Affinity-matured MICA antibodies and their applications

Abstract

The present invention provides an antibody or an antigen-binding fragment thereof and its application. Among them, the antibody or the antigen-binding fragment thereof includes: the CDR1, CDR2, and CDR3 sequences of the heavy-chain variable region shown by the amino acid sequences such as SEQ ID NO: 1, 2, and 3 or amino acid sequences having at least 80% identity with SEQ ID NO: 1, 2, and 3; and the CDR1, CDR2, and CDR3 sequences of the light-chain variable region shown by the amino acid sequences such as SEQ ID NO: 4, 5, and 6 or amino acid sequences having at least 80% identity with SEQ ID NO: 4, 5, and 6. The antibody or the antigen-binding fragment thereof can bind to human and monkey MICA proteins with high affinity and promote the killing of tumors by PBMC (human peripheral blood mononuclear cells).

Description

Technical Field

[0001] The present invention relates to the field of antibodies. Specifically, the present invention relates to an affinity-matured MICA antibody and its applications. More specifically, the present invention relates to an antibody or its antigen-binding fragment, a nucleic acid molecule, an expression vector, a method for preparing an antibody or its antigen-binding fragment, a recombinant cell, a composition and its uses, and a drug and its uses. Background Art

[0002] Cancer is a major disease affecting human survival and development. According to the latest data, there are about 19 million newly diagnosed cancer cases globally each year, and about 10 million cancer deaths each year, and the incidence and mortality rates are on the rise.

[0003] In addition to surgical resection, traditional cancer treatment methods such as chemotherapy and radiotherapy have large side effects and are prone to recurrence. In recent years, immunotherapy, including tumor-targeting antibodies, immune checkpoint antibodies, bispecific antibodies, etc., has become a new hot spot and hope for cancer treatment.

[0004] In recent years, immunotherapy represented by PD-1 / L1 has shown great potential. However, it cannot be ignored that even the overall response rate of the currently approved PD-1 / L1 therapy with the widest range of indications is still only 30%, and more patients still cannot benefit from it. Immune checkpoint molecules are inhibitory molecules expressed on the surface of immune cells including T, NK, mononuclear macrophages, etc. After binding to the corresponding ligands, they transmit inhibitory signals into immune cells and inhibit the anti-cancer functions of immune cells. Due to the very large heterogeneity of the expression of immune checkpoint ligands in tumors and tumor-infiltrating lymphocytes, a single type of immune checkpoint therapy cannot be applicable to all patients, and most patients cannot benefit from it. On the other hand, some patients who have received immune checkpoint therapy will relapse with tumors and develop tolerance to this immune checkpoint therapy, and continued administration will not produce therapeutic effects. In addition, T cells recognize neo-antigens (i.e., antigens mutated in tumors) through the T cell receptors (TCRs) on their surfaces, and some tumors have a low frequency of gene mutations and few neo-antigens, which are called "cold tumors". Current immune checkpoint therapies such as PD-1 / L1 therapy achieve the anti-cancer goal by restoring the function of T cells themselves. However, in "cold tumors", T cells cannot effectively recognize tumors, resulting in the ineffectiveness of immune checkpoint therapy for cold tumors.

[0005] NK cells are another very important anti-cancer immune cells. NK cells have a different recognition mechanism from T cells, and a series of activating receptors are expressed on their surfaces, which recognize and bind to ligands expressed on the surface of tumors. For example, NK cells recognize and bind to ligands such as MICA and MICB on the surface of tumors through the activating receptor NKG2D, and activate and kill tumor cells.

[0006] MICA and MICB (major histocompatibility complex class I-related chain A and chain B polypeptides) are proteins encoded by autologous genes and are not expressed in normal tissues and cells. When cells undergo malignant transformation into tumor cells, MICA and MICB will be expressed on the surface of tumor cells. NK cells recognize and kill tumors through the NKG2D-MICA / B interaction.

[0007] Tumors will down-regulate the expression of MICA / B on the cell surface through various mechanisms, including matrix metalloproteinase-mediated shedding of MICA / B. MICA / B is a type I transmembrane protein, and its extracellular domain includes three domains: α1, α2, and α3, and binds to NKG2D through the α1 and α2 domains. Under the action of matrix metalloproteinase, most of the amino acids in the extracellular domain of MICA / B (including the α1 and α2 domains and a part of the α3 domain) are shed from the surface of tumor cells. NKG2D cannot bind to the remaining part of the α3 domain on the cell surface, so tumors can escape NK cell immune surveillance.

[0008] Studies have shown that MICA antibodies that bind to the α3 domain can inhibit the shedding of MICA from the tumor surface, thereby promoting NKG2D-MICA / B-mediated NK cell recognition and inhibiting tumor immune escape. So far, only one MICA monoclonal antibody drug has entered the phase I clinical stage (CLN-619). Therefore, the development of monoclonal antibody drugs targeting MICA is of great significance for the treatment of tumors. Summary of the Invention

[0009] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0010] This application is based on the inventor's discovery of the following problems and facts:

[0011] MICA is not expressed in most normal human tissues, is only lowly expressed in tissues such as the ovary and testis, and is widely highly expressed in tumor tissues, which makes MICA a very promising tumor treatment target. So far, only one MICA monoclonal antibody is in clinical trials, namely CLN-619; in addition, Genentech has also developed MICA antibodies (1D5V11 in this patent comes from Genentech patents). In summary, MICA is a promising treatment target, and the development of monoclonal antibody drugs targeting MICA is very valuable.

[0012] Thus, in a first aspect of the present invention, the present invention provides an antibody or an antigen-binding fragment thereof. According to an embodiment of the present invention, the antibody or the antigen-binding fragment thereof comprises: heavy chain variable region CDR1, CDR2, and CDR3 sequences respectively shown as SEQ ID NO: 1, 2, and 3 or amino acid sequences having at least 80% identity with SEQ ID NO: 1, 2, and 3; and light chain variable region CDR1, CDR2, and CDR3 sequences respectively shown as SEQ ID NO: 4, 5, and 6 or amino acid sequences having at least 80% identity with SEQ ID NO: 4, 5, and 6.

[0013] According to an embodiment of the present invention, the antibody or the antigen-binding fragment can bind to MICA with high affinity.

[0014] According to an embodiment of the present invention, the antibody or the antigen-binding fragment may further comprise at least one of the following additional technical features:

[0015] According to an embodiment of the present invention, the antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region CDR1 sequence shown as SEQ ID NO: 1, a heavy chain variable region CDR2 shown as SEQ ID NO: 2, a heavy chain variable region CDR3 shown as SEQ ID NO: 3, a light chain variable region CDR1 shown as SEQ ID NO: 4, a light chain variable region CDR2 shown as SEQ ID NO: 5, and a light chain variable region CDR3 shown as SEQ ID NO: 6.

[0016] According to an embodiment of the present invention, the antibody or the antigen-binding fragment thereof specifically recognizes MICA.

[0017] According to an embodiment of the present invention, the antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 8.

[0018] According to an embodiment of the present invention, the antibody is a humanized antibody.

[0019] According to an embodiment of the present invention, the antibody or the antigen-binding fragment thereof contains a heavy chain framework region sequence and a light chain framework region sequence, and at least a part of at least one of the heavy chain framework region sequence and the light chain framework region sequence is derived from at least one of a murine antibody, a human antibody, a primate antibody, or a mutant thereof.

[0020] According to an embodiment of the present invention, the antibody or the antigen-binding fragment thereof contains at least one of a heavy chain constant region and a light chain constant region, and at least a part 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, a murine antibody, or a mutant thereof.

[0021] According to an embodiment of the present invention, both the light chain constant region and the heavy chain constant region are derived from murine IgG1 antibody, IgG2a antibody or mutants thereof, or human IgG1 antibody, IgG2 antibody, IgG3 antibody, IgG4 antibody or mutants thereof.

[0022] According to an embodiment of the present invention, the antibody or its antigen-binding fragment is at least one of a single-chain antibody, a multimeric antibody, a CDR-grafted antibody, a Fab antibody, and an Fv antibody.

