Anti-hNKG2D antibodies and their applications

Abstract

The present invention relates to the field of antibodies, and in particular to anti-hNKG2D antibodies and their uses. The present invention provides human NKG2D (hNKG2D) antibodies and their uses. The present invention also provides mouse-derived and human-mouse chimeric antibody forms of anti-hNKG2D antibodies. These antibodies have high affinity for hNKG2D protein and can be used to prevent or treat diseases such as cancer, inflammation or autoimmune diseases.

Description

Technical Field

[0001] The present invention relates to the field of antibodies, in particular to anti-hNKG2D antibodies and applications thereof. Background Art

[0002] Natural killer cells (NK) are important immune cells in the body. They are not only related to tumors, viral infections and immune regulation, but also participate in the occurrence and development of hypersensitivity reactions and autoimmune diseases. NK cells have similar functions to cytotoxic T cells and have an extremely fast response rate to virus-infected cells and tumor cells. The difference is that they do not require tumor-specific antigen presentation, so NK cells have a broader spectrum of anti-cancer effects.

[0003] NK cells kill target cells mainly through the following three mechanisms: 1. Release perforin / granzyme or death receptors to mediate the killing of tumor cells; 2. Secrete cytokines and chemokines to regulate the immune system and coordinate T cells to kill tumor cells; 3. The CD16 molecules on the surface of NK cells bind to the Fc segment of the antibody, mediate antibody-dependent cell-mediated cytotoxicity (ADCC), and recognize and kill tumor cells bound by antibodies.

[0004] The activity of NK cells is coordinated and regulated by activating and inhibitory receptors on the cell surface. Inhibitory receptors include killer cell immunoglobulin-like receptors, c-type lectin receptors (NKG2A / CD94), leukocyte immunoglobulin-like receptors and immune checkpoint receptors (PD-1, TIM-3, LAG-3, TIGIT), etc., which recognize and bind to major histocompatibility complex (MHC) molecules. When the expression of MHC-1 molecules in tumor cells is downregulated, causing tumor cells to escape immune system, the activity of the inhibitory receptor pathway of NK cells decreases, negative feedback is downregulated, NK cells are activated, and then kill tumor cells that do not express MHC-I.

[0005] Activated receptors on the surface of NK cells include: natural cytotoxicity triggering receptors, SLAM family receptors, c-type lectins (NKG2D), and CD16 (FcγRIII). The NKG2D molecule is encoded by the Klrk1 (killer cell lectin-like receptor subfamily member 1) gene and is expressed on the membrane surface of NK cells, NKT cells, and some CD8+ and CD4+ T cells. Because its intracellular region is relatively short, it requires the partner molecule DAP10 to coordinate signal transmission. When NKG2D binds to the ligand, the downstream PI3K (phosphoinositide 3-kinase, PI3K) and / or GRB2 (growth factor receptor-bound protein 2, GRB2) signaling pathways are activated, leading to NK cell activation, proliferation, cytokine secretion, and activation of the immune system. NKG2D ligands include the MHC class I-related molecule family (MICA / B) and the human cytomegalovirus protein UL16 binding protein (ULBP1-6) family. The above ligands are expressed on cells infected with viruses / bacteria and malignant transformation. Studies have shown that the above ligands are also abnormally expressed in inflammatory sites or tissues of autoimmune patients, such as rheumatoid arthritis, diabetes, celiac disease, Crohn's disease, atherosclerosis, alopecia and asthma (Guerra et al. Clin Immunol 2013; 149: 432-9).

[0006] The soluble ligands of NKG2D produced by tumor cells can internalize NKG2D molecules, reduce NK cell-mediated tumor cell lysis, and lead to tumor immune escape. Therefore, NKG2D molecules have become one of the new targets for tumor immunotherapy. At present, drug development for NKG2D is still in the clinical research stage. Studies have found that anti-NKG2D antibodies can enhance the cytotoxicity and cytokine secretion of NK cells; other articles have reported that antibodies against NKG2D ligands MICA / MICB can block the ligand from falling off the cell membrane and stabilize the number of membrane-type ligands, thereby enhancing the activity of NK cells and enhancing tumor killing activity. The above studies show that antibodies targeting NKG2D can play a role in regulating NK cell activity, suggesting that NKG2D can become a target for the treatment of tumors, inflammation or autoimmune diseases. Summary of the invention

[0007] In view of this, the present invention provides an anti-hNKG2D antibody and its application. The antibody has a high affinity to hNKG2D protein and can be used to prevent or treat diseases such as cancer, inflammation or autoimmune diseases.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides an antibody or an antigen-binding portion thereof that specifically binds to hNKG2D, comprising a heavy chain variable region, wherein the heavy chain variable region comprises any one or more of HCDR1, HCDR2 or HCDR3;

[0010] (I) The HCDR1 sequence comprises an amino acid sequence as shown in SEQ ID No.1 or SEQ ID No.2; and / or

[0011] The HCDR2 sequence comprises the amino acid sequence shown in SEQ ID No.3 or SEQ ID No.4; and / or

[0012] The HCDR3 sequence comprises the amino acid sequence shown in SEQ ID No.5 or SEQ ID No.6;

[0013] or

[0014] (II) An amino acid sequence obtained by replacing, deleting or adding an amino acid to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I);

[0015] or

[0016] (III). An amino acid sequence having 95% or more identity with the amino acid sequence described in (I) or (II).

[0017] In some specific embodiments of the present invention, the antibody or antigen-binding portion thereof further comprises a light chain variable region, wherein the light chain variable region comprises any one or more of LCDR1, LCDR2 or LCDR3,

[0018] (I) The LCDR1 sequence comprises an amino acid sequence as shown in SEQ ID No.7 or SEQ ID No.8; and / or

[0019] The LCDR2 sequence comprises the amino acid sequence shown in SEQ ID No.9 or SEQ ID No.10; and / or

[0020] The LCDR3 sequence comprises the amino acid sequence shown in SEQ ID No.11 or SEQ ID No.12;

[0021] or

[0022] (II) An amino acid sequence obtained by replacing, deleting or adding an amino acid to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I);

[0023] or

[0024] (III). An amino acid sequence having 95% or more identity with the amino acid sequence described in (I) or (II).

[0025] In some specific embodiments of the present invention, the antigen binding portion is selected from: a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a scFv fragment, a Fd fragment or a single domain antibody.

