B7-H3 chimeric antigen receptor modified T cells and their applications

Abstract

The present invention provides a B7-H3 chimeric antigen receptor modified T cell and its application. Specifically, the present invention provides an antibody targeting B7-H3, and a chimeric antigen receptor and an engineered immune cell prepared using the antibody. The present invention also provides a preparation method and application of antibodies and immune cells. The engineered immune cells of the present invention can effectively control the growth of tumor cells such as gastric cancer, osteosarcoma, breast cancer, neuroblastoma, pancreatic cancer, colon cancer, etc., and no obvious toxic side effects occur in in vivo experiments. It has very good safety and is expected to become an effective immunotherapy for the treatment of various solid tumors.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to a B7-H3 chimeric antigen receptor modified T cell and application thereof. Background Art

[0002] The concept of chimeric antigen receptor (CAR) modified T cells for cancer treatment was proposed in the late 1980s. After more than 30 years of development, CAR-T immunotherapy has achieved many remarkable achievements in the field of cancer treatment. In recent years, CAR-T cells have made major breakthroughs in the treatment of blood tumors, among which CAR-T therapy targeting CD19 antigen for the treatment of B lymphoid leukemia is the most representative. In 2017, the US FDA approved two drugs for adolescent relapsed and refractory chronic B lymphocytic leukemia (Kymriah) and adult relapsed and refractory large B-cell lymphoma (Yescarta). The significant research results of CAR-T cell therapy in the field of blood tumor treatment have further stimulated its research and application in the field of solid tumor treatment. However, so far, CAR-T cell therapy has not achieved the same satisfactory effect in the field of solid tumor treatment as in the treatment of blood tumors. One of the main reasons is that the antigens of solid tumors are highly heterogeneous, and the selection of targets is difficult, and there is a lack of ideal therapeutic targets like CD19.

[0003] B7-H3 molecules, also known as CD276 molecules, are a recently discovered type I transmembrane glycoprotein belonging to the B7 immune co-stimulatory and co-inhibitory globulin family. Human B7-H3 molecules have two subtypes, 2Ig-B7-H3 and 4Ig-B7H3, while mouse B7-H3 has only one subtype, 2Ig-B7-H3, which has 88% amino acid sequence homology with human 2Ig-B7-H3 subtype. At present, the function of B7-H3 molecules still needs to be further elucidated, but existing studies have shown that B7-H3 molecules have both immune activation and immunosuppression. On the one hand, B7-H3 molecules can play an immune-suppressive role by reducing the secretion of type I cytokine IFN-γ. Many research results support that B7-H3 has a negative immune regulatory effect in models of graft-versus-host disease, cardiac transplant rejection, airway inflammation, and autoimmune encephalomyelitis; on the other hand, B7-H3 has also been found to play a role in providing immune co-stimulatory signals for T cells in autoimmune diseases in vivo and in vitro.

[0004] Numerous studies have shown that B7-H3 is overexpressed in a variety of human tumors, including leukemia, gastric cancer, breast cancer, non-small cell lung cancer, ovarian cancer, osteosarcoma, neuroblastoma, pancreatic cancer, breast cancer, etc., but it is only limitedly expressed in normal tissues. At the same time, it has also been reported to be expressed in important tumor microenvironment components such as tumor stem cells, tumor stromal cells and tumor angiogenesis. Overexpression of B7-H3 usually reduces the infiltration of tumor-associated lymphocytes in the tumor, accelerates disease progression, and is often closely related to the poor prognosis of cancers such as pancreatic ductal carcinoma, prostate cancer, ovarian cancer, lung cancer and clear cell renal carcinoma.

[0005] Gastric cancer is a very common malignant tumor worldwide, which is ranked as the fifth most common cancer and the third most common cause of cancer death. Patients with gastric cancer have no obvious symptoms in the early stage and are mostly diagnosed in the late stage. In addition, GC cells are prone to metastasis and proliferation, and relapse frequently after surgery. In the past few decades, although the treatment of gastric cancer has improved, the median overall survival (mOS) is only 6.6 to 18.4 months. The FDA has approved trastuzumab (anti-ERBB2 / HER2) and ramucirumab (anti-KDR / VEGFR2) for the treatment of advanced or metastatic gastric cancer, but the survival rate is still not ideal and drug resistance has developed. The B7-H3 molecule has been reported to be highly expressed in the tumor tissues of gastric cancer patients. At the same time, the expression of the B7-H3 molecule is closely related to the metastasis of gastric cancer cells and the poor prognosis of patients.

[0006] Triple-negative breast cancer is a type of refractory breast cancer characterized by the lack of expression of progesterone receptors, estrogen receptors, and HER2 antigens. It is common in young people, African Americans, Hispanics, or those with BRCA1 gene mutations, accounting for approximately 10-15% of confirmed breast cancer cases. Due to the lack of these three types of receptors, conventional treatments such as targeted drugs for estrogen receptors, progesterone receptors, and HER-2 antigens are unable to effectively treat it. Currently, combined treatment with surgery, chemotherapy, and radiotherapy is still an effective option for the treatment of triple-negative breast cancer. The five-year survival rate of triple-negative breast cancer is 83%, which is lower than other forms of breast cancer, and triple-negative breast cancer is more likely to recur after treatment than other types of breast cancer. Studies have reported that the B7-H3 antigen is highly expressed in metastatic and malignant breast cancer tissues.

[0007] Osteosarcoma, also known as osteogenic sarcoma, is a tumor that occurs frequently in children and adolescents, accounting for about 3% of childhood tumors. Osteosarcoma is usually caused by the canceration of new bone growth cells, so in children and adolescents it usually occurs at the ends of long bones where the bones grow fastest. Currently, the main treatments for osteosarcoma are surgery, radiotherapy, and chemotherapy. For osteosarcoma patients who have not metastasized, the cure rate is usually high, and about three-quarters of patients can be cured, but the survival rate of patients whose osteosarcoma cells have metastasized is only 30%. Studies have shown that the B7-H3 molecule is highly expressed in the tumor tissues of more than 90% of osteosarcoma patients, and the expression of B7-H3 is closely related to the recurrence of osteosarcoma and the survival of patients.

[0008] Pancreatic cancer refers to a malignant tumor that occurs in the pancreas, which is divided into exocrine pancreatic malignant tumors and endocrine pancreatic malignant tumors. Exocrine pancreatic malignant tumors are mainly derived from adenocarcinomas of the pancreatic ductal epithelium, accounting for more than 90% of pancreatic cancer. Therefore, pancreatic cancer usually refers to pancreatic ductal epithelial carcinoma. Other common exocrine malignant tumors include acinar cell carcinoma and cystadenocarcinoma of the pancreas. Some pancreatic cancers also come from neuroendocrine cells, such as malignant insulinomas and gastrinomas, which may have symptoms caused by increased hormones. Pancreatic cancer is very malignant, accounting for about 1%-2% of all malignant tumors, and there is a trend of increasing year by year. In pancreatic cancer cells, B7-H3 activates the TLR4 / NF-kb signaling pathway, increases the expression of IL-8 and VEGF, and then promotes the proliferation, migration and infiltration of pancreatic cancer cells; therefore, B7-H3 is an ideal target for targeted treatment of pancreatic cancer.

[0009] Neuroblastoma is a malignant tumor originating from the sympathetic nerves. It is mainly composed of undifferentiated neuroblasts and is prone to occur in children. Nearly half of neuroblastomas occur in infants under 2 years old. The condition is relatively complicated, the tumor boundaries are relatively clear, the texture is relatively hard, and there are often bleeding, necrosis or cystic changes. The clinical manifestations are relatively complex and can occur in different parts of the human body, with different manifestations depending on the different parts. Neuroblastoma mainly occurs in the chest, abdomen and neck. The treatment methods are relatively complicated and the prognosis is also different. In general, neuroblastoma is highly malignant and difficult to cure. Studies have found that neuroblastoma cell lines highly express the B7-H3 molecule.

[0010] Colon cancer is a common malignant tumor of the digestive tract, ranking second in gastrointestinal tumors. The most common sites are the rectum and the junction of the rectum and the sigmoid colon, accounting for 60%. Most cases occur after the age of 40, with a male-female ratio of 2:1. The incidence of colon cancer is mainly related to a high-fat and low-fiber diet. Chronic inflammation of the colon makes the incidence of colon cancer higher than that of the general population. The incidence of colon cancer in people with colon polyps is 5 times that of those without colon polyps. The incidence of canceration in familial multiple intestinal polyposis is even higher. Genetic factors may also be involved in the onset of colon cancer. The early symptoms of colon cancer are obvious, and in the middle and late stages, they may manifest as abdominal distension and indigestion, followed by changes in bowel habits, abdominal pain, mucus or bloody stools. After tumor ulceration, blood loss, and toxin absorption, symptoms such as anemia, low fever, fatigue, weight loss, and lower limb edema often occur. Studies have shown that B7-H3 is also highly expressed in colon cancer cell lines.

[0011] Due to the above characteristics of B7-H3 molecules, it has become one of the hot targets for tumor immunotherapy in recent years. Currently, two monoclonal antibodies targeting B7-H3, 8H9 and MGA271, have been clinically proven to be safe and effective. However, there are still many deficiencies in the existing research on B7-H3 monoclonal antibodies, especially B7-H3 chimeric antigen receptor-modified T cells. The field needs to develop new B7-H3 monoclonal antibodies and related CAR-T cells and applications. Summary of the invention

[0012] The purpose of the present invention is to provide a B7-H3 chimeric antigen receptor modified T cell and its application.

[0013] In a first aspect of the present invention, an antibody targeting B7-H3 is provided, characterized in that the antibody has a heavy chain variable region and / or a light chain variable region;

[0014] Wherein, the heavy chain variable region includes the following complementarity determining region CDR:

[0015] CDR1 shown in SEQ ID NO:2,

[0016] CDR2 shown in SEQ ID NO:3, and

[0017] CDR3 shown in SEQ ID NO:4;

[0018] The light chain variable region includes the following complementarity determining regions CDR:

[0019] CDR1' shown in SEQ ID NO:6,

[0020] CDR2' shown in SEQ ID NO:7, and

[0021] CDR3' shown in SEQ ID NO:8.

[0022] In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO:

[0023] As shown in 1, 9 and 11.

[0024] In another preferred embodiment, the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO:

[0025] As shown in Figures 5, 10 and 12.

[0026] In another preferred example, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 5.

[0027] In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:10.

[0028] In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 12.

[0029] In another preferred embodiment, any one of the above amino acid sequences also includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain the B7-H3 binding affinity.

[0030] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids is 1-5 (such as 1-3, preferably 1-2, more preferably 1).

[0031] In another preferred embodiment, the derivative sequence that has been added, deleted, modified and / or substituted at least one amino acid and can retain B7-H3 binding affinity is an amino acid sequence with a homology or sequence identity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0032] In another preferred embodiment, the antibody further comprises a heavy chain constant region and / or a light chain constant region.

