Anti-CD94 antibodies and their applications

Abstract

Provided is an anti-CD94 antibody and its use, the antibody comprising heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, the amino acid sequences of which are at least 80% identical to the sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and the amino acid sequences of which are at least 80% identical to the sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively. The antibody can bind to human CD94 protein, relieve CD94 / NKG2A-mediated immunosuppression, promote T cell activation, and effectively treat or prevent CD94-mediated related diseases.

Description

[Technical Field]

[0001] The present invention relates to the biomedical field, specifically to anti-CD94 antibodies and applications. [Background technology]

[0002] Cancer poses a serious threat to the life and health of humans. In addition to conventional treatments such as surgical resection, radiation therapy, and chemotherapy, various cancer treatment methods are currently under development, including small molecule targeted therapy, immune checkpoint therapy, and cell and gene therapy.

[0003] In recent years, immunotherapy, represented by PD-1 / L1, has shown great potential, and three immunotherapies, namely PD-1 / L1, CTLA-4, and LAG-3, have already been approved. However, what cannot be ignored is that even though the overall response rate of PD-1 / L1 therapy, which has the widest range of currently approved indications, is still only 30%, more patients will not be able to benefit from it.

[0004] Immune checkpoint molecules are inhibitory molecules found on the surface of immune cells, including T, NK, monocytes, and macrophages. After binding to their corresponding ligands, they transmit inhibitory signals into immune cells, suppressing their anti-cancer functions.

[0005] Because immune checkpoint ligand expression in tumors and tumor-infiltrating lymphocytes is highly heterogeneous, a single immune checkpoint therapy cannot be applied to all patients, and many patients will not benefit from it. In addition, some patients who have received immune checkpoint therapy will experience tumor recurrence and develop resistance to the immune checkpoint therapy, resulting in no therapeutic effect even after continued administration. For these two reasons, the development of immune checkpoint antibodies with more targets is necessary.

[0006] CD94 is expressed on the surface of NK cells and T cells and forms heterodimers with NKG2A and NKG2C, both of which are ligands for HLA-E. CD94 / NKG2A is an inhibitory receptor, while CD94 / NKG2C is an activating receptor. However, tumor-infiltrating NK cells and T cells predominantly express CD94 / NKG2A, rather than non-CD94 / NKG2C, on their surface. Therefore, immune checkpoint therapies targeting CD94 and NKG2A are potentially valuable.

[0007] To date, no immune checkpoint therapy targeting CD94 has been publicly reported or entered clinical trials worldwide, and the most rapid progress in NKG2A-targeting therapy is Innate Pharma's huZ270 antibody. Therefore, the development of immune checkpoint therapy targeting CD94 is of great significance for the treatment and prevention of diseases. Summary of the Invention [Problem to be solved by the invention]

[0008] The present application has been filed based on the inventor's discovery of the following problems and facts.

[0009] CD94 is expressed on the surface of NK cells and T cells, forming heterodimers with NKG2A and NKG2C. Tumor-infiltrating NK cells and T cells primarily express CD94 / NKG2A on their surface, and CD94 / NKG2A is an inhibitory receptor. Therefore, immune checkpoint therapies targeting CD94 and NKG2A are both potentially valuable. However, there are not many tumor treatment and research tools for molecules such as CD94.

[0010] The present inventors have successfully screened for a mouse-derived anti-CD94 monoclonal antibody with high binding activity to human CD94 protein. The inventors then humanized the constant region of the monoclonal antibody, retaining the CDRs of the mouse-derived anti-CD94 monoclonal antibody to obtain a chimeric antibody. Furthermore, the inventors further humanized the framework regions of the light chain variable region or heavy chain variable region of the chimeric antibody to obtain a fully humanized anti-CD94 antibody. This humanized antibody specifically targets and binds to human CD94 protein and has the advantages of low immunogenicity, allowing it to effectively treat and / or prevent CD94-mediated diseases. The present invention compared the in vitro activity of a CD94 antibody with the NKG2A antibody huZ270, and found that the CD94 antibody was more potent than the NKG2A antibody in activating Jurkat T cells in a reporter system. [Means for solving the problem]

[0011] Thus, in a first aspect, the present invention provides an antibody or antigen-binding fragment, according to an embodiment of the present invention, comprising a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, and a light chain CDR1, a light chain CDR2 and a light chain CDR3, the amino acid sequence of the heavy chain CDR1 has at least 80% identity with the sequence shown in SEQ ID NO: 1; the amino acid sequence of the heavy chain CDR2 has at least 80% identity with the sequence shown in SEQ ID NO: 2; the amino acid sequence of the heavy chain CDR3 has at least 80% identity with the sequence shown in SEQ ID NO: 3; the amino acid sequence of the light chain CDR1 has at least 80% identity with the sequence set forth in SEQ ID NO: 4; the amino acid sequence of the light chain CDR2 has at least 80% identity to the sequence set forth in SEQ ID NO: 5; The amino acid sequence of the light chain CDR3 has at least 80% identity to the sequence set forth in SEQ ID NO: 6. The antibodies or antigen-binding fragments according to embodiments of the present invention can bind to human CD94 protein and can effectively treat or prevent CD94-mediated related diseases, and can be used in related scientific research.

[0012] According to an embodiment of the present invention, the antibody or antigen-binding fragment may further comprise at least one of the following additional technical features:

[0013] According to an embodiment of the invention, the antibody or antigen-binding fragment comprises at least one of a heavy chain FR region and a light chain FR region.

[0014] According to an embodiment of the present invention, at least a portion of at least one of the heavy chain FR region and the light chain FR region is derived from at least one of a human-derived antibody, a primate-derived antibody, a mouse-derived antibody, or a variant thereof.

[0015] According to an embodiment of the present invention, the antibody or antigen-binding fragment has at least one of the heavy chain framework regions HFR1, HFR2, HFR3, and HFR4 sequences shown in SEQ ID NOs: 7 to 10, respectively, or at least one of the heavy chain framework regions HFR1, HFR2, HFR3, and HFR4 sequences shown in SEQ ID NOs: 15 to 18, respectively.

[0016] According to an embodiment of the present invention, the antibody or antigen-binding fragment has at least one of the light chain framework regions LFR1, LFR2, LFR3, and LFR4 sequences shown in SEQ ID NOs: 11 to 14, respectively, or at least one of the light chain framework regions LFR1, LFR2, LFR3, and LFR4 sequences shown in SEQ ID NOs: 19 to 22, respectively.

[0017] According to an embodiment of the present invention, the antibody or antigen-binding fragment has at least one of the heavy chain framework regions HFR1, HFR2, HFR3, and HFR4 sequences shown in SEQ ID NOs: 7 to 10, respectively, and at least one of the light chain framework regions LFR1, LFR2, LFR3, and LFR4 sequences shown in SEQ ID NOs: 11 to 14, respectively, or at least one of the heavy chain framework regions HFR1, HFR2, HFR3, and HFR4 sequences shown in SEQ ID NOs: 15 to 18, respectively, and at least one of the light chain framework regions LFR1, LFR2, LFR3, and LFR4 sequences shown in SEQ ID NOs: 19 to 22, respectively.

[0018] According to an embodiment of the invention, the antibody or antigen-binding fragment comprises a heavy chain variable region set forth in SEQ ID NO:23 or SEQ ID NO:25 and / or a light chain variable region set forth in SEQ ID NO:24 or SEQ ID NO:26.

[0019] According to an embodiment of the invention, the antibody or antigen-binding fragment comprises 1) a heavy chain variable region set forth in SEQ ID NO:23 and a light chain variable region set forth in SEQ ID NO:24, or 2) a heavy chain variable region set forth in SEQ ID NO:25 and a light chain variable region set forth in SEQ ID NO:26.

[0020] According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises at least one of a heavy chain constant region and a light chain constant region, and at least a portion of the heavy chain constant region and / or the light chain constant region is derived from at least one of a human-derived antibody, a primate-derived antibody, a mouse-derived antibody, or a variant thereof.

[0021] According to an embodiment of the present invention, the light chain constant region and the heavy chain constant region are both derived from a mouse-derived IgG antibody or a mutant thereof, or a human-derived IgG antibody or a mutant thereof.

[0022] According to an embodiment of the present invention, the light chain constant region and the heavy chain constant region are both derived from a mouse-derived IgG1 or IgG4 antibody or a mutant thereof, or a human-derived IgG1 or IgG4 antibody or a mutant thereof.

[0023] According to an embodiment of the present invention, the light chain constant region comprises a human-derived or mouse-derived kappa or lambda chain or a variant thereof.

[0024] According to an embodiment of the invention, the antibody comprises a heavy chain constant region having the amino acid sequence shown in SEQ ID NO: 27, 29 or 31 and / or a light chain constant region having the amino acid sequence shown in SEQ ID NO: 28, 30 or 32.

[0025] According to an embodiment of the invention, the antibody has a heavy chain constant region of the amino acid sequence shown in SEQ ID NO:27 and a light chain constant region of the amino acid sequence shown in SEQ ID NO:28.

[0026] According to an embodiment of the invention, the antibody has a heavy chain constant region of the amino acid sequence shown in SEQ ID NO:29 and a light chain constant region of the amino acid sequence shown in SEQ ID NO:30.

[0027] According to an embodiment of the invention, the antibody has a heavy chain constant region of the amino acid sequence shown in SEQ ID NO:31 and a light chain constant region of the amino acid sequence shown in SEQ ID NO:32.

[0028] According to an embodiment of the invention, the antibody or antigen-binding fragment has a heavy chain having an amino acid sequence set forth in any one of SEQ ID NOs: 33, 35, 36, and 38, and a light chain having an amino acid sequence set forth in any one of SEQ ID NOs: 34, 37, and 39.

