Use of mageb16 modulator in preparation of product for treating autoimmune disease or resisting tumor

Abstract

The present invention relates to the field of biomedicine, and specifically relates to the use of an MAGEB16 modulator in the preparation of a product for treating an autoimmune disease or resisting a tumor. Provided is the use of an MAGEB16 modulator in the preparation of a product for treating an autoimmune disease or resisting a tumor. Further provided is an antibody that binds to the surface of a tumor cell, or an antigen-binding fragment thereof. It is found that the MAGEB16 protein can be expressed on the surface of tumor cells. This enables the direct targeting of MAGEB16 on the surface of tumor cells by means of antibodies or nucleic acid molecules, and makes a series of technical solutions targeting MAGEB16 on the surface of tumor cells become feasible technical solutions, thereby providing new technical solutions for efficiently resisting tumors.

Description

Use of MAGEB16 modulator in preparation of products for treating autoimmune diseases or anti-tumor TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular to the use of MAGEB16 modulator in preparation of products for treating autoimmune diseases or anti-tumor. BACKGROUND

[0002] Malignant tumor is still a difficult problem to be solved at present, and is one of the biggest threats to human health. Traditional methods for treating malignant tumor, including surgery, radiotherapy and chemotherapy, have their own shortcomings and limited efficacy. Recent studies have found that immunotherapy has good effect in anti-tumor. Monoclonal antibody therapy, immune checkpoint blockade therapy, antibody drug conjugate (ADC) therapy and other immunotherapy have made new breakthroughs in the field of malignant tumor treatment. Antibody drug conjugate is a new treatment form that connects a drug with biological activity and an antibody through a chemical linker. Compared with traditional anti-tumor drugs, antibodies and antibody drug conjugates can precisely target tumor cells and reduce the impact on non-tumor cells. However, these methods still have limitations such as limited target points and indications, which need to be broken through. Therefore, it is urgent to develop antibodies and their drug conjugates targeting new target points with good efficacy and safety in clinic, so as to broaden the indications for effective treatment.

[0003] MAGE family is highly expressed in various cancer tissues and plays an important role in tumor occurrence and growth.

[0004] MAGE-B subfamily belongs to a large family of cancer and testicular antigens, and there are few reports on MAGEB16.

[0005] Previous studies have found that MAGEB16 protein is mainly expressed in the cytoplasm, and existing technologies have found that immunotherapeutic yeast expressing MAGEB16 tumor antigen on its wall can be used for targeting dendritic cells, but there is no report on efficient control of tumor occurrence and development by inhibiting MAGEB16. SUMMARY

[0006] Based on the defects of the prior art, the present application provides a scheme for efficiently controlling the occurrence and development of tumors by inhibiting MAGEB16 of tumor cells. It should be understood that those skilled in the art can make many modifications and changes according to the concept of the present application without creative labor. Therefore, any technical solution that can be obtained by logical analysis, reasoning or limited experiments by those skilled in the art on the basis of the prior art according to the concept of the present application shall be within the protection scope defined by the claims. The present application obtains antibodies or antigen-binding fragments thereof by immunizing mice with different tumor cells, and the target is MAGEB16. It is found that MAGEB16 is overexpressed on the surface of a variety of tumor cells, and a series of technical solutions for directly targeting MAGEB16 on the surface of tumor cells by antibodies and nucleic acid molecules can inhibit tumor cells. It is also found that MAGEB16 protein can effectively inhibit T cell function, and can be used for regulating immune response and treating autoimmune diseases by using MAGEB16 recombinant protein or gene therapy of overexpressing MAGEB16.

[0007] The first aspect of the present application provides the use of a MAGEB16 modulator in the preparation of a product for treating autoimmune diseases or anti-tumor.

[0008] The MAGEB16 modulator is selected from any one or more of the following:

[0009] 1) a gene therapy drug overexpressing MAGEB16;

[0010] 2) an isolated MAGEB16 recombinant protein;

[0011] 3) a nucleic acid molecule that reduces or increases the expression level of MAGEB16;

[0012] 4) an antibody or antigen-binding fragment thereof capable of binding to the surface of tumor cells, or a chimeric antigen receptor thereof, or a T cell receptor thereof;

[0013] 5) an antibody drug conjugate or polypeptide drug conjugate capable of binding to the surface of tumor cells;

[0014] 6) a small molecule inhibitor of MAGEB16.

[0015] The second aspect of the present application provides an antibody or an antigen-binding fragment thereof binding to a tumor cell surface antigen, the MAGEB16 antibody or the antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the MAGEB16 antibody or the antigen-binding fragment thereof comprising a HCDR1 as shown in any one of SEQ ID NO: 11, 17, 23, 29, a HCDR2 as shown in any one of SEQ ID NO: 12, 18, 24, 30, a HCDR3 as shown in any one of SEQ ID NO: 13, 19, 25, 31, a LCDR1 as shown in any one of SEQ ID NO: 14, 20, 26, 32, a LCDR2 as shown in any one of SEQ ID NO: 15, 21, 27, 33, and a LCDR3 as shown in any one of SEQ ID NO: 16, 22, 28, 34.

[0016] The third aspect of the present application provides a chimeric antigen receptor or a T cell receptor, the chimeric antigen receptor or the T cell receptor comprising the antibody or the antigen-binding fragment thereof of the second aspect described above.

[0017] The fourth aspect of the present application provides a polynucleotide encoding the antibody or the antigen-binding fragment thereof of the second aspect described above, or the chimeric antigen receptor or the T cell receptor of the third aspect described above.

[0018] The fifth aspect of the present application provides a vector containing the polynucleotide of the fourth aspect described above.

[0019] The sixth aspect of the present application provides a host cell containing the vector of the fifth aspect described above or the polynucleotide of the fourth aspect described above integrated into the genome of the host cell.

[0020] The seventh aspect of the present application provides an antibody drug conjugate or a polypeptide drug conjugate, the antibody drug conjugate or the polypeptide drug conjugate comprising the antibody or the antigen-binding fragment thereof of the second aspect described above and a therapeutic agent.

[0021] The eighth aspect of the present application provides a nucleic acid molecule for reducing the expression level of MAGEB16, the nucleic acid molecule being an siRNA comprising a nucleotide sequence as shown in any one or more of SEQ ID NO: 35-40, preferably, the sequence of the sense strand of the siRNA comprising a sequence as shown in any one of SEQ ID NO: 35, 37, 39, and the sequence of the antisense strand comprising a sequence as shown in any one of SEQ ID NO: 36, 38, 40.

[0022] The ninth aspect of the present application provides an immunomodulator comprising an effective amount of the MAGEB16 modulator in the use of the first aspect, or the antibody or antigen binding fragment thereof of the second aspect, or the chimeric antigen receptor or T cell receptor of the third aspect, or the polynucleotide of the fourth aspect, or the vector of the fifth aspect, or the host cell of the sixth aspect, or the antibody drug conjugate or polypeptide drug conjugate of the seventh aspect, or the nucleic acid molecule of the eighth aspect.

[0023] The tenth aspect of the present application provides a pharmaceutical composition or kit comprising an effective amount of the MAGEB16 modulator in the use of the first aspect, or the antibody or antigen binding fragment thereof of the second aspect, or the chimeric antigen receptor or T cell receptor of the third aspect, or the polynucleotide of the fourth aspect, or the vector of the fifth aspect, or the host cell of the sixth aspect, or the antibody drug conjugate or polypeptide drug conjugate of the seventh aspect, or the nucleic acid molecule of the eighth aspect, or the immunomodulator of the ninth aspect.

[0024] The eleventh aspect of the present application provides the use of the antibody or antigen binding fragment thereof of the second aspect in the preparation of a tumor or autoimmune disease diagnostic product or a companion diagnostic product of the MAGEB16 modulator in the use of the first aspect.

[0025] The twelfth aspect of the present application provides the use of the antibody or antigen binding fragment thereof of the second aspect, or the chimeric antigen receptor or T cell receptor of the third aspect, or the polynucleotide of the fourth aspect, or the vector of the fifth aspect, or the host cell of the sixth aspect, or the antibody drug conjugate or polypeptide drug conjugate of the seventh aspect, or the nucleic acid molecule of the eighth aspect, or the immunomodulator of the ninth aspect in the preparation of a product for treating autoimmune diseases or anti-tumor.

[0026] The beneficial effects of the present application are:

[0027] The present application finds that the MAGEB16 protein can be expressed on the surface of tumor cells, so that it is feasible to directly target the MAGEB16 on the surface of tumor cells by antibodies or nucleic acid molecules and a series of technical solutions for targeting MAGEB16 on the surface of tumor cells, thereby providing new technical solutions for efficient anti-tumor.

[0028] The present application finds that the MAGEB16 protein can effectively inhibit T cell function, and can be used for treating autoimmune diseases by using MAGEB16 recombinant protein or gene therapy for overexpressing MAGEB16.

[0029] The present application creatively develops a targeting antibody capable of binding to MAGEB16 expressed on the surface of tumor cells with high affinity, which can neutralize the function of MAGEB16, inhibit the binding of MAGEB16 to one or more ligands, thereby relieving the immune escape of tumor cells, promoting the killing of tumor cells by immune cells in vivo and in vitro, or inhibiting the function of T cells in autoimmune diseases, and alleviating the progression of autoimmune diseases. The antibody drug has high specificity, affinity, can carry ADC conjugated drugs, and can be modified into CAR, thereby making it possible to construct CAR-T cell drugs. The antibody drug conjugate provided by the present application has extremely high drug efficacy and has obvious inhibitory effect on tumors. The antibody and antibody drug conjugate provided by the present application can be used as an effective component to prepare a product for treating tumors, and provides a new effective solution for tumor treatment. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 is a result graph of flow staining of the antibodies 1A5, 3F6 and 6F44 screened in Example 1 of the present application on mouse colon cancer CT26 and breast cancer 4T1.

[0031] Figure 2 is a result of binding of different mouse hybridoma antibodies to human MAGEB16 protein in Examples 1-2 of the present application. Figure 2A is an ELISA statistical graph of reaction of 5 micrograms per milliliter of two hybridoma antibodies with 5 micrograms per milliliter of MAGEB16 protein in Example 1, and Figure 2B is an ELISA statistical graph of reaction of 5 micrograms per milliliter of five hybridoma antibodies with 5 micrograms per milliliter of MAGEB16 protein in Example 2.

[0032] Figure 3 is a result graph of flow cytometry of surface staining of IgG and 6F44 in PBMC in Example 1 of the present application.

[0033] Figure 4 is a result graph of binding of mouse hybridoma MAGEB16 antibodies 6F44 and 3F6 to various tumor cells expressing MAGEB16 in Example 2 of the present application. Specifically, it is a graph of immunohistochemistry of lung cancer, gastric cancer, lymphoma and renal cancer using MAGEB16 antibody (3F6) (the right is an enlarged view), and it can be seen that MAGEB16 is expressed on the tumor cell membrane. Figure 4B is a result graph of flow cytometry of surface staining of IgG control and MAGEB16 antibody (6F44) in different tumor cells.

[0034] Figure 5 is a result graph of subcutaneous tumor transplantation experiment of human melanoma A375 in NCG mice in Example 4 of the present application. Figure 5A is a volume growth curve of A375 tumor after treatment with 10 mg / kg MAGEB16 antibody (6F44) (IgG group is a negative control), and Figure 5B is a picture of solid tumor after treatment and tumor weight statistics.

[0035] Figure 6 is a result of subcutaneous transplantation tumor experiment of human non-small cell lung cancer cell NCI-H1299 in Example 4 of the present application. Figure 6A is the volume growth curve of NCI-H1299 tumor after treatment with 10 mg / kg MAGEB16 antibody (6F44), and Figure 6B is the picture of solid tumor after treatment and tumor weight statistics.

[0036] Figure 7 is the binding verification of 6F44 humanized antibody and MAGEB16 in Example 3 of the present application. Figure 7A is an ELISA experiment for antibody antigen affinity detection between MAGEB16 and different humanized antibodies. Figure 7B is the detection of MAGEB16 expression of NCI-H1299 by flow cytometry using different concentrations of H24 antibody and chimeric antibody.

[0037] Figure 8 is a result of binding of 6F44 humanized antibody H24 to a plurality of tumor cells expressing MAGEB16 in Example 3 of the present application. Figure 8A is the flow cytometry results of IgG control and antibody group of a plurality of tumor cells, respectively, and Figure 8B is a statistical chart of the proportion of MAGEB16 positive cell population.

[0038] Figure 9 is a result of subcutaneous transplantation tumor experiment of human ovarian cancer cell A2780 in Example 4 of the present application. Figure 9A is the volume growth curve of A2780 tumor after treatment with 10 mg / kg H24 antibody, and Figure 9B is the picture of solid tumor after treatment and tumor weight statistics.

[0039] Figure 10 is a result of functional experiment of MAGEB16 protein in Example 5 of the present application. Figure 10A is the flow cytometry result of MAGEB16 protein promoting PD-1 expression of T cells, and Figures 10B and 10C are ELISA charts of MAGEB16 protein regulating IL10, IFN-γ and TNF-a.

[0040] Figure 11 is a result of detection of H24 antibody promoting PBMC killing tumor cells in Example 6 of the present application. Figure 11A is the flow cytometry result of H24 antibody promoting PBMC killing tumor cells, and Figure 11B is the statistical result of PBMC killing rate. The detection results of adding 20 micrograms per milliliter of IgG or H24 antibody in the co-culture system of tumor cells and PBMC, respectively.

[0041] Figure 12 is the effect of PBMC on the killing of different tumor cells after knocking down MAGEB16 in Example 6 of the present application. Figure 12A is the effect of knocking down MAGEB16 in MDA-MB-231 cells verified by Western Blot experiment, Figure 12B is the quantitative statistical result of Figure 10A, and Figures 12C and 12D are the analysis of the killing efficiency of PBMC on tumor cells after knocking down MAGEB16 (siMAGEB16) by small interfering RNA.

