Anti-GITR antibodies and uses thereof

Abstract

The present invention relates to novel anti-GITR antibodies and their uses for immune enhancement, anti-cancer and the prevention and / or treatment of immune-related diseases.

Description

[Technical field]

[0001] Cross-reference to related applications This application claims the benefit of priority based on Korean Patent Application No. 10-2021-0060012 dated May 10, 2021, and all contents disclosed in the documents of said Korean patent application are incorporated herein by reference.

[0002] The present invention relates to novel anti-GITR antibodies and their uses for immune enhancement, anti-cancer and the prevention and / or treatment of immune-related diseases. [Background technology]

[0003] Cancer immunotherapy is a method of treating cancer using the body's immune system and is called the fourth cancer treatment method after chemotherapy, surgery, and radiation therapy. Cancer immunotherapy requires a mechanism to activate the immune system to increase recognition and response to tumor cells. Immune system activation is a complex mechanism involving the functions of various cells, such as antigen-presenting cells, which are important in initiating antigen-specific responses, and effector cells, which are responsible for destroying tumor cells.

[0004] On the other hand, GITR (glucocorticoid-inducible TNFR-related protein) is a member of the tumor necrosis factor receptor (TNFR) superfamily and plays a role in activating T cells and promoting immune responses.

[0005] Therefore, there is a demand for the development of drugs that activate GITR function and have immune enhancing effects. Summary of the Invention

[0006] Provided are anti-GITR antibodies or antigen-binding fragments thereof that bind to GITR, and pharmaceutical uses thereof.

[0007] The anti-GITR antibodies or antigen-binding fragments thereof provided herein may act as agonists for GITR.

[0008] One example provides an anti-GITR antibody or antigen-binding fragment thereof that binds to GITR.

[0009] The anti-GITR antibody or antigen-binding fragment thereof is a heavy chain variable region comprising heavy chain complementarity determining regions (CDRs) or heavy chain complementarity determining regions, including a polypeptide comprising the amino acid sequence of SEQ ID NO:1 (CDR-H1), a polypeptide comprising the amino acid sequence of SEQ ID NO:2 (CDR-H2), and a polypeptide comprising the amino acid sequence of SEQ ID NO:3 (CDR-H3); and A light chain variable region comprising a light chain complementarity determining region or a light chain complementarity determining region comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 (CDR-L1), a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 (CDR-L2), and a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 (CDR-L3). It may include.

[0010] In one example, a polypeptide comprising the amino acid sequence of SEQ ID NO:1 (CDR-H1) may comprise the amino acid sequence of SEQ ID NO:7, 8 or 9, and a polypeptide comprising the amino acid sequence of SEQ ID NO:5 (CDR-L2) may comprise the amino acid sequence of SEQ ID NO:10, 11 or 12.

[0011] In one embodiment, the anti-GITR antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13, 14, 15, 16, 17, or 18; and A light chain variable region comprising the amino acid sequence of SEQ ID NO: 19, 20, 21, 22, 23, or 24. It may include.

[0012] Another embodiment provides an immune enhancing agent or a pharmaceutical composition for immune enhancement, comprising an anti-GITR antibody or an antigen-binding fragment thereof as an active ingredient.

[0013] Another embodiment provides a pharmaceutical composition for preventing and / or treating an immune-related disease, comprising an anti-GITR antibody or an antigen-binding fragment thereof as an active ingredient.

[0014] Another example provides an anti-cancer agent or a pharmaceutical composition for preventing and / or treating cancer, comprising an anti-GITR antibody or an antigen-binding fragment thereof as an active ingredient.

[0015] Another example provides a method of immune enhancement comprising administering a pharma- ceutically effective amount of an anti-GITR antibody or antigen-binding fragment thereof to a subject in need of immune enhancement.

[0016] Another example provides a method for preventing and / or treating an immune-related disease, comprising administering a pharma- tically effective amount of an anti-GITR antibody or antigen-binding fragment thereof to a subject in need of such prevention and / or treatment.

[0017] Another example provides a method for preventing and / or treating cancer, comprising administering a pharma- tically effective amount of an anti-GITR antibody or antigen-binding fragment thereof to a subject in need of such treatment.

[0018] Other examples provide uses of anti-GITR antibodies or antigen-binding fragments thereof for immune enhancement or for the manufacture of immune enhancement agents.

[0019] Other examples provide the use of an anti-GITR antibody or an antigen-binding fragment thereof for the prevention and / or treatment of an immune-related disease, or for the manufacture of a pharmaceutical composition for the prevention and / or treatment of an immune-related disease.

[0020] Other examples provide the use of an anti-GITR antibody or an antigen-binding fragment thereof for the prevention and / or treatment of cancer, or for the manufacture of a medicament for the prevention and / or treatment of cancer.

[0021] Other examples provide nucleic acid molecules encoding heavy chain complementarity determining regions, heavy chain variable regions or heavy chains of anti-GITR antibodies.

[0022] Another example provides a nucleic acid molecule encoding the light chain complementarity determining region, the light chain variable region, or the light chain of an anti-GITR antibody.

[0023] Another example provides a recombinant vector that contains a nucleic acid molecule encoding a heavy chain complementarity determining region, a heavy chain variable region, or a heavy chain of an anti-GITR antibody and a nucleic acid molecule encoding a light chain complementarity determining region, a light chain variable region, or a light chain of an anti-GITR antibody, either together in one vector or in separate vectors. The recombinant vector may be an expression vector for expressing the nucleic acid molecule.

[0024] Another example provides a recombinant cell comprising the recombinant vector.

[0025] Another example provides a method for producing an anti-GITR antibody or antigen-binding fragment thereof, comprising expressing a nucleic acid molecule in a host cell. Expressing the nucleic acid molecule in a host cell may comprise culturing a cell containing the nucleic acid molecule or a recombinant vector comprising the same. The method may additionally comprise isolating and / or purifying the antibody from the culture medium after the expression step.

[0026] The present specification provides an anti-GITR antibody or an antigen-binding fragment thereof that binds to GITR, and medical uses thereof. The anti-GITR antibody or an antigen-binding fragment thereof acts as an agonist against GITR, and has the function of activating immunity (e.g., enhancing effector T cell function, controlling Treg activity, increasing cytokine secretion, etc.), and can be used as a variety of immune activators and / or immunotherapeutics.

[0027] As used herein, the term "GITR (glucocorticoid-induced TNFR-related protein)" refers to a receptor that is a member of the TNF-receptor superfamily that binds to GITR ligand (GITR-L). GITR can also be referred to as tumor necrosis factor receptor superfamily, member 18 (TNFRSF18), AITR, and CD357. GITR can include GITR naturally expressed by a cell, as well as any mutant or isoform thereof. In one example, GITR may be human GITR or an isoform thereof, such as, but not limited to, UniProt Accession No. Q9Y5U5-1 (coded by NCBI Accession No. NP_004186.1; NM_004195.3), NCBI Accession No. NP_683699.1 (coded by NM_148901.2), NCBI Accession No. NP_683700.1 (coded by NM_148902.1), etc. [Brief description of the drawings]

[0028] [Figure 1a] 1 shows the sequence alignment results of the heavy chain (1a) and light chain (1b) of a chimeric and humanized antibody according to one embodiment. [Figure 1b] Same as above [Diagram 2] 1 shows the results of epitope mapping of an anti-GITR antibody according to one embodiment. [Figure 3a] 1 is a graph showing the binding affinity of an anti-GITR antibody according to one embodiment to an antigen (human GITR). [Figure 3b] 1 is a graph showing the binding level of an anti-GITR antibody according to one embodiment to GITR in human primary immune cells. [Figure 4] 1 is a graph showing the efficacy of an anti-GITR antibody against GITR according to one embodiment. [Figure 5a] 1 is a graph showing cytokine production levels of human primary T cells upon treatment with an anti-GITR antibody according to one embodiment, showing IL-2 levels in CD4+ T cells. [Figure 5b] 1 is a graph showing cytokine production levels of human primary T cells when treated with an anti-GITR antibody according to one embodiment, showing IFN-gamma levels in CD4+ T cells. [Figure 5c] 1 is a graph showing cytokine production levels of human primary T cells when treated with an anti-GITR antibody according to one embodiment, showing IL-2 levels in CD8+ T cells. [Figure 5d] 1 is a graph showing cytokine production levels of human primary T cells when treated with an anti-GITR antibody according to one embodiment, showing IFN-gamma levels in CD8+ T cells. [Figure 6] 1 is a graph showing the effect of an anti-GITR antibody according to one embodiment on increasing cytokine production in human T cells. [Figure 7a] 1 shows the NK cell proliferation effect of an anti-GITR antibody according to one embodiment, showing the proliferation level of isolated NK cells. [Figure 7b] 1 shows the NK cell proliferation effect of an anti-GITR antibody according to one embodiment, showing the proliferation level of NK cell-containing PBMC. [Figure 8] 1 is a graph showing the T cell proliferation effect of an anti-GITR antibody according to one embodiment. [Figure 9a] 1 is a graph showing the expression level of CD107a on the surface of NK cells when treated with an anti-GITR antibody according to one embodiment. [Figure 9b] 1 is a graph showing the number of Treg cells upon treatment with an anti-GITR antibody according to one embodiment. [Figure 10a] 1 is a graph showing the tumor cell (ovarian cancer cell) killing effect of an anti-GITR antibody according to one embodiment. [Figure 10b] Same as above [Figure 11] 1 is a graph showing the effect of an anti-GITR antibody according to one embodiment on increasing cytokine production in T cells isolated from a colon cancer patient. [Figure 12] 1 is a graph showing the effect of an anti-GITR antibody according to one embodiment on increasing cytokine production in T cells isolated from a lung cancer patient. [Figure 13]1 is a graph showing the effect of an anti-GITR antibody according to one embodiment on increasing cytokine production in T cells isolated from a liver cancer patient. [Figure 14] 1 is a graph showing changes in tumor size in a tumor animal model upon treatment with an anti-GITR antibody according to one embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] This will be explained in more detail below.

[0030] Antibody or antigen-binding fragment One example provides an anti-GITR antibody or antigen-binding fragment thereof that binds to GITR. The anti-GITR antibody or antigen-binding fragment thereof is A polypeptide comprising the amino acid sequence of SEQ ID NO:1 (CDR-H1), A polypeptide comprising the amino acid sequence of SEQ ID NO:2 (CDR-H2), A polypeptide comprising the amino acid sequence of SEQ ID NO:3 (CDR-H3), A polypeptide comprising the amino acid sequence of SEQ ID NO:4 (CDR-L1), A polypeptide comprising the amino acid sequence of SEQ ID NO:5 (CDR-L2), and Polypeptide comprising the amino acid sequence of SEQ ID NO:6 (CDR-L3) It may include.

[0031] In one example, the anti-GITR antibody or antigen-binding fragment thereof is A polypeptide comprising the amino acid sequence of SEQ ID NO: 7, 8 or 9 (CDR-H1); A polypeptide comprising the amino acid sequence of SEQ ID NO:2 (CDR-H2), A polypeptide comprising the amino acid sequence of SEQ ID NO:3 (CDR-H3), A polypeptide comprising the amino acid sequence of SEQ ID NO:4 (CDR-L1), A polypeptide comprising the amino acid sequence of SEQ ID NO: 10, 11, or 12 (CDR-L2), and Polypeptide comprising the amino acid sequence of SEQ ID NO:6 (CDR-L3) It may include.

[0032] In a specific example, the anti-GITR antibody or antigen-binding fragment thereof comprises: (1) CDR-H1 comprising the amino acid sequence of SEQ ID NO:7, CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, CDR-H3 comprising the amino acid sequence of SEQ ID NO:3, CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, CDR-L2 comprising the amino acid sequence of SEQ ID NO:10, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:6; (2) CDR-H1 comprising the amino acid sequence of SEQ ID NO:8, CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, CDR-H3 comprising the amino acid sequence of SEQ ID NO:3, CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, CDR-L2 comprising the amino acid sequence of SEQ ID NO:11, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:6; or (3) CDR-H1 comprising the amino acid sequence of SEQ ID NO:9, CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, CDR-H3 comprising the amino acid sequence of SEQ ID NO:3, CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, CDR-L2 comprising the amino acid sequence of SEQ ID NO:12, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:6 It may include.

