Antibodies that specifically bind to human DR3 and their applications

Abstract

The present invention relates to an antibody that specifically binds to human DR3 and its use. The antibody specifically binds to a specific epitope of DR3, selectively acts on regulatory T cells, and exhibits effector activity. + T cells and CD8 + It is possible to minimize the activity of T cells while strongly activating only regulatory T cells. By inducing such selective regulatory T cell activity, it is possible to effectively prevent or treat graft-versus-host disease, autoimmune diseases, and type 2 diabetes, and thus it can be usefully used as a therapeutic agent for various diseases related thereto.

Description

[Technical Field]

[0001] This invention relates to an antibody that specifically binds to human DR3 and its applications.

[0002] This application claims priority based on Republic of Korea Patent Application No. 10-2022-0074078 filed on 17 June 2022 and Republic of Korea Patent Application No. 10-2023-0077023 filed on 15 June 2023, and all content disclosed in the specifications and drawings of said applications is incorporated herein by reference. [Background technology]

[0003] Death receptor 3 (DR3, tumor necrosis factor receptor superfamily 25, TNFRSF25) is one of the components of TNFRSF. DR3 consists of 417 amino acids, has a molecular weight of 47 kDa, and is homotrimer. DR3 is highly expressed in regulatory T cells (Tregs) under steady state, but its expression also increases when conventional T cells are activated. In addition, DR3 is known to be expressed in epidemic cells such as NK cells, ILCs, B cells, and mononuclear cells, as well as non-epidemic cells such as osteocytes and endothelial cell colony-forming cells.

[0004] TL1A (tumor necrosis factor-like ligand 1A), a ligand for DR3, is primarily expressed by activated T cells, dendritic cells, macrophages, mononuclear cells, and endothelial cells. TL1A activates NF-κB signaling by binding to DR3, thereby functioning as a costimulation factor for T cells. According to previously reported studies, selective activation of regulatory T cells can be induced using DR3 agonists. Unlike IL-2, which activates effector T cells in addition to regulatory T cells, DR3 agonists selectively activate only regulatory T cells. Activation of effector T cells can be a major obstacle to treatment when addressing various inflammatory diseases.

[0005] DR3 agonists may be TL1A, a ligand for DR3, or agonistic anti-DR3 antibodies. However, TL1A has a natural inhibitor, DcR3 (decoy receptor), and natural cytokine inhibitors are usually regulated by negative feedback. Therefore, inducing DR3 activation using TL1A is difficult in terms of efficient drug delivery, and its homotrimer cytokine characteristics can increase the difficulty of the new drug development process. On the other hand, using agonistic anti-DR3 antibodies can circumvent negative feedback by DcR3 and induce efficient DR3 activation.

[0006] On the other hand, while research into the clinical applicability of antibody drugs has been continuously conducted since the development of the first monoclonal antibody in 1975, the fundamental core of the potential for antibody drug development lies in their mechanism of action, which utilizes the properties of antibodies—strong binding affinity and high binding specificity—to control specific biological reactions very strongly and selectively. In particular, compared to existing therapeutic agents based on chemically synthesized substances, antibody drugs have relatively fewer side effects and superior therapeutic efficacy due to their high binding specificity and stability in the human body. Therefore, the field of antibody-based therapeutic agent development is attracting attention as a next-generation core field of new drug research and development.

[0007] However, no antibody drug has yet been developed that specifically binds to DR3, activates regulatory T cells, and exhibits excellent therapeutic effects for graft-versus-host disease, autoimmune diseases, or type 2 diabetes. [Overview of the project] [Problems that the invention aims to solve]

[0008] One object of the present invention is to provide an anti-DR3 antibody or its antigen-binding fragment that specifically binds to death receptor 3 (DR3).

[0009] Another object of the present invention is to provide a nucleic acid sequence for encoding the antibody or antigen-binding fragment.

[0010] Another object of the present invention is to provide a vector containing the nucleic acid sequence.

[0011] Another object of the present invention is to provide a transformant transformed with the vector.

[0012] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of graft-versus-host disease, autoimmune disease, or type 2 diabetes, comprising an anti-DR3 antibody or an antigen-binding fragment thereof as an active ingredient, which specifically binds to the cell death receptor 3 (DR3).

[0013] Another object of the present invention is to provide a kit for the prevention or treatment of graft-versus-host disease, autoimmune disease, or type 2 diabetes, comprising the vector or composition and a manual.

[0014] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other problems not mentioned should be clearly understood by a person with ordinary skill in the art to which the present invention pertains from the following description. [Means for solving the problem]

[0015] The present invention provides an anti-DR3 antibody or an antigen-binding fragment thereof that specifically binds to the cell death receptor 3 (DR3).

[0016] The present invention provides a nucleic acid sequence for encoding the antibody or antigen-binding fragment.

[0017] The present invention provides a vector containing the nucleic acid sequence.

[0018] The present invention provides a transformant transformed with the aforementioned vector.

[0019] The present invention provides a composition for the prevention or treatment of graft-versus-host disease, autoimmune disease, or type 2 diabetes, comprising an anti-DR3 antibody or its antigen-binding fragment as an active ingredient, which specifically binds to the aforementioned cell death receptor 3 (DR3).

[0020] The present invention provides a kit for the prevention or treatment of graft-versus-host disease, autoimmune disease, or type 2 diabetes, comprising the vector or composition and a manual.

[0021] In one embodiment of the present invention, the antibody or the antigen-binding fragment may be selected from, but is not limited to, any one of the following groups:

[0022] i) A heavy chain variable region containing heavy chain CDR1 as described by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 as described by the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 as described by the amino acid sequence of SEQ ID NO: 3; and an antibody or antigen-binding fragment containing a light chain variable region containing light chain CDR1 as described by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 as described by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 as described by the amino acid sequence of SEQ ID NO: 6; ii) A heavy chain variable region including heavy chain CDR1 described by the amino acid sequence of SEQ ID NO: 7, heavy chain CDR2 described by the amino acid sequence of SEQ ID NO: 8, and heavy chain CDR3 described by the amino acid sequence of SEQ ID NO: 9; and an antibody or antigen-binding fragment including a light chain variable region including light chain CDR1 described by the amino acid sequence of SEQ ID NO: 10, light chain CDR2 described by the amino acid sequence of SEQ ID NO: 11, and light chain CDR3 described by the amino acid sequence of SEQ ID NO: 12; iii) A heavy chain variable region including heavy chain CDR1 as described in the amino acid sequence of SEQ ID NO: 13, heavy chain CDR2 as described in the amino acid sequence of SEQ ID NO: 14, and heavy chain CDR3 as described in the amino acid sequence of SEQ ID NO: 15; and an antibody or antigen-binding fragment including a light chain variable region including light chain CDR1 as described in the amino acid sequence of SEQ ID NO: 16, light chain CDR2 as described in the amino acid sequence of SEQ ID NO: 17, and light chain CDR3 as described in the amino acid sequence of SEQ ID NO: 18; iv) A heavy chain variable region including heavy chain CDR1 described by the amino acid sequence of SEQ ID NO: 19, heavy chain CDR2 described by the amino acid sequence of SEQ ID NO: 20, and heavy chain CDR3 described by the amino acid sequence of SEQ ID NO: 21; and an antibody or antigen-binding fragment including a light chain variable region including light chain CDR1 described by the amino acid sequence of SEQ ID NO: 22, light chain CDR2 described by the amino acid sequence of SEQ ID NO: 23, and light chain CDR3 described by the amino acid sequence of SEQ ID NO: 24; and v) A heavy chain variable region comprising a heavy chain CDR1 described by the amino acid sequence of SEQ ID NO: 25, a heavy chain CDR2 described by the amino acid sequence of SEQ ID NO: 26, and a heavy chain CDR3 described by the amino acid sequence of SEQ ID NO: 27; and a light chain variable region comprising a light chain CDR1 described by the amino acid sequence of SEQ ID NO: 28, a light chain CDR2 described by the amino acid sequence of SEQ ID NO: 29, and a light chain CDR3 described by the amino acid sequence of SEQ ID NO: 30, or an antigen-binding fragment.