[0023] In a second aspect of the present invention, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the antibody or its antigen-binding fragment described in the first aspect.

[0024] 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 it actually includes either any one of the complementary double strands or both. For convenience, in this specification and claims, although only one strand is given in most cases, the other complementary strand is actually also disclosed. In addition, the nucleic acid sequences in the present application include DNA form or RNA form, and disclosing one means that the other is also disclosed.

[0025] In a third aspect of the present invention, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the nucleic acid molecule described in the second aspect. The expression vector may include optional control sequences, and the control sequences are operably linked to the nucleic acid molecule. Among them, the control sequence is one or more control sequences that can direct the expression of the nucleic acid molecule in a host. The expression vector provided by the embodiment of the present invention can highly express the antibody or antigen-binding fragment in a suitable host cell.

[0026] The expression vector provided by the embodiment of the present invention can highly express the aforementioned antibody or its antigen-binding fragment in a suitable recipient cell. The antibody or its antigen-binding fragment designed by the present invention has stronger specificity and higher safety.

[0027] In a fourth aspect of the present invention, the present invention provides a method for preparing the above-mentioned antibody or its antigen-binding fragment. According to an embodiment of the present invention, the method includes: introducing the expression vector described in the third aspect into a cell; culturing the cell under conditions suitable for protein expression and secretion to obtain the antibody or its antigen-binding fragment.

[0028] The inventors found that the antibody or its antigen-binding fragment prepared according to the embodiment of the present invention can more efficiently culture a high-purity antibody or its antigen-binding fragment, and the operation steps are simple and the cost is low.

[0029] According to some specific embodiments of the present invention, the cells are not particularly limited, and prokaryotic cells or eukaryotic cells can be used.

[0030] According to some specific embodiments of the present invention, the cells are eukaryotic cells.

[0031] According to some specific embodiments of the present invention, the eukaryotic cells are mammalian cells. According to some specific examples of the present invention, when the cells are eukaryotic cells, such as mammalian cells, the expression efficiency of the recombinant antibody is relatively high.

[0032] In the fifth aspect of the present invention, the present invention provides a recombinant cell. According to the embodiments of the present invention, the recombinant cell expresses the antibody or its antigen-binding fragment described in the first aspect, and carries the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect. 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 abundantly express the above-mentioned antibody or antigen-binding fragment under suitable conditions.

[0033] According to some specific examples of the present invention, the recombinant cell can efficiently and abundantly express an antibody or its antigen-binding fragment under suitable conditions. The antibody or its antigen-binding fragment has stronger specificity, a longer half-life and higher potency, and can deliver the antibody drug to target cells at a lower dosage, thereby effectively treating or preventing MICA-mediated diseases with low toxicity and side effects and higher safety.

[0034] It should be noted that the recombinant cells described in the present invention are not particularly limited and can be prokaryotic cells, eukaryotic cells or phages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, etc., insect cells such as Spodoptera frugiperda, plant cells such as Nicotiana tabacum, and mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc. In some embodiments, the recombinant cells described in the present invention are preferably mammalian cells, including BHK cells, CHO cells, NSO cells or COS cells, and do not include animal germ cells, fertilized eggs or embryonic stem cells.

[0035] It should be noted that the "suitable conditions" described in the specification of this application refer to the 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 an antibody or antigen-binding fragment include, but are not limited to, appropriate transformation or transfection methods, appropriate transformation or transfection conditions, healthy host cell states, appropriate host cell densities, suitable cell culture environments, and suitable cell culture times. The "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.

[0036] In a sixth aspect of the present invention, the present invention provides a composition. According to an embodiment of the present invention, the composition comprises: the antibody or its antigen-binding fragment according to the first aspect, the nucleic acid molecule according to the second aspect, the expression vector according to the third aspect, or the recombinant cell according to the fifth aspect.

[0037] As mentioned above, the antibody or antigen-binding fragment in some specific embodiments of the present invention can effectively bind to human MICA protein and effectively inhibit the proliferation of tumor cells. Therefore, the composition containing the above substances can also effectively bind to human MICA protein and has a good effect on preventing and / or treating MICA-mediated diseases. The type of the composition is not particularly limited and can be a food composition or a pharmaceutical composition.

[0038] The compositions of the present invention can also be administered in combination with each other or in combination with one or more other therapeutic compounds. For example, they can be administered in combination with chemotherapeutic agents. Therefore, the composition can also contain a chemotherapeutic agent. The antibody or its antigen-binding fragment, nucleic acid molecule, expression vector, or recombinant cell of the present invention can also be combined with a second therapeutic agent. Exemplary agents of the second therapeutic agent include, but are not limited to, other agents that inhibit MICA activity (including other antibodies or their antigen-binding fragments, peptide inhibitors, small molecule antagonists, etc.) and / or agents that interfere with MICA upstream or downstream signal transduction.

[0039] In certain embodiments, the composition comprises a combination that is separated in time and / or space, as long as they can act 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, the individual components can be administered to the subject simultaneously or sequentially.

[0040] Typically, the antibody or its antigen-binding fragment is administered in an effective amount, i.e., an amount sufficient to achieve the desired therapeutic and / or prophylactic effect. For example, an amount that causes prevention or alleviation of symptoms associated with the disease being treated, such as a disease associated with MICA. The effective amount of the composition administered to a subject will depend on the type and severity of the disease, as well as on the characteristics of the individual, such as general health status, age, gender, weight, and tolerance to the drug; it will also depend on the severity and type of the disease, and those skilled in the art will be able to determine an appropriate dose based on these factors and others.

[0041] According to an embodiment of the present invention, the composition of the present invention can achieve the same therapeutic effect as an antibody or its antigen-binding fragment, a nucleic acid molecule, an expression vector, and a recombinant cell when entering an organism.

[0042] In a seventh aspect of the present invention, there is provided the use of the antibody or its antigen-binding fragment according to the first aspect, the nucleic acid molecule according to the second aspect, the expression vector according to the third aspect, the recombinant cell according to the fifth aspect, or the composition according to the sixth aspect in the preparation of a medicament. According to an embodiment of the present invention, the medicament is used for preventing and / or treating MICA-mediated diseases. As mentioned above, the antibody or antigen-binding fragment in some specific embodiments of the present invention can effectively bind to human MICA protein. Therefore, a medicament containing an effective amount of the antibody or antigen-binding fragment or a series of substances thereof can also effectively bind to human MICA protein and has a good effect in preventing and / or treating MICA-mediated diseases, such as cancer.

[0043] According to an embodiment of the present invention, the MICA-mediated disease is cancer.

[0044] According to an embodiment of the present invention, the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0045] In an eighth aspect of the present invention, there is provided a medicament. According to an embodiment of the present invention, the medicament comprises: the antibody or its antigen-binding fragment according to the first aspect, the nucleic acid molecule according to the second aspect, the expression vector according to the third aspect, the recombinant cell according to the fifth aspect, or the composition according to the sixth aspect, and the medicament is used for preventing and / or treating MICA-mediated diseases.

[0046] According to an embodiment of the present invention, the MICA-mediated disease is cancer.

[0047] According to an embodiment of the present invention, the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0048] According to an embodiment of the present invention, the drug may further include a pharmaceutically acceptable carrier.

[0049] The effective amount of the antibody or antigen-binding fragment described in the present invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by those of ordinary skill in the art according to various factors (e.g., through clinical trials). Such factors include, but are not limited to: the pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the patient, the patient's weight, the patient's immune status, the route of administration, etc. For example, due to the urgency of the treatment situation, several separate doses may be administered daily, or the dose may be proportionally reduced.

[0050] In a ninth aspect of the present invention, the present invention provides the use of the antibody or its antigen-binding fragment described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fifth aspect in the preparation of a kit. According to an embodiment of the present invention, the kit is used for detecting MICA. As mentioned above, the antibody or antigen-binding fragment of some specific embodiments of the present invention can effectively bind to the human MICA protein. Therefore, the antibody or antigen-binding fragment can be used to prepare a kit for detecting the MICA protein, and the kit can effectively perform qualitative or quantitative detection of the human MICA protein.