[0033] In the present specification, the complementarity determining region (CDR) is defined based on the CDR definition according to the Kabat system.

[0034] In one embodiment, the six CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) that may be included in the anti-GITR antibody or antigen-binding fragment thereof provided herein are summarized in Table 1 below.

[0035] [Table 1]

[0036] In one example, the anti-GITR antibody or antigen-binding fragment thereof is a heavy chain variable region comprising CDR-H1 of SEQ ID NO:1 (e.g., SEQ ID NO:7, 8, or 9), CDR-H2 of SEQ ID NO:2, and CDR-H3 of SEQ ID NO:3; and A light chain variable region comprising CDR-L1 of SEQ ID NO:4, CDR-L2 of SEQ ID NO:5 (e.g., SEQ ID NO:10, 11, or 12), and CDR-L3 of SEQ ID NO:6. It may include.

[0037] In one embodiment, the anti-GITR antibody or antigen-binding fragment thereof comprises: (1) a heavy chain variable region comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO:7, CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and CDR-H3 comprising the amino acid sequence of SEQ ID NO:3; and a light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, CDR-L2 comprising the amino acid sequence of SEQ ID NO:10, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:6; (2) a heavy chain variable region comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO:8, CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and CDR-H3 comprising the amino acid sequence of SEQ ID NO:3; and a light chain variable region comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:11, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:6; or (3) a heavy chain variable region comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO:9, CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and CDR-H3 comprising the amino acid sequence of SEQ ID NO:3; and A light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, CDR-L2 comprising the amino acid sequence of SEQ ID NO:12, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:6. It may include.

[0038] In one example, the anti-GITR antibody or antigen-binding fragment thereof is a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13, 14, 15, 16, 17, or 18; and A light chain variable region comprising the amino acid sequence of SEQ ID NO: 19, 20, 21, 22, 23, or 24. It may include.

[0039] In one embodiment, the anti-GITR antibody or antigen-binding fragment thereof may comprise: A heavy chain variable region of SEQ ID NO: 13 and a light chain variable region of SEQ ID NO: 19; A heavy chain variable region of SEQ ID NO: 14 and a light chain variable region of SEQ ID NO: 20; A heavy chain variable region of SEQ ID NO: 15 and a light chain variable region of SEQ ID NO: 21; A heavy chain variable region of SEQ ID NO: 16 and a light chain variable region of SEQ ID NO: 22; A heavy chain variable region of SEQ ID NO: 17 and a light chain variable region of SEQ ID NO: 23; or A heavy chain variable region of SEQ ID NO:18 and a light chain variable region of SEQ ID NO:24.

[0040] Optionally (eg, when recombinantly produced), the heavy chain variable region and / or the light chain variable region can additionally comprise a suitable signal sequence at the N-terminus.

[0041] The amino acid sequences of the heavy chain variable region and the light chain variable region that can be included in the anti-GITR antibodies or antigen-binding fragments thereof provided herein are illustrated in Table 2 below.

[0042] [Table 2]

[0043] In one example, the anti-GITR antibodies or antigen-binding fragments thereof provided herein can bind to one or more amino acids within a region of amino acid residues 92-132 of a GITR protein, e.g., a region of amino acid residues 26-241 of a human GITR protein (amino acid residues 1-25 of SEQ ID NO:33; the region is a signal sequence), such as amino acid residues 92-96 (SGTFS, SEQ ID NO:34), 107-116 (TDCTQFGFLT, SEQ ID NO:35), and 122-132 (KTHNAVCVPGS, The target polypeptide may bind to one or more regions and / or one or more amino acids within the region selected from SEQ ID NO: 36, and may bind to at least one or more selected from the group consisting of 92S (Ser(S) at the 92nd amino acid residue; hereinafter, the same will be interpreted), 96S, 107T, 110T, 116T, 122K, and 132S, but is not limited thereto (the amino acid residue positions of human GITR are counted from the 26th amino acid residue excluding the signal sequence (1-25)).

[0044] [Human GITR (UniProt Q9Y5U5-1, SEQ ID NO: 33, the amino acid residues 1-25 region (in italics) is the signal sequence)] [ka]

[0045] As used herein, the term "antibody" is a general term for a protein that specifically binds to a specific antigen, and may be a protein produced in the immune system by stimulation with an antigen or a chemically synthesized or recombinantly produced protein, with no particular limitation on the type. The antibody may be non-naturally produced, for example, recombinantly or synthetically produced. The antibody may be an animal antibody (e.g., a mouse antibody), a chimeric antibody, a humanized antibody, or a human antibody. The antibody may be a monoclonal antibody or a polyclonal antibody.

[0046] In the anti-GITR antibody or antigen-binding fragment thereof provided herein, the heavy chain CDR and light chain CDR regions defined above, or the remaining regions excluding the heavy chain variable region and the light chain variable region, may be derived from any subtype of immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, or IgG4), IgM, etc.), for example, from the framework regions, and / or the light chain constant region and / or the heavy chain constant region of any subtype of immunoglobulin. In one example, the anti-GITR antibody provided herein may be a human IgG type antibody, for example, an IgG1, IgG2, IgG3, or IgG4 type antibody, but is not limited thereto.

[0047] A complete antibody (e.g., IgG type) has two full-length light chains and two full-length heavy chains, each of which is linked to a heavy chain by a disulfide bond. The constant region of an antibody is divided into a heavy chain constant region and a light chain constant region, and the heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, with subclasses of gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The light chain constant region has kappa (κ) and lambda (λ) types.

[0048] In one example, the anti-GITR antibody provided herein can comprise an IgG constant region as the heavy chain constant region and a kappa constant region as the light chain constant region, but is not limited thereto.

[0049] In one embodiment, the constant region of IgG (e.g., human IgG1) may be a wild type, while in another embodiment, the constant region of IgG may be a mutant type including, but not limited to, a mutation of N298A (the 298th amino acid, N, is replaced with A) based on human IgG1.

[0050] The term "heavy chain" refers to a variable region domain V that contains an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. H and three constant region domains C H1 , C H2 , and C H3 The term "light chain" is intended to include a full-length heavy chain, including the heavy chain and hinge, and any fragment thereof. The term "light chain" is intended to include a variable region domain V that contains an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. L and constant region domain C L It is to be understood as meaning to include all full length light chains and fragments thereof.

[0051] The term "CDR (complementarity determining region)" refers to a site in an antibody variable region that confers binding specificity to an antigen, and refers to an amino acid sequence in the hypervariable region of an immunoglobulin heavy chain and light chain. Each of the heavy chain and light chain can contain three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). CDRs can provide key contact residues in the binding of an antibody to an antigen or epitope. Meanwhile, in this specification, the terms "specifically bind" and "specifically recognize" have the same meaning as commonly known to those skilled in the art, and mean that an antigen and an antibody specifically interact with each other to cause an immunological reaction.

[0052] The complementarity determining regions (CDRs) described herein are determined based on the CDR definition according to the Kabat system.

[0053] Unless otherwise specified, the antibody used herein can be understood to include an antigen-binding fragment of an antibody that retains antigen-binding ability.

[0054] The term "antigen-binding fragment" refers to any form of polypeptide that contains a portion capable of binding to an antigen (e.g., the six CDRs as defined herein), such as, but not limited to, an scFv, scFv-Fc, (scFv)2, Fab, Fab', or F(ab')2 of an antibody.

[0055] Among the antigen-binding fragments, Fab consists of the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region of the heavy chain (C H1 ) and has one antigen-binding site.

[0056] Fab' is the heavy chain C H1 It differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the domain.

[0057] F(ab')2 antibodies are produced by forming disulfide bonds between cysteine ​​residues in the hinge region of Fab'. Fv is the minimum antibody fragment that contains only the heavy chain variable region and the light chain variable region, and recombinant techniques for producing Fv fragments are widely known in the art.

[0058] In a two-chain Fv, the heavy chain variable region and the light chain variable region are linked by a non-covalent bond, while in a single-chain Fv, the heavy chain variable region and the single chain variable region are generally linked by a covalent bond via a peptide linker or directly at the C-terminus, so that they can form a dimer-like structure like the two-chain Fv.

[0059] Antigen-binding fragments can be obtained using proteolytic enzymes (for example, whole antibodies can be digested with papain to obtain Fab fragments, or with pepsin to obtain F(ab')2 fragments) or can be produced through recombinant gene techniques.

[0060] The term "hinge region" refers to a region contained in an antibody heavy chain, which is located between the CH1 and CH2 regions and functions to provide flexibility to the antigen-binding site in the antibody.

[0061] The antibody provided herein may be a monoclonal antibody. The monoclonal antibody can be produced according to methods well known in the art. For example, it can be produced using phage display technology. Alternatively, the antibody can be produced from a monoclonal antibody derived from an animal (e.g., mouse) by conventional methods.

[0062] Meanwhile, individual monoclonal antibodies can be screened based on their binding ability to the receptor-binding domain of GITR using a typical ELISA (Enzyme-Linked ImmunoSorbent Assay) format. The binding partners can be assayed for inhibitory activity through functional assays such as competitive ELISA to assay molecular interactions or cell-based assays. Then, the monoclonal antibody members selected based on their strong inhibitory activity can be assayed for their respective affinity (Kd value) for the receptor-binding domain of GITR.

[0063] The final selected antibody can be prepared and used as a humanized antibody, or an antibody in which the remaining portion except for the antigen-binding site is converted to a human immunoglobulin antibody. Methods for preparing humanized antibodies are well known in the art.

[0064] The antigen-binding fragment of the anti-GITR antibody provided herein means a fragment derived from the anti-GITR antibody and retains the binding ability to the antigen (GITR), and may be any polypeptide containing the six CDRs of the anti-GITR antibody, such as, but not limited to, scFv, scFv-Fc, scFv-Ck (kappa constant region), scFv-Cλ (lambda constant region), (scFv)2, Fab, Fab', or F(ab')2. In one example, the antigen-binding fragment may be, but is not limited to, an scFv, or a fusion polypeptide (scFv-Fc) in which the scFv is fused to the Fc portion of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4, etc.) or a fusion polypeptide (scFv-Ck or scFv-Cλ) in which the scFv is fused to the constant region of the light chain (e.g., kappa or lambda).

[0065] As used herein, an antibody (e.g., a CDR, a variable region, or a heavy chain / light chain, an antigen-binding fragment, etc.) "comprises, consists of, or is expressed by a particular amino acid sequence" may mean both cases in which the amino acid sequence is essentially contained, and cases in which meaningless mutations (e.g., substitution, deletion, and / or addition of amino acid residues) that do not significantly affect antibody activity (e.g., antigen affinity, pharmacological activity, etc.) have been introduced into the amino acid sequence.