[0033] In another preferred embodiment, the heavy chain constant region is of human or mouse origin.

[0034] In another preferred embodiment, the heavy chain constant region is a human antibody heavy chain IgG1 constant region.

[0035] In another preferred embodiment, the light chain constant region is of human or mouse origin.

[0036] In another preferred embodiment, the light chain constant region is a human antibody light chain kappa constant region.

[0037] In another preferred embodiment, the antibody is selected from: animal-derived antibody, chimeric antibody, humanized antibody, fully human antibody, or a combination thereof.

[0038] In another preferred embodiment, the ratio of the immunogenicity Z1 of the chimeric antibody in humans to the immunogenicity Z0 of the non-chimeric antibody (such as mouse antibody) in humans (Z1 / Z0) is 0-0.5, preferably 0-0.2, and more preferably 0-0.05 (such as 0.001-0.05).

[0039] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0040] In another preferred embodiment, the antibody is a full-length antibody protein or an antigen-binding fragment.

[0041] In another preferred embodiment, the antibody may be a bispecific antibody or a multispecific antibody.

[0042] In a second aspect of the present invention, a scFv targeting B7-H3 is provided, wherein the heavy chain variable region of the scFv comprises the following complementarity determining regions CDR:

[0043] CDR1 shown in SEQ ID NO:2,

[0044] CDR2 shown in SEQ ID NO:3, and

[0045] CDR3 shown in SEQ ID NO:4;

[0046] And the light chain variable region of the scFv includes the following complementarity determining regions CDR:

[0047] CDR1' shown in SEQ ID NO:6,

[0048] CDR2' shown in SEQ ID NO:7, and

[0049] CDR3' shown in SEQ ID NO:8.

[0050] In another preferred embodiment, the scFv further comprises a connecting peptide between the heavy chain variable region and the light chain variable region.

[0051] In another preferred embodiment, the connecting peptide is 1-4 consecutive sequences shown in SEQ ID NO: 17 (GGGGS), preferably 3-4, and more preferably 4.

[0052] In another preferred embodiment, the scFv is shown in the following formula A or formula B:

[0053] V H -V L ,(A);V L -V H ,(B)

[0054] Where V H V is the variable region of the antibody heavy chain; L is the antibody light chain variable region; “-” is a connecting peptide or peptide bond.

[0055] In another preferred embodiment, the scFv is as shown in Formula A (V H -V L ) as shown.

[0056] In another preferred embodiment, the amino acid sequence of the scFv is shown in SEQ ID NO: 13, 14 or 18.

[0057] In another aspect of the present invention, a recombinant protein is provided, wherein the recombinant protein comprises:

[0058] (i) the antibody according to the first aspect of the present invention; and

[0059] (ii) optionally a tag sequence to facilitate expression and / or purification.

[0060] In another preferred embodiment, the tag sequence includes a 6His tag.

[0061] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0062] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0063] In the third aspect of the present invention, a chimeric antigen receptor (CAR) is provided, which comprises the scFv described in the second aspect of the present invention.

[0064] In another preferred example, the antigen binding domain of the CAR includes the antibody heavy chain variable region shown in SEQ ID NO: 1, 9 or 11, and the antibody light chain variable region shown in SEQ ID NO: 5, 10 or 12.

[0065] In another preferred embodiment, the chimeric antigen receptor has the following structure:

[0066] L-scFv-H-TM-C-CD3ζ (I)

[0067] In the formula,

[0068] Each "-" is independently a connecting peptide or a peptide bond;

[0069] L is none or a signal peptide sequence;

[0070] scFv is a scFv targeting B7-H3;

[0071] H is an optional hinge region;

[0072] TM is the transmembrane domain;

[0073] C is a co-stimulatory signal molecule;

[0074] CD3ζ is the CD3ζ cytoplasmic signaling sequence.

[0075] In another preferred embodiment, the L is a signal peptide selected from the following histones: EF1, CD8, GM-CSF, CD4, CD137, or a combination thereof.

[0076] In another preferred embodiment, the L is a signal peptide derived from EF1.

[0077] In another preferred embodiment, the scFv is shown in the following formula A or formula B:

[0078] V H -V L ,(A);V L -V H ,(B)

[0079] Where V H V is the variable region of the antibody heavy chain; L is the antibody light chain variable region; “-” is a connecting peptide or peptide bond.

[0080] In another preferred embodiment, the scFv is as shown in Formula A (V H -V L ) as shown.

[0081] In another preferred embodiment, the H is a hinge region of a protein selected from the group consisting of CD8, CD28, CD137, Fc, or a combination thereof.

[0082] In another preferred embodiment, the H is a hinge region derived from CD8 or Fc.

[0083] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0084] In another preferred embodiment, the TM is a transmembrane region derived from CD8.

[0085] In another preferred embodiment, the TM is a transmembrane region derived from ICOS.

[0086] In another preferred embodiment, C is a co-stimulatory signal molecule of a protein selected from the following group: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.

[0087] In another preferred embodiment, C is a co-stimulatory signal molecule derived from 4-1BB.

[0088] In another preferred embodiment, C is a co-stimulatory signal molecule derived from ICOS and 4-1BB.

[0089] In another preferred embodiment, the CAR (preferably the C-terminus) also includes a cell suicide element.

[0090] In another preferred embodiment, the CAR is connected to the cell suicide element via a self-cleavage element.

[0091] In another preferred embodiment, the cell suicide element is connected to the CD3ζ of the CAR via T2A.

[0092] In another preferred embodiment, the cell suicide element is selected from the following group: HSV-TK, iCasp9, ΔCD20, mTMPK, ΔCD19, RQR8, EGFRt, or a combination thereof.

[0093] In another preferred embodiment, the cell suicide element is tEGFR.

[0094] In another preferred example, the amino acid sequence of tEGFR is shown in SEQ ID NO:15.

[0095] In another preferred example, the amino acid sequence of the CAR is shown in SEQ ID NO:16.

[0096] In another preferred embodiment, in the chimeric antigen receptor, H is the hinge region derived from Fc, TM is the transmembrane region derived from ICOS, and C is the co-stimulatory signal molecule derived from ICOS and 4-1BB.

[0097] In another preferred example, the amino acid sequence of the CAR is shown in SEQ ID NO:19.

[0098] In a fourth aspect of the present invention, a polynucleotide is provided, wherein the polynucleotide encodes a polypeptide selected from the group consisting of:

[0099] (1) The antibody according to the first aspect of the present invention;

[0100] (2) the scFv according to the second aspect of the present invention; and

[0101] (3) The chimeric antigen receptor as described in the third aspect of the present invention.

[0102] In the fifth aspect of the present invention, a vector is provided, wherein the vector contains the polynucleotide described in any one of the fourth aspect of the present invention.

[0103] In another preferred embodiment, the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.

[0104] In another preferred embodiment, the vector comprises a lentiviral vector.

[0105] In the sixth aspect of the present invention, a genetically engineered host cell is provided, wherein the host cell contains the vector described in the fifth aspect of the present invention, or the polynucleotide described in the fourth aspect of the present invention is integrated into the genome, or expresses the antibody described in the first aspect of the present invention, or expresses the scFv described in the second aspect of the present invention, or expresses the chimeric antigen receptor described in the third aspect of the present invention.

[0106] In another preferred embodiment, the cell is an isolated cell, and / or the cell is a genetically engineered cell.

[0107] In another preferred embodiment, the immune cells are from humans or non-human mammals (such as mice).

[0108] In another preferred embodiment, the cells include T cells and NK cells.

[0109] In another preferred embodiment, the host cell is an engineered immune cell.

[0110] In another preferred embodiment, the immune cells express exogenous cell suicide elements.

[0111] In another preferred embodiment, CAR and cell suicide elements are co-expressed in the immune cells.

[0112] In another preferred embodiment, the engineered immune cells include T cells or NK cells, preferably (i) chimeric antigen receptor T cells (CAR-T cells); or (ii) chimeric antigen receptor NK cells (CAR-NK cells).

[0113] In the seventh aspect of the present invention, an antibody conjugate is provided, the antibody conjugate comprising:

[0114] (a) an antibody portion, wherein the antibody portion is selected from the group consisting of: an antibody as described in the first aspect of the present invention; and

[0115] (b) a conjugated moiety conjugated to the antibody portion, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0116] In another preferred embodiment, the antibody portion and the coupling portion are coupled via a chemical bond or a linker.

[0117] In another preferred embodiment, the antibody conjugate is an antibody-drug conjugate.

[0118] In the eighth aspect of the present invention, a method for preparing an engineered cell is provided, wherein the engineered cell expresses the antibody described in the first aspect of the present invention, the scFv described in the second aspect of the present invention, or the chimeric antigen receptor described in the third aspect of the present invention, comprising the following steps: transducing the polynucleotide described in the fourth aspect of the present invention or the vector described in the fifth aspect of the present invention into a host cell, thereby obtaining the engineered cell.

[0119] In another preferred embodiment, the method further comprises the step of: introducing an expression cassette for expressing a cell suicide element into the immune cell.

[0120] In another preferred embodiment, the method further comprises the step of testing the function and effectiveness of the obtained engineered cells.

[0121] In the ninth aspect of the present invention, a pharmaceutical composition is provided, which comprises the antibody described in the first aspect of the present invention, the scFv protein described in the second aspect of the present invention, the CAR described in the third aspect of the present invention, the vector described in the fifth aspect of the present invention, the host cell described in the sixth aspect of the present invention, or the antibody conjugate described in the seventh aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0122] In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

[0123] In another preferred embodiment, the pharmaceutical composition is an injection.

[0124] In another preferred embodiment, the pharmaceutical composition further comprises a second anti-tumor active ingredient, preferably a second antibody or a chemotherapeutic agent.

[0125] In another preferred embodiment, the chemotherapeutic agent is selected from the following group: docetaxel, carboplatin, or a combination thereof.

[0126] In the tenth aspect of the present invention, a use of an active ingredient is provided, wherein the active ingredient is selected from the following group: the antibody described in the first aspect of the present invention, the scFv described in the second aspect of the present invention, the CAR described in the third aspect of the present invention, the vector described in the fifth aspect of the present invention, the host cell described in the sixth aspect of the present invention, or the antibody conjugate described in the seventh aspect of the present invention, for (a) preparing a diagnostic reagent or kit; and / or (b) preparing a drug or preparation for preventing and / or treating cancer or tumors.

[0127] In another preferred embodiment, the tumor is a B7-H3 positive tumor.

[0128] In another preferred embodiment, the tumor is selected from the following group: a blood tumor, a solid tumor, or a combination thereof.

[0129] In another preferred embodiment, the blood tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

[0130] In another preferred embodiment, the solid tumor is selected from the group consisting of gastric cancer, peritoneal metastasis of gastric cancer, liver cancer, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer, testicular cancer, colorectal cancer, urinary tract tumor, thyroid cancer, or a combination thereof.

