammatory disease using the measured level of OX40L protein may be carried out by comparing the measured level of OX40L protein with the protein level of typical healthy individuals and / or patients.

[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, comprising the steps of: (a) measuring the level of at least one of OX40L protein and TNFα protein in an isolated sample from an individual suspected of having an autoimmune disease using the bispecific antibody that specifically binds to OX40L and TNFα; and (b) determining the presence of an autoimmune disease or an inflammatory disease using the level of at least one of OX40L protein and TNFα protein measured in step (a).

[0194] For example, the step of determining whether an autoimmune disease or an inflammatory disease is present using the measured level of at least one of OX40L protein and TNFα protein may be carried out by comparing the measured level of at least one of OX40L protein and TNFα protein with the levels of at least one of OX40L protein and TNFα protein in typical healthy individuals and / or patients.

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

[0196] The anti-OX40L antibody or antigen-binding fragment thereof, or the bispecific antibody that specifically binds to OX40L and TNFα, autoimmune disease, inflammatory disease, individual, diagnosis, and step (method) of measuring the protein level are the same as those described above, unless inconsistent.

[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 intercellular fluid, which have different expression levels of OX40L in patients with autoimmune diseases. [Effects 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 receptor-receptor binding, but also has excellent immunosuppressive ability and can demonstrate 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 OX40L and TNFα exhibits strong affinity not only for OX40L but also for TNFα, and maintains its binding for a long period of time in the human body, thereby exhibiting excellent immunosuppressive ability and demonstrating significantly excellent effects in the fields of treatment and diagnosis of autoimmune diseases and inflammatory diseases. [Brief explanation of the drawings]

[0200] [Figure 1] Figure 1 shows exemplary structures of an anti-OX40L antibody and a bispecific antibody capable of simultaneously binding to OX40L and TNFα. In Figure 1, CH1, CH2, and CH3 represent heavy chain constant regions, CL represents the light chain constant region, and the striped (or diagonal) portions represent the variable regions of each chain (CDR regions (white) and framework regions (colored)). [Figure 2] FIG. 2 shows the results of SDS-PAGE analysis of anti-OX40L antibodies and bispecific antibodies capable of simultaneously binding to OX40L and TNFα. [Figure 3] Figure 3 shows the results of epitope mapping of the OX40L antigen in response to anti-OX40L antibodies, analyzed by HDX-MS. [Figure 4]Figure 4 shows the results of Biacore analysis to determine whether a bispecific antibody can simultaneously bind to the antigens OX40L and TNFα. The horizontal axis of the graph in Figure 4 represents time (0 = capture level), and the vertical axis represents response (0 = capture level). [Figure 5] FIG. 5 shows the results of an in vitro blockade assay of an anti-OX40L antibody and a bispecific antibody capable of simultaneously binding to OX40L and TNFα. [Figure 6] FIG. 6 shows the results of examining the effects of anti-OX40L antibody and a bispecific antibody capable of simultaneously binding to OX40L and TNFα on IL-2 secretion from T cells. DETAILED DESCRIPTION OF THE INVENTION

[0201] The present invention will be described in detail below with reference to examples, but the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0202] Example 1: Selection of OX40L-specific antibody clones

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

[0204] The antigen of human OX40L is derived from the extracellular domain of the human OX40L, which is located at positions 51 to 183 of the amino acid sequence of human OX40L (SEQ ID NO: 1) in Accession No. NP_003317. Human OX40L protein (Cat# OXL-H52Q8) provided by AcroBiosystems, Inc., which was prepared by fusing a histidine tag to the N-terminus of the amino acid sequence (Q51 to L183), was obtained and used.

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

[0206] [Table 2]

[0207] Example 1-2: Construction of human library phage

[0208] Diverse human-derived scFv library cells 7.5 x 10 10 The cells were cultured in 2xYT-glucose-MgCl2-chloramphenicol (CM) medium at 37°C until the absorbance of the culture solution reached OD 600 The culture was continued until the pH reached 0.5 to 0.7.

[0209] The cells were infected with helper phage and cultured at 37°C for approximately 1 hour. After centrifugation (5000 rpm, 4°C, 10 minutes), the cultured cells were remixed with 2xYT-IPTG-MgCl2-kanamycine (KM)-CM medium and cultured in a shaking incubator at 30°C for 16 hours. The cultured cells were centrifuged (5000 rpm, 4°C, 10 minutes). The supernatant was thoroughly dissolved in 4% PEG (Sigma, 81253) and 3% NaCl (Junsei, 1905-0350) and incubated on ice for approximately 1 hour. The cells were again centrifuged (7500 rpm, 4°C, 30 minutes). The pellet was dissolved in DPBS (Wellgene, LB001-02) and then centrifuged (10000 rpm, 4°C, 10 minutes) to obtain the supernatant containing the library phage. The supernatant was then placed in a new tube and stored at 5±3°C.