[0026] In some specific embodiments of the present invention, the antibody is a murine antibody;

[0027] (I) The heavy chain variable region of the murine antibody comprises the amino acid sequence shown in SEQ ID No.13 or SEQ ID No.14, and the light chain variable region of the murine antibody comprises the amino acid sequence shown in SEQ ID No.15 or SEQ ID No.16;

[0028] or

[0029] (II) An amino acid sequence obtained by substituting, deleting or adding one amino acid to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I);

[0030] or

[0031] (III). An amino acid sequence that is 95% or more identical to the amino acid sequence described in (I) or (II).

[0032] In some specific embodiments of the present invention, the antibody is a chimeric antibody;

[0033] (I) The heavy chain variable region of the murine antibody comprises the amino acid sequence shown in SEQ ID No.13 or SEQ ID No.14, and the light chain variable region of the murine antibody comprises the amino acid sequence shown in SEQ ID No.15 or SEQ ID No.16;

[0034] or

[0035] (II) An amino acid sequence obtained by substituting, deleting or adding one amino acid to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I);

[0036] or

[0037] (III). An amino acid sequence that is 95% or more identical to the amino acid sequence described in (I) or (II).

[0038] In some specific embodiments of the present invention, the substitution, deletion or addition of a plurality of amino acids is 2, 3, 4 or 5.

[0039] The present invention also provides nucleic acid molecules encoding the antibodies or antigen-binding portions thereof.

[0040] In some specific embodiments of the present invention, the nucleic acid molecule is a nucleic acid sequence of a mouse antibody, including but not limited to the following:

[0041] (I). The nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID No.17 or SEQ ID No.18; and / or

[0042] The nucleotide sequence encoding the light chain variable region is shown in SEQ ID No.19 or SEQ ID No.20;

[0043] or

[0044] (II) The complementary sequence of the nucleotide sequence as shown in (I); or

[0045] (III) A sequence encoding the same protein as the nucleotide sequence of (I) or (II) but differing from the nucleotide sequence of (I) or (II) due to the degeneracy of the genetic code; or

[0046] (IV). A sequence that is at least 80% identical to the sequence described in (I), (II) or (III).

[0047] In addition, the present invention also provides an expression vector, comprising the nucleic acid molecule.

[0048] The present invention also provides a host cell transformed or transfected with the expression vector.

[0049] The present invention also provides a method for preparing the antibody or its antigen binding portion, comprising: culturing the host cell to induce the expression of the antibody.

[0050] In addition, the present invention also provides a conjugate comprising the chemically labeled or biologically labeled antibody or antigen-binding portion thereof.

[0051] Based on the above research, the present invention also provides a conjugate prepared by coupling the antibody or antigen-binding portion thereof or the conjugate with a solid medium or a semi-solid medium.

[0052] More importantly, the present invention also provides a pharmaceutical composition comprising the antibody or the antigen-binding portion thereof.

[0053] In some specific embodiments of the present invention, the pharmaceutical composition further comprises one or more active ingredients, including but not limited to agents for treating tumors.

[0054] Based on the above research, the present invention also provides the use of the antibody or antigen-binding portion thereof, the nucleic acid molecule, the expression vector, the host cell, the conjugate, the conjugate and / or the pharmaceutical composition in the preparation of a drug for preventing and / or treating a disease;

[0055] The diseases include tumors, inflammatory or autoimmune diseases;

[0056] The tumor is selected from one or more of the following: bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, blood cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer and prostate cancer;

[0057] The inflammatory or autoimmune disease is selected from one or more of the following: multiple sclerosis, rheumatoid arthritis, type I diabetes, Crohn's disease, ulcerative colitis, myasthenia gravis, systemic lupus erythematosus, scleroderma, ankylosing spondylitis, graft-versus-host disease, organ transplantation, Sjögren's syndrome or autoimmune hepatitis.

[0058] More importantly, the present invention provides a drug, including the antibody or antigen-binding portion thereof, the conjugate, the coupling and / or the pharmaceutical composition and a pharmaceutically acceptable excipient.

[0059] The present invention also provides a method for preventing and / or treating a disease, wherein the drug is administered;

[0060] The diseases include tumors, inflammatory or autoimmune diseases;

[0061] The tumor is selected from one or more of the following: bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, blood cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer and prostate cancer;

[0062] The inflammatory or autoimmune disease is selected from one or more of the following: multiple sclerosis, rheumatoid arthritis, type I diabetes, Crohn's disease, ulcerative colitis, myasthenia gravis, systemic lupus erythematosus, scleroderma, ankylosing spondylitis, graft-versus-host disease, organ transplantation, Sjögren's syndrome or autoimmune hepatitis.

[0063] The present invention also provides the use of the antibody or its antigen binding portion, the conjugate and / or the conjugate in preparing a product for detecting hNKG2D; the product includes a detection reagent, a detection kit, a detection chip or a detection device.

[0064] More importantly, the present invention also provides a detection reagent, a detection kit, a detection chip or a detection device, including the antibody or its antigen-binding portion, the conjugate and / or the coupling, and an auxiliary agent acceptable in the detection.

[0065] The present invention also provides a method for diagnosing a disease, using the detection reagent, detection kit, detection chip or detection device to detect hNKG2D expression, and judging whether the disease exists according to the expression level of hNKG2D;

[0066] The diseases include tumors, inflammatory or autoimmune diseases;

[0067] The tumor is selected from one or more of the following: bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, blood cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer and prostate cancer;

[0068] The inflammatory or autoimmune disease is selected from one or more of the following: multiple sclerosis, rheumatoid arthritis, type I diabetes, Crohn's disease, ulcerative colitis, myasthenia gravis, systemic lupus erythematosus, scleroderma, ankylosing spondylitis, graft-versus-host disease, organ transplantation, Sjögren's syndrome or autoimmune hepatitis.