[0131] In another preferred embodiment, the tumor is gastric cancer, breast cancer, osteosarcoma, neuroblastoma, pancreatic cancer, or colon cancer.

[0132] In another preferred embodiment, the diagnostic reagent is a test piece or a test plate.

[0133] In another preferred embodiment, the diagnostic reagent or kit is used for:

[0134] (1) detecting B7-H3 protein in a sample; and / or

[0135] (2) detecting endogenous B7-H3 protein in tumor cells; and / or

[0136] (3) Detection of tumor cells expressing B7-H3 protein.

[0137] In the eleventh aspect of the present invention, a method for in vitro detection (including diagnostic or non-diagnostic) of B7-H3 protein in a sample is provided, the method comprising the steps of:

[0138] (1) contacting the sample with the antibody described in the first aspect of the present invention, the recombinant protein described in the second aspect of the present invention, or the antibody conjugate described in the seventh aspect of the present invention in vitro;

[0139] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of B7-H3 protein in the sample.

[0140] In the twelfth aspect of the present invention, a detection plate is provided, which comprises: a substrate (support plate) and a test strip, wherein the test strip contains the antibody described in the first aspect of the present invention, the recombinant protein described in the second aspect of the present invention, or the antibody conjugate described in the seventh aspect of the present invention, or a combination thereof.

[0141] In the thirteenth aspect of the present invention, a kit is provided, comprising:

[0142] (1) a first container, wherein the first container contains the antibody according to the first aspect of the present invention; and / or

[0143] (2) a second container, wherein the second container contains a secondary antibody against the antibody described in the first aspect of the present invention;

[0144] or,

[0145] The kit contains the detection plate described in the twelfth aspect of the present invention.

[0146] In a fourteenth aspect of the present invention, a method for preparing a recombinant polypeptide is provided, the method comprising:

[0147] (a) culturing the host cell according to the sixth aspect of the present invention under conditions suitable for expression;

[0148] (b) isolating a recombinant polypeptide from the culture, wherein the recombinant polypeptide is the antibody according to the first aspect of the present invention or the scFv according to the second aspect of the present invention.

[0149] In the fifteenth aspect of the present invention, a method for treating a disease is provided, comprising administering to a subject in need thereof an effective amount of the antibody described in the first aspect of the present invention, or the scFv described in the second aspect of the present invention, or the antibody conjugate described in the seventh aspect of the present invention, or the host cell described in the sixth aspect of the present invention, or the pharmaceutical composition described in the ninth aspect of the present invention, or a combination thereof.

[0150] In another preferred embodiment, the disease is a B7-H3 positive tumor.

[0151] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0152] Figure 1A The map of the humanized CD276 antibody light chain and heavy chain expression vectors is shown.

[0153] Figure 1B The binding of different cloned humanized antibodies to CD276 (B7-H3) overexpressing cell line CD276-CHO-K1 cells is shown.

[0154] Figure 2 The binding of the B7-H3 antibody of the present invention to two subclasses of human B7-H3 antigens and mouse B7-H3 antigen is shown. The mB7-H3 Ab antibody refers to the mouse CD276 hybridoma antibody against human B7-H3 of the present invention, the αhB7-H3 Ab refers to the humanized CD276 antibody of the present invention, and the hB7-H3 Ab antibody refers to the commercial B7-H3 antibody (Biolegend, cat: 351006).

[0155] Figure 3A The CAR positivity rate of αhB7-H3 CAR-Jurkat T cells detected by flow cytometry is shown.

[0156] Figure 3B The expression of B7-H3 on the cell surface of three gastric cancer cell lines HGC-27, AGS and KATO-III was shown. The results showed that HGC-27 and AGS were B7-H3 high-expressing cell lines, and KATO-III cells were B7-H3 low-expressing cell lines.

[0157] Figure 3C The expression of CD69, CD25, and PD1 after incubation of B7-H3 CAR-Jurkat T cells with target cells is shown.

[0158] Figure 3DThe expression of cytokines (IL2, granzyme B) in the supernatant after incubation of B7-H3 CAR-Jurkat T cells with target cells is shown.

[0159] Figure 4A A schematic diagram of the structure of the second-generation CAR vector constructed using the mouse B7-H3 antibody sequence and the humanized B7-H3 antibody sequence targeting human B7-H3 is shown.

[0160] Figure 4B A schematic diagram of the structure of the lentiviral vector used for CAR expression is shown.

[0161] Figure 4C The transfection efficiency of humanized B7-H3-CAR-T cells (αhB7-H3 CAR-T, βhB7-H3 CAR-T) and mouse B7-H3-CAR-T cells (mB7-H3-CAR-T) is shown.

[0162] Figure 5A The expression of B7-H3 antigen on the cell surface of two pancreatic cancer lines, SW1990 and PANC-1, is shown.

[0163] Figure 5B The specific inhibitory effects of three B7-H3-CAR-T cells on pancreatic cancer cell lines SW1990 and PANC-1 were shown. The effector-target ratios were 1:1 and 4:1, respectively.

[0164] Figure 5C The release of cytokines (IL-2, Granzyme-B, IFN-γ) after incubation of three B7-H3-CAR-T cells with pancreatic cancer cell lines SW1990 and PANC-1 is shown.

[0165] Figure 6 The results show that αhB7-H3 CAR-T cells kill Raji cells (mB7-H3-Raji cells) that overexpress mouse B7-H3 antigen in vitro. Raji is a B7-H3 negative target cell.

[0166] Fig. 7A The results showed that αhB7-H3 CAR-T cells could kill gastric cancer cell line HGC-27 (B7-H3 positive cell line) and KATOIII (B7-H3 negative cell line) in vitro.

[0167] Figure 7B The release of cytokines after incubation of αhB7-H3 CAR-T with gastric cancer cell lines HGC-27 and KATOIII cells is shown.

[0168] Fig. 8AThe experimental flow chart of αhB7-H3 CAR-T cell therapy for the HGC-27 cell subcutaneously transplanted tumor NSG mouse model is shown.

[0169] Figure 8B The in vivo inhibitory effect of αhB7-H3 CAR-T cells on HGC-27 cells was shown.

[0170] Figure 8C The in vivo inhibitory effect of αhB7-H3 CAR-T cells on HGC-27 cells was shown.

[0171] Fig.8D Shown are the weight changes of HGC-27 tumor-bearing mice treated with αhB7-H3 CAR-T cells compared with the control group.

[0172] Fig. 8E Shown are the changes in body weight of mice in the αhB7-H3 CAR-T group compared with the control group.

[0173] Fig.8F The changes in organ coefficients of mice in the αhB7-H3 CAR-T group compared with those in the control group are shown.

[0174] Figure 8G The changes in blood routine parameters of mice in the αhB7-H3 CAR-T group compared with those in the control group are shown.

[0175] Fig. 9A The expression of B7-H3 on the cell surface of breast cancer cell lines T47D and MDA-MB-468 is shown.

[0176] Fig. 9B The in vitro inhibitory effect of αhB7-H3 CAR-T cells on breast cancer cell lines (T47D and MDA-MB-468) was shown.

[0177] Fig. 9C The release of cytokines (IL-2, Granzyme-B, IFN-γ, TNF-α) after incubation of αhB7-H3 CAR-T cells with breast cancer cell lines is shown.

[0178] Fig. 10A The expression of B7-H3 on the surface of three osteosarcoma cell lines (SJSA-1, MG63, and HOS cells) is shown.

[0179] Fig. 10B The results show that αhB7-H3 CAR-T cells have an in vitro inhibitory effect on three osteosarcoma cell lines (SJSA-1, MG63, and HOS cells).

[0180] Fig. 10CThe release of cytokines (IL-2, Granzyme-B, IFN-γ) after incubation of αhB7-H3 CAR-T cells with osteosarcoma cell lines is shown.

[0181] Fig.11A The experimental flow chart of αhB7-H3 CAR-T cell therapy for MG-63 cell subcutaneously transplanted tumor NSG mouse model is shown.

[0182] Fig. 11B It was shown that compared with the control group, αhB7-H3 CAR-T cells could significantly inhibit the growth and proliferation of tumor cells in mice.

[0183] Fig. 11C Shown are the changes in body weight of mice in the αhB7-H3 CAR-T cell group compared with the control group.

[0184] Fig. 12A The inhibitory effect of αhB7-H3 CAR-T cells on AGS and HGC27 cells was evaluated using the RTCA method.

[0185] Fig. 12B The release of cytokines after incubation of αhB7-H3 CAR-T cells with AGS and HGC27 cells is shown.

[0186] Fig.13A The experimental flow chart of αhB7-H3 CAR-T cells for treating the NSG mouse model of subcutaneously transplanted tumors of the neuroblastoma SK-N-AS cell line is shown.

[0187] Fig. 13B Shown are the changes in tumor size in tumor-bearing mice treated with αhB7-H3 CAR-T cells compared with the control group (T cell group).

[0188] Fig. 13C The figure shows the comparison of tumor size between the CAR-T group and the control group mice after mouse autopsy.

[0189] Fig.13D The weight changes of mice in the control group (T cell group) and the αhB7-H3 CAR-T cell group are shown.

[0190] Fig.14 The structural diagrams of three generations of B7-H3-CAR vectors are shown.

[0191] Fig.15 The expression of B7-H3 on the surface of colon cancer cell line SW620 is shown.

[0192] Fig.16AThe in vitro inhibitory effects of three generations of B7-H3-CAR-T cells on SW620 were shown.

[0193] Fig. 16B The release of cytokines after three generations of B7-H3-CAR-T cells were incubated with SW620 is shown.

[0194] Fig.17A The experimental process of three generations of B7-H3-CAR-T cells in treating SW620 tumor-bearing NCG mice is shown.

[0195] Fig. 17B Shown are the results of in vivo imaging experiments of mice in the experimental and control groups.

[0196] Fig. 17C The changes in tumor fluorescence signal values ​​between the experimental and control group mice are shown.

[0197] Fig.17D Survival curves of mice in the experimental and control groups are shown.

[0198] Fig.18A The expression of B7-H3 in four pancreatic cancer cell lines is shown.

[0199] Fig.18B The experimental procedure of local X-ray irradiation of tumor sites in experimental mice is shown.

[0200] Fig. 18C Shows the changes in tumor size in experimental mice. DETAILED DESCRIPTION

[0201] After extensive and in-depth research, the inventors unexpectedly discovered for the first time a B7-H3 chimeric antigen receptor modified T cell and its application. Specifically, the present invention provides an antibody targeting B7-H3, and a chimeric antigen receptor and an engineered immune cell prepared using the antibody. The present invention also provides a method for preparing antibodies and immune cells and their application. The engineered immune cells of the present invention can effectively control the growth of tumor cells such as gastric cancer, osteosarcoma, and triple-negative breast cancer, and no obvious toxic side effects occur in in vivo experiments, and have very good safety, and are expected to become an effective immunotherapy for the treatment of various solid tumors. The present invention was completed on this basis.