[0210] Example 1-3: Construction of immune library phage

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

[0212] Ten 7-week-old Balb / C mice and four 8-week-old male SD rats were purified and immunized with human OX40L antigen on days 0, 21, 42, and 63. On day 84, the spleens were removed and RNA was extracted. cDNA was synthesized using the eluted RNA, and the heavy and light chain variable regions were amplified. The heavy and light chain variable regions were mixed, and the amplified DNA in the form of scFv was inserted into a phage vector (pYG100) to generate RAT-derived immune scFv library cells. The cells were cultured in 2xYT-glucose-MgCl2-chloramphenicol (CM) medium at 37°C until the absorbance of the culture medium reached OD . 600 The culture was continued until the pH reached 0.5 to 0.7.

[0213] The cultured cells were infected with helper phage and incubated for 37 The cells were then cultured at 4°C for approximately 1 hour. After centrifugation (5000 rpm, 4°C, 10 minutes), the cultured cells were remixed with 2xYT-IPTG-MgCl2-kanamycine (KM)-CM medium and cultured in a shaking incubator at 30°C for 16 hours. The cultured cells were then centrifuged (5000 rpm, 4°C, 10 minutes). The supernatant was thoroughly dissolved in 4% PEG (Sigma, 81253) and 3% NaCl (Junsei, 1905-0350) and incubated on ice for approximately 1 hour. The cells were again centrifuged (7500 rpm, 4°C, 30 minutes). The pellet was dissolved in DPBS (Wellgene, LB001-02) and centrifuged (10000 rpm, 4°C, 10 minutes) to obtain the supernatant containing the library phage. The supernatant was then placed in a new tube and stored at 5±3°C.

[0214] Examples 1-4: Phage display panning

[0215] To select OX40L antibodies that bind 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. The OX40L protein was allowed to adsorb to the surface of the immunotube overnight at 5±3°C. A 1% solution of bovine serum albumin was then added to the tube to protect the surface to which OX40L had not been adsorbed. After emptying the tube, 10% bovine serum albumin dispersed in the 1% bovine serum albumin solution was added. 12 CFU of the human antibody phage library (Example 1-2) or immune phage library (Example 1-3) was placed in a test tube and allowed to bind to the antigen. Non-specifically bound phages were washed 5 to 20 times with PBS-T (Phosphate buffered saline-0.05% Tween 20) solution, and then washed 1 to 5 times with DPBS. The remaining antigen-specific phage antibodies were then recovered using 0.1 M TAE solution.

[0216] The recovered phages were neutralized with 1 M Tris buffer (pH 7.5) and then infected with XL1Blue E. coli at 37°C for 1 hour. The infected E. coli was smeared on an SOBCG plate using glass beads and cultured in an incubator at 37°C for approximately 16 hours. The next day, the cultured E. coli was suspended in 4 ml of SB (superbroth) carbenicillin culture medium, and 15% glycerol was added. A portion was stored at -80°C, and 50 μl of the remaining E. coli was added to 20 ml of SB carbenicillin culture medium and cultured at 37°C in a 2% glucose solution. When the absorbance of the culture medium reached 0.6 at 600 nm, the medium was centrifuged to remove the medium. This was then resuspended in 20 ml of SB carbenicillin culture medium and cultured for 10 minutes. 12 PFU M13 helper phage was added and cultured at 37°C with gentle stirring. The next day, the culture medium was centrifuged to remove the culture medium alone, which was then precipitated with polyethylene glycol and sodium chloride (NaCl) at 4°C for 30 minutes and centrifuged again. The supernatant was removed, and the precipitated phage was suspended in 1 ml of PBS. This was used as a library, and the panning process was repeated 3 to 5 times to amplify and enrich antigen-specific clones.

[0217] Examples 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, single colonies were obtained by smearing on agar medium, which were then inoculated into 1-1.5 mL of culture medium and cultured. After induction with IPTG, the scFv protein was expressed in E. coli. The E. coli culture was centrifuged to obtain the supernatant, which was used to confirm the binding of the recombinant human OX40L antigen to the scFv by ELISA (Steinberger, Rader and Barbas III, 2000. Phage display vectors. In: Phage Display Laboratory Manual. Published. Cold Spring Harbor Laboratory, 2003). (YPress. NY. USA. pp. 11.9-11.12). Bound scFv was detected using HRP (horseradish peroxidase)-anti-His antibody and tetramethylbenzidine (TMB) substrate.

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

[0221] Example 2: Generation of anti-OX40L antibodies

[0222] Based on the sequences for the antibody variable regions (scFv) obtained in Examples 1-5, the heavy chain variable region was linked to the heavy chain constant region (SEQ ID NO: 8), and the light chain variable region was linked to the light chain constant region (SEQ ID NO: 10) to produce antibodies. 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 comprises a heavy chain CDR1 represented by SEQ ID NO: 12; a heavy chain CDR2 represented by SEQ ID NO: 15; a heavy chain CDR3 represented by SEQ ID NO: 19; a light chain CDR1 represented by SEQ ID NO: 23; a light chain CDR2 represented by SEQ ID NO: 25; and a light chain CDR3 represented by SEQ ID NO: 27.