[0042] Figure 13 is the expression of MAGEB16 on the surface of different tumor cells after knocking down HLA-A (siHLA-A) in Example 6 of the present application. Figure 13A is the effect of knocking down HLA-A in NCI-H1299 cells verified by Western Blot experiment, and Figure 13B is the quantitative statistical result of Figure 13A. Figure 13C is the analysis and detection result graph of the cell membrane expression of MAGEB16 in different tumor cells after knocking down HLA-A.

[0043] Figure 14 is the ADCC effect result graph of MAGEB16 humanized antibody H24 in Example 6 of the present application.

[0044] Figure 15 is a comparison graph of the affinity of MAGEB16 humanized antibody (H24) and its drug conjugate (H24-ADC) for human colorectal cancer cell RKO in Example 7 of the present application.

[0045] Figure 16 is a co-localization effect graph of MAGEB16 humanized antibody drug conjugate (H24-ADC) with early endosome marker EEA1, late endosome marker RAB7, recycling endosome marker RAB11, and lysosome marker LAMP1 at 30, 120, and 240 minutes, respectively, in Example 8 of the present application.

[0046] Figure 17 is the killing effect of antibody drug conjugate in Example 9 of the present application. Figure 17A is the in vitro killing effect of MAGEB16 humanized antibody (H24) and its drug conjugate (H24-ADC), control IgG1 antibody drug conjugate (control IgG1-ADC) on RKO cell line. Figure 17B is a comparison graph of the killing effect of MAGEB16 humanized antibody drug conjugate (H24-ADC) on MDA-MB-231 and A2780 cells.

[0047] Figure 18 is a comparison graph of the effect of blocking cell cycle and inducing apoptosis of MAGEB16 humanized antibody drug conjugate (H24-ADC) and control IgG1 antibody drug conjugate (control IgG1-ADC) after different treatment times in Example 9 of the present application.

[0048] Figure 19 is a graph showing the efficacy of MAGEB16 humanized antibody drug conjugate (H24-ADC) on human colorectal cancer cell RKO in BALB / c Nude mice subcutaneous tumor xenograft. Figure 19A is the tumor volume growth curve after treatment with 1 mg / kg H24-ADC (PBS and 1 mg / kg control IgG1-ADC are negative controls), and Figure 19B is the picture of solid tumor after treatment and tumor weight statistics.

[0049] Figure 20 is a graph showing the efficacy of MAGEB16 humanized antibody drug conjugate (H24-ADC) on mouse melanoma cell B16-F10 in C57 mice subcutaneous tumor xenograft. Figure 20A is the tumor volume growth curve after treatment with 3 mg / kg H24-ADC (3 mg / kg IgG1-ADC is negative control), and Figure 20B is the picture of solid tumor after treatment and tumor weight statistics.

[0050] Figure 21 is a graph showing the expression of MAGEB16 in different tumors. Figure 21A is a graph showing the expression of MAGEB16 in different tumors (T) and paracancer (N) in TCGA database. Figure 21B is a graph showing the immunohistochemistry of tissue chip using MAGEB16 antibody, Figure 21C is a graph showing the immunohistochemistry results of different pathological sections, and Figure 21D is a graph showing the immunohistochemistry score statistics of different cancer types. DETAILED DESCRIPTION

[0051] The specific embodiments of the application will now be described in detail with specific reference being made to the figures. It is to be understood that other advantages can be attained with the disclosed application and that modifications can be made without departing from the scope of the application. The application is not to be limited to the specific embodiments disclosed and making use of specific designs, which are given as illustrative only. It will be apparent to those skilled in the art that various modifications and variations can be made in the present application without departing from the spirit or scope of the application. Thus, it is intended that the present application cover the modifications and variations of this application provided they come within the scope of the appended claims and their equivalents.

[0052] Before further description of the application, it is to be understood that the application is not limited to the particular specific embodiments described herein; it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting; as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise.

[0053] When the embodiments give a numerical range, it should be understood that, unless otherwise specified in the present application, both ends of each numerical range and any one numerical value between the two ends can be selected. Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as generally understood by those skilled in the art. In addition to the specific methods, devices, materials used in the embodiments, any method, device and material of the prior art similar or equivalent to the method, device and material described in the embodiments of the present application can also be used to implement the present application according to the master of the prior art of those skilled in the art and the description of the present application.

[0054] The present application obtains antibodies or antigen-binding fragments thereof by immunizing mice with different tumor cells, and the target is identified as MAGEB16. The present application also finds that MAGEB16 protein can regulate the function of T cells, and the modulator against MAGEB16 can be used to prepare products for treating autoimmune diseases or anti-tumor.

[0055] MAGE gene is a proto-oncogene belonging to the cancer-testis antigen (CTA) family, which was first discovered in melanoma. More than 60 members of the MAGE gene family have been found so far. The largest common feature of the MAGE gene family is that there is a MAGE homology domain (MHD) in the amino acid sequence. MHD generally contains 165-171 amino acid residues. According to the structure prediction of its amino acid arrangement, it may contain 4 alpha helices and 5 beta sheet structures, but its real structure and function are not very clear at present. According to the specific location of MAGE gene on the chromosome and its different expression patterns, MAGE gene is divided into two subfamilies, MAGE-I class antigen and MAGE-II class antigen. MAGE-I class antigen belongs to a large family of cancer-testis antigens, which can be further divided into MAGE-A, B and C subfamilies, which are mainly distributed in germ cells and trophoblasts. MAGE-II class antigen includes MAGE-D, nerve cell growth inhibitor antigen, reticuloendothelial system stimulator antigen, etc., which are mainly expressed in neural tissue and other normal tissues.

[0056] The present application analyzes the expression of MAGEB16 in different tumor tissues, including ACC (adrenal cortical carcinoma), BLCA (bladder urothelial carcinoma), BRCA (breast invasive carcinoma), CESC (cervical squamous cell carcinoma and adenocarcinoma), CHOL (cholangiocarcinoma), COAD (colon adenocarcinoma), DLBC (diffuse large B-cell lymphoma), ESCA (esophageal carcinoma), GBM (glioblastoma multiforme), HNSC (head and neck squamous cell carcinoma), KICH (kidney chromophobe), KIRC (kidney renal clear cell carcinoma), KIRP (kidney renal papillary cell carcinoma), LAML (acute myeloid leukemia), LGG (brain lower grade glioma), LIHC (liver hepatocellular carcinoma), LUAD (lung adenocarcinoma), LUSC (lung squamous cell carcinoma), MESO (mesothelioma), OV (ovarian carcinoma), PAAD (pancreatic adenocarcinoma), PCPG (pheochromocytoma and paraganglioma), PRAD (prostate adenocarcinoma), READ (rectum adenocarcinoma), SARC (sarcoma), SKCM (cutaneous melanoma), STAD (stomach adenocarcinoma), TGCT (testicular germ cell tumors), THCA (thyroid carcinoma), THYM (thymoma), UCEC (uterine corpus endometrioid carcinoma), UCS (uterine carcinosarcoma), and UVM (uveal melanoma), and the results show that the expression of MAGEB16 in tumor tissues is higher than that in the adjacent tissues.

[0057] As known by those skilled in the art, MAGE family is originally an intracellular protein, which is degraded in the cell and then presented on the cell surface by MHC (Major Histocompatibility Complex), including HLA (human leukocyte antigen) system. The main function of HLA molecule is to present antigens (such as protein fragments of viruses, bacteria or tumor cells) to the immune system. That is, according to the prior art in the art, MAGEB16 can be presented on the cell surface by HLA and recognized as an antigen. However, the present application unexpectedly found that MAGEB16 protein can be expressed on the surface of tumor cells. In the present application, the antibody can bind to MAGEB16 without breaking the tumor cells, which indicates that MAGEB16 protein itself is expressed on the cell membrane surface. Moreover, the present application found that knocking down the expression of HLA does not affect the expression of MAGEB16, which further indicates that MAGEB16 protein itself is expressed on the cell membrane surface, and is not transported to the cell membrane by the antigen presentation pathway. Therefore, this finding makes it possible to directly target MAGEB16 on the surface of tumor cells by antibodies and nucleic acid molecules, and a series of technical solutions for targeting MAGEB16 on the surface of tumor cells. The present application also found that MAGEB16 protein can significantly inhibit the function of T cells, and thus can be used for treating autoimmune diseases.

[0058] Firstly, the present application provides the use of MAGEB16 modulator in the preparation of products for treating autoimmune diseases or anti-tumor products, the tumor is MAGEB16 positive tumor, the MAGEB16 modulator is selected from any one or more of the following:

[0059] 1) nucleic acid molecules that reduce or increase the expression level of MAGEB16;

[0060] 2) gene therapy drugs that overexpress MAGEB16;

[0061] 3) isolated MAGEB16 recombinant protein;

[0062] 4) antibodies or antigen-binding fragments thereof, or chimeric antigen receptors thereof, or T cell receptors thereof capable of binding to the surface of tumor cells;

[0063] 5) antibody drug conjugates or polypeptide drug conjugates capable of binding to the surface of cells;

[0064] 6) small molecule inhibitors of MAGEB16.

[0065] In the detailed description of the present application, the antibody is a MAGEB16 antibody, which can bind to MAGEB16 on the surface of tumor cells.

[0066] In the detailed description of the present application, the antibody drug conjugate or polypeptide drug conjugate can bind to MAGEB16 on the surface of tumor cells.

[0067] Based on the MAGEB16 target discovered in the present application and the effect of the MAGEB16 target on tumor cells, it can be known that any substance based on the target MAGEB16 according to the prior art for the regulation of the target MAGEB16 can be used as a MAGEB16 modulator for anti-tumor, not limited to the list in the present application.

[0068] Based on the effect of MAGEB16 on T cells discovered in the present application, it can be known that any substance based on the target MAGEB16 according to the prior art for the regulation of the target MAGEB16 can be used as a MAGEB16 modulator for autoimmune diseases, not limited to the list in the present application.

[0069] In the present application, the product can be a drug, a pharmaceutical composition, a reagent, a kit, etc.

[0070] The regulation of the modulator can be up-regulation, increase or promotion, or down-regulation, decrease or inhibition, etc. In the specific embodiments of the present application, the MAGEB modulator can increase or decrease the level of MAGEB16 in the body, can bind to the cell surface expressed MAGEB16, or promote or inhibit the function of the cell surface expressed MAGEB16, or increase or decrease the level of the cell surface expressed MAGEB16. In some embodiments, the cell can be a tumor cell.

[0071] In the specific embodiments of the present application, the modulator can also be used as a raw material to prepare an anti-tumor product that regulates MAGEB16, such as an isolated MAGEB16 recombinant protein as a raw material to immunize mice to prepare a MAGEB16 antibody, etc., or an isolated MAGEB16 recombinant protein as a raw material to prepare a vaccine to induce the production of a corresponding antibody in the body to achieve an anti-tumor effect. The isolated MAGEB16 recombinant protein can also inhibit the function of T cells to achieve a therapeutic effect on autoimmune diseases.

[0072] In the specific embodiments of the present application, the tumor is a MAGEB16 positive and high expression tumor, and MAGEB16 is expressed on the surface of tumor cells. Specifically, the tumor is selected from any one of ovarian cancer, breast cancer, lung cancer, colorectal cancer, melanoma, liver cancer, pancreatic cancer, sarcoma, nervous system tumor, leukemia, urothelial carcinoma, cervical cancer, prostate cancer, esophageal cancer, thymoma, squamous cell carcinoma, lymphoma, gastric cancer, testicular cancer, thyroid cancer, endometrial cancer, head and neck cancer, renal cancer, adrenal cortex cancer, bile duct cancer, mesothelioma, pheochromocytoma, or paraganglioma.

[0073] In the specific embodiments of the present application, the autoimmune disease is selected from any one of systemic lupus erythematosus and lupus nephritis, rheumatoid arthritis, Sjogren's syndrome and related kidney disease, systemic sclerosis, mixed connective tissue disease, antiphospholipid syndrome, adult Still's disease, type I diabetes, Graves' disease, Hashimoto's thyroiditis, Addison's disease, celiac disease, autoimmune hepatitis, primary biliary cholangitis, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, Guillain-Barre syndrome, psoriasis, vitiligo, pemphigus, autoimmune hemolytic anemia, idiopathic thrombocytopenia purpura, autoimmune neutropenia, pernicious anemia, Goodpasture's syndrome, autoimmune ear disease, ankylosing spondylitis, arthritis, polyarteritis nodosa, granulomatous polyangiitis, giant cell arteritis, Behcet's disease, relapsing polychondritis, IgG4-related disease, Vogt-Koyanagi-Harada syndrome, anti-glomerular basement membrane disease, Goodpasture's syndrome, ANCA-associated vasculitis renal damage, or anti-phospholipase A2 receptor antibody-associated membranous nephropathy.

[0074] The present application screens antibodies that bind to tumor cells. Therefore, the present application also provides an antibody or antigen-binding fragment thereof that binds to the surface of a tumor cell.

[0075] In the present application, the antibody or antigen-binding fragment thereof described above includes a heavy chain variable region and a light chain variable region, and the antibody or antigen-binding fragment thereof includes HCDR1 as shown in any one of SEQ ID NOs: 11, 17, 23, and 29, HCDR2 as shown in any one of SEQ ID NOs: 12, 18, 24, and 30, HCDR3 as shown in any one of SEQ ID NOs: 13, 19, 25, and 31, LCDR1 as shown in any one of SEQ ID NOs: 14, 20, 26, and 32, LCDR2 as shown in any one of SEQ ID NOs: 15, 21, 27, and 33, and LCDR3 as shown in any one of SEQ ID NOs: 16, 22, 28, and 34.

