Anti-CD94 antibodies and their applications

Abstract

The present invention provides an anti-CD94 antibody and its application, the antibody comprises heavy chain CDR1, CDR2 and CDR3, and light chain CDR1, CDR2 and CDR3; wherein: the amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 have at least 80% homology with the sequences shown in SEQ ID NO: 1, 2, 3, respectively, and the amino acid sequences of the light chain CDR1, CDR2 and CDR3 have at least 80% homology with the sequences shown in SEQ ID NO: 4, 5, 6, respectively. The antibody of the embodiment of the present invention can bind to the human CD94 protein, relieve CD94 / NKG2A-mediated immunosuppression, promote T cell activation, and can effectively treat and / or prevent CD94-mediated related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an anti-CD94 antibody and application thereof. Background Art

[0002] Cancer seriously threatens human life and health. In addition to traditional treatments such as surgical resection, radiotherapy, and chemotherapy, a variety of cancer treatments such as small molecule targeted therapy, immune checkpoint therapy, cell and gene therapy are also being developed.

[0003] In recent years, immunotherapy represented by PD-1 / L1 has demonstrated great potential, and three types of immunotherapy, PD-1 / L1, CTLA-4, and LAG-3, have been approved as drugs. However, it cannot be ignored that even the PD-1 / L1 therapy, which has the broadest approved indications, has an overall response rate of only 30%, and there are still more patients who cannot benefit from it.

[0004] Immune checkpoint molecules are inhibitory molecules expressed on the surface of immune cells including T, NK, monocytes and macrophages. After binding with the corresponding ligands, they transmit inhibitory signals into the immune cells, inhibiting the anti-cancer function of the immune cells.

[0005] Due to the great heterogeneity of immune checkpoint ligand expression in tumors and tumor-infiltrating lymphocytes, a single type of immune checkpoint therapy cannot be applied to all patients, and most patients cannot benefit from it. On the other hand, some patients who have received immune checkpoint therapy will have tumor recurrence and develop tolerance to the immune checkpoint therapy, and continued administration will not produce therapeutic effects. For these two reasons, it is necessary to develop immune checkpoint antibodies targeting more targets.

[0006] CD94 is expressed on the surface of NK cells and T cells, and forms heterodimers with NKG2A and NKG2C. The ligands of these heterodimers are all HLA-E. CD94 / NKG2A is an inhibitory receptor, while CD94 / NKG2C is an activating receptor. However, tumor-infiltrating NK cells and T cells mainly express CD94 / NKG2A, rather than CD94 / NKG2C, so immune checkpoint therapies targeting CD94 and NKG2A have potential value.

[0007] So far, there are no publicly reported or clinical trial-stage immune checkpoint therapies targeting CD94 in the world. The fastest-progressing therapy targeting NKG2A is the huZ270 antibody from Innate Pharma. Therefore, the development of immune checkpoint therapies targeting CD94 is of great significance for the treatment and prevention of diseases. Summary of the invention

[0008] This application is filed based on the inventor's discovery of the following issues and facts:

[0009] CD94 is expressed on the surface of NK cells and T cells, and forms heterodimers with NKG2A and NKG2C. CD94 / NKG2A is mainly expressed on the surface of tumor-infiltrating NK cells and T cells, and CD94 / NKG2A is an inhibitory receptor. Therefore, immune checkpoint therapies targeting CD94 and NKG2A have potential value. However, there are not many tumor treatments and research methods targeting the CD94 molecule.

[0010] The inventors of the present application have successfully screened a mouse anti-CD94 monoclonal antibody, which has a high binding activity with human CD94 protein. In addition, the inventors humanized the constant region of the above monoclonal antibody, retaining the CDR of the mouse anti-CD94 monoclonal antibody to obtain a chimeric antibody. Furthermore, the framework region in the light chain variable region or the heavy chain variable region of the chimeric antibody is humanized to obtain a fully humanized anti-CD94 antibody, which can specifically target and bind to human CD94 protein, has the characteristics of low immunogenicity, and can effectively treat and / or prevent CD94-mediated related diseases. The present invention compares the in vitro activity of CD94 antibody and NKG2A antibody huZ270. In the reporting system, the activation ability of CD94 antibody on JurkatT cells is better than that of NKG2A antibody.

[0011] Therefore, in the first aspect of the present invention, the present invention provides an antibody or antigen-binding fragment. According to an embodiment of the present invention, it comprises 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; wherein:

[0012] The amino acid sequence of the heavy chain CDR1 has at least 80% homology with the sequence shown in SEQ ID NO: 1,

[0013] The amino acid sequence of the heavy chain CDR2 has at least 80% homology with the sequence shown in SEQ ID NO: 2,

[0014] The amino acid sequence of the heavy chain CDR3 has at least 80% homology with the sequence shown in SEQ ID NO: 3,

[0015] The amino acid sequence of the light chain CDR1 has at least 80% homology with the sequence shown in SEQ ID NO:4,

[0016] The amino acid sequence of the light chain CDR2 has at least 80% homology with the sequence shown in SEQ ID NO:5,

[0017] The amino acid sequence of the light chain CDR3 has at least 80% homology with the sequence shown in SEQ ID NO: 6. The antibodies or antigen-binding fragments according to the embodiments of the present invention can bind to human CD94 protein, effectively treat or prevent CD94-mediated related diseases, and conduct related scientific research.

[0018] According to an embodiment of the present invention, the above-mentioned antibody or antigen-binding fragment may further include at least one of the following additional technical features:

[0019] According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises: at least one of a heavy chain FR region and a light chain FR region.

[0020] 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 antibody, a primate antibody, a murine antibody, or a mutant thereof.

[0021] According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises: at least one of the heavy chain framework region HFR1, HFR2, HFR3 and HFR4 sequences shown in SEQ ID NOs: 7 to 10, respectively; or at least one of the heavy chain framework region HFR1, HFR2, HFR3 and HFR4 sequences shown in SEQ ID NOs: 15 to 18, respectively.

[0022] According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises: at least one of the light chain framework region LFR1, LFR2, LFR3 and LFR4 sequences shown in SEQ ID NOs: 11 to 14, respectively; or at least one of the light chain framework region LFR1, LFR2, LFR3 and LFR4 sequences shown in SEQ ID NOs: 19 to 22, respectively.

[0023] According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises: at least one of the heavy chain framework region HFR1, HFR2, HFR3 and HFR4 sequences as shown in SEQ ID NOs: 7 to 10, respectively; at least one of the light chain framework region LFR1, LFR2, LFR3 and LFR4 sequences as shown in SEQ ID NOs: 11 to 14, respectively; or at least one of the heavy chain framework region HFR1, HFR2, HFR3 and HFR4 sequences as shown in SEQ ID NOs: 15 to 18, respectively; at least one of the light chain framework region LFR1, LFR2, LFR3 and LFR4 sequences as shown in SEQ ID NOs: 19 to 22, respectively.

[0024] According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises: a heavy chain variable region as shown in SEQ ID NO: 23 or SEQ ID NO: 25; and / or a light chain variable region as shown in SEQ ID NO: 24 or SEQ ID NO: 26.

[0025] According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises: 1) a heavy chain variable region as shown in SEQ ID NO: 23, and a light chain variable region as shown in SEQ ID NO: 24; or 2) a heavy chain variable region as shown in SEQ ID NO: 25, and a light chain variable region as shown in SEQ ID NO: 26.

[0026] According to an embodiment of the present invention, the antibody or antigen-binding fragment contains 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 antibody, a primate antibody and a murine antibody or a mutant thereof.

[0027] According to an embodiment of the present invention, the light chain constant region and the heavy chain constant region are both derived from a murine IgG antibody or a mutant thereof or a human IgG antibody or a mutant thereof.

[0028] According to an embodiment of the present invention, the light chain constant region and the heavy chain constant region are both derived from mouse IgG1, IgG4 antibodies or mutants thereof or human IgG1, IgG4 antibodies or mutants thereof.

[0029] According to an embodiment of the present invention, the light chain constant region comprises a human or mouse κ, λ chain or a variant thereof.