[0051] According to an embodiment of the present invention, the kit can detect MICA more efficiently and accurately, saving time and exploration costs for clinical treatment.

[0052] In a tenth aspect of the present invention, the present invention provides a kit. According to an embodiment of the present invention, the kit contains the antibody or its antigen-binding fragment described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fifth aspect.

[0053] As described above, the antibodies or antigen-binding fragments of some specific embodiments of the present invention can effectively bind to human MICA protein. Therefore, the kits containing the antibodies or antigen-binding fragments can effectively qualitatively or quantitatively detect human MICA protein. The kits provided by the present invention can be used, for example, in immunoblotting, immunoprecipitation and other kits that involve detecting by using the specific binding of human MICA and antibodies. These kits may include any one or more of the following: antagonists, anti-MICA antibodies or drug reference materials; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents; instructions or literature, etc. Anti-MICA antibodies can be used in different types of diagnostic tests. For example, they can detect the presence of various diseases, drugs, toxins or other proteins in vitro or in vivo. For example, by detecting the serum or blood of a subject, they can be used to test related diseases, and can also be used for scientific research to detect human MICA protein in a sample to be tested by using the kit. Such related diseases may include MICA-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 diseases, etc. For the above application scenarios, the binding molecules are equally applicable and will not be elaborated here.

[0054] According to some specific embodiments of the present invention, the kit may further include those conventionally used for detecting MICA, such as coating solutions, etc.

[0055] In the eleventh aspect of the present invention, the present invention provides a method for treating or preventing MICA-mediated related diseases. According to an embodiment of the present invention, the method includes administering to a subject at least one of the following: 1) the antibody or antigen-binding fragment described in the first aspect; 2) the nucleic acid molecule described in the second aspect; 3) the expression vector described in the third aspect; 4) the recombinant cell described in the fifth aspect; 5) the composition described in the sixth aspect; and 6) the drug described in the eighth aspect. As described above, the antibody or antigen-binding fragment can bind to human MICA protein and can effectively treat or prevent MICA-mediated related diseases, preferably cancer. Therefore, the method according to the embodiment of the present invention can effectively treat or prevent MICA-mediated related diseases.

[0056] 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.

[0057] In a twelfth aspect of the present invention, the present invention provides a method for diagnosing MICA-mediated related diseases. According to an embodiment of the present invention, the method includes detecting MICA in a test sample using at least one of the following: 1) the antibody or antigen-binding fragment described in the first aspect; 2) the nucleic acid molecule described in the second aspect; 3) the expression vector described in the third aspect; and 4) the recombinant cell described in the fifth aspect. Based on the detection result of MICA, the content of MICA in the test sample is determined. The antibody or antigen-binding fragment proposed in the present application, or the antibody or antigen-binding fragment expressed by the nucleic acid molecule, expression vector, or recombinant cell can effectively bind to the human MICA protein, or the antibody or antigen-binding fragment expressed by the nucleic acid molecule, expression vector, or recombinant cell can effectively bind to MICA. Therefore, the method described in the present application can effectively detect the content of MICA in a test sample derived from a subject individual and can effectively diagnose related diseases caused by MICA.

[0058] 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:

[0059] According to an embodiment of the present invention, the indication that the content of MICA in the test sample is not lower than the minimum standard for disease is that the test sample is derived from a patient suffering from a MICA-mediated related disease. The value of the minimum standard can be determined by differential comparative analysis and verification of the content of MICA in test samples from a large number of individuals suffering from the MICA-mediated related disease and a large number of healthy individuals.

[0060] According to an embodiment of the present invention, the test sample includes at least one of the following: blood, saliva, sweat, tissue, cells, blood, serum, plasma, feces, and urine.

[0061] According to an embodiment of the present invention, the MICA-mediated related diseases include cancer.

[0062] 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.

[0063] In a thirteenth aspect of the present invention, the present invention provides a method for staging MICA-mediated related diseases. According to an embodiment of the present invention, it includes detecting MICA in a test sample using at least one of the following: 1) the antibody or antigen-binding fragment described in the first aspect; 2) the nucleic acid molecule described in the second aspect; 3) the expression vector described in the third aspect; and 4) the recombinant cell described in the fifth aspect. Based on the detection result of MICA, the content of MICA in the test sample is determined. The antibody or antigen-binding fragment proposed in the present application, or the antibody or antigen-binding fragment expressed by the nucleic acid molecule, expression vector, or recombinant cell can effectively bind to human MICA. Therefore, the method described in the present application can effectively detect the content of MICA in the test sample derived from the subject individual, and evaluate the stage of the related diseases caused by MICA based on the content of MICA.

[0064] According to an embodiment of the present invention, the method for staging the disease may further include at least one of the following additional technical features:

[0065] According to an embodiment of the present invention, the content of MICA in the test sample not being lower than the standard level of a patient with stage IV tumor is an indication that the test sample is derived from a patient with stage IV tumor, and the content of MICA in the test sample being between the standard levels of stage IV and stage III tumors is an indication that the test sample is derived from a patient with stage III tumor; the content of MICA in the test sample being between the standard levels of stage III and stage II tumors is an indication that the test sample is derived from a patient with stage II tumor; the content of MICA in the test sample being between the standard levels of stage I and stage II tumors is an indication that the test sample is derived from a patient with stage I tumor. Those skilled in the art can understand that the level of MICA during stage I, II, III, and IV of the tumor varies according to the type of tumor. To determine the stage of the tumor, it is only necessary to compare the content of MICA in the test sample with the corresponding standard level of MICA at this tumor stage, or compare the content of MICA in the test sample with the content of MICA in the samples from individuals or groups with known disease stages. The values of the standard levels of stage I, II, III, and IV of the tumor can be determined by comparing and analyzing the differences in the content of MICA in the test samples of a large number of individuals with MICA-mediated related diseases and a large number of healthy individuals, as well as verification.

[0066] According to an embodiment of the present invention, the test sample includes at least one of the following: blood, saliva, sweat, tissue, cells, blood, serum, plasma, feces, and urine.

[0067] According to an embodiment of the present invention, the MICA-mediated related diseases include cancer.

[0068] 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.

[0069] In the fourteenth aspect of the present invention, the present invention provides a method for evaluating the prognosis of MICA-mediated related diseases. According to an embodiment of the present invention, it includes detecting MICA in a test sample using at least one of the following: 1) the antibody or antigen-binding fragment described in the first aspect; 2) the nucleic acid molecule described in the second aspect; 3) the expression vector described in the third aspect; and 4) the recombinant cell described in the fifth aspect. Based on the detection result of MICA, the content of MICA in the test sample is determined. As mentioned above, the content of MICA has an important impact on cancer. After an individual with a related disease is treated, by monitoring the content of MICA in their tissues or excreta, such as peripheral blood, urine, etc., the prognosis of such diseases can be effectively evaluated. For example, by comparing the content of MICA in the subject before and after treatment, or by comparing the content of MICA in the subject after treatment with the MICA level of a normal individual or a diseased individual, etc. The antibody or antigen-binding fragment described in the present application, or the antibody or antigen-binding fragment expressed by the nucleic acid molecule, expression vector, or recombinant cell can effectively bind to human MICA. Therefore, the method described in the present application can effectively detect the content of MICA in the test sample derived from a subject individual and evaluate the prognosis of MICA-induced related diseases based on the content of MICA.

[0070] 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:

[0071] According to an embodiment of the present invention, the test sample is derived from a patient with MICA-mediated related diseases before or after treatment.

[0072] According to an embodiment of the present invention, the test sample includes at least one of the following: blood, saliva, sweat, tissue, cells, blood, serum, plasma, feces, and urine.

[0073] According to an embodiment of the present invention, based on the content of MICA in the test sample of a patient with MICA-mediated related diseases before or after treatment, the prognosis effect of MICA-mediated related diseases is determined.

[0074] According to an embodiment of the present invention, the MICA-mediated related diseases include cancer.