[0023] In one embodiment of the present invention, the antibody or the antigen-binding fragment may be any one selected from the group consisting of, but not limited to: i) An antibody or antigen-binding fragment comprising a heavy chain variable region described by the amino acid sequence of SEQ ID NO: 63 and a light chain variable region described by the amino acid sequence of SEQ ID NO: 64; ii) An antibody or antigen-binding fragment comprising a heavy chain variable region described by the amino acid sequence of SEQ ID NO: 67 and a light chain variable region described by the amino acid sequence of SEQ ID NO: 68; iii) An antibody or antigen-binding fragment comprising a heavy chain variable region described by the amino acid sequence of SEQ ID NO: 71 and a light chain variable region described by the amino acid sequence of SEQ ID NO: 72; iv) An antibody or antigen-binding fragment comprising a heavy chain variable region described by the amino acid sequence of SEQ ID NO: 75 and a light chain variable region described by the amino acid sequence of SEQ ID NO: 76; and v) An antibody or antigen-binding fragment comprising a heavy chain variable region described by the amino acid sequence of SEQ ID NO: 79 and a light chain variable region described by the amino acid sequence of SEQ ID NO: 80.

[0024] In one embodiment of the present invention, the antibody or the antigen-binding fragment may comprise a heavy chain described by the amino acid sequence of SEQ ID NO: 83 and a light chain described by the amino acid sequence of SEQ ID NO: 84, but is not limited thereto.

[0025] In one embodiment of the present invention, the antibody or the antigen-binding fragment may comprise, but is not limited to, Fab.

[0026] In one embodiment of the present invention, the antibody or antigen-binding fragment may be an IgG1 or IgG4 subclass, but is not limited thereto.

[0027] In one embodiment of the present invention, the antigen-binding fragment may be any one selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabody, but is not limited thereto.

[0028] In one embodiment of the present invention, the antibodies or antigen-binding fragments described in iii), iv), and v) can specifically bind to DR3 competitively with TL1A, but are not limited thereto.

[0029] In one embodiment of the present invention, the antibody or antigen-binding fragment may be characterized by one or more of the following, but is not limited thereto: Selectively activate regulatory T cells or increase their survival rate; and Activates NF-κB.

[0030] In one embodiment of the present invention, the autoimmune disease may be one or more selected from the group consisting of graft-versus-host disease, hemophagocytic lymphohistiocytosis, systemic lupus erythematosus, Kikuchi disease, adult-onset Still's disease, Behcet's disease, IgG4-associated disease, type 1 diabetes mellitus, systemic sclerosis, psoriasis, multiple sclerosis, and Graves' hyperthyroidism, but is not limited thereto.

[0031] The present invention also provides a method for the prevention or treatment of graft-versus-host disease, autoimmune disease, or type 2 diabetes, comprising the step of administering an anti-DR3 antibody or its antigen-binding fragment, or a composition containing the same as an active ingredient, to an individual in need.

[0032] Furthermore, the present invention provides an anti-DR3 antibody or its antigen-binding fragment that binds to death receptor 3 (DR3), or a composition containing the same as an active ingredient, for use in the prevention or treatment of graft-versus-host disease, autoimmune diseases, or type 2 diabetes.

[0033] Furthermore, the invention provides applications for manufacturing graft-versus-host disease, autoimmune diseases, or type 2 diabetes mellitus formulations using anti-DR3 antibodies or antigen-binding fragments thereof that bind to death receptor 3 (DR3), or compositions containing these as active ingredients. [Effects of the Invention]

[0034] The antibody that specifically binds to human DR3 and its applications involve specifically binding to a specific epitope of human DR3, selectively acting on regulatory T cells, minimizing the activity of effector CD4+ T cells and CD8+ T cells, and potentially improving the division or survival rate of regulatory T cells. Due to this activity, it exhibits remarkablely superior preventive or therapeutic effects against related diseases, making it useful as a therapeutic agent for graft-versus-host disease, autoimmune diseases, or type 2 diabetes. [Brief explanation of the drawing]

[0035] [Figure 1] Figures 1(a) and 1(b) show experimental results of the process of discovering anti-DR3 scFv clones that specifically bind to DR3 via phage display. [Figure 2] Figures 2(a) to 2(c) show the binding specificity and affinity of anti-DR3 monoclonal antibodies to DR3. [Figure 3]Figures 3(a) and 3(b) show the results confirming that the binding site of the anti-DR3 4-4 antibody against DR3 is superimposed on the binding site of TL1A. [Figure 4] Figure 4 shows the NF-κB activation level of the anti-DR3 antibody of the present invention. [Figure 5] Figure 5 shows the results of comparing the affinity of anti-DR3 4-4 antibodies to DR3 based on IgG subclass. [Figure 6] Figure 6 shows the results of a comparative analysis of NF-κB activation levels of anti-DR3 4-4 antibodies based on IgG subclasses. [Figure 7] Figure 7 shows the effect of anti-DR34-4IgG4 antibody on increasing the survival rate of regulatory T cells in the absence of TCR signaling. [Figure 8] Figure 8 shows the effect of anti-DR3 4-4 IgG4 antibody on increasing the division and survival rate of regulatory T cells, as measured by TCR signal intensity. [Modes for carrying out the invention]

[0036] In this invention, we discovered an anti-DR3 antibody that binds to the natural form of DR3 using a stable cell line that fully displays human phage and expresses DR3. We confirmed that the anti-DR3 antibody of this invention can function as an agonist by activating T cells through competitive binding of TL1A to DR3 expressed by human T cells. Furthermore, we manufactured antibodies with different isotypes, such as IgG1, IgG2, and IgG4, from clone 4-4, which most strongly activates T cells, and compared the degree of T cell activation for each antibody isotype. We confirmed that the anti-DR3 IgG4 antibody specifically binds to the natural form of DR3 and activates T cells most strongly. Through this, we confirmed that the anti-DR3 4-4 IgG4 antibody, which was confirmed to be the most superior, increases the viability of regulatory T cells in the absence of TCR stimulation and effectively increases the division or viability of regulatory T cells when there is TCR signal stimulation of a certain intensity or higher, thereby completing the present invention.