[0069] The antibody provided by the present invention has high affinity to hNKG2D protein and can be used to prevent or treat diseases such as cancer, inflammation or autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0071] Figure 1 The binding of the mouse anti-hNKG2D antibody of the present application (antibodies secreted by hybridomas 4A3 and 7C5) to HEK293 cells 293T-hNKG2D expressing hNKG2D is shown;

[0072] Figure 2 It shows that the anti-hNKG2D monoclonal antibodies secreted by 4A3 and 7C5 hybridomas compete with the hNKG2D ligand hMICA for binding;

[0073] Figure 3 The chimeric anti-hNKG2D antibody of the present application binds to HEK293 cells 293T-hNKG2D overexpressing hNKG2D. DETAILED DESCRIPTION

[0074] The present invention discloses anti-hNKG2D antibodies and their applications. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve them. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0075] definition

[0076] As used herein, the term "antigen binding portion" of an antibody refers to a portion or segment of a complete antibody molecule that is responsible for binding to an antigen. The antigen binding portion may include a heavy chain variable region (VH), a light chain variable region (VL), or both. The antigen binding portion of an antibody can be prepared from a complete antibody molecule using any suitable standard technique, including proteolytic digestion or recombinant genetic engineering techniques, etc. Non-limiting examples of antigen binding portions include: Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single-chain Fv (scFv) molecules, single-domain antibodies, dAb fragments, and the minimum recognition unit (e.g., isolated CDR) composed of amino acid residues that mimic the hypervariable region of an antibody. The term "antigen binding portion" also includes other engineered molecules, such as double antibodies, three antibodies, four antibodies, and mini antibodies, etc. For example, the Fd fragment described herein refers to an antibody fragment consisting of VH and CH1 domains; the Fv fragment consists of VL and VH domains in a single arm of an antibody; and the dAb fragment (Ward et al., Nature 1989; 341: 544-546) consists of a VH domain.

[0077] It is well known to those skilled in the art that the complementary determining region (CDR, usually CDR1, CDR2 and CDR3) is the region in the variable region that has the greatest impact on the affinity and specificity of the antibody. There are two common ways to define the CDR sequence of VH or VL, namely the Kabat definition and the Chothia definition, for example, see Kabat et al., "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, MD. (1991); Al-Lazikani et al., J Mol Biol 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). For the variable region sequence of a given antibody, the CDR region sequence in the VH and VL sequences can be determined according to the Kabat definition or the Chothia definition. In the embodiment of the present application, the Kabat definition of CDR sequence is used. Herein, CDR1, CDR2 and CDR3 of the heavy chain variable region are referred to as HCDR1, HCDR2 and HCDR3, respectively; CDR1, CDR2 and CDR3 of the light chain variable region are referred to as LCDR1, LCDR2 and LCDR3, respectively.

[0078] For a given antibody variable region sequence, the CDR region sequence in the variable region sequence can be analyzed in a variety of ways, for example, it can be determined using the online software Abysis (http: / / www.abysis.org / ).

[0079] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigen epitope, such as the ability of an antibody to bind to a specific antigen with an affinity at least twice greater than its affinity for a non-specific antigen. However, it should be understood that an antibody can specifically bind to two or more antigens related to its sequence. For example, the antibodies of the present application can specifically bind to hNKG2D.

[0080] As used herein, the term "KD" is intended to refer to the equilibrium dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd / ka) and is expressed as a molar concentration (M). The KD value of an antibody can be determined using well-established methods in the art. A preferred method for determining the KD of an antibody is by using surface plasmon resonance, preferably using a biosensor system (e.g., an SPR system) or flow cytometry and Scatchard analysis.

[0081] As used herein, the term "high affinity" for IgG antibodies refers to antibodies that have a 10 -8 M or less, preferably 10-9 M or less, and more preferably 10 -10 However, for other antibody isotypes, "high affinity" binding may vary. For example, "high affinity" binding for an IgM isotype means that the antibody has a KD of 10 -7 M or less, preferably 10 -8 M or smaller KD.

[0082] As used herein, the term staining index is applied in flow cytometry, and its calculation formula is: staining index = (median fluorescence intensity of sample - median fluorescence intensity of negative control) / (2×standard deviation of negative control).

[0083] As used herein, the term "cancer" refers to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can form malignant tumors or cells that invade adjacent tissues and can metastasize to distant parts of the body via the lymphatic system or bloodstream.

[0084] The inventors screened and obtained two hybridoma cells, the antibodies in the supernatants of which can bind to cells expressing hNKG2D. The present application provides two novel anti-hNKG2D antibodies or antigen-binding portions thereof that can specifically bind to hNKG2D.

[0085] The inventors have also prepared chimeric antibodies from mouse anti-hNKG2D antibodies by genetic engineering. These antibodies can specifically bind to hNKG2D expressed on the cell surface, thereby effectively inducing hNKG2D-mediated immune responses and playing a role in preventing or treating diseases such as tumors, inflammatory diseases or autoimmune diseases.

[0086] In a first aspect, the present application provides an antibody or an antigen-binding portion thereof that specifically binds to hNKG2D, which comprises a heavy chain variable region, wherein the heavy chain variable region comprises any one or more of HCDR1, HCDR2 and HCDR3 sequences, wherein the HCDR1 sequence is GYSFTSYW (SEQ ID NO: 1) or GFTFSDYY (SEQ ID NO: 2); the HCDR2 sequence comprises the amino acid sequence IHPSDSET (SEQ ID NO: 3) or ISDGGRYT (SEQ ID NO: 4); and / or the HCDR3 sequence comprises the amino acid sequence ARHYYGYVDFMDY (SEQ ID NO: 5) or ARDGRLHDGYYGRFSY (SEQ ID NO: 6).

[0087] In a preferred embodiment, the heavy chain variable region of the antibody or antigen binding portion thereof described herein comprises HCDR1 shown in SEQ ID NO:1 or SEQ ID NO:2, HCDR2 shown in SEQ ID NO:3 or SEQ ID NO:4, and HCDR3 sequence shown in SEQ ID NO:5 or SEQ ID NO:6.

[0088] In some embodiments, the antibodies or antigen-binding portions thereof described herein further comprise a light chain variable region comprising any one or more of LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1 sequence is QEISGY (SEQ ID NO:7) or SSVSY (SEQ ID NO:8); the LCDR2 sequence is AAS (SEQ ID NO:9) or DTS (SEQ ID NO:10); the LCDR3 sequence is LQYVAYPWT (SEQ ID NO:11) or QQWISYPPT (SEQ ID NO:12).

[0089] In a preferred embodiment, the light chain variable region of the antibodies or antigen-binding portions thereof described herein comprises LCDR1 as shown in SEQ ID NOs:7-8, LCDR2 as shown in SEQ ID NOs:9-10, and LCDR3 as shown in SEQ ID NOs:11-12.