[0076] In the detailed description of the present application, the antibody or antigen-binding fragment thereof is a MAGEB16 antibody or antigen-binding fragment thereof that can bind to MAGEB16 on the surface of a tumor cell. Based on the finding that MAGEB16 is expressed on the surface of a tumor cell in the present application, the MAGEB16 antibody can directly and efficiently bind to MAGEB16 on the surface of the cell without the need for HLA antigen presentation, and thus the binding of the MAGEB16 antibody to MAGEB16 can be more efficient.

[0077] In the detailed description of the present application, the HCDR1 of the MAGEB16 antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 11, the HCDR2 is as shown in SEQ ID NO: 12, the HCDR3 is as shown in SEQ ID NO: 13, the LCDR1 is as shown in SEQ ID NO: 14, the LCDR2 is as shown in SEQ ID NO: 15, and the LCDR3 is as shown in SEQ ID NO: 16.

[0078] In another embodiment of the present application, the HCDR1 of the MAGEB16 antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 17, the HCDR2 is as shown in SEQ ID NO: 18, the HCDR3 is as shown in SEQ ID NO: 19, the LCDR1 is as shown in SEQ ID NO: 20, the LCDR2 is as shown in SEQ ID NO: 21, and the LCDR3 is as shown in SEQ ID NO: 22.

[0079] In yet another embodiment of the present application, the HCDR1 of the MAGEB16 antibody or antigen binding fragment thereof is set forth in SEQ ID NO: 23; the HCDR2 is set forth in SEQ ID NO: 24; the HCDR3 is set forth in SEQ ID NO: 25; the LCDR1 is set forth in SEQ ID NO: 26; the LCDR2 is set forth in SEQ ID NO: 27; and the LCDR3 is set forth in SEQ ID NO: 28.

[0080] In yet another embodiment of the present application, the HCDR1 of the MAGEB16 antibody or antigen binding fragment thereof is set forth in SEQ ID NO: 29; the HCDR2 is set forth in SEQ ID NO: 30; the HCDR3 is set forth in SEQ ID NO: 31; the LCDR1 is set forth in SEQ ID NO: 32; the LCDR2 is set forth in SEQ ID NO: 33; and the LCDR3 is set forth in SEQ ID NO: 34.

[0081] In the present application, the heavy chain variable region of the MAGEB16 antibody or antigen binding fragment thereof comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 7, or 9; and the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 6, 8, or 10.

[0082] In an embodiment of the present application, the heavy chain variable region of the MAGEB16 antibody or antigen binding fragment thereof is set forth in SEQ ID NO: 1, and the light chain variable region is set forth in SEQ ID NO: 2.

[0083] In another embodiment of the present application, the heavy chain variable region of the MAGEB16 antibody or antigen binding fragment thereof is set forth in SEQ ID NO: 3, and the light chain variable region is set forth in SEQ ID NO: 4.

[0084] In yet another embodiment of the present application, the heavy chain variable region of the MAGEB16 antibody or antigen binding fragment thereof is set forth in SEQ ID NO: 5, and the light chain variable region is set forth in SEQ ID NO: 6.

[0085] In yet another embodiment of the present application, the heavy chain variable region of the MAGEB16 antibody or antigen binding fragment thereof is set forth in SEQ ID NO: 7, and the light chain variable region is set forth in SEQ ID NO: 8.

[0086] In yet another embodiment of the present application, the heavy chain variable region of the MAGEB16 antibody or antigen binding fragment thereof is set forth in SEQ ID NO: 9, and the light chain variable region is set forth in SEQ ID NO: 10.

[0087] In the preferred embodiments of the present application, the heavy chain variable region of the MAGEB16 antibody is as shown in SEQ ID NO: 1 or 9, and the light chain variable region is as shown in SEQ ID NO: 2 or 10, which have the best binding ability to MAGEB16. Among them, SEQ ID NO: 9 and 10 are obtained by mammalian codon optimization of the amino acid sequences shown in SEQ ID NO: 1 and 2.

[0088] As some specific embodiments of the present application, the MAGEB16 antibody can be a polyclonal antibody, a monoclonal antibody, a single-chain antibody, an antigen binding domain, a bispecific antibody, a multispecific antibody, or an antigen binding part in a chimeric antigen receptor.

[0089] The MAGEB16 antibody can be a murine antibody or a humanized antibody, and the humanized antibody can be a chimeric antibody. Specifically, the murine antibody means that the coding gene of the antibody is completely derived from a mouse. The humanized antibody means that part of the constant region of the antibody or the whole antibody is encoded by human antibody genes. The humanized antibody can greatly reduce the immune side effects of heterologous antibodies on the human body. The humanized antibody includes chimeric antibodies, modified antibodies, and fully humanized antibodies, etc. The chimeric antibody is a monoclonal antibody produced by inserting the light chain and heavy chain variable regions of a murine monoclonal antibody into a vector containing the constant region of a human antibody, transforming mammalian cells for expression. The degree of humanization of the chimeric antibody can reach 70%, completely retaining the variable region of the murine monoclonal antibody and the parent activity, and the introduction of the constant region of the human antibody reduces the immunogenicity.

[0090] As known by those skilled in the art, antibodies in non-intact tetramer forms including but not limited to Fab, Fab', F(ab') or F(ab')2, nanobodies (VHH), single-chain antibodies (scFv), BsFv, dsFv, (dsFv)2, or Fv, etc. can also have the effect of specifically binding to antigens, and therefore the present application also includes antigen binding fragments of the MAGEB16 antibody.

[0091] In general cases of the present application, the antibody further includes a heavy chain constant region and a light chain constant region, which can be derived from human or murine sources. The heavy chain constant region is selected from IgG, IgA, IgM, IgD or IgE type; and / or, the light chain constant region is selected from κ or λ type. IgG is a globulin that is necessary for maintaining immunity in vivo, and human IgG has four subtypes, namely IgG1, IgG2, IgG3 and IgG4. For example, in specific embodiments of the present application, the heavy chain constant region can be of IgG1 type, and the light chain constant region can be of κ type.

[0092] The MAGEB16 antibody or antigen-binding fragment thereof in the present application can bind to MAGEB16 expressed on the cell membrane surface, thereby being capable of specifically binding to tumor cells, so as to achieve the regulation of MAGEB16. The regulation is specifically down-regulation or inhibition.

[0093] The antibody of the present application can also be engineered into a chimeric antigen receptor, i.e., CAR, and therefore the present application also provides a chimeric antigen receptor comprising an antigen binding domain, a transmembrane domain and an intracellular signaling domain. The antigen binding domain comprises the antibody or antigen-binding fragment thereof as described above.

[0094] The CAR further comprises a transmembrane domain, which is a key region connecting the extracellular antigen recognition part and the intracellular signaling part of the CAR. The transmembrane domain is usually composed of a hydrophobic amino acid sequence, which enables the CAR to be anchored on the cell membrane of T cells or other immune cells.

[0095] In the specific embodiments of the present application, the transmembrane domain can be the transmembrane region of CD8a, CD28, CD3e, CD3zeta, CD45, CD4, CD5, CD9, CD16, CD20, CD22, CD27, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD152, CD154, DAP10, DAP12, PD-1, a, b or z chain of T cell receptor, etc.

[0096] The CAR further comprises an intracellular signaling domain, which is responsible for transmitting an activation signal to the T cell after the CAR binds to the tumor cell surface antigen, thereby activating the T cell and triggering an immune response. The intracellular signaling domain further comprises a primary signaling domain and a costimulatory signaling domain. The primary signaling domain contains immunoreceptor tyrosine-based activation motifs (ITAMs), which can trigger downstream signaling pathways after receptor activation, thereby activating T cells; the costimulatory signaling domain provides the necessary second signal to enhance T cell activation, proliferation and survival.

[0097] In the specific embodiments of the present application, the intracellular signaling domain comprises at least one costimulatory signaling domain and a primary signaling domain in sequence from N-terminus to C-terminus.

[0098] In the specific embodiments of the present application, the primary signaling domain can be the intracellular signaling domain of CD3zeta, FcRgamma, FcRbeta, CD3gamma, CD3delta, CD3epsilon, CD22, CD79a, DAP10, CD79b or CD66d, etc.

[0099] In embodiments of the application, the co-stimulatory signaling domain can be an intracellular signaling domain of CD137 (4-1BB), CD27, CD28, ICOS, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, or the like, or a combination thereof.

[0100] The CAR further comprises a hinge region between the C-terminus of the antigen binding domain and the N-terminus of the transmembrane domain, which is a domain between the antigen recognition domain and the transmembrane region, and is usually derived from the hinge region of an antibody. The main role of the hinge region is to provide the necessary flexibility and flexibility, so that the CAR can move freely in space, so as to effectively recognize and bind to the antigen on the surface of the tumor cell.

[0101] In embodiments of the application, the hinge region can be a hinge region of CD8a, CD28, IgG1, or IgG4.

[0102] The CAR further comprises a signal peptide at the N-terminus of the chimeric antigen receptor polypeptide, which is a short peptide chain, and can enhance the expression level of the CAR on the surface of the T cell, affect the surface stability and antigen recognition efficiency of the CAR-T cell, and affect the immune activation and effector function of the CAR-T cell.

[0103] In embodiments of the application, the signal peptide can be derived from HLA-A, CD8a, CD33, IgK, IL-2, or GM-CSFRa, or the like.

[0104] In embodiments of the application, the chimeric antigen receptor CAR comprises, in order from the N-terminus to the C-terminus, the signal peptide, the antigen binding domain, the hinge region, the transmembrane domain, the co-stimulatory signaling domain, and the primary signaling domain.

[0105] The antibody of the application can also be engineered into a T cell receptor, i.e., a TCR, and thus the application also provides a T cell receptor comprising an antibody or antigen binding fragment thereof as described above.

[0106] In embodiments of the application, the T cell receptor is an aP chain receptor and / or a gD chain receptor.

[0107] In embodiments of the application, the variable region of the T cell receptor comprises the heavy chain variable region and / or the light chain variable region of the MAGEB16 antibody or antigen binding fragment thereof described above.

[0108] In addition, the skilled person in the art knows that the above antibody or antigen-binding fragment thereof, chimeric antigen receptor or T cell receptor can be encoded by a polynucleotide, and therefore based on the above MAGEB16 antibody or antigen-binding fragment thereof, or chimeric antigen receptor or T cell receptor, the present application also provides a polynucleotide encoding the above antibody or antigen-binding fragment thereof, or the above chimeric antigen receptor or T cell receptor.

[0109] The method for preparing the polynucleotide is known in the art, and can be selected according to the specific circumstances, for example, it can be prepared by automatic DNA synthesis, or it can be obtained by recombinant DNA technology, or it can be isolated from a suitable natural source.

[0110] The polynucleotide refers to a polymer of nucleotides usually linked from one deoxyribose or ribose to another deoxyribose or ribose, and the polynucleotide in the present application has no size limit and can include polynucleotides containing modifications, especially modified nucleotides. In some embodiments, the polynucleotide can be RNA, DNA or cDNA, etc.

[0111] In the detailed description of the present application, the polynucleotide comprises the nucleotide sequence as shown in SEQ ID NOs. 46-47.

[0112] Based on the above-mentioned polynucleotide, the polynucleotide can be inserted into a vector using the prior art to express it, and therefore the present application also provides a vector containing the above-mentioned polynucleotide.

[0113] In some embodiments of the present application, the vector contains a polynucleotide encoding the antibody or antigen-binding fragment thereof.

[0114] In some other embodiments of the present application, the vector contains a polynucleotide encoding the chimeric antigen receptor or T cell receptor.

[0115] The "vector" in the present application refers to a polynucleotide capable of carrying at least one polynucleotide fragment, and the vector can exist in a circular or linear (linearized) form, and also includes a vector fragment, and can be an artificial chromosome or similar individual polynucleotide containing a means for allowing the transfer of an exogenous nucleic acid fragment. The vector can contain at least one expression cassette containing a regulatory sequence for the correct expression of the polynucleotide incorporated therein. The polynucleotide to be introduced into the cell (such as a polynucleotide encoding a product of interest or a selection marker) can be inserted into the expression cassette of the vector for expression therefrom. The vector delivers the polynucleotide to the host cell for expression. The vector can be a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR / Cas9 vector or a viral vector, etc.

[0116] In the detailed description of the present application, the vector is an expression vector, and specifically, can be a pcDNA3.1-based expression vector, such as pcDNA3.1-IgG1 Fc, pcDNA3.1-IgK vector.

[0117] The vector is introduced into a host cell to achieve expression of the polynucleotide in the vector, and thus the present application also provides a host cell containing the above-mentioned vector or having the above-mentioned polynucleotide integrated into the genome.

[0118] In some embodiments of the present application, the host cell can contain a vector containing a polynucleotide encoding the antibody or antigen-binding fragment thereof, or the genome of the host cell can have a polynucleotide encoding the antibody or antigen-binding fragment thereof integrated therein. The host cell can be selected from bacterial cells, yeast cells, filamentous fungal cells, mammalian cells, etc., and specifically, can be selected from E. coli, Streptomyces, Salmonella typhimurium, yeast, filamentous fungi, Drosophila S2 or Sf9 cells, CHO cells, COS cells, 293 cells, Bowes melanoma cells, etc.

[0119] In some embodiments of the present application, the host cell can contain a vector containing a polynucleotide encoding the antibody or antigen-binding fragment thereof, or the genome of the host cell can have a polynucleotide encoding the antibody or antigen-binding fragment thereof integrated therein. The host cell can be selected from bacterial cells, yeast cells, filamentous fungal cells, mammalian cells, etc., and specifically, can be selected from E. coli, Streptomyces, Salmonella typhimurium, yeast, filamentous fungi, Drosophila S2 or Sf9 cells, CHO cells, COS cells, 293 cells, Bowes melanoma cells, etc.