[0037] The present invention provides an anti-DR3 antibody or its antigen-binding fragment that specifically binds to death receptor 3 (DR3).

[0038] In this invention, DR3 (TNFRSF25, member 25 of the TNF receptor superfamily, cell death receptor 3) is one of the tumor death factor receptor superfamily and is expressed at high levels by regulatory T cells, but can also be induced from pre-existing T cells upon TCR stimulation. In this invention, DR3 may include, but is not limited to, DR3, hDR3, etc. DR3 signaling can promote the activation and proliferation of regulatory T cells under normal conditions, as demonstrated in mice with agonist anti-DR3 antibodies, and can induce regulatory T cell proliferation with only small amounts without proliferation of non-target immune cell populations, which is a key difference from the IL-2 complex, which also promotes the division of CD4 and CD8 T cells. Therefore, since the division of conventional T cells (Tconv) can be a major obstacle to the treatment of various inflammatory diseases, anti-DR3 antibodies can be used as a novel method to selectively activate regulatory T cells.

[0039] In one embodiment of the present invention, the antibody or antigen-binding fragment may be any one selected from the group consisting of the following, but is not limited thereto: i) A heavy chain variable region containing heavy chain CDR1 as described by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 as described by the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 as described by the amino acid sequence of SEQ ID NO: 3; and an antibody or antigen-binding fragment containing a light chain variable region containing light chain CDR1 as described by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 as described by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 as described by the amino acid sequence of SEQ ID NO: 6; ii) A heavy chain variable region including heavy chain CDR1 described by the amino acid sequence of SEQ ID NO: 7, heavy chain CDR2 described by the amino acid sequence of SEQ ID NO: 8, and heavy chain CDR3 described by the amino acid sequence of SEQ ID NO: 9; and an antibody or antigen-binding fragment including a light chain variable region including light chain CDR1 described by the amino acid sequence of SEQ ID NO: 10, light chain CDR2 described by the amino acid sequence of SEQ ID NO: 11, and light chain CDR3 described by the amino acid sequence of SEQ ID NO: 12; iii) A heavy chain variable region including heavy chain CDR1 as described in the amino acid sequence of SEQ ID NO: 13, heavy chain CDR2 as described in the amino acid sequence of SEQ ID NO: 14, and heavy chain CDR3 as described in the amino acid sequence of SEQ ID NO: 15; and an antibody or antigen-binding fragment including a light chain variable region including light chain CDR1 as described in the amino acid sequence of SEQ ID NO: 16, light chain CDR2 as described in the amino acid sequence of SEQ ID NO: 17, and light chain CDR3 as described in the amino acid sequence of SEQ ID NO: 18; iv) A heavy chain variable region including heavy chain CDR1 described by the amino acid sequence of SEQ ID NO: 19, heavy chain CDR2 described by the amino acid sequence of SEQ ID NO: 20, and heavy chain CDR3 described by the amino acid sequence of SEQ ID NO: 21; and an antibody or antigen-binding fragment including a light chain variable region including light chain CDR1 described by the amino acid sequence of SEQ ID NO: 22, light chain CDR2 described by the amino acid sequence of SEQ ID NO: 23, and light chain CDR3 described by the amino acid sequence of SEQ ID NO: 24; and v) A heavy chain variable region containing heavy chain CDR1 as described by the amino acid sequence of SEQ ID NO: 25, heavy chain CDR2 as described by the amino acid sequence of SEQ ID NO: 26, and heavy chain CDR3 as described by the amino acid sequence of SEQ ID NO: 27; and an antibody or antigen-binding fragment containing a light chain variable region containing light chain CDR1 as described by the amino acid sequence of SEQ ID NO: 28, light chain CDR2 as described by the amino acid sequence of SEQ ID NO: 29, and light chain CDR3 as described by the amino acid sequence of SEQ ID NO: 30.

[0040] In one embodiment of the present invention, the antibody or the antigen-binding fragment may be selected from, but is not limited to, any one of the following groups: i) An antibody or antigen-binding fragment containing the heavy chain variable region described by the amino acid sequence of SEQ ID NO: 63 and the light chain variable region described by the amino acid sequence of SEQ ID NO: 64; ii) An antibody or antigen-binding fragment containing the heavy chain variable region described by the amino acid sequence of SEQ ID NO: 67 and the light chain variable region described by the amino acid sequence of SEQ ID NO: 68; iii) An antibody or antigen-binding fragment containing the heavy chain variable region described by the amino acid sequence of SEQ ID NO: 71 and the light chain variable region described by the amino acid sequence of SEQ ID NO: 72; iv) An antibody or antigen-binding fragment containing the heavy chain variable region described by the amino acid sequence of SEQ ID NO: 75 and the light chain variable region described by the amino acid sequence of SEQ ID NO: 76; and v) An antibody or antigen-binding fragment containing a heavy chain variable region described by the amino acid sequence of SEQ ID NO: 79 and a light chain variable region described by the amino acid sequence of SEQ ID NO: 80.

[0041] The terms "percent identity," "sequence identity," "percent similarity," "sequence similarity," and "percent sequence identity" used in this application in relation to amino acid sequences and / or nucleic acid sequences may refer to, but are not limited to, a measure that maximizes the similarity between aligned amino acid residues or nucleotides and is determined by comparing the degree of similarity between two sequences based on the number of identical or similar residues or nucleotides, the total number of residues or nucleotides, and sequence alignment as a function of the presence and length of gaps in sequence alignment.

[0042] A portion of a polynucleotide or polypeptide sequence may contain additions or additions (i.e., gaps) compared to a reference sequence (which does not contain additions or additions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions in which the same nucleic acid base or amino acid residue appears in both sequences, calculating the number of matching positions, dividing the number of matching positions by the total number of positions on the comparison screen, and multiplying the result by 100 to calculate the sequence identity percentage.

[0043] In relation to two or more nucleic acid or polypeptide sequences, the terms “identical” or percentage “identical” refer to two or more sequences or subsequences that have the same or identical percentage of embodied amino acid residues or nucleotides (i.e., approximately 60% identity with respect to an embodied region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity when compared and aligned for maximum correspondence to a comparison screen or displayed region) (see, for example, the NCBI website http: / / www.ncbi.nlm.nih.gov / BLAST / ). Such sequences are also referred to as “substantially identical.”

[0044] In the present invention, the amino acid sequence or nucleotide sequence may include a nucleotide sequence having 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more sequence homology with the amino acid sequence or nucleotide sequence indicated by each sequence number. For example, it may include, but is not limited to, sequences having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence homology.

[0045] In this invention, the naturally occurring "antibody" is a glycoprotein containing at least two heavy chains (H) and two light chains (L) linked by disulfide bonds between the chains. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain invariant region. The heavy chain invariant region consists of three domains CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain invariant region. The light chain invariant region consists of the domain CL.

[0046] The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), separated by more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.