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

[0227] Suspension FreeStyle™ 293-F animal cells transduced with an expression vector containing the gene encoding the anti-OX40L antibody 02C09 using PEI, a polymer that increases the efficiency of intracellular gene delivery, were cultured in 500 mL Erlenmeyer culture flasks (Corning) at 200 mL per flask, and then cultured in large quantities as needed.

[0228] Transduced FreeStyle™ 293-F cells were cultured in suspension at 37°C and 8% CO2 in FreeStyle™ 293 Expression Medium AGT™ (Invitrogen, AG1000D9P1). For overexpression, 500 μg of PEI (Polysciences, 23966-2) and 125 μg of DNA to be overexpressed were mixed in 5 mL of culture medium. Approximately 24 hours after the addition of DNA-PEI, 10 mL of 10% soytone (BD, 212488) was added, and the cells were further cultured for approximately 5 days. The supernatant was then used for antibody purification.

[0229] To purify the antibodies, they were first purified from the culture medium using a recombinant Protein-A Sepharose column. If further purification was required, the primary purified product was subjected to secondary purification using Superdex 200 gel filtration chromatography or hydroxyapatite chromatography.

[0230] [Table 4]

[0231] [Table 5]

[0232] (2) Anti-OX40L antibody Hu3F07

[0233] The Hu3F07 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.

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

[0235] [Table 6]

[0236] [Table 7]

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

[0238] The 10H07 antibody comprises 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 expressed in the expression vector pcDNA3.1 (Invitrogen The antibody was produced using the animal cell lines (n) and FreeStyle™ 293-F (Invitrogen). The specific culture conditions, culture method, and purification method were essentially the same as those described in (1) Anti-OX40L antibody 02C09 above.

[0240] [Table 8]

[0241] [Table 9]

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

[0243] The 21G07 antibody comprises 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] Anti-OX40L antibody 21G07 was produced using the expression vector pcDNA3.1 (Invitrogen) and the FreeStyle™ 293-F (Invitrogen) animal cell line. The specific culture conditions, culture method, and purification method were essentially the same as those described for (1) anti-OX40L antibody 02C09 above.

[0245] [Table 10]

[0246] [Table 11]

[0247] (5) Anti-OX40L antibody I3F07

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

[0249] The anti-OX40L antibody I3F07 was produced using the expression vector pcDNA3.1 (Invitrogen) and the FreeStyle™ 293-F (Invitrogen) animal cell line. The specific culture conditions, culture method, and purification method were essentially the same as those described for (1) anti-OX40L antibody 02C09 above.

[0250] [Table 12]

[0251] [Table 13]

[0252] Example 3: Generation of bispecific antibodies targeting OX40L and TNFα

[0253] The antibodies (02C09, Hu3F07, I3FO7) that bind to human OX40L prepared in Example 2 or their fragments (scFv) were linked to an anti-TNFα antibody (TNFαi antibody (Humira)) or its fragment (scFv) using a linker to prepare a bispecific antibody that can also bind to human TNFα (see Figure 1).

[0254] Specifically, the bispecific antibodies were prepared in the following forms: (i) a form in which the heavy chain variable region and light chain variable region of an anti-TNFα antibody are linked via a linker to the C-terminus of the heavy chain constant region of an anti-OX40L antibody; (ii) a form in which the heavy chain variable region and light chain variable region of an anti-TNFα antibody are linked via a linker to the C-terminus of the light chain constant region of an anti-OX40L antibody; (iii) a form in which the heavy chain variable region and light chain variable region of an anti-OX40L antibody are linked via a linker to the C-terminus of the heavy chain constant region of an anti-TNFα antibody; and (iv) a form in which the heavy chain variable region and light chain variable region of an anti-OX40L antibody are linked via a linker to the C-terminus of the light chain constant region of an anti-TNFα antibody. These antibodies were designated 02C09-TNFαi HC, 02C09-TNFαi LC, hu3F07-TNFαi HC, and hu3F07-TNFαi, respectively, in the order of the IgG-scFv-binding site. They were named LC, TNFαi-02C09 HC, TNFαi-02C09 LC, TNFαi-hu3F07 HC, TNFαi-hu3F07 LC, I3F07-TNFαi HC, I3F07-TNFαi LC, TNFαi-I3F07 HC, and TNFαi-I3F07 LC.

[0255] The linker used was GGGGSGGGSGGGGS as shown in SEQ ID NO:31.

[0256] [Table 14]

[0257] [Table 15]

[0258] The TNFαi antibody was produced by a method substantially similar to the method for producing (preparing) the anti-OX40L antibody in Example 2, in which the heavy chain variable region of the TNFα antibody was linked to a heavy chain constant region (sequence number 8), and the light chain variable region was linked to a light chain constant region (sequence number 10).

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

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

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

[0262] [Table 16-1] [Table 16-2]

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

[0264] 02C09-TNFαi LC is a TNFαi antibody binding fragment (ScFv) in which the Humira heavy chain variable region (sequence number 35) and Humira light chain variable region (sequence number 36) are linked by a linker (sequence number 32) to the C-terminus of the light chain constant region of the anti-OX40L antibody 02C09 via a linker (sequence number 31).