[0030] According to an embodiment of the present invention, the antibody has a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO: 27, 29 or 31 and / or a light chain constant region with an amino acid sequence as shown in SEQ ID NO: 28, 30 or 32.

[0031] According to an embodiment of the present invention, the antibody has a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:27 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:28.

[0032] According to an embodiment of the present invention, the antibody has a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:29 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:30.

[0033] According to an embodiment of the present invention, the antibody has a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:31 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:32.

[0034] According to an embodiment of the present invention, the antibody or antigen-binding fragment has a heavy chain having an amino acid sequence shown in any one of SEQ ID NOs: 33, 35, 36 and 38 and a light chain having an amino acid sequence shown in any one of SEQ ID NOs: 34, 37 and 39.

[0035] According to an embodiment of the present invention, the antibody or antigen-binding fragment has a heavy chain with an amino acid sequence as shown in SEQ ID NO:33 and a light chain with an amino acid sequence as shown in SEQ ID NO:34; the antibody or antigen-binding fragment has a heavy chain with an amino acid sequence as shown in SEQ ID NO:35 and a light chain with an amino acid sequence as shown in SEQ ID NO:37; the antibody or antigen-binding fragment has a heavy chain with an amino acid sequence as shown in SEQ ID NO:36 and a light chain with an amino acid sequence as shown in SEQ ID NO:37; or the antibody or antigen-binding fragment has a heavy chain with an amino acid sequence as shown in SEQ ID NO:38 and a light chain with an amino acid sequence as shown in SEQ ID NO:39.

[0036] According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises a monoclonal antibody or a polyclonal antibody.

[0037] According to an embodiment of the present invention, the monoclonal antibody includes at least one of Fv, single-chain antibody, Fab, single-domain antibody and minimum recognition unit.

[0038] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof is capable of binding to the amino acid sequence shown in SEQ ID NO:40.

[0039] In the second aspect of the present invention, the present invention provides a nucleic acid molecule encoding the antibody or antigen-binding fragment described in the first aspect. According to some specific embodiments of the present invention, the antibody or antigen-binding fragment encoded by the nucleic acid molecule can bind to the human CD94 protein and effectively treat or prevent CD94-mediated related diseases.

[0040] According to an embodiment of the present invention, the above nucleic acid molecule may further include at least one of the following additional technical features:

[0041] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0042] It should be noted that, for the nucleic acids mentioned in the specification and claims of the present invention, those skilled in the art should understand that they actually include any one or both of the complementary double strands. For convenience, in the specification and claims, although only one strand is given in most cases, the other strand complementary thereto is actually disclosed. In addition, the nucleic acid sequence in the present application includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.

[0043] In the third aspect of the present invention, the present invention proposes an expression vector, the expression vector carries the nucleic acid molecule described above. The expression vector may include an optional control sequence, and the control sequence is operably connected to the nucleic acid molecule. Wherein, the control sequence is one or more control sequences that can guide the expression of the nucleic acid molecule in the host. The expression vector proposed in the embodiment of the present invention can efficiently express the antibody or antigen-binding fragment in a large amount in a suitable host cell.

[0044] Herein, "operably connected" means connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their intended function of regulating the transcription and translation of the exogenous gene. When the above-mentioned nucleic acid molecules are connected to the vector, the nucleic acid molecules can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecules. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself. It can be understood by those skilled in the art that the nucleic acid molecules used to encode antibodies or antigen-binding fragments can be inserted into different vectors separately and independently, and it is common to insert them into the same vector. Commonly used vectors can be, for example, plasmids, bacteriophages, etc. For example, Plasmid-X plasmid.

[0045] In a fourth aspect of the present invention, the present invention provides a method for preparing the above-mentioned antibody or antigen-binding fragment, comprising: introducing the above-mentioned expression vector into cells; culturing the cells under conditions suitable for protein expression and secretion to obtain the antibody or antigen-binding fragment. The method proposed according to some specific embodiments of the present invention can effectively obtain the antibody or antigen-binding fragment in large quantities in vitro.

[0046] According to some specific embodiments of the present invention, the method for preparing the above-mentioned antibody or antigen-binding fragment may also include at least one of the following additional technical features:

[0047] According to some specific embodiments of the present invention, the cells are not particularly limited, and either prokaryotic cells or eukaryotic cells can be used.

[0048] According to some specific embodiments of the present invention, the cell is a eukaryotic cell.

[0049] According to some specific embodiments of the present invention, the eukaryotic cell is a mammalian cell. According to some specific embodiments of the present invention, when the cell is a eukaryotic cell, such as a mammalian cell, the expression efficiency of the recombinant antibody is higher.

[0050] In a fifth aspect of the present invention, the present invention provides a recombinant cell, wherein the recombinant cell contains 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 transfecting or transforming the expression vector. According to some specific embodiments of the present invention, the recombinant cell can efficiently and massively express the above-mentioned antibody or antigen-binding fragment under appropriate conditions.

[0051] It should be noted that the recombinant cells of 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, etc. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, insect cells such as armyworms, plant cells such as tobacco, mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc. In some embodiments, the recombinant cells of the present invention are preferably mammalian cells, including BHK cells, CHO cells, NSO cells or COS cells, and do not include animal germ cells, fertilized eggs or embryonic stem cells.

[0052] It should be noted that the "suitable conditions" described in the specification of this application refer to conditions suitable for the expression of the antibody or antigen-binding fragment described in this application. It is easy for those skilled in the art to understand that the conditions suitable for the expression of the antibody or antigen-binding fragment include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell states, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the antibody or antigen-binding fragment according to the specific environment of the laboratory.

[0053] In the sixth aspect of the present invention, the present invention provides an immunoconjugate, comprising the above-mentioned antibody or antigen-binding fragment, and a therapeutic agent. As mentioned above, the antibody or antigen-binding fragment of the embodiment of the present invention can effectively bind to the CD94 protein, and effectively treat or prevent CD94-related diseases. Therefore, the immunoconjugate containing the antibody or antigen-binding fragment can also bind to the human CD94 protein, and the immunoconjugate has a good effect of preventing and / or treating CD94-mediated diseases.

[0054] In the seventh aspect of the present invention, the present invention proposes a composition, including the above-mentioned antibody or antigen-binding fragment, nucleic acid molecule, expression vector or recombinant cell. As mentioned above, the antibody or antigen-binding fragment of some specific embodiments of the present invention can effectively bind to human CD94 protein and effectively inhibit the proliferation of tumor cells. Therefore, the composition containing the above substances can also effectively bind to human CD94 protein and has a good effect of preventing and / or treating CD94-mediated diseases. The type of the composition is not particularly limited and can be a food composition or a pharmaceutical composition.

[0055] The compositions of the present invention may also 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 composition may also contain a chemotherapeutic agent. The antibodies or antigen-binding fragments thereof, or immunoconjugates of the present invention may also be combined with a second therapeutic agent, exemplary agents 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 CD94 upstream or downstream signal transduction.

[0056] It should be noted that the composition includes combinations separated in time and / or space, as long as they can work together to achieve the purpose of the present invention. For example, the components contained in the composition can be administered to the subject as a whole, or separately. When the components contained in the composition are administered to the subject separately, each component can be administered to the subject simultaneously or sequentially.

[0057] In the eighth aspect of the present invention, the present invention provides a drug, including the above-mentioned antibody or antigen-binding fragment, nucleic acid molecule, expression vector, recombinant cell or composition. As mentioned above, the antibody or antigen-binding fragment of some specific embodiments of the present invention can effectively bind to human CD94 protein, therefore, a drug containing an effective amount of the active ingredient of the antibody or antigen-binding fragment or a series of substances thereof can also effectively bind to human CD94 protein, and has a good effect of preventing and / or treating CD94-mediated diseases.

[0058] According to an embodiment of the present invention, the above-mentioned medicine may further include at least one of the following additional technical features:

[0059] According to an embodiment of the present invention, the drug may further include a pharmaceutically acceptable carrier.

[0060] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals.

[0061] The effective amount of the antibody or antigen-binding fragment of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. For example, several divided doses may be administered daily, or the dose may be reduced proportionally, depending on the urgency of the treatment situation.