[0202] Specifically, the present invention successfully obtained the mB7-H3 antibody by multiple rounds of immunization of Balb / c mice using h2IgB7-H3-CHO-K1 recombinant cells, after hybridoma cell fusion and two monoclonal screenings. Subsequently, it was humanized by the CDR transplantation method, and two humanized B7-H3 chimeric antibodies with equivalent activity to the mouse antibody were obtained: αhB7-H3 and βhB7-H3. The corresponding second-generation CAR-T cells were constructed using the obtained mouse anti-human mB7-H3, humanized αhB7-H3 and βhB7-H3 antibodies. In vitro cytotoxicity experiments have shown that αhB7-H3-CAR-T cells have a strong growth inhibitory effect on tumor cells and a stronger cytokine secretion ability, so its anti-tumor activity has been further studied. The present invention studies the anti-tumor effects of αhB7-H3 CAR-T cells on gastric cancer, osteosarcoma, and triple-negative breast cancer. The experimental results in vivo and in vitro show that αhB7-H3-CAR-T cells can effectively control the growth of tumor cells such as gastric cancer, osteosarcoma, and triple-negative breast cancer, which fully demonstrates the effectiveness of αhB7-H3-CAR-T cells in treating these solid tumors. At the same time, human 2Ig-B7-H3 molecules and mouse 2Ig-B7-H3 molecules have a high degree of homology. Antibody binding experiments show that both antigens can be recognized by αhB7-H3 antibodies, but mice in each αhB7-H3 CAR-T cell treatment group did not show obvious toxic side effects during the treatment process. This result strongly illustrates the safety of αhB7-H3 CAR-T cells in treating solid tumors. In summary, αhB7-H3-CAR-T cells are expected to become an effective immunotherapy for the treatment of a variety of solid tumors.

[0203] In recent years, CAR-T therapy has made breakthrough progress in the field of tumor immunotherapy. CAR-T therapy targeting CD19 has achieved remarkable results in the treatment of B lymphocyte tumors, bringing good news to many patients with relapsed and refractory B lymphocyte leukemia. However, CAR-T cell immunotherapy has not achieved satisfactory results in the treatment of solid tumors. This is mainly because compared with blood tumors, many characteristics of solid tumors themselves hinder the effect of CAR-T therapy. First, the antigen expression of solid tumors is highly heterogeneous and lacks ideal therapeutic targets; secondly, it is difficult for CAR-T cells to effectively home to the tumor site; thirdly, solid tumors have a highly inhibitory tumor microenvironment that can impair the function of CAR-T cells.

[0204] The present invention successfully obtained the mB7-H3 mouse monoclonal antibody by using recombinant h2IgB7-H3-CHO-K1 cells to immunize Balb / c mice for multiple rounds, and then by hybridoma cell screening, multiple rounds of hybridoma cell cloning screening and hybridoma cell sequencing. Subsequently, the mB7-H3 antibody was humanized, and the amino acid sequences of the antibody heavy chain and light chain were humanized by CDR transplantation scheme. Then, two humanized B7-H3 antibodies with similar activity to the mB7-H3 antibody were screened from the obtained humanized antibodies: αhB7-H3 antibody and βhB7-H3 antibody. Then, the binding ability of αhB7-H3 antibody and tumor cells expressing human h2IgB7H3 and h4IgB7H3 and mouse m2IgB7H3 molecules on the cell surface was further studied. The results showed that the αhB7-H3 antibody can simultaneously recognize and bind to two human B7-H3 molecular subtypes and mouse B7-H3 molecules. In addition, αhB7-H3 CAR-T cells also showed specific cytotoxic effects on m2IgB7H3-Raji cells. The above experimental results provide important experimental basis for evaluating the safety of CAR-T cells in mouse models.

[0205] In order to verify whether αhB7-H3 CAR can mediate antigen-dependent effector cell-specific activation, Jurkat T cells with high expression of αhB7-H3 CAR (αhB7-H3 CAR- Jurkat T) were constructed by lentiviral transduction. Since Jurkat T cells are immortalized T lymphocyte leukemia cell lines with good characteristics, constructing CAR-Jurkat T cells can more simply and reliably confirm whether CAR can mediate cell-specific activation. The results of co-incubation of αhB7-H3 CAR-Jurkat T and gastric cancer cells showed that co-incubation with B7-H3 antigen-positive tumor cells could significantly upregulate the expression of activation molecules CD25 and CD69 and inhibitory signal molecule PD-1 of CAR-Jurkat T cells, but co-incubation with B7-H3 antigen-negative KATO III cells only limitedly increased the expression of these molecules, and this phenomenon is most likely caused by the high degree of activation of αhB7-H3CAR- Jurkat T cells themselves. The detection of cytokine content also obtained similar results, and the cytokine secretion of αhB7-H3 CAR-Jurkat T cells was positively correlated with the antigen expression of tumor cells co-incubated with them. These results show that the αhB7-H3 CAR molecule constructed by the present invention can mediate specific activation of effector cells in an antigen-dependent manner.

[0206] The present invention uses the obtained B7-H3 antibody to construct mB7-H3 CAR-T cells, αhB7-H3 CAR-T cells and βhB7-H3 CAR-T cells, respectively, and finds that mB7-H3 CAR-T cells and αhB7-H3 CAR-T cells have better anti-tumor activity through in vitro cytotoxicity experiments. Therefore, in the subsequent anti-tumor activity study, the in vivo and in vitro anti-tumor activity of αhB7-H3 CAR-T cells on solid tumors such as gastric cancer, breast cancer, and osteosarcoma was studied. The experimental results show that αhB7-H3 CAR-T cells can specifically lyse various tumor cells such as gastric cancer, breast cancer, osteosarcoma, etc. that are positive for B7-H3 antigen in vitro, but have no obvious cytotoxic effect on tumor cells that are negative for B7-H3 antigen. At the same time, it was also found that the cytotoxic effect of αhB7-H3 CAR-T cells on tumor cells was positively correlated with the amount of tumor cell antigen expression. The higher the tumor cell antigen density, the stronger the ability to stimulate αhB7-H3 CAR-T cells to secrete cytokines. This result also shows that antigen density can affect the cytotoxic effect of αhB7-H3 CAR-T cells, once again confirming the idea of ​​Majzner et al. that CAR-T cells have an antigen-dependent therapeutic window.

[0207] In the evaluation of the in vivo anti-tumor activity of αhB7-H3 CAR-T cells, various xenograft tumor models were constructed using NSG mice: gastric cancer cell subcutaneous tumor model, breast cancer cell metastasis tumor model, and osteosarcoma cell subcutaneous tumor model, to evaluate the in vivo anti-tumor activity of αhB7-H3 CAR-T cells and the safety of their treatment. The results showed that αhB7-H3 CAR-T cells can effectively inhibit the growth of tumor cells in various tumor xenograft models, and no obvious toxic side effects were caused to mice during the treatment process. The weight, organ coefficient, and blood routine of the mice in the treatment group were not significantly different from those in normal groups. The results of the antigen binding experiment showed that the αhB7-H3 antibody can simultaneously recognize two subtypes of human B7-H3 molecules and mouse B7-H3 molecules, but the treatment of αhB7-H3 CAR-T cells did not cause obvious toxic side effects in mice, indicating that αhB7-H3 CAR-T cells will not produce cytotoxic effects on normal mouse tissues and cells expressing B7-H3 antigen. The reason may be that the amount of B7-H3 antigen expressed in normal tissues is not enough to trigger αhB7-H3 CAR-T cells to produce specific cytotoxic effects. The above experimental results provide an important safety reference for the future clinical application of αhB7-H3 CAR-T cells in humans.

[0208] In summary, the present invention successfully obtained a B7-H3 monoclonal antibody with good activity through mouse immune screening, and obtained two humanized B7-H3 monoclonal antibodies through humanized transformation. Three second-generation CAR-T cells were constructed using single-chain variable regions derived from mouse antibodies and two humanized antibodies, and the anti-tumor activity and safety of αhB7-H3 CAR-T cells with good activity were studied in depth. The results showed that αhB7-H3CAR-T cells had good anti-tumor activity against gastric cancer, breast cancer, and osteosarcoma in vivo and in vitro. At the same time, αhB7-H3 CAR-T cells did not cause serious toxic side effects to treated mice. These results all show that αhB7-H3 CAR-T cells have good and reliable safety in the treatment of solid tumors. The above results show that CAR-T immunotherapy targeting B7-H3 molecules has great application prospects in the treatment of various solid tumors, and is expected to bring good news to the majority of patients with solid tumors.

[0209] Antibody

[0210] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons with identical structural features, which consists of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by a covalent disulfide bond, while the number of disulfide bonds between heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0211] As used herein, the term "variable" means that some parts of the variable region in an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three fragments called complementary determining regions (CDRs) or hypervariable regions in the variable regions of light and heavy chains. The more conservative parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are roughly in a β-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)). The constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0212] The "light chains" of vertebrate antibodies (immunoglobulins) can be classified into one of two distinct classes (called kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be divided into different classes based on the amino acid sequence of their heavy chain constant regions. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known in the art.

[0213] Generally, the antigen binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs). This segment is divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. These CDRs form a ring structure, and the β-folds formed by the FRs in between are close to each other in spatial structure. The CDRs on the heavy chain and the CDRs on the corresponding light chains constitute the antigen binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR region.

[0214] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0215] In the present invention, antibodies include mouse, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human parts, can be obtained by standard DNA recombinant techniques, and they are all useful antibodies. A chimeric antibody is a molecule in which different parts are from different animal species, such as a chimeric antibody having a variable region from a mouse monoclonal antibody and a constant region from a human immunoglobulin (see, for example, U.S. Pat. No. 4,816,567 and U.S. Pat. No. 4,816,397, which are hereby incorporated by reference in their entirety). A humanized antibody refers to an antibody molecule derived from a non-human species, having one or more complementary determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Pat. No. 5,585,089, which is hereby incorporated by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombinant techniques well known in the art.

[0216] In the present invention, the antibodies may be monospecific, bispecific, trispecific, or more multispecific.

[0217] In the present invention, the antibody of the present invention also includes its conservative variants, which refers to a polypeptide formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids of similar or similar properties compared to the amino acid sequence of the antibody of the present invention. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.

[0218] Table A

[0219]

[0220]

[0221] Anti-B7-H3 Antibody

[0222] The present invention provides a highly specific and high affinity antibody against B7-H3, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region (VH) amino acid sequence, and the light chain comprises a light chain variable region (VL) amino acid sequence.