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

[0266] [Table 17-1] [Table 17-2]

[0267] (3) Bispecific antibody Hu3F07-TNFαi HC Hu3F07-TNFαi HC is a heavy chain constant region C of the anti-OX40L antibody hu3F07. At the end, a binding fragment (ScFv) of a TNFαi antibody, in which the heavy chain variable region (sequence number 35) of a TNFαi antibody and the light chain variable region (sequence number 36) of a TNFαi antibody are linked by a linker (sequence number 32), is linked by a linker (sequence number 31).

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

[0269] [Table 18-1] [Table 18-2]

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

[0271] Hu3F07-TNFαi LC is a TNFαi antibody binding fragment (ScFv) in which the heavy chain variable region (sequence number 35) of a TNFαi antibody and the light chain variable region (sequence number 36) of a TNFαi antibody are linked by a linker (sequence number 32) to the C-terminus of the light chain constant region of the anti-OX40L antibody hu3F07 via a linker (sequence number 31).

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

[0273] [Table 19-1] [Table 19-2]

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

[0275] TNFαi-02C09 HC is a binding fragment (ScFv) of anti-OX40L antibody 02C09, in which the heavy chain variable region (sequence number 37) of anti-OX40L antibody 02C09 and the light chain variable region (sequence number 38) of anti-OX40L antibody 02C09 are linked by a linker (sequence number 32) to the C-terminus of the heavy chain constant region of TNFαi antibody (Humira) via a linker (sequence number 31).

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

[0277] [Table 20-1] [Table 20-2]

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

[0279] TNFαi-02C09 LC is a binding fragment (ScFv) of anti-OX40L antibody 02C09, in which the heavy chain variable region (sequence number 37) of anti-OX40L antibody 02C09 and the light chain variable region (sequence number 38) of anti-OX40L antibody 02C09 are linked by a linker (sequence number 32) to the C-terminus of the light chain constant region of the TNFαi antibody via a linker (sequence number 31).

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

[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) of the anti-OX40L antibody hu3F07 and the light chain variable region (SEQ ID NO: 42) of the anti-OX40L antibody hu3F07 are linked to the C-terminus of the heavy chain constant region of the TNFαi antibody via a linker (SEQ ID NO: 32). hu3F07 The binding fragments (ScFv) of the above are linked by a linker (SEQ ID NO: 31).

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

[0285] [Table 22-1] [Table 22-2]

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

[0287] TNFαi-hu3F07 LC is a TNFαi antibody light chain constant region C-terminus linked by a linker (sequence number 31) to a binding fragment (ScFv) of the anti-OX40L antibody hu3F07, in which the heavy chain variable region (sequence number 41) of the anti-OX40L antibody hu3F07 and the light chain variable region (sequence number 42) of the anti-OX40L antibody hu3F07 are linked by a linker (sequence number 32).

[0288] The bispecific antibody TNFαi-02C09 LC was produced using the expression vector pcDNA3.1 (Invitrogen) and the FreeStyle™ 293-F animal cell line. The specific culture conditions, culture method, and purification method were essentially the same as those described for (1) anti-OX40L antibody 02C09 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 (Humira) in which the heavy chain variable region (SEQ ID NO: 35) of the TNFαi antibody (Humira) and the light chain variable region (SEQ ID NO: 36) of the TNFαi antibody (Humira) are linked via a linker (SEQ ID NO: 32) to the C-terminus of the heavy chain constant region of the anti-OX40L antibody I3F07 via a linker (SEQ ID NO: 31).

[0292] The bispecific antibody I3F07-TNFαi HC was produced using the expression vector pcDNA3.1 (Invitrogen) and the FreeStyle™ 293-F (Invitrogen) animal cell line. The specific culture conditions, culture method, and purification method were essentially the same as those described for (1) anti-OX40L antibody 02C09 above.

[0293] [Table 24-1] [Table 24-2]

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

[0295] I3F07-TNFαi LC is a TNFαi antibody binding fragment (ScFv) in which the heavy chain variable region of Humira (sequence number 35) and the light chain variable region of Humira (sequence number 36) are linked by a linker (sequence number 32) to the C-terminus of the light chain constant region of the anti-OX40L antibody I3F07 via a linker (sequence number 31).

[0296] The bispecific antibody I3F07-TNFαi LC was produced using the expression vector pcDNA3.1 (Invitrogen) and the FreeStyle™ 293-F (Invitrogen) animal cell line. The specific culture conditions, culture method, and purification method were essentially the same as those described for (1) anti-OX40L antibody 02C09 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) of the anti-OX40L antibody I3F07 and the light chain variable region (SEQ ID NO: 54) of the anti-OX40L antibody I3F07 are linked to the C-terminus of the heavy chain constant region of the TNFαi antibody via a linker (SEQ ID NO: 32). I3F07 The binding fragments (ScFv) of the above are linked by a linker (SEQ ID NO: 31).