[0062] As used herein, "pharmaceutically acceptable" ingredients are suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), i.e., substances with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

[0063] The medicine of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Usually, the drug preparation should match the mode of administration, wherein the mode of administration can be oral administration, nasal administration, intradermal administration, subcutaneous administration, intramuscular administration, intravenous administration or intraperitoneal administration, and the dosage form of the medicine of the present invention is injection, oral preparation (tablet, capsule, oral liquid), transdermal agent, sustained release agent. For example, it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The medicine is preferably manufactured under sterile conditions. The antibody or antigen-binding fragment can be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.

[0064] Of course, the anti-CD94 monoclonal antibody herein can also be made into a part of a kit or other diagnostic reagent as required.

[0065] In the ninth aspect of the present invention, the present invention proposes a kit, the kit containing the above-mentioned antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector or recombinant cell. As mentioned above, the antibody or antigen-binding fragment of some specific embodiments of the present invention can effectively bind to the human CD94 protein, so the kit containing the antibody or antigen-binding fragment can effectively detect the human CD94 protein qualitatively or quantitatively. The kit provided by the present invention can be used for immunoblotting, immunoprecipitation and other kits involving the use of human CD94 and antibody specific binding properties for detection. These kits may contain any one or more of the following: antagonists, anti-CD94 antibodies or drug reference materials; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents; instructions or literature, etc. Anti-CD94 antibodies can be used for different types of diagnostic tests, for example, various diseases or the presence of drugs, toxins or other proteins can be detected in vitro or in vivo, for example, by testing the serum or blood of the subject to test related diseases, and scientific research can also be carried out, using the kit to detect human CD94 protein in the sample to be tested. 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 radioimmunoassay and radioimmunotherapy of the above-mentioned diseases, etc. For the above-mentioned application scenarios, the binding molecules are also applicable and will not be described here.

[0066] The kit may also include conventional materials for detecting CD94, such as coating fluid.

[0067] In the tenth aspect of the present invention, the present invention proposes the use of the above-mentioned antibody or antigen-binding fragment nucleic acid molecule, expression vector, recombinant cell or composition in the preparation of a drug, which is used to prevent and / or treat CD94-mediated related diseases. As mentioned above, the antibody or antigen-binding fragment of some specific embodiments of the present invention can effectively bind to human CD94 protein, so a drug containing an effective amount of the antibody or antigen-binding fragment or a series of substances thereof can also effectively bind to human CD94 protein, and has a good effect of preventing and / or treating CD94-mediated diseases.

[0068] According to an embodiment of the present invention, the above-mentioned use of preparing a medicine may also 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.

[0070] According to an embodiment of the present invention, the autoimmune disease includes at least one of the following: 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, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.

[0071] According to an embodiment of the present invention, the cancer includes at least one of the following: 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.

[0072] In the eleventh aspect of the present invention, the present invention proposes the use of the above-mentioned antibody or antigen-binding fragment, nucleic acid molecule, expression vector or recombinant cell in preparing a kit for detecting CD94. As mentioned above, the antibody or antigen-binding fragment of some specific embodiments of the present invention can effectively bind to human CD94 protein and block the binding of the CD94 protein to its receptor. Therefore, the antibody or antigen-binding fragment can be used to prepare a kit for detecting CD94 protein, and the kit can effectively perform qualitative or quantitative detection of human CD94 protein.

[0073] In the twelfth aspect of the present invention, the present invention proposes a method for treating or preventing CD94-mediated related diseases. 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 the human CD94 protein, and can effectively treat or prevent CD94-mediated related diseases, preferably autoimmune diseases or cancer. Therefore, the method according to the embodiment of the present invention can effectively treat or prevent CD94-mediated related diseases.

[0074] According to an embodiment of the present invention, the above method for treating or preventing a disease may further include at least one of the following additional technical features:

[0075] According to an embodiment of the present invention, the CD94-mediated related diseases include transplant rejection, autoimmune diseases, infectious diseases and cancer. Those skilled in the art will appreciate that CD94 antibodies are highly expressed on the surface of NK cells, and excessive activation of NK cells in sterile inflammation (transplant rejection, autoimmune diseases) and bacterial inflammation (infectious diseases) can lead to tissue and organ damage, so CD94 antibodies can be used as clearance antibodies to treat or prevent the above diseases.

[0076] According to an embodiment of the present invention, the autoimmune disease includes at least one of the following: 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, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.

[0077] According to an embodiment of the present invention, the cancer includes at least one of the following: 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.

[0078] In the thirteenth aspect of the present invention, the present invention proposes a method for diagnosing CD94-mediated related diseases. According to an embodiment of the present invention, it includes using at least one of the following to detect CD94 in the sample to be tested: 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 based on the detection result of the CD94, the content of CD94 in the sample to be tested is determined. The antibody or antigen-binding fragment, or the antibody or antigen-binding fragment expressed by the nucleic acid molecule, expression vector, or recombinant cell proposed in this application can effectively bind to the human CD94 protein, or the nucleic acid molecule, expression vector, or the antibody or antigen-binding fragment expressed by the recombinant cell can effectively bind to CD94. Therefore, the method described in this application can effectively detect the content of CD94 in the sample to be tested from the subject, and can effectively diagnose the related diseases caused by CD94.

[0079] According to an embodiment of the present invention, the above method for diagnosing a disease may further include at least one of the following additional technical features:

[0080] According to an embodiment of the present invention, the CD94 content in the test sample is not less than the minimum standard for disease, which indicates that the test sample is derived from a patient suffering from a disease related to CD94. The value of the minimum standard can be determined by performing a comparative analysis and verification of the CD94 content in the test samples of a large number of individuals suffering from the disease related to CD94 and a large number of healthy individuals.

[0081] According to an embodiment of the present invention, the sample to be tested includes at least one of the following: blood, saliva, sweat, tissue, cell, blood, serum, plasma, feces and urine.

[0082] According to an embodiment of the present invention, the CD94-mediated related diseases include transplant rejection, autoimmune diseases, infectious diseases and cancer.

[0083] According to an embodiment of the present invention, the autoimmune disease includes at least one of the following: 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, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.

[0084] According to an embodiment of the present invention, the cancer includes at least one of the following: 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.

[0085] In the fourteenth aspect of the present invention, the present invention proposes a method for staging CD94-mediated related diseases. According to an embodiment of the present invention, it includes using at least one of the following to detect CD94 in the sample to be tested: 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 based on the detection result of the CD94, the content of CD94 in the sample to be tested is determined. The antibody or antigen-binding fragment, or the nucleic acid molecule, expression vector, and antibody or antigen-binding fragment expressed by the recombinant cell proposed in this application can effectively bind to human CD94. Therefore, the method described in this application can effectively detect the content of CD94 in the sample to be tested from the subject, and evaluate the stage of the related disease caused by CD94 based on the content of CD94.

[0086] 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:

[0087] According to an embodiment of the present invention, the content of CD94 in the sample to be tested is not less than the standard level of tumor stage IV, which indicates that the sample to be tested is derived from a patient with tumor stage IV; the content of CD94 in the sample to be tested is between the standard levels of tumor stage IV and stage III, which indicates that the sample to be tested is derived from a patient with tumor stage III; the content of CD94 in the sample to be tested is between the standard levels of tumor stage III and stage II, which indicates that the sample to be tested is derived from a patient with tumor stage II; the content of CD94 in the sample to be tested is between the standard levels of stage I and stage II, which indicates that the sample to be tested is derived from a patient with tumor stage I. Those skilled in the art can understand that the level of CD94 in the tumor stage I, stage II, stage III, and stage IV varies according to the type of tumor, and the stage of the tumor can be determined by comparing the content of CD94 in the sample to be tested with the standard level of CD94 corresponding to the tumor stage, or comparing the content of CD94 in the sample to be tested with the content of CD94 in a sample of an individual or group of known disease stages. The values ​​of the standard levels of tumor stages I, II, III, and IV can be determined by comparative analysis and verification of the differences in the CD94 content in the test samples of a large number of individuals suffering from the CD94-related diseases and a large number of healthy individuals.

[0088] According to an embodiment of the present invention, the sample to be tested includes at least one of the following: blood, saliva, sweat, tissue, cell, blood, serum, plasma, feces and urine.