[0075] 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.

[0076] In a fifteenth aspect of the present invention, the present invention provides the use of the antibody or antigen-binding fragment described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fifth aspect, the composition described in the sixth aspect, or the drug described in the eighth aspect in the treatment or prevention of MICA-mediated related diseases. As described above, the antibody or antigen-binding fragment can effectively bind to human MICA and can effectively treat or prevent MICA-mediated related diseases.

[0077] According to an embodiment of the present invention, the above use may further include at least one of the following additional technical features:

[0078] According to an embodiment of the present invention, the MICA-mediated related diseases include cancer.

[0079] 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.

[0080] In a sixteenth aspect of the present invention, the present invention provides the use of the antibody or antigen-binding fragment described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fifth aspect in the diagnosis of MICA-mediated related diseases, staging of MICA-mediated related diseases, or assessment of the prognosis of MICA-mediated related diseases. As described above, the antibody or antigen-binding fragment proposed in the present application, or the antibody or antigen-binding fragment expressed by the nucleic acid molecule, expression vector, or recombinant cell can effectively bind to human MICA. Therefore, the method described in the present application can effectively detect the content of MICA in a test sample derived from a subject individual and can effectively diagnose, stage, and evaluate the prognosis of MICA-mediated related diseases.

[0081] According to an embodiment of the present invention, the above use may further include at least one of the following additional technical features:

[0082] According to an embodiment of the present invention, the MICA-mediated related diseases include cancer.

[0083] 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.

[0084] The "subject" or "individual" involved in the present invention generally refers to mammals, such as primates and / or rodents, especially humans, monkeys or mice.

[0085] Advantages of the present invention:

[0086] 1) The affinity matured antibody h5A1002 obtained in the present invention has stronger binding activity compared to the parental antibody h5A1 and the same type of antibody CLN-619 (Cullinan, in the phase I clinical stage).

[0087] 2) The affinity matured antibody h5A1002 obtained in the present invention has stronger activity in promoting the killing of tumors by PBMC (human peripheral blood mononuclear cells) compared to the parental antibody h5A1 and the same type of antibody CLN-619 (Cullinan, in the phase I clinical stage).

[0088] 3) The affinity matured antibody h5A1002 obtained in the present invention has weaker endocytosis activity compared to the same type of antibody CLN-619 (Cullinan, in the phase I clinical stage), and has better drug-forming properties as a monoclonal antibody or bispecific antibody. Description of the drawings

[0089] The above and / or additional aspects and advantages of the present invention will become apparent and be easily understood from the description of the embodiments in conjunction with the following drawings, where:

[0090] Figure 1 It is a SPR result graph for the determination of the affinity between the parental humanized antibody h5A1 antibody and the MICA protein according to an embodiment of the present invention;

[0091] Figure 2 It is a SPR result graph for the determination of the affinity between the affinity matured antibody h5A1002 antibody and the MICA protein according to an embodiment of the present invention;

[0092] Figure 3 It is an ELISA result graph for the binding of the parental antibody h5A1 and the affinity matured antibody h5A1002 to the MICA*002-Fc protein according to an embodiment of the present invention;

[0093] Figure 4 It is an ELISA result graph for the binding of the parental antibody h5A1 and the affinity matured antibody h5A1002 to the MICA*005-Fc protein according to an embodiment of the present invention;

[0094] Figure 5 ELISA result graph showing the binding of the parental antibody h5A1, affinity matured antibody h5A1002 according to an embodiment of the present invention to MICA*008-Fc protein;

[0095] Figure 6 ELISA result graph showing the binding of the parental antibody h5A1, affinity matured antibody h5A1002 according to an embodiment of the present invention to MICA*004-Fc protein;

[0096] Figure 7 ELISA result graph showing the binding of the parental antibody h5A1, affinity matured antibody h5A1002 according to an embodiment of the present invention to MICB*001-Fc protein;

[0097] Figure 8 ELISA result graph showing the binding of the parental antibody h5A1, affinity matured antibody h5A1002 according to an embodiment of the present invention to MICB*005-Fc protein;

[0098] Figure 9 Flow cytometry result graph showing the binding of the parental antibody h5A1, affinity matured antibody h5A1002 according to an embodiment of the present invention to human malignant melanoma A-375 cells;

[0099] Figure 10 Flow cytometry result graph showing the binding of the parental antibody h5A1, affinity matured antibody h5A1002 according to an embodiment of the present invention to human colorectal cancer HCT-15 cells;

[0100] Figure 11 ELISA result graph showing the binding of the affinity matured antibody h5A1002, isotype antibodies CLN-619 (Cullinan) and 1D5V11 (Genentech) according to an embodiment of the present invention to MICA*002-Fc protein;

[0101] Figure 12 ELISA result graph showing the binding of the affinity matured antibody h5A1002, isotype antibodies CLN-619 (Cullinan) and 1D5V11 (Genentech) according to an embodiment of the present invention to MICA*005-Fc protein;

[0102] Figure 13 ELISA result graph showing the binding of the affinity matured antibody h5A1002, isotype antibodies CLN-619 (Cullinan) and 1D5V11 (Genentech) according to an embodiment of the present invention to MICA*008-Fc protein;

[0103] Figure 14ELISA results of the affinity matured antibody h5A1002, isotype antibody CLN-619 (Cullinan), and 1D5V11 (Genentech) binding to MICA*004-Fc protein according to an embodiment of the present invention;

[0104] Figure 15 ELISA results of the affinity matured antibody h5A1002, isotype antibody CLN-619 (Cullinan), and 1D5V11 (Genentech) binding to MICB*001-Fc protein according to an embodiment of the present invention;

[0105] Figure 16 ELISA results of the affinity matured antibody h5A1002, isotype antibody CLN-619 (Cullinan), and 1D5V11 (Genentech) binding to MICB*005-Fc protein according to an embodiment of the present invention;

[0106] Figure 17 Flow cytometry results of the affinity matured antibody h5A1002, isotype antibody CLN-619 (Cullinan), and 1D5V11 (Genentech) binding to human malignant melanoma A-375 cells according to an embodiment of the present invention;

[0107] Figure 18 Flow cytometry results of the affinity matured antibody h5A1002, isotype antibody CLN-619 (Cullinan), and 1D5V11 (Genentech) binding to human non-small cell lung cancer A549 cells according to an embodiment of the present invention;

[0108] Figure 19 Flow cytometry results of the affinity matured antibody h5A1002, isotype antibody CLN-619 (Cullinan), and 1D5V11 (Genentech) binding to human colorectal cancer HCT-15 cells according to an embodiment of the present invention;

[0109] Figure 20 Flow cytometry results of the affinity matured antibody h5A1002, isotype antibody CLN-619 (Cullinan), and 1D5V11 (Genentech) binding to human breast cancer MDA-MB-231 cells according to an embodiment of the present invention;

[0110] Figure 21Flow cytometry results showing the competitive binding of the affinity - matured antibody h5A1002, the isotype antibody CLN - 619 (Cullinan), and 1D5V11 (Genentech) according to the embodiments of the present invention to human colorectal cancer HCT - 15 cells with the h5A1002 - Biotin antibody;

[0111] Figure 22 Flow cytometry results showing the endocytosis of the affinity - matured antibody h5A1002, the isotype antibody CLN - 619 (Cullinan), and 1D5V11 (Genentech) according to the embodiments of the present invention by human breast cancer MDA - MB - 231 cells;

[0112] Figure 23 Results showing that the affinity - matured antibody h5A1002 according to the embodiments of the present invention promotes the killing of human malignant melanoma A - 375 cells by PBMC;

[0113] Figure 24 Flow cytometry results showing that the affinity - matured antibody h5A1002, the isotype antibody CLN - 619 (Cullinan), and 1D5V11 (Genentech) according to the embodiments of the present invention promote the killing of human colorectal cancer HCT - 15 cells by PBMC;

[0114] Figure 25 Flow cytometry results showing that the affinity - matured antibody h5A1002, the isotype antibody CLN - 619 (Cullinan), and 1D5V11 (Genentech) according to the embodiments of the present invention promote the killing of human malignant melanoma A - 375 cells by PBMC. Detailed implementation manners

[0115] The embodiments of the present invention will be described in detail below. The following described embodiments are exemplary and are only used to explain the present invention, and should not be construed as a limitation of the present invention.