[0223] In another preferred embodiment, the antibody has a heavy chain variable region and / or a light chain variable region;

[0224] Wherein, the heavy chain variable region includes the following complementarity determining region CDR:

[0225] CDR1 shown in SEQ ID NO:2,

[0226] CDR2 shown in SEQ ID NO:3, and

[0227] CDR3 shown in SEQ ID NO:4;

[0228] The light chain variable region includes the following complementarity determining regions CDR:

[0229] CDR1' shown in SEQ ID NO:6,

[0230] CDR2' shown in SEQ ID NO:7, and

[0231] CDR3' shown in SEQ ID NO:8.

[0232] In another preferred embodiment, any one of the above amino acid sequences also includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain the B7-H3 binding affinity.

[0233] In another preferred embodiment, the sequence formed by adding, deleting, modifying and / or replacing at least one amino acid sequence is preferably an amino acid sequence with a homology of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%. More preferably, the number of amino acids added, deleted, modified and / or substituted can be 1-7, more preferably 1-5, more preferably 1-3, and more preferably 1-2.

[0234] The antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from animal-derived antibodies, chimeric antibodies, and humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and more preferably fully humanized antibodies.

[0235] The antibody derivatives of the present invention may be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2 or other antibody derivatives known in the art, as well as any one or more of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies.

[0236] Wherein, the animal is preferably a mammal, such as a mouse.

[0237] The antibody of the present invention may be a chimeric antibody, a humanized antibody, a CDR-grafted and / or modified antibody targeting human B7-H3.

[0238] Antibody preparation

[0239] The sequence of the DNA molecule of the antibody of the present invention or its fragment can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences of the light chain and the heavy chain can be fused together to form a single-chain antibody.

[0240] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0241] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0242] At present, the DNA sequence encoding the antibody (or its fragment, or its derivative) of the present invention can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0243] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0244] The host cell can be a prokaryotic cell, such as a bacterial cell, a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO-S, HEK-293 cells.

[0245] Typically, the transformed host cells are cultured under conditions suitable for the expression of the antibodies of the present invention, and then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc., conventional separation and purification means well known to those skilled in the art to obtain the antibodies of the present invention.

[0246] The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of the monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibodies can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0247] The antibodies of the present invention can be expressed in cells, on cell membranes, or secreted outside cells. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting out method), centrifugation, osmotic sterilization, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0248] Antibody-drug conjugates (ADCs)

[0249] The present invention also provides an antibody-drug conjugate (ADC) based on the antibody of the present invention.

[0250] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, wherein the antibody is coupled to the effector molecule, and preferably chemically coupled. The effector molecule is preferably a drug with therapeutic activity. In addition, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug or a radionuclide.

[0251] The antibody of the present invention and the effector molecule can be coupled via a coupling agent. Examples of the coupling agent can be any one or more of a non-selective coupling agent, a coupling agent utilizing a carboxyl group, a peptide chain, and a coupling agent utilizing a disulfide bond. The non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde. The coupling agent utilizing a carboxyl group can be any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (the coupling site is an acylhydrazone).

[0252] Certain residues on antibodies (such as Cys or Lys, etc.) are used to connect to a variety of functional groups, including imaging agents (such as chromophores and fluorescent groups), diagnostic agents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymers) and therapeutic agents. Antibodies can be coupled to functional agents to form antibody-functional agent conjugates. Functional agents (such as drugs, detection agents, stabilizers) are coupled (covalently linked) to antibodies. Functional agents can be directly or indirectly linked to antibodies through linkers.

[0253] Antibodies can be coupled to drugs to form antibody drug conjugates (ADCs). Typically, ADCs contain a linker between the drug and the antibody. The linker can be a degradable or non-degradable linker. Degradable linkers are typically easily degraded in the intracellular environment, such as degradation of the linker at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (such as lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronide-containing linkers that can be degraded by glucuronidase. Peptide linkers can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (such as linkers that hydrolyze when pH is less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (such as disulfide linkers). Non-degradable linkers typically release drugs under conditions where the antibody is hydrolyzed by proteases.

[0254] Prior to attachment to the antibody, the linker has an active reactive group capable of reacting with certain amino acid residues, and attachment is achieved through the active reactive group. Thiol-specific active reactive groups are preferred and include: for example, maleimide compounds, halogenated amides (e.g., iodinated, brominated or chlorinated); halogenated esters (e.g., iodinated, brominated or chlorinated); halogenated methyl ketones (e.g., iodinated, brominated or chlorinated), benzyl halides (e.g., iodinated, brominated or chlorinated); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, maleimide attached to the antibody via thiosuccinimide.

[0255] The drug can be any cytotoxic, cytostatic or immunosuppressive drug. In an embodiment, a linker connects the antibody and the drug, and the drug has a functional group that can form a bond with the linker. For example, the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group that can form a bond with the linker. In the case where the drug is directly connected to the linker, the drug has a reactive group before being connected to the antibody.

[0256] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folate antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazol idinobenzodiazepines), and vinca alkaloids.

[0257] In the present invention, drug-linkers can be used to form ADCs in one simple step. In other embodiments, bifunctional linker compounds can be used to form ADCs in a two-step or multi-step process. For example, a cysteine ​​residue is reacted with a reactive moiety of a linker in a first step, and in a subsequent step, a functional group on the linker reacts with a drug to form an ADC.

[0258] Typically, the functional groups on the linker are selected to react specifically with the appropriate reactive groups on the drug moiety. As a non-limiting example, an azide-based moiety can be used to react specifically with a reactive alkynyl group on the drug moiety. The drug is covalently attached to the linker by a 1,3-dipolar cycloaddition between the azide and the alkynyl group. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other attachment strategies, such as those described in Bioconjugation Technology, 2nd Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that when a complementary pair of reactive functional groups is selected for selective reaction of the drug moiety and the linker, each member of the complementary pair can be used for both the linker and the drug.

[0259] The present invention also provides a method for preparing ADC, which may further comprise: combining an antibody with a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).

[0260] In certain embodiments, the methods of the invention include: combining the antibody with the bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the invention further include: combining the antibody-linker conjugate with the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody via the linker.

[0261] In some embodiments, the antibody drug conjugate ADC has the following molecular formula:

[0262]

[0263] in:

[0264] Ab is antibody,

[0265] LU is the connector;

[0266] D is for medicine;

[0267] And the subscript p is a value selected from 1 to 8.

[0268] Chimeric Antigen Receptor

[0269] The chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen binding domain). The intracellular domain includes a costimulatory signaling region and a ζ chain portion. The costimulatory signaling region refers to a portion of the intracellular domain including a costimulatory molecule. Costimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens, rather than antigen receptors or their ligands.

[0270] Between the extracellular domain and the transmembrane domain of CAR, or between the cytoplasmic domain and the transmembrane domain of CAR, a joint may be incorporated. As used herein, the term "joint" generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane domain to the extracellular domain or cytoplasmic domain of a polypeptide chain. The joint may include 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

[0271] In a preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention includes an antigen binding domain targeting B7-H3. When the CAR of the present invention is expressed in T cells, it is possible to perform antigen recognition based on antigen binding specificity. When it binds to its associated antigen, it affects tumor cells, causing tumor cells not to grow, to be caused to die or otherwise affected, and causes the patient's tumor load to be reduced or eliminated. The antigen binding domain is preferably fused with one or more intracellular domains from a costimulatory molecule and a ζ chain. Preferably, the antigen binding domain is fused with an intracellular domain of a combination of a 4-1BB signaling domain and a CD3ζ signaling domain.

[0272] As used herein, "antigen binding domain" and "single-chain antibody fragment" all refer to Fab fragments, Fab' fragments, F(ab')2 fragments, or single Fv fragments with antigen binding activity. Fv antibodies contain the variable region of the antibody heavy chain and the variable region of the light chain, but no constant region, and are the smallest antibody fragments with all antigen binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains, and are capable of forming the structure required for antigen binding. The antigen binding domain is usually scFv (single-chain variable fragment). The size of scFv is generally 1 / 6 of a complete antibody. A single-chain antibody is preferably an amino acid chain sequence encoded by a nucleotide chain. As a preferred embodiment of the present invention, the scFv specifically recognizes B7-H3.

[0273] For hinge region and transmembrane region (transmembrane domain), CAR can be designed to include a transmembrane domain fused to the extracellular domain of CAR. In one embodiment, a transmembrane domain naturally associated with one of the domains in CAR is used. In some examples, a transmembrane domain can be selected, or modified by amino acid replacement to avoid binding such a domain to the transmembrane domain of the same or different surface membrane proteins, So as to minimize the interaction with other members of the receptor complex.

[0274] The intracellular domain in the CAR of the present invention includes the signaling domain of 4-1BB and the signaling domain of CD3ζ.

[0275] Carrier

[0276] The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening a library from a cell expressing the gene, by obtaining the gene from a known vector comprising the gene, or by directly isolating from cells and tissues comprising the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically.

[0277] The present invention also provides vectors into which the expression cassette of the present invention is inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia viruses because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.

[0278] In brief summary, the expression cassette or nucleic acid sequence of the present invention is usually operably connected to a promoter and incorporated into an expression vector. The vector is suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

[0279] The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0280] The nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be cloned into such vectors, which include but are not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors.

[0281] Further, expression vectors can be provided to cells in the form of viral vectors. Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and slow viruses. Typically, suitable vectors are included in at least one organism that works on a replication origin, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent number 6,326,193).

[0282] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to a subject's cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0283] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. Typically, these are located in the 30-110bp region upstream of the start site, although recently it has been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible, so that when an element is inverted or moved relative to another, the promoter function is maintained. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50bp apart, and activity begins to decline. Depending on the promoter, it is shown that a single element can work cooperatively or independently to start transcription.

[0284] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence that can drive any polynucleotide sequence operably connected thereto to express at a high level. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr (Epstein-Barr) virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the present invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered to be part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0285] In order to evaluate the expression of CAR polypeptides or parts thereof, the expression vector introduced into the cell may also include any one or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a cell population seeking to be transfected or infected by a viral vector. In other aspects, selectable markers may be carried on a single DNA segment and used for co-transfection procedures. Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences so that they can be expressed in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

[0286] Reporter gene is used to identify the cells of potential transfection and to evaluate the functionality of regulatory sequences. Generally, reporter gene is the following gene: it is not present in or expressed by a receptor organism or tissue, and it encodes a polypeptide whose expression is clearly represented by some easily detectable properties such as enzymatic activity. After DNA has been introduced into the receptor cell, the expression of the reporter gene is measured at the appropriate time. Suitable reporter gene can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein (for example, Ui-Tei et al., 2000FEBS Letters479:79-82). Suitable expression systems are well known and can be prepared using known techniques or commercially available. Generally, the construct with minimum 5 flanking regions showing the highest level of reporter gene expression is identified as a promoter. Such a promoter region can be connected to a reporter gene and used to evaluate the ability of a reagent to regulate promoter-driven transcription.