[0047] In the present invention, antibodies may be derived from a variety of species, including but are not limited to mice, rats, rabbits, and guinea pigs. The present invention also includes chimeric antibodies having an antibody-invariant region from one species (preferably) that possesses an antigen-binding site from another species. Furthermore, the antibodies may include antibodies having an antigen-binding site from a non-human species that possesses an origin-invariant region and a framework region. The antibodies described herein may be monoclonal antibodies, i.e., antibody molecules having a single molecular composition, but are not limited to this. Systems, immunological operations, and techniques for producing and isolating hybridomas that secrete monoclonal antibodies are well known in the art.

[0048] In this invention, “antigen” refers to a substance containing a binding site (epitope) that induces and / or produces an immune response to the epitope. Antigens may include, but are not limited to, peptides, proteins, glycoproteins, polysaccharides, and lipids, their portions, and combinations thereof. Non-restrictive exemplary antigens include tumor antigens or pathogenic antigens. The term “antigen” may also refer to a molecule that elicits an immune response. Such immune responses may include antibody production or activation of specific immunologically active cells. It should be understood by those skilled in the art that virtually any macromolecule, including all proteins or peptides, can be used as an antigen.

[0049] In this invention, “binding site” or “epitope” may refer to an antigen-deterministic cluster in a molecule, i.e., a portion of the molecule recognized by the immune system (e.g., by an antibody), but is not limited thereto. In this invention, “epitope” may mean a portion to which DR3 and TL1A or DR3 and an anti-DR3 antibody bind, but is not limited thereto.

[0050] In one embodiment of the present invention, the antibody or antigen-binding fragment may include, but is not limited to, the heavy chain described by the amino acid sequence of SEQ ID NO: 83 and the light chain described by the amino acid sequence of SEQ ID NO: 84.

[0051] As a result of manufacturing the 4-4 IgF4 antibody, which has been confirmed to be the most effective in the present invention, for application to graft-versus-host disease, autoimmune diseases, or type 2 diabetes, in one embodiment of the present invention, the antibody or the antigen-binding fragment may, but is not limited to, contain Fab.

[0052] Therapeutic antibodies are typically immunoglobulin G (IgG), which is divided into four subclasses (IgG1, IgG2, IgG3, and IgG4). Since each IgG subclass has a different effector function, it is important to select the appropriate IgG subclass based on the indication for the therapeutic antibody. In this laboratory, we confirmed that not only the effector function but also the antibody's binding affinity and activation function to the target antigen differ depending on the IgG class. Furthermore, the strength of binding affinity may vary depending on the IgG subclass and the type of antigen-antibody combination.

[0053] As a result of confirming the most functionally superior ISOTYPE compared to 4-4, which was confirmed to be the most effective in the present invention, in one embodiment of the present invention, the antibody or the antigen-binding fragment may be an IgG4 subclass, but is not limited thereto.

[0054] In one embodiment of the present invention, the antibody or antigen-binding fragment may be an IgG1 or IgG4 subclass, but is not limited thereto.

[0055] In the present invention, "antigen-binding fragment" refers to one or more fragments of an antibody that possesses specific antigen-binding ability, for example, (i) a monovalent fragment composed of a Fab fragment, VL, VH, CL, and CH domains; (ii) a bivalent fragment containing an F(ab')2 fragment and two Fab fragments linked by a disulfide bond in the hinge region; (iii) an Fd fragment composed of VH and CH domains; (iv) an Fv fragment composed of the VL and VH domains of a single arm of the antibody; (v) a single-domain antibody fragment composed of a VH domain; (vi) a conjugate fragment containing a combination of two or more isolated CDRs selectively linked by a synthetic linker. This also includes a single-chain antibody (or single-chain Fv(scFv)) formed by using a recombinant method to link the two domains VL and VH of the FV segment by a synthetic linker.

[0056] In one embodiment of the present invention, the antigen-binding fragment may be any one selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabody, but is not limited thereto.

[0057] In this invention, in order to develop an agonistic anti-DR3 antibody for activating the target antigen human DR3, we discovered an anti-DR3 antibody having a binding site that overlaps with TL1A, a ligand that activates human DR3, through epitope mapping. The antibody or antigen-binding fragment of the present invention may have the same or overlapping epitopes that bind to TL1A and DR3. In this case, it can competitively and specifically bind to DR3 and induce regulatory T cell activity, etc., but is not limited to this. Furthermore, if the epitopes that bind to TL1A and DR3 do not overlap, it can bind to DR3 together with TL1A and activate DR3 effectively even non-competitively. Therefore, the anti-DR3 antibody of the present invention can exhibit preventive or therapeutic effects on graft-versus-host disease, autoimmune diseases, or type 2 diabetes by binding to DR3 competitively and / or non-competitively with TL1A and activating the function of DR3.

[0058] In one embodiment of the present invention, the antibodies or antigen-binding fragments described in iii), iv), and v) can specifically bind to DR3 competitively with TL1A, but are not limited thereto. That is, the binding sites (epitopes) of the antibodies or antigen-binding fragments described in iii), iv), and v) to DR3 can overlap with the binding sites of TL1A to DR3, but are not limited thereto.

[0059] In one embodiment of the present invention, the antibody or antigen-binding fragment may be characterized by one or more of the following, but is not limited thereto: Selectively activate regulatory T cells or increase their survival rate; and Activates NF-κB.

[0060] In the present invention, the anti-DR3 antibody can exhibit an activation efficiency of approximately 10% to 80% for CD3+CD4+NF-κB depending on the concentration, and an activation efficiency of approximately 1% to 60% for CD3+CD8+NF-κB, but is not limited thereto. In particular, the anti-DR3 4-4 IgG1 antibody of the present invention can increase the activation efficiency of NF-κB cells by inducing phosphorylation processes of up to approximately 80% and 60% for CD3+CD4+NF-κB and CD3+CD8+NF-κB, respectively, depending on the antibody treatment concentration, but is not limited thereto. In the present invention, the anti-DR3 antibody is CD3 + CD4 + The anti-DR3 antibody can exhibit activation efficiencies of 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, and 81% for NF-κB, and preferably 80% for 10, but is not limited thereto. + CD8 + For NF-κB, activation efficiencies of 100% (exceeding 0), 95% (exceeding 0), 90% (exceeding 0), 85% (exceeding 0), 80% (exceeding 0), 75% (exceeding 0), 70% (exceeding 0), 69% (exceeding 0), 68% (exceeding 0), 67% (exceeding 0), 66% (exceeding 0), 65% (exceeding 0), 64% (exceeding 0), 63% (exceeding 0), 62% (exceeding 0), and 61% (exceeding 0) can be observed. Preferably, it may be 60% (exceeding 0), but it is not limited to this.

[0061] In this invention, anti-DR3 antibodies can induce potent expansion of regulatory T cells in vivo. For example, the number and percentage of regulatory T cells can increase in lymph nodes and the spleen, and this may occur selectively for regulatory T cells, but is not limited to this.