[0089] According to an embodiment of the present invention, the CD94-mediated related diseases include transplant rejection, autoimmune diseases, infectious diseases and cancer.

[0090] According to an embodiment of the present invention, the autoimmune disease includes at least one of the following: 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, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.

[0091] According to an embodiment of the present invention, the cancer includes at least one of the following: 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.

[0092] In the fifteenth aspect of the present invention, the present invention provides a method for evaluating the prognosis of a CD94-mediated disease. According to an embodiment of the present invention, the method comprises detecting CD94 in a sample to be tested using 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; and 4) the recombinant cell described above, and determining the content of CD94 in the sample to be tested based on the detection result of CD94. As mentioned above, the content of CD94 has an important influence on cancer. After individuals with related diseases are treated, the prognosis of such diseases can be effectively evaluated by monitoring the content of CD94 in their tissues or excreta, such as peripheral blood, urine, etc. For example, the content of CD94 in the subject before and after treatment is compared, or the content of CD94 in the subject after treatment is compared with the CD94 level of a normal individual or a diseased individual. The antibodies or antigen-binding fragments, or nucleic acid molecules, expression vectors, and antibodies or antigen-binding fragments expressed by recombinant cells proposed in the present application can effectively bind to human CD94. Therefore, the method described in the present application can effectively detect the content of CD94 in the test sample derived from the subject, and evaluate the prognosis of related diseases caused by CD94 based on the content of CD94.

[0093] According to an embodiment of the present invention, the above method for evaluating disease prognosis may further include at least one of the following additional technical features:

[0094] According to an embodiment of the present invention, the sample to be tested is derived from a patient suffering from a CD94-mediated related disease before or after treatment.

[0095] According to an embodiment of the present invention, the sample to be tested includes at least one of the following: blood, saliva, sweat, tissue, cell, blood, serum, plasma, feces and urine.

[0096] 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 test sample of a patient suffering from a CD94-mediated related disease before or after the treatment.

[0097] According to an embodiment of the present invention, a decrease in the level of CD94 in a test sample of a patient suffering from a CD94-mediated disease after treatment is an indication of a good prognosis for the patient.

[0098] According to an embodiment of the present invention, the CD94-mediated related diseases include transplant rejection, autoimmune diseases, infectious diseases and cancer.

[0099] According to an embodiment of the present invention, the autoimmune disease includes at least one of the following: 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, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.

[0100] According to an embodiment of the present invention, the cancer includes at least one of the following: 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.

[0101] In the sixteenth aspect of the present invention, the present invention proposes the use of the above-mentioned antibody or antigen-binding fragment, nucleic acid molecule, expression vector, recombinant cell, composition or drug in treating or preventing CD94-mediated related diseases. As mentioned above, the antibody or antigen-binding fragment can effectively bind to human CD94 and can effectively treat or prevent CD94-mediated related diseases.

[0102] According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features:

[0103] According to an embodiment of the present invention, the CD94-mediated related diseases include transplant rejection, autoimmune diseases, infectious diseases and cancer.

[0104] According to an embodiment of the present invention, the autoimmune disease includes at least one of the following: 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, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.

[0105] According to an embodiment of the present invention, the cancer includes at least one of the following: 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.

[0106] In the seventeenth aspect of the present invention, the present invention proposes the use of the above-mentioned antibodies or antigen-binding fragments, nucleic acid molecules, expression vectors or recombinant cells in diagnosing CD94-mediated related diseases, staging CD94-mediated related diseases or evaluating the prognosis of CD94-mediated related diseases. As mentioned above, the antibodies or antigen-binding fragments, or nucleic acid molecules, expression vectors, antibodies or antigen-binding fragments expressed by recombinant cells proposed in the present application can effectively bind to human CD94. Therefore, the method described in the present application can effectively detect the content of CD94 in the sample to be tested from the subject, and can effectively diagnose, stage and evaluate the prognosis of CD94-mediated related diseases.

[0107] According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features:

[0108] According to an embodiment of the present invention, the CD94-mediated related diseases include transplant rejection, autoimmune diseases, infectious diseases and cancer.

[0109] According to an embodiment of the present invention, the autoimmune disease includes at least one of the following: 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, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.

[0110] According to an embodiment of the present invention, the cancer includes at least one of the following: 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.

[0111] The "subject" or "individual" referred to in the present invention generally refers to a mammal, such as a primate and / or a rodent, in particular a human, a monkey or a mouse.

[0112] Beneficial effects of the present invention:

[0113] 1) So far, there are no patents or research reports on the use of blocking CD94 antibodies in anti-cancer research.

[0114] 2) The CD94 antibody obtained by the present invention has high binding activity with the CD94 protein, and in in vitro functional experiments, the activity of the CD94 antibody is superior to that of the NKG2A antibody in the clinical stage.

[0115] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0116] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0117] Figure 1 This is a diagram showing the binding result of human-mouse 15C10 chimeric antibody to 293T-CD94 / NKG2A cells according to an embodiment of the present invention;

[0118] Figure 2 This is a diagram showing the result of human-mouse 15C10 chimeric antibody blocking HLA-E binding to 293T-CD94 / NKG2A cells according to an embodiment of the present invention;

[0119] Figure 3 This is a graph showing the results of human-mouse 15C10 chimeric antibody and humanized 15C10 antibody binding to 293T-CD94 / NKG2A cells according to an embodiment of the present invention;

[0120] Figure 4 This is a graph showing the results of humanized 15C10 antibody according to an embodiment of the present invention, and NKG2A antibodies huZ199 and huZ270 as controls binding to 293T-CD94 / NKG2A cells;

[0121] Figure 5 This is a diagram showing the result of humanized 15C10 antibody blocking HLA-E binding to 293T-CD94 / NKG2A cells according to an embodiment of the present invention;

[0122] Figure 6 This is a diagram showing the ELISA result of the binding of the humanized 15C10 antibody to the CD94 / NKG2A-Fc heterodimer protein according to an embodiment of the present invention;

[0123] Figure 7 This is a result diagram showing that the humanized 15C10 antibody promotes the activation of Jurkat-NFAT-lucia-CD94 / NKG2AT cells according to an embodiment of the present invention;

[0124] Figure 8 This is a graph showing the results of humanized 15C10 antibody promoting anti-cancer in mice reconstituted with the immune system according to an embodiment of the present invention. DETAILED DESCRIPTION

[0125] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0126] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0127] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0128] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. The abbreviations for amino acid residues are standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0129] The antibody or antigen-binding fragment of the present invention is usually prepared by a biosynthetic method. According to the nucleotide sequence of the present invention, a person skilled in the art can easily prepare the encoding nucleic acid of the present invention by various known methods. These methods include but are not limited to: PCR, DNA artificial synthesis, etc. For specific methods, please refer to J. Sambrook, "Molecular Cloning Laboratory Guide". As an embodiment of the present invention, the encoding nucleic acid sequence of the present invention can be constructed by synthesizing the nucleotide sequence in segments and then performing overlap extension PCR. Wherein, the antibody or antigen fragment is numbered and defined using the Kabat numbering system.

[0130] The antibodies of the present invention include murine antibodies, chimeric antibodies, and humanized antibodies, preferably humanized antibodies.

[0131] The term "murine antibody" in the present invention refers to a monoclonal antibody against human CD94 prepared according to the knowledge and skills in the art. During preparation, a test subject is injected with an antigen, and then a hybridoma expressing an antibody having the desired sequence or functional properties is isolated. In a preferred embodiment of the present invention, the murine CD94 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine κ, λ chain or a variant thereof, or further comprise a heavy chain constant region of a murine IgG1, IgG2, IgG3 or a variant thereof.

[0132] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse antibody. The variable region gene is cloned from mouse hybridoma cells, and then the constant region gene of the human antibody is cloned as needed. The mouse variable region gene and the human constant region gene are connected into a chimeric gene and inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic expression system or a 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 κ, λ chain or a variant thereof. The antibody heavy chain of the CD94 chimeric antibody further comprises a heavy chain constant region of a human IgG1, IgG2, IgG3, IgG4 or a variant thereof.