[0287] Methods for introducing genes into cells and expressing genes into cells are known in the art. In the context of expression vectors, vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical or biological means.

[0288] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0289] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used methods for inserting genes into mammalian cells such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0290] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0291] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. Consider using lipid formulations to introduce nucleic acid into a host cell (in vitro, ex vivo or in vivo). On the other hand, The nucleic acid can be associated with a lipid. Nucleic acids associated with lipids can be encapsulated in the aqueous interior of the liposome, Dispersed in the lipid bilayer of the liposome, attached to the liposome through a connecting molecule associated with both the liposome and the oligonucleotide, trapped in the liposome, compounded with the liposome, dispersed in a solution comprising lipids, mixed with lipids, combined with lipids, included in lipids as a suspension, included in micelles or compounded with micelles, Or otherwise associated with lipids. The lipids, lipid / DNA or lipid / expression vectors associated with the composition are not limited to any specific structure in the solution. For example, they can be present in a bilayer structure, as micelles or have a "collapsed (collapsed)" structure. They can also be simply dispersed in the solution, and may form aggregates of size or shape inhomogeneity. Lipid is a fatty substance, which can be a naturally occurring or synthetic lipid. For example, lipids include fat droplets that occur naturally in the cytoplasm as well as compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0292] In a preferred embodiment of the present invention, the vector is a lentiviral vector.

[0293] Therapeutic applications of CART

[0294] The present invention includes therapeutic applications of cells (e.g., T cells) transduced with a lentiviral vector (LV) encoding the expression cassette of the present invention. The transduced T cells can target the tumor cell marker B7-H3, synergistically activate T cells, and induce a T cell immune response, thereby significantly improving its efficiency in killing tumor cells.

[0295] Therefore, the present invention also provides a method for stimulating a T cell-mediated immune response to a target cell population or tissue in a mammal, comprising the following steps: administering the CAR-T cell of the present invention to the mammal.

[0296] In one embodiment, the present invention includes a type of cell therapy that separates the patient's autologous T cells (or allogeneic donors), activates and genetically modifies them to produce CAR-T cells, which are then injected into the same patient. This approach has a very low probability of graft-versus-host disease, and antigens are recognized by T cells in an MHC-free manner. In addition, one CAR-T can treat all cancers that express the antigen. Unlike antibody therapy, CAR-T cells can replicate in vivo, producing long-term persistence that can lead to sustained tumor control.

[0297] In one embodiment, the CAR-T cells of the present invention can undergo robust in vivo T cell expansion and can sustain an extended amount of time. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy procedure, wherein the CAR-modified T cells induce an immune response specific to the antigen binding domain in the CAR. For example, anti-B7-H3 CAR-T cells cause a specific immune response against cells expressing B7-H3.

[0298] Although the data disclosed herein specifically discloses a lentiviral vector comprising an anti-B7-H3 scFv, hinge and transmembrane regions, and 4-1BB and CD3ζ signaling domains, the present invention should be construed to include any number of variations to each of the construct components.

[0299] Treatable cancers include tumors that are not vascularized or substantially not vascularized, and vascularized tumors. Cancer may include non-solid tumors (such as hematological tumors, such as leukemia and lymphoma) or may include solid tumors. The types of cancer treated with the CAR of the present invention include, but are not limited to, cancer, blastoma and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, cancers and melanomas. Also included are adult tumors / cancers and childhood tumors / cancers.

[0300] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, myelo-monocytic, monocytic, and erythroleukemias), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphomas, Hodgkin's disease, non-Hodgkin's lymphomas (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0301] Solid tumors are abnormal masses of tissue that usually do not contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcomas, myxosarcoma, liposarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer, ovarian cancer, etc.

[0302] The CAR-modified T cells of the present invention can also be used as a type of vaccine for ex vivo immunization and / or in vivo therapy of mammals. Preferably, the mammal is a human.

[0303] For ex vivo immunization, at least one of the following occurs in vitro prior to administering the cells into a mammal: i) expanding the cells, ii) introducing a nucleic acid encoding a CAR into the cells, and / or iii) cryopreserving the cells.

[0304] Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from mammals (preferably humans) and genetically modified (i.e., in vitro transduction or transfection) with vectors expressing the CAR disclosed herein. CAR-modified cells can be administered to a mammalian recipient to provide therapeutic benefits. The mammalian recipient can be a human, and the CAR-modified cells can be autologous relative to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic relative to the recipient.

[0305] In addition to the use of cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

[0306] The present invention provides a method for treating a tumor, comprising administering a therapeutically effective amount of the CAR-modified T cells of the present invention to a subject in need thereof.

[0307] The CAR-modified T cells of the present invention may be administered alone or as a pharmaceutical composition with a diluent and / or in combination with other components such as IL-2, IL-17 or other cytokines or cell populations. Simply put, The pharmaceutical composition of the present invention may include a target cell population as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The composition of the present invention is preferably formulated for intravenous administration.

[0308] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease-although the appropriate dosage can be determined by clinical trials.

[0309] When an "immunologically effective amount", "anti-tumor effective amount", "tumor-suppressive effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, who takes into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. It can be generally stated that the pharmaceutical composition comprising the T cells described herein can be administered in an amount of 10 4 Up to 10 9 The dosage is preferably 10 cells / kg body weight. 5 Up to 10 6 The T cell composition can also be administered at these doses multiple times. The cells can be administered by using well-known injection techniques in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dose and treatment regimen for a specific patient can be easily determined by a medical technician by monitoring the patient's disease signs and adjusting the treatment accordingly.

[0310] Administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous (iv) injection or intraperitoneally. In one embodiment, the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by iv injection. The composition of the T cell can be injected directly into the tumor, lymph node or infection site.

[0311] In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art to expand T cells to therapeutic levels are administered to patients in combination with any number of related treatment forms (e.g., before, at the same time, or after), including but not limited to treatment with the following agents: agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or efavirenz treatment for psoriasis patients or other treatments for PML patients. In further embodiments, the T cells of the present invention can be used in combination with chemotherapy, radiation, immunosuppressants, such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunotherapeutic agents. In further embodiments, the cell compositions of the present invention are administered to patients in combination with bone marrow transplantation, using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide (e.g., before, at the same time, or after). For example, in one embodiment, the subject can undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives an infusion of the expanded immune cells of the invention following transplantation. In an additional embodiment, the expanded cells are administered prior to or after surgery.

[0312] The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The dosage ratio for human administration can be implemented according to practices accepted in the art. Typically, 1×10 6 to 1×10 10 The modified T cells of the present invention are administered to the patient, for example, by intravenous infusion.

[0313] CART preparations

[0314] The present invention provides a preparation containing the CAR-T cells of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the preparation is a liquid preparation. Preferably, the preparation is an injection. Preferably, the concentration of the CAR-T cells in the preparation is 1×10 3 -1×10 8 cells / ml, preferably 1×10 4 -1×10 7 cells / ml.

[0315] In one embodiment, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, etc.; a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or an amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The formulation of the present invention is preferably formulated for intravenous administration.

[0316] Pharmaceutical composition

[0317] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein or its ADC or corresponding CAR-T cell, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary with the properties of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0318] The antibody of the present invention can also be expressed in cells by nucleotide sequences for cell therapy, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.

[0319] The pharmaceutical composition of the present invention can be directly used to bind to TF protein molecules, and thus can be used to prevent and treat diseases such as tumors. In addition, other therapeutic agents can also be used simultaneously.

[0320] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned monoclonal antibody (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used with other therapeutic agents.

[0321] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms / kg body weight, and in most cases does not exceed about 50 milligrams / kg body weight, preferably the dosage is about 10 micrograms / kg body weight to about 20 milligrams / kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

[0322] Test Uses and Kits

[0323] The antibodies or conjugates thereof of the present invention can be used in detection applications, such as for detecting samples, thereby providing diagnostic information.

[0324] In the present invention, the sample (specimen) used includes cells, tissue samples and biopsy specimens. The term "biopsy" used in the present invention should include all types of biopsies known to those skilled in the art. Therefore, the biopsy used in the present invention may include, for example, a resection sample of a tumor, a tissue sample prepared by an endoscopic method or a puncture or needle biopsy of an organ.

[0325] Samples used in the present invention include fixed or preserved cell or tissue samples.

[0326] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further comprises a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be fixed to a detection plate.

[0327] The present invention also provides uses of the antibodies of the present invention, such as use in preparing diagnostic preparations, or preparing drugs for preventing and / or treating B7-H3 positive tumors.

[0328] The main advantages of the present invention include:

[0329] (a) The CAR-T cells of the present invention have good anti-tumor activity against gastric cancer, breast cancer, osteosarcoma, neuroblastoma, colon cancer and pancreatic cancer in vivo and in vitro;

[0330] (b) The CAR-T cells of the present invention have good and reliable safety in treating solid tumors;

[0331] (c) The CAR-T cells of the present invention have a high degree of activation, and co-incubation with B7-H3 antigen-positive tumor cells can significantly upregulate the expression of CAR-T cell activation molecules CD25 and CD69 and inhibitory signaling molecule PD-1.

[0332] (d) The CAR-T cells of the present invention can significantly inhibit the growth and proliferation of tumor cells in mice bearing gastric cancer, osteosarcoma, neuroblastoma, and colon cancer cell lines, and have a significant in vivo anti-tumor effect.

[0333] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0334] General Materials and Methods:

[0335] Cell lines

[0336] K562 cells, Raji cells, h4IgB7H3-Raji cells, h2IgB7H3-K562 cells, m2IgB7H3-Raji cells and h2IgB7-H3-CHO-K1 recombinant cells were provided by Aikangde Biomedical Technology (Suzhou) Co., Ltd. 293T cells, T47D cells, MDA-MB-231 cells, MDA-MB-468 cells and AGS cells were purchased from ATCC. HGC-27 cells, KATO III cells, SUN-1 cells, MG63 cells, HOS cells, and cells were purchased from the Institute of Cell Biology, Chinese Academy of Sciences, Shanghai. K562 cells, Raji cells, h4IgB7H3- Raji cells, h2IgB7H3- K562 cells, m2IgB7H3- Raji cells, h2IgB7H3- CHO-K1 recombinant cells, HGC-27 cells, KATO III cells, and SUN-1 cells were cultured in 1640 medium containing 10% fetal bovine serum. 293T cells, T47D cells, MDA-MB-231 cells, MDA-MB-468 cells, HOS cells, MG63 cells, and AGS cells were cultured in DMEM medium containing 10% fetal bovine serum. All of the above cells were cultured in a 37°C incubator containing 5% CO2.