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

[0301] [Table 26-1] [Table 26-2]

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

[0303] TNFαi-I3F07 LC is a binding fragment (ScFv) of anti-OX40L antibody I3F07, in which the heavy chain variable region (sequence number 53) of anti-OX40L antibody I3F07 and the light chain variable region (sequence number 54) of anti-OX40L antibody I3F07 are linked by a linker (sequence number 32) to the C-terminus of the light chain constant region of the TNFαi antibody via a linker (sequence number 31).

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

[0305] [Table 27-1] [Table 27-2]

[0306] Furthermore, the VH and VL sequences of the anti-OX40L antibody oxelumab (SEQ ID NO: 3) were generated in a manner substantially similar to the method for producing (preparing) the anti-OX40L antibody in Example 2. A reference antibody, anti-OX40L antibody (hereinafter, Ref. Ab or O4L), was produced using the VH and VL sequences (SEQ ID NOs: 35 and 36) of the anti-TNFα antibody Humira.

[0307] [Table 28]

[0308] [Table 29]

[0309] Using the antibody O4L and Ref. TNFαi thus prepared, anti-OX40L-TNFαi HC and LC antibodies (hereinafter referred to as O4L-TNFαi HC and O4L-TNFαi LC, respectively) were prepared in a manner substantially similar to the method for producing (manufacturing) the bispecific antibody in Example 3. ) was produced.

[0310] [Table 30-1] [Table 30-2]

[0311] [Table 31-1] [Table 31-2]

[0312] Experimental Example 1: Antibody Analysis

[0313] The anti-OX40L antibody obtained in Example 2 and the bispecific antibody obtained in Example 3 were subjected to SDS-PAGE analysis, and the results are shown in FIG.

[0314] Experimental Example 2: Epitope of OX40L antigen against anti-OX40L antibody

[0315] The epitope of the OX40L antigen in response to the anti-OX40L antibody was analyzed by HDX-MS. A pepsin column was used, labeled with a DO-based buffer solution, for five time points. The data were processed using PLGS and DynamX, and the results are shown in Figure 3.

[0316] Experimental Example 3: Antibody characteristics

[0317] Experimental Example 3-1. Equilibrium dissociation constants (K D )analysis

[0318] The antigen affinities of the anti-OX40L antibodies and the bispecific antibodies that simultaneously regulate OX40L and TNFα isolated and purified in Examples 2 and 3 were analyzed as follows.

[0319] Of these antibodies, the anti-OX40L antibody was confirmed to have binding ability to human OX40L (SEQ ID NO: 2), and the OX40L / TNFα bispecific antibody was confirmed to have binding ability to human OX40L (SEQ ID NO: 2) and human TNFα (TNF-H5228, Acrobiosystems) (Table 32).

[0320] SPR (Surface Plasmon Resonance) analysis was performed using a Bicore T200, with HBS-EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.15% surfactant P20) as the running buffer. Human antibody capture kit (anti-hFc antibody) was immobilized on the surface of a CM5 chip by the amine coupling method. Human OX40L or human TNFα was run. After diluting the antibody to 10 nM with buffer, it was serially diluted in half and analyzed at five concentration intervals. The antibody concentration was confirmed by sterilizing the antibody through a 0.2 μm filter and measuring the absorbance (A280). Analytical samples were prepared at high purity and concentration with a minimum dilution factor of 100 or more to minimize the buffer effect. A regeneration step was performed between all analyses to maintain a constant baseline for the experiment. The Biacore analysis results are shown in Table 32 and Figure 4.

[0321] In FIG. 4, Ag1 represents human OX40L, and Ag2 represents human TNFα.

[0322] [Table 32]

[0323] As can be seen from Table 32, the anti-OX40L antibody of Example 2 was confirmed to strongly bind to OX40L, and the bispecific antibody of Example 3 was confirmed to strongly bind to both OX40L and TNFα. Specifically, it was confirmed that both the anti-OX40L antibody and the bispecific antibody had nM-level binding affinity to OX40L, and in particular, the bispecific antibody also had nM-level binding affinity to TNFα. These results suggest that the bispecific antibody maintains high levels of binding ability to each antigen without interference.

[0324] Experimental Example 3-2. Thermal stability test of anti-OX40L antibody and bispecific antibody

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

[0326] The antibody was diluted in DPBS to a concentration of 3 μM / 45 μL, then mixed with 5 μL of 200× Sypro Orange dye (#S6650, Thermo Scientific) and dispensed into 50 μL qPCR tubes (#B77009, B57651, Bioplastics). qPCR was performed using a Biorad CFX96 real-time PCR instrument. The qPCR conditions were: 25°C for 30 seconds, followed by 1°C increments up to 99°C for 1 minute at each temperature, and finally 10 seconds at 25°C before termination. The melting temperature (Tm) was used as the rate constant for antibody unwinding. The results are shown in Table 33.

[0327] [Table 33]

[0328] As can be seen from the table above, the melting temperatures of the anti-OX40L antibody and the bispecific antibody are between 59 and 65°C, and the results indicate that the bispecific antibody also has similar thermal stability to the anti-OX40L antibody.