[0062] In this invention, the anti-DR3 antibody exhibits superior binding affinity, with a significantly lower binding-dissociation constant to DR3 compared to TL1A, which is a ligand for DR3. The anti-DR3 antibody of the present invention is approximately 2 times stronger than 10,000 times stronger than TL1A, approximately 2 times stronger than 9,800 times stronger than 9,600 times stronger than 9,400 times stronger than 9,200 times stronger than 9,200 times stronger than 9,200 times stronger than 10,0 It is possible to exhibit bonding strengths of 9800 times, approximately 9600 times (more than approximately 8 times), approximately 9400 times (more than approximately 8 times), approximately 9200 times (more than approximately 8 times), approximately 10000 times (more than approximately 10 times), approximately 9800 times (more than approximately 10 times), approximately 9600 times (more than approximately 10 times), approximately 9400 times (more than approximately 10 times), approximately 9200 times (more than approximately 10 times), approximately 10000 times (more than approximately 12 times), approximately 9800 times (more than approximately 12 times), approximately 9600 times (more than approximately 12 times), and approximately 9400 times (more than approximately 12 times). Preferably, it is possible to exhibit bonding strengths of approximately 9200 times or more (more than approximately 12 times), but it is not limited thereto.

[0063] The present invention provides a nucleic acid sequence for encoding the antibody or antigen-binding fragment.

[0064] In this invention, "genome" may mean the genetic material of an organism (e.g., chromosomes), and "nucleic acid sequence" or "gene" may mean, but are not limited to, a nucleic acid (e.g., DNA or RNA) sequence containing an encrypted sequence necessary for the production of polypeptides or precursors (e.g., proinsulin).

[0065] The “nucleotide sequence” or “base sequence” of the present invention refers to a nucleotide sequence (e.g., RNA or DNA) whose manipulation is considered preferable for a predetermined reason (e.g., treating or improving a disease, expressing the protein of the present invention in a host cell, or conferring the expression of a ribozyme) by one of the ordinary art. Such nucleotide sequences may include, but are not limited to, encoded sequences of structural genes (e.g., reporter genes, selection marker genes, oncogenes, drug resistance genes, growth factors, etc.) and unencoded regulatory sequences that do not encode mRNA or protein products (e.g., promoter sequences, polyadenylation sequences, termination sequences, enhancer sequences, etc.).

[0066] The present invention provides a vector containing the nucleic acid sequence.

[0067] The "vector" of this invention refers to a genetic element such as a plasmid, phage, transposon, cosmid, chromosome, virus, or virion, which can replicate when ligated with appropriate regulatory elements and can move gene sequences between cells, but is not limited to these.

[0068] In this invention, “recombinant expression vector” means a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. Generally, any plasmid and vector can be used as long as it can replicate and stabilize within the host.

[0069] The recombinant expression vector and the expression vector containing the appropriate transcription / translation regulatory signal can be constructed by methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombinant techniques.

[0070] Furthermore, both viral and non-viral vectors can be used for recombination. For example, viral vectors can include, but are not limited to, lentivirus, retrovirus, adenovirus, herpesvirus, or avipoxvirus vectors.

[0071] Furthermore, the recombinant protein may include, but is not limited to, additional genes such as tagging genes to increase the production of recombinant proteins, tagging genes to maintain the structural stability of recombinant proteins, tagging genes to facilitate the isolation of recombinant proteins, and selection marker genes such as antibiotic resistance genes for selecting transformants.

[0072] The present invention provides a transformant transformed with the aforementioned vector.

[0073] In this invention, "transformation" is a general term for the alteration of an organism's genetic properties due to injected DNA, and "transgenic organism" is a living organism produced by injecting external genes using molecular genetic methods, preferably a cell or living organism transformed by the recombinant expression vector of the present invention, but is not limited thereto.

[0074] In certain cases, the aforementioned living organisms may include, without limitation, any living organism such as microorganisms, eukaryotic cells, insects, animals, and plants, and are preferably, but not limited to, intestinal bacteria, Salmonella, Bacillus, yeast, animal cells, mice, rats, dogs, monkeys, pigs, horses, cattle, Agrobacterium tumefaciens, and plants.

[0075] The transformants may be produced by methods such as transformation, transfection, Agrobacterium-mediated transformation, particle gun bombardment, sonication, electroporation, and PEG (polyethylene glycol)-mediated transformation, but are not limited thereto. They may also be introduced into cells together with a transmission reagent containing G-fectin, Mirus TransIT-TKO lipid affinity reagent, lipofectin, lipofectamine, cellfectin, cationic phospholipid nanoparticles, cationic polymers, cationic micelles, cationic emulsions, or liposomes, or their intracellular absorption may be increased by conjugating them with biocompatible polymers such as polyethylene glycol, but are not limited thereto.

[0076] When transforming eukaryotic cells with the vector of the present invention, the host cell may be yeast (Saccharomyce cerevisiae), insect cells, human cells (e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN, and MDCK cell lines), or plant cells, and preferably, Agrobacterium may be used.

[0077] The vector of the present invention can be delivered into host cells by methods such as the CaCl2 method, the Hanahan method (Hanahan, D, J Mol Biol, 166:557-580 (1983)), and electroporation when the host cell is a prokaryotic cell. When the host cell is a eukaryotic cell, the vector can be injected into the host cell by methods such as microinjection, calcium phosphate precipitation, electroporation, liposome-mediated phenotypic infection, DEAE-dextran treatment, and gene bombardment.

[0078] The present invention provides a pharmaceutical composition for the prevention or treatment of transplantation host disease, autoimmune disease, or type 2 diabetes, comprising an anti-DR3 antibody or its antigen-binding fragment as an active ingredient, which specifically binds to the aforementioned cell death receptor 3 (DR3).

[0079] In one embodiment of the present invention, the antibody or antigen-binding fragment may include, but is not limited to, the heavy chain described by the amino acid sequence of SEQ ID NO: 83 and the light chain described by the amino acid sequence of SEQ ID NO: 84.

[0080] In one embodiment of the present invention, the autoimmune disease may be one or more selected from the group consisting of graft-versus-host disease, hemophagocytic lymphohistiocytosis, systemic lupus erythematosus, Kikuchi disease, adult-onset Still's disease, Behcet's disease, IgG4-associated disease, type 1 diabetes mellitus, systemic sclerosis, psoriasis, multiple sclerosis, and Graves' hyperthyroidism, but is not limited thereto.

[0081] The "pharmaceutical composition" according to the present invention may further include suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. The excipient may be one or more selected from the group consisting of, for example, diluents, binders, disintegrants, lubricants, adsorbents, humectants, film-coating substances, and controlled-release additives.

[0082] The pharmaceutical compositions according to the present invention may be used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, elsilics, emulsions, suspensions, alcoholic preparations, lozenges, aromatic preparations, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric-coated capsules, pills, tinctures, softened extracts, dried extracts, liquid extracts, injections, capsules, perfusion solutions, ointments, lotions, pastes, sprays, inhalants, patches, sterile injections, or aerosols, respectively, by conventional methods. The external preparations may also have dosage forms such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasms.

[0083] Examples of carriers, excipients, and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0084] When formulating, the product is typically prepared using fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, or other diluents or excipients.