[0337] Antibody Screening

[0338] Five Balb / c mice were intraperitoneally injected with 5×10 6The mice were immunized with h2IgCHO-K1-CD276 recombinant cells (provided by Aikangde Biomedical Technology (Suzhou) Co., Ltd.), and the injection was repeated every 14 days. Ten days after the third intraperitoneal injection, 100 μl of blood was collected from each mouse through the tail vein, and the B7-H3 antibody immune titer in the serum of each mouse was detected using the Elisa method. The mice with the best immune titer were selected and 5×10 6 Recombinant cell lines were used for impact immunization. Three days after immunization, the mouse spleen was isolated for hybridoma fusion. The mouse with the best immune titer test was killed by cervical dislocation, and the mouse spleen was obtained under sterile conditions. The B cell single cell suspension was prepared and mixed with SP2 / 0 myeloma cells at a ratio of 1:1. The cells were fused using the Bio-rad Gene Pulser electroporation system. After fusion, the cells were resuspended in DMEM medium containing 20% ​​fetal bovine serum and HAT, and inoculated into 96-well plates. About 10 days after fusion, the HT medium was changed. After two days of culture, the supernatant was taken to detect specific antibodies. The removed culture supernatant was incubated with the ELISA well plate pre-coated with the target antigen, and identified according to the standard ELISA operation steps. The wells with higher OD values ​​were selected for the second and third rounds of subcloning screening, and the B7-H3 antibody titer in the supernatant of each round of screening cells was detected by ELISA. Take 5×10 6 Total RNA was extracted from the hybridoma cells obtained by the final screening. After the total RNA was reverse transcribed into cDNA samples using a reverse transcription kit, the heavy and light chain variable regions of the antibody were amplified by PCR using hybridoma sequencing primers, and then TA cloning was performed. The fragments obtained by PCR were subcloned into the pMD-19T vector. After blue-white screening, 10 clones were selected for each chain for sequencing. The antibody sequences finally obtained were analyzed.

[0339] Antibody humanization

[0340] Humanized antibody sequence design: Humanized design was performed based on the amino acid sequence information of the heavy and light chains of the target antibody obtained by the above sequencing, and the CDR region sequence of the original antibody was kept unchanged. According to the results of germline alignment and the results of antibody structure simulation, different humanized antibody templates were selected for the heavy and light chains, and the framework regions after humanization were reverse mutated. Five light chain and five heavy chain humanized antibody candidate sequences were designed, and the heavy and light chain antibody sequences were subcloned into pcDNA3.1-IgG1Fc and pcDNA3.1-IgKc expression vectors respectively after gene synthesis. The constructed light and heavy chain humanized vectors were respectively transferred into 293F-SVP16 cells using the LVTransm transfection method. After three days of continuous culture, the culture supernatant was collected and filtered through a 0.45μm filter membrane, and the filtrate was transferred to a sterile centrifuge tube for flow cytometry detection. Each humanized antibody was incubated with CHO-K1 cells and h2IgB7-H3-CHO-K1 recombinant cells at room temperature for 1h. Wash with PBS three times, then add 2μL PE-labeled Anti-human IgG to the corresponding groups, mix thoroughly, and incubate at room temperature in the dark for 30min. After incubation, wash with PBS three times. Add 500μL PBS to each group to resuspend the cells, and then perform flow cytometry analysis. Based on the flow cytometry results, humanized antibodies with better activity were screened and their affinity was tested. The B7-H3-His recombinant protein was fixed on the CM5 chip using 10mM acetate coupling buffer, and the humanized antibodies and human-mouse chimeric antibodies prepared by flow cytometry were used as the mobile phase to detect the binding ability of the antibodies before and after humanization to the target protein B7-H3 antigen.

[0341] Vector construction

[0342] In this experiment, mouse anti-human B7-H3 antibodies were successfully screened, and two humanized antibodies with equivalent activity were obtained through humanization: αhB7-H3 and βhB7-H3. Using the mouse antibody and the single-chain variable regions of these two humanized antibodies, the mB7-H3-CAR, αhB7-H3 CAR and βhB7-H3 CAR structures were designed, respectively, and synthesized by gene synthesis. Subsequently, the synthesized mB7-H3-CAR, αhB7-H3 CAR and βhB7-H3 CAR sequences were subcloned into the pCAR-AT-Free lentiviral vector, respectively, and named αhB7-H3 CAR vector lentivirus and βhB7-H3 CAR lentiviral vector, respectively.

[0343] Lentivirus preparation

[0344] The mB7-H3-CAR, αhB7-H3 CAR lentivirus and βhB7-H3 CAR lentivirus were prepared using the 293T-S suspension cell PEI lentivirus packaging method, and the lentivirus stock solution was harvested 48 hours after plasmid transfection. After centrifugation of the stock solution, it was filtered using 0.22μm. The filtered virus was concentrated by ultracentrifugation. After concentration, the supernatant was discarded, and DMEM medium containing 0.5% DNase I was added, and the virus was resuspended by vortexing. The virus reselection solution was collected, centrifuged to remove impurities, and the virus resuspension solution was aliquoted and frozen in a -80℃ refrigerator for later use.

[0345] Construction of mB7-H3-CAR, αhB7-H3 CAR-T cells and βhB7-H3CAR-T cells

[0346] The peripheral blood used in the present invention is provided by healthy volunteers. PBMC is extracted by Ficoll gradient centrifugation. The obtained PBMC is resuspended in TexMACA GMP Medium containing IL-7 and IL-15 and inoculated into a 24-well plate, and 20μl TransAct reagent is added to each well for activation. After 48h, the activated and amplified T cells are collected, centrifuged and resuspended and inoculated into a 48-well plate, and the corresponding amount of virus is added with an MOI of 20, and the volume is adjusted to 100μl. After 12h, the supernatant is removed by centrifugation, and fresh culture medium is added to continue culturing the cells. Seven days after transfection, the cells are collected and washed 3 times with PBS. 100μl of tEGFR antibody (final antibody concentration: 1ug / ml) diluted in advance with PBS at 1:1000 is added, and the cells are incubated at room temperature for 30min after resuspending. After the incubation is completed, the supernatant is discarded by centrifugation and washed 3 times with PBS. 500μl PBS is added to resuspend the cells and detected by flow cytometry.

[0347] αhB7-H3 CAR-Jurkat T cell construction

[0348] 5×10 5 Jurkat T cells were inoculated in a 48-well plate, and the corresponding amount of virus was added at an MOI of 20, and the culture system was adjusted to 200μl. After 12h, the cells were collected by centrifugation, resuspended in fresh culture medium, and re-inoculated in a 24-well plate for continued culture. Five days after transfection, the cells were collected and washed three times with PBS. 100μl of tEGFR antibody pre-diluted with PBS at 1:1000 was added, resuspended and incubated at room temperature for 30min. After the incubation, centrifuged and discarded the supernatant, and washed three times with PBS. 500μl of PBS was added to resuspend the cells and detected by flow cytometry.

[0349] αhB7-H3 CAR-Jurkat T cell specific activation experiment

[0350] HGC-27, AGS and KATO III cells were collected by trypsin digestion, washed three times with PBS, and resuspended in CFSE solution diluted with PBS 1:1000, and incubated at 37°C for 15 min. The cells were centrifuged and discarded the supernatant, washed three times with PBS, and then resuspended in culture medium and the cell density was adjusted to 4×10 5 / ml. The above tumor cell suspension was inoculated into a 24-well plate at 500μl / well. The corresponding effector cells were added to each well at a ratio of 1:1, and the final culture system was adjusted to 1ml / well, and placed in a CO2 incubator for co-culture for 24h. Three replicate wells were set up for each experimental group. The cells in each well were collected for 24h, and 100μl of the supernatant from each well was collected after centrifugation for cytokine detection. The remaining supernatant was discarded, washed 3 times with PBS, and then resuspended with 100μl PBS respectively. 1μl of PD-1 antibody, CD69 antibody and CD25 antibody were added to the corresponding tubes, mixed, and incubated at 37°C for 30min. After 30min, centrifuge and discard the supernatant, wash 3 times with PBS, add 500μl PBS to each tube to resuspend the cells, and perform detection and analysis on a flow cytometer.

[0351] Tumor cell antigen expression B7-H3 antigen expression detection

[0352] After collecting each cell to be tested by centrifugation, discard the supernatant and wash 3 times with PBS. Resuspend the cells with 100μl PBS solution, add 1μl αhB7-H3 antibody, and incubate at 37℃ for 30min. After 30min, centrifuge and discard the supernatant, wash three times with PBS, then add 100μl IgG1-Fc antibody pre-diluted with PBS 1:1000 to resuspend the cells and incubate at 37℃ for 30min. The experiment was designed with blank control tube, secondary antibody control tube and detection tube. After 30 min, centrifuge and discard the supernatant, wash 3 times with PBS. Resuspend the cells with 500μl PBS and analyze on flow cytometer.

[0353] In vitro cytotoxicity assay

[0354] CFSE / 7-AAD flow cytometry: Collect tumor target cells, wash three times with PBS, label with pre-diluted CFSE (hydroxyfluorescein diacetate succinimidyl lipid) solution, and incubate at 37°C for 15 min. Collect cells by centrifugation, discard the supernatant, and wash three times with PBS. Add culture medium to resuspend cells and adjust the cell density to 2×10 5 / mL, inoculated in a 24-well plate. According to the set effector-target ratio, add the corresponding effector cells, mix well, and culture in a CO2 incubator for 24 hours. Set up 3 replicates for each experimental group. Collect the cells after 24 hours, centrifuge and discard the supernatant, wash 3 times with PBS, and then resuspend the cells with 500μl PBS. Add 1μl 7-AAD (7-aminoactinomycin D) to the corresponding tube and perform detection and analysis on a flow cytometer. CFSE and 7-AAD double-positive cells represent dead tumor cells, based on which the killing efficiency of the corresponding effector cells can be calculated.

[0355] RTCA method: collect target cells by centrifugation and adjust the cell density to 2×10 5 / ml. Add culture medium to the E-Plate at 50μl / well for baseline balance. After balance, add target cell suspension to the E-Plate at 50μl / well and let it stand in the biosafety cabinet for 30min. After 30min, put it into the RTCA instrument and collect data every 5 minutes. After 24 hours, add effector cells according to the E:T ratio, adjust the cell culture system to 200μl per well, put it back into the RTCA instrument for continued monitoring, and collect data every 15min.

[0356] Cytokine detection

[0357] The cell co-culture supernatants in each cytotoxicity experiment were collected, and the secretion of interferon gamma (IFN-γ), granzyme B, tumor necrosis factor alpha (TNF-α), and IL-2 of each effector cell was detected and analyzed according to the CBA detection method. The human granzyme B-CBA Flex Set D7 kit, human IFN-γ CBA Flex Set E7 kit, human IL-2 CBA Flex Set A4, and human TNF-CBA Flex Set D9 used for cytokine detection were purchased from BD.

[0358] Animal models

[0359] The present invention constructs a variety of xenogeneic tumor transplantation NOD-Prkdcs-cidIl2rgtm1 / Bcgen (B-NSG) mouse models to evaluate the effectiveness and safety of αhB7-H3 CAR-T cell therapy. All experimental animals used in the study were 6-8 week old magnetic B-NSG mice.