[0029] According to embodiments of the invention, the antibody or antigen-binding fragment has a heavy chain having the amino acid sequence set forth in SEQ ID NO:33 and a light chain having the amino acid sequence set forth in SEQ ID NO:34; the antibody or antigen-binding fragment has a heavy chain having the amino acid sequence set forth in SEQ ID NO:35 and a light chain having the amino acid sequence set forth in SEQ ID NO:37; the antibody or antigen-binding fragment has a heavy chain having the amino acid sequence set forth in SEQ ID NO:36 and a light chain having the amino acid sequence set forth in SEQ ID NO:37; or the antibody or antigen-binding fragment has a heavy chain having the amino acid sequence set forth in SEQ ID NO:38 and a light chain having the amino acid sequence set forth in SEQ ID NO:39.

[0030] According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises a monoclonal antibody or a polyclonal antibody.

[0031] According to an embodiment of the present invention, the monoclonal antibody comprises at least one of an Fv, a single chain antibody, an Fab, a single domain antibody, and a minimal recognition unit.

[0032] According to an embodiment of the invention, the antibody or antigen-binding fragment thereof is capable of binding to the amino acid sequence shown in SEQ ID NO:40.

[0033] In a second aspect, the present invention provides a nucleic acid molecule encoding the antibody or antigen-binding fragment according to the first aspect. In some specific embodiments of the present invention, the antibody or antigen-binding fragment encoded by the nucleic acid molecule binds to human CD94 protein and can effectively treat or prevent CD94-mediated related diseases.

[0034] According to an embodiment of the present invention, the nucleic acid molecule may further include at least one of the following additional technical features:

[0035] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0036] It should be understood by those skilled in the art that the nucleic acids referred to in the present specification and claims actually include either or both of the complementary strands. For convenience, the specification and claims often refer to only one strand, but in reality, the complementary strand is also disclosed. Furthermore, references to nucleic acid sequences in this application include DNA and RNA forms, and disclosure of one means disclosure of the other.

[0037] In a third aspect, the present invention provides an expression vector carrying the aforementioned nucleic acid molecule. The expression vector may include a selectable control sequence, which is operably linked to the nucleic acid molecule. The control sequence may be one or more control sequences capable of directing expression of the nucleic acid molecule in a host. The expression vectors mentioned in the examples of the present invention are capable of efficiently expressing the antibody or antigen-binding fragment in a suitable host cell in large amounts.

[0038] As used herein, "operably linked" refers to the ability of a foreign gene to be connected to a vector, allowing control elements within the vector, such as transcriptional and translational control sequences, to function in regulating the transcription and translation of the intended foreign gene. When the nucleic acid molecule is connected to a vector, the nucleic acid molecule can be directly or indirectly linked to the control elements on the vector, and these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can be derived directly from the vector itself or can be exogenous, i.e., not necessarily derived from the vector itself. As will be appreciated by those skilled in the art, nucleic acid molecules encoding antibodies or antigen-binding fragments can be inserted independently into different vectors, but are typically inserted into the same vector. Commonly used vectors include plasmids and bacteriophages, such as Plasmid-X plasmids.

[0039] In a fourth aspect, the present invention provides a method for preparing the aforementioned antibody or antigen-binding fragment, comprising the steps of introducing the aforementioned expression vector into cells and culturing the cells under conditions suitable for protein expression and secretion, thereby obtaining the antibody or antigen-binding fragment. The method according to some specific embodiments of the present invention allows for the effective ex vivo production of large amounts of the antibody or antigen-binding fragment.

[0040] According to some specific embodiments of the present invention, the above-mentioned method for preparing an antibody or antigen-binding fragment may further include at least one of the following additional technical features:

[0041] According to some specific embodiments of the present invention, the cells are not particularly limited and may be prokaryotic or eukaryotic cells.

[0042] According to some specific embodiments of the invention, the cell is a eukaryotic cell.

[0043] According to some specific embodiments of the present invention, the eukaryotic cells are mammalian cells. According to some specific examples of the present invention, when the cells are eukaryotic cells, for example mammalian cells, the expression efficiency of the recombinant antibody is high.

[0044] In a fifth aspect, the present invention provides a recombinant cell that carries the aforementioned nucleic acid or expression vector or is capable of expressing the aforementioned antibody or antigen-binding fragment. The recombinant cell is obtained by transfection or transformation with the aforementioned expression vector. According to some specific embodiments of the present invention, the recombinant cell efficiently expresses the antibody or antigen-binding fragment in large amounts under appropriate conditions.

[0045] The recombinant cells described in the present invention are not particularly limited and may be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells may be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis. The eukaryotic cells may be fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Trichoderma; insect cells such as grass rotifers; plant cells such as tobacco; or mammalian cells such as BHK cells, CHO cells, COS cells, or myeloma cells. In some embodiments, the recombinant cells described in the present invention are preferably mammalian cells, including BHK cells, CHO cells, NSO cells, or COS cells, but do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0046] The term "appropriate conditions" as used herein refers to conditions suitable for the expression of the antibody or antigen-binding fragment described herein. As those skilled in the art will readily understand, suitable conditions for the expression of an antibody or antigen-binding fragment include, but are not limited to, a suitable transformation or transfection method, suitable transformation or transfection conditions, a healthy host cell state, a suitable host cell density, a suitable cell culture environment, and a suitable cell culture time. The "appropriate conditions" are not particularly limited, and those skilled in the art will optimize the optimal conditions for the expression of the antibody or antigen-binding fragment according to the specific laboratory environment.

[0047] In a sixth aspect, the present invention provides an immunoconjugate, comprising the above-described antibody or antigen-binding fragment and a therapeutic agent. As described above, the antibodies or antigen-binding fragments according to embodiments of the present invention can effectively bind to the CD94 protein and effectively treat or prevent CD94-mediated diseases. Therefore, immunoconjugates comprising the antibodies or antigen-binding fragments can similarly bind to human CD94 protein, and the immunoconjugates have good efficacy in preventing and / or treating CD94-mediated diseases.

[0048] In a seventh aspect, the present invention provides a composition comprising the above-described antibody or antigen-binding fragment, nucleic acid molecule, expression vector, or recombinant cell. As described above, the antibody or antigen-binding fragment according to some specific embodiments of the present invention can effectively bind to human CD94 protein and inhibit tumor cell proliferation. Therefore, compositions comprising the above substances can also effectively bind to human CD94 protein and effectively treat or prevent CD94-mediated diseases. The type of the composition is not particularly limited and may be a food composition or a pharmaceutical composition.

[0049] The compositions of the present invention can be administered in combination with each other or with one or more other therapeutic compounds, for example, in combination with a chemotherapeutic agent. Thus, the compositions may further comprise a chemotherapeutic agent. The antibodies or antigen-binding fragments thereof, or immunoconjugates of the present invention may be combined with a second therapeutic agent, examples of which include, but are not limited to, other agents that inhibit CD94 activity (including other antibodies or antigen-binding fragments thereof, peptide inhibitors, small molecule antagonists, etc.) and / or agents that interfere with signal transduction upstream or downstream of CD94.

[0050] The composition includes combinations of components separated in time and / or space as long as they can act cooperatively to achieve the objectives of the present invention. For example, the components contained in the composition can be administered to a subject as a whole or separately. When the components contained in the composition are administered to a subject separately, the components can be administered to the subject simultaneously or sequentially.

[0051] In an eighth aspect of the present invention, the present invention includes a drug comprising the above-described antibody or antigen-binding fragment, nucleic acid molecule, expression vector, recombinant cell, or composition. As described above, the antibody or antigen-binding fragment according to some specific embodiments of the present invention can bind to human CD94 protein, and therefore, an active ingredient or a drug of a series of substances comprising an effective amount of the antibody or antigen-binding fragment can also effectively bind to human CD94 protein and effectively treat or prevent CD94-mediated diseases.

[0052] According to an embodiment of the present invention, the drug may further include at least one of the following additional technical features:

[0053] According to an embodiment of the present invention, the medicament may comprise a pharmaceutically acceptable vector.

[0054] As used herein, "effective amount" or "effective dose" means an amount that produces a function or activity in humans and / or animals and is acceptable to humans and / or animals.

[0055] The effective amount of the antibody or antigen-binding fragment described in the present invention can vary depending on the mode of administration and the severity of the disease being treated. The selection of a preferred effective amount can be determined by one of skill in the art based on various factors (e.g., clinical trials). These factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life, the severity of the disease being treated in the patient, the patient's body weight, the patient's immune status, and the route of administration. For example, the antibody or antigen-binding fragment may be administered in divided doses daily or the dose may be proportionally reduced depending on the exigencies of the therapeutic situation.

[0056] As used herein, a "pharmaceutically acceptable" component is a substance that can be administered to humans and / or mammals without undue side effects (e.g., toxicity, irritation, allergies, etc.), i.e., a substance that has a reasonable benefit / risk ratio. The term "pharmaceutically acceptable vector" refers to a vector used to administer a therapeutic agent, including various excipients and diluents.

[0057] The medicament of the present invention comprises a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable vector. Such vectors include, but are not limited to, saline, buffer, glucose, water, glycerin, ethanol, and combinations thereof. Typically, the pharmaceutical formulation is tailored to the mode of administration, which may be oral, nasal, intradermal, subcutaneous, intramuscular, intravenous, or intraperitoneal. The dosage form of the medicament of the present invention may be an injection, oral preparation (tablet, capsule, oral liquid), transdermal preparation, or sustained-release preparation. For example, it may be prepared by conventional methods using saline or an aqueous solution containing glucose and other excipients. The medicament is preferably manufactured under sterile conditions. The antibody or antigen-binding fragment may be administered by intravenous infusion or injection, or intramuscular or subcutaneous injection.

[0058] Of course, the anti-CD94 monoclonal antibodies herein may be prepared as part of a kit or other diagnostic reagent, if desired.