[0090] In a preferred embodiment, the antibody or antigen-binding portion thereof described herein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 shown in SEQ ID NOs: 1-2, HCDR2 shown in SEQ ID NOs: 3-4, and HCDR3 sequences shown in SEQ ID NOs: 5-6, and the light chain variable region comprises LCDR1 shown in SEQ ID NOs: 7-8, LCDR2 shown in SEQ ID NOs: 9-10, and LCDR3 sequences shown in SEQ ID NOs: 11-12.

[0091] In some more specific embodiments, the antibodies disclosed herein may be anti-hNKG2D monoclonal antibodies. The anti-hNKG2D antibody type and subtype may be determined by any method known in the art.

[0092] In some embodiments, the antibody described herein is a murine antibody, preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:13-14, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:15-16.

[0093] In a preferred embodiment, the murine antibody described herein comprises a heavy chain variable region set forth in SEQ ID NOs: 13-14 and a light chain variable region set forth in SEQ ID NOs: 15-16.

[0094] In some embodiments, the antibodies described herein are chimeric antibodies. The chimeric antibodies described herein comprise the variable region of a murine antibody (including the heavy chain variable region VH and / or the light chain variable region VL) and the constant region of a human antibody.

[0095] In a preferred embodiment, the chimeric antibodies described herein comprise the variable regions of a murine antibody (including a heavy chain variable region and a light chain variable region) and the constant regions of a human antibody.

[0096] Preferably, the chimeric antibody comprises the heavy chain variable region shown in SEQ ID NO: 13-14 and the human antibody heavy chain constant region IgG1, and / or the light chain variable region shown in SEQ ID NO: 15-16 and the human antibody light (Kappa) chain constant region.

[0097] In a preferred embodiment, the chimeric antibody described herein comprises the heavy chain variable region shown in SEQ ID NO: 13-14 and the human antibody heavy chain constant region IgG1, and / or the light chain variable region shown in SEQ ID NO: 15-16 and the human antibody light (Kappa) chain constant region.

[0098] The antibodies described herein may also include mouse or human antibody constant regions. Mouse antibody constant regions include the heavy chain constant regions of mouse IgG1, IgG2a, IgG2b or IgG3 and the κ or λ type light chain constant regions, etc. Human antibody constant regions include the heavy chain constant regions of human IgG1, IgG2, IgG3 or IgG4 and the κ or λ type light chain constant regions, etc.

[0099] In some embodiments, the hNKG2D described herein is a primate NKG2D. Preferably, the primate NKG2D described herein is selected from hNKG2D or monkey NKG2D.

[0100] In some embodiments, the antigen binding portion described herein is selected from: a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a scFv fragment, a Fd fragment, or a single domain antibody.

[0101] In a second aspect, the present application provides a pharmaceutical composition comprising the antibody or antigen-binding portion thereof according to the first aspect and a pharmaceutically acceptable carrier.

[0102] Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and / or dispersion media. The use of such carriers and diluents is well known in the art. Some non-limiting examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10 ) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; (22) C2-C 12 Alcohols, such as ethanol; and (23) other non-toxic compatible substances used in pharmaceutical preparations. Wetting agents, colorants, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives and antioxidants may also be present in the pharmaceutical preparations.

[0103] In some embodiments, the pharmaceutical composition described herein further comprises one or more other active ingredients, for example, for treating hNKG2D-related diseases such as tumors, inflammatory diseases or autoimmune diseases.

[0104] In a third aspect, the present application provides a nucleic acid molecule encoding the antibody or antigen-binding portion thereof described in the first aspect.

[0105] In a preferred embodiment, the nucleic acid described herein can be a codon-optimized nucleic acid suitable for expression in a host cell, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID No. 17 or 18; the nucleotide sequence encoding the light chain variable region is shown in SEQ ID No. 19 or 20. For example, according to the degeneracy of the codons, they still encode the same protein. The method of codon optimization according to the host cell used is well known to those skilled in the art.

[0106] In a fourth aspect, the present application provides an expression vector comprising the nucleic acid molecule described in the third aspect.

[0107] Any suitable expression vector can be used. For example, prokaryotic cloning vectors include plasmids from Escherichia coli, such as colE1, pCR1, pBR322, pMB9, pUC, pKSM and RP4. Prokaryotic vectors also include derivatives of phage DNA such as M13 and other filamentous single-stranded DNA phages. The example of a vector that can be used for yeast is a 2 μg plasmid. Suitable vectors for expression in mammalian cells include the following well-known derivatives: SV-40, adenovirus, retrovirus-derived DNA sequences and shuttle vectors derived from functional mammalian vectors (such as those described above) and a combination of functional plasmids and phage DNA.

[0108] Additional eukaryotic expression vectors are known in the art (e.g., P J. Southern & P. ​​Berg, J. Mol. Appl. Genet, 1:327-341 (1982); Subramani et al., Mol. Cell. Biol, 1:854-864 (1981); Kaufinann & Sharp, "Amplification And Expression of Sequences Cotransfected with a Modular Dihydrofolate Reductase Complementary DNA Gene," J. Mol. Biol, 159:601-621 (1982); Kaufhiann & Sharp, Mol. Cell. Biol, 159:601-664 (1982); Scahill et al., "Expression And Characterization Of The Product Of A Human Immune Interferon DNA Gene In Chinese Hamster Ovary Cells," Proc. Nat'l Acad. Sci. USA, 80:4654-4659 (1983); Urlaub & Chasin, Proc. Nat'l Acad. Sci USA, 77:4216-4220, (1980), which are incorporated herein by reference in their entireties).

[0109] The expression vector that can be used for the present application contains at least one expression control sequence, which is operably connected to the DNA sequence or fragment to be expressed. The control sequence is inserted into the vector to control and regulate the expression of the cloned DNA sequence. Examples of useful expression control sequences are the lac system, the trp system, the tac system, the trc system, the major operator and promoter region of bacteriophage λ, the control region of the fd coat protein, the glycolytic promoter of yeast, such as the promoter of 3-phosphoglycerate kinase, the promoter of yeast acid phosphatase, such as Pho5, the promoter of yeast α-mating factor, and promoters derived from polyoma viruses, adenoviruses, retroviruses and simian viruses, such as the early and late promoters of SV40 and other sequences known to control the gene expression of prokaryotic or eukaryotic cells and their viruses or their combinations.