[0120] The method of introducing the vector into the host cell can be selected according to the actual situation, for example, microinjection, gene gun, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation, etc.

[0121] The present application also provides a method for preparing an antibody or antigen-binding fragment thereof, which comprises culturing the above-mentioned host cell under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the culture of the host cell; wherein the genome of the host cell has the above-mentioned nucleotide sequence encoding the antibody or antigen-binding fragment thereof integrated therein, or the host cell contains a vector containing a polynucleotide encoding the antibody or antigen-binding fragment thereof.

[0122] The present application also provides a method for preparing an immune effector cell, which comprises introducing the above-mentioned polynucleotide encoding the chimeric antigen receptor or T cell receptor or the vector containing the polynucleotide encoding the chimeric antigen receptor or T cell receptor into the immune effector cell.

[0123] Based on the MAGEB16 antibody prepared in the present application, a therapeutic agent is coupled with the antibody to obtain an antibody drug conjugate. The present application also provides an antibody drug conjugate or a polypeptide drug conjugate, which comprises the antibody or antigen binding fragment thereof as described above and a therapeutic agent. The antibody drug conjugate (ADC) is a small molecule drug with biological activity connected to a monoclonal antibody through a chemical chain, and the monoclonal antibody serves as a carrier to target and transport the small molecule drug into target cells.

[0124] In the present application, the therapeutic agent can be selected from cytotoxic molecules, immune enhancers and radioisotopes; the cytotoxic molecules include but are not limited to microtubulin inhibitors or DNA damaging agents; the microtubulin inhibitors include but are not limited to Dolastatin and its Auristatin derivatives MMAE, MMAF, Maytansine and Maytansine derivatives DM1, DM4; the DNA damaging agents include but are not limited to calicheamicin, duocarmysin, anthramycin PBD, camptothecins and camptothecin derivatives SN-38, Dxd; preferably, the therapeutic agent is MMAE. The immune enhancers include but are not limited to TLR agonists, STING agonists, BTK inhibitors. The radioisotopes include but are not limited to Lutetium-177, Rhenium-186, Rhenium-188, Iodine-131, Yttrium-90, Radium-223, Rhenium-177, Gallium-68.

[0125] In addition to the antibody drug conjugate or polypeptide drug conjugate, a linker is also included to conjugate the antibody or antigen binding fragment thereof with the therapeutic agent. In the detailed description of the present application, the linker can be a cleavable linker or a non-cleavable linker. The cleavable linker is stable in the blood circulation for a long time and selectively releases the payload in the tumor microenvironment, which can specifically include an acid-cleavable linker, a reducible linker, an enzyme-cleavable linker, etc. The non-cleavable linker is divided into two categories, namely a thioether or a maleimide-based hexanoyl group (MC). They are composed of stable bonds, which prevent proteolytic cleavage and ensure greater stability than their cleavable counterparts. As certain embodiments of the present application, the linker is an enzyme-cleavable linker, which includes a peptide unit containing 2-20 amino acids; the peptide unit can contain 2-4, 4-6, 4-8, 8-10, 10-12, 12-14, 14-16, 16-18, 18-20 amino acids. Preferably, the peptide unit is selected from any one or a combination of more than one of -valine-citrulline- (-Val-Cit-), -glycine-glycine-phenylalanine-glycine- (-Gly-Gly-Phe-Gly-), -valine-alanine- (-Val-Ala-). In a more preferred embodiment, the linker of the present application is Mc-Val-Cit-PAB.

[0126] As certain embodiments of the present application, the general structure of the antibody drug conjugate is A-(L-U)n, wherein: A is a MAGEB16 antibody or an antigen binding fragment thereof; U is a therapeutic agent; L is a linker; n is an integer selected from 1 to 8. The A can be connected with 1, 2, 3, 4, 5, 6, 7, 8 active drug units U through one or more linkers L.

[0127] In some embodiments of the present application, the linker can be connected with the antibody or antigen binding fragment thereof by any means known in the art. Preferably, the linker is connected with the amino acid residue or the sulfhydryl residue on the antibody or antigen binding fragment thereof. In a more preferred embodiment, the linker of the present application is covalently connected with the sulfhydryl residue formed after the opening of the interchain disulfide bond of the anti-MAGEB16 antibody.

[0128] The present application also provides a nucleic acid molecule for reducing the expression level of MAGEB16, which is an siRNA including the nucleotide sequence shown in any one or more of SEQ ID NOs: 35-40.

[0129] In the present application, the nucleic acid molecule for reducing the expression level of MAGEB16 gene knocks down or silences the MAGEB16 gene through the RNA interference pathway, thereby inhibiting the transcription or translation of the MAGEB16 gene, reducing the expression of the MAGEB16 gene, which can be siRNA, shRNA, miRNA, antisense RNA, etc.; in the specific embodiments of the present application, the nucleic acid molecule for reducing the expression level of the MAGEB16 gene is siRNA; the sequence of the sense strand of the siRNA includes the sequence shown in any one of SEQ ID NO: 35, 37, 39, and the sequence of the antisense strand includes the sequence shown in any one of SEQ ID NO: 36, 38, 40, and the specific sequences are shown below.

[0130] In the present application, based on the MAGEB16 target, siRNA for knocking down the expression of MAGEB16 is designed, and it is proved by examples that the use of siRNA can reduce the expression of MAGEB16 through RNA interference, thereby playing an anti-tumor role, i.e., it is proved that any RNA molecule sequence capable of reducing the expression of MAGEB16 through RNA interference can play the same role through the same or similar mechanism, therefore, those skilled in the art can synthesize RNA molecules capable of reducing the expression level of the MAGEB16 gene other than the sequences of the present application based on the existing technology, which are all within the scope of the present application.

[0131] For the MAGEB16 target expressed on the cell membrane surface discovered in the present application, in addition to the MAGEB16 antibody that can bind to it for regulation, or the nucleic acid molecule that reduces the expression of MAGEB16, other regulation methods known to those skilled in the art can also achieve the regulation of MAGEB16 on the surface of tumor cells, thereby inhibiting MAGEB16-positive tumors, for example, including but not limited to, gene therapy drugs for regulating the MAGEB16 gene; gene editing systems for knocking out or mutating the MAGEB16 gene in cells; nucleic acid molecules for introduction into cells; isolated MAGEB16 recombinant proteins; small molecule inhibitors of MAGEB16, etc.

[0132] Therefore, based on the discovery of the present application that MAGEB16 can be expressed on the cell membrane surface, those skilled in the art can also conceive, according to the existing technology, the use of the above-mentioned gene therapy drugs, gene editing systems, nucleic acid molecules, recombinant proteins, small molecule inhibitors, etc. to regulate MAGEB16 and thereby regulate the immune response and anti-tumor.

[0133] The present application also provides a pharmaceutical composition or a kit comprising an effective amount of the MAGEB16 modulator, or the antibody or antigen-binding fragment thereof, or the chimeric antigen receptor or T cell receptor, or the polynucleotide, or the vector, or the host cell, or the immune effector cell, or the antibody drug conjugate or polypeptide drug conjugate, or the nucleic acid molecule described above.

[0134] An effective amount refers to an amount of a pharmaceutical compound or composition that causes a measurable clinical, biological, or pharmacological change or response in a biomarker, cell, tissue, system, or patient.

[0135] In addition, the pharmaceutical composition can further comprise a pharmaceutically acceptable carrier or excipient. Specific examples of some substances that can be used as the pharmaceutically acceptable carrier or excipient are sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methyl cellulose, ethyl cellulose, and methyl cellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa oil; polyhydric alcohols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; coloring agents; flavoring agents; tabletting agents, stabilizers, antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffer solutions, etc. The dosage form of the pharmaceutical composition is not limited, and the appropriate carrier or excipient can be selected according to the dosage form.

[0136] In the detailed description of the present application, in the pharmaceutical composition, the antibody or antigen-binding fragment thereof can be a single effective ingredient, or can be combined with other one or more other active ingredients having therapeutic effects on tumors or regulating immune responses to form a combined preparation, and the efficacy and safety of each component do not conflict with each other. The content of each component in the combined preparation is usually a safe and effective amount, which can be adjusted based on the actual use situation (e.g., patient weight, application type, disease condition, severity).

[0137] The subject of administration of the pharmaceutical composition is preferably a mammal, such as but not limited to a human, a primate, livestock (such as a sheep, a cow, a horse, a donkey, a pig), a pet (such as a dog, a cat), a laboratory test animal (such as a mouse, a rabbit, a rat, a guinea pig, a hamster), or a captured wild animal (such as a fox, a deer); preferably, the subject is a primate; more preferably, the subject is a human.

[0138] In the detailed description of the present application, the kit can also include reagents required for flow cytometry or immunohistochemistry, which can be used to verify MAGEB16 expression using flow cytometry or immunohistochemistry, for predicting the therapeutic effect of MAGEB16 antibodies, antibody drug conjugates or polypeptide drug conjugates, MAGEB16 modulators, etc.

[0139] The present application also provides the use of the above-mentioned antibodies or antigen-binding fragments thereof in the preparation of a tumor or autoimmune disease diagnostic product or a companion diagnostic product, which is a companion diagnostic product of the MAGEB16 modulator in the above-mentioned use. Companion diagnostics is an in vitro diagnostic technology that guides drug regimens, and is usually developed together with target drugs. In the present application, by detecting MAGEB16, a patient population most likely to respond to drugs targeting MAGEB16 is screened, thereby improving treatment prognosis (efficacy, risk, etc.). The companion diagnostic product can include reagents required for ELISA, and MAGEB16 present in a biological sample is detected by ELISA, thereby screening suitable MAGEB16 modulators.

[0140] The present application also provides the use of the above-mentioned MAGEB16 modulators, the above-mentioned antibodies or antigen-binding fragments thereof, or the above-mentioned chimeric antigen receptors or T cell receptors, or the above-mentioned polynucleotides, or the above-mentioned vectors, or the above-mentioned host cells, or the above-mentioned antibody drug conjugates or polypeptide drug conjugates, or the above-mentioned nucleic acid molecules, or the above-mentioned pharmaceutical compositions or kits in the preparation of a product for treating autoimmune diseases or anti-tumor.

[0141] In the present application, the product can be a drug, a pharmaceutical composition, a reagent, a kit, etc.

[0142] In the present application, the cancer can be a solid tumor or a hematological tumor, for example, can be a MAGEB16 highly-expressed cancer such as ovarian cancer, breast cancer, lung cancer, colorectal cancer, melanoma, liver cancer, pancreatic cancer, sarcoma, nervous system tumor, leukemia, urothelial carcinoma, cervical cancer, prostate cancer, esophageal cancer, thymoma, squamous cell carcinoma, lymphoma, gastric cancer, testicular cancer, thyroid cancer, endometrial cancer, head and neck cancer, kidney cancer, adrenal cortex cancer, bile duct cancer, mesothelioma, pheochromocytoma, or paraganglioma, or other MAGEB16-related cancer.

[0143] In the present application, the autoimmune disease is selected from any one of systemic lupus erythematosus and lupus nephritis, rheumatoid arthritis, Sjogren's syndrome and related nephropathy, systemic sclerosis, mixed connective tissue disease, antiphospholipid syndrome, adult Still's disease, type 1 diabetes, Graves' disease, Hashimoto's thyroiditis, Addison's disease, celiac disease, autoimmune hepatitis, primary biliary cholangitis, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, Guillain-Barre syndrome, psoriasis, vitiligo, pemphigus, autoimmune hemolytic anemia, idiopathic thrombocytopenia purpura, autoimmune neutropenia, pernicious anemia, Goodpasture's syndrome, autoimmune ear disease, ankylosing spondylitis, arthritis, polyarteritis nodosa, granulomatosis polyangiitis, giant cell arteritis, Behcet's disease, relapsing polychondritis, IgG4-related disease, Vogt-Koyanagi-Harada syndrome, anti-glomerular basement membrane disease, Goodpasture's syndrome, ANCA-associated vasculitis renal damage, or anti-phospholipase A2 receptor antibody-associated membranous nephropathy.

[0144] The present application also provides a method for modulating immune response, which comprises administering to a patient an effective amount of the MAGEB16 modulator for use described above, or the antibody or antigen binding fragment thereof described above, or the chimeric antigen receptor or T cell receptor described above, or the antibody drug conjugate or polypeptide drug conjugate described above, or the nucleic acid molecule described above, or the pharmaceutical composition or kit described above.

[0145] The method for modulating immune response can also be combined with other treatment methods, and the treatment can be performed simultaneously or sequentially. Other methods for modulating immune response can be administering immunomodulators, such as microbial-derived drugs (e.g., bacillus Calmette-Guerin), human or animal immune system products (e.g., monoclonal antibodies, thymosin, transfer factor, interferon, interleukin, etc.), chemically synthesized drugs (e.g., levamisole, polyinosinic acid), traditional Chinese medicines (e.g., ginseng, astragalus, etc.), and others.

[0146] The present application also provides a method for treating cancer, which comprises administering to a patient an effective amount of the MAGEB16 modulator for use described above, or the antibody or antigen binding fragment thereof described above, or the chimeric antigen receptor or T cell receptor described above, or the antibody drug conjugate or polypeptide drug conjugate described above, or the nucleic acid molecule described above, or the pharmaceutical composition or kit described above.

[0147] The method for treating cancer can also be combined with other treatment methods, and the treatment can be performed simultaneously or sequentially. Other methods for treating tumors include, but are not limited to, surgical treatment, radiotherapy, chemotherapy, targeted therapy, immunotherapy, hormone therapy, stem cell transplantation.