[0059] In a ninth aspect of the present invention, a kit is provided, comprising the aforementioned antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, or recombinant cell. As described above, antibodies or antigen-binding fragments according to some specific embodiments of the present invention can bind to human CD94 protein, and kits containing the antibodies or antigen-binding fragments can effectively detect human CD94 protein qualitatively or quantitatively. Kits according to the present invention may be used for detection utilizing the specific binding ability of antibodies to human CD94, such as immunoblotting or immunoprecipitation. These kits may also include one or more of antagonists, anti-CD94 antibody or drug reference materials, protein purification columns, immunosphere protein affinity purification buffers, cell assay diluents, specifications, literature, etc. Anti-CD94 antibodies can be used in various types of diagnostic tests, for example, to detect the presence of various diseases, drugs, toxins, or other proteins in vitro or in vivo. For example, they can be used to test for related diseases and conduct scientific research by detecting human CD94 protein in a sample to be detected. Such related diseases may include CD94-related diseases, such as cancer. Of course, the antibodies or antigen-binding fragments provided herein can also be used for radioimmunodetection and radioimmunotherapy of the above diseases. The binding molecules are similarly applicable to the above application scenarios, and therefore will not be described again here.

[0060] The kit may also include materials commonly used for detecting CD94, such as coating solutions.

[0061] In a tenth aspect, the present invention provides use of the aforementioned antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, recombinant cell, or composition in the preparation of a medicament for preventing and / or treating a CD94-mediated related disease. As described above, the antibody or antigen-binding fragment according to some specific embodiments of the present invention can bind to human CD94 protein, and therefore a medicament comprising an effective amount of the antibody or antigen-binding fragment, or a series of substances thereof, can similarly effectively bind to human CD94 protein and effectively treat or prevent a CD94-mediated disease.

[0062] According to an embodiment of the present invention, the use of preparing the above-mentioned drug may further include at least one of the following additional technical features:

[0063] According to an embodiment of the present invention, the CD94-mediated associated diseases include transplant rejection, autoimmune diseases, infectious diseases and cancer.

[0064] According to an embodiment of the present invention, the autoimmune disease comprises at least one of systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, chronic atrophic gastritis with pernicious anemia, pulmonary hemorrhagic nephritic syndrome, pemphigus vulgaris, pemphigoid, primary biliary cholangitis, multiple sclerosis, and acute idiopathic polyneuropathy.

[0065] According to an embodiment of the present invention, the cancer comprises at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0066] In an eleventh aspect, the present invention provides use of the aforementioned antibody or antigen-binding fragment, nucleic acid molecule, expression vector, or recombinant cell in the preparation of a kit for detecting CD94. As described above, the antibodies or antigen-binding fragments according to some specific embodiments of the present invention bind to human CD94 protein and inhibit the binding of the CD94 protein to its receptor, and therefore the antibodies or antigen-binding fragments can be used to prepare a kit for detecting CD94 protein, which can effectively detect human CD94 protein qualitatively or quantitatively.

[0067] In a twelfth aspect, the present invention provides a method for treating or preventing a CD94-mediated related disease. According to an embodiment of the present invention, the method comprises administering to a subject at least one of the following: 1) the antibody or antigen-binding fragment described above, 2) the nucleic acid molecule described above, 3) the expression vector described above, 4) the recombinant cell described above, 4) the composition described above, and 6) the drug described above. As described above, the antibody or antigen-binding fragment can bind to human CD94 protein and can effectively treat or prevent a CD94-mediated related disease, preferably an autoimmune disease or cancer. Therefore, the method according to an embodiment of the present invention can effectively treat or prevent a CD94-mediated related disease.

[0068] According to an embodiment of the present invention, the method for treating or preventing the above diseases may further include at least one of the following additional technical features:

[0069] According to an embodiment of the present invention, the CD94-mediated related diseases include transplant rejection, autoimmune diseases, infectious diseases, and cancer. As will be understood by those skilled in the art, CD94 antibodies are highly expressed on the surface of NK cells. However, excessive activation of NK cells in sterile inflammation (transplant rejection, autoimmune diseases) and bacterial inflammation (infectious diseases) can lead to tissue and organ damage. Therefore, CD94 antibodies can be used as depleting antibodies to treat or prevent the above diseases.

[0070] According to an embodiment of the present invention, the autoimmune disease comprises at least one of systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, chronic atrophic gastritis with pernicious anemia, pulmonary hemorrhagic nephritic syndrome, pemphigus vulgaris, pemphigoid, primary biliary cholangitis, multiple sclerosis, and acute idiopathic polyneuropathy.

[0071] According to an embodiment of the present invention, the cancer comprises at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0072] In a thirteenth aspect, the present invention provides a method for diagnosing a CD94-mediated related disease. According to one embodiment of the present invention, the method comprises the steps of detecting CD94 in a sample to be detected using at least one of 1) the antibody or antigen-binding fragment described above, 2) the nucleic acid molecule described above, 3) the expression vector described above, and 4) the recombinant cell described above, and determining the amount of CD94 in the sample to be detected based on the CD94 detection result. Because the antibody or antigen-binding fragment described herein, or the antibody or antigen-binding fragment expressed in the nucleic acid molecule, expression vector, or recombinant cell, can effectively bind to human CD94 protein, or because the antibody or antigen-binding fragment expressed in the nucleic acid molecule, expression vector, or recombinant cell can effectively bind to CD94 protein, the method described herein can effectively detect the amount of CD94 in a sample to be detected from a subject, thereby enabling the effective diagnosis of a CD94-mediated related disease.

[0073] According to an embodiment of the present invention, the method for diagnosing the disease may further include at least one of the following additional technical features:

[0074] According to an embodiment of the present invention, the CD94 content in the test sample is equal to or greater than the minimum threshold for a disease, which indicates that the test sample is derived from a patient with a CD94-related disease. The minimum threshold value can be determined by comparing and verifying the difference in CD94 content in the test sample between a number of individuals with the CD94-related disease and a number of healthy individuals.

[0075] According to an embodiment of the present invention, the detection target sample includes at least one of blood, saliva, sweat, tissue, cells, blood, serum, plasma, feces, and urine.

[0076] According to an embodiment of the present invention, the CD94-mediated associated diseases include transplant rejection, autoimmune diseases, infectious diseases and cancer.

[0077] According to an embodiment of the present invention, the autoimmune disease comprises at least one of systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, chronic atrophic gastritis with pernicious anemia, pulmonary hemorrhagic nephritic syndrome, pemphigus vulgaris, pemphigoid, primary biliary cholangitis, multiple sclerosis, and acute idiopathic polyneuropathy.

[0078] According to an embodiment of the present invention, the cancer comprises at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0079] In a fourteenth aspect, the present invention provides a method for staging a CD94-mediated related disease. According to one embodiment of the present invention, the method comprises the steps of detecting CD94 in a sample to be detected using at least one of 1) the antibody or antigen-binding fragment described above, 2) the nucleic acid molecule described above, 3) the expression vector described above, and 4) the recombinant cell described above, and determining the amount of CD94 in the sample to be detected based on the CD94 detection result. Because the antibodies or antigen-binding fragments described herein, or antibodies or antigen-binding fragments expressed in nucleic acid molecules, expression vectors, or recombinant cells, can effectively bind to human CD94, the method described herein can be used to effectively detect the amount of CD94 in a sample to be detected from a subject, and the stage of a CD94-mediated related disease can be assessed based on the amount of CD94.

[0080] According to an embodiment of the present invention, the above method for staging a disease may further include at least one of the following additional technical features:

[0081] According to an embodiment of the present invention, the content of CD94 in the sample to be detected is equal to or greater than the reference level for stage IV tumor disease, which is an indication that the sample to be detected is derived from a patient with stage IV tumor; the content of CD94 in the sample to be detected is between the reference levels for stage IV tumor and stage III tumor disease, which is an indication that the sample to be detected is derived from a patient with stage III tumor; the content of CD94 in the sample to be detected is between the reference levels for stage III tumor and stage II tumor disease, which is an indication that the sample to be detected is derived from a patient with stage II tumor; and the content of CD94 in the sample to be detected is between the reference levels for stage I tumor and stage II tumor disease, which is an indication that the sample to be detected is derived from a patient with stage I tumor. As will be understood by those skilled in the art, the level of CD94 varies depending on the type of tumor during stage I, stage II, stage III, and stage IV disease, and the stage of a tumor can be determined by comparing the CD94 content in the sample to the corresponding reference level of CD94 for that tumor stage, or by comparing the CD94 content in the sample to the CD94 content in a sample from an individual or population with a known stage of the disease. The reference levels for stage I, stage II, stage III, and stage IV tumors can be determined by comparing and verifying the difference in CD94 content in the sample from a number of individuals with the CD94-related disease and a number of healthy individuals.

[0082] According to an embodiment of the present invention, the detection target sample includes at least one of blood, saliva, sweat, tissue, cells, blood, serum, plasma, feces, and urine.

[0083] According to an embodiment of the present invention, the CD94-mediated associated diseases include transplant rejection, autoimmune diseases, infectious diseases and cancer.

[0084] According to an embodiment of the present invention, the autoimmune disease comprises at least one of systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, chronic atrophic gastritis with pernicious anemia, pulmonary hemorrhagic nephritic syndrome, pemphigus vulgaris, pemphigoid, primary biliary cholangitis, multiple sclerosis, and acute idiopathic polyneuropathy.

[0085] According to an embodiment of the present invention, the cancer comprises at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0086] In a fifteenth aspect, the present invention provides a method for assessing the prognosis of a CD94-mediated related disease, which includes the steps of detecting CD94 in a sample to be detected using at least one of 1) the antibody or antigen-binding fragment described above, 2) the nucleic acid molecule described above, 3) the expression vector described above, and 4) the recombinant cell described above, and determining the amount of CD94 in the sample to be detected based on the CD94 detection result. As described above, the content of CD94 has an important effect on cancer. After treating an individual with a related disease, monitoring the content of CD94 in their tissues or excretions, such as peripheral blood and urine, can effectively assess the prognosis of such diseases. For example, this can be done by comparing the content of CD94 in the subject's body before and after treatment, or by comparing the content of CD94 in the subject's body after treatment with the CD94 level in normal or diseased individuals. Because the antibodies or antigen-binding fragments of the present application, as well as antibodies or antigen-binding fragments expressed in nucleic acid molecules, expression vectors, or recombinant cells, can effectively bind to human CD94, the methods described herein can be used to effectively detect the content of CD94 in a sample derived from a subject, and to assess the prognosis of CD94-related diseases based on the content of CD94.