[0329] Experimental Example 3-3. PK (Pharmacokinetics analysis) of anti-OX40L antibody and bispecific antibody

[0330] Analysis was performed to determine the pharmacokinetics of the bispecific antibody of Example 3 and the anti-OX40L antibody of Example 2 upon administration (Table 34).

[0331] Male Sprague-Dawley rats, widely used for pharmacokinetic studies due to their consistent drug response and stable supply system, were used. The study was conducted without fasting. Three 7-week-old male Sprague-Dawley (SD) rats were administered a single intravenous bolus of 5 mpk (2.5–2.4 mg / ml) in each group. After administration, blood samples were collected from the jugular vein (approximately 150 μl / time point) at 3 min, 3, 8, 24, 48, 72, 96, 120, 144, and 168 h. Approximately 70 μl of plasma was isolated using sodium heparin as an anticoagulant. The samples were centrifuged (12,000 rpm, 3 min) to obtain approximately 70 μl of plasma, which was then immediately stored in a deep-freezer. General observations were performed at least once daily throughout the experiment. After the final blood collection, the experimental animals were euthanized by CO2 inhalation.

[0332] The plasma obtained from the PK study was analyzed using the Gyrolab xPlore® (Cat. #P0020300, GYROS PROTEIN) analyzer and the Gyrolab PK kit (Cat. #P0020499, GYROS PROTEIN). The results from Gyrolab were used to obtain parameter values such as AUC(last), AUC(inf), Cmax, Tmax, and Half life using BA Calc 2007 1.0.0 or PK Solver 2.0 programs. The results are shown in Table 34.

[0333] [Table 34]

[0334] The half-lives of the anti-OX40L antibody of Example 2 were 3.6 days and 4.0 days, respectively, and the half-life of the bispecific antibody of Example 3 was observed to be 3.6 to 6.0 days. Therefore, it can be assumed that the drug will also exhibit a half-life of approximately two weeks or more in humans.

[0335] Experimental Example 4: Antibody efficacy

[0336] Experimental Example 4-1. Evaluation of antibody signal suppression ability (1)

[0337] The anti-OX40L antibody of Example 2 and the bispecific antibody of Example 3 (Hu3F07-TNFαi Activity evaluation experiments of the OX40L / OX40 blocking bioassay kit (Promega CS197706) and the TNFα / TNFα Rc blocking bioassay kit (Promega CS177503) were performed (Table 35, Figure 5).

[0338] (1) The activities of the anti-OX40L antibody of Example 2 and the bispecific antibody of Example 3 were evaluated using an OX40L / OX40 blocking bioassay kit.

[0339] NFκB-luc2 / OX40 Jurkat cells were immersed in RPMI1640 (10% FBS) culture medium and cultured overnight in a 37°C, 5% CO2 incubator. The next day, antigens and antibodies to be reacted with the cells were prepared. OX40L antigen was prepared in RPMI1640 (10% FBS) culture medium so that the final concentration when introduced into the cells would be 15 ng / mL. The anti-OX40L antibody, bispecific antibody, and control antibody were diluted so that the final starting concentration when introduced into the cells would be 33 μg / mL, and then serially diluted 1 / 3 to prepare 9 levels. The 10th concentration was prepared by replacing the culture medium with 0, so that a total of 10 levels of concentration gradient were prepared. 25 μL of the prepared antigen dilution and antibody dilution were each dispensed onto the cells. Each sample was dispensed in triplicate. After dispensing, the total volume of the cells, antigen dilution, and antibody dilution was adjusted to 100 μL. The reaction was allowed to proceed for approximately 5 hours in a CO2 incubator. I did.

[0340] Bio-Glo was dispensed in 75 μL aliquots and reacted for approximately 10 minutes. The samples were analyzed for luminescence using a microplate reader, and then analyzed using a 4-parameter (X-axis log(concentration)) analysis.

[0341] IC in μg / mL, the antibody concentration unit 50 The analytical results were converted to nM and the final IC 50 of This was done to observe under the same conditions, since the anti-OX40L antibody is 150 kDa and the bispecific antibody is 200 kDa.

[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 cultured at 37°C in a 5% CO2 incubator. The following was prepared. TNFα antigen was diluted in DMEM (10% FBS) culture medium to a final concentration of 3 ng / mL when reacted with cells. The bispecific antibody and the control antibody Humira (Ref. TNFαi) were diluted in DMEM (10% FBS) culture medium to a starting concentration of 10 nM, and then serially diluted in half to prepare eight levels. When reacted with cells, the final starting concentration was 0.8 nM.

[0344] The prepared diluted antigen solution and diluted antibody solution were mixed in a 3:2 ratio and dispensed into the cells in 20 μL portions. Each sample was dispensed in duplicate. After dispensing, the mixture was incubated in a CO2 incubator for approximately 4 hours. I made him respond.

[0345] Bio-Glo was dispensed in 100 μL aliquots and reacted. The samples were analyzed by luminescence using a microplate reader, and then analyzed using a 4-parameter (X-axis log(concentration)) to determine the IC 50 asked for.