[0148] The present application also provides a method for treating a tumor, which comprises administering to a patient an effective amount of the MAGEB16 modulator, or the antibody or antigen binding fragment thereof, or the chimeric antigen receptor or T cell receptor, or the antibody drug conjugate or polypeptide drug conjugate, or the nucleic acid molecule, or the pharmaceutical composition or kit for use according to the above use, which can also be combined with other therapeutic methods, administered simultaneously or sequentially.

[0149] The present application also provides a method for treating an autoimmune disease, which comprises administering to a patient an effective amount of the MAGEB16 modulator, or the antibody or antigen binding fragment thereof, or the chimeric antigen receptor or T cell receptor, or the antibody drug conjugate or polypeptide drug conjugate, or the nucleic acid molecule, or the pharmaceutical composition or kit for use according to the above use, which can also be combined with other therapeutic methods, administered simultaneously or sequentially.

[0150] In the present application, the subject to be administered with the MAGEB16 modulator, antibody or antigen binding fragment thereof, chimeric antigen receptor or T cell receptor, antibody drug conjugate or polypeptide drug conjugate, nucleic acid molecule, pharmaceutical composition or kit, product or method is a mammal. The subject to be administered with the method for treating a cancer is preferably a mammal, such as but not limited to a human, a primate, a livestock (such as a sheep, a cow, a horse, a donkey, a pig), a pet (such as a dog, a cat), a laboratory test animal (such as a mouse, a rabbit, a rat, a guinea pig, a hamster) or a captured wild animal (such as a fox, a deer); preferably, the subject is a primate; more preferably, the subject is a human.

[0151] The present application is illustrated by the following specific examples. Unless otherwise indicated, the experimental methods, detection methods, preparation methods disclosed in the present application all adopt the conventional techniques in the field of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields. These techniques have been well described in the existing literature, see Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (P. M. Wassarman and A. P. Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (P. B. Becker, ed.) Humana Press, Totowa, 1999, etc. Unless otherwise specified, the instruments, reagents, materials used in the examples can be obtained by conventional means.

[0152] Preparation and validation of antibodies that specifically bind to tumor cells

[0153] 1. Preparation of monoclonal antibodies that bind to tumor cells

[0154] (1) Immunization: 8-week-old male BALB / c mice were immunized by injecting tumor cell lysates of different origins at multiple sites on their backs and footpads. Specifically, melanoma A375, lung cancer NCI-H1299, ovarian cancer A2780, breast cancer MDA-MB-231, and liver cancer HuH-7 tumor cell lysates were used. The immunization procedure involved one injection every 21 days for a total of 5 immunizations, with only one cell lysate used per injection. Subsequently, spleen cells were collected and fused with SP2 / 0 (mouse myeloma cells).

[0155] (2) Primary screening: The tumor cell lysates were plated, and monoclonal antibodies were screened by enzyme-linked immunosorbent assay (ELISA). Wells with high OD values during primary screening were selected for further culture.

[0156] (3) Secondary screening: After two days, 100 μl of culture supernatant was added to the coated 96-well plate, and 2, 4, 8, and 16-fold dilutions were performed. The plate was incubated and the OD value was detected, and the clones were selected again. After subcloning and expansion, the cell supernatant was used to express and purify the protein.

[0157] 2. Purification of monoclonal antibodies using Protein A

[0158] The purification steps for monoclonal antibodies are as follows: The water, buffers, and ascites used were centrifuged at 13,000 RPM and 4°C for 15 min before use, and filtered with a 0.45 μm filter. Protein A beads were loaded into a suitable chromatography column, and equilibrated with 5 column volumes of equilibration buffer (20 mM Na2HPO4, 0.15 M NaCl, pH 7.0). The sample was added to the equilibrated Protein A beads, and the flow-through was collected. The column was washed with 10 column volumes of wash buffer (20 mM Na2HPO4, 0.15 M NaCl, pH 7.0), and the wash buffer was collected. The column was eluted with 10 column volumes of elution buffer (0.1 M glycine, pH 3.0), and the eluate was collected. The eluate was neutralized with 1 / 10 the volume of neutralization buffer (1 M Tris-HCl, pH 8.5). The samples obtained after purification (including the flow-through, wash, and elution fractions) and the original sample were analyzed by SDS-PAGE to assess the purification efficiency. The antibodies were stored at -80°C for future use. Through the above steps, antibody clones 1A5, 3F6, and 6F44 were obtained, which could bind to various tumor cell lysates. Immunoprecipitation combined with mass spectrometry was performed on the tumor cell lysates using the above antibodies, and it was identified that the common antigen of clones 1A5, 3F6, and 6F44 was MAGEB16 protein. Further flow cytometry staining was performed on mouse colon cancer CT26 and breast cancer 4T1, and clones 3F6 and 6F44 were selected, as shown in Figure 1. The specific operation method is the same as that of Example 2.3.

[0159] 3. Enzyme-linked immunosorbent assay (ELISA) to verify the binding of hybridoma antibodies to MAGEB16

[0160] To verify the binding of each hybridoma antibody to MAGEB16, direct binding of each antibody to MAGEB16 protein was confirmed by ELISA. ELISA-specific plates were used. First, the plates were coated with 100 μL of ELISA coating solution containing 5 μg / mL of human MAGEB16-His recombinant protein, and the negative control was coated with 100 μL of coating solution without MAGEB16 recombinant protein. The coating was performed overnight at 4°C. After washing with PBST, 100 μL of 5% BSA dissolved in PBS was used for blocking, and the blocking was performed at 37°C for 90 minutes. After washing with PBST, MAGEB16 mouse hybridoma antibodies were added, and the binding was performed at 37°C for 60 minutes. After washing with PBST, HRP-labeled specific anti-mouse Fc fragment antibodies (Invitrogen, USA) diluted with PBS were incubated and bound at 37°C for 30 minutes. After washing with PBST, color developing solution was prepared, 100 μL per well, and placed in an incubator for 15 minutes, and then a termination solution was added, and the reading was performed at 450 nm under an enzyme labeler. As shown in the ELISA results of FIG. 2A, 3F6 and 6F44 monoclonal antibodies can effectively bind to human MAGEB16.

[0161] The CDR1 / 2 / 3 sequences of the heavy chain of 6F44 are shown in SEQ ID NO: 11 / 12 / 13, and the CDR1 / 2 / 3 sequences of the light chain are shown in SEQ ID NO: 14 / 15 / 16; the CDR1 / 2 / 3 sequences of the heavy chain of 3F6 are shown in SEQ ID NO: 17 / 18 / 19, and the CDR1 / 2 / 3 sequences of the light chain are shown in SEQ ID NO: 20 / 21 / 22; as shown in Table 1 below.

[0162] The amino acid sequence of the variable region of the heavy chain of 6F44 is shown in SEQ ID NO: 1, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 2; the amino acid sequence of the variable region of the heavy chain of 3F6 is shown in SEQ ID NO: 3, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 4; as shown in Table 2 below.

[0163] Table 1 CDR region sequences of 6F44 and 3F6

[0164] Table 2 Variable region sequences of 6F44 and 3F6

[0165] 4. Flow cytometry (FACS) to detect the binding of hybridoma antibodies to human peripheral blood mononuclear cell (PBMC) surface antigens

[0166] The specific method is the same as Example 2.3. As shown in Figure 3, the 6F44 antibody does not significantly bind to PBMCs without breaking tumor cells, which indicates that the 6F44 antibody has no blood toxicity and has the potential to distinguish tumor and normal tissues.

[0167] Example 2: Murine hybridoma antibodies can specifically bind to human or mouse MAGEB16 protein

[0168] 1. Preparation of MAGEB16 mouse hybridoma antibodies

[0169] (1) Immunization: 8-week-old male BALB / c mice were injected with 50 μg of MAGEB16 protein (diluted with PBS) at multiple sites on the back and foot bottom, immunized once every 21 days, and immunized a total of 3 times. Subsequently, the spleen cells were collected, and the spleen cells and SP2 / 0 (mouse myeloma cells) were fused.

[0170] (2) Primary screening: human MAGEB16 protein was coated on plates, and monoclonal antibodies were screened by enzyme-linked immunosorbent assay (ELISA), and wells with high OD values were selected for further culture.

[0171] (3) Secondary screening: 100 μL of culture supernatant was taken after two days and added to the coated 96-well plate, and 2, 4, 8, and 16-fold dilutions were made, incubated, and the OD value was detected, and the clones were selected again. After subculturing and amplifying, the cell supernatant was taken to express and purify the protein.

[0172] (4) Protein A purification of monoclonal antibodies: the specific method is the same as Example 1.2.

[0173] 2. ELISA verification of the binding of each hybridoma antibody to MAGEB16

[0174] The specific method is the same as Example 1.3. Figure 2B is the ELISA experimental result. It is the ELISA result of the binding of five hybridoma antibodies to MAGEB16 protein. The experiment shows that the binding strength of each antibody to MAGEB16 is different. Among these hybridoma antibodies, the 27C12 and 48G7 antibodies bind to MAGEB16 protein more strongly.

[0175] The CDR1 / 2 / 3 sequences of the heavy chain of 27C12 are shown as SEQ ID NO: 23 / 24 / 25, and the CDR1 / 2 / 3 sequences of the light chain are shown as SEQ ID NO: 26 / 27 / 28; the CDR1 / 2 / 3 sequences of the heavy chain of 48G7 are shown as SEQ ID NO: 29 / 30 / 31, and the CDR1 / 2 / 3 sequences of the light chain are shown as SEQ ID NO: 32 / 33 / 34; as shown in Table 3 below.

[0176] 27C12 heavy chain variable region amino acid sequence is shown in SEQ ID NO: 5, light chain variable region amino acid sequence is shown in SEQ ID NO: 6; 48G7 heavy chain variable region amino acid sequence is shown in SEQ ID NO: 7, light chain variable region amino acid sequence is shown in SEQ ID NO: 8; as shown in Table 4 below.

[0177] Table 3 CDR region sequences of 27C12 and 48G7

[0178] Table 4 Variable region sequences of 27C12 and 48G7

[0179] 3. Flow cytometry (FACS) verification of binding of each hybridoma antibody to MAGEB16

[0180] To further verify the binding of each hybridoma antibody to MAGEB16, flow cytometry was used to confirm the direct binding of each antibody (27C12, 48G7, 3F6, 6F44) to MAGEB16 protein on the surface of tumor cells. The following four cell lines (ATCC, USA) were used: melanoma A375, lung cancer NCI-H1299, ovarian cancer A2780, breast cancer MDA-MB-231, and the number of cells was adjusted to 1 x 10 6 , and hybridoma antibodies or control IgG were added at a final concentration of 20 micrograms per milliliter, and incubated at 4 degrees for 45 minutes. Then the cells were resuspended and washed by adding staining buffer, and after centrifugation for 5 minutes, the supernatant was discarded. The centrifugation and washing were repeated. The anti-mouse Fc segment-Alexa Fluor 488 antibody (Invitrogen, CA, USA) was diluted with cell staining buffer and added to the cells, and incubated at 4 degrees for 30 minutes. The centrifugation and washing were repeated. Finally, an appropriate amount of cell staining buffer was added, and the MAGEB16 positive population was detected on a cell flow cytometer.

[0181] Without the need to break the tumor cells, this experiment showed that the binding of different antibodies to MAGEB16 varied in strength, and the results of this FACS experiment are shown in Figure 4, in which 6F44 is the strongest antibody, proving that MAGEB16 antibodies can bind to MAGEB16 positive melanoma cells, lung cancer cells, ovarian cancer cells, and breast cancer cells expressing MAGEB16 on the cell surface, indicating that the antibodies have an inhibitory effect on MAGEB16 positive tumors such as melanoma, lung cancer, ovarian cancer, and breast cancer.

[0182] Example 3 Humanized antibodies can specifically bind to MAGEB16

[0183] 1. Expression and purification of humanized monoclonal antibodies

[0184] The amino acid sequence of the humanized 6F44 monoclonal antibody was obtained by humanizing the 6F44 monoclonal antibody.

[0185] The humanized monoclonal antibody 6F44 heavy chain variable region sequence (SEQ ID NO:1) encoding DNA was codon-optimized for mammalian use by General Biotechnology (Anhui) Co., Ltd., and cloned into the pcDNA3.1-IgG1Fc expression vector to obtain plasmid 44F_5XKU_vH. The humanized monoclonal antibody 6F44 light chain variable region sequence (SEQ ID NO:2) encoding DNA was also codon-optimized for mammalian use and cloned into the pcDNA3.1-IgK expression vector to obtain plasmid 44F_5XKU_vL. Both plasmids were co-transfected into Expi293F cells to express the antibody. Expi293F cells were diluted to a density of 1.5 × 10⁶ cells / year. 6 / mL, cell density after overnight culture was approximately 3.2×10⁹ / mL. 6 / mL, diluted with culture medium to 3×10 6 / mL, for later use. Prepare a 25mL cell transfection system. ① Take 1.0 μg of plasmid DNA (the sum of the light and heavy chains, with a ratio of 1:1) to 1mL, add it to the culture medium, and mix well; ② Take 3.2mL of PEI and add it to the culture medium, mix well, and let it stand for 5min. ③ Add ① to ② and mix well. ④ After the PEI and plasmid DNA mixture has been incubated at room temperature for 15min, slowly add it to the cell culture flask while gently rotating the flask. ⑤ Place the culture flask in a 37℃, 8% CO2 orbital shaker for incubation. 24h after transfection, add 1mM sodium butyrate and continue culturing; collect the supernatant on day 6. Purify the humanized 6F44 monoclonal antibody with Protein A (method as in Example 1.2).

[0186] Table 5. Nucleotide sequence of the 6F44 variable region

[0187] 2. ELISA validation of the binding of different humanized antibodies against 6F44 to MAGEB16

[0188] The experimental method was the same as in Example 1.3, except that a specific anti-human Fc fragment antibody with an HRP tag was used for secondary antibody incubation. As shown in Figure 7A, different humanized antibodies (H24, H53, H55) of 6F44 all bound to MAGEB16, with H24 exhibiting the strongest binding affinity. The amino acid sequence of the heavy chain variable region of H24 is shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:10, as shown in Table 6 below.