[0087] According to an embodiment of the present invention, the method for assessing disease prognosis may further include at least one of the following additional technical features:

[0088] According to an embodiment of the present invention, the sample to be detected is derived from a patient with a CD94-mediated associated disease before or after treatment.

[0089] According to an embodiment of the present invention, the detection target sample includes at least one of blood, saliva, sweat, tissue, cells, blood, serum, plasma, feces, and urine.

[0090] According to an embodiment of the present invention, the prognostic effect of a CD94-mediated related disease is determined based on the content of CD94 in a sample to be detected from a patient with a CD94-mediated related disease before or after the treatment.

[0091] According to an embodiment of the present invention, a decrease in the amount of CD94 in a sample detected from a patient with a CD94-mediated associated disease after treatment is indicative of a favorable prognosis for the patient.

[0092] According to an embodiment of the present invention, the CD94-mediated associated diseases include transplant rejection, autoimmune diseases, infectious diseases and cancer.

[0093] According to an embodiment of the present invention, the autoimmune disease comprises at least one of systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, chronic atrophic gastritis with pernicious anemia, pulmonary hemorrhagic nephritic syndrome, pemphigus vulgaris, pemphigoid, primary biliary cholangitis, multiple sclerosis, and acute idiopathic polyneuropathy.

[0094] According to an embodiment of the present invention, the cancer comprises at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0095] In a sixteenth aspect, the present invention proposes the use of the aforementioned antibodies or antigen-binding fragments, nucleic acid molecules, expression vectors, recombinant cells, compositions, or drugs in the treatment or prevention of CD94-mediated related diseases. As described above, the antibodies or antigen-binding fragments can effectively bind to human CD94 and can effectively treat or prevent CD94-mediated related diseases.

[0096] According to an embodiment of the present invention, the above use may further include at least one of the following additional technical features:

[0097] According to an embodiment of the present invention, the CD94-mediated associated diseases include transplant rejection, autoimmune diseases, infectious diseases and cancer.

[0098] According to an embodiment of the present invention, the autoimmune disease comprises at least one of systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, chronic atrophic gastritis with pernicious anemia, pulmonary hemorrhagic nephritic syndrome, pemphigus vulgaris, pemphigoid, primary biliary cholangitis, multiple sclerosis, and acute idiopathic polyneuropathy.

[0099] According to an embodiment of the present invention, the cancer comprises at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0100] In a seventeenth aspect, the present invention provides use of the aforementioned antibodies or antigen-binding fragments, nucleic acid molecules, expression vectors, or recombinant cells in the diagnosis of CD94-related diseases, staging of CD94-related diseases, or evaluation of the prognosis of CD94-related diseases. As described above, the antibodies or antigen-binding fragments of the present application, or antibodies or antigen-binding fragments expressed in nucleic acid molecules, expression vectors, or recombinant cells, can effectively bind to human CD94. Therefore, by employing the methods described herein, the content of CD94 in a test sample derived from a subject can be effectively detected, and effective diagnosis, disease staging, and prognosis evaluation of CD94-related diseases can be performed.

[0101] According to an embodiment of the present invention, the above use may further include at least one of the following additional technical features:

[0102] According to an embodiment of the present invention, the CD94-mediated associated diseases include transplant rejection, autoimmune diseases, infectious diseases and cancer.

[0103] According to an embodiment of the present invention, the autoimmune disease comprises at least one of systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, chronic atrophic gastritis with pernicious anemia, pulmonary hemorrhagic nephritic syndrome, pemphigus vulgaris, pemphigoid, primary biliary cholangitis, multiple sclerosis, and acute idiopathic polyneuropathy.

[0104] According to an embodiment of the present invention, the cancer comprises at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0105] A "subject" or "individual" in the context of the present invention generally refers to a mammal, such as a primate and / or rodent, particularly a human, monkey, or mouse.

[0106] The beneficial effects of the present invention are as follows: 1) To date, there have been no patents or research reports on the application of inhibitory CD94 antibodies to anti-cancer research. 2) The CD94 antibody and CD94 protein obtained in the present invention have high binding activity, and in in vitro functional experiments, the activity of the CD94 antibody is superior to that of NKG2A antibodies in clinical trials.

[0107] Additional aspects and advantages of the present invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the present invention.

[0108] The above and / or additional aspects and advantages of the present invention will become apparent and easier to understand from the following description of the embodiments taken in conjunction with the drawings. [Brief explanation of the drawings]

[0109] [Figure 1] FIG. 1 shows the results of binding between the human-mouse 15C10 chimeric antibody and 293T-CD94 / NKG2A cells according to an embodiment of the present invention. [Figure 2] FIG. 1 shows the results of the human-mouse 15C10 chimeric antibody according to an embodiment of the present invention inhibiting the binding of HLA-E to 293T-CD94 / NKG2A cells. [Figure 3] FIG. 1 shows the results of binding between the human-mouse 15C10 chimeric antibody and the humanized 15C10 antibody according to an embodiment of the present invention and 293T-CD94 / NKG2A cells. [Figure 4] FIG. 1 shows the results of binding between the humanized 15C10 antibody according to an example of the present invention, and the control NKG2A antibodies huZ199 and huZ270 and 293T-CD94 / NKG2A cells. [Figure 5] FIG. 1 shows the results of the humanized 15C10 antibody according to an embodiment of the present invention inhibiting the binding of HLA-E to 293T-CD94 / NKG2A cells. [Figure 6]FIG. 1 shows the ELISA results of the binding of the humanized 15C10 antibody according to an example of the present invention to the CD94 / NKG2A-Fc heterodimer protein. [Figure 7] FIG. 10 shows the results of the humanized 15C10 antibody according to an embodiment of the present invention promoting Jurkat-NFAT-lucia-CD94 / NKG2A T cell activation. [Figure 8] FIG. 1 shows the results of the humanized 15C10 antibody according to an embodiment of the present invention promoting anti-cancer activity in immune system-reconstituted mice. DETAILED DESCRIPTION OF THE INVENTION

[0110] The following examples of the present invention will be described in detail. The examples described below are illustrative and are used only to explain the present invention, and should not be understood as limitations on the present invention.

[0111] It should be noted that the terms "first," "second," etc. are for descriptive purposes only and cannot be considered to indicate or imply relative importance or to imply a specific number of technical features. Thus, a feature qualified as "first" or "second" may expressly or implicitly include one or more of the feature. Furthermore, in the present description, unless specifically stated otherwise, "plurality" means two or more than two.

[0112] The endpoints of ranges and any value disclosed herein should be understood to be not limited to such exact ranges or values, but to include values ​​close to those ranges or values. In the case of ranges of numerical values, values ​​between the endpoints of each range, between the endpoints of each range and any single point value, and between any single point value can be combined with each other to create one or more new numerical ranges, and these numerical ranges are considered to be specifically disclosed in the specification.

[0113] To facilitate understanding of the present invention, certain technical and scientific terms are defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the meaning commonly understood by those of ordinary skill in the art. Abbreviations for amino acid residues refer to the standard three-letter and / or one-letter codes used in the art to represent one of the 20 commonly used L-amino acids.

[0114] The antibodies or antigen-binding fragments described in the present invention are typically prepared by biosynthesis. Using the nucleotide sequences described in the present invention, those skilled in the art can conveniently prepare the encoding nucleic acids of the present invention by various known methods. These methods include, but are not limited to, PCR and DNA synthesis. For specific methods, see J. Sambrook, "A Laboratory Guide to Molecular Cloning." In one embodiment of the present invention, the encoding nucleic acid sequences of the present invention are constructed by segmented synthesis of nucleotide sequences followed by overlap extension PCR. The antibody or antigen-binding fragments are numbered and defined according to the Kabat numbering system.

[0115] The antibody of the present invention includes a mouse-derived antibody, a chimeric antibody, and a humanized antibody, and preferably a humanized antibody.

[0116] The term "mouse-derived antibody" in the present invention refers to a monoclonal antibody against human CD94 prepared according to the knowledge and skill of the art. During preparation, a test subject is injected with an antigen, and then hybridomas expressing antibodies with the desired sequence or functional characteristics are isolated. In a preferred embodiment of the present invention, the mouse-derived CD94 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a mouse-derived kappa or lambda chain or a variant thereof, or a heavy chain constant region of a mouse-derived IgG1, IgG2, or IgG3 chain or a variant thereof.

[0117] The term "chimeric antibody" refers to an antibody in which the variable region of a mouse-derived antibody is fused with the constant region of a human antibody, and can reduce the immune response elicited by mouse-derived antibodies. The variable region genes are cloned from mouse hybridoma cells, and if necessary, the constant region genes of a human antibody are cloned. The mouse variable region genes and the human constant region genes are ligated to form a chimeric gene, which is then inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic expression system. In a preferred embodiment of the present invention, the antibody light chain of the CD94 chimeric antibody further comprises a light chain Fc region of a human-derived kappa or lambda chain or a mutant thereof. The antibody heavy chain of the CD94 chimeric antibody further comprises a heavy chain constant region of a human-derived IgG1, IgG2, IgG3, IgG4, or a mutant thereof.

[0118] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody generated by grafting mouse CDR sequences onto a human antibody variable region framework, i.e., using a different type of human species-specific antibody framework sequence. Such framework sequences can be obtained from public DNA databases of species-specific antibody gene sequences or published references. Species-specific DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human species-specific sequence database. To avoid reduced activity due to reduced immunogenicity, minimal back mutations or restorative mutations are made to the human antibody variable region framework sequences to maintain activity.