[0066] The anti-GITR antibodies or antigen-binding fragments thereof provided herein have a binding affinity (K D) may be, for example, 10 mM or less, 5 mM or less, 1 mM or less, 0.5 mM or less, 0.2 mM or less, 0.1 mM or less, 0.05 mM or less, 0.01 mM or less, 0.005 mM or less, or 0.001 mM or less, based on the case measured by surface plasmon resonance (SPR; e.g., Biacore), and may be, for example, 0.0001 nM to 10 mM, 0.0005 nM to 10 mM, 0.001 nM to 10 mM, 0.005 nM to 10 mM, 0.01 nM to 10 mM, 0.05 nM to 10 mM, 0.1 nM to 10 mM, 0.5 nM to 10 mM, 1 nM to 10 mM M, 0.0001nM~5mM, 0.0005nM~5mM, 0.001nM~5mM, 0.005nM~5mM, 0.01nM~5 mM, 0.05nM~5mM, 0.1nM~5mM, 0.5nM~5mM, 1nM~5mM, 0.0001nM~1mM, 0.0005 nM~1mM, 0.001nM~1mM, 0.005nM~1mM, 0.01nM~1mM, 0.05nM~1mM, 0.1nM~1 mM, 0.5nM~1mM, 1nM~1mM, 0.0001nM~0.5mM, 0.0005nM~0.5mM, 0.001nM~0. 5mM, 0.005nM~0.5mM, 0.01nM~0.5mM, 0.05nM~0.5mM, 0.1nM~0.5mM, 0.5n M~0.5mM, 1nM~0.5mM, 0.0001nM~0.2mM, 0.0005nM~0.2mM, 0.001nM~0.2m M, 0.005nM~0.2mM, 0.01nM~0.2mM, 0.05nM~0.2mM, 0.1nM~0.2mM, 0.5nM~ 0.2mM, 1nM~0.2mM, 0.0001nM~0.1mM, 0.0005nM~0.1mM, 0.001nM~0.1mM, 0 .005nM~0.1mM, 0.01nM~0.1mM, 0.05nM~0.1mM, 0.1nM~0.1mM, 0.5nM~0.1 mM, 1nM~0.1mM, 0.0001nM~0.05mM, 0.0005nM~0.05mM, 0.001nM~0.05mM, 0 .005nM~0.05mM, 0.01nM~0.05mM, 0.05nM~0.05mM, 0.1nM~0.05mM, 0.5nM ~0.05mM, 1nM~0.05mM, 0.0001nM~0.01mM, 0.0005nM~0.01mM, 0.001nM~0.The concentration may be, but is not limited to, 0.01 mM, 0.005 nM to 0.01 mM, 0.01 nM to 0.01 mM, 0.05 nM to 0.01 mM, 0.1 nM to 0.01 mM, 0.5 nM to 0.01 mM, or 1 nM to 0.01 mM.

[0067] In another example, a polypeptide molecule is provided that comprises a heavy chain complementarity determining region (CDR-H1, CDR-H2, CDR-H3, or a combination thereof), a light chain complementarity determining region (CDR-L1, CDR-L2, CDR-L3, or a combination thereof), or a combination thereof; or a heavy chain variable region, a light chain variable region, or a combination thereof, of the anti-GITR antibody described above.

[0068] Polypeptide molecules can be used not only as antibody precursors for antibody production, but also as components (e.g., CDRs or variable regions) of protein scaffolds (e.g., peptibodies, nanobodies, etc.) having a structure similar to that of antibodies, bispecific antibodies, and multispecific antibodies having a similar structure.

[0069] The term "peptide+antibody" refers to a fusion protein in which a peptide is fused to all or part of a constant region, such as the Fc region, of an antibody, and has a similar structure and function to an antibody, in which one or more of the peptides described above can act as antigen-binding sites (heavy and / or light chain CDRs or variable regions).

[0070] The term "nanobody", also known as single-domain antibody, refers to an antibody fragment that contains a single variable domain of an antibody in a monomeric form and has the property of selectively binding to a specific antigen, similar to an intact antibody. The molecular weight of nanobodies is generally about 12 kDa to about 15 kDa, which is very small compared to the typical molecular weight of an intact antibody (containing two heavy chains and two light chains) (about 150 kDa to about 160 kDa), and in some cases is smaller than Fab fragments or scFv fragments.

[0071] The term "multimeric antibody" (meaning a dual-binding antibody) refers to an antibody that recognizes and / or binds to two or more different antigens or recognizes and / or binds to different sites on the same antigen, and one antigen-binding site of the multimeric antibody may comprise a polypeptide, antibody, or antigen-binding fragment that binds to GITR as described above.

[0072] The polypeptides, antibodies, or antigen-binding fragments that bind to GITR provided herein can be used in the form of a conjugate conjugated to one or more useful polymers, labeling substances, and the like.

[0073] Useful polymers may be, for example, non-protein polymers that increase the in vivo half-life of polypeptides, antibodies, and / or antigen-binding fragments, and examples thereof include, but are not limited to, one or more hydrophilic polymers selected from the group consisting of polyethylene glycol (PEG) (e.g., PEG having a molecular weight of 2 kDa, 5 kDa, 10 kDa, 12 kDa, 20 kDa, 30 kDa, or 40 kDa), dextran, monomethoxypolyethylene glycol (mPEG), and the like.

[0074] The labeling substance may be, but is not limited to, one or more fluorescent or chemiluminescent small molecule chemicals selected from the group consisting of rare earth chelators, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanates, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, 152Eu, dansyl, umbelliferone, luciferin, luminal label, isoluminal label, aromatic acridinium ester label, imidazole label, acridinium salt label, oxalate ester label, aequorin label, 2,3-dihydrophthalazinedione, biotin / avidin, spin label, stable free radical, and the like, radioisotopes, and the like.

[0075] Medical Use The anti-GITR antibody or antigen-binding fragment thereof provided herein acts as an agonist against GITR (GITR activation) and activates immunity (e.g., enhancing effector T cell function, controlling Treg activity, increasing cytokine secretion, etc.). Therefore, the anti-GITR antibody or antigen-binding fragment thereof can be usefully applied to enhance immunity, prevent and / or treat immune-related diseases, particularly prevent and / or treat cancer.

[0076] Another embodiment provides an immune enhancing agent or a pharmaceutical composition for immune enhancement, comprising an anti-GITR antibody or an antigen-binding fragment thereof as an active ingredient.

[0077] Another embodiment provides a pharmaceutical composition for preventing and / or treating an immune-related disease, comprising an anti-GITR antibody or an antigen-binding fragment thereof as an active ingredient.

[0078] Another example provides an anti-cancer agent or a pharmaceutical composition for preventing and / or treating cancer, comprising an anti-GITR antibody or an antigen-binding fragment thereof as an active ingredient.

[0079] Another example provides a method of immune enhancement comprising administering a pharma- tically effective amount of an anti-GITR antibody or antigen-binding fragment thereof to a subject in need of immune enhancement. The immune enhancement method can additionally comprise the step of identifying a subject in need of immune enhancement prior to the administering step.

[0080] Another example provides a method for preventing and / or treating an immune-related disease, comprising administering a pharma- tically effective amount of an anti-GITR antibody or antigen-binding fragment thereof to a subject in need of such prevention and / or treatment. The method for preventing and / or treating an immune-related disease may additionally comprise, prior to the administering step, identifying a subject in need of such prevention and / or treatment.

[0081] Another example provides a method for preventing and / or treating cancer, comprising administering a pharma- ceutical effective amount of an anti-GITR antibody or antigen-binding fragment thereof to a subject in need of such treatment. The method for preventing and / or treating cancer may additionally comprise, prior to the administering step, identifying a subject in need of such treatment.

[0082] Other examples provide uses of anti-GITR antibodies or antigen-binding fragments thereof for immune enhancement or for the manufacture of immune enhancement agents.

[0083] Other examples provide the use of an anti-GITR antibody or an antigen-binding fragment thereof for the prevention and / or treatment of an immune-related disease, or for the manufacture of a pharmaceutical composition for the prevention and / or treatment of an immune-related disease.

[0084] Other examples provide the use of an anti-GITR antibody or an antigen-binding fragment thereof for the prevention and / or treatment of cancer, or for the manufacture of a medicament for the prevention and / or treatment of cancer.

[0085] As used herein, the term "immune enhancement (or immune strengthening)" may mean inducing an initial immune response to an antigen or increasing an existing immune response, and may be interchangeably used with terms such as immune stimulation, immune enhancement, and immune activation. In one example, immune enhancement may mean one or more selected from the group consisting of enhanced (activation) and / or proliferation of immune cells (effector T cells, e.g., cytotoxic T cells; CD3+ T cells, CD4+ T cells, CD8+ T cells, etc.), suppression and / or depletion of regulatory T cell (Treg) activity, and increased production and / or secretion of immune proteins (e.g., cytokines, etc.), but is not limited thereto.

[0086] As used herein, the term "immune-related disease" encompasses all diseases caused by impaired and / or insufficient activity of the immune system, and may be, for example, one or more selected from the group consisting of cancer, infectious diseases, autoimmune diseases, inflammatory diseases, etc., but is not limited thereto.

[0087] The cancer may be a solid cancer or a blood cancer, including, but not limited to, one or more selected from the group consisting of squamous cell carcinoma, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, etc.), peritoneal cancer, skin cancer, melanoma (e.g., cutaneous or intraocular melanoma, etc.), rectal cancer, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, liver cancer, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, breast cancer, colorectal cancer (e.g., colon cancer, rectal cancer, colorectal cancer, etc.), endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulva cancer, head and neck cancer, brain cancer, osteosarcoma, etc.

[0088] The preventive and / or therapeutic effect of cancer includes not only the effect of inhibiting the occurrence and / or growth of cancer cells, but also the effect of inhibiting the progression of cancer due to migration, invasion, metastasis, and the like.

[0089] The infectious disease, autoimmune disease, and inflammatory disease may be selected from all infectious diseases, autoimmune diseases, and inflammatory diseases that can be treated, alleviated, and / or prevented by the immune enhancement described above (e.g., enhancement (activation) and / or proliferation of immune cells (effector T cells, e.g., cytotoxic T cells; CD3+ T cells, CD4+ T cells, CD8+ T cells, etc.), suppression and / or depletion of regulatory T cell (Treg) activity, increased production and / or secretion of immune proteins (e.g., cytokines (IL-2, IFN-gamma, etc.)), etc.). For example, infectious disease is a general term for diseases that occur when disease-causing pathogens such as viruses, bacteria, fungi, and parasites spread and invade living organisms (e.g., animals including humans), and may be infection or a disease caused by infection (infectious disease) with one or more pathogens selected from the group consisting of viruses, bacteria, fungi, parasites, etc. The autoimmune disease can be selected from the group consisting of, but is not limited to, rheumatoid arthritis, type 1 diabetes, Crohn's disease, ulcerative colitis, Behcet's syndrome, lupus, Sjogren's syndrome, myasthenia gravis, scleroderma, hypothyroidism, hyperthyroidism, psoriasis, vitiligo, multiple sclerosis, autoimmune hepatitis, autoimmune nephritis, autoimmune pancreatitis, autoimmune encephalitis, cytokine storm, and the like.Inflammatory diseases are a general term for inflammation (e.g., chronic inflammation or acute inflammation) or diseases caused by inflammation, such as cardiac inflammation (e.g., coronary artery disease, angina pectoris, myocardial infarction, pericarditis, myocarditis, etc.), vascular inflammation (e.g., atherosclerosis, vasculitis, disseminated intravascular coagulation (DIC), immune thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), anemia, etc.), upper respiratory tract inflammation (e.g., acute nasopharyngitis, allergic rhinitis, sinusitis, pharyngitis, tonsillitis, laryngitis, etc.), lower respiratory tract and / or pulmonary inflammation (e.g., bronchitis, bronchiectasis, asthma, chronic obstructive pulmonary disease (COPD), pneumonia, epileptic pulmonary disease, tuberculosis, etc.), upper gastrointestinal inflammation (e.g., gastritis, esophagitis, etc.), lower gastrointestinal inflammation (e.g., enteritis, ulcerative colitis, Crohn's disease, celiac disease, diverticulitis, irritable bowel syndrome, appendicitis, anal fistula, etc.), liver, biliary tract and / or pancreatic inflammation (e.g., hepatitis, fatty liver, cholangitis, cholecystitis, pancreatitis, type 1 diabetes, etc.), kidney (upper urinary tract) inflammation (e.g., pyelonephritis, glomerulonephritis, urinary tract infection, etc.), lower urinary tract inflammation (e.g., urinary tract infection, ureteral inflammation, urethritis, cystitis, prostatitis / chronic pelvic pain syndrome, etc.), thyroid and / or parathyroid inflammation (e.g. thyroiditis, parathyroiditis, etc.), adrenal inflammation (e.g. adrenalitis, etc.), reproductive organ inflammation (e.g. pelvic inflammatory disease, oophoritis, orchitis, epididymitis, etc.), bone and / or joint inflammation (e.g. osteoarthritis, rheumatoid arthritis, osteomyelitis, synovitis, etc.), skin inflammation (e.g. skin: cellulitis, erysipelas, tinea versicolor, athlete's foot, acne, etc.), muscle inflammation (e.g. myositis, etc.), brain inflammation (e.g. encephalitis, major depressive disorder, The inflammation may be one or more selected from the group consisting of, but is not limited to, neuroinflammation (e.g., neuritis in various sites such as the eye and ear, complex regional pain syndrome, Guillain-Barre syndrome, etc.), eye inflammation (e.g., styes, uveitis, conjunctivitis, etc.), ear inflammation (e.g., otitis media, mastoiditis, etc.), oral inflammation (e.g., stomatitis, periodontitis, gingivitis, etc.), systemic inflammation (e.g., systemic inflammatory response syndrome (sepsis, etc.), metabolic syndrome-related diseases, etc.), peritonitis, reperfusion injury, transplant rejection, hypersensitivity reaction, and the like.