[0189] Table 6 Variable region sequence of H24

[0190] 3. FACS to compare the affinity of H24 antibody or chimeric antibody to MAGEB16

[0191] To further verify the binding of H24 antibody to MAGEB16, FACS was used to compare the affinity of H24 antibody or chimeric antibody to MAGEB16 protein. Non-small cell lung cancer NCI-H1299 with high expression of MAGEB16 on cell membrane was used. Gradient-diluted antibodies were added respectively, and incubated at 4°C for 45 minutes. After washing, anti-human Fc segment-Alexa Fluor 488 antibody (Invitrogen, USA) was added and incubated at 4°C for 30 minutes. Other specific methods were the same as those in Example 2.3. As shown in FIG. 7B, both humanized antibody H24 and chimeric antibody had significant binding to MAGEB16, and the affinity was similar. Humanized antibody H24 had significant binding to MAGEB-positive non-small cell lung cancer NCI-H1299, indicating that it had the effect of inhibiting MAGEB16-positive tumors such as non-small cell lung cancer.

[0192] 4. FACS to verify the affinity of H24 antibody to MAGEB16 in different tumor cells

[0193] In different tumor cells, FACS was used to verify the direct binding of each antibody to MAGEB16 protein. MAGEB16-positive tumor cells with MAGEB16 expression on cell membrane were used, including melanoma A375, lung cancer NCI-H1299, ovarian cancer A2780, breast cancer MDA-MB-231, colon cancer RKO, liver cancer HuH-7, glioma U-87MG, pancreatic cancer PANC-1, osteosarcoma KHOS-240S, embryonic kidney cells HEK-293T and lymphoma Raji. Antibodies or control IgG with a final concentration of 20 micrograms per milliliter were added respectively, and other specific methods were the same as those in Example 3.3. As shown in FIG. 8, humanized antibody H24 had significant binding to MAGEB16 protein on the surface of melanoma A375, lung cancer NCI-H1299, ovarian cancer A2780, breast cancer MDA-MB-231, colon cancer RKO, liver cancer HuH-7, glioma U-87MG, pancreatic cancer PANC-1, osteosarcoma KHOS-240S, embryonic kidney cells HEK-293T and lymphoma Raji cells, indicating that humanized antibody H24 had the effect of inhibiting MAGEB-positive tumors such as melanoma, lung cancer, ovarian cancer, breast cancer, colon cancer, liver cancer, glioma, pancreatic cancer, osteosarcoma and lymphoma.

[0194] Example 4. Pharmacodynamic detection of MAGEB16 murine hybridoma antibody or humanized antibody in different tumor models

[0195] The cells to be inoculated were expanded, the logarithmically growing cells were digested and resuspended with PBS, and the NCG severely immunodeficient mice were prepared for inoculation. Human peripheral blood mononuclear cells (PBMCs) were also prepared. The cells were resuspended and mixed well, and MAGEB16-positive melanoma A375 cells and non-small cell lung cancer NCI-H1299 cells were injected at a dose of 1.8 x 10 6 cells per mouse, and the PBMCs were 6 x 10 5 cells per mouse. When the tumors grew to 50 mm 3 , they were divided into an Anti-IgG group and an Anti-MAGEB16 group (6F44), and the antibody was administered at a dose of 10 mg / kg, intraperitoneally every three days, for a total of 3-4 times. The long diameter and short diameter of the tumor were measured before each administration. Finally, the tumor was dissected, photographed, and weighed.

[0196] As shown in FIGS. 5 and 6, the results of the pharmacodynamic test show that the Anti-MAGEB16 group can significantly inhibit the growth of MAGEB16-positive tumors such as xenotransplanted melanoma and non-small cell lung cancer, indicating that antagonizing the MAGEB16 target can significantly inhibit the growth of MAGEB16-positive tumor cells in vivo.

[0197] 2. Anti-tumor pharmacodynamic test of MAGEB16 humanized antibody (H24)

[0198] The specific method was the same as in Example 4.1, and ovarian cancer A2780 cells were used, and the antibody group (H24) was administered at a dose of 10 mg / kg. FIG. 9 shows the results of the experiment, which prove that the MAGEB16 humanized antibody (H24) can significantly inhibit the growth of MAGEB16-positive tumors such as transplanted A2780 ovarian cancer, indicating that MAGEB16 can be an important target for inhibiting the growth of MAGEB16-positive tumors.

[0199] Example 5. MAGEB16 protein inhibits T cell function

[0200] 1. FACS detection of MAGEB16 protein regulation of T cells

[0201] To explore the inhibitory effect of MAGEB16 protein on T cell function, the effect of MAGEB16 protein on the expression of PD-1 on the surface of CD8+ T cells was analyzed by FACS. The sequence of MAGEB16 protein is as follows:

[0202] Freshly thawed human peripheral blood mononuclear cells (PBMCs) were stimulated for 48 h at 37 °C in 5% CO2 to activate T cells in RPMI-1640 complete medium (containing 10% FBS) containing 1 μg / mL Anti-CD3 / CD28 antibodies (Invitrogen, USA). Then the supernatant was discarded by centrifugation, and 0, 2.5, 5, 10 μg / mL recombinant MAGEB16 protein was added respectively to continue incubation for 12 h. After collecting the cells, they were washed twice with pre-cooled PBS, and then dead cells were excluded using APC / Cy7-labeled Live / Dead dye (Thermofisher, USA), white blood cells were labeled with APC-CD45 (BioLegend, USA), T cells were labeled with BV421-CD3 (BD Biosciences, USA), and CD8 + T cell subsets were distinguished using FITC-CD8 (eBioscience, USA), and PD-1 expression levels were detected by BV605-PD-1 (BioLegend, USA). As shown in FIG. 10, the proportion of PD-1 positive cells in the live CD45 + CD3 + CD8 + population was significantly increased in a concentration-dependent manner in the MAGEB16 protein treatment group, indicating that it can directly induce T cell exhaustion phenotype.

[0203] 2. ELISA detection of the effect of MAGEB16 protein on cytokines

[0204] The regulatory effect of MAGEB16 protein on cytokine secretion was evaluated by ELISA. After treating PBMCs according to the method of Example 5.1, the culture supernatant was collected, and the levels of different cytokines IL-10, IFN-γ and TNF-α were detected using an ELISA kit (Jointek, China). The specific operation is as follows: standard and sample were added to the pre-coated antibody 96-well plate, incubated at room temperature, then washed, and then biotinylated detection antibody, HRP conjugated streptavidin and TMB color developing substrate were added in turn, and the absorbance was measured at 450 nm wavelength after the reaction was terminated. The results shown in FIG. 10B show that MAGEB16 protein significantly promotes IL-10 secretion. In addition, as shown in FIG. 10C, the expression levels of pro-inflammatory factors IFN-γ and TNF-α were significantly decreased in the culture system containing 2.5 μg / mL MAGEB16 protein. These results show that MAGEB16 protein can significantly inhibit the function of T cells.

[0205] Example 6. Functional experiments of MAGEB16 antibody and RNA molecule

[0206] 1. Apoptosis experiment to determine the promotion of MAGEB16 antibody (H24) on PBMC cell killing tumor cells

[0207] The recovered PBMC cells were activated 48 hours in advance with 1 microgram per milliliter of Anti-CD3 / CD28 (Invitrogen, USA). The MAGEB16 positive tumor cells with cell membrane expressing MAGEB16: lung cancer NCI-H1299, breast cancer MDA-MB-231, liver cancer HuH-7, embryonic kidney cells HEK-293T, glioma U-87MG, melanoma A375 were counted, and the PBMC cells were adjusted to 1 x 10 6 / mL, respectively, 500 μL was added to the 24-well plate, and different concentrations of monoclonal antibodies were added, mixed and placed in the incubator for 18 hours of incubation. The cells were resuspended and washed with cell staining buffer, and FACS detection was performed using an apoptosis detection kit. As shown in Figure 11, compared with the control, the H24 humanized antibody binding to MAGEB16 protein can promote the killing of PBMC on lung cancer NCI-H1299, breast cancer MDA-MB-231, liver cancer HuH-7, embryonic kidney cells HEK-293T, glioma U-87MG, melanoma A375 and other tumor cells, indicating that the H24 humanized antibody binding to MAGEB16 protein has the effect of inhibiting MAGEB16 positive tumors such as lung cancer, breast cancer, liver cancer, glioma, melanoma, etc.

[0208] 2. siRNA can knock down MAGEB16 and promote PBMC killing tumor cells

[0209] GP-transfect-Mate transfection reagent (Gibima, China) was used to transfect MAGEB16 siRNA in different tumor cells. The sequence is shown in Table 7 as follows:

[0210] Table 7 Sequence of siRNA of MAGEB16

[0211] The cell strains used were MAGEB16-positive tumor cells with cell membrane expressing MAGEB16: breast cancer MDA-MB-231, liver cancer HuH-7, pancreatic cancer PANC-1, and bladder cancer UM-UC-3. The recovered PBMC cells were activated with 1 microgram per milliliter of Anti-CD3 / CD28. After 48 hours, the PBMC cells were co-cultured with the tumor cells at a cell number of 1:1 for 18 hours. The cells were resuspended and washed with cell staining buffer, and FACS detection was performed using an apoptosis detection kit. As shown in FIG. 12, compared with the control (NC group), knockdown of MAGEB16 can significantly promote the killing of PBMC on MAGEB16-positive tumor cells with cell membrane expressing MAGEB16, such as breast cancer MDA-MB-231, liver cancer HuH-7, pancreatic cancer PANC-1, and bladder cancer UM-UC-3, indicating that knockdown of MAGEB16 has an inhibitory effect on MAGEB16-positive tumors such as breast cancer, liver cancer, pancreatic cancer, and bladder cancer.

[0212] 3. siRNA can knock down HLA-A but cannot affect the expression of MAGEB16 on the cell membrane

[0213] GP-transfect-Mate transfection reagent (Gibco, China) was used to transfect siRNA of HLA-A in different tumor cells. The sequence is shown in Table 8 below:

[0214] Table 8: Sequence of siRNA of HLA-A

[0215] The cell strains used were lung cancer NCI-H1299, leukemia M-07e, bladder cancer UM-UC-3, and cervical cancer Hela. After 48 hours, 20 micrograms per milliliter of H24 antibody or control IgG was added, and incubation was performed at 4 degrees for 45 minutes. After washing, anti-human Fc segment-Alexa Fluor 488 antibody was added, and flow cytometry detection was performed after incubation at 4 degrees for 30 minutes. As shown in FIG. 13, siRNA can knock down HLA-A but cannot affect the expression of MAGEB16 on the cell membrane, suggesting that the MAGEB16 protein itself is expressed on the cell membrane surface of MAGEB16-positive tumor cells such as lung cancer NCI-H1299, leukemia M-07e, bladder cancer UM-UC-3, and cervical cancer Hela, and is not transported to the cell membrane through the antigen presentation pathway.

[0216] 4. ADCC effect detection

[0217] NCI-H1299 target cells were plated and gradient dilution of MAGEB16 humanized antibody was added, negative control was IgG4 antibody without ADCC effect, antibody concentration was 300, 75, 18.75, 4.688, 1.172, 0.293, 0.073 and 0.018 microgram per milliliter. Then effector cells Jurkat-CD16a (Sino Biological, China) were added, incubated at 37℃ for 5 hours, and finally ONE-Glo TM Luciferase reagent (Promega, USA) was added, color development was performed for 10 minutes, and reading was performed on a microplate reader. The results are shown in Figure 14, which show that MAGEB16 humanized antibody H24 has ADCC effect.

[0218] Example 7. Preparation of antibody drug conjugate and affinity determination

[0219] 1. Preparation of antibody drug conjugate

[0220] A conjugation solution was prepared, which had the following components: 25 mM Na2B4O7, 25 mM NaCl, 1 mM DPTA, and the pH was adjusted to 7.4; 1 mM TCEP was prepared with pure water; and a 10 mM Mc-Vc-PAB-MMAE solution was prepared with dimethyl sulfoxide (DMSO). During conjugation, MAGEB16 humanized antibody H24 was first exchanged from PBS to the conjugation solution by ultrafiltration, and the antibody concentration was adjusted to 2.5 milligrams per milliliter after ultrafiltration. 1 mM reducing agent TCEP was added to the antibody solution, the molar ratio of TCEP to antibody was 3:1, and incubation was performed at room temperature for 2 hours. Then Mc-Vc-PAB-MMAE solution (diluted with DMSO) was added, the molar ratio of Mc-Vc-PAB-MMAE to antibody was 7:1, and incubation was performed at room temperature for 2 hours. The product was exchanged to PBS by ultrafiltration to remove unreacted Mc-Vc-PAB-MMAE free substance. Then the drug-antibody ratio (DAR) of MAGEB16 humanized antibody drug conjugate (H24-Mc-Vc-PAB-MMAE, i.e. H24-ADC) was analyzed by hydrophobic high performance liquid chromatography, and the average DAR was 4. In addition, a conjugate of human IgG1 and Mc-Vc-PAB-MMAE (control IgG1-Mc-Vc-PAB-MMAE, i.e. control IgG1-ADC) was also prepared, and the average DAR was 4.