[0119] As used herein, the term "monoclonal antibody" refers to an antibody that has a single antigen-binding site.

[0120] As used herein, the term "polyclonal antibody" refers to an antibody having two or more different antigen-binding sites.

[0121] As used herein, the term "mutant" or "variant" refers to any naturally occurring or engineered molecule and includes molecules resulting from mutations involving one or more nucleotides or amino acids.

[0122] The term "complementarity determining region" or "CDR" or "CDR sequence" refers to the amino acid sequence responsible for antigen binding in an antibody, typically consisting of amino acid residues 23-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region, and amino acid residues 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable region (Kabat et al., Human Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)), and / or "high-variability loops" (e.g., amino acid residues 26-32 (LI), 50-52 (L2), and 91-96 (L3) in the light chain variable region, and amino acid residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable region (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987))

[0123] As used herein, the term "homology," when describing an amino acid sequence or a nucleic acid sequence relative to a reference sequence, refers to the percentage of identical amino acids or nucleotides between two amino acid or nucleic acid sequences determined by conventional methods, e.g., Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York), and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5:Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). There are many algorithms for aligning sequences and measuring sequence homology, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443, the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482, the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444, the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997), and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs utilizing these algorithms can also be obtained, and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)), or GAP, BESTFIT, BLAST (Altschul et al.), FASTA, and TFASTA, available from Genetics Computing Group (GCG) Bag, Version 8, Madison, Wisconsin, USA, and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0124] Those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acids in the sequences of the present invention, obtaining variants of the antibody or functional fragment thereof, provided that the antibody activity is not substantially affected (retaining at least 95% activity). These variants are considered to be within the scope of the present invention. For example, amino acids with similar properties can be substituted in the variable region. The variant sequences described in the present invention have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence. Sequence identity described in the present invention can be measured using sequence analysis software, such as the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. All amino acid sequences referred to in the present invention are presented in N- to C-terminal order.

[0125] As described above, the monoclonal antibodies of the present invention may be full-length antibodies, may include only functional fragments (e.g., Fab, F(ab')2, or scFv fragments), or may be modified to affect function. The present invention includes anti-CD94 antibodies with modified glycosylation patterns. For some applications, it may be useful to modify them to remove undesired glycosylation sites, or to eliminate fucose moieties on the oligosaccharide chains, for example, to enhance antibody-dependent cellular cytotoxicity (ADCC) function. For other applications, galactosylation modifications can be performed to alter complement-dependent cytotoxicity (CDC).

[0126] As used herein, the term "full antibody" refers to a tetrameric structure consisting of two identical light chains and two identical heavy chains connected by interchain disulfide bonds, such as immunosphere protein G (IgG), immunosphere protein A (IgA), immunosphere protein M (IgM), immunosphere protein D (IgD), or immunosphere protein E (IgE). The same immunosphere protein can also be classified into different subclasses, such as IgG1, IgG2, IgG3, and IgG4, based on amino acid composition. The light chains of immunosphere proteins are classified into κ chains and λ chains depending on the constant region.

[0127] The term "functional fragment" as used herein refers, inter alia, to antibody fragments, such as CDR-grafted antibodies, Fab, Fab', F(ab')2, Fv or scFv, nanobodies, or any fragment whose half-life can be extended by chemical modification or liposome encapsulation, such as the addition of a poly(alkylene)diol, e.g., polyethylene glycol ("pegylated, PEGylated") (referred to as pegylated fragments of Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG or Fab'-PEG) ("PEG" stands for polyethylene glycol), and which retain CD94-binding activity. Preferably, such functional fragments consist of or comprise a subsequence of the heavy or light chain variable region of the antibody from which they are derived, sufficient to retain the same binding specificity and sufficient affinity for CD94 as the antibody from which they are derived, preferably at least 1 / 100th, and in a more preferred embodiment at least 1 / 10th, of the affinity of the antibody from which they are derived. Such functional fragments comprise at least 3 amino acids, and preferably 5, 10, 15, 25, 50 and 100 contiguous amino acids of the antibody sequence from which it is derived.

[0128] In the present invention, unless otherwise specified, the term "antigen-binding fragment" as used generally refers to an antigen-binding antibody fragment, which may comprise a portion of an intact antibody, typically the antigen-binding or variable region, including, by way of example, CDR-grafted antibodies, Fab, Fab', F(ab')2, Fv or scFv, nanobodies, etc.

[0129] As used herein, the term "CDR-grafted antibody" refers to the transplantation of the CDRs of a monoclonal antibody of one species onto the variable region of an antibody of another species. For example, the CDRs of a mouse-derived monoclonal antibody can be transplanted into the variable region of a human-derived antibody, replacing the CDRs of the human-derived antibody, so that the human-derived antibody acquires the antigen-binding specificity of the mouse-derived monoclonal antibody while reducing its heterogeneity.

[0130] As used herein, the term "Fab antibody" or "Fab" generally refers to an antibody or fragment containing only the Fab molecule, which consists of the VH and CH1 of the heavy chain and an intact light chain, with a single disulfide bond connecting the light and heavy chains.

[0131] As used herein, the term "nanobody" (single domain antibody or VHH antibody) was originally described as an antigen-binding immunoglobulin (variable) domain of a "heavy chain antibody" (i.e., an antibody lacking light chains) (Hamers-Casterman C, Atharhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)), which contains a heavy chain variable region (VH) and the usual CH2 and CH3 regions, and specifically binds to an antigen specific for the heavy chain variable region.

[0132] As used herein, the term "Fv antibody" generally refers to an antibody in which a light chain variable region (VL) and a heavy chain variable region (VH) are non-covalently connected, and is the smallest functional fragment of an antibody molecule that retains the complete antigen-binding site.

[0133] As used herein, the term "single-chain antibody" or "scFv" refers to a fragment in which the antibody heavy chain variable region and light chain variable region are connected via a short peptide.

[0134] The amino acid or nucleic acid sequences relevant to the present invention are detailed in Table 1. [Table 1] TIFF2026503988000002.tif254170TIFF2026503988000003.tif255169TIFF2026503988000004.tif255169TIFF2026503988000005.tif25516 8TIFF2026503988000006.tif255169TIFF2026503988000007.tif255169TIFF2026503988000008.tif255169TIFF2026503988000009.tif39170

[0135] The present invention will be described in detail with reference to the following examples. In the examples or test examples, unless specific experimental conditions are specified, the experimental methods are carried out under ordinary conditions.

[0136] The following examples are used in combination to illustrate the present invention. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If specific techniques or conditions are not specified in the examples, they should be carried out according to the techniques or conditions described in the literature in the field or according to the product specifications. If the manufacturer of the reagents or equipment used is not specified, they are conventional products that can be purchased commercially.

[0137] Example 1: Preparation of anti-human CD94 hybridoma monoclonal antibody

[0138] 1.1 Screening of hybridoma cells

[0139] In this example, a mouse-derived monoclonal antibody against human CD94 was obtained. The immunizing antigen was purified recombinant CD94 extracellular domain Fc fusion protein (CD94-Fc) (recombinant CD94 extracellular domain Fc fusion protein, amino acid sequence shown in SEQ ID NO: 54). C57BL / 6 mice (9 weeks old, purchased from Shanghai Resc, weighing approximately 20 g) were immunized.

[0140] The immunized mice were intraperitoneally immunized three times using purified antigen and complete Freund's adjuvant, and the immune response was detected after blood collection from the tail vein. The serum was screened using ELISA and flow cytometry according to the usual procedure to obtain mice with anti-human CD94 immunoglobulin protein. Splenocytes were collected from the mice with the highest levels of anti-CD94 immunoglobulin protein and fused with mouse myeloma cells SP2 / 0 (ATCC number CRL-1581). Antibody screening was performed on the fused hybridoma cells to obtain mouse monoclonal antibodies. The specific procedure is as follows: The cells were resuspended in HAT complete medium (RPMI-1640 medium containing 20% ​​FBS, 1x HAT, and 1x OPI), dispensed into a 96-well cell culture plate, and incubated at 37°C and 5% CO2. On day 5 after fusion, 50 μL of HAT complete medium was added per well. On days 7–8 after fusion, a complete liquid exchange was performed depending on the cell growth density. The medium that was replaced was 200 μL of HT complete medium (RPMI-1640 medium containing 20% ​​FBS, 1x HT, and 1x OPI) per well.

[0141] On day 10-11 after fusion, flow cerebrospinal fluid (FCE) binding detection is performed depending on the cell growth density. The medium in positive wells is replaced, and the cells are expanded to 24-well plates depending on the cell density. The cell lines transferred to the 24-well plates are re-measured, then stored and subjected to the first round of subcloning. Those that screened positive in the first round of subcloning are stored and subjected to the second round of subcloning. Those that screened positive in the second round of subcloning are stored and subjected to protein expression. Antibodies are further prepared using serum-free cell culture methods, purified by protein G affinity chromatography, and used for subsequent functional activity detection.

[0142] 1.2 Sequencing of hybridoma cells

[0143] The total number of candidate hybridoma cells screened in part 1.1 above was 10 6The cells were cultured at 10°C for 10 minutes and centrifuged at 800 rpm to harvest the cells. Total RNA was extracted using a Trizol kit (Invitrogen). A cDNA library (Invitrogen) was then synthesized by reverse transcription using the total RNA as a template. The cDNA was then used as a template for PCR amplification of the CD94 antibody variable region nucleic acid sequence corresponding to the hybridoma cells. The primer sequences used in the PCR amplification reaction were complementary to the first framework region or signal peptide region and constant region of the antibody variable region (see Larrick, J.W., et al. (1990) Scand. J. Immunol., 32, 121-128 and Coloma, J.J., et al. (1991) BioTechniques, 11, 152-156 for specific sequences). PCR amplification was performed in a 50 μL reaction system containing 2 μL of cDNA, 5 μL of 10× PCR buffer, 2 μL of upstream and downstream primers (5 μM), 2 μL of dNTPs, 1 μL of Taq enzyme (Takara, Ex Taq), and 38 μL of HO. The PCR was pre-denatured at 95°C for 5 minutes, followed by temperature cycling. The reaction conditions were 30 s denaturation at 94°C, 45 s annealing at 58°C, and 50 s extension at 72°C for a total of 32 cycles, followed by 7 min extension at 72°C. The sequences of the heavy chain variable region (SEQ ID NO: 23) and light chain variable region (SEQ ID NO: 24) of the anti-CD94 mouse monoclonal antibody were obtained by sequencing the amplified product.