[0110] In the fifth aspect, the present application provides a host cell, which comprises the nucleic acid molecule described in the third aspect or the expression vector described in the fourth aspect.

[0111] In some embodiments, the host cells described herein are mammalian cells. Mammalian cells may include, but are not limited to, CHO cells, NS0 cells, SP2 / 0 cells, HEK293 cells, COS cells, and PER.C6 cells. Those skilled in the art can select suitable host cells as needed.

[0112] The preparation method of the anti-hNKG2D monoclonal antibody disclosed herein may include: culturing host cells under expression conditions to express the anti-hNKG2D monoclonal antibody; isolating and purifying the expressed anti-hNKG2D monoclonal antibody. Using the above method, a crude anti-hNKG2D monoclonal antibody can be obtained. The anti-hNKG2D monoclonal antibody is then purified into a substantially uniform substance, such as a single band on SDS-PAGE electrophoresis, by a purification method including hNKG2D-based affinity purification, non-denaturing gel purification, HPLC or RP-HPLC, molecular exclusion, purification on a protein A column, or any combination of these techniques.

[0113] In the sixth aspect, the present application provides the use of the antibody or antigen-binding portion thereof described in the first aspect, the pharmaceutical composition described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, or the host cell described in the fifth aspect in the preparation of a medicament for preventing and / or treating related diseases such as tumors, inflammatory diseases or autoimmune diseases.

[0114] In the seventh aspect, the present application provides a method for preventing and / or treating related diseases such as tumors, inflammatory diseases or autoimmune diseases, which comprises administering to an individual in need thereof a therapeutically effective amount of the antibody or antigen-binding portion thereof described in the first aspect, the pharmaceutical composition described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, or the host cell described in the fifth aspect.

[0115] In some embodiments, the method further comprises administering a second agent for preventing and / or treating a related disease such as a tumor, an inflammatory disease, or an autoimmune disease.

[0116] In the eighth aspect, the present application provides the use of the antibody or antigen-binding portion thereof described in the first aspect, the pharmaceutical composition described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, or the host cell described in the fifth aspect for preventing and / or treating related diseases such as tumors, inflammatory diseases or autoimmune diseases.

[0117] In any one of the embodiments of the sixth to eighth aspects, the related disease is a tumor, an inflammatory disease, an autoimmune disease or the like.

[0118] In some embodiments, the tumor described herein can be any tumor, including but not limited to bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, blood cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer and prostate cancer. The inflammatory disease or autoimmune disease includes but is not limited to multiple sclerosis, rheumatoid arthritis, type I diabetes, Crohn's disease, ulcerative colitis, myasthenia gravis, systemic lupus erythematosus, scleroderma, ankylosing spondylitis, graft-versus-host disease, organ transplantation, Sjögren's syndrome or autoimmune hepatitis.

[0119] In embodiments of any aspect, the methods, uses and pharmaceutical compositions described herein may further comprise administering a second agent and / or treatment to the individual, for example as part of a combination therapy. Non-limiting examples of second agents and / or treatments can include radiation therapy, surgery, gemcitabine, cilastatin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin, and carboplatin; vinblastine; vindesine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib) that reduce cell proliferation. ) and VEGF-A inhibitors and any pharmaceutically acceptable salts, acids or derivatives thereof.

[0120] In a ninth aspect, the present application provides a detection reagent or kit comprising the antibody or antigen-binding portion thereof described in the first aspect.

[0121] Antibodies or their antigen-binding portions as described herein can be combined with a detectable portion. Exemplary detectable portions include, but are not limited to, radioisotopes such as iodine 125, iodine-131, cesium-137, iridium 192, and cobalt 60, horseradish peroxidase, fluorescein isothiocyanate, biotin, alkaline phosphatase, chemiluminescent agents such as luminols, etc. Those skilled in the art can select suitable detectable portions to be combined with antibodies or their antigen-binding portions of the present application as needed, thereby achieving different detection purposes.

[0122] Throughout the specification and claims, the words “comprises,” “comprising,” and “containing” mean “including but not limited to,” and are not intended to exclude other parts, additives, components, or steps.

[0123] It will be appreciated that features, characteristics, components or steps described in conjunction with a particular aspect, embodiment or example of the present application may be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0124] The present invention discloses antibodies that specifically bind to NKG2D of mammals (humans, primates, etc.), and the present invention provides applications of such proteins in treatment, screening, and detection, such as use in cancer treatment. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the scope of the present invention. Those skilled in the art can modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the present invention.

[0125] The following examples are used to illustrate the present application, but are not intended to limit the scope of the present application. Without departing from the spirit and substance of the present application, modifications or substitutions made to the methods, steps or conditions of the present application are within the scope of the present application.

[0126] Unless otherwise specified, the chemical reagents used in the examples are conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0127] The examples do not include a detailed description of conventional methods, such as those used to construct vectors and plasmids, insert protein-encoding genes into vectors and plasmids, or introduce plasmids into host cells. Such methods are well known to those of ordinary skill in the art and are described in many publications, for example, see Sambrook, J., Fritsch, EF. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press.

[0128] The present invention will be further described below in conjunction with embodiments:

[0129] Example 1. Construction of hNKG2D high expression cell line

[0130] The hNKG2D / DAP10 high-expression cell line was constructed using a stable cell line construction platform. The specific steps are as follows:

[0131] Construction of plasmids pcDNA3.1-Hygro-hNKG2D and pcDNA3.1-G418-DAP10: hNKG2D cDNA (UniProtKB / Swiss-Prot: P26718.1, Pro72-Val216) and DAP10 cDNA (GenBank: AAH35931.1, Met1-Gly93) (synthesized by Universal Biosystems (Anhui) Co., Ltd.) was used as a template, and two small fragments of about 800 bp were amplified with primers and recovered by gel; the pcDNA3.1-Hygro and pcDNA3.1-G418 plasmids were digested with NheI / XbaI, and a large fragment of 7450 bp was recovered by gel; the two small fragments obtained above were respectively connected to the large fragments of pcDNA3.1-Hygro and pcDNA3.1-G418 plasmids digested with NheI / XbaI and recovered by gel, and the positive clones were selected for sequencing. The plasmids with correct sequencing were named pcDNA3.1-Hygro-hNKG2D and pcDNA3.1-G418-DAP10, respectively.