[0085] Excipients for tablets, powders, granules, capsules, pills, and lozenges according to the present invention include: corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, monocalcium phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, Primozel, etc.; gelatin, gum arabic, ethanol, agar powder, cellulose phthalate acetate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium casein Binders such as ammonium bicarbonate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, purified shellac, starch paste, hydroxypropylcellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, and polyvinylpyrrolidone may be used; and disintegrants such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, calcium carboxymethylcellulose, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropylcellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, acacia gum, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, D-sorbitol solution, and hard anhydrous silicic acid;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium powder, kaolin, petrolatum, sodium stearate, cocoa butter, sodium salicylate, magnesium salicylate, polyethylene glycol 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and hard anhydrous silicic acid may be used.

[0086] Possible additives for the liquid formulation according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, and the like.

[0087] The syrup according to the present invention may contain a solution of sucrose, other sugars, or sweeteners, and may, if necessary, contain fragrances, colorants, preservatives, stabilizers, suspending agents, emulsifiers, viscosity modifiers, etc.

[0088] Purified water may be used in the emulsion of the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0089] The suspending agent according to the present invention may use suspending agents such as acacia, tragacantha, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropyl methylcellulose, HPMC1828, HPMC2906, HPMC2910, and, if necessary, surfactants, preservatives, stabilizers, colorants, and fragrances may be used.

[0090] The injectable preparations according to the present invention include solvents such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactating Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; solubilizers such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidinedione, propylene glycol, twins, nijonthinamide, hexamine, and dimethylacetamide; weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), and organic compounds. It may contain buffering agents such as compounds, proteins, albumin, peptones, and gums; isotonic agents such as sodium chloride; stabilizers such as sodium sulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; sulfurizing agents such as 0.1% sodium bisulfide, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, and sodium acetone bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium CMC, sodium alginate, Twin 80, and aluminum monostearate.

[0091] The suppositories according to the present invention contain cocoa butter, lanolin, vitepsol, polyethylene glycol, glycerol gelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, coconut oil, cocoa butter + cholesterol, lecithin, lanette wax, glycerol monostearate, twin or span, Imhausen, monolen (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, and Cebes Pharma 16. 16) Hexalide Base 95, Cotomar, Hydrocoat SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydrocoat 25, Hydrocoat 711, Idropostal, Massa Estralium Bases such as estrarium (A, AS, B, C, D, E, I, T), Masa-MF, Maspol, Maspol-15, Neospostal-en, Paramount-B, Sposyl (OSI, OSIX, A, B, C, D, H, L), Suppository base type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Spostal (N, Es), Wecobi (W, R, S, M, Fs), and Tezestr triglyceride base (TG-95, MA, 57) may be used.

[0092] Solid preparations for oral administration include tablets, pills, powders, granules, and capsules. Such solid preparations are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose, or lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium styrate talc are also used.

[0093] Liquid formulations for oral administration include suspensions, liquid preparations, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients may be included, such as humectants, sweeteners, fragrances, and preservatives. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspension solvents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0094] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined by factors including the type and severity of the patient’s disease, the activity of the drug, the patient’s sensitivity to the drug, the time of administration, the route of administration and elimination ratio, the duration of treatment, drugs used concurrently, and other factors well known in the medical field.

[0095] The pharmaceutical compositions according to the present invention may be administered as individual therapeutic agents, in combination with other therapeutic agents, sequentially or simultaneously with conventional therapeutic agents, or administered as a single or multiple agent. Considering all of the above factors, it is important to administer an amount that provides the greatest effect with the minimum amount without side effects, which can be easily determined by a person of the skill in the art to which the present invention belongs.

[0096] The pharmaceutical compositions of the present invention can be administered to individuals by various routes. All possible methods of administration are predictable, but for example, they may be administered orally, by subcutaneous injection, intraperitoneal injection, intravenous injection, intramuscular injection, intradural injection, sublingual injection, buccal injection, rectal insertion, vaginal insertion, ocular injection, ear injection, nasal injection, inhalation, spraying through the mouth or nose, skin administration, transdermal administration, etc.

[0097] The pharmaceutical composition of the present invention is determined by the type of drug that is the active ingredient, along with various related factors such as the disease being treated, the route of administration, the patient's age, sex, weight, and the severity of the disease. Specifically, the effective amount of the composition according to the present invention can vary depending on the patient's age, sex, and weight, and is generally 0.001 to 150 mg per kg of body weight, preferably 0.01 to 100 mg, administered daily or every other day, or divided into 1 to 3 doses per day. However, the dosage can be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, etc., so the aforementioned dosage does not limit the scope of the present invention in any way.

[0098] In this invention, "individual" means a subject requiring treatment for a disease, and more specifically, it means a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow.

[0099] In the present invention, “administration” means providing a predetermined composition of the present invention to an individual by any suitable method. In the present invention, “prevention” means all actions that suppress or delay the onset of the disease of interest; “treatment” means all actions by which the disease of interest and its associated metabolic abnormalities are improved or favorably altered by the administration of a pharmaceutical composition according to the present invention; and “improvement” means all actions by which the parameters associated with the disease of interest, such as the severity of symptoms, are reduced by the administration of a composition according to the present invention.

[0100] The present invention provides a kit for the prevention or treatment of graft-versus-host disease, autoimmune disease, or type 2 diabetes, comprising the vector or composition and a manual.

[0101] The "kit" of the present invention may also include, in addition to the above-described components, a method for expressing the vector of the present invention, other components, apparatus, and substances that are normally necessary for the storage, management, and enhancement of the composition of the present invention. Furthermore, all components included in the kit can be used one or more times without limitation, there is no restriction on the order in which each substance is used, and the application of each substance may proceed simultaneously or at the end.

[0102] The kit of the present invention may include a container in addition to the formulation and manual. The container may serve to package the components, or to store and secure them. The material of the container may take the form of, for example, a bottle, tube, sachet, envelope, tube, ampoule, etc., which may be formed in part or all from plastic, glass, paper, foil, wax, etc. The container may be fitted with a stopper that is initially part of the container or can be attached to the container by mechanical, adhesive or other means, which can be completely or partially detachable, and may also be fitted with a stopper that allows access to the contents with an injection needle. The kit may include an external package, which may, but is not limited to, instructions on how to use the components. <Modes for carrying out the invention>

[0103] The following are preferred embodiments to aid in understanding the present invention. However, the following embodiments are provided only to facilitate understanding of the present invention, and the scope of the present invention is not limited by these embodiments. [Examples]

[0104] <Example 1. Discovery of anti-DR3 clones using a phage display> We used phage display to identify clones that specifically bind to DR3. First, we selectively amplified scFv clones that bind to DR3 through three panning steps using a human scFv library and recombinant DR3. At each stage, we confirmed the amplification of scFv clones that bind to recombinant DR3 by ELISA using the phage obtained after panning. Subsequently, we infected the single clones identified by colony PCR with phage, confirmed the single clones that bind to DR3 by ELISA, and identified the CDR region of each single clone by sequencing.

[0105] As a result, five clones that bind to DR3 were identified (Figure 1(a) and Figure 1(b)), and the five antibodies were named 2-10, 3-18, 4-4, 6-2, and 6-15, respectively. The five antibodies were sequenced to obtain the complementarity-determining regions (CDRs) of each clone. Tables 1 and 2 show the amino acid and nucleotide sequences of CDR1-CDR3 in the heavy chain and variable regions of each antibody. Table 3 shows the nucleotide and amino acid sequences of the heavy chain and variable regions of each antibody. In each table, bold text indicates the CDR sequence by IMGT, and underlined text indicates the CDR sequence by KABAT.