[0144] Example 2: Flow cerebrospinal fluid binding experiments of CD94 chimeric antibodies

[0145] Flow cytometry experiments were used to detect the binding properties of CD94 chimeric antibodies. 293T cells (ATCC No. CRL-3216) were overexpressed with CD94 and NKG2A proteins (293T-CD94 / NKG2A). After antibody addition, the signal intensity was used to determine the binding properties of the chimeric antibodies with CD94.

[0146] 2.1 Construction of 293T cells overexpressing human CD94

[0147] 5 × 10 HEK293T cells 5 Cells were plated in a 6-well plate at 100 cells / well and cultured overnight in DMEM medium without double antibody. Before transfection, discard the medium and add 1 mL of fresh DMEM medium without double antibody. pLVX-EF1a-CD94-IRES-puro (a coding sequence (SEQ ID NO: 41) of the human CD94 protein (SEQ ID NO: 40) inserted between the EcoRI and BamHI enzyme sites of the pLVX-EF1a-IRES-puro vector) or pLVX-EF1a-NKG2A-IRES-puro (a coding sequence (SEQ ID NO: 42) of the NKG2A protein (SEQ ID NO: 43) inserted between the EcoRI and BamHI enzyme sites of the pLVX-EF1a-IRES-puro vector) were used. 43) was added to 200 μL of serum-free DMEM medium at a 2:1:1 ratio with pMD2G and psPAX2 vectors (3 μg total), and 12 μg of polyetherimide (PEI, Polysciences) was added. After uniform mixing, the mixture was left to stand for 16 minutes, and then the entire liquid was added to a 6-well plate containing HEK293T cells. After 6 hours of incubation, the medium was discarded and fresh complete DMEM medium was added and incubated. 48 hours after transfection, the cell culture supernatant was collected and passed through a 0.45 μm filter (Millipore), i.e., the virus supernatant. The virus supernatant was then diluted to 1 × 10 4 The cells were added to a 6-well plate of 293T cells, and polybrene (Sigma) was added at a final concentration of 4 μg / mL. The cells were then cultured for 12 hours. The supernatant was then completely removed and fresh complete DMEM medium was added. The resulting cells were 293T-CD94 / NKG2A cells.

[0148] 2.2 Detection of the binding activity of chimeric antibody CD94

[0149] The CD94 chimeric antibody 15C10-hIgG1mut used in this section was expressed in our laboratory, and its heavy chain amino acid sequence is shown in SEQ ID NO: 33, and its light chain amino acid sequence is shown in SEQ ID NO: 34. The inhibitory NKG2A antibody huZ270-hIgG1mut used in this section was also expressed in our laboratory, and its heavy chain amino acid sequence is shown in SEQ ID NO: 44, and its light chain amino acid sequence is shown in SEQ ID NO: 45.

[0150] 2 × 10 293T-CD94 / NKG2A cells in PBS 6 The cells were diluted to 1 mL / mL and added to 1.5 mL EP tubes at a volume of 100 μL per tube. 10 μL of mouse serum was added per tube and blocked at 4°C for 30 min. Different concentration gradients of the CD94 chimeric antibody 15C10-hIgG1mut or the inhibitory NKG2A antibody huZ270-hIgG1mut were added separately and incubated at 4°C for 30 min. 1 mL of PBS was added to the EP tubes, and the cells were centrifuged at 3500 rpm for 5 min at 4°C. The supernatant was completely removed and the cells were washed once with PBS. After centrifugation, the supernatant was completely removed and the cells were resuspended in 100 μL per tube of PBS. 1 μL per tube of Alexa-647-labeled mouse anti-human Fc secondary antibody (Biolegend) was added and incubated at 4°C for 30 min in the dark. The cells were washed twice with PBS, centrifuged, and the supernatant was completely removed. The cells were resuspended in 200 μL / tube of PBS and detected by flow cytometry. The results, as shown in FIG. 1, further demonstrated that the 15C10-hIgG1mut chimeric antibody of the present invention can bind to CD94.

[0151] Example 3: Detection of the inhibitory capacity of CD94 chimeric antibodies

[0152] CD94 antibodies inhibit the binding of CD94 / NKG2A to its ligand HLA-E by binding to the extracellular domain of the CD94 protein, and flow cytometry experiments are used to detect the inhibition of HLA-E binding of 293T-CD94 / NKG2A by CD94 antibodies.

[0153] 293T-CD94 / NKG2A cells (same as in Example 2) were diluted to 2 × 10 6 The cells were diluted to 1 mL / mL and added to 1.5 mL EP tubes at a volume of 100 μL per tube. 10 μL of mouse serum was added per tube and blocked at 4°C for 30 minutes. The CD94 chimeric antibody 15C10-hIgG1mut and the inhibitory NKG2A antibody huZ270-hIgG1mut were added separately and incubated at 4°C for 30 minutes. 0.1 μg / tube of APC-labeled HLA-E tetramer protein was added and incubated at 4°C for 30 minutes. The cells were washed twice with PBS, centrifuged, and the supernatant was completely removed. The cells were resuspended in 200 μL / tube of PBS and analyzed by flow cytometry. The results, shown in Figure 2, demonstrate that the CD94 antibodies of the present invention can inhibit the binding of HLA-E to CD94 / NKG2A, with inhibitory activity comparable to that of the NKG2A antibody huZ270.

[0154] Example 4: Humanization of mouse-derived anti-human CD94 monoclonal antibodies

[0155] Based on Examples 1 and 2, by comparing the IMGT human antibody heavy and light chain variable region gene database with MOE software, heavy and light chain variable region gene sequences with high homology to mouse monoclonal antibodies were selected as templates, and the CDRs of the mouse monoclonal antibodies were grafted onto the corresponding human templates to form variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Amino acid residues were identified and annotated according to the Kabat numbering system.

[0156] To maintain the conformation of the CDR region, based on the three-dimensional structure of the mouse-derived antibody, restoration mutations were made to the residues at the VL and VH binding interface, the residues buried inside the protein close to the CDR, and the residues that directly interact with the CDR, to obtain a humanized antibody without affecting the activity of the variable region. The sequence of the humanized CD94 antibody heavy chain variable region is shown in SEQ ID NO: 25, and the heavy chain variable region sequence is shown in SEQ ID NO: 26.

[0157] Example 5: Flow cerebrospinal fluid binding experiment of CD94 chimeric antibody and human-derived CD94 antibody

[0158] 293T cells (ATCC number 10111) were overexpressed with CD94 and NKG2A proteins (293T-CD94 / NKG2A), and ELISA experiments were performed to detect the binding properties of CD94 chimeric antibodies and human-derived CD94 antibodies to 293T-CD94 / NKG2A.

[0159] 2 × 10 293T-CD94 / NKG2A cells overexpressing CD94 protein and NKG2A protein obtained in Example 2 were added to PBS. 6The antibody was diluted to 1 / mL and added to 1.5 mL EP tubes at a volume of 100 μL per tube. 10 μL of mouse serum was added per tube and blocked at 4°C for 30 minutes. Gradient concentrations (3-fold dilutions, up to a final concentration of 10 μg / mL) of CD94 chimeric antibody 15C10-hIgG4 (expressed in our laboratory, heavy chain sequence SEQ ID NO: 36, light chain sequence SEQ ID NO: 37) and humanized antibody h15C10-hIgG4 (expressed in our laboratory, heavy chain sequence SEQ ID NO: 38, light chain sequence SEQ ID NO: 39) were added separately and incubated at 4°C for 30 minutes. 1 mL of PBS was added to the EP tubes, and the mixture was centrifuged at 3500 rpm for 5 minutes at 4°C. The supernatant was completely removed and the tubes were washed once with PBS. After centrifugation, the supernatant was completely removed, and the cells were resuspended in 100 μL / tube of PBS. 0.1 μL / tube of Alexa-647-labeled mouse anti-human Fc secondary antibody (Biolegend) was added and incubated at 4°C for 30 minutes in the dark. After washing twice with PBS, the supernatant was completely removed after centrifugation. The cells were resuspended in 200 μL / tube of PBS and detected by flow cytometry. As shown in Figure 3, both the humanized and chimeric antibodies of the present invention had good binding ability, with the humanized antibody having a superior binding ability to the chimeric antibody.

[0160] Example 6: Human-derived CD94 antibody ELISA binding experiment

[0161] CD94 and NKG2A proteins (293T-CD94 / NKG2A) were overexpressed in 293T cells (ATCC number 293T-CD94 / NKG2A), and ELISA experiments were performed to detect the binding properties of the human CD94 and NKG2A antibodies obtained in the above examples to 293T-CD94 / NKG2A.