[0221] 2. Affinity comparison of MAGEB16 humanized antibody H24 and its drug conjugate

[0222] MAGEB16 positive colon cancer RKO cells expressing MAGEB16 on cell membrane were fixed with formaldehyde at room temperature for 15 minutes, then H24 antibody and H24-ADC (diluted from 40 micrograms per milliliter to 0.0098 micrograms per milliliter according to a 4-fold concentration gradient) were added, and after incubation on ice for 60 minutes, the cells were washed twice, diluted with cell staining buffer, and resuspended, and incubated on ice for 30 minutes, and then detected by flow cytometry. As shown in Figure 15, the relative geometric mean fluorescence intensity represents the percentage of the geometric mean fluorescence intensity under each antibody concentration staining relative to the geometric mean fluorescence intensity under the highest antibody concentration staining. The results show that both H24 antibody and H24-ADC have strong binding affinity to MAGEB16 positive RKO tumor cells expressing MAGEB16 on cell membrane, and the EC50 values are 1.560 and 1.305 micrograms per milliliter, respectively, indicating that the antibody drug conjugate produced in this conjugation mode does not reduce the affinity of the antigen binding site, indicating that H24 antibody and H24-ADC have the effect of inhibiting MAGEB16 positive tumors such as colon cancer.

[0223] Example 8. Endocytosis experiment of MAGEB16 humanized antibody drug conjugate

[0224] MAGEB16 positive breast cancer MDA-MB-231 cells expressing MAGEB16 on cell membrane were plated at 1x105 cells per well in a 20 mm confocal culture dish. 10 micrograms per milliliter of H24-ADC was added to each well, and incubated on ice for 1 hour. After washing with pre-cooled PBS, complete medium was added, and after different time points, the culture was taken out from the incubator and placed on ice to terminate endocytosis. PBS was washed twice, 4% paraformaldehyde was added to each well for room temperature fixation for 30 minutes, then 0.3% Triton and 3% BSA were added to the transmembrane blocking solution, and incubated at room temperature for 1 hour. Then the corresponding organelle marker antibody (rabbit EEA1 antibody for early endosome marker; rabbit RAB7 antibody for late endosome marker; rabbit LAMP1 antibody for lysosome marker; rabbit RAB11 antibody for recycling endosome marker) diluted with transmembrane blocking solution was added, and incubated at 4 degrees overnight. Then, different fluorescently labeled secondary antibodies (anti-human 488 and anti-rabbit 594) were added to each well, and incubated at room temperature for 2 hours. Finally, DAPI was stained for 5 minutes, and then placed under a confocal microscope for imaging. The results are shown in Figure 16. The results show that H24-ADC has obvious co-localization with intracellular organelles, indicating that the MAGEB16 humanized antibody drug conjugate provided by the application has good endocytosis effect, can be efficiently delivered to the lysosome of target cells, and can play a role in inhibiting MAGEB16 positive tumors such as breast cancer.

[0225] Example 9. In vitro cytotoxic activity and cell cycle arrest experiment of MAGEB16 humanized antibody drug conjugate

[0226] Different MAGEB16 positive cell colon cancer RKO, breast cancer MDA-MB-231 and melanoma A2780 with different MAGEB16 cell membrane surface expression levels were inoculated in 96-well plates and incubated in a cell incubator overnight. H24 antibody, H24-ADC, control IgG1-ADC were added to the microplate with a two-fold concentration gradient dilution starting from 10 micrograms per milliliter. After 5 days of incubation in a cell incubator, CCK8 detection solution (APExBIO, USA) was added and the absorbance at 450 nm was detected. The results are shown in Figure 17. The results show that the H24 antibody has no obvious killing effect on cells, and the control IgG1-ADC shows weak killing effect at high concentration, but the H24-ADC shows significant killing activity at low concentration, with IC 50 0.218, 0.681 and 1.084 micrograms per milliliter, respectively, indicating that the MAGEB16 humanized antibody drug conjugate has an inhibitory effect on MAGEB16 positive tumors such as colon cancer, breast cancer, melanoma, etc. Different killing effects on different cells are affected by the amount of MAGEB16 expression and also related to the sensitivity of different cells to small molecule toxin MMAE.

[0227] 2. Cell cycle arrest experiment of MAGEB16 humanized antibody drug conjugate

[0228] Different concentrations of H24-ADC and control IgG1-ADC were added to MAGEB16 positive colon cancer RKO cells expressing MAGEB16 on the cell membrane surface in different treatment wells, and placed in a cell incubator for culture. Cell cycle detection was performed according to the instructions of the cell cycle detection kit (Link Biotech, China). As shown in Figure 18, at 12 hours, higher concentrations of H24-ADC can block the cell cycle at the G2 phase, showing an increase in the proportion of G2 phase cells. At 24 hours, low concentrations of H24-ADC can effectively block the cell cycle at the G2 phase. At 48 hours, the cells blocked in the division cycle turned to apoptosis, indicating a significant increase in the proportion of subG1 peak of apoptotic cells; compared with H24-ADC, the ability of control IgG1-ADC at the same concentration to block the cell cycle and induce cell apoptosis was significantly weakened.

[0229] Example 10. Pharmacodynamic detection of MAGEB16 humanized antibody drug conjugate in different subcutaneous xenograft models

[0230] 1. MAGEB16 humanized antibody drug conjugate on human colorectal cancer cell line RKO in BALB / c Nude mice subcutaneous transplanted tumor efficacy

[0231] MAGEB16 positive colorectal cancer RKO cells expressing MAGEB16 on the surface of cell membrane were subcutaneously inoculated in BALB / c Nude mice (5x10 6 individuals), and when the tumor grew to 100mm 3 , the PBS group, the control IgG1-ADC group (1 mg / kg) and the H24-ADC group (1 mg / kg) were divided, and the drug was given once every 3 days, all intraperitoneally, a total of four times. At the same time of administration, the long diameter and short diameter of the tumor were measured. Finally, the tumor was dissected, photographed and weighed, and the results are shown in Figure 19. The experiment proved that compared with the PBS and control IgG1-ADC groups, the same dose of H24-ADC had a significant inhibitory effect on RKO transplanted colorectal cancer and other MAGEB16 positive tumors.

[0232] 2. MAGEB16 humanized antibody drug conjugate on mouse melanoma B16-F10 in C57 mice subcutaneous transplanted tumor efficacy

[0233] MAGEB16 positive melanoma B16-F10 cells expressing MAGEB16 on the surface of cell membrane were subcutaneously inoculated in C57BL / 6J mice (3x10 5 individuals), and when the tumor grew to 20-50mm 3 , the control IgG1-ADC group (3 mg / kg) and the H24-ADC group (3 mg / kg) were divided, and the drug was given once every 2 days, a total of four times. Other specific methods are the same as in Example 10.1. As shown in Figure 20, compared with the control group, H24-ADC had a significant inhibitory effect on B16-F10 transplanted melanoma and other MAGEB16 positive tumors.

[0234] Example 11. Expression of MAGEB16 in different tumors

[0235] 1. Analysis of MAGEB16 expression in different tumors using TCGA database

[0236] MAGEB16 expression in different tumors was analyzed by TCGA database. The tumors include: BLCA (bladder urothelial carcinoma), BRCA (breast invasive carcinoma), ESCA (esophageal carcinoma), HNSC (head and neck squamous cell carcinoma), KICH (kidney renal clear cell carcinoma), LIHC (liver hepatocellular carcinoma), LUAD (lung adenocarcinoma), LUSC (lung squamous cell carcinoma), SKCM (skin cutaneous melanoma) and STAD (stomach adenocarcinoma). The results showed that MAGEB16 was expressed at a higher level in tumor tissues than in paracancer tissues, as shown in FIG. 19A, indicating that bladder urothelial carcinoma, breast invasive carcinoma, esophageal carcinoma, head and neck squamous cell carcinoma, kidney renal clear cell carcinoma, liver hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, skin cutaneous melanoma, and stomach adenocarcinoma are all MAGEB16-positive tumors (OV, ovarian cancer; BRCA, Breast Invasive Carcinoma; CSEC, Cervical squamous cell carcinoma and endocervical adenocarcinoma; PRAD, Prostate adenocarcinoma; LUAD, Lung adenocarcinoma; STES, Stomach and Esophageal carcinoma; LSCC, laryngeal squamous cell cancer; BLCA, Bladder Urothelial Carcinoma; PAAD, Pancreatic adenocarcinoma; SARC, Sarcoma; SKCM, Skin Cutaneous Melanoma; NHL, non-Hodgkin lymphoma).

[0237] 2. Immunohistochemistry was performed on tissues specifically using antibodies against MAGEB16 (3F6 hybridoma antibody)

[0238] The experimental procedure is as follows:

[0239] 1) Reagent preparation

[0240] Inactivated enzyme reagent: 3% H2O2-methanol solution, 1 mL of 30% H2O2 was added to 9 mL of methanol

[0241] PBST washing solution: 1000 mL of pH 7.4 PBS + 3 mL of Triton mixed

[0242] 1% hydrochloric acid-ethanol solution: 1 mL of hydrochloric acid was dissolved in 99 mL of ethanol

[0243] Antigen renaturation agent pH 6.0 citric acid buffer: 810 mL of 0.1 M citric acid solution + 190 mL of 0.1 M trisodium citrate solution.

[0244] 2) Baking: 60°C oven for 30 minutes.

[0245] 3) Dewaxing and rehydrating: two jars of xylene for 10 minutes each, two jars of absolute ethanol for 7 minutes each, one jar of 95% ethanol for 5 minutes, one jar of 75% ethanol for 5 minutes, one jar of 50% ethanol for 5 minutes, and ddH2O for 5 minutes, and washing for three times.

[0246] 4) Antigen retrieval using citrate high temperature and high pressure method, and washing.

[0247] 5) Inactivation of enzyme: 50 μL of inactivation enzyme reagent was added to each slice, and the slices were treated at room temperature for 15 minutes in the dark. After washing, 50 μL of PBST blocking solution was added, and the slices were blocked at room temperature for 30 minutes in a wet box.

[0248] 6) Addition of primary antibody: the blocking solution was discarded, and MAGEB16 primary antibody (mouse 3F6 hybridoma antibody, final concentration 5 μg / mL) was added to each slice, and the slices were incubated at 4°C overnight in a wet box.

[0249] 7) Addition of enzyme-labeled secondary antibody: HRP-labeled anti-mouse IgG was added to each slice, and the slices were incubated at 37°C for 30 minutes.

[0250] 8) Color development (DAB method): after washing, color developing solution was added, and the slices were developed in a wet box for 5 minutes. The color development reaction was terminated using distilled water.

[0251] 9) Counterstaining: 50 μL of hematoxylin cell staining solution was added to each slice, and the slices were stained for 10 minutes.

[0252] 10) Bleaching and returning to blue: 1% hydrochloric acid-ethanol was added, and the slices were quickly placed in ddH2O within 3 seconds to terminate the reaction, and then the slices were placed in PBST for 15 minutes to return to blue.

[0253] 11) Mounting: the slices were immersed in 75% ethanol and absolute ethanol for 2 minutes, respectively, and then were immersed in xylene, and neutral balsam was added to the slices, and a cover glass was added.

[0254] As shown in FIGS. 21B and 21C, the slices used in the figures were from different cancer types, and the slices were from Zhongshan Hospital Affiliated to Fudan University. The results of immunohistochemical staining using the antibody specific to MAGEB16 showed that MAGEB16 was positively expressed in various tumor tissues, such as ovarian cancer, breast cancer, cervical cancer, prostate cancer, lung cancer, esophageal cancer, thymoma, colorectal cancer, squamous cell carcinoma, urothelial carcinoma, pancreatic cancer, sarcoma, melanoma, lymphoma, gastric cancer, and liver cancer.

[0255] The above embodiments are only illustrative of the principles of the present application and its effects, and are not intended to limit the present application. Any modification or change made by any person skilled in the art without departing from the spirit and scope of the present application shall be covered by the claims of the present application.

Claims

1. Use of a MAGEB16 modulator in the preparation of a product for treating autoimmune diseases or anti-tumor.

2. Use according to claim 1, characterized in that, The MAGEB16 modulator is selected from any one or more of the following: 1) a gene therapy drug overexpressing MAGEB16; 2) an isolated MAGEB16 recombinant protein; 3) a nucleic acid molecule that reduces or increases the expression level of MAGEB16; 4) an antibody or antigen-binding fragment thereof, or a chimeric antigen receptor thereof, or a T cell receptor thereof, capable of binding to the surface of a tumor cell; preferably, the antibody is a MAGEB16 antibody, capable of binding to MAGEB16 on the surface of a tumor cell; 5) an antibody drug conjugate or polypeptide drug conjugate capable of binding to the surface of a tumor cell; preferably, the antibody drug conjugate or polypeptide drug conjugate is capable of binding to MAGEB16 on the surface of a tumor cell; 6) a small molecule inhibitor of MAGEB16.