[0133] The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting the mouse CDR sequence into the human antibody variable region framework, i.e., different types of human germline antibody framework sequences. Such framework sequences can be obtained from public DNA databases or published references including germline antibody gene sequences. For example, the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database. In order to avoid a decrease in immunogenicity and a decrease in activity, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity.

[0134] As used herein, the term "monoclonal antibody" refers to an antibody that has a single antigen binding site.

[0135] Herein, the term "polyantibody" refers to an antibody having two or more different antigen binding sites.

[0136] As used herein, the term "mutant" or "variant" may refer to any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid mutations.

[0137] The term "complementarity determining region" or "CDR" or "CDR sequence" refers to the amino acid sequence in an antibody that is responsible for antigen binding, for example, generally including: amino acid residues around 23-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable region, and around 31-35B (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable region (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health). of Health, Bethesda, MD. (1991)); and / or from "hypervariable loops" (e.g., amino acid residues around 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable region, and 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)).

[0138] As used herein, the term "identity" is used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see 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 Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, 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 BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs utilizing these algorithms are also available 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, BLASTAltschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0139] Without substantially affecting the activity of the antibody (retaining at least 95% of the activity), those skilled in the art may replace, add and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids in the sequence of the present invention to obtain variants of the sequence of the antibody or its functional fragment. They are all considered to be included in the scope of protection of the present invention. For example, amino acids with similar properties are replaced in the variable region. The variant sequence of the present invention may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity (or homology) with the reference sequence. The sequence identity of the present invention can be measured using sequence analysis software. For example, the computer program BLAST with default parameters is used, especially BLASTP or TBLASTN. The amino acid sequences described in the present invention are all shown in an N-terminal to C-terminal manner.

[0140] As previously described, the monoclonal antibody of the present invention may be a full-length antibody or may only contain a functional fragment thereof (e.g., Fab, F(ab')2 or scFv fragment), or may be modified to affect function. The present invention includes anti-CD94 antibodies with modified glycosylation patterns. In some applications, it may be useful to modify to remove undesirable glycosylation sites, or antibodies in which fucose moieties are absent on the oligosaccharide chains, for example, to enhance antibody-dependent cellular cytotoxicity (ADCC) function. In other applications, galactosylation modification may be performed to alter complement-dependent cytotoxicity (CDC).

[0141] In this article, the "full-length antibody" is a tetrapeptide chain structure composed of two identical light chains and two identical heavy chains connected by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD) or immunoglobulin E (IgE). The same type of immunoglobulin can also be divided into different subclasses according to the amino acid composition, such as IgG1, IgG2, IgG3, IgG4. Immunoglobulin light chains are divided into κ chains or λ chains according to different constant regions.

[0142] The term "functional fragment" as used herein refers in particular to antibody fragments such as CDR-grafted antibodies, Fab, Fab', F(ab')2, Fv or scFv, nanobodies, or any fragments that should be able to increase half-life by chemical modification, such as the addition of poly(alkylene) glycols, such as polyethylene glycol ("PEGylation, PEGylation") (called PEGylated fragments of Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG or Fab'-PEG) ("PEG" is polyethylene glycol), or by incorporation into liposomes, wherein the fragment has CD94 binding activity. Preferably, the functional fragment will be composed of or contain a partial sequence of the heavy chain variable region or light chain variable region of the antibody from which it is derived, and the partial sequence is sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived, preferably at least 1 / 100 of the affinity of the antibody from which it is derived, and in a more preferred manner at least 1 / 10. Such functional fragments will contain a minimum of 3 amino acids, and preferably 5, 10, 15, 25, 50 and 100 consecutive amino acids of the antibody sequence from which they are derived.

[0143] In the present invention, unless otherwise specified, the term "antigen-binding fragment" used generally refers to an antigen-binding antibody fragment, which may include a portion of a complete antibody, generally an antigen-binding region or a variable region, such as, for example, CDR-grafted antibodies, Fab, Fab', F(ab')2, Fv or scFv, nanobodies, etc.

[0144] In this article, the term "CDR transplanted antibody" refers to transplanting the CDR of a monoclonal antibody of one species to the variable region of an antibody of another species. For example, the CDR of a mouse monoclonal antibody can be transplanted to the variable region of a human antibody to replace the human antibody CDR, so that the human antibody obtains the antigen binding specificity of the mouse monoclonal antibody while reducing its heterology.

[0145] As used herein, the term "Fab antibody" or "Fab" generally refers to an antibody containing only Fab molecules, which are composed of VH and CH1 of the heavy chain and a complete light chain, with the light chain and the heavy chain connected by a disulfide bond.

[0146] As used herein, the term "nanoantibody" (single domain antibody or VHH antibody), which was originally described as an antigen-binding immunoglobulin (variable) domain of a "heavy chain antibody" (i.e., "antibody lacking a light chain") (Hamers-Casterman C, Atarhouch 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)), comprises only a heavy chain variable region (VH) and conventional CH2 and CH3 regions, which specifically bind to an antigen through the heavy chain variable region.

[0147] As used herein, the term "Fv antibody" generally refers to an antibody consisting only of a light chain variable region (VL) and a heavy chain variable region (VH) linked by non-covalent bonds, and is the smallest functional fragment of an antibody that retains a complete antigen binding site.

[0148] As used herein, the term "single-chain antibody" or "scFv" refers to a fragment consisting of the variable regions of the heavy and light chains of an antibody connected by a short peptide.

[0149] The amino acid or nucleic acid sequences involved in the present invention are shown in Table 1.

[0150] Table 1:

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157] The present invention will be described in detail below through examples. In the examples or test examples, experimental methods without specific conditions are all carried out under conventional conditions.

[0158] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0159] Example 1 Preparation of anti-human CD94 hybridoma monoclonal antibody

[0160] 1.1 Hybridoma cell screening

[0161] This example is used to obtain a murine monoclonal antibody against human CD94, wherein the immunizing antigen is a purified recombinant CD94 extracellular region Fc fusion protein (CD94-Fc) (recombinant CD94 extracellular region Fc fusion protein, the amino acid sequence of which is shown in SEQ ID NO: 54), and C57bl / 6 mice (9 weeks old, purchased from Shanghai Lesk, weighing about 20 g) are immunized.

[0162] Immunized mice were intraperitoneally immunized three times with purified antigen and complete Freund's adjuvant, and the immune response was detected after bleeding from the tail vein. The serum was screened by ELISA and flow cytometry through routine operation steps to obtain mice with anti-human CD94 immunoglobulin. The spleen cells of the mice with the highest anti-CD94 immunoglobulin were taken out and fused with mouse myeloma cells SP2 / 0 cells (ATCC No. CRL-1581). The fused hybridoma cells were screened for antibodies to obtain mouse monoclonal antibodies. The specific operation was as follows:

[0163] Resuspend with HAT complete medium (RPMI-1640 medium containing 20% ​​FBS, 1×HAT and 1×OPI), distribute in 96-well cell culture plates, and incubate at 37°C, 5% CO2. Add HAT complete medium at 50 μL / well on the 5th day after fusion. On the 7th to 8th day after fusion, according to the cell growth density, replace the medium with HT complete medium (RPMI-1640 medium containing 20% ​​FBS, 1×HT and 1×OPI), 200 μL / well.

[0164] On the 10th-11th day after fusion, flow cytometry binding detection was performed according to the cell growth density. The positive wells were replaced with medium and expanded to 24-well plates in time according to the cell density. The cell lines transferred into the 24-well plates were retested for seed preservation and the first subcloning. The first subclone screening was positive for seed preservation and the second subcloning was performed. The second subclone was positive for seed preservation and protein expression. Antibodies were further prepared by serum-free cell culture method, purified by protein G affinity chromatography, and used for subsequent functional activity detection.