[0162] 2 × 10 293T-CD94 / NKG2A cells overexpressing CD94 protein and NKG2A protein obtained in Example 2 were added to PBS. 6The mixture was diluted to 1 / mL and added to 1.5 mL EP tubes at a volume of 100 μL / tube, to which 10 μL / tube of mouse serum was added, followed by blocking treatment for 30 minutes at 4° C. Gradient concentrations (3-fold dilutions, up to a final concentration of 30 μg / mL) of humanized antibody h15C10-hIgG4 (as in Example 5), non-inhibitory NKG2A antibody huZ199-hIgG4 (expressed in our laboratory, heavy chain sequence SEQ ID NO: 46, and light chain sequence SEQ ID NO: 47), inhibitory NKG2A antibody huZ270-hIgG4 (expressed in our laboratory, heavy chain sequence SEQ ID NO: 48, and light chain sequence SEQ ID NO: 49), or control antibody hIgG4 were added individually and incubated at 4° C. for 30 minutes. 1 mL of PBS was added to the EP tube, and the cells were centrifuged at 3500 rpm for 5 minutes at 4°C. The supernatant was completely removed and washed once with PBS. After centrifugation, the supernatant was completely removed and the cells were resuspended in 100 μL of PBS per tube. 0.1 μL of Alexa-647-labeled mouse anti-human Fc secondary antibody (Biolegend) was added per tube and incubated at 4°C for 30 minutes in the dark. The cells were washed twice with PBS, centrifuged, and the supernatant was completely removed. The cells were resuspended in 200 μL of PBS per tube and detected by flow cytometry. The results, shown in Figure 4, demonstrate that the humanized antibodies of the present invention exhibited superior binding ability to 293T-CD94 / NKG2A cells compared to the NKG2A antibodies huZ199 and huZ270.

[0163] Example 7: Detection of the inhibitory ability of CD94 humanized antibodies

[0164] CD94 antibodies inhibit the binding of CD94 / NKG2A to its ligand HLA-E by binding to the CD94 extracellular domain. Flow cerebrospinal fluid (FPE) experiments were used to detect the inhibition of the binding of 293T-CD94 / NKG2A to HLA-E.

[0165] 2 × 10 293T-CD94 / NKG2A cells (as above) in PBS 6The cells were diluted to 1 / mL and added to 1.5 mL EP tubes at a volume of 100 μL per tube. 10 μL of mouse serum was added per tube and blocked at 4°C for 30 minutes. The CD94 humanized antibody 15C10-hIgG4, NKG2A antibody huZ270-hIgG4, or control antibody hIgG4 used in the above examples was added and incubated at 4°C for 30 minutes. 0.1 μg / tube of APC-labeled HLA-E tetramer protein was added and incubated at 4°C for 30 minutes. The cells were washed twice with PBS, centrifuged, and the supernatant was completely removed. The cells were resuspended in 200 μL / tube of PBS and analyzed by flow cytometry. The results, shown in Figure 5, demonstrate that the CD94 humanized antibodies of the present invention can effectively inhibit the binding of HLA-E to CD94 / NKG2A, with inhibitory activity comparable to that of the NKG2A antibody huZ270.

[0166] Example 8: ELISA binding experiments of CD94 antibodies

[0167] ELISA experiments are used to detect the binding properties of CD94 antibodies. A heterodimer protein of CD94 extracellular region Fc fusion protein and NKG2A extracellular region Fc fusion protein is coated on a 96-well plate, and the signal intensity after addition of the antibody is used to determine the binding properties of the antibody to the CD94 protein.

[0168] CD94 extracellular region Fc fusion protein and NKG2A extracellular region Fc fusion protein (CD94 / NKG2A-Fc) heterodimer fusion protein (expressed in this laboratory; the amino acid sequences of CD94-Fcmutknob and NKG2A-Fcmuthole are shown in SEQ ID NO: 50 and SEQ ID NO: 51, respectively) were diluted to 2 μg / mL in PBS buffer, added to a 96-well plate at a volume of 100 μL / well, and incubated overnight at 4°C. The PBS buffer in the 96-well plate was then aspirated and the plate was washed six times with PBST (pH 7.2, PBS containing 0.1% Tween 20). 200 μL / well of PBS / 10% BSA was added and incubated at 37°C for 2 hours for blocking. After removing the blocking solution and washing six times with PBST, the target CD94 humanized antibody h15C10-hIgG4 (NKG2A antibody huZ270-hIgG4, as in Example 5) diluted to the appropriate concentration in PBST / 0.05% BSA was added at 100 μL / well and incubated at 37°C for 1 hour. The reaction mixture was removed, and the plate was washed six times with PBST. Then, 100 μL / well of HRP (horse radish peroxidase)-labeled anti-human IgG-Fab secondary antibody was diluted in PBST / 0.05% BSA and incubated at 37°C for 1 hour. After washing the plate six times with PBST, 80 μL / well of TMB (tetramethylbenzidine) was added and incubated at room temperature for 3 minutes. The reaction was stopped by adding 80 μL / well of 4M sulfuric acid. Absorbance values ​​were read at 450 nm using a microplate reader. The results, as shown in Figure 6, demonstrated that the CD94 antibody of the present invention can bind to the CD94 / NKG2A dimer protein, and that the binding is stronger than that of the NKG2A antibody huZ270.

[0169] Example 9: Experiment on promotion of Jurkat T cell activation by CD94 antibody

[0170] Reporter cell experiments are used to detect the functional activity of CD94 antibodies. K56233220-HLA-E cells are co-incubated with Jurkat-NFAT-lucia-CD94 / NKG2A cells, and the chemiluminescence intensity after antibody addition is used to determine the functional activity of CD94 antibodies in promoting T cell activation.

[0171] 5 × 10 HEK293T cells 5 Cells were plated in a 6-well plate at 100 cells / well and cultured overnight in DMEM medium without double antibody. Before transfection, the medium was discarded and 1 mL of fresh DMEM medium without double antibody was added. pLVX-EF1a-CD94-IRES-puro (a coding sequence (SEQ ID NO: 41) of the CD94 protein (SEQ ID NO: 40) inserted between the EcoRI and BamHI enzyme sites of the pLVX-EF1a-IRES-puro vector) or pLVX-EF1a-NKG2A-IRES-puro (a coding sequence (SEQ ID NO: 42) of the NKG2A protein (SEQ ID NO: 43) inserted between the EcoRI and BamHI enzyme sites of the pLVX-EF1a-IRES-puro vector) were used. 43), pMD2G, and psPAX2 vectors (3 μg total) were added to 200 μL of serum-free DMEM medium at a 2:1:1 ratio, followed by 12 μg of polyetherimide (PEI, purchased from Polysciences). After uniform mixing, the mixture was left to stand for 16 minutes, and then the entire liquid was added to a 6-well plate containing HEK293T cells. After 6 hours of incubation, the medium was discarded and fresh complete DMEM medium was added and incubated. 48 hours after transfection, the cell culture supernatant was collected and passed through a 0.45 μm filter (Millipore), i.e., the virus supernatant. All virus supernatants were diluted to 1 × 10 4 The cells were added to a 6-well plate containing Jurkat-NFAT-lucia cells (purchased from Invivogen), and polybrene (Sigma) was added at a final concentration of 4 μg / mL. The cells were then cultured for 12 hours. The supernatant was then completely removed, and fresh complete IMDM medium was added. The resulting cells were Jurkat-NFAT-lucia-CD94 / NKG2A cells.

[0172] 5 × 10 HEK293T cells 5 Cells were plated at 1000 cells / well in a 6-well plate and cultured overnight in DMEM medium without the double antibody. Before transfection, the medium was discarded and 1 mL of fresh DMEM medium without the double antibody was added. pLVX-EF1a-HLA-E-IRES-puro (the coding sequence (SEQ ID NO: 53) of the HLA-E protein (SEQ ID NO: 52) inserted between the EcoRI and BamHI enzyme sites of the pLVX-EF1a-IRES-puro vector), pMD2G, and psPAX2 vectors (3 μg in total) were added to 200 μL of serum-free DMEM medium at a 2:1:1 ratio, and 12 μg of polyetherimide (PEI, purchased from Polysciences) was added. After uniform mixing, the mixture was left to stand for 16 minutes, and then the entire liquid was added to a 6-well plate containing HEK293T cells. After 6 hours of incubation, the medium was discarded and fresh complete DMEM medium was added and incubated. 48 hours after transfection, the cell culture supernatant was collected and passed through a 0.45 μm filter (Millipore), i.e., the virus supernatant. All virus supernatants were collected at 1 × 10 4 Polybrene (Sigma) was added to a 6-well plate containing K56233220 cells at a final concentration of 4 μg / mL and cultured for 12 hours. The supernatant was then completely removed and fresh complete IMDM medium was added. The resulting cells were K56233220-HLA-E cells. (1) 1 × 10 Jurkat-NFAT-lucia-CD94 / NKG2A cells in complete IMDM medium 5 Dilute to 100µL / mL and add 100µL / well to a 96-well plate. (2) CD94 humanized antibody h15C10-hIgG4, NKG2A antibody huZ270-hIgG4, and control antibody hIGg were diluted in complete IMDM medium in a gradient series and added to a 96-well plate at 20 μL / well. (3) 7.5 × 10 K56233220-HLA-E cells in complete IMDM medium 5 Dilute to 100µL / mL and add 80µL / well to a 96-well plate. (4) After culturing for 24 hours in a 37°C, 5% CO2 incubator, 50 μL of the supernatant was aspirated, 50 μL of substrate was added, and chemiluminescence was detected.

[0173] The results, as shown in Figure 7, further demonstrated that the CD94 antibody of the present invention can promote the activation of Jurkat-NFAT-lucia-CD94 / NKG2A T cells, and its activation function is stronger than that of the NKG2A antibody huZ270.

[0174] Example 10: Anti-cancer effects of humanized CD94 antibody in mice

[0175] In vivo efficacy experiments were conducted to examine whether the affinity-matured humanized CD94 antibody h15C10-hIgG4 obtained in Example 6 promoted the anti-cancer function in immune reconstituted mice. (1) On day 1, human PBMCs (purchased from Saikasa Bio) were injected into the tail vein of NCG mice (purchased from Suichuan Pharmaceutical Co., Ltd.) at an injection volume of 1 × 10 7 / animals, (2) On day 0, tumors developed subcutaneously on the right flank of NCG mice, with 1 × 10 6 tumour cells were injected, and the mice were randomly divided into groups. (3) On days 0, 3, 6, and 9, mice were intraperitoneally injected with the humanized antibody h15C10-hIgG4 and the control antibody hIgG at 250 μg per mouse. (4) After the injection of the above antibodies, the tumor volume was measured once every three days.