[0116] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly indicating the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise stated, the meaning of "a plurality" is two or more.

[0117] In this article, the term "comprising" or "including" is an open - ended expression, that is, it includes the content specified by the present invention, but does not exclude other aspects of the content.

[0118] In this document, the terms "optionally", "optional" or "option" generally mean that the subsequent described event or condition may but does not necessarily occur, and this description includes the cases where the event or condition occurs and the cases where the event or condition does not occur.

[0119] To facilitate the understanding of the present invention, 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 pertains. The abbreviations of amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 common L-amino acids.

[0120] The antibodies or antigen-binding fragments described in the present invention are generally prepared by biosynthetic methods. Based on the nucleotide sequences described in the present invention, those skilled in the art can conveniently obtain the encoding nucleic acids of the present invention by various known methods. These methods include, for example, but are not limited to: PCR, DNA artificial synthesis, etc. For specific methods, reference can be made to J. Sambrook, "Molecular Cloning: A Laboratory Manual". As an embodiment of the present invention, the encoding nucleic acid sequence of the present invention can be constructed by segmentally synthesizing the nucleotide sequence and then performing overlap extension PCR. Among them, the antibodies or antigen fragments are numbered and defined using the Kabat numbering system.

[0121] In this document, the term "monoclonal antibody" refers to an antibody having a single antigen-binding site.

[0122] In this document, the term "polyclonal antibody" refers to an antibody having two or more different antigen-binding sites.

[0123] In this document, the terms "mutant" or "variant" may refer to a molecule obtained by including one or more nucleotide or amino acid mutations in any naturally occurring or engineered molecule.

[0124] The term "complementary determining region" or "CDR" or "CDR sequence" refers to the amino acid sequence in an antibody responsible for antigen binding. For example, it generally includes: amino acid residues near positions 23 - 34 (L1), 50 - 56 (L2), and 89 - 97 (L3) in the light chain variable region, and near positions 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, Bethesda, MD. (1991)); and / or from "hypervariable loops" (e.g., amino acid residues near positions 26 - 32 (LI), 50 - 52 (L2), and 91 - 96 (L3) in the light chain variable region, and near positions 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)).

[0125] As used herein, "operably linked" means that an exogenous gene is linked to a vector such that control elements within the vector, such as transcriptional control sequences and translational control sequences, etc., can perform their intended functions of regulating the transcription and translation of the exogenous gene. When the above nucleic acid molecule is linked to a vector, the nucleic acid molecule can be directly or indirectly linked to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule, etc. Of course, these control elements can be directly from the vector itself or can be exogenous, i.e., not from the vector itself. Those skilled in the art will understand that nucleic acid molecules encoding antibodies or antigen - binding fragments can be inserted into different vectors separately and independently, and commonly are inserted into the same vector. Commonly used vectors can be, for example, plasmids, phages, etc. For example, Plasmid - X plasmid.

[0126] In this text, when the terms "identity", "homology", or "similarity" are used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, 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, D.C.)). 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 the 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, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, 8th edition, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0127] Without substantially affecting the antibody activity (retaining at least 95% of the activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the sequences of the present invention to obtain variants of the sequences of the antibody or its functional fragments. They are all considered to be included within the scope of protection of the present invention. For example, amino acids with similar properties are substituted in the variable region. The variant sequences of the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence. The sequence identity described in the present invention can be measured using sequence analysis software. For example, using the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The amino acid sequences described in the present invention are all shown in the N-terminal to C-terminal manner.

[0128] As mentioned above, the monoclonal antibody of the present invention can be a full-length antibody or can only contain its functional fragments (e.g., Fab, F(ab’)2 or scFv fragments), or can be modified to affect the function. The present invention includes anti-MICA antibodies with modified glycosylation patterns. In some applications, it may be useful to perform modifications to remove undesired glycosylation sites, or antibodies without fucose moieties on the oligosaccharide chains to enhance, for example, antibody-dependent cell cytotoxicity (ADCC) function. In other applications, galactosylation modifications can be performed to alter complement-dependent cytotoxicity (CDC).

[0129] As used herein, the "full-length antibody" is a four-peptide chain structure formed by two identical light chains and two identical heavy chains linked by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE). The same class of immunoglobulins 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 the different constant regions.

[0130] As used herein, the term "functional fragment" particularly refers to antibody fragments such as CDR-grafted antibodies, Fab, Fab', F(ab')2, Fv or scFv, nanobodies, or any fragment that should be able to increase the half-life through chemical modification or incorporation into liposomes. The chemical modification such as adding poly(alkylene) glycol, such as polyethylene glycol ("PEGylation"), (PEGylated fragments called Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG or Fab'-PEG) ("PEG" is polyethylene glycol), and the fragment has MICA binding activity. Preferably, the functional fragment will consist of or contain partial sequences of the heavy chain variable region or the light chain variable region of its source antibody, and the partial sequences are sufficient to retain the same binding specificity and sufficient affinity as its source antibody. For MICA, it is preferably at least equal to 1 / 100 of the affinity of its source antibody, and in a more preferred manner at least equal to 1 / 10. Such a functional fragment will contain at least 3 amino acids, preferably 5, 10, 15, 25, 50 and 100 consecutive amino acids of the antibody sequence of its source.

[0131] In the present invention, unless otherwise stated, the term "antigen-binding fragment" generally refers to an antigen-binding antibody fragment, which may include a part of a complete antibody, generally the antigen-binding region or variable region. Exemplarily, it includes CDR-grafted antibodies, Fab, Fab', F(ab')2, Fv or scFv, nanobodies, etc.

[0132] As used herein, the term "CDR-grafted antibody" refers to grafting the CDRs of a monoclonal antibody of one species into the variable region of an antibody of another species. For example, the CDRs of a murine monoclonal antibody can be grafted into the variable region of a human antibody to replace the human CDRs, enabling the human antibody to acquire the antigen-binding specificity of the murine monoclonal antibody while reducing its heterogenicity.

[0133] As used herein, the term "Fab antibody" or "Fab" generally refers to an antibody containing only the Fab molecule, which consists of VH and CH1 of the heavy chain and the complete light chain, and the light chain and the heavy chain are connected by a disulfide bond.

[0134] In this text, the term "nanobody" (single-domain antibody or VHH antibody), which was initially described as the antigen-binding immunoglobulin (variable) domain of "heavy-chain antibody" (i.e., "antibody lacking 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)), contains only the variable heavy-chain region (VH) and the conventional CH2 and CH3 regions, and specifically binds to the antigen through the variable heavy-chain region.

[0135] In this text, the term "Fv antibody" generally refers to an antibody composed only of the variable light-chain region (VL) and the variable heavy-chain region (VH) connected by non-covalent bonds, which is the smallest functional fragment of the antibody molecule that retains the complete antigen-binding site.

[0136] In this text, the term "single-chain antibody" or "scFv" is a fragment formed by connecting the variable heavy-chain region and the variable light-chain region of the antibody with a short peptide.

[0137] In this text, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity in humans and / or animals and is acceptable to humans and / or animals.

[0138] In this text, a "pharmaceutically acceptable" component is a substance that is suitable for humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, a substance with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier used for administering a therapeutic agent, including various excipients and diluents.

[0139] The drug 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 solution, glucose, water, glycerol, ethanol, and their combinations. Usually, the pharmaceutical preparation should match the administration method. Among them, the administration methods can be oral administration, nasal administration, intradermal administration, subcutaneous administration, intramuscular administration, intravenous administration, or intraperitoneal administration. The dosage form of the drug 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 said drug should be manufactured under sterile conditions. The said antibody or antigen-binding fragment can be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.