[0132] 293T cells (from Concord Cell Bank) were inoculated into two T25 culture flasks, with 2×10 cells in each culture flask. 6 . On the second day, the 293T cell culture medium was replaced with 4 mL Opti-MEM (Thermofisher Scientific Cat31985070). 5 μg of plasmids pcDNA3.1-Hygro-hNKG2D and pcDNA3.1-G418-DAP10, which cloned human hNKG2D and human DAP10, were added to Opti-MEM, with a final volume of 500 μL. Another 500 μL Opti-MEM was prepared, and 7 μL of transfection reagent PEI (3 μg / mL) was added thereto. The two were mixed and allowed to stand at room temperature for 20 minutes before being added to the above-cultured 4 ml 293T cells. On the third day, the cell culture medium was replaced with 5 mL DMEM high-glucose medium. On the fourth day, 100 μg / mL Hygro and 200 μg / mL G418 were added for screening. After 2-3 days, a large number of cells died. Fresh medium was replaced until the cells grew stably, and then single clone screening was performed, amplification culture was performed, and cryopreservation was performed. The constructed cell line stably expressing the target gene was named 293T-hNKG2D cells.

[0133] Example 2. Preparation of hybridomas producing anti-hNKG2D antibodies

[0134] 1. Take 8-10 week old Balb / c mice and inject hNKG2D protein (50μg / dose / mouse) subcutaneously. The extracellular region of hNKG2D protein (DNA sequence is UniProtKB / Swiss-Prot: P26718.1, protein sequence is extracellular region Phe78-Val 216, C-terminus contains His tag), and its molecular weight is 16.1kDa verified by SDS-PAGE. Repeat this dose 5-7 times within 3-8 weeks. Four days before fusion, give mice intraperitoneal injection of hNKG2D protein for 3 consecutive days.

[0135] 2. One day before fusion, take the peritoneal macrophages of ordinary Kunming mice as the trophoblast layer and inoculate them into a 96-well plate.

[0136] 3. The spleens from the immunized mice were fused with the non-secretory myeloma SP2 / 0 cell line, and the cells were added to a 96-well plate pre-coated with a trophoblast layer, and the fused cells were subjected to HAT selection (Galfre and Milstein, Methods Enzymol 1981; 73: 3-46).

[0137] 4. Recover a group of hybridoma cells that all secrete anti-hNKG2D specific antibodies. The primary screening is to determine the expression of anti-hNKG2D antibodies secreted by the hybridoma using enzyme-linked immunosorbent assay (ELISA). Table 1 shows the test results of the supernatants of 5 candidate hybridomas, clone numbers 1E8, 4A3, 9D6, 9D9, 7C5.

[0138] Table 1 Anti-hNKG2D hybridoma initial screening results (ELISA)

[0139] Initial hybridoma clone number OD450 1E8 0.225 4A3 2.111 9D6 0.392 9D9 1.085 7C5 1.328

[0140] 5. Anti-hNKG2D hybridoma rescreening (FACS)

[0141] 293T-hNKG2D cells were collected, washed once with FACS buffer, and centrifuged at 2-5 × 10 5 Cells / well were plated into a 96-well deep-well plate, and 50 μL of hybridoma supernatant was added to each well and incubated for 1 hour. Immunized mouse serum was selected as the positive control antibody and washed twice with FACS buffer. APC anti-mouse IgG Fc secondary antibody (Biolegend) was added, and the cells were washed twice with FACS buffer after incubation for 1 hour and detected by flow cytometry.

[0142] The results are shown in Table 2, and five candidate monoclonal antibodies were obtained, which were labeled as 1E8, 4A3, 9D9, 7E10, and 7C5. The two monoclonal antibodies 4A3 and 7C5 with the strongest binding activity (highest chromatin number) to 293T-hNKG2D were preferred.

[0143] Table 2 Anti-hNKG2D hybridoma rescreening results (FACS)

[0144] Initial hybridoma clone number Staining Index 1E8 0.00 4A3 7.96 9D6 0.00 9D9 0.02 7C5 2.99

[0145] Example 3. Binding activity of anti-hNKG2D monoclonal antibodies secreted by 4A3 and 7C5 hybridomas and hNKG2D-positive cells

[0146] The corresponding antibodies were purified from the supernatants of 4A3 and 7C5 hybridomas using affinity purification. Take one T75 cell culture flask containing 293T-hNKG2D cells, wash once with FACS buffer, resuspend and count with FACS buffer, and add 2×10 5 Each cell was mixed with 1 μg of antibody in 100 μL FACS buffer system, incubated at room temperature for 1 hour, and washed twice with FACS buffer. APC anti-mouse IgG Fc secondary antibody was added, incubated for 1 hour, washed twice with FACS buffer, and detected by flow cytometry. The results are shown in Table 3 and Figure 1 The results in Table 3 show that the anti-hNKG2D monoclonal antibodies 4A3, 7C5 and the positive control antibody (APC-labeled anti-hNKG2D antibody purchased from Biolegend) all have binding activity with 293T-hNKG2D cells, and the staining indexes are 5.53, 2.11 and 4.23, respectively, indicating that the positive clones 4A3 and 7C5 we screened have binding activity with cells expressing NKG2D.

[0147] Table 3 Binding activity of anti-hNKG2D antibodies to hNKG2D positive cells

[0148] Initial hybridoma clone number Staining Index Blank control 0 Positive Control 4.23 Secondary Antibody Control 0 4A3 5.53 7C5 2.11

[0149] Example 4. Ligand competition binding experiment of anti-hNKG2D monoclonal antibodies secreted by 4A3 and 7C5 hybridomas

[0150] 293T-hNKG2D cells were collected and washed once with PBS buffer, and 2×10 5 Each cell was mixed with 1 μg of antibody 4A3 or 7C5 and 1 μg of ligand hMICA-hFc (human Fc fusion protein) in 100 μL FACS buffer system, and blank, secondary antibody control and single ligand MICA control groups were set up and incubated for 1 hour. FACS buffer was washed twice, and APC anti-human IgG Fc secondary antibody (Biolegend) was added. After incubation for 1 hour, flow cytometry was used for detection.