[0090] The subject to which the anti-GITR antibodies, antigen-binding fragments thereof, and / or pharmaceutical compositions containing the same provided herein are administered may be any animal or cell, for example, an animal selected from mammals including humans, primates such as monkeys, and rodents such as rats and mice, or cells, tissues, body fluids (e.g., serum) or cultures thereof derived from animals (isolated), for example, humans or cells, tissues, body fluids (e.g., serum) isolated from humans.

[0091] In addition to the anti-GITR antibody or antigen-binding fragment thereof as an active ingredient, the pharmaceutical composition may additionally contain a pharma- ceutically acceptable carrier, which may be one or more commonly used in the formulation of protein drugs, including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. The pharmaceutical composition may also additionally contain one or more commonly used in the preparation of pharmaceutical compositions, including diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0092] The administration of anti-GITR antibodies, antigen-binding fragments thereof and / or pharmaceutical compositions can be via oral or parenteral routes. Parenteral administration can be by intravenous, subcutaneous, intramuscular, peritoneal, intradermal, topical, intranasal, pulmonary and intrarectal administration. During oral administration, proteins or peptides are digested, so oral compositions must be formulated to coat or protect the active agent from degradation in the stomach.

[0093] In addition, the anti-GITR antibody, its antigen-binding fragment and / or pharmaceutical composition may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or may be formulated into the form of an extract, powder, powder, granule, tablet or capsule, injection, etc., and may additionally contain a dispersant or stabilizer for formulation.

[0094] The content of the anti-GITR antibody or antigen-binding fragment thereof, which is the active ingredient in the pharmaceutical composition, can be variously formulated depending on factors such as the formulation method, administration method, age, weight, sex, pathological condition, food, administration time, administration interval, administration route, excretion rate, and reaction sensitivity. The daily dosage of the pharmaceutical composition is 0.00001 to 1000 mg / kg, 0.00001 to 500 mg / kg, 0.00001 to 100 mg / kg, 0.00001 to 50 mg / kg, 0.0001 to 1000 mg / kg, 0.0001 to 500 mg / kg, 0.0001 to 100 mg / kg, 0.0001 to 50 mg / kg, 0.0001 to 10 ... The range may be, but is not limited to, 0.001-500mg / kg, 0.001-100mg / kg, 0.001-50mg / kg, 0.01-1000mg / kg, 0.01-500mg / kg, 0.01-100mg / kg, 0.01-50mg / kg, 0.1-1000mg / kg, 0.1-500mg / kg, 0.1-100mg / kg, or 0.1-50mg / kg. The daily dosage may be formulated as a single formulation in a unit dosage form, or may be formulated by appropriately dividing it into portions, or may be manufactured in a multi-dose container. In addition, the pharmacologic effective amount in this specification means the amount of the active ingredient that can exhibit the desired pharmacological activity, and may be a dosage range.

[0095] Polynucleotides, Expression Vectors, and Antibody Production Another example provides a nucleic acid molecule encoding a heavy chain complementarity determining region, heavy chain variable region or heavy chain of an anti-GITR antibody. In one example, the nucleic acid molecule may comprise the nucleic acid sequence of SEQ ID NO:8 or SEQ ID NO:12.

[0096] Another example provides a nucleic acid molecule encoding a light chain complementarity determining region, a light chain variable region, or a light chain of an anti-GITR antibody. In one example, the nucleic acid molecule may comprise the nucleic acid sequence of SEQ ID NO:10 or SEQ ID NO:14.

[0097] Another example provides a recombinant vector that contains a nucleic acid molecule encoding a heavy chain complementarity determining region, a heavy chain variable region, or a heavy chain of an anti-GITR antibody and a nucleic acid molecule encoding a light chain complementarity determining region, a light chain variable region, or a light chain of an anti-GITR antibody, either together in one vector or in separate vectors. The recombinant vector may be an expression vector for expressing the nucleic acid molecule.

[0098] Another example provides a recombinant cell comprising the recombinant vector.

[0099] The term "vector" refers to a means for expressing a gene of interest in a host cell. For example, it includes a plasmid vector, a cosmid vector, and a virus vector such as a bacteriophage vector, a lentivirus vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector. The vector that can be used as a recombinant vector can be produced by manipulating a plasmid (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19, etc.), a phage (e.g., λgt4λB, λ-Charon, λΔz1, and M13, etc.), or a virus (e.g., SV40, etc.) that is sometimes used in the art.

[0100] In a recombinant vector, the nucleic acid molecule can be operably linked to a promoter. The term "operatively linked" refers to a functional connection between a nucleotide expression control sequence (e.g., a promoter sequence) and another nucleotide sequence. A control sequence can regulate the transcription and / or translation of another nucleotide sequence by being "operatively linked".

[0101] Recombinant vectors can typically be constructed as vectors for cloning or vectors for expression. Expression vectors can be any vector commonly used in the art to express foreign proteins in plants, animals, or microorganisms. Recombinant vectors can be constructed by a variety of methods known in the art.

[0102] The recombinant vector can be constructed in a prokaryotic or eukaryotic host. For example, when the vector used is an expression vector and the host is a prokaryotic cell, a strong promoter capable of transcription (e.g., pL λ Generally, the vector contains a ribosome binding site for the initiation of translation and a transcription / translation termination sequence (e.g., promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.), a ribosome binding site for the initiation of translation, and a transcription / translation termination sequence. When a eukaryotic cell is used as a host, the replication origin contained in the vector that operates in a eukaryotic cell includes, but is not limited to, the f1 replication origin, SV40 replication origin, pMB1 replication origin, adeno replication origin, AAV replication origin, and BBV replication origin. In addition, a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, and HSV tk promoter) can be used, and generally contains a polyadenylation sequence as a transcription termination sequence.

[0103] The recombinant cell may be obtained by introducing a recombinant vector into a suitable host cell. The host cell may be any host cell known in the art as a cell capable of stably and continuously cloning or expressing a recombinant vector. Examples of prokaryotic cells include Bacillus strains such as E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and enterobacteria and strains such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species. In the case of transformation into eukaryotic cells, examples of the host cell include yeast (Saccharomyce cerevisiae), insect cells, plant cells, and animal cells, such as Sp2 / 0, CHO (Chinese hamster ovary) K1, CHO DG44, CHO S, CHO DXB11, CHO Cell lines that can be used include, but are not limited to, GS-KO, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, and MDCK.

[0104] The delivery (introduction) of a nucleic acid molecule or a recombinant vector containing the same into a host cell can be performed by a delivery method well known in the art, for example, when the host cell is a prokaryotic cell, the CaCl2 method or the electroporation method can be used, and when the host cell is a eukaryotic cell, the microinjection method, the calcium phosphate precipitation method, the electroporation method, the liposome-mediated transfection method, the gene bombardment method, etc. can be used, but are not limited to these.

[0105] The method of selecting transformed host cells can be easily carried out by a method widely known in the art using the phenotype expressed by the selection marker. For example, when the selection marker is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic.

[0106] Another example provides a method for producing an anti-GITR antibody or antigen-binding fragment thereof, comprising expressing a nucleic acid molecule in a host cell. Expressing the nucleic acid molecule in a host cell may comprise culturing a cell containing the nucleic acid molecule or a recombinant vector containing the same. After the culturing step, the production method may optionally further comprise isolating and / or purifying the antibody or antigen-binding fragment from the culture medium.

[0107] The anti-GITR antibody or antigen-binding fragment thereof provided herein acts as an agonist against GITR to activate the immune system (e.g., enhance effector T cell function, control Treg activity, increase cytokine secretion, etc.), and can be used as a variety of immune activating and / or immunotherapeutic agents.

[0108] The present invention will be described in more detail below with reference to examples, but these are merely illustrative and are not intended to limit the scope of the present invention. It is obvious to those skilled in the art that the following examples can be modified without departing from the essential gist of the invention. EXAMPLES

[0109] Example 1: Preparation of anti-GITR antibody 1.1. Generation of anti-GITR monoclonal antibodies Ten mice (5 Balb / c and 5 C57 / bl6) were immunized with the immunogen (GITR protein, >90% pure, mammalian expressed; Genscript). The immunization schedule is as follows: Primary immunization: sc injection of 50 μg of human GITR protein per mouse (Q9Y5U5-1); 1st booster: T = 14 days, ip injection of 25 μg of human GITR protein per animal; Second booster: T=28 days, ip injection of 25 μg of human GITR protein per mouse; and Final booster: T=56±7 days, ip injection of 25 μg human GITR protein per animal.

[0110] Cell fusion and screening B lymphocytes were collected from the spleens of immunized mice and fused with SP2 / 0 myeloma cells (cell fusion) to produce hybridomas. Cell fusion was performed by electrical cell fusion. The fusion efficiency was observed to be approximately 1 hybridoma / 3000 B cells. All fused cells obtained from each cell fusion were plated in 96-well plates. A maximum of 50 plates were used for each fusion.

[0111] Then, protein primary screening and FACS confirmation screening were performed. Protein primary screening was performed by ELISA against the immunization antigen (soluble GITR protein). FACS was performed using a cell line (Genscript) stably expressing GITR, and positive supernatants were screened from the primary screening results.

[0112] Ligand binding screening was then performed. The screened positive clones were screened by ELISA to check whether they blocked or promoted the binding of GITR to GITRL. Specific positive clones were selected for the next one-round subcloning.

[0113] Subcloning and expansion First round of subcloning and collection of hybridoma culture supernatant: Monoclonal cells of each selected clone (parent clone) were selected as test samples. Culture supernatant of each expanded clone was collected from the 24-well plate in an amount of 1 ml, and the antibody concentration in the culture supernatant was measured.

[0114] Subcloning: Positive primary clones obtained from each selected fusion were subcloned by limiting dilutions to ensure that the subclones were derived from a single parent cell. Clones were propagated for up to three generations.

[0115] Subcloning and screening: The subclones were screened by ELISA or HTP FACS.

[0116] Hybridoma tissue culture supernatants were screened against the immunogen (Genscript) using two rounds of indirect ELISA. Positive clones were additionally tested for ligand binding inhibition through a competition assay using indirect ELISA. Selected clones were cultured with GITR-overexpressing CHO cells and FACS was performed to test for GITR binding.

[0117] Complete subcloning: Selected monoclones obtained from each cell fusion were subjected to two additional rounds of subcloning to obtain stable subclones.

[0118] Antibody sequence sequencing Total RNA was isolated from hybridoma cells using TRIzol® Reagent (Ambion, Cat. 15596-026) according to the manufacturer's technical instructions. TM Total RNA was reverse transcribed into cDNA using a universal primer or an isotype-specific antisense primer using the 1st Strand cDNA Synthesis Kit (Takara, Cat. 6110A) according to the manufacturer's technical instructions. Heavy and light chain antibody fragments were amplified and cloned into standard cloning vectors, respectively. Colony PCR was performed to select clones with the correct size insert.