[0140] In a preferred embodiment of the present invention, in order to further improve the biocompatibility of the antibody, the antibody can also be humanized, that is, the antibody is a chimeric antibody or a humanized antibody. The term "chimeric antibody" refers to a recombinant antibody obtained by using recombinant DNA technology to replace the amino acid sequence of the constant region of a monoclonal antibody from one species (such as a mouse) with the constant region of an antibody from another species (such as a human). The term "humanized antibody" refers to a recombinant antibody obtained by using recombinant DNA technology to replace all the amino acid sequences of the constant region and the non-CDR (Fv framework region (FR)) of the variable region of a monoclonal antibody from one species (such as a mouse) with the non-CDR amino acid sequences of the constant region and the variable region of an antibody from another species (such as a human). That is, when the constant region of an antibody is humanized, it is called a chimeric antibody, and when all the non-CDR amino acid sequences of the constant region and the variable region are humanized, it is called a humanized antibody. The method of humanization can be referred to the conventional antibody engineering technology and will not be elaborated here.

[0141] In this article, the antibody affinity maturation described in the present invention refers to an immune function state that normally exists in the body. In humoral immunity, the average affinity of the antibodies produced in the secondary response is higher than that in the primary immune response, and this phenomenon is called antibody affinity maturation. This functional state of the body is the result of long-term evolution and continuous adaptation to the external environment, and it has very important significance for the body's defense and maintenance of its own immune surveillance. The present invention utilizes the in vitro antibody affinity maturation technology. Specifically, it is a process of mutating the CDR amino acids of the antibody by using molecular biology technology and screening from the mutated antibody library to obtain an antibody with significantly improved affinity.

[0142] The amino acid or nucleic acid sequences involved in the present invention are shown in Table 1 in detail.

[0143] Table 1

[0144]

[0145]

[0146]

[0147]

[0148] 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 construed as a limitation of the present invention. For those not specified in the embodiments regarding specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For those reagents or instruments not specified as to the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0149] Example 1: Production of Antibody

[0150] The production of the antibody was carried out as follows: (1) ExpiCHO cells (purchased from Thermo Fisher) were cultured using ExpiCHO Expression Medium (purchased from Thermo Fisher), and the cell concentration was adjusted to 6×10 6 / mL to obtain an ExpiCHO cell solution. (2) The pcDNA3.4 vectors containing the heavy chain and light chain of the antibody (synthesized by Nanjing Genscript) were added to 2 mL of OptiSFM medium (purchased from Thermo Fisher) at a ratio of 1:1 to obtain Solution A. (3) 160 μL of ExpiFectamine CHO transfection reagent (purchased from Thermo Fisher) was added to 2 mL of OptiSFM medium (purchased from Thermo Fisher) to obtain Solution B. (4) Then, Solution A and Solution B were mixed to obtain a transfection mixture, and the entire transfection mixture was added to 50 mL of the ExpiCHO cell solution within 5 minutes. (5) After culturing for 1 day at 37°C and 5% CO2, 8 mL of Feed and 300 μL of Enhancer (purchased from Thermo Fisher) were added, and the culture was transferred to 32°C and 5% CO2 for 9 days, and the culture supernatant was harvested, with 8 mL of Feed added on the 5th day. (6) The parental humanized antibody h5A1 antibody was obtained by affinity purification from the culture supernatant using a Protein A purification column (purchased from Nano-Micro).

[0151] Example 2: Antibody Affinity Maturation

[0152] During the affinity maturation of natural antibodies, somatic hypermutation mainly occurs in the CDR regions. By performing single-site saturation mutagenesis at each site in the CDR regions through in vitro experiments, sufficient mutation diversity can be obtained without disrupting the protein structure, and this approach can achieve the most similar in vitro reproduction of somatic hypermutation in natural antibodies in vivo.

[0153] Single-site saturation mutagenesis was performed at each amino acid site in the CDR regions to construct an unbiased single-site saturation mutagenesis plasmid library of the parental antibody. ELISA was used to screen for mutant sites with enhanced specific binding to the antigen, and then these sites were combined and screened to obtain candidate antibody mutant sequences.

[0154] The parental humanized antibody h5A1 (prepared by the method described in Example 1, with a heavy chain variable region SEQ ID NO: 9 and a light chain variable region SEQ ID NO: 10, from Patent CN114369162A) was affinity matured through this technical route to obtain the high-affinity MICA monoclonal antibody h5A1002 (prepared by the method described in Example 1), which has the sequences of a heavy chain variable region (SEQ ID NO: 7) and a light chain variable region (SEQ ID NO: 8). The comparison of the CDR sequences of the parental humanized antibody h5A1 and the affinity matured antibody h5A1002 is shown in Table 2.

[0155] Table 2

[0156] HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 h5A1 GFSLTTYGV IWTDGTT ARKGHGYYYAMDY SSVSSSY STSNLA HQYHRSPFT h5A1002 <![CDATA[GFSLTTYG G > <![CDATA[IWTDG W T]]> ARKGHGYYYAMDY SSVSSSY <![CDATA[STSNL I > HQYHRSPFT

[0157] Example 3: Antibody Affinity Detection

[0158] Biacore is a method for analyzing biomolecular interactions based on the principle of optical surface plasmon resonance (SPR). It can not only detect the specific binding between antigens and antibodies, but also obtain very important data in drug research and development such as the association rate constant (Ka), dissociation rate constant (Kd), and equilibrium dissociation constant (KD) of molecules, thereby calculating the affinity of the antibody.

[0159] In the Biacore 8K ((Cytiva) system, the antibody was diluted to 10 μg / mL with running buffer (HBS-EP), and the antibody was conjugated to a protein A ((Cytiva, 29127556) chip at a flow rate of 10 μL / min. Under the condition of a flow rate of 30 μL / min, the kinetic and affinity data of the antigen-antibody binding were detected, with a set binding time of 120 s and a dissociation time of 800 s.

[0160] The kinetic and affinity data of the parental antibody h5A1 and the affinity matured antibody h5A1002 binding to MICA were detected. The results are shown in Table 3. Compared with the parental antibody h5A1, the affinity of the affinity matured antibody h5A1002 for MICA was increased by approximately 39-fold.

[0161] Table 3

[0162] Ab Ka(1 / Ms) Kd(1 / s) KD(M) h5A1 2.97E+03 6.41E-04 2.15E-07 h5A1002 2.38E+04 1.29E-04 5.40E-09

[0163] Note: Ka represents the association rate constant (the larger the value, the stronger the affinity); Kd represents the dissociation rate constant (the smaller the value, the stronger the affinity), which reflects the affinity of the compound for the target; KD represents Kd / Ka, the equilibrium dissociation constant (affinity constant). The smaller the KD, the less dissociation, indicating the stronger the affinity.

[0164] Example 4: MICA antibody ELISA binding experiment

[0165] (1) Use ELISA to detect the binding characteristics of the parental antibody h5A1, affinity matured antibody h5A1002, and the α3 domains of MICA and MICB. The inventors coated the extracellular α3 domains (α3 domains of MICA-002, 005, 008, 004, and α3 domains of MICB-001, 005) Fc fusion proteins of various MICA and MICB variants obtained onto 96-well plates, and the strength of the signal after adding the antibody was used to judge the binding characteristics of the antibody to MICA and MICB.