[0165] 1.2 Hybridoma cell sequencing

[0166] Cultivate the total number of candidate hybridoma cells screened in Section 1.1 above to 10 6 , centrifuged at 800 rpm for 10 minutes to collect the cells, and extracted total RNA using Trizol kit (Invitrogen); using total RNA as template, reverse transcribed to synthesize cDNA library (Invitrogen), and using cDNA as template, PCR amplified the variable region nucleic acid sequence of CD94 antibody corresponding to the hybridoma cells. The primer sequences used in the PCR amplification reaction are complementary to the first framework region or signal peptide region and constant region of the antibody variable region (specific sequence selection refers to Larrick, JW, et al., (1990) Scand. J. Immunol., 32, 121-128 and Coloma, JJ et al., (1991) BioTechniques, 11, 152-156). PCR amplification was performed in a 50 μL reaction system, and 2 μL of cDNA, 5 μL of 10×PCR buffer, 2 μL of upstream and downstream primers (5 μM), 2 μL of dNTP, 1 μL of Taq enzyme (Takara, ExTaq), and 38 μL of H2O were added respectively; pre-denaturation at 95°C for 5 min, and temperature cycle was entered for PCR amplification. The reaction conditions were: denaturation at 94°C for 30S, annealing at 58°C for 45S, extension at 72°C for 50S, a total of 32 cycles, and then extension at 72°C for 7 min. After sequencing the amplified product, the heavy chain variable region (SEQ ID NO: 23) and light chain variable region (SEQ ID NO: 24) sequences of the mouse monoclonal antibody against CD94 were obtained.

[0167] Example 2 CD94 chimeric antibody flow cytometry binding experiment

[0168] Flow cytometry experiments were used to detect the binding properties of CD94 chimeric antibodies. CD94 protein and NKG2A protein were overexpressed in 293T cells (ATCC No. CRL-3216) (293T-CD94 / NKG2A). The strength of the signal after the addition of the antibody was used to determine the binding properties of the chimeric antibody and CD94.

[0169] 2.1 Construction of 293T cells overexpressing human CD94

[0170] HEK293T cells were cultured at 5 × 10 5Cells / well were plated in a six-well plate and cultured overnight in DMEM medium without dual antibodies. Before transfection, the medium was discarded and 1 mL of fresh DMEM medium without dual antibodies was added. The coding sequence (SEQ ID NO: 41) of human CD94 protein (SEQ ID NO: 40) was inserted between the restriction sites EcoRI and BamHI of the pLVX-EF1a-IRES-puro vector, or the coding sequence (SEQ ID NO: 42) of NKG2A protein (SEQ ID NO: 43) was inserted between the restriction sites EcoRI and BamHI of the pLVX-EF1a-IRES-puro vector. IDNO: 43) were added to 200 μL serum-free DMEM medium with pMD2G and psPAX2 vectors (3 μg in total) at a ratio of 2:1:1, and 12 μg of polyetherimide (PEI, Polysciences Co., Ltd.) was added. After mixing, let it stand for 16 minutes, and then all the liquid was added to a six-well plate with HEK293T cells. After 6 hours of culture, the culture medium was discarded and fresh complete DMEM medium was added for culture. After 48 hours of transfection, the cell culture supernatant was collected and filtered through a 0.45 μm filter (Millipore), which was the viral supernatant. All the viral supernatant was added to a 1×10 4 Add polybrene (Sigma) at a final concentration of 4 μg / mL to a 6-well plate of 293T cells and culture for 12 hours. Then discard the supernatant and add fresh complete DMEM medium. The resulting cells are 293T-CD94 / NKG2A cells.

[0171] 2.2 Detection of chimeric antibody CD94 binding activity

[0172] The CD94 chimeric antibody 15C10-hIgG1mut used in this section was expressed in this laboratory, wherein the heavy chain amino acid sequence is shown in SEQ ID NO:33, and the light chain amino acid sequence is shown in SEQ ID NO:34. In addition, the blocking NKG2A antibody huZ270-hIgG1mut used was also expressed in this laboratory, wherein the heavy chain amino acid sequence is shown in SEQ ID NO:44, and the light chain amino acid sequence is shown in SEQ ID NO:45.

[0173] Dilute 293T-CD94 / NKG2A cells to 2×10 6 / mL, add it to a 1.5mL EP tube at a volume of 100μL / tube, add 10μL / tube of mouse serum, and block it at 4℃ for 30min. Add different concentration gradients of CD94 chimeric antibody 15C10-hIgG1mut or blocking NKG2A antibody huZ270-hIgG1mut separately and incubate at 4℃ for 30min. Add 1mL PBS to the EP tube, centrifuge at 4℃, 3500rpm×5min, discard the supernatant, and wash it again with PBS. After centrifugation, discard the supernatant, resuspend the cells with 100μL / tube PBS, add 1μL / tube Alexa-647-labeled mouse anti-human Fc antibody secondary antibody (Biolegend), and incubate at 4℃ in the dark for 30min. Wash twice with PBS, discard the supernatant after centrifugation. Resuspend the cells with 200μL / tube PBS and detect them by flow cytometry. The results are as follows Figure 1 As shown, it is further demonstrated that the 15C10-hIgG1mut chimeric antibody of the present invention is able to bind to CD94.

[0174] Example 3 Detection of blocking ability of CD94 chimeric antibody

[0175] The CD94 antibody blocks the binding of CD94 / NKG2A and its ligand HLA-E by binding to the extracellular region of the CD94 protein. Flow cytometry experiments were used to detect the blocking effect of the CD94 antibody on HLA-E binding to 293T-CD94 / NKG2A.

[0176] 293T-CD94 / NKG2A cells (same as in Example 2) were diluted to 2×10 6 / mL, add it to a 1.5mL EP tube at a volume of 100μL / tube, add 10μL / tube of mouse serum, and block it at 4℃ for 30min. Add the above-mentioned CD94 chimeric antibody 15C10-hIgG1mut and blocking NKG2A antibody huZ270-hIgG1mut separately, and incubate at 4℃ for 30min. Add 0.1μg / tube APC-labeled HLA-E tetramer protein and incubate at 4℃ for 30min. Wash twice with PBS, discard the supernatant after centrifugation. Resuspend the cells with 200μL / tube PBS and detect them by flow cytometry. The results are as follows Figure 2 As shown, it can be seen that the CD94 antibody of the present invention can block the binding of HLA-E to CD94 / NKG2A, and the blocking ability is comparable to that of the NKG2A antibody huZ270.

[0177] Example 4 Humanization of mouse anti-human CD94 monoclonal antibody

[0178] On the basis of Examples 1 and 2, by comparing the IMGT human antibody heavy and light chain variable region germline gene database and MOE software, the heavy chain or light chain variable region germline genes with high homology to the mouse monoclonal antibody were selected as templates, and the CDRs of the mouse monoclonal antibody were transplanted into the corresponding human templates to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The amino acid residues were determined and annotated by the Kabat numbering system.

[0179] In order to maintain the conformation of the CDR region, based on the three-dimensional structure of the mouse antibody, the residues on the VL and VH binding interface, the residues close to the CDR and embedded in the protein, and the residues that directly interact with the CDR were back mutated to ensure that the activity of the variable region would not be affected, thereby obtaining a humanized antibody. The sequence of the heavy chain variable region of the humanized CD94 antibody is shown in SEQ ID NO: 25, and the sequence of the heavy chain variable region is shown in SEQ ID NO: 26.

[0180] Example 5 Flow cytometry binding experiment of CD94 chimeric antibody and human CD94 antibody

[0181] CD94 protein and NKG2A protein were overexpressed in 293T cells (ATCC number) (293T-CD94 / NKG2A), and ELISA experiments were used to detect the binding properties of CD94 chimeric antibodies and human CD94 antibodies to 293T-CD94 / NKG2A.

[0182] The 293T-CD94 / NKG2A cells overexpressing CD94 and NKG2A obtained in Example 2 were diluted to 2×10 6 / mL, add 100μL / tube to 1.5mL EP tube, add 10μL / tube mouse serum, and block at 4℃ for 30min. Add gradient concentrations (3-fold dilution, final concentration up to 10μg / mL) of CD94 chimeric antibody 15C10-hIgG4 (expressed in this laboratory, heavy chain sequence SEQ ID NO:36, and light chain sequence SEQ ID NO:37) and humanized antibody h15C10-hIgG4 (expressed in this laboratory, heavy chain sequence SEQ ID NO:38, and light chain sequence SEQ ID NO:39) separately, and incubate at 4℃ for 30min. Add 1mL PBS to the EP tube, centrifuge at 4℃, 3500rpm×5min, discard the supernatant, and wash again with PBS. After centrifugation, discard the supernatant, resuspend the cells with 100 μL / tube PBS, add 0.1 μL / tube Alexa-647 labeled mouse anti-human Fc antibody secondary antibody (Biolegend), and incubate at 4°C in the dark for 30 min. Wash twice with PBS, centrifuge and discard the supernatant. Resuspend the cells with 200 μL / tube PBS and detect with flow cytometer. The results are shown in Figure 3 As shown, both the humanized antibody and the chimeric antibody of the present invention have good binding ability, wherein the binding ability of the humanized antibody is better than that of the chimeric antibody.