[0119] As a representative of the selected clones, the DNA sequence and amino acid sequence analysis results of the light chain and heavy chain variable regions of the anti-GITR monoclonal antibody clone 51B are shown in Table 3 below:

[0120] [Table 3-1] [Table 3-2]

[0121] 1.2. Humanization of mouse anti-GITR clone Structural models of antibody variable regions (V regions) were generated using the Swiss PDB and analyzed to identify key "constraining" amino acids in the V regions that may be essential for the binding properties of the antibody. Most residues contained within the CDRs (using both Kabat and Chothia definitions) along with a number of framework residues were assessed as critical.

[0122] Based on the structural analysis, human sequence fragments that can be used to generate humanized variants were selected and analyzed using iTope-AI technology (ABZENA plc) for in silico analysis of peptide binding to human MHC class II alleles. For the CDR extra- and flanking regions, a wide selection of human sequence fragments was identified as possible building blocks for new humanized variable regions, whereas for the CDR regions, the selection of available sequences was narrowed. Where possible, sequence fragments identified as significant non-human germline binding to human MHC class II were discarded. This reduced the fragment set, and these combinations were reanalyzed by the method to ensure that the junctions between the fragments did not contain potential T cell epitopes.

[0123] Selected sequence fragments were assembled to generate complete variable region sequences that were free or (where possible) reduced in significant T cell epitopes. Five heavy chain (VH1-VH5) and five light chain (VL1-VL5) sequences were designed for gene synthesis and pairing for mammalian cell expression (Table 4).

[0124] Exemplary combinations of heavy and light chains to generate one chimeric antibody (VH0 / VL0) and 25 humanized antibodies are shown in Table 4 below.

[0125] [Table 4]

[0126] The sequences of the chimeric antibody (VH0xVL0) and humanized antibody obtained by applying the combinations in Table 4 to the 51B clone in Table 3 are shown in Table 5, Table 6, and Figures 1a and 1b. In the sequences in Table 5, Table 6, and Figures 1a (heavy chain variable region) and 1b (light chain variable region), the amino acid sequence numbering and CDR sites were determined according to the Kabat definition, and the CDRs determined as described above in Figures 1a and 1b and the amino acid residues that were changed in the parent sequence (VH0 or VL0) are shown shaded.

[0127] [Table 5]

[0128] [Table 6]

[0129] In the Examples below, identical light chain clones may all be designated by VL or Vk, e.g., VL0, VL1, VL2, VL3, VL4, and VL5 refer to clones identical to Vk0, Vk1, Vk2, Vk3, Vk4, and Vk5, respectively.

[0130] Among the antibodies produced, an Fc recombinant mutant was produced by introducing an N298A point mutation into the Fc region of the heavy chain of the 51B VH0 / VL0 chimeric antibody.

[0131] The heavy and light chain sequences of antibodies including Fc engineered variants are listed in Table 7.

[0132] [Table 7]

[0133] 1.3. Transient expression of chimeric and humanized IgG1 antibodies The VH0 / VL0 chimeric antibody-encoding DNA, two control antibody-encoding DNAs, and a combination of humanized heavy and light chain-encoding DNAs (total of 25 humanized pairings) were transiently transfected into HEK293 EBNA adherent cells (LGC Standards, Teddington, UK) in 6-well plates using the PEI transfection method and then cultured for 7 days. Samples were collected and antibody concentrations were measured on an Octet QK384 using a Protein A biosensor (Molecular Devices, Wokingham, Berkshire, UK) based on human IgG1 antibody.

[0134] The results obtained are shown in Table 8 below.

[0135] [Table 8]

[0136] Table 8 above shows the IgG concentrations (μg / mL) in the supernatants after expression of the chimeric antibody (VH0 / VL0) and the 25 humanized antibodies in HEK cells.

[0137] As shown in Table 8, all antibodies tested were found to be generally well expressed.

[0138] 1.4. Single cycle kinetic analysis of chimeric and humanized anti-GITR antibodies To evaluate the binding of all selected mutants (humanized antibodies) to the human GITR antigen and to select the lead humanized IgG antibody with the closest affinity to the chimeric (VH0 / VL0) antibody, single-cycle kinetic analysis was performed on the supernatants of the transfected cell cultures. Kinetic experiments were performed at 25°C using a Biacore 8K running Biacore 8K Control software V3.0.12.15655 and Biacore Insight Evaluation software V3.0.12.15655 (Cytiva, Uppsala, Sweden).

[0139] HBS-P+ (Cytiva, Uppsala, Sweden) supplemented with 1% BSA w / v (Sigma, Dorset, UK) (to reduce non-specific binding to the reference surface) was used as the running buffer and was also used for dilution of the ligand and analyte. The IgG-containing supernatant was diluted to 1 μg / mL in running buffer. At the start of each cycle, it was loaded onto Fc2 on channels 1–8 of a Protein A sensor chip (Cytiva, Little Chalfont, UK). IgG antibodies were captured at a flow rate of 10 μl / min to achieve an immobilisation level (RL) of ~130 RU. The surface was then allowed to stabilise.

[0140] Single-cycle kinetic data were obtained using recombinant human GITR (Cat No. 13643-H08H, Lot No. LC12JA2509, Sino Biological, Beijing, China) as the analyte, injected at a flow rate of 40 μl / min to minimize potential mass transfer effects. Antigen was used in 2-fold dilutions (five points) ranging from 0.6125 nM to 10 nM concentrations with no regeneration between concentrations. A 200-second association phase was monitored for each injection of five points of increasing antigen concentration, and a single dissociation phase was measured for 600 seconds after the last antigen injection. The sensor chip surface was regenerated by a single injection of 10 mM glycine (pH 1.5).

[0141] The double-based sensorgrams were fitted with a Langmuir (1:1) binding model, and the closeness of the data to the model was assessed using the Chi-squared value, which describes the deviation between the experimental and fitted (observed and predicted) curves. The fitting algorithm was designed to minimize the Chi-squared value.

[0142] The results are shown in Table 9.

[0143] [Table 9]

[0144] Table 9 shows the single cycle kinetic parameters of chimeric (VH0 / VL0) and humanized variants (tested in cell culture supernatants) binding to the human GITR antigen measured using Biacore 8K. Relative K D is the K of the humanized mutant D The K of VH0 / VL0 chimeric antibodies analyzed in the same experiment D This was calculated by dividing it by .

[0145] As shown in Table 9, the K associated with binding to human GITR for all humanized variants (antibodies) tested was DValues ​​were shown to be within 5.9-fold compared to VH0 / VL0. Relative expression levels, percentage of humanization, MHC class II binding risk, and relative K D Considering the values ​​(obtained by Biacore single cycle kinetic analysis on the supernatants), six humanized variants (VH3 / VL1, VH3 / VL3, VH3 / VL4, VH3 / VL5, VH4 / VL3, and VH4 / VL4) were selected for further thermostability analysis.

[0146] 1.5. Thermal Stability assessment A thermal ramp stability experiment (Tm and Tagg) was performed to obtain information on the thermal stability of the antibody from the temperature at which it unfolds from the native state to the denatured state. This unfolding process occurs in a narrow temperature range, and the midpoint of this transition is called the 'melting temperature' or 'Tm'. At this time, the protein undergoes a conformational change, so the melting temperature of the protein was determined by measuring the fluorescence of Sypro Range (which binds to the exposed hydrophobic regions of the protein).

[0147] Samples were diluted in PBS to a final test concentration of 0.5 mg / ml and incubated with Sypro TM Orange (160x stock solution; Sigma-Aldrich) was added to a final concentration of 20x solution. 9 μL of each sample mixture was loaded in duplicate into UNi microcuvettes. A temperature gradient from 15 to 95 °C was performed on the samples (gradient rate 0.3 °C / min and excitation at 473 nm). Total emission spectra were collected from 250 to 720 nm, and the area under the curve between 510 and 680 nm was used to calculate the inflection points (Tonset and Tm) of the transition curve. Protein aggregation was detected by monitoring static light scattering (SLS) at 473 nm, and Tagg (onset of aggregation) was calculated from the resulting SLS profile. Data analysis was performed by UNi microcuvettes. TM The experiment was performed using software version 4.0 (ABZENA).

[0148] The thermal stability values ​​of humanized and chimeric antibodies obtained using the UNcle biostability platform as described above are shown in Table 10.

[0149] [Table 10]

[0150] As seen in Table 10, most of the humanized antibodies showed thermal stability equal to or greater than that of the chimeric antibodies.

[0151] 1.6. Affinity determination of humanized variants using multi-cycle kinetic analysis To measure the binding affinity of the chimeric antibody and six lead humanized variants (VH3 / VL1, VH3 / VL3, VH3 / VL4, VH3 / VL5, VH4 / VL3, and VH4 / VL4) to the human GITR antigen, multicycle kinetic analysis was performed on the purified proteins (antibodies). Kinetic experiments were performed at 25°C using Biacore 8K Control software V3.0.12.15655 and Biacore Insight Evaluation software V3.0.12.15655 (Cytiva, Uppsala, Sweden).

[0152] HBS-EP+ (Cytiva, Uppsala, Sweden) supplemented with 1% BSA w / v (Sigma, Dorset, UK) was used as the running buffer and was also used for ligand and analyte dilution. Purified lead antibody was diluted to 1 μg / mL in running buffer and loaded onto Fc2 on channels 1–8 of a Protein A sensor chip (Cytiva, Little Chalfont, UK) at the start of each cycle. Antibody was captured at a flow rate of 10 μl / min to achieve an immobilized level (RL) of ∼120 RU. The surface was then allowed to stabilize.

[0153] Multicycle kinetic data were obtained using recombinant human GITR (Sino Biological, Beijing, China) as the analyte, injected at a flow rate of 30 μl / min to minimize potential mass transfer effects. The antigen (GITR) was diluted two-fold (six points) in the running buffer over the concentration range of 1.25 nM to 40 nM. For each concentration, the association phase was monitored for 210 s and the dissociation phase was measured for 1200 s. The sensor chip surface was regenerated between cycles with a single injection of 10 mM glycine (pH 1.5). To confirm the stability of the surface and all analytes over the kinetic cycles, multiple replicates of the blank and replicates of two concentrations of the analyte were programmed into the kinetic run.

[0154] The results obtained for the chimeric antibody (VH0 / VL0) and the six lead antibodies are shown in Table 11.

[0155] [Table 11]

[0156] Table 11 above shows the binding parameters for the six purified humanized and chimeric antibodies (VH0 / VL0) to human GITR antigen, measured using Biacore 8K.

[0157] As shown in Table 11, all of the tested antibodies were shown to have excellent binding affinity to human GITR, and all of the humanized antibodies showed binding affinity within 1.5-fold higher than that of the VH0 / VL0 chimeric antibody.

[0158] 1.7. Epitope Mapping To determine the epitopes of the GITR / 51B complex at high resolution, the protein complex was incubated with a deuterium crosslinker and subjected to multi-enzymatic cleavage. After enrichment of the crosslinked peptides in this way, the samples were analyzed by a high-resolution mass spectrometer (nLC-Q-Exactive MS) and the generated data were analyzed using XQuest and Stavrox software.

[0159] The analysis was performed using nLC chromatography coupled with a Q-Exactive mass spectrometer.

[0160] A mixture of hGITR (containing a His tag; Cat. GIR-H5228, Acrobiosystems) and 51B antibody was prepared at the following concentrations.