[0151] The results are shown in Table 4 and Figure 2As shown, when hMICA-hFc was incubated with 293T-hNKG2D cells alone, binding activity was detected, and the staining index was 3.22. When antibodies 4A3 or 7C5 were incubated with hMICA-hFc and 293T-hNKG2D cells at the same time, hMICA-hFc could not bind to 293T-hNKG2D cells, and the staining index was 0.21 or 0.04, indicating that antibodies 4A3 and 7C5 can specifically block the binding of the ligand MICA protein to 293T-hNKG2D, proving that the two anti-hNKG2D antibodies are blocking antibodies.

[0152] Table 4 Binding activity of anti-hNKG2D antibodies to hNKG2D positive cells

[0153] Initial hybridoma clone number Staining Index Blank control -0.02 Secondary Antibody Control 0.00 hMICA-hFc group 3.22 Antibody 4A3 and hMICA-hFc mixed group 0.21 Antibody 7C5 and hMICA-hFc mixed group 0.04

[0154] Example 5. Binding activity of anti-hNKG2D chimeric antibody and hNKG2D positive cells (FACS detection)

[0155] Preparation of anti-hNKG2D chimeric antibody:

[0156] The cDNA of 4A3 and 7C5 hybridoma cells was obtained, and the antibody primers stored in this laboratory were used for PCR amplification and sequencing, and finally the nucleotide sequences encoding the heavy chain variable region and light chain variable region of the mouse anti-hNKG2D antibody 4A3 and 7C5 were obtained. The PCR amplification products of the above variable region sequences were connected into the expression vector by homologous recombination seamless cloning method, the heavy chain variable region was connected into pQKX1LALA, and the light chain variable region was connected into pQKX2, and the heavy chain expression vectors pQK ChimAb 4A3-NKG2D-H, pQKChimAb 4A3-NKG2D-L, pQK ChimAb4A3-NKG2D-H and pQK ChimAb 4A3-NKG2D-L were obtained.

[0157] FreeStyle TM 293-F cells (Thermo Fisher) were diluted to 1.5×10 6cells / mL, the final volume is 200 ml, and cultured at 37°C in a shaking incubator for 24 hours. Plasmids pQK Ab4A3-NKG2D-H and pQKAb 4A3-NKG2D-L were diluted with fresh culture medium at 10% of the transfection volume to a concentration of 1 μg / mL calculated based on the volume of transfected cells. Add 200 μl of 3 mg / mL PEI to the diluted plasmid at 1 / 1000 of the cell volume, vortex immediately for 10 seconds, leave at room temperature for 15 minutes, add the plasmid / PEI mixture dropwise to the cell culture medium, gently shake the culture bottle while adding, and culture on a shaker. Collect the culture supernatant after 7 days, filter with a 0.4 μm filter membrane, and use a ProA purification column to capture antibodies from the culture supernatant. The flow rate is set to 3 ml / min, and the purification column is equilibrated with 5 column volumes of 20 mM PB + 150 mM Nacl, PH7.4 equilibrium solution. After the column is balanced and stable, load the sample. After loading, select 20 mM PB + 150 mM Nacl, PH7.4 for elution. After elution, use 50 mM citric acid pH3.0 eluent for elution. Collect the antibody with 1 M The eluted mouse antibody Chim-Ab-NKG2D-4A3 (the amino acid sequences of its heavy chain and light chain are shown in SEQ ID NO:13 and SEQ ID NO:15, respectively) was neutralized with Tris-HCl, pH 9.0, and the anti-NKG2D chimeric antibody Chim-Ab-NKG2D-7C5 (the amino acid sequences of its heavy chain and light chain are shown in SEQ ID NO:14 and SEQ ID NO:16, respectively) was obtained by the same method.

[0158] Collect 293T-hNKG2D cells from a T75 cell culture flask, wash once with FACS buffer, resuspend and count with FACS buffer, and count 2×10 5 Each cell was mixed with 1 μg of antibody in 100 μL of FACS buffer system and incubated at room temperature for 1 hour.

[0159] The cells were washed twice with FACS buffer, APC anti-human IgG Fc secondary antibody (Biolegend) was added, incubated for 1 hour, washed twice with FACS buffer, and detected by flow cytometry. The results are shown in Tables 5 and Figure 3 The results in Table 5 show that the anti-hNKG2D chimeric antibodies Chim-Ab-NKG2D-4A3 and Chim-Ab-NKG2D-7C5 both specifically bind to 293T-hNKG2D cells, with staining indices of 3.77 and 3.71, respectively, proving that the chimeric antibodies still have binding activity to cells expressing NKG2D.

[0160] Table 5 Binding activity of anti-hNKG2D chimeric antibodies and hNKG2D positive cells

[0161] sample Staining Index Blank control 0.00 Positive Control 4.23 Secondary Antibody Control 0.00 Chim-Ab-NKG2D-4A3 3.77 Chim-Ab-NKG2D-7C5 3.71

[0162] Example 6. Determination of Kinetic Parameters of Antibodies

[0163] The kinetic parameters of hNKG2D binding to anti-hNKG2D chimeric antibodies (4A3 and 7C5) were determined using the Fortebio molecular interaction instrument. Each monoclonal antibody was captured and fixed with a ProA sensor, and after equilibration in PBS, it was bound to the gradient diluted antigen hNKG2D. The parameter detection results are shown in Table 6. The KD values ​​of all candidate antibodies were below 1 nM, proving that antibodies 4A3 and 7C5 have high affinity for hNKG2D.

[0164] Table 6 Kinetic parameters of hNKG2D binding to anti-hNKG2D chimeric antibodies

[0165] Antibody ID KD(M) KD Error kon(1 / Ms) Kon Error kdis(1 / s) R^2 4A3 <1.0E-12 3.52E-09 9.40E+03 2.47E+02 <1.0E-07 0.9938 7C5 <1.0E-12 3.44E-09 1.01E+04 2.77E+02 <1.0E-07 0.9925