[0166] Dilute the fusion protein (produced in this laboratory. The amino acid sequence of MICA002α3-Fc is shown in SEQ ID NO:22; the amino acid sequence of MICA005α3-Fc is shown in SEQ ID NO:24; the amino acid sequence of MICA008α3-Fc is shown in SEQ ID NO:25; the amino acid sequence of MICA004α3-Fc is shown in SEQ ID NO:23; the amino acid sequence of MICB001α3-Fc is shown in SEQ ID NO:26; the amino acid sequence of MICB005α3-Fc is shown in SEQ ID NO:27) to 1 μg / ml with PBS buffer, and add it to 96-well plates at a volume of 100 μL / well, and place it overnight at 4°C. Aspirate the PBS buffer in the 96-well plates, wash the plates 6 times with PBST (i.e., PBS containing 0.1% by volume of Tween 20 at pH 7.2) buffer, then add 200 μL / well of PBS containing 10% BSA, and incubate at 37°C for 2 h for blocking. Remove the blocking solution, wash the plates 6 times with PBST, then add 100 μL / well of h5A1 antibody (heavy chain sequence SEQ ID NO:11 and light chain sequence SEQ ID NO:12), h5A1002 antibody (heavy chain sequence SEQ ID NO:13 and light chain sequence SEQ ID NO:14), and control IgG1 (purchased from Bio Ying) gradient diluted with PBST containing 0.05% BSA (the highest working concentration is 20000 ng / ml, 5-fold dilution, 8 gradients), and incubate at 37°C for 1 h. Aspirate the reaction system in the wells, wash the plates 6 times with PBST, then dilute HRP (horseradish peroxidase)-labeled anti-human antibody secondary antibody (Fab specific) (purchased from Sigma) with PBST containing 0.05% BSA at 100 μL / well, and incubate at 37°C for 1 h. Aspirate the secondary antibody in the wells, wash the plates 6 times with PBST, then add 80 μL / well of TMB (tetramethylbenzidine), incubate at room temperature for 3 min, and add 80 μL / well of 4 M sulfuric acid to terminate the reaction. Read the absorbance value at 450 mm with an enzyme-linked immunosorbent assay reader.

[0167] The results are asFigure 3 and 4 As shown in Figures 5, 6, 7, 8, it shows that the antibodies of the present invention can bind to MICA*002, MICA*005, MICA*008, MICA*004, MICB*001, MICB*005, and the affinity matured antibody h5A1002 binds stronger to MICA*002, MICA*005, MICA*008 than the parental humanized antibody h5A1.

[0168] (2) ELISA was used to detect the binding characteristics of the affinity matured antibody h5A1002, the isotype antibody CLN-619 (Cullinan, the sequence is from Patent US20210253711A1, prepared by the method described in Example 1), 1D5V11 (Genentech, the sequence is from Patent US20200055939A1, prepared by the method described in Example 1) and the α3 domains of MICA and MICB. The inventors coated the extracellular domain α3 domains (α3 domains of MICA-002, 005, 008, 004, MICB-001, 005) Fc fusion proteins of various MICA and MICB variants obtained onto a 96-well plate, and the strength of the signal after adding the antibody was used to judge the binding characteristics of the antibody and MICA and MICB.

[0169] The experimental procedure was as described in step (1), and the results were as shown in Figure 11 and 12 Figures 13, 14, 15, 16, showing that the antibodies of the present invention can bind to MICA*002, MICA*005, MICA*008, MICA*004, MICB*001, MICB*005, and the affinity matured antibody h5A1002 binds stronger to MICA*002, MICA*005, MICA*008 than the isotype antibody CLN-619 (Cullinan).

[0170] Example 5: Flow cytometry binding experiment of MICA antibody

[0171] (1) Dilute the tumor cells with PBS to 2×10 6 / mL, add it to a 1.5 ml EP tube at a volume of 100 μL / tube. Add 10 μL / tube of goat serum to it and incubate at 4°C for 30 min. Add gradient-diluted (the highest working concentration is 50 μg / ml, 10 gradients, each gradient is diluted 3-fold) h5A1 antibody (heavy chain sequence SEQ ID NO:11 and light chain sequence SEQ ID NO:12), h5A1002 antibody (heavy chain sequence SEQ ID NO:13 and light chain sequence SEQ ID NO:14), control IgG1 (purchased from Bio Ying), and incubate at 4°C for 30 min. Add 1 mL of PBS to the EP tube, centrifuge at 3500 rpm for 5 min at 4°C, discard all the supernatant, and wash once with PBS. After centrifugation, discard all the supernatant, resuspend the cells with 100 μl / tube of PBS, add 1 μL / tube of Alexa-647-labeled goat anti-human antibody secondary antibody (purchased from Jackson lab), and incubate at 4°C in the dark for 30 min. Wash twice with PBS, and after centrifugation, discard all the supernatant. Resuspend the cells with 200 μL / tube of PBS and detect with a flow cytometer. The results are as Figure 9 、 10 shown, indicating that the affinity-matured antibody h5A1002 of the present invention binds to A-375 melanoma cells and HCT-15 colorectal cancer cells stronger than the parental humanized antibody h5A1.

[0172] (2) Dilute the tumor cells with PBS to 2×10 6 / mL, add it to a 1.5 ml EP tube at a volume of 100 μL / tube. Add 10 μL / tube of goat serum to it and incubate at 4°C for 30 min. Add gradient-diluted (the highest working concentration is 50 μg / ml, 10 gradients, each gradient is diluted 3-fold) h5A1002 antibody (heavy chain sequence SEQ ID NO:13 and light chain sequence SEQ ID NO:14), CLN-619 antibody (heavy chain sequence SEQ ID NO:17 and light chain sequence SEQ ID NO:18), 1D5V11 antibody (heavy chain sequence SEQ ID NO:19 and light chain sequence SEQ ID NO:20), control IgG1 (purchased from Bio Ying), and incubate at 4°C for 30 min. Add 1 mL of PBS to the EP tube, centrifuge at 3500 rpm for 5 min at 4°C, discard all the supernatant, and wash once with PBS. After centrifugation, discard all the supernatant, resuspend the cells with 100 μl / tube of PBS, add 1 μL / tube of Alexa-647-labeled goat anti-human antibody secondary antibody (purchased from Jackson lab), and incubate at 4°C in the dark for 30 min. Wash twice with PBS, and after centrifugation, discard all the supernatant. Resuspend the cells with 200 μL / tube of PBS and detect with a flow cytometer. The results are as Figure 17 、 18, as shown in FIGS. 19 and 20, it is shown that the affinity matured antibody h5A1002 of the present invention binds to tumor cells A-375 melanoma cells, A-549 non-small cell lung cancer cells, HCT-15 colorectal cancer cells, and MDA-MB-231 breast cancer cells stronger than the same target antibody CLN-619 (Cullinan).

[0173] Example 6: Epitope competition experiment

[0174] Flow cytometry was used to detect the antigen epitope competition between the affinity matured antibody h5A1002 and the same class antibodies CLN-619 (Cullinan) and 1D5V11 (Genentech).

[0175] Dilute HCT-15 cells with PBS to 2×10 6 / mL, add it to a 1.5 ml EP tube at a volume of 100 μL / tube, add 10 μL / tube of goat serum to it, and incubate at 4°C for 30 min. Add gradient-diluted (the highest working concentration is 300 μg / ml, 10 gradients, and each gradient is diluted 3 times) affinity matured antibody h5A1002 (heavy chain sequence SEQ ID NO: 13 and light chain sequence SEQ ID NO: 14), CLN-619 antibody (heavy chain sequence SEQ ID NO: 17 and light chain sequence SEQ ID NO: 18), 1D5V11 antibody (heavy chain sequence SEQ ID NO: 19 and light chain sequence SEQ ID NO: 20), hIgG1 (purchased from Bio Ying Biotechnology), and incubate at 4°C for 30 min. Add biotin-labeled affinity matured antibody Biotin-h5A1002 (heavy chain sequence SEQ ID NO: 13 and light chain sequence SEQ ID NO: 14), and incubate at 4°C for 30 min. Add 1 mL of PBS to the EP tube, centrifuge at 3500 rpm for 5 min at 4°C, discard all the supernatant, and wash once with PBS. After centrifugation, discard all the supernatant, resuspend the cells with 100 μl / tube of PBS, add 1 μL / tube of Alexa-647-labeled Streptavidin secondary antibody (purchased from Biolegend) to it, and incubate at 4°C in the dark for 30 min. Wash twice with PBS, and discard all the supernatant after centrifugation. Resuspend the cells with 200 μL / tube of PBS, and detect with a flow cytometer. The results are as Figure 21 shown, showing that the antigen epitope bound by the affinity matured antibody h5A1002 of the present invention overlaps with that of CLN-619 (Cullinan), while h5A1002 binds to a different antigen epitope from 1D5V11 (Genentech).