[0176] The results, as shown in FIG. 8, demonstrated that the CD94 antibody of the present invention can effectively promote anti-cancer activity of PBMC.

[0177] As can be seen from the above experimental results, the antibody obtained in the present invention can bind to CD94, inhibit the interaction between CD94 / NKG2A and HLA-E, and promote the anti-cancer activity of immune cells.

[0178] In the description herein, a statement referring to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples" means that a particular feature, structure, material, or characteristic described with reference to that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, general references to such terms do not necessarily refer to the same embodiment or example. In addition, a particular feature, structure, material, or characteristic described may be incorporated in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art can combine and combine the various embodiments or examples described herein and the features of the various embodiments or examples without mutual contradiction.

[0179] Although the embodiments of the present invention have been shown and described, the above embodiments are illustrative and should not be construed as limiting the present invention. Those skilled in the art will understand that various changes, modifications, substitutions and variations can be made to the above embodiments within the scope of the present invention.

Claims

1. an antibody or antigen-binding fragment, comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3; the amino acid sequence of the heavy chain CDR1 has at least 80% identity with the sequence shown in SEQ ID NO: 1; the amino acid sequence of the heavy chain CDR2 has at least 80% identity with the sequence shown in SEQ ID NO: 2; the amino acid sequence of the heavy chain CDR3 has at least 80% identity with the sequence shown in SEQ ID NO: 3; the amino acid sequence of the light chain CDR1 has at least 80% identity with the sequence set forth in SEQ ID NO: 4; the amino acid sequence of the light chain CDR2 has at least 80% identity with the sequence set forth in SEQ ID NO: 5; An antibody or antigen-binding fragment thereof, wherein the amino acid sequence of said light chain CDR3 has at least 80% identity with the sequence shown in SEQ ID NO:

6.

2. a heavy chain variable region as set forth in SEQ ID NO: 23 or SEQ ID NO: 25, and / or The antibody or antigen-binding fragment thereof according to claim 1, characterized in that it comprises a light chain variable region shown in SEQ ID NO: 24 or SEQ ID NO:

26.

3. 1) a heavy chain variable region set forth in SEQ ID NO: 23 and a light chain variable region set forth in SEQ ID NO: 24, or 2) The antibody or antigen-binding fragment thereof according to claim 1, characterized in that it comprises a heavy chain variable region shown in SEQ ID NO: 25 and a light chain variable region shown in SEQ ID NO:

26.

4. The antibody or antigen-binding fragment according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment comprises at least one of a heavy chain constant region and a light chain constant region, and at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a human-derived antibody, a primate-derived antibody, a mouse-derived antibody, or a mutant thereof.

5. The antibody or antigen-binding fragment of claim 4, wherein the light chain constant region and the heavy chain constant region are both derived from a mouse-derived IgG antibody or a mutant thereof or a human-derived IgG antibody or a mutant thereof.

6. The antibody or antigen-binding fragment of claim 5, wherein the light chain constant region and the heavy chain constant region are both derived from a mouse-derived IgG1 or IgG4 antibody or a mutant thereof, or a human-derived IgG1 or IgG4 antibody or a mutant thereof.

7. the antibody or antigen-binding fragment has a heavy chain constant region of the amino acid sequence set forth in SEQ ID NO: 27, 29, or 31 and / or a light chain constant region of the amino acid sequence set forth in SEQ ID NO: 28, 30, or 32; the antibody or antigen-binding fragment has a heavy chain constant region of the amino acid sequence set forth in SEQ ID NO: 27 and a light chain constant region of the amino acid sequence set forth in SEQ ID NO: 28; the antibody or antigen-binding fragment has a heavy chain constant region having the amino acid sequence set forth in SEQ ID NO: 29 and a light chain constant region having the amino acid sequence set forth in SEQ ID NO: 30; The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment has a heavy chain constant region of the amino acid sequence shown in SEQ ID NO: 31 and a light chain constant region of the amino acid sequence shown in SEQ ID NO:

32.

8. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment has a heavy chain having an amino acid sequence set forth in any one of SEQ ID NOs: 33, 35, 36, and 38, and a light chain having an amino acid sequence set forth in any one of SEQ ID NOs: 34, 37, and 39.

9. the antibody or antigen-binding fragment has a heavy chain having the amino acid sequence set forth in SEQ ID NO: 33 and a light chain having the amino acid sequence set forth in SEQ ID NO: 34; the antibody or antigen-binding fragment has a heavy chain having the amino acid sequence set forth in SEQ ID NO: 35 and a light chain having the amino acid sequence set forth in SEQ ID NO: 37; the antibody or antigen-binding fragment has a heavy chain of the amino acid sequence set forth in SEQ ID NO:36 and a light chain of the amino acid sequence set forth in SEQ ID NO:37; or The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment has a heavy chain having the amino acid sequence set forth in SEQ ID NO: 38 and a light chain having the amino acid sequence set forth in SEQ ID NO:

39.

10. the antibody or antigen-binding fragment comprises a monoclonal antibody or a polyclonal antibody; The monoclonal antibody comprises at least one of a full-length antibody, an Fv, a single-chain antibody, an Fab, a single-domain antibody, and a minimal recognition unit; The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment is capable of binding to the amino acid sequence shown in SEQ ID NO:

40.

11. A nucleic acid molecule encoding the antibody or antigen-binding fragment of any one of claims 1 to 10.

12. An expression vector carrying the nucleic acid molecule of claim 11.

13. A recombinant cell characterized in that it carries the nucleic acid molecule of claim 11, the expression vector of claim 12, or is capable of expressing the antibody or antigen-binding fragment of any one of claims 1 to 10.

14. The recombinant cell is obtained by introducing the expression vector according to claim 12 into a host cell, The recombinant cell of claim 13, wherein the recombinant cell is a eukaryotic cell.

15. The recombinant cell of claim 14, wherein the recombinant cell is a mammalian cell.

16. A composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 10, a nucleic acid molecule according to claim 11, an expression vector according to claim 12 or a recombinant cell according to any one of claims 13 to 15.

17. A drug comprising the antibody or antigen-binding fragment of any one of claims 1 to 10, the nucleic acid molecule of claim 11, the expression vector of claim 12, the recombinant cell of any one of claims 13 to 15, or the composition of claim 16.

18. A kit comprising the antibody or antigen-binding fragment of any one of claims 1 to 10, the nucleic acid molecule of claim 11, the expression vector of claim 12, or the recombinant cell of any one of claims 13 to 15.

19. Use of the antibody or antigen-binding fragment of any one of claims 1 to 10, the nucleic acid molecule of claim 11, the expression vector of claim 12, the recombinant cell of any one of claims 13 to 15 or the composition of claim 16 in the preparation of a medicament for preventing and / or treating a CD94-mediated associated disease, comprising: The CD94-mediated related diseases include transplant rejection, autoimmune diseases, infectious diseases and cancer; the autoimmune disease comprises at least one of systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, chronic atrophic gastritis with pernicious anemia, pulmonary hemorrhagic nephritic syndrome, pemphigus vulgaris, pemphigoid, primary biliary cholangitis, multiple cerebrospinal sclerosis, and acute idiopathic polyneuropathy; The cancer comprises at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

20. Use of the antibody or antigen-binding fragment of any one of claims 1 to 10, the nucleic acid molecule of claim 11, the expression vector of claim 12 or the recombinant cell of any one of claims 13 to 15 in the preparation of a kit for detecting CD94.

21. Use of the antibody or antigen-binding fragment of any one of claims 1 to 10, the nucleic acid molecule of claim 11, the expression vector of claim 12, the recombinant cell of any one of claims 13 to 15, the composition of claim 16 or the drug of claim 17 in the treatment or prevention of a CD94-mediated related disease.

22. Use of the antibody or antigen-binding fragment of any one of claims 1 to 10, the nucleic acid molecule of claim 11, the expression vector of claim 12 or the recombinant cell of any one of claims 13 to 15 in diagnosing a CD94-mediated related disease, staging a CD94-mediated related disease or assessing the prognosis of a CD94-mediated related disease.

23. 1. A method for treating or preventing a CD94-mediated related disease, comprising administering to a subject: The antibody or antigen-binding fragment of any one of claims 1 to 10. The nucleic acid molecule of claim 11 . The expression vector according to claim 12. The recombinant cell according to any one of claims 13 to 15. The composition of claim 16, and 20. A method comprising administering at least one of the drugs of claim 17.

24. 1. A method for assessing the prognosis of a CD94-mediated associated disease, comprising: The antibody or antigen-binding fragment of any one of claims 1 to 10. The nucleic acid molecule of claim 11 . The expression vector of claim 12, and Detecting CD94 in a sample to be detected using at least one recombinant cell according to any one of claims 13 to 15; determining the content of CD94 in the sample to be detected based on the detection result of CD94; and determining the prognostic effect of the CD94-mediated related disease based on the content of the CD94 in the sample to be detected before or after treatment.

25. The CD94-mediated related diseases include transplant rejection, autoimmune diseases, infectious diseases and cancer; the autoimmune disease comprises at least one of systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, chronic atrophic gastritis with pernicious anemia, pulmonary hemorrhagic nephritic syndrome, pemphigus vulgaris, pemphigoid, primary biliary cholangitis, multiple cerebrospinal sclerosis, and acute idiopathic polyneuropathy; The use according to claims 21 to 22 or the method according to claims 23 to 24, wherein the cancer comprises at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

26. the sample to be detected is derived from a patient with a CD94-mediated associated disease before or after treatment, The detection target sample includes at least one of blood, saliva, sweat, tissue, cells, blood, serum, plasma, feces, and urine, 25. The method of claim 24, wherein a decrease in the CD94 content in the sample to be detected from a patient with a CD94-mediated associated disease after treatment is indicative of a good prognosis for the patient.

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