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

[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 of Examples 2 and 3 was 1.3 x 10 -9 M (nM) or less, and in particular, the OX40L blocking ability of the anti-OX40L antibody was 0.9 × 10 -9 The bispecific antibody showed a TNFα blocking activity of 1×10 -9These results show that the anti-OX40L antibodies of the present invention exhibit excellent OX40L blocking ability at a level of M (nM) or less, and that the bispecific antibodies prepared using these antibodies exhibit excellent blocking ability not only against OX40L but also against TNFα.

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

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

[0351] [Table 36]

[0352] As can be seen from Table 36, the TNFα blocking ability of the bispecific antibodies 02C09-TNFαi LC, TNFαi-02C09 HC, and TNFαi 02C09 LC of Example 3 was 1×10 -9 The levels were below M (nM), indicating excellent blocking ability.

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

[0354] The bispecific antibody of Example 3 (I3F07-TNFαi HC, I3F07-TNFαi The activity of TNFαi-I3F07 LC, TNFαi-I3F07 HC, and TNFαi-I3F07 LC was evaluated using an OX40L / OX40 blocking bioassay kit (Promega CS197706) and a TNFα / TNFα Rc blocking bioassay kit (Promega CS177503) (Table 37). Specific evaluation conditions and methods were essentially the same as those described in Experimental Example 4-1. Evaluation of antibody signal suppression ability (1).

[0355] [Table 37]

[0356] As can be seen from Table 37, the OX40L blocking ability of the bispecific antibody of Example 3 was 1×10 -9 M (nM) or less, and the TNFα blocking ability was 1×10 -9 It was found that it exhibited excellent blocking ability at levels below M (nM).

[0357] Experimental Example 4-4. Evaluation of antibody's ability 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, their ability to inhibit immune cell activity was confirmed using peripheral blood mononuclear cells (PBMCs) from healthy individuals and PBMCs from rheumatoid arthritis (RA) patients ( FIG. 6 ).

[0359] Using the above method, T cells were isolated from PBMCs of healthy individuals and PBMCs of RA patients, and the reduced level of secretion of IL-2, a cytokine secreted upon activation of T cells, was analyzed.

[0360] To separate T cells from PBMCs of healthy donors and patients, anti-CD3 (R&D Systems, MAB100) was diluted to 100 ng / well and allowed to adhere to 96-well plates for approximately 16 hours (5 ± 3 min). After removing the adherent solution, the plates were washed with PBS. DNase I was diluted to 20 U / mL in LGM-3 (10% FBS, 1% P / S), and PBMCs from healthy donors were added to prepare a mixture. This mixture was then centrifuged (200 g, 15 min), the supernatant was removed, and the healthy donor PBMCs were resuspended in LGM-3 (10% FBS, 1% P / S) until 1 × 10 6 The samples were diluted to 100 cells / 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 added in 50 μL portions to PBMCs from healthy donors. Anti-OX40L antibodies and bispecific antibodies were prepared at 800 ng / mL in LGM-3 (10% FBS, 1% P / S) and then diluted to 1 nM. Fifty μL portions were dispensed into plates containing T cells isolated from PBMCs, human OX40L, and human TNFα. After approximately 24 to 72 hours, the supernatants were collected and IL-2 assayed. The results are shown in Figure 6.

[0361] In FIG. 6, A shows the results of a T cell assay derived from PBMCs of a healthy individual; B shows the results of a T cell assay derived from the patient's PBMC.

[0362] As can be seen from Figure 6, administration of the bispecific antibody of the present invention shows a tendency for IL-2 to decrease. In particular, looking at the results for T cells derived from PBMC of RA patients, it can be seen that the bispecific antibody of the present invention significantly reduced 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 can be particularly effective in RA patients who are refractory to Humira.

[0363] Therefore, it can be seen that the anti-OX40L antibody and bispecific antibody of the present invention can calm an overactivated 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 an OX40 receptor.

2. The anti-OX40L antibody or its antigen-binding fragment according to claim 1, wherein the anti-OX40L antibody or its antigen-binding fragment binds to one or more epitopes set forth in an amino acid sequence 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 .gtoreq..times ...

4. The anti-OX40L antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising: a heavy chain CDR1 set forth in an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, and 14; a heavy chain CDR2 set forth in an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 16, 17, and 18; and a heavy chain CDR3 set forth in an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, 21, and 22; and An anti-OX40L antibody or its antigen-binding fragment according to claim 1 or 2, comprising a light chain variable region comprising: a light chain CDR1 set forth in one amino acid sequence selected from the group consisting of SEQ ID NOs: 23 and 24; a light chain CDR2 set forth in one amino acid sequence selected from the group consisting of SEQ ID NOs: 25 and 26; and a light chain CDR3 set forth in one amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, 29, and 30.

5. The anti-OX40L antibody or antigen-binding fragment thereof comprises: (i) an antibody or antigen-binding fragment thereof comprising 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 heavy chain CDR1 set forth in SEQ ID NO: 23; a heavy chain CDR2 set forth in SEQ ID NO: 25; and a light chain CDR3 set forth in SEQ ID NO: 27; (ii) an antibody or antigen-binding fragment thereof comprising 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; (iii) an antibody or antigen-binding fragment thereof comprising 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; or (iv) An anti-OX40L antibody or an antigen-binding fragment thereof according to claim 1 or 2, which is an antibody or an antigen-binding fragment thereof comprising 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.