[0183] Example 6 Human CD94 Antibody ELISA Binding Experiment

[0184] CD94 protein and NKG2A protein were overexpressed in 293T cells (ATCC number) (293T-CD94 / NKG2A), and ELISA experiments were used to detect the binding properties of the human CD94 antibody and NKG2A antibody obtained in the above example to 293T-CD94 / NKG2A.

[0185] The 293T-CD94 / NKG2A cells overexpressing CD94 and NKG2A obtained in Example 2 were diluted to 2×10 6 / mL, added to a 1.5mL EP tube at a volume of 100μL / tube, 10μL / tube of mouse serum was added thereto, and blocked at 4°C for 30min. Humanized antibody h15C10-hIgG4 (same as Example 5), non-blocking NKG2A antibody huZ199-hIgG4 (expressed in this laboratory, heavy chain sequence SEQ ID NO:46, and light chain sequence SEQ ID NO:47) and blocking NKG2A antibody huZ270-hIgG4 (expressed in this laboratory, heavy chain sequence SEQ ID NO:48, and light chain sequence SEQ ID NO:49) or control antibody hIgG4 were added separately at gradient concentrations (3-fold dilution, final concentration up to 30μg / mL) and incubated at 4°C for 30min. 1mL PBS was added to the EP tube, centrifuged at 4°C, 3500rpm×5min, the supernatant was discarded, and then washed once with PBS. After centrifugation, discard the supernatant, resuspend the cells with 100 μL / tube PBS, add 0.1 μL / tube Alexa-647 labeled mouse anti-human Fc antibody secondary antibody (Biolegend), and incubate at 4°C in the dark for 30 min. Wash twice with PBS, centrifuge and discard the supernatant. Resuspend the cells with 200 μL / tube PBS and detect with flow cytometer. The results are shown in Figure 4 As shown, the ability of the humanized antibody of the present invention to bind to 293T-CD94 / NKG2A cells is superior to that of the NKG2A antibodies huZ199 and huZ270.

[0186] Example 7: Detection of blocking ability of humanized CD94 antibody

[0187] CD94 antibody blocks the binding of CD94 / NKG2A to its ligand HLA-E by binding to the extracellular region of CD94. Flow cytometry experiments were used to detect the blocking effect of CD94 antibody on HLA-E binding to 293T-CD94 / NKG2A.

[0188] 293T-CD94 / NKG2A cells (same as above) were diluted to 2×10 6 / mL, add it to a 1.5ml EP tube at a volume of 100μL / tube, add 10μl / tube of mouse serum, and block it at 4℃ for 30min. Add the CD94 humanized antibody 15C10-hIgG4, NKG2A antibody huZ270-hIgG4 or control antibody hIgG4 used in the above example, and incubate at 4℃ for 30min. Add 0.1μg / tube APC-labeled HLA-E tetramer protein and incubate at 4℃ for 30min. Wash twice with PBS, discard the supernatant after centrifugation. Resuspend the cells with 200μL / tube PBS and detect them by flow cytometry. The results are as follows Figure 5As shown, it can be seen that the CD94 humanized antibody of the present invention can effectively block the binding of HLA-E to CD94 / NKG2A, and the blocking ability is equivalent to that of the NKG2A antibody huZ270.

[0189] Example 8: CD94 antibody ELISA binding experiment

[0190] ELISA experiments were used to detect the binding properties of CD94 antibodies. The heterodimer protein of CD94 extracellular region Fc fusion protein and NKG2A extracellular region Fc fusion protein was coated into a 96-well plate, and the strength of the signal after the addition of the antibody was used to determine the binding properties of the antibody and CD94 protein.

[0191] The CD94 extracellular region Fc fusion protein and the NKG2A extracellular region Fc fusion protein (CD94 / NKG2A-Fc) heterodimer fusion protein (expressed in this laboratory, the amino acid sequence of CD94-Fcmutknob is shown in SEQ ID NO: 50, and the amino acid sequence of NKG2A-Fcmuthole is shown in SEQ ID NO: 51) were diluted to 2 μg / mL with PBS buffer, added to a 96-well plate at a volume of 100 μL / well, and placed at 4°C overnight. The PBS buffer in the 96-well plate was aspirated, and the plate was washed 6 times with PBST (pH 7.2 PBS containing 0.1% Tween 20) buffer, and then 200 μL / well PBS / 10% BSA was added and incubated at 37°C for 2h for blocking. Remove the blocking solution, wash the plate 6 times with PBST, add 100 μL / well of the CD94 humanized antibody h15C10-hIgG4 (same as Example 5) and NKG2A antibody huZ270-hIgG4 to be tested diluted to an appropriate concentration with PBST / 0.05% BSA, and incubate at 37°C for 1 hour. Remove the reaction system, wash the plate 6 times with PBST, dilute the HRP (horseradish peroxidase) labeled anti-human IgG-Fab secondary antibody with 100 μL / well of PBST / 0.05% BSA, and incubate at 37°C for 1 hour. Wash the plate 6 times with PBST, add 80 μl / well TMB (tetramethylbenzidine), incubate at room temperature for 3 minutes, and add 80 μL / well 4M sulfuric acid to terminate the reaction. Read the absorbance at 450 mm using an enzyme reader. The results are as follows. Figure 6 This indicates that the CD94 antibody of the present invention can bind to the CD94 / NKG2A dimer protein, and the binding is stronger than that of the NKG2A antibody huZ270.

[0192] Example 9: CD94 antibody promotes Jurkat T cell activation experiment

[0193] The reporter cell experiment was used to detect the functional activity of CD94 antibody. K56233220-HLA-E cells were co-incubated with Jurkat-NFAT-lucia-CD94 / NKG2A cells. The intensity of chemiluminescence after the addition of the antibody was used to determine the functional activity of CD94 antibody in promoting T cell activation.

[0194] HEK293T cells were cultured at 5 × 10 5 Cells / well were plated in a six-well plate and cultured overnight in DMEM medium without dual antibodies. Before transfection, the medium was discarded and 1 mL of fresh DMEM medium without dual antibodies was added. The coding sequence (SEQ ID NO: 41) of the CD94 protein (SEQ ID NO: 40) was inserted between the restriction sites EcoRI and BamHI of the pLVX-EF1a-IRES-puro vector or the coding sequence (SEQ ID NO: 42) of the NKG2A protein (SEQ ID NO: 43) was inserted between the restriction sites EcoRI and BamHI of the pLVX-EF1a-IRES-puro vector. IDNO: 43), pMD2G, and psPAX2 vectors (3 μg in total) were added to 200 μL serum-free DMEM medium at a ratio of 2:1:1, and 12 μg of polyetherimide (PEI, purchased from Polysciences) was added. After mixing, let it stand for 16 minutes, and then all the liquid was added to a six-well plate with HEK293T cells. After 6 hours of culture, the culture medium was discarded and fresh complete DMEM medium was added for culture. After 48 hours of transfection, the cell culture supernatant was collected and filtered through a 0.45 μm filter (Millipore), which was the viral supernatant. All the viral supernatants were added to a 1×10 4 Jurkat-NFAT-lucia cells (purchased from Invivogen) were added to a 6-well plate with a final concentration of 4 μg / mL of polybrene (Sigma) and cultured for 12 hours. The supernatant was then discarded and fresh complete IMDM medium was added. The resulting cells are Jurkat-NFAT-lucia-CD94 / NKG2A cells.