[0106] [Table 1]

[0107] [Table 2]

[0108] [Table 3-1]

[0109] [Table 3-2]

[0110] [Table 3-3]

[0111] <Example 2. Confirmation of the excellent DR3-specific binding affinity of five antibodies> The specific binding affinity of the five antibodies developed in Example 1 to DR3 was confirmed through ELISA, SPR, and FACS experiments.

[0112] Example 2-1. Confirmation of specific binding affinity to DR3 using ELISA. ELISA was performed using the monoclonal antibody from the clone selected in Example 1. Specifically, 0.4 μg / ml of DR3-His tag was added to PBS and mixed, and the total 50 μl of the mixture was coated onto a Ni plate. Then, to prevent non-specific binding, the plate was treated with PBS containing 200 μl of 5% FBS and incubated at room temperature for 30 minutes, and the monoclonal antibody concentration was titrated and incubated for another 30 minutes. The degree of binding between the coated DR3 and the monoclonal antibody was detected with anti-human IgG Fc HRP. After each step, the plate was washed five times with 250 μl of 0.1% Tween 20. An isotype control was used as the negative control group.

[0113] As a result, the five antibodies of the present invention exhibited superior specific binding affinity to DR3 compared to the control group, and among them, antibodies 4-4, 6-2, and 6-15 were confirmed to maintain remarkably high binding specificity to DR3 even at low antibody concentrations (Figure 2(a)).

[0114] Example 2-2. Confirmation of specific binding affinity to DR3 using SPR. SPR was carried out using the monoclonal antibody of the clone selected in Example 1. After immobilizing a 10 μg / ml DR3-His tag on a CM5 chip, binding specificity and binding strength were measured using a Biacore T100 under conditions of a contact time of 30 sec and a flow rate of 30 μl / min.

[0115] As a result, it was confirmed that the five antibodies from Example 1 had significantly lower binding-dissociation constants for DR3 compared to TL1A, a DR3 agonist, exhibiting a binding affinity of approximately 12 times and over 9200 times (Figure 2(b)).

[0116] Example 2-3. Confirmation of specific binding affinity to DR3 using FACS. FACS was performed using the monoclonal antibody of the clone selected in Example 1. Specifically, 0.2 x 10⁻¹⁰ 6 The DR3 stable cell line was mixed with 2 μg / ml of monoclonal antibody and incubated at 4°C for 30 minutes. The degree of binding between the DR3 stable cell line and the monoclonal antibody was confirmed by FACS analysis using anti-human IgG PE. Cells were washed once with 200 μl of PBS at each stage. An isotype control was used as the negative control group.

[0117] As a result, it was confirmed that antibodies 4-4, 6-2, and 6-15 strongly bind to DR3 (Figure 2(c)).

[0118] <Example 3. Epitope mapping of 5 antibodies against DR3> Example 3-1. Epitope mapping using ELISA ELISA was performed to confirm whether the rDR3 epitope to which the anti-DR3 4-4 antibody from Example 1 binds superimposed on TL1A, the ligand for rDR3, or other monoclonal antibodies. Specifically, 0.4 μg / ml of DR3-His tag was added to PBS and mixed, and then 50 μl of the mixture was coated onto a Ni plate. Subsequently, to prevent non-specific binding, the mixture was mixed with 200 μl of PBS composed of 5% FBS and incubated at room temperature for 30 minutes. The concentrations of TL1A and the monoclonal antibody were then titrated and incubated for another 30 minutes. After that, 200 ng / ml of biotinylated anti-DR3 4-4 antibody was incubated for 30 minutes without washing, and the degree of binding between DR3 and the biotinylated anti-DR3 4-4 antibody was detected using anti-human IgG Fc HRP. Except for the stage without washing, the plate was washed five times with 250 μl 0.1% Tween 20 after each stage. An isotype control was used for the negative control group.

[0119] Example 3-2. Epitope mapping using FACS FACS was performed to confirm whether the epitopes to which five selected antibodies bind to DR3 expressed on the cell surface are superimposed on TL1A, the ligand for human DR3, or other monoclonal antibodies. 0.2X10 6 The DR3 stable cell line was mixed with 2 μg / ml monoclonal antibody and incubated at 4°C for 30 minutes. Then, without washing, 0.625 μg / ml TL1A His tag was mixed in and incubated at 4°C for 30 minutes. The degree of binding between the DR3 stable cell line and the TL1A-His tag was detected using anti-His tag AF647 and confirmed by FACS. Except for the step without washing, the cells were washed once with 200 μl PBS after each step. An isotype control was used as the negative control group.

[0120] As a result, it was confirmed that clones 4-4, 6-2, and 6-15 bind to the epitope to which DR3 and TL1A bind, while clones 2-10 and 3-18 do not bind to the same epitope (Figures 3(a) and 3(b)).

[0121] <Example 4. Confirmation of excellent NF-κB activity effect by selected antibodies> To confirm the NF-κB activity of the antibodies selected in Example 1, phosphorylated NF-κB (pNF-κB) levels were compared and analyzed by FACS. Specifically, clone4-4 IgG1, clone2-10 IgG1, clone3-18 IgG1, and clone6-2 IgG1 monoclonal antibodies were activated with rhTL1A. 1X10 6 hPBMCs were mixed with rhTL1A-His tagged or titrated monoclonal antibody, and then incubated at 37°C for 10 minutes. The activation level of NF-κB (pNF-κB) was analyzed using FACS with anti-human phospho-NF-κB p65(Ser529)eFlour 660.

[0122] As a result, it was confirmed that 4-4(IgG1) produced the highest level of NF-κB activation, followed by 6-2. This was investigated to be because the site on which the antibody binds to DR3 overlaps with TL1A, strongly phosphorylating NF-κB (Figure 4).

[0123] <Example 5. Comparison of binding strength by IgG subclass of Clone4-4> The functionally superior subclass of the anti-DR3 4-4 antibody, which was confirmed to be the best in Example 2, was analyzed. To this end, clone 4-4 IgG1, clone 4-4 IgG2, and clone 4-4 IgG4 monoclonal antibodies were prepared by classifying the anti-DR3 4-4 antibody into IgG subclasses, and ELISA was performed to compare their binding affinity to DR3. Specifically, 0.4 μg / ml of DR3-His tag was mixed in PBS, and a total of 50 μl of the mixture was coated onto a Ni plate. Then, to prevent non-specific binding, the mixture was mixed with 200 μl of PBS consisting of 5% FBS and incubated at room temperature for 30 minutes, followed by titration of the monoclonal antibody and incubation for another 30 minutes. The degree of binding between the coated DR3 and the monoclonal antibody was detected with anti-human IgG Fc HRP. After each step, the plate was washed five times with 250 μl of 0.1% Tween 20.

[0124] As a result, it was confirmed that the anti-DR3 4-4 antibody exhibited superior affinity for DR3 in the order of IgG1, IgG4, and IgG2 subclasses (Figure 5).