6. The anti-OX40L antibody or antigen-binding fragment thereof comprises: a 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 comprises: (a) a heavy chain variable region set forth in SEQ ID NO: 37 and a light chain variable region set forth in SEQ ID NO: 38; (b) a heavy chain variable region set forth in SEQ ID NO: 41 and a light chain variable region set forth in SEQ ID NO: 42; (c) a heavy chain variable region set forth in SEQ ID NO: 45 and a light chain variable region set forth in SEQ ID NO: 46; (d) a heavy chain variable region set forth in SEQ ID NO: 49 and a light chain variable region set forth in SEQ ID NO: 50; or (e) a heavy chain variable region set forth in SEQ ID NO: 53 and a 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, comprising:

8. The anti-OX40L antibody or antigen-binding fragment thereof comprises: A 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 an antigen-binding fragment thereof that specifically binds to OX40L; and an anti-TNFα antibody or an antigen-binding fragment thereof that specifically binds to TNFα.

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

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

12. The anti-OX40L antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising a heavy chain CDR1 set forth in an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, and 14; a heavy chain CDR2 set forth in an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 16, 17, and 18; and a heavy chain CDR3 set forth in an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, 21, and 22; and 11. The bispecific antibody of claim 10, comprising a light chain variable region comprising a light chain CDR1 set forth in an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 and 24; a light chain CDR2 set forth in an amino acid sequence selected from the group consisting of SEQ ID NOs: 25 and 26; and a light chain CDR3 set forth in an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, 29, and 30.

13. The anti-OX40L antibody or antigen-binding fragment thereof comprises: (i) an antibody or antigen-binding fragment thereof comprising 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 heavy chain CDR1 set forth in SEQ ID NO: 23; a heavy chain CDR2 set forth in SEQ ID NO: 25; and a light chain CDR3 set forth in SEQ ID NO: 27; (ii) an antibody or antigen-binding fragment thereof comprising 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; (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 CDR1 set forth in SEQ ID NO: 24; a light chain CDR2 set forth in SEQ ID NO: 26; and a light chain CDR set forth in SEQ ID NO: 29 an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising R3; or (iv) The bispecific antibody of claim 10, which is an antibody or antigen-binding fragment thereof, comprising 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.

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

15. The anti-OX40L antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region set forth in SEQ ID NO: 37 and a light chain variable region set forth in SEQ ID NO: 38; (b) a heavy chain variable region set forth in SEQ ID NO: 41 and a light chain variable region set forth in SEQ ID NO: 42; (c) a heavy chain variable region set forth in SEQ ID NO: 45 and a light chain variable region set forth in SEQ ID NO: 46; (d) a heavy chain variable region set forth in SEQ ID NO: 49 and a light chain variable region set forth in SEQ ID NO: 50; (e) a heavy chain variable region set forth in SEQ ID NO: 53 and a light chain variable region set forth in SEQ ID NO: 54; or (f) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 33 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 34; The bispecific antibody of claim 10, comprising:

16. The anti-TNFα antibody or antigen-binding fragment thereof comprises: 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 16. The bispecific antibody according to any one of claims 10 to 15, comprising 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.

17. The bispecific antibody of claim 16 , wherein the anti-TNFα antibody or antigen-binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO: 35 and a light chain variable region set forth in SEQ ID NO:

36.

18. The bispecific antibody was incubated with human OX40L at 1.5 x 10 －９ K below M Ｄ and 1 × 10 for human TNFα. －９ K below M Ｄ wherein said K Ｄ is measured by surface plasmon resonance (Biacore) analysis.

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

21. The bispecific antibody has an O The bispecific antibody according to claim 16, which is linked to an anti-OX40L antibody or an antigen-binding fragment thereof that specifically binds to X40L.

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

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

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

32.

25. A nucleic acid encoding the antibody or 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. 26. An expression vector comprising the nucleic acid of 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 fragment thereof, or a bispecific antibody using the transformant described in claim 27.

29. A pharmaceutical composition for preventing or treating an autoimmune disease or an inflammatory disease, comprising the antibody or 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.

30. 30. The pharmaceutical composition according to claim 29, wherein the pharmaceutical composition is for preventing or treating rheumatoid arthritis.

31. A method for providing information for diagnosing an autoimmune disease or an inflammatory disease, using the antibody or 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.

32. The antibody or 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. A kit that provides information for diagnosing an autoimmune or inflammatory disease.

33. A method for preventing or treating an autoimmune disease or an inflammatory disease, comprising administering a pharmaceutically effective amount of the antibody or 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.

34. Use of the antibody or 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, in the manufacture of a medicament for the prevention or treatment of an autoimmune disease or an inflammatory disease.

35. Use of the antibody or 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, for the prevention or treatment of an autoimmune disease or an inflammatory disease.

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