[0166] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. Sequence Listing <110> Beijing Immune Ark Pharmaceutical Technology Co., Ltd. <120> Anti-hNKG2D antibodies and their applications <130> MP21001078 <160> 20 <170> SIPOSequenceListing 1.0 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <400> 1 Gly Tyr Ser Phe Thr Ser Tyr Trp 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <400> 2 Gly Phe Thr Phe Ser Asp Tyr Tyr 1 5 <210> 3 <211> 8 <212> PRT <213> Artificial Sequence <400> 3 Ile His Pro Ser Asp Ser Glu Thr 1 5 <210> 4 <211> 8 <212> PRT <213> Artificial Sequence <400> 4 Ile Ser Asp Gly Gly Arg Tyr Thr 1 5 <210> 5 <211> 13 <212> PRT <213> Artificial Sequence <400> 5 Ala Arg His Tyr Tyr Gly Tyr Val Asp Phe Met Asp Tyr 1 5 10 <210> 6 <211> 16 <212> PRT <213> Artificial Sequence <400> 6 Ala Arg Asp Gly Arg Leu His Asp Gly Tyr Tyr Gly Arg Phe Ser Tyr 1 5 10 15 <210> 7 <211> 6 <212> PRT <213> Artificial Sequence <400> 7 Gln Glu Ile Ser Gly Tyr 1 5 <210> 8 <211> 5 <212> PRT <213> Artificial Sequence <400> 8 Ser Ser Val Ser Tyr 1 5 <210> 9 <211> 3 <212> PRT <213> Artificial Sequence <400> 9 Ala Ala Ser 1 <210> 10 <211> 3 <212> PRT <213> Artificial Sequence <400> 10 Asp Thr Ser 1 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <400> 11 Leu Gln Tyr Val Ala Tyr Pro Trp Thr 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <400> 12 Gln Gln Trp Ile Ser Tyr Pro Pro Thr 1 5 <210> 13 <211> 120 <212> PRT <213> Artificial Sequence <400> 13 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Val Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Tyr Tyr Gly Tyr Val Asp Phe Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 14 <211> 123 <212> PRT <213> Artificial Sequence <400> 14 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Asp Gly Gly Arg Tyr Thr Tyr Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asp Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Arg Leu His Asp Gly Tyr Tyr Gly Arg Phe Ser Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 15 <211> 108 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 15 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr 20 25 30 Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Phe Val Asp Tyr Tyr Cys Leu Gln Tyr Val Ala Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Asn Arg 100 105 <210> 16 <211> 106 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 16 Glu Leu Gln Met Thr Gln Ser Pro Ala Thr Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Leu Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ile Ser Tyr Pro Pro Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 17 <211> 360 <212> DNA <213> Artificial Sequence <400> 17 caggtgcagc tgcagcagcc tggggctgag ctggtgaggc ctggagcttc agtgaagctg 60 tcctgcaagg cttctggcta ctccttcacc agttactgga tgaactgggt gaagcagagg 120 cctggacaag gccttgagtg gattggcatg attcatcctt ccgatagtga aactaggtta 180 aatcagaagt tcaaggacaa ggccacattg actgtagaca aatcctccag tacagtctac 240 atgcaactca gcagcccgac atctgaggac tctgcggtct attactgtgc aagacattac 300 tacggctacg tggattttat ggactactgg ggtcaaggaa cctcagtcac cgtctcctca 360 <210> 18 <211> 369 <212> DNA <213> Artificial Sequence <400> 18 gaggtccagc ttgtcgagtc agggggaggc ttagtgaagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cactttcagt gactattata tgtattgggt tcgccagact 120 ccggaaaaga ggctggagtg ggtcgcaacc attagtgatg gtggtcgtta cacctactat 180 tcagacagtg tgaaggggcg attcaccatc tccagagaca atgccaagaa cgacctgtac 240 ctgcaaatga gcagtctgaa gtctgaggac acagccatgt attactgtgc aagagatggg 300 aggctccatg atggttacta cgggaggttt tcttactggg gccaagggac tctggtcact 360 gtctctgca 369 <210> 19 <211> 324 <212> DNA <213> Artificial Sequence <400> 19 gatattcaga tgacccagag tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60 ctcacttgtc gggcaagtca ggaaattagt ggttacttaa gctggcttca gcaaaaacca 120 gatgggacta ttaaacgcct gatctacgcc gcatccactt tagattctgg tgtcccaaaa 180 aggttcagtg gcagtaggtc tgggtcagat tattctctca ccatcagcag ccttgagtct 240 gaagattttg tagactatta ctgtctacaa tatgttgctt atccgtggac gttcggtgga 300 ggcaccaagc tggaaatcaa ccgg 324 <210> 20 <211> 318 <212> DNA <213> Artificial Sequence <400> 20 gagctccaga tgacacagtc tccagcaacc atgtctgcat ctccagggga gaaggtcacc 60 atgacctgca gtgccagctc aagtgtcagt tacatgtatt ggtaccagca gaagccagga 120 tcctccccca gactcctgat ttatgacaca tccaacctgg cttctggagt ccctgttcgc 180 ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagccgact ggaggctgaa 240 gatgctgcca cttattactg ccagcagtgg attagttacc cacccacgtt cggctcgggg 300 acaaagttgg aaataaaa 318

Claims

1. An antibody or antigen-binding portion thereof that specifically binds to hNKG2D, characterized in that: It comprises a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3; The amino acid sequence of the HCDR1 is shown in SEQ ID No. 1; The amino acid sequence of the HCDR2 is shown in SEQ ID No. 3; and The amino acid sequence of the HCDR3 is shown in SEQ ID No.5; The antibody or antigen binding portion thereof that specifically binds to hNKG2D further comprises a light chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3; The amino acid sequence of LCDR1 is shown in SEQ ID No.7; The amino acid sequence of the LCDR is shown in SEQ ID No. 9; and The amino acid sequence of the LCDR3 is shown in SEQ ID No.

11.

2. The antibody or antigen-binding portion thereof according to claim 1, wherein The antigen binding portion is selected from the group consisting of: a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment or a scFv fragment.

3. A nucleic acid molecule encoding the antibody or antigen-binding portion thereof as claimed in claim 1 or 2.

4. An expression vector, characterized in that Comprising the nucleic acid molecule as described in claim 3.

5. A host cell transfected or transformed with the expression vector according to claim 4.

6. A drug or a pharmaceutical composition, characterized in that Comprising the antibody or antigen-binding portion thereof as claimed in claim 1 or 2.

7. Use of the antibody or antigen-binding portion thereof according to claim 1 or 2 in the preparation of a product for detecting hNKG2D; The products include detection reagents, detection kits, detection chips or detection devices.

8. A detection reagent, a detection kit, a detection chip or a detection device, comprising the antibody or the antigen-binding portion thereof according to claim 1 or 2 and an auxiliary agent acceptable in the detection.

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