[0195] HEK293T cells were cultured at 5 × 10 5Cells / well were plated in a six-well plate and cultured overnight in DMEM medium without dual antibodies. Before transfection, the medium was discarded and 1 mL of fresh DMEM medium without dual antibodies was added. pLVX-EF1a-HLA-E-IRES-puro (the coding sequence (SEQ ID NO: 53) of HLA-E protein (SEQ ID NO: 52) was inserted between the restriction sites EcoRI and BamHI of the pLVX-EF1a-IRES-puro vector), pMD2G, and psPAX2 vector (3 μg in total) were added to 200 μL serum-free DMEM medium at a ratio of 2:1:1, and 12 μg of polyetherimide (PEI, purchased from Polysciences) was added. After mixing, the mixture was allowed to stand for 16 minutes, and then all the liquid was added to a six-well plate with HEK293T cells. After 6 hours of culture, the culture medium was discarded and fresh complete DMEM culture medium was added for culture. After 48 hours of transfection, the cell culture supernatant was collected and filtered through a 0.45 μm filter (Millipore) to obtain the viral supernatant. All the viral supernatants were added to a 1×10 4 Add polybrene (Sigma) at a final concentration of 4 μg / mL to a 6-well plate of K56233220 cells and culture for 12 hours. Then discard the supernatant and add fresh complete IMDM medium. The resulting cells are K56233220-HLA-E cells.

[0196] (1) Dilute Jurkat-NFAT-lucia-CD94 / NKG2A cells to 1×10 5 / mL, added to 96-well plate, 100 μL / well;

[0197] (2) CD94 humanized antibody h15C10-hIgG4, NKG2A antibody huZ270-hIgG4, and control antibody hIGg were diluted into a series of gradients using complete IMDM medium and added to a 96-well plate at 20 μL / well;

[0198] (3) Dilute K56233220-HLA-E cells to 7.5×10 5 / mL, added to 96-well plate, 80 μL / well;

[0199] (4) After culturing in a 37°C, 5% CO2 incubator for 24 h, 50 μL of supernatant was aspirated, 50 μL of substrate was added, and chemiluminescence was detected.

[0200] The results are as follows Figure 7 As shown, it is further shown that the CD94 antibody of the present invention can promote the activation of Jurkat-NFAT-lucia-CD94 / NKG2AT cells, and the activation function is stronger than that of the NKG2A antibody huZ270.

[0201] Example 10: Effect of humanized CD94 antibody on anti-cancer in mice

[0202] The in vivo efficacy experiment was used to detect the anti-cancer function of the affinity-matured humanized CD94 antibody h15C10-hIgG4 obtained in Example 6 in promoting immune reconstitution mice.

[0203] (1) On day -1, human PBMC (purchased from Sai Li Biotechnology) was transfused into NCG mice (purchased from Jicui Pharmaceutical) via the tail vein at an injection volume of 1×10 7 / Only;

[0204] (2) On day 0, NCG mice were subcutaneously injected with 1×10 6 tumor cells, and the mice were randomly divided into groups;

[0205] (3) On days 0, 3, 6, and 9, the humanized antibody h15C10-hIgG4 and the control antibody hIgG were intraperitoneally injected into the mice, 250 μg per mouse;

[0206] (4) Tumor volume was measured every 3 days after injection of the above antibodies.

[0207] The results are as follows Figure 8 As shown, it can be seen that the CD94 antibody of the present invention can effectively promote the anti-cancer effect of PBMC.

[0208] It can be seen from the above experimental results that the antibodies obtained by the present invention can bind to CD94; can block the interaction between CD94 / NKG2A and HLA-E; and can promote immune cells to fight cancer.

[0209] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0210] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. An anti-CD94 antibody or an antigen-binding fragment thereof, characterized in that: are heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, and light chain CDR1, light chain CDR2 and light chain CDR3; wherein: The amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID NO: 1, The amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID NO: 2, The amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID NO: 3, The amino acid sequence of the light chain CDR1 is as shown in SEQ ID NO: 4, The amino acid sequence of the light chain CDR2 is as shown in SEQ ID NO: 5, The amino acid sequence of the light chain CDR3 is as shown in SEQ ID NO:

6.

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

26.

3. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, characterized in that: The antibody or antigen-binding fragment thereof contains 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 antibody and a murine antibody or a mutant thereof.

4. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that: The light chain constant region and the heavy chain constant region are both derived from mouse IgG antibody or its mutant or human IgG antibody or its mutant.

5. The antibody or antigen-binding fragment thereof according to claim 4, characterized in that: The light chain constant region and the heavy chain constant region are both derived from mouse IgG1, IgG4 antibodies or their mutants or human IgG1, IgG4 antibodies or their mutants.

6. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof has a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO: 27, 29 or 31 and / or a light chain constant region with an amino acid sequence as shown in SEQ ID NO: 28, 30 or 32.

7. The antibody or antigen-binding fragment thereof according to claim 6, characterized in that: The antibody or antigen-binding fragment thereof has a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:27 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:

28.

8. The antibody or antigen-binding fragment thereof according to claim 6, characterized in that: The antibody or antigen-binding fragment thereof has a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:29 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:

30.

9. The antibody or antigen-binding fragment thereof according to claim 6, characterized in that: The antibody or antigen-binding fragment thereof has a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:31 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:

32.

10. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof has a heavy chain having an amino acid sequence shown in any one of SEQ ID NOs: 33, 35, 36 and 38 and a light chain having an amino acid sequence shown in any one of SEQ ID NOs: 34, 37 and 39.

11. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof has a heavy chain with an amino acid sequence as shown in SEQ ID NO: 33 and a light chain with an amino acid sequence as shown in SEQ ID NO: 34; The antibody or antigen-binding fragment thereof has a heavy chain with an amino acid sequence as shown in SEQ ID NO: 35 and a light chain with an amino acid sequence as shown in SEQ ID NO: 37; The antibody or antigen-binding fragment thereof has a heavy chain with the amino acid sequence shown in SEQ ID NO: 36 and a light chain with the amino acid sequence shown in SEQ ID NO: 37; or The antibody or antigen-binding fragment thereof has a heavy chain with the amino acid sequence shown in SEQ ID NO:38 and a light chain with the amino acid sequence shown in SEQ ID NO:

39.

12. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof includes monoclonal antibody or polyclonal antibody.

13. The antibody or antigen-binding fragment thereof according to claim 12, characterized in that: The monoclonal antibody includes at least one of a full-length antibody, Fv, a single-chain antibody, Fab, and a single-domain antibody.

14. The antibody or antigen-binding fragment thereof according to claim 13, characterized in that: The antibody or antigen-binding fragment thereof is capable of binding to the amino acid sequence shown in SEQ ID NO:

40.

15. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14.

16. An expression vector, characterized in that: Carrying the nucleic acid molecule of claim 15.

17. A recombinant cell, characterized in that Carrying the nucleic acid molecule of claim 15, the expression vector of claim 16, or being capable of expressing the antibody or antigen-binding fragment thereof of any one of claims 1 to 4; The recombinant cell is obtained by introducing the expression vector according to claim 16 into a host cell; The recombinant cell is a eukaryotic cell, and the eukaryotic cell is a non-plant cell.

18. The recombinant cell according to claim 17, characterized in that The recombinant cell is a mammalian cell.

19. A composition, characterized in that The method comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, the nucleic acid molecule according to claim 15, the expression vector according to claim 16, or the recombinant cell according to any one of claims 17 to 18.

20. A drug, characterized in that The method comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, the nucleic acid molecule according to claim 15, the expression vector according to claim 16, the recombinant cell according to any one of claims 17 to 18, or the composition according to claim 19.

21. A kit, characterized in that: The kit contains the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, the nucleic acid molecule according to claim 15, the expression vector according to claim 16, or the recombinant cell according to any one of claims 17 to 18.

22. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, the nucleic acid molecule according to claim 15, the expression vector according to claim 16, the recombinant cell according to any one of claims 17 to 18, or the composition according to claim 19 in the preparation of a medicament for treating cancer; The cancer includes at least one of the following: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer.

23. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, the nucleic acid molecule according to claim 15, the expression vector according to claim 16, or the recombinant cell according to any one of claims 17 to 18 in preparing a kit for detecting CD94.

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