3. Use according to claim 2, characterized in that, The nucleic acid molecule is selected from sgRNA, siRNA, shRNA, miRNA, antisense RNA, IncRNA, aptamer; preferably, the sequence of the nucleic acid molecule comprises any one or more of SEQ ID NOs: 35-40; more preferably, the nucleic acid molecule is siRNA, and more preferably, the sequence of the sense strand of the siRNA comprises any one of SEQ ID NOs: 35, 37, 39, and the sequence of the antisense strand comprises any one of SEQ ID NOs: 36, 38, 40; more preferably, the sequence of the sense strand of the siRNA is as set forth in SEQ ID NO: 35, and the sequence of the antisense strand is as set forth in SEQ ID NO: 36; or, the sequence of the sense strand of the siRNA is as set forth in SEQ ID NO: 37, and the sequence of the antisense strand is as set forth in SEQ ID NO: 38; or, the sequence of the sense strand of the siRNA is as set forth in SEQ ID NO: 39, and the sequence of the antisense strand is as set forth in SEQ ID NO: 40; and / or, the MAGEB16 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, and the MAGEB16 antibody or antigen-binding fragment thereof comprises HCDR1 as set forth in any one of SEQ ID NOs: 11, 17, 23, 29, HCDR2 as set forth in any one of SEQ ID NOs: 12, 18, 24, 30, HCDR3 as set forth in any one of SEQ ID NOs: 13, 19, 25, 31, LCDR1 as set forth in any one of SEQ ID NOs: 14, 20, 26, 32, LCDR2 as set forth in any one of SEQ ID NOs: 15, 21, 27, 33, and LCDR3 as set forth in any one of SEQ ID NOs: 16, 22, 28, 34; Preferably, the MAGEB16 antibody or antigen-binding fragment thereof has any one of the following characteristics: 1) the MAGEB16 antibody or antigen-binding fragment thereof comprises HCDR1-3 as set forth in SEQ ID NOs: 11-13; LCDR1-3 as set forth in SEQ ID NOs: 14-16; 2) the MAGEB16 antibody or antigen-binding fragment thereof comprises HCDR1-3 as set forth in SEQ ID NOs: 17-19; LCDR1-3 as set forth in SEQ ID NOs: 20-22; 3) the MAGEB16 antibody or antigen-binding fragment thereof comprises HCDR1-3 as set forth in SEQ ID NOs: 23-25; LCDR1-3 as set forth in SEQ ID NOs: 26-28; 4) the MAGEB16 antibody or antigen-binding fragment thereof comprises HCDR1-3 as set forth in SEQ ID NOs: 29-31; LCDR1-3 as set forth in SEQ ID NOs: 32-34; More preferably, the heavy chain variable region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 9; and the light chain variable region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 2, 4, 6, 8, 10; More preferably, the MAGEB16 antibody or antigen-binding fragment thereof has any one of the following characteristics: 1) the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 1, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 2; 2) the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 3, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 4; 3) the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 5, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 6; 4) the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 7, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 8; 5) the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 9, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO:

10.

4. Use according to claim 1, characterized in that, The tumor is selected from any one of ovarian cancer, breast cancer, lung cancer, colorectal cancer, melanoma, liver cancer, pancreatic cancer, sarcoma, nervous system tumor, leukemia, urothelial carcinoma, cervical cancer, prostate cancer, esophageal cancer, thymoma, squamous cell carcinoma, lymphoma, gastric cancer, testicular cancer, thyroid cancer, endometrial cancer, head and neck cancer, kidney cancer, adrenal cortex cancer, cholangiocarcinoma, mesothelioma, pheochromocytoma, paraganglioma. and / or the autoimmune disease is selected from any one of systemic lupus erythematosus and lupus nephritis, rheumatoid arthritis, Sjogren's syndrome and associated renal disease, systemic sclerosis, mixed connective tissue disease, antiphospholipid syndrome, adult onset still's disease, type 1 diabetes mellitus, Graves' disease, Hashimoto's thyroiditis, Addison's disease, celiac disease, autoimmune hepatitis, primary biliary cholangitis, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, Guillain-Barre syndrome, psoriasis, vitiligo, pemphigus, autoimmune hemolytic anemia, idiopathic thrombocytopenia purpura, autoimmune neutropenia, pernicious anemia, Goodpasture's syndrome, autoimmune ear disease, ankylosing spondylitis, arthritis, polyarteritis nodosa, granulomatosis with polyangiitis, giant cell arteritis, Behcet's disease, relapsing polychondritis, IgG4-related disease, Vogt-Koyanagi-Harada syndrome, anti-glomerular basement membrane disease, Goodpasture's syndrome, ANCA-associated vasculitis renal damage, and anti-phospholipase A2 receptor antibody-associated membranous nephropathy.

5. Use according to claim 1, characterized in that, The MAGEB16 modulator increases or decreases the level of MAGEB16 in the body, promotes or inhibits the function of MAGEB16 expressed on the cell surface, or increases or decreases the level of MAGEB16 expressed on the cell surface, preferably the cell is a tumor cell.

6. An antibody or antigen-binding fragment thereof that binds to a tumor cell surface, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the antibody or antigen-binding fragment thereof comprising HCDR1 as set forth in any one of SEQ ID NOs: 11, 17, 23, 29, HCDR2 as set forth in any one of SEQ ID NOs: 12, 18, 24, 30, HCDR3 as set forth in any one of SEQ ID NOs: 13, 19, 25, 31, LCDR1 as set forth in any one of SEQ ID NOs: 14, 20, 26, 32, LCDR2 as set forth in any one of SEQ ID NOs: 15, 21, 27, 33, and LCDR3 as set forth in any one of SEQ ID NOs: 16, 22, 28, 34.

7. The antibody or antigen-binding fragment thereof of claim 6, wherein, The antibody or antigen-binding fragment thereof is a MAGEB16 antibody or antigen-binding fragment thereof having any one of the following characteristics: 1) the MAGEB16 antibody or antigen-binding fragment thereof comprises HCDR1-3 as set forth in SEQ ID NOs: 11-13, and LCDR1-3 as set forth in SEQ ID NOs: 14-16; 2) the MAGEB16 antibody or antigen-binding fragment thereof comprises HCDR1-3 as set forth in SEQ ID NOs: 17-19, and LCDR1-3 as set forth in SEQ ID NOs: 20-22; 3) the MAGEB16 antibody or antigen-binding fragment thereof comprises HCDR1-3 as set forth in SEQ ID NOs: 23-25; LCDR1-3 as set forth in SEQ ID NOs: 26-28; 4) the MAGEB16 antibody or antigen-binding fragment thereof comprises HCDR1-3 as set forth in SEQ ID NOs: 29-31; LCDR1-3 as set forth in SEQ ID NOs: 32-34; and / or, the MAGEB16 antibody is selected from any one of a polyclonal antibody, a monoclonal antibody, a single chain antibody, an antigen binding domain, a bispecific antibody, a multispecific antibody, or an antigen binding portion in a chimeric antigen receptor; and / or, the antigen-binding fragment is selected from any one of a scFv, a BsFv, a dsFv, a (dsFv)2, a Fab, a Fab', a F(ab')2, or a Fv; Preferably, the heavy chain variable region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 9; and the light chain variable region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 2, 4, 6, 8, 10; More preferably, the MAGEB16 antibody or antigen-binding fragment thereof has any one of the following characteristics: 1) the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 1, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 2; 2) the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 3, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 4; 3) the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 5, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 6; 4) the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 7, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 8; 5) the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 9, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO:

10.

8. A chimeric antigen receptor or T cell receptor comprising the antibody or antigen-binding fragment thereof of any one of claims 6-7.

9. An immunoconjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 6-7.

10. A polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 6-7, or the chimeric antigen receptor or T cell receptor of claim 8.

11. A vector comprising the polynucleotide of claim 10.

12. A host cell comprising the vector of claim 11 or having integrated into its genome the polynucleotide of claim 10; Preferably, the host cell is an immune effector cell; More preferably, the host cell is a T cell.

13. A method of producing an antibody or antigen-binding fragment thereof, chimeric antigen receptor, or T cell receptor, comprising culturing a host cell containing the vector of claim 11 or a host cell into which a polynucleotide of claim 10 has been introduced exogenously under conditions permitting expression of the antibody or antigen-binding fragment thereof, chimeric antigen receptor, or T cell receptor, and recovering the antibody or antigen-binding fragment thereof, chimeric antigen receptor, or T cell receptor from the cultured host cell culture.

14. An antibody drug conjugate or polypeptide drug conjugate, comprising the antibody or antigen-binding fragment thereof of any one of claims 6-7 and a therapeutic agent.

15. The antibody drug conjugate or polypeptide drug conjugate of claim 14, wherein, The antibody drug conjugate or polypeptide drug conjugate further comprises a linker connecting the antibody or antigen-binding fragment thereof and the therapeutic agent; Preferably, the antibody drug conjugate or polypeptide drug conjugate further comprises any one or more of the following features: 1) the general structure of the antibody drug conjugate or polypeptide drug conjugate is A-(L-U)n, wherein: A is the antibody or antigen-binding fragment thereof; U is the therapeutic agent; L is the linker; and n is an integer selected from 1 to 8; 2) the linker is connected to an amino acid residue or a thiol residue on the antibody or antigen-binding fragment thereof; 3) the linker comprises a cleavable linker or a non-cleavable linker, and the cleavable linker comprises a peptide unit; 4) the therapeutic agent is selected from a cytotoxic molecule, or an immune enhancer, or a radioisotope; More preferably, the antibody drug conjugate or polypeptide drug conjugate further comprises any one or more of the following features: 1) the peptide unit comprises 2-20 amino acids; more preferably, the peptide unit is selected from any one or more of a combination of -valine-citrulline-, -glycine-glycine-phenylalanine-glycine-, -valine-alanine-; 2) the cytotoxic molecule is selected from a tubulin inhibitor or a DNA damaging agent; more preferably, the tubulin inhibitor is selected from a dolastatin or an auristatin derivative MMAE, MMAF, or a maytansinoid and a maytansinoid derivative DM1, DM4, and the DNA damaging agent is selected from a duocarmycin, or a calicheamicin, or an anthramycin drug PBD, or a camptothecin and a camptothecin derivative SN-38, Dxd.

16. A nucleic acid molecule for reducing the expression level of MAGEB16, the nucleic acid molecule being an siRNA comprising a nucleotide sequence as set forth in any one or more of SEQ ID NOs: 35-40, preferably, the sequence of the sense strand of the siRNA comprises a sequence as set forth in any one of SEQ ID NOs: 35, 37, 39, and the sequence of the antisense strand comprises a sequence as set forth in any one of SEQ ID NOs: 36, 38, 40.

17. A pharmaceutical composition or a kit comprising an effective amount of the MAGEB16 modulator for use according to any one of claims 1 to 5, or the antibody or antigen-binding fragment thereof according to any one of claims 6 to 7, or the chimeric antigen receptor or T cell receptor according to claim 8, or the immunoconjugate according to claim 9, or the polynucleotide according to claim 10, or the vector according to claim 11, or the host cell according to claim 12, or the antibody-drug conjugate or polypeptide-drug conjugate according to any one of claims 14 to 15, or the nucleic acid molecule according to claim 16. Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient. Preferably, the kit further comprises reagents required for flow cytometry or immunohistochemistry.

18. Use of the antibody or antigen-binding fragment thereof according to any one of claims 6 to 7 in the manufacture of a diagnostic product for a tumor or autoimmune disease, or a companion diagnostic product for the MAGEB16 modulator for use according to any one of claims 1 to 5.

19. Use of the antibody or antigen-binding fragment thereof according to any one of claims 6 to 7, or the chimeric antigen receptor or T cell receptor according to claim 8, or the immunoconjugate according to claim 9, or the polynucleotide according to claim 10, or the vector according to claim 11, or the host cell according to claim 12, or the antibody-drug conjugate or polypeptide-drug conjugate according to any one of claims 14 to 15, or the nucleic acid molecule according to claim 16 in the manufacture of a product for the treatment of an autoimmune disease or anti-tumor.

20. Use according to claim 19, characterized in that, The tumor is selected from any one of ovarian cancer, breast cancer, lung cancer, colorectal cancer, melanoma, liver cancer, pancreatic cancer, sarcoma, nervous system tumor, leukemia, urothelial cancer, cervical cancer, prostate cancer, esophageal cancer, thymoma, squamous cell carcinoma, lymphoma, gastric cancer, testicular cancer, thyroid cancer, endometrial cancer, head and neck cancer, renal cancer, adrenal cortex cancer, cholangiocarcinoma, mesothelioma, pheochromocytoma, paraganglioma. and / or, the autoimmune disease is selected from any one of systemic lupus erythematosus and lupus nephritis, rheumatoid arthritis, Sjogren's syndrome and associated renal disease, systemic sclerosis, mixed connective tissue disease, antiphospholipid syndrome, adult onset still's disease, type 1 diabetes mellitus, Graves' disease, Hashimoto's thyroiditis, Addison's disease, celiac disease, autoimmune hepatitis, primary biliary cholangitis, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, Guillain-Barre syndrome, psoriasis, vitiligo, pemphigus, autoimmune hemolytic anemia, idiopathic thrombocytopenia purpura, autoimmune neutropenia, pernicious anemia, Goodpasture's syndrome, autoimmune ear disease, ankylosing spondylitis, arthritis, polyarteritis nodosa, granulomatosis with polyangiitis, giant cell arteritis, Behcet's disease, relapsing polychondritis, IgG4-related disease, Vogt-Koyanagi-Harada syndrome, anti-glomerular basement membrane disease, Goodpasture's syndrome, ANCA-associated vasculitis renal damage, and anti-phospholipase A2 receptor antibody-associated membranous nephropathy.

21. Use of an antibody specifically recognizing MAGEB16 for marking the border of primary cancerous tissue and paracancerous tissue, cancerous cells metastasized to lymph nodes and normal lymphatic tissue, cancerous cells with distant metastasis and normal tissue of the metastatic organ, and cancerous tissue live cells in other biological specimens. Preferably, the cancer is a MAGEB16 -related cancer. Preferably, the antibody is the antibody of any one of claims 6-7.

22. A method of treating a tumor, comprising administering to a patient an effective amount of the MAGEB16 modulator for use of any one of claims 1-5, or the antibody or antigen binding fragment thereof of any one of claims 6-7, or the chimeric antigen receptor or T cell receptor of claim 8, or the immunoconjugate of claim 9, or the antibody drug conjugate or polypeptide drug conjugate of any one of claims 14-15, or the nucleic acid molecule of claim 16, or the pharmaceutical composition or kit of claim 17.

23. A method of treating an autoimmune disease, comprising administering to a patient an effective amount of the MAGEB16 modulator for use of any one of claims 1-5, or the antibody or antigen binding fragment thereof of any one of claims 6-7, or the chimeric antigen receptor or T cell receptor of claim 8, or the immunoconjugate of claim 9, or the antibody drug conjugate or polypeptide drug conjugate of any one of claims 14-15, or the nucleic acid molecule of claim 16, or the pharmaceutical composition or kit of claim 17.

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