[0176] Example 7: MICA antibody endocytosis experiment

[0177] The affinity matured antibody h5A1002, together with the isotype antibodies CLN-619 (Cullinan) and 1D5V11 (Genentech), was labeled with pHrodo (purchased from Thermo) and added to the cell culture system. After incubation for 24 h, cell fluorescence was detected, and the strength of the fluorescence signal reflected the endocytosis of the antibody.

[0178] MDA-MB-231 cells were diluted with PBS to 1×10 5 / mL and added to a 96-well plate at a volume of 200 μL / well. Gradient-diluted (the highest working concentration was 50 μg / ml, 10 gradients, each gradient diluted 3-fold) h5A1002-pHrodo (heavy chain sequence SEQ ID NO: 13 and light chain sequence SEQ ID NO: 14), CLN-619-pHrodo (heavy chain sequence SEQ ID NO: 17 and light chain sequence SEQ ID NO: 18), and 1D5V11-pHrodo (heavy chain sequence SEQ ID NO: 19 and light chain sequence SEQ ID NO: 20) were added thereto. They were cultured at 37 °C and 5% CO2 for 24 h, and then flow cytometry was used to detect antibody endocytosis. The results are as Figure 22 shown, indicating that the endocytosis of the affinity matured antibody h5A1002 of the present invention was weaker than that of the isotype antibodies CLN-619 and 1D5V11, suggesting that its druggability as a monoclonal antibody was better.

[0179] Example 8: MICA antibody promotes PBMC to kill tumor cells

[0180] The ability of MICA monoclonal antibody to promote PBMC to kill A-375 melanoma cells and HCT-15 colorectal cancer cells was detected.

[0181] (1) Complete RPMI-1640 medium was added to a 16-well RTCA plate at a volume of 50 μL / well and calibrated on the machine;

[0182] (2) Tumor cells were diluted with complete RPMI-1640 medium to 2×10 5 / mL and separately added to the RTCA plate obtained in step (1) at a volume of 50 μL / well. Then, the cell index was detected for 24 h using an xCELLigence RTCA MP device under the conditions of 37 °C and 5% CO2;

[0183] (3) Gradient dilute h5A1002 (heavy chain sequence SEQ ID NO: 13 and light chain sequence SEQ ID NO: 14), h5A1002-hIgG1LALA (prepared by the method described in Example 1; heavy chain sequence SEQ ID NO: 15 and light chain sequence SEQ ID NO: 14), h5A1002-hIgG1DLE (prepared by the method described in Example 1; heavy chain sequence SEQ ID NO: 16 and light chain sequence SEQ ID NO: 14), CLN-619 antibody (heavy chain sequence SEQ ID NO: 17 and light chain sequence SEQ ID NO: 18), 1D5V11 antibody (heavy chain sequence SEQ ID NO: 19 and light chain sequence SEQ ID NO: 20), and hIgG1 (purchased from Bio Ying) with complete RPMI-1640 medium ( Figure 23 The highest concentration is 10 μg / ml, with 8 gradients, and each gradient is diluted 3-fold; Figure 24 、 25 The highest concentration is 50 μg / ml, with 12 gradients, and each gradient is diluted 5-fold), add them to the RTCA plate obtained in step (2), and the added volume is 20 μL / well;

[0184] (4) Dilute PBMC (purchased from MiaoShun Bio) with complete RPMI-1640 medium to 1.25×10 6 cells / mL, add it to the RTCA plate obtained in step (3), and the added volume is 80 μL / well;

[0185] (5) Detect the cell index of the reaction system obtained in step (4) at 37 °C and 5% CO2 for 24 h using the xCELLigence RTCA MP device.

[0186] As Figure 23 shown, the MICA antibody of the present invention promotes the killing of tumors by PBMC, and the hIgG1 subtype antibodies h5A1002, the antibody h5A1002-hIgG1LALA without ADCC function, and the ADCC-enhanced antibody h5A1002-hIgG1DLE all have promoting functions.

[0187] As Figure 24 、 25 shown, the h5A1002 antibody of the present invention promotes the killing of tumors by PBMC, and the hIgG1 subtype antibody (h5A1002) has a better tumor-killing function than the same-target antibody CLN-619 (also an hIgG1 subtype); the ADCC-enhanced antibody h5A1002-hIgG1DLE has the strongest promoting function, stronger than all other antibodies.

[0188] In the description of this specification, the descriptions referring to terms such as "one embodiment", "some embodiments", "examples", "specific examples", or "some examples", etc., mean that the specific features, structures, materials, or characteristics described in connection 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, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0189] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. An antibody or an antigen-binding fragment thereof, characterized in that, Comprising: The heavy chain variable region CDR1 sequence shown in SEQ ID NO: 1, the heavy chain variable region CDR2 shown in SEQ ID NO: 2, the heavy chain variable region CDR3 shown in SEQ ID NO: 3, the light chain variable region CDR1 shown in SEQ ID NO: 4, the light chain variable region CDR2 shown in SEQ ID NO: 5, and the light chain variable region CDR3 shown in SEQ ID NO: 6; The antibody or its antigen-binding fragment specifically recognizes MICA.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

8.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody is a humanized antibody.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-2, characterized in that The antibody or its antigen-binding fragment contains a heavy chain framework region sequence and a light chain framework region sequence, and at least a part of at least one of the heavy chain framework region sequence and the light chain framework region sequence is derived from at least one of a murine antibody and a human antibody.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-2, characterized in that, The antibody or its antigen-binding fragment contains at least one of a heavy chain constant region and a light chain constant region, and at least a part 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.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein, Both the light chain constant region and the heavy chain constant region are derived from a murine IgG1 antibody, a murine IgG2a antibody, a human IgG1 antibody, a human IgG2 antibody, a human IgG3 antibody, or a human IgG4 antibody.

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or its antigen-binding fragment is at least one of a single-chain antibody, a CDR-grafted antibody, a Fab antibody, and an Fv antibody.

8. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or its antigen-binding fragment according to any one of claims 1-7.

9. An expression vector, characterized in that, Carrying the nucleic acid molecule according to claim 8.

10. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1-7, characterized in that, Comprising: Introducing the expression vector according to claim 9 into a cell; Culturing the cell under conditions suitable for protein expression and secretion to obtain the antibody or its antigen-binding fragment.

11. The method according to claim 10, wherein The cell is a prokaryotic cell or a eukaryotic cell.

12. The method according to claim 11, wherein The cell is a eukaryotic cell.

13. A recombinant cell, characterized in that, The recombinant cell expresses the antibody or its antigen-binding fragment according to any one of claims 1-7, and carries the nucleic acid molecule according to claim 8 or the expression vector according to claim 9.

14. A composition, characterized in that, Comprising: The antibody or its antigen-binding fragment according to any one of claims 1-7, the nucleic acid molecule according to claim 8, the expression vector according to claim 9, or the recombinant cell according to claim 13.

15. Use of the antibody or its antigen-binding fragment according to any one of claims 1-7, the nucleic acid molecule according to claim 8, the expression vector according to claim 9, the recombinant cell according to claim 13, or the composition according to claim 14 in the preparation of a medicament for treating MICA-mediated diseases; The MICA-mediated diseases are at least one of colon cancer and melanoma.

16. A drug, characterized in that, Comprising: The antibody or antigen-binding fragment thereof according to any one of claims 1-7, the nucleic acid molecule according to claim 8, the expression vector according to claim 9, the recombinant cell according to claim 13 or the composition according to claim 14, wherein the drug is used for treating MICA-mediated diseases; The MICA-mediated diseases are at least one of colon cancer and melanoma.

17. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-7, the nucleic acid molecule according to claim 8, the expression vector according to claim 9 or the recombinant cell according to claim 13 in the preparation of a kit for detecting MICA.

18. A kit, characterized in that, The kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1-7, the nucleic acid molecule according to claim 8, the expression vector according to claim 9 or the recombinant cell according to claim 13.

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