[0161] [Table 12]

[0162] 20 μL of the GITR / 51B mixture prepared as described above was mixed with 2 μL of DSS d0 / d12 (2 mg / mL, DMF) before incubation for 180 min at room temperature. After incubation, 1 μL of ammonium bicarbonate (final concentration 20 mM) was added to stop the reaction, and then incubated at room temperature for 1 h. The solution was then dried in a speedvac, and 8 M urea was suspended in H2O (20 μL). After mixing, 2 μl of DTT (500 mM) was added to the above solution. The mixture was then incubated at 37 °C for 1 h. Then, 2 μl of iodoacetamide (1 M) was added, followed by incubation for 1 h in the dark and at room temperature. After incubation, 80 μl of protein buffer was added. In the protein buffers, trypsin buffer contains 50 mM Ambic pH 8.5 and 5% acetonitrile; chymotrypsin buffer contains 100 mM Tris HCl and 10 mM CaCl2 pH 7.8; ASP-N buffer contains 50 mM phosphate buffer pH 7.8; elastase buffer contains 50 mM Tris HCl pH 8.0; thermolysin buffer contains 50 mM Tris HCl and 0.5 mM CaCl2 pH 9.0.

[0163] The proteolytic enzyme reactions were carried out as follows. -Trypsin Proteolysis 100 μl of the prepared reduced / alkylated GITR / 51B mixture was mixed with 0.7 μl of trypsin (Promega) in a ratio of 1 / 100. The proteolysis mixture was incubated overnight at 37° C.

[0164] -Chymotrypsin Proteolysis 100 μl of the prepared reduced / alkylated GITR / 51B mixture was mixed with 0.5 μl of chymotrypsin (Promega) in a ratio of 1 / 200. The proteolysis mixture was incubated overnight at 25° C.

[0165] -ASP-N Proteolysis 100 μl of the prepared reduced / alkylated GITR / 51B mixture was mixed with 0.5 μl of ASP-N (Promega) in a ratio of 1 / 200. The proteolysis mixture was incubated overnight at 37°C.

[0166] -Elastase Proteolysis 100 μl of the prepared reduced / alkylated GITR / 51B mixture was mixed with 0.7 μl of elastase (Promega) in a ratio of 1 / 100. The proteolytic mixture was incubated overnight at 37° C.

[0167] -Thermolysis Proteolysis 100 μl of the prepared reduced / alkylated GITR / 51B mixture was mixed with 1.4 μl of Thermolysin (Promega) in a ratio of 1 / 50. The proteolysis mixture was incubated at 70° C. overnight.

[0168] After digestion, formic acid was added to a final concentration of 1% (v / v).

[0169] Cross-linked peptides were analyzed using Xquest version 2.0.

[0170] After trypsin, chymotrypsin, ASP-N, elastase, and thermolysin proteolysis (using deuterated d0d12) of the protein complex GITR / 51B, we detected 12 cross-linked peptides between GITR and 51B through nLC-orbitrap MS / MS analysis.

[0171] As a result of the analysis, it was confirmed that 92S (Ser(S), the 92nd amino acid residue counting from the 26th amino acid of SEQ ID NO:33, hereinafter interpreted as the same) of the GITR amino acid sequence (amino acid sequence of the 26th to 241st positions of SEQ ID NO:33), 96S, 107T, 110T, 116T, 122K, and 132S were specifically crosslinked with the 51B antibody, and the following amino acid sequences were confirmed as epitopes on GITR: 1) Amino acid residues 92-96 (SGTFS, SEQ ID NO:34) 2) Amino acid residues 107-116 (TDCTQFGFLT, SEQ ID NO:35) 3) Amino acid residues 122-132 (KTHNAVCVPGS, SEQ ID NO:36)

[0172] The identified epitope regions on GITR are shown in FIG. 2. FIG. 2 shows the interaction site between GITR and 51B. The epitope sites are darkly shaded in the GITR PDB structure 7KHD. The darkly shaded amino acids in the GITR protein structure correspond to 92-96 (SGTPS, SEQ ID NO: 34), 107-116 (TDCTQFGFLT, SEQ ID NO: 35), and 122-132 (KTHNAVCVPGS, SEQ ID NO: 36) of the GITR sequence. In FIG. 2, A, B, C, D, and E respectively show the ribbon / surface shapes of the front view (A), back view (B), side view 1 (C), side view 2 (D), and top view (E), while F, G, H, I, and J respectively show the ribbon shapes of the front view (F), back view (G), side view 1 (H), side view 2 (I), and top view (J).

[0173] 1.8. Measurement of specificity and binding affinity of anti-GITR antibodies to human GITR protein 1.8.1. Binding Ability Analysis of Anti-GITR Antibodies to Human GITR (hGITR) Protein (by ELISA) ELISA plates were coated with recombinant human GITR protein (in PBS) at a concentration of 1 μg / ml and incubated overnight at 4°C. Plates were blocked with blocking buffer (3% skim milk in PBS) for 1 hour, and serially diluted anti-GITR 51B VH0 / Vk0 (chimeric aGITR 51B mAb) and anti-GITR 51B VH3 / Vk4 (humanized anti-GITR 51B mAb) antibodies (starting concentration 3 μg / ml; 1:3 serial dilutions) were added to the coated plates. After incubation, anti-mouse / human IgG-HRP antibody (Abcam) (1:10000 dilution) was added to each well. TMB solution was added to the plates and stopped with 1N HCl. OD values ​​were measured on a GloMax® Explorer Full Loaded at 450 nM.

[0174] The results are shown in Figure 3a. As shown in Figure 3a, the specificity of the hGITR protein binding of the tested anti-GITR 51B VH0 / Vk0 chimeric antibody and anti-GITR 51B VH3 / Vk4 humanized antibody was confirmed. Both the chimeric and humanized antibodies showed substantial binding to the hGITR protein in a dose-dependent manner.

[0175] 1.8.2. Specificity and Binding Affinity of Anti-GITR Antibodies to hGITR on Primary Human Immune Cells To measure anti-GITR antibody binding to the GITR molecule expressed on human primary lymphocytes, including T cells, NK cells, and NKT cells, APEX TM抗体Anti-GITR 51B VH0 / Vk0 (chimeric mAb) and 51BVH3 / Vk4 (humanized mAb) were conjugated with Alexa Fluor® 488 (AF488) using a labeling kit (Invitrogen) according to the manufacturer's instructions. Human peripheral blood mononuclear cells (PBMCs) isolated from healthy donors were purchased from StemCell technologies and cultured for 2 days with 10% (v / v) FBS and 400 U / ml human IL-2 in the presence of 1 μg / ml anti-CD3 monoclonal antibody (BD Biosciences) and 1 μg / ml anti-CD28 monoclonal antibody (BD Biosciences) at 5% CO2 and 37°C. During the culture, IL-2 400 U / ml and IL-15 20 ng / ml were added every 2 or 3 days. After culture, cells were stained with fluorophore-conjugated antibodies against T cell surface markers (CD3, CD4, and CD8) and NK or NKT cell surface markers (CD56) and various doses (50, 250, 750, 1250, and 2500 ng / ml) of AF488-labeled anti-GITR antibody. Cells were analyzed with a flow cytometer (CytoFLEX, Beckman Coulter) and data were analyzed using Flowjo software (BD). Total T cells (CD3 + ), CD4 + T cells (CD3 + CD4 + CD8 - ), CD8 + T cells (CD3 + CD8 + CD4 - ), CD56 + NK cells (CD3 - CD56 + ) and NKT cells (CD3 + CD56 + ) GITR binding of anti-GITR antibodies was measured.

[0176] The results are shown in Figure 3b. Anti-GITR 51B chimeric and humanized antibodies were confirmed to bind to primary T cells, NK and NKT cells in a dose-dependent manner. The results indicate the specificity of anti-GITR 51B antibodies for the GITR protein expressed in immune cells.

[0177] Example 2. Biological activity (anti-GITR agonist activity) testing of anti-GITR antibodies through cell-based reporter assays The Promega GITR bioassay kit was purchased from Promega Corporation (Madison, WI, USA). One vial (0.5 mL) of GITR effector cells (Promeg) was added to 9.5 mL of assay buffer (99% RPMI, 1% FBS), and the cell suspension (50 μL) was transferred to a 96-well white flat-bottom assay plate (Greiner Bio-one, Chonburi, Thailand). Serial dilutions of anti-GITR antibodies (starting concentration: 5 μg / mL) were made using assay buffer, and then further diluted 2.5-fold with assay buffer to a final antibody concentration of 0.0032768 μg / mL. 25 μL of diluted anti-GITR antibodies were added to the assay plate. After incubation at 37°C and 5% CO2 for 6 h, Bio-Glo Reagent was added and incubated at ambient temperature for 10 min. Luminescence was measured using Promega TM Luminescence was measured using a GloMax® plate reader. RLU vs. Log 10 [Antibody] and Fold Induction vs. Log 10[Antibody] Data was displayed in a graph. Fit curves and EC50 values ​​of antibody reactivity were measured using curve fitting software (such as GraphPad Prism® software). Throughout the experiment, the GITR-mediated luminescence level detected after the addition of GITR agonistic antibody (test antibody) was measured to confirm the agonist activity of the test antibody. As comparative antibodies, anti-GITR 28F3 antibody (BMS; see WO2015 / 187835), anti-GITR 110416 (R&D Systems; see “Heine et al. Oncoimmunology 2017 and Schmiedel et al, Molecular Therapy 2013”) and positive control anti-GITR antibody (Promega Cat. # K1171) were used.

[0178] The results are shown in Figure 4. It was confirmed that cross-linking of anti-GITR 51B VH3 / Vk5 antibody (referred to as anti-GITR 51B) increased T cell activation signal transduction through the agonist activity of the anti-GITR antibody. This efficacy was shown to be significantly superior to that of comparative antibodies.

[0179] Example 3: Cytokine production effect of anti-GOTR antibody 3.1. Confirmation of dose-dependent induction of anti-tumor cytokine production by anti-GITR antibody in human primary T cells For cytokine production, human PBMCs (see Example 1.8.2) were cultured at 0.5 × 10 in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U / ml penicillin, and 100 μg / ml streptomycin in 96-well round-bottom plates. 6The cells were cultured at 1000 x 1000 cells / well. The cells were activated with anti-CD3 monoclonal antibody 0.2 μg / ml and anti-CD28 monoclonal antibody 1 μg / ml for 19 hours at 37°C and 5% CO2 in the presence of anti-GITR antibodies (chimeric and six lead humanized antibodies, 10 μg / ml and 20 μg / ml, respectively). Anti-GITR antibody 110416 (R&D Systems) was used as a reference antibody, and medium alone (no antibody) was used as a negative control. Brefeldin A was added to cells treated with costimulatory antibodies (anti-CD3 and anti-CD28 antibodies) to block cytokine secretion. The harvested cells were stained with Zombie Aqua fixed dead cell staining solution (Invitrogen) to measure cell viability for 20 minutes at room temperature. The cells were stained with fluorophore-conjugated antibodies against surface markers CD3, CD4, and CD8 (all from Biolegend) for 30 minutes on ice. eBioscience TM Intracellular cytokine staining was performed after cell fixation and permeabilization with Foxp3 / Transcription Factor Fixation / Permeabilization Concentrate and Diluent kit (Thermofisher). Then, cells were stained with fluorophore-conjugated antibodies against IL-2 and IFN-γ, respectively, for intracellular staining (all from Biolegend) in permeabilization buffer for 30 min at room temperature. After that, cells were measured with a flow cytometer (CytoFLEX, Beckman Coulter) and data were analyzed using flowjo software (BD). Anti-GITR 110416 (R&D Systems) was used as a reference antibody.

[0180] The results are shown in Figure 5a (IL-2 levels in CD4+ T cells), 5b (IFN-gamma levels in CD4+ T cells), 5c (IL-2 levels in CD8+ T cells), and 5d (IFN-gamma levels in CD8+ T cells). In Figures 5a to 5d, of the two results for each antibody, the left side shows the results when the antibody concentration was 10 μg / ml, and the right side shows the results when the antibody concentration was 20 μg / ml.