[0125] <Example 6. Confirmation of excellent NF-κB activity effect by Clone4-4 IgG subclass> To compare NF-κB activation levels by IgG subclasses of Clone4-4, FACS was performed after stimulation with clone4-4 IgG1, clone4-4 IgG2, and clone4-4 IgG4 monoclonal antibodies. Specifically, 1X10 6 hPBMCs were mixed with rhTL1A-His tagged or titrated monoclonal antibody, and then incubated at 37°C for 10 minutes. NF-κB activation levels were analyzed using FACS with anti-human phospho-NF-κB p65(Ser529)eFlour 660.

[0126] As a result, it was confirmed that for antibody 4-4, the NF-κB activation levels were high in the order of IgG4, IgG1, and IgG2 subclasses (Figure 6).

[0127] <Example 7. Effect of anti-DR3 4-4 IgG4 antibody on increasing the survival rate of regulatory T cells without TCR signal stimulation> To confirm whether the anti-DR3 4-4 IgG4 antibody, which was confirmed to be the most excellent in Example 6, can increase the survival rate of human regulatory T cells when there is no TCR signal stimulation, CD4 T cell culture was performed using the anti-DR3 4-4 IgG4 antibody. Specifically, after separating CD4 T cells from hPBMC using the Human CD4 T cell Enrichment Kit, 20 ng / ml of IL-2, 15 ng / ml of TL1A, or 1 μg / ml of the anti-DR3 4-4 IgG4 antibody was added and cultured for 3 days, followed by analysis through FACS.

[0128] As a result, it was confirmed that in an environment without TCR signal stimulation, the anti-DR3 4-4 IgG4 antibody can increase the survival rate of regulatory T cells at a level similar to that of the DR3 ligand TL1A (Figure 7).

[0129] <Example 8. Effect of anti-DR3 4-4 IgG4 antibody on promoting the division and increasing the survival rate of regulatory T cells depending on TCR signal intensity> To confirm the degree of promotion of the division of human regulatory T cells by the anti-DR3 4-4 IgG4 antibody depending on the intensity of TCR signal stimulation for the anti-DR3 4-4 IgG4 antibody, which was confirmed to be the most excellent in Example 6, CD4 T cell culture was performed using the anti-hCD3 antibody and the anti-DR3 4-4 IgG4 antibody. Specifically, one day before cell culture, 1.5 μg / ml, 0.5 μg / ml, 0.25 μg / ml, 0.125 μg / ml, and 0 μg / ml of the anti-hCD3ε antibody were respectively coated on a 96-well cell culture plate. Then, on the day of cell culture, CD4 T cells were separated from hPBMC using the Human CD4 T Cell Enrichment Kit. After that, 0.1×10 6CD4 T cells were cultured and incubated for 6 days with 20 ng / ml of IL-2, 0.5 ng / ml of TGF-β, or anti-DR3 4-4 IgG4 antibody, according to the group's needs, and then analyzed by FACS.

[0130] As a result, we confirmed that when TCR signaling stimulation of a certain intensity or higher was applied, the anti-DR3 4-4 IgG4 antibody effectively increased the division or survival rate of regulatory T cells (Figure 8).

[0131] <Example 9. Sequencing of anti-DR3 4-4 IgG4 monoclonal antibody> For application in the treatment of graft-versus-host disease, autoimmune diseases, and type 2 diabetes, the anti-DR3 4-4 IgG4 monoclonal antibody that demonstrated the best function in Examples 3-8 was sequenced. Specifically, based on sequence analysis data, the Fab region of the 4-4 mAb was cloned into an IgG4 expression vector, and the DNA and amino acid sequences of the cloned vector are shown in Table 4. Table 4 shows the DNA and amino acid sequences of the complete 4-4 (fully human 4-4).

[0132] [Table 4]

[0133] The above description of the present invention is illustrative, and any person with ordinary skill in the art to which the invention pertains can easily modify it into other specific forms without altering the technical idea or essential features of the invention. Therefore, the above-described embodiments should be understood to be illustrative and not limiting in all respects. [Industrial applicability]

[0134] Antibodies that specifically bind to human DR3 specifically bind to the epitope to which the TL1A ligand of human DR3 binds, selectively acting on regulatory T cells to minimize the activity of effector CD4+ T cells and CD8+ T cells, while also improving the division or survival rate of regulatory T cells. Therefore, they can be usefully utilized as therapeutic agents for graft-versus-host disease, autoimmune diseases, or type 2 diabetes, and thus have industrial applicability.

Claims

1. An anti-DR3 antibody or its antigen-binding fragment, characterized by its specific binding to the cell death receptor 3 (DR3), The antibody or antigen-binding fragment comprises a heavy chain variable region including a heavy chain CDR1 described by the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 described by the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 described by the amino acid sequence of SEQ ID NO: 3; and a light chain variable region including a light chain CDR1 described by the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 described by the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 described by the amino acid sequence of SEQ ID NO: 6, and is an anti-DR3 antibody or its antigen-binding fragment.

2. The anti-DR3 antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region described by the amino acid sequence of SEQ ID NO: 63 and a light chain variable region described by the amino acid sequence of SEQ ID NO:

64.

3. The anti-DR3 antibody or antigen-binding fragment according to claim 1, characterized in that the antibody or antigen-binding fragment comprises Fab.

4. The anti-DR3 antibody or antigen-binding fragment according to claim 1, characterized in that the antibody or antigen-binding fragment is an IgG1 or IgG4 subclass.

5. The anti-DR3 antibody or antigen-binding fragment according to claim 1, characterized in that the antigen-binding fragment is one selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabody.

6. The anti-DR3 antibody or antigen-binding fragment according to claim 1 is characterized by having one or more of the following features: Selectively activate regulatory T cells or increase their survival rate; and Activates NF-κB.

7. A nucleic acid characterized by encoding the antibody or antigen-binding fragment described in claim 1.

8. A vector characterized by comprising the nucleic acid described in Claim 7.

9. A transformant characterized by being transformed into the vector described in Claim 8.

10. A pharmaceutical composition for the prevention or treatment of graft-versus-host disease, autoimmune disease, or type 2 diabetes, characterized by comprising, as an active ingredient, an anti-DR3 antibody or an antigen-binding fragment thereof that specifically binds to the cell death receptor 3 (DR3) described in claim 1.

11. The aforementioned autoimmune diseases include graft-versus-host disease, hemophagocytic lymphohistiocytosis, systemic lupus erythematosus, Kikuchi disease, adult-onset Still's disease, Behcet's disease, IgG4-associated disease, type 1 diabetes, systemic sclerosis, psoriasis, multiple sclerosis, and Graves' hyperthyroidism. The pharmaceutical composition according to claim 10, characterized in that it is one or more selected from the group consisting of (hyperthyroidism).

12. Use of a composition comprising an anti-DR3 antibody or an antigen-binding fragment thereof that specifically binds to the cell death receptor 3 (DR3) described in Claim 1 as an active ingredient, for the production of a prophylactic or therapeutic agent for graft-versus-host disease, autoimmune disease, or type 2 diabetes.

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