[0181] As shown in Figures 5a-5d, all tested anti-GITR 51B antibodies (chimeric and humanized) upregulate Th1 / Tc1 cytokine production in CD4+ and CD8+ T cells through cross-linking with GITR on the surface of immune cells, thereby enhancing immune responses. The levels of cytokines (IL-2 and IFN-gamma) upon treatment with anti-GITR 51B antibodies were significantly higher than those of the control antibody (clone 110416, R&D System). These results indicate that all tested anti-GITR 51B antibodies potently and dose-dependently enhance T cell function.

[0182] 3.2. Confirmation of the effect of anti-GITR antibody on increasing cytokine production in human T cells Cytokine production was measured in human PBMC (see Example 1.8.2). Cells (500,000 cells / reaction) were cultured with anti-CD3 monoclonal antibody (BD Biosciences, cat no. 555336; 0.2 μg / ml) and anti-CD28 monoclonal antibody (BD Biosciences, cat no 555725; 1 μg / ml) in the presence of anti-GITR 51B VH3 / Vk4-IgG1 humanized antibody (test antibody, 10 μg / ml). As a comparison antibody, the anti-GITR antibody 28F3 (BMS) described above was used in the same volume (10 μg / ml) as the test antibody. After incubation, cells were spun down at 400×g for 10 min at 4° C. and cytokine levels in CD4+ T cells were measured. For this, intracellular cytokines were stained in the following manner: cells were stained with Zombie Aqua fixable dead cell dye solution (Invitrogen) and labeled with fluorophore-conjugated antibodies against CD4+ and CD8+ T cell surface markers for 30 min on ice (anti-CD3 antibody (Biolegend), anti-CD4 antibody (Biolegend), and anti-CD8 antibody (Biolegend) were used to stain the CD4+ and CD8+ T cell surface). eBioscience TM Cells were fixed and permeabilized using the Foxp3 / Transcription Factor Fixation / Permeabilization Concentrate and Diluent kit (ThermoFisher) according to the manufacturer's instructions. Cells were stained with fluorophore-conjugated antibodies against intracellular IL-2 and IFNγ. Cells were analyzed on a flow cytometer (CytoFLEX, Beckman Coulter) and data were analyzed with Flowjo software (BD).

[0183] The results are shown in Figure 6. As shown in Figure 6, anti-GITR 51BVH3 / Vk4-IgG1 antibody upregulates Th1 / Tc1 cytokine production in CD4+ T cells, thereby enhancing immune responses. In addition, the cytokine production level of anti-GITR 51BVH3 / Vk4-IgG1 antibody was higher than that of the comparative antibody 28F3. These results suggest that anti-GITR 51BVH3 / Vk5-IgG1 antibody increases effector T cell function more potently than the comparative antibody 28F3.

[0184] Example 4. Effect of anti-GITR antibody on immune cell proliferation 4.1. GITR cross-linking enhances NK cell proliferation in response to IL-15 ELISA plates were coated with anti-GITR 51B humanized antibodies (VH3 / Vk1, VH3 / Vk3, VH3 / Vk4) at 10 μg / ml. NK cells were isolated from human PBMCs (see Example 1.8.2) using an NK isolation kit (Miltenyi Biotec) according to the manufacturer's instructions and stained with cell tracker violet (BioLegend) for 20 min at 37°C. Isolated NK cells (see Figure 7a) or PBMCs containing NK cells (see Figure 7b, 500,000 cells / reaction) were added to the anti-GITR antibody-coated plates and then cultured with IL-15 (5 ng / ml) for 6 days. After culture, the cells were stained with a live / dead reagent (ThermoFisher LIVE / DEAD TM The cells were stained with CD3 (Biolegend) and CD56 (Biolegend) antibodies and analyzed using a flow cytometer (CytoFlex, Beckman Coulter). Data were then analyzed using Flowjo software (BD).

[0185] The results are shown in Figures 7a (isolated NK cells) and 7b (PBMCs containing NK cells). When GITR was cross-linked, IL-15-induced proliferation was increased in isolated NK cells (A) and in PBMCs (B). All tested anti-GITR antibodies increased NK cell proliferation in human primary T cells. These results demonstrate that anti-GITR antibodies have enhanced immune functions through cross-linking with GITR, suggesting that they may have anti-tumor activity.

[0186] 4.2. Anti-GITR antibody enhances CD8+ T cell proliferation CD8+ T cells, Treg, and NK cells were isolated from human PBMCs (see Example 1.8.2) using Miltenyi kits according to the manufacturer's instructions (Miltenyi kits (Miltenyi Biotec): CD8+ T Cell Isolation Kit, human (130-096-495), CD4+ CD25+ CD127-dim Treg cell isolation kit II (130-094-775), and NK Cell Isolation Kit, human (130-092-657)). The number of isolated Treg cells was determined by trypan blue exclusion and LUNA-II immunofluorescence assay. TMAn Automated Cell Counter was used. Cells were seeded at a ratio of 4:4:1 NK:CD8:Treg cells, and anti-GITR antibodies 51B VH0 / Vk0, VH3 / Vk5, VH4 / Vk3, and VG4 / Vk4 were added to the wells at 10 μg / mL each. The prepared cell and anti-GITR antibody mixture was cultured with CD3 / CD28 Dynabeads (ThermoFisher) in the presence of IL-2 (50 ng / ml) for 96 h. After culture, cells were stained for CD3, CD8, CD4, and live / dead markers, then fixed and permeabilized using CytoFix / CytoPerm (BD n.554722) for 20 min at 4°C. Afterwards, intracellular staining was performed with Ki67 antibody for detection of Ki67, and cells were analyzed by flow cytometer (CytoFlex, Beckman Coulter).

[0187] The results are shown in Figure 8. As shown in Figure 8, anti-GITR antibody increased CD8+ T cell proliferation in human primary PBMCs, suggesting that anti-GITR antibody can improve human immune cell function.

[0188] 4.3. Immunomodulation of T regulatory cells and NK cells by anti-GITR antibodies After incubation of PBMCs (see Example 1.8.2) with anti-GITR 51B VH3 / Vk4 antibody, CD107a was measured on NK cells to measure NK cell activation (see FIG. 9a), and the effect of anti-GITR antibody on Treg depletion was evaluated (see FIG. 9b). More specifically, anti-GITR 51B VH3 / Vk4 antibody (5 μg / ml and 10 μg / ml) was co-cultured with human PBMCs in the presence of CD3 and CD28 antibodies, and the frequency of Treg cells was evaluated by flow cytometry. The number of Treg cells was compared with the control group, which was not treated with anti-GITR.

[0189] The results (antibody concentration 10 μg / ml) are shown in Figure 9a (CD107a level) and 9b (Treg depletion). Cross-linking of GITR through anti-GITR 51B VH3 / Vk4 antibody induces NK cell surface expression of CD107a. Thus, anti-GITR 51B VH3 / Vk4 antibody significantly activates NK cells (see Figure 9a). Furthermore, co-culture of human PBMC with anti-GITR antibody (anti-GITR 51B VH3 / Vk4) reduced the number of Treg (see Figure 9b), suggesting that cross-linking of GITR antibody with GITR induces Treg elimination by NK cells. Thus, anti-GITR antibody induces NK cell activation and promotes Treg cytotoxicity of NK cells against Treg. Considering that an increase in the number and function of Treg cells in the tumor microenvironment suppresses immune function and inhibits anti-tumor effects, the tested anti-GITR antibody can reduce the number of Treg cells and improve anti-tumor effects.

[0190] Example 5. Cancer cell killing effect of anti-GITR antibody The cytotoxicity of anti-GITR antibody humanized variants (anti-GITR 51B VH3 / Vk4 and 51B VH3 / Vk3 antibodies) against SKOV-3 ovarian cancer cells was tested. To this end, SKOV-3 cells (ATCC, 2.5 × 10 64-cell cultures) were cultured at an effector to target cell ratio (E:T) of 10:1. 4 T cells / well) and PBMCs (see Example 1.8.2) were co-cultured with anti-GITR 51B antibodies (10 μg / mL each). To activate T cells, Cytostim (Miltenyi) was added to each well. As a comparison antibody, the previously described anti-GITR antibody 110416 (R&D System) was used to test in the same manner. Target cells were counted using IncuCyte native software.

[0191] The results (anti-GITR 51B VH3 / Vk4 antibody) are shown in Figures 10a and 10b. The tumor cell killing effect of all tested anti-GITR 51B antibodies was significantly higher than that of the control group, the cytostim-treated group, and the comparative antibody-treated group. The degree of ovarian cancer cell killing was increased by treatment with the test antibodies anti-GITR 51B VH3 / Vk4 and anti-GITR 51B VH3 / Vk3, suggesting that the anti-GITR 51B acting antibody (test antibody) exhibits strong antitumor activity. GITR cross-linking by anti-GITR 51B antibody (test antibody) induces higher cytotoxicity against SKOV3 cell line (ovarian cancer) compared with the comparative antibody.

[0192] Example 6. Potent anti-tumor cytokine-inducing effect of anti-GITR antibody in human T cells from cancer patients 6.1. Increased cytokine production in T cells from colorectal cancer (CRC) patients Because GITR-expressing immune cells are dysfunctional in cancer patients, we investigated whether anti-GITR antibody therapy could enhance immune responses and induce antitumor effects in cancer (CRC) patients. To confirm the effect of anti-GITR antibody on cytokine production in cancer patients, human PBMCs (500,000 cells / reaction) derived from colorectal cancer (CRC) patients were cultured with anti-CD3 antibody (0.2 μg / ml, BD Biosciences) and anti-CD28 antibody (1 μg / ml, BD Biosciences) in the presence of anti-GITR 51B VH3 / Vk4 antibody (10 μg / mL) or the same volume of a comparison antibody (anti-GITR 28F3; BMS). PBMCs from CRC patients were obtained from Cureline, Inc. Intracellular cytokine production was measured as described in Example 3.

[0193] The results are shown in FIG. 11 (background: cells only (no treatment with CD3 / CD28 antibody or anti-GITR antibody), stimulated: treatment with CD3 / CD28 antibody and no treatment with anti-GITR antibody). As shown in FIG. 11, the test anti-GITR 51B VH3 / Vk4 antibody (denoted as 51B VH3 / Vk4) increased cytokine production in CD4+ and CD8+ T cells at higher levels in PBMCs of CRC patients compared to the comparative antibody 28F3 (ref-28F3). IL-2 and IFN-γ are cytokines that have a core function in the immune system by increasing the proliferation and tumor killing activity of NK and T cells. Therefore, the results indicate that the test anti-GITR antibody has excellent anti-cancer efficacy for cancer (CRC) patients.

[0194] 6.2. Increased cytokine production in T cells from lung cancer (non-small cell lung cancer; NSCLC) patients Because GITR-expressing immune cells are dysfunctional in cancer patients, we investigated whether anti-GITR antibody therapy could enhance immune responses and induce antitumor effects in cancer (NSCLC) patients.

[0195] To confirm the effect of anti-GITR antibody on cytokine production in cancer patients, human PBMCs (500,000 cells / reaction) derived from non-small-cell lung carcinoma (NSCLC) patients were cultured with anti-CD3 antibody (0.2 μg / ml, BD Biosciences) and anti-CD28 antibody (1 μg / ml, BD Biosciences) in the presence of anti-GITR 51B VH3 / Vk4 antibody (10 μg / mL) or the same volume of a comparison antibody (anti-GITR 28F3; BMS). PBMCs from NSCLC patients were obtained from Cureline, Inc. Intracellular cytokine production was measured with reference to Example 3.

[0196] The results are shown in FIG. 12 (background: cells only (no treatment with CD3 / CD28 antibody or anti-GITR antibody), stimulated: treatment with CD3 / CD28 antibody and no treatment with anti-GITR antibody). As shown in FIG. 12, the test anti-GITR 51B VH3 / Vk4 antibody (denoted as 51B VH3 / Vk4) increased cytokine production in CD4+ and CD8+ T cells at higher levels in PBMCs of NSCLC patients compared to the comparative antibody 28F3 (ref-28F3). IL-2 and IFN-γ are cytokines that have a core function in the immune system by increasing the proliferation and tumor killing activity of NK and T cells. Therefore, the results indicate that the test anti-GITR antibody has excellent anti-cancer efficacy for cancer (NSCLC) patients.

[0197] 6.3. Increased cytokine production in T cells from liver cancer (hepatocellular carcinoma; HCC) patients Because GITR-expressing immune cells are dysfunctional in cancer patients, we investigated whether anti-GITR antibody therapy could enhance immune responses and induce antitumor effects in cancer (HCC) patients.

[0198] To confirm the effect of anti-GITR antibody on cytokine production in cancer patients, human PBMCs (500,000 cells / reaction) derived from hepatocellular carcinoma (HCC) patients were cultured with anti-CD3 antibody (0.2 μg / ml, BD Biosciences) and anti-CD28 antibody (1 μg / ml, BD Biosciences) in the presence of anti-GITR 51B VH3 / Vk4 antibody (10 μg / mL) or the same volume of a comparison antibody (anti-GITR 28F3; BMS). PBMCs from HCC patients were obtained from Cureline, Inc. Intracellular cytokine production was measured with reference to Example 3.

[0199] The results are shown in FIG. 13 (background: cells only (no treatment with CD3 / CD28 antibody or anti-GITR antibody), stimulated: treatment with CD3 / CD28 antibody and no treatment with anti-GITR antibody). As shown in FIG. 13, the test anti-GITR 51B VH3 / Vk4 antibody (indicated as 51B VH3 / Vk4) increased cytokine production in CD4+ and CD8+ T cells at higher levels in PBMCs of HCC patients compared to the comparative antibody 28F3 (ref-28F3). IL-2 and IFN-γ are cytokines that have a core function in the immune system by increasing the proliferation and tumor killing activity of NK and T cells. Therefore, the results indicate that the test anti-GITR antibody has excellent anti-cancer efficacy for cancer (HCC) patients.

[0200] The results confirm that the tested anti-GITR antibody induces potent anti-tumor cytokine production in human T cells from a broad range of cancer patients.

[0201] Example 7. Confirmation of anti-cancer efficacy of anti-GITR antibody in MC38 colon carcinoma model (in vivo) MC38 tumor cells (Biocytogen) were maintained in vitro in DMEM + 4 μg / ml puromycin medium supplemented with 10% (v / v) fetal bovine serum (FBS) at 5% CO2 in air and 37°C. Cells in exponential growth phase were harvested and cell numbers were determined using a cell counter prior to tumor inoculation.

[0202] C57BL6 hGITR knock-in mice (Jiangsu Biocytogen Co., Ltd.) were injected with 5 × 10 5 Tumors were generated by subcutaneously inoculating 100 x 100 cells (in 0.1 mL of PBS solution). The average tumor size was 100-150 mm. 3 Randomization was performed when the mice reached the age of 18. Each group consisted of 8 mice. The randomization date was recorded as Day 0.

[0203] After tumor cell inoculation, animals were monitored daily for morbidity, mortality, and weight gain / loss (body weights were measured three times per week after randomization). Mortality and observed clinical signs were recorded in detail for each individual animal. Tumor volumes were measured in two dimensions using calipers three times per week after randomization, and volumes were calculated in mm using the following formula: 3 The calculation was done as follows. V=(L×W×xW) / 2 [V is the tumor volume (mm 3 ), L is the tumor length (the long axis length of the tumor), and W is the tumor width (the longest perpendicular length of L)].

[0204] Dosage, tumor and body weight measurements were performed using a Laminar Flow Cabinet. Body weight and tumor volume were measured using StudyDirector. TM Measurements were performed using software (version 3.1.399.19).

[0205] Treatment was started immediately after grouping. The control group (group 1) was treated with vehicle (PBS solution), while groups 2 and 3 were treated with anti-GITR 51B VH3 / Vk4 antibody produced in CHO cells and INCAGN1876 (Biocytogen) at a dose of 20 mg / kg, respectively (see Table 13).

[0206] [Table 13]

[0207] The measured tumor size (volume) is shown in Figure 14. As shown in Figure 14, the anti-GITR 51B VH3 / Vk4 antibody (Group 2) showed a significant tumor growth inhibitory effect compared to the PBS-treated Group 1 (control group). For example, on day 24, the tumor size of the anti-GITR 51B VH3 / Vk4 antibody-treated test group (Group 2) was significantly reduced compared to the control group (Group 1). In addition, the anti-GITR 51B VH3 / Vk4 antibody-treated test group (Group 2) showed a significantly higher tumor reduction efficacy compared to the comparative antibody (INCAGN1876)-treated test group (Group 3).

[0208] In addition, tumor growth inhibition (ΔTGI) was calculated using the following formula. ΔTGI(Average % of ΔInhibition)=((Average(C)-Average(C0))-(Average(T)-Average(T0))) / (Average(C)-Average(C0))*100% [T-value of the current group / T0-initial value of the current group / C-value of the control group / C0-initial value of the control group] [T: tumor size at the time of measurement of the test group, T0: initial tumor size of the test group; C: Tumor size at the time of measurement in the control group; C0: Initial tumor size in the control group]

[0209] The tumor growth inhibition (ΔTGI) of the anti-GITR 51B VH3 / Vk4 antibody was 64.8%, demonstrating an increased inhibitory effect compared to the INCAGN1876 analog (ΔTGI=44.7%).

[0210] The test animals used in this example tolerated the antibody at a dose of 20 mg / kg (iv), and no BWL was observed during the study.

[0211] Statistical Processing Statistical significance was assessed by conventional one-way ANOVA (paired or unpaired Student's t-test using GraphPad Prism 9 software). Data are presented as mean ± SEM. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns=not significant. [Sequence List Free Text]

[0212] SEQ ID NO:1: Synthetic_CDR-H1, where X is E, N or D SEQ ID NO:2: synthetic_CDR-H2 SEQ ID NO: 3: synthetic_CDR-H3 SEQ ID NO: 4: synthetic_CDR-L1 SEQ ID NO: 5: synthetic_CDR-L2, where X is A, Q or E SEQ ID NO: 6: synthetic_CDR-L3 SEQ ID NO: 7: synthetic_CDR-H1 SEQ ID NO: 8: synthetic_CDR-H1 SEQ ID NO: 9: synthetic_CDR-H1 SEQ ID NO: 10: synthetic_CDR-L2 SEQ ID NO: 11: synthetic_CDR-L2 SEQ ID NO: 12: synthetic_CDR-L2 SEQ ID NO: 13: Synthetic heavy chain variable region SEQ ID NO: 14: Synthetic heavy chain variable region SEQ ID NO: 15: Synthetic heavy chain variable region SEQ ID NO: 16: Synthetic heavy chain variable region SEQ ID NO: 17: Synthetic heavy chain variable region SEQ ID NO: 18: Synthetic heavy chain variable region SEQ ID NO: 19: Synthetic light chain variable region SEQ ID NO: 20: Synthetic light chain variable region SEQ ID NO: 21: Synthetic light chain variable region SEQ ID NO: 22: Synthetic light chain variable region SEQ ID NO: 23: Synthetic light chain variable region SEQ ID NO: 24: Synthetic light chain variable region SEQ ID NO: 25: Synthesis_Heavy chain constant region SEQ ID NO: 26: Synthetic light chain constant region SEQ ID NO: 27: Synthetic_51B chimeric N298A_VH (heavy chain) SEQ ID NO: 28: Synthetic_51B chimera N298A_VL (light chain) SEQ ID NO: 29: Synthetic 51B heavy chain variable region containing signal sequence, where the amino acid sequence at positions 1 to 19 is the signal sequence. SEQ ID NO: 30: DNA encoding the 51B heavy chain variable region including synthetic signal sequence SEQ ID NO: 31: Synthetic signal sequence-containing 51B light chain variable region, in which the amino acid sequence at positions 1 to 22 is the signal sequence SEQ ID NO: 32: DNA encoding the 51B light chain variable region including synthetic signal sequence SEQ ID NO: 33: synthetic human GITR, the amino acid sequence of positions 1 to 25 is a signal sequence SEQ ID NO: 34: Synthetic epitope region on human GITR SEQ ID NO: 35: Synthetic epitope region on human GITR SEQ ID NO: 36: Synthetic epitope region on human GITR

Claims

1. A polypeptide comprising the amino acid sequence of SEQ ID NO:1 (CDR-H1), A polypeptide comprising the amino acid sequence of SEQ ID NO:2 (CDR-H2); A polypeptide comprising the amino acid sequence of SEQ ID NO:3 (CDR-H3); A polypeptide comprising the amino acid sequence of SEQ ID NO:4 (CDR-L1), A polypeptide comprising the amino acid sequence of SEQ ID NO:5 (CDR-L2), and Polypeptide (CDR-L3) comprising the amino acid sequence of SEQ ID NO:6 An anti-GITR antibody or an antigen-binding fragment thereof comprising:

2. A polypeptide comprising the amino acid sequence of SEQ ID NO: 7, 8 or 9 (CDR-H1); A polypeptide comprising the amino acid sequence of SEQ ID NO:2 (CDR-H2); A polypeptide comprising the amino acid sequence of SEQ ID NO:3 (CDR-H3); A polypeptide comprising the amino acid sequence of SEQ ID NO:4 (CDR-L1), A polypeptide (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 10, 11, or 12, and Polypeptide (CDR-L3) comprising the amino acid sequence of SEQ ID NO:6 The anti-GITR antibody or antigen-binding fragment thereof of claim 1 .

3. (1) CDR-H1 comprising the amino acid sequence of SEQ ID NO:7, CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, CDR-H3 comprising the amino acid sequence of SEQ ID NO:3, CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, CDR-L2 comprising the amino acid sequence of SEQ ID NO:10, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:6; (2) CDR-H1 comprising the amino acid sequence of SEQ ID NO:8, CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, CDR-H3 comprising the amino acid sequence of SEQ ID NO:3, CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, CDR-L2 comprising the amino acid sequence of SEQ ID NO:11, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:6; or (3) CDR-H1 comprising the amino acid sequence of SEQ ID NO:9, CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, CDR-H3 comprising the amino acid sequence of SEQ ID NO:3, CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, CDR-L2 comprising the amino acid sequence of SEQ ID NO:12, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:

6. The anti-GITR antibody or antigen-binding fragment thereof of claim 1 .

4. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13, 14, 15, 16, 17, or 18; and A light chain variable region comprising the amino acid sequence of SEQ ID NO: 19, 20, 21, 22, 23, or 24. The anti-GITR antibody or antigen-binding fragment thereof of claim 1 .

5. The anti-GITR antibody or antigen-binding fragment thereof according to claim 1, which binds to one or more amino acid residues selected from amino acid residues 92-132 of the GITR protein.

6. The anti-GITR antibody or antigen-binding fragment thereof according to claim 1 , wherein the anti-GITR antibody is an animal antibody, a chimeric antibody, or a humanized antibody.

7. The anti-GITR antibody or antigen-binding fragment thereof of claim 1, wherein the anti-GITR antibody or antigen-binding fragment thereof is an antigen-binding fragment, and the antigen-binding fragment is an scFv, scFv-Fc, (scFv)2, Fab, Fab', or F(ab')2 of the anti-GITR antibody.

8. A pharmaceutical composition for preventing or treating cancer, comprising the anti-GITR antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

9. The pharmaceutical composition for preventing or treating cancer according to claim 8 , wherein the cancer is a solid cancer or a blood cancer.

10. A pharmaceutical composition for enhancing immunity, comprising the anti-GITR antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

11. The pharmaceutical composition for enhancing immunity according to claim 10, having one or more activities selected from the group consisting of immune cell activation, regulatory T cell suppression, and increased immune protein production.

12. A pharmaceutical composition for preventing or treating an immune-related disease, comprising the anti-GITR antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

13. The pharmaceutical composition for preventing or treating an immune-related disease according to claim 12, wherein the immune-related disease is an infectious disease, an inflammatory disease, or an autoimmune disease.

Images