[0360] Establishment of HGC-27 gastric cancer xenograft NSG mouse tumor model: 1×10 6 HGC-27 cells were inoculated subcutaneously on the right side of the back of mice, and the weight and tumor size of mice were measured twice a week.3 At the same time, the mice were randomly divided into 3 groups: PBS group, T cell group, and αhB7-H3 CAR-T cell group, with 5 mice in each group. Then, 1x10 7 The corresponding effector cells were injected into the tumors of mice in the PBS group at the same volume as the effector cell suspension. 3 At 14:00, mice in each group were killed and dissected to observe the changes in organ coefficients and tumor size. In addition, before killing mice, blood was collected from the orbital vein of each group of mice for five-category routine blood analysis.

[0361] Establishment of MG63 osteosarcoma xenograft NSG mouse tumor model: 5×10 6 MG63 cells mixed with matrix gel were inoculated subcutaneously on the right side of the back of mice, and the mice were weighed twice a week. 3 At the same time, the mice were randomly divided into 3 groups: PBS group, T cell group and αhB7-H3 CAR-T cell group, with 5 mice in each group. Then, 1×10 7 The corresponding effector cells were injected into the tumors of mice in the PBS group with an equal volume of PBS as the effector cell suspension. The weight changes and survival period were continuously observed. On the 108th day of treatment, the mice in each group were killed and the number and size of tumors in the mice were counted.

[0362] Data analysis

[0363] The data obtained in the present invention were statistically analyzed using GraphPad Prism5 software. Student's test was used for analysis, and a P value less than 0.05 was considered to be statistically significant. *, **, *** and **** represent P<0.05, <0.01, <0.001 and <0.0001, respectively, to show the degree of difference between the corresponding two groups of data.

[0364] Example 1 Screening and humanization of B7-H3 monoclonal antibodies

[0365] Five Balb / c mice were immunized every 14 days using B7-H3-CHO-K1 recombinant cells. Ten days after the third immunization, the B7-H3 antibody titer in the serum of each mouse was tested, and the mouse with the best antibody titer was screened out, and its spleen cells were fused with hybridoma cells. Subsequently, through two rounds of cloning screening, mouse anti-human mB7-H3 antibodies with strong binding ability to the antigen were successfully obtained, and their antibody sequences were obtained by hybridoma cell sequencing.

[0366] According to the amino acid sequences of the heavy and light chains of the mB7-H3 antibody, the humanized design was performed using a CDR transplantation scheme. The designed humanized candidate antibodies were gene synthesized, and the humanized heavy chain variable region was subcloned into the pcDNA3.1-IgG1Fc expression vector, and the humanized light chain variable region was subcloned into the pcDNA3.1-IgKc expression vector ( Figure 1A ). The constructed humanized light chain and heavy chain expression vectors were combined in pairs, and transiently co-transfected into 293F cells (cultured without serum). After 72 hours of transfection, the expression supernatant was collected for FACS detection.

[0367] The results showed that the humanized antibodies constructed in this example can bind to the recombinant 2hIgB7-H3-CHO-K1 cells ( Figure 1B ), and the binding force was comparable to that of the chimeric antibody. Two humanized antibodies, T1H1-T1L1 and T2H1-T2L1, were selected for affinity testing, and the results are shown in Table 1. The T1H1-T1L1 and T2H1-T2L1 antibodies were named αhB7-H3 and βhB7-H3 antibodies, respectively.

[0368] Table 1

[0369] Ka(M-1s-1) Kd(s-1) KD(M) mB7-H3 <![CDATA[3.643×10 4 ]]> 0.002581 <![CDATA[7.083×10 -8 ]]> B7-H3 T1H1-T1L1 <![CDATA[2.482×10 4 ]]> 0.002823 <![CDATA[1.138×10 -7 ]]> B7-H3 T2H1-T2L1 <![CDATA[3.966×10 4 ]]> 0.002887 <![CDATA[7.279×10 -8 ]]>

[0370] The sequence numbers of the main antibodies involved in this example are shown in Table 2.

[0371] Table 2

[0372]

[0373] Example 2 Recognition of human B7-H3 antigen and mouse B7-H3 antigen by αhB7-H3 antibody

[0374] After obtaining the antibody αhB7-H3, h4IgB7H3-Raji cells, h2IgB7H3-K562 cells and m2IgB7H3-Raj i cells, which express two subtypes of human B7-H3 and one subtype of mouse B7-H3, were used to study whether the αhB7-H3 antibody could bind to various antigens.

[0375] The results are as follows Figure 2 As shown, the commercial B7-H3 antibody (Biolegend, cat: 351006) can only bind to the corresponding B7-H3 subtype, while the B7-H3 antibody of the present invention can simultaneously recognize two B7-H3 antigen subtypes of humans and the B7-H3 antigen of mice. This result shows that the B7-H3 of the present invention has a wider antigen recognition ability, which can greatly facilitate the verification of its safety in mouse models.

[0376] Example 3 αhB7-H3 CAR-Jurkat T cells can specifically activate B7-H3 antigen-positive tumor cells

[0377] To further study the specificity of αhB7-H3 CAR molecules, αhB7-H3 CAR-Jurkat T cells were constructed by lentiviral transfection, and the positive rate of flow cytometry was 92.84% ( Figure 3A ).

[0378] In this experiment, three gastric cancer cells, HGC-27, AGS and KATO-III, were used to verify the specificity of αhB7-H3 CAR- Jurkat T cells. Figure 3B shown.

[0379] Jurkat T and αhB7-H3-CAR-Jurkat T cells were co-incubated with CFSE-labeled HGC-27, AGS, and KATO-III cells at a target ratio of 1:1 for 24 hours. Jurkat T cells and αhB7-H3-CAR-Jurkat cells in each group were then collected to detect the expression of CD69, CD25, and PD-1 on the cell surface.

[0380] The results showed that after co-incubation with HGC-27 cells and AGS cells, the expression of CD69, CD25 and PD-1 molecules were significantly upregulated compared with Jurkat cells, while after co-incubation with KATOⅢ cells, the expression of CD69, CD25 and PD-1 on Jurkat T and B7-H3 CAR-JurkatT cells was not significantly upregulated (Figure 3C). In addition, the results of cytokine content detection in the supernatant showed that after co-incubation with HGC-27 cells, the secretion capacity of IL-2 and granzyme B of αhB7-H3 CAR-Jurkat T cells was significantly enhanced, while this phenomenon was not found in the KOTA Ⅲ co-incubation group ( Figure 3D ). These results indicate that the activation of αhB7-H3-CAR-Jurkat T cells is highly antigen-dependent.

[0381] Example 4 Construction of B7-H3 CAR-T cells

[0382] The second-generation mB7-H3 CAR, αhB7-H3 CAR, and βhB7-H3 CAR were constructed using the single-chain variable regions of the mouse mB7-H3 antibody, humanized αB7-H3, and βhB7-H3 antibodies, respectively. Their structures are shown in Figure 4A As shown, tEGFR protein can be used to detect the expression rate of CAR molecules, which are then subcloned into a lentiviral vector ( Figure 4B ). Subsequently, mB7-H3 CAR-T cells, αhB7-H3 CAR- T cells, and βhB7-H3 CAR T cells were constructed by lentiviral transfection. On the tenth day of transfection, mouse anti-human tEGFR antibody was used to stain each B7-H3 CAR-T cell, and its positive rate was detected by flow cytometry.

[0383] The results showed that the positive rates of mB7-H3 CAR-T cells, αhB7-H3 CAR-T cells, and βhB7-H3 CAR T cells were 19.13%, 17.78%, and 17.94%, respectively. Figure 4C ).

[0384] Example 5 Comparison of cytotoxicity of αhB7-H3 CAR-T cells and βhB7-H3 CAR-T cells

[0385] The RTCA method was used to compare the growth inhibitory effects of αhB7-H3 CAR-T cells and βhB7-H3 CAR-T cells on pancreatic cancer SW1990 cells and PANC-1.

[0386] The antigen expression of B7-H3 in two pancreatic cancer lines is as follows Figure 5A The results showed that compared with T cells, mB7-H3CAR-T cells, αhB7-H3 CAR-T cells and βhB7-H3 CAR-T cells had specific inhibitory effects on SW1990 cells and PANC-1, and mB7-H3 CAR-T cells and αhB7-H3 CAR-T cells had stronger inhibitory effects ( Figure 5B ).

[0387] The results of the cytokine content test in the experimental supernatant also showed that mB7-H3 CAR-T cells and αhB7-H3CAR-T cells had stronger cytokine secretion ability than βhB7-H3 CAR-T cells ( Figure 5C). The above results show that αhB7-H3 CAR-T cells have better anti-tumor activity than βhB7-H3 CAR-T cells. Therefore, αhB7-H3 CAR-T cells were selected for further study in subsequent experiments.

[0388] Example 6 αB7-H3 CAR-T cells have specific cytotoxic effects on m2IgB7H3-Raji cells

[0389] The results of the antibody binding experiment showed that the αhB7-H3 antibody can recognize both human and mouse B7-H3 antigens, thus further verifying the cytotoxic effect of αB7-H3 CAR-T cells on Raji cells and m2IgB7H3-Raji cells. Raji cells naturally express the CD19 antigen, so CD19 CAR-T cells were selected as positive control cells.

[0390] The results showed that αhB7-H3 CAR-T cells only showed significant specific cytotoxicity against m2IgB7H3-Raji cells, while CD19-CAR T cells showed specific cytotoxicity against both Raji cells and m2IgB7H3-Raji cells ( Figure 6 ). The above experimental results show that αhB7-H3 CAR-T cells can specifically recognize and kill tumor cells expressing mouse B7-H3 antigen, providing an important basis for subsequent in vivo mouse experiments to evaluate the safety of αhB7-H3 CAR-T cells.

[0391] Example 7 Treatment of Gastric Cancer with αB7-H3 CAR-T Cells

[0392] B7-H3 molecules are highly expressed in tumor tissues of gastric cancer patients, and the expression of B7-H3 molecules is closely related to the metastasis of gastric cancer cells and the poor prognosis of patients. Therefore, the anti-tumor activity of αhB7-H3 CAR-T cells against gastric cancer cells was evaluated.

[0393] In the in vitro experiments, HGC-27 and KATO-III cells were co-incubated with T cells and αhB7-H3 CAR-T cells for 24 hours, and the effector-target ratios were set to 1:1, 5:1, and 10:1, respectively.

[0394] The results showed that compared with T cells, αhB7-H3 CAR-T cells had a significant specific enhanced cytotoxicity against B7-H3-positive HGC-27 cells, while αhB7-H3 CAR-T cells had no specific enhanced cytotoxicity against B7-H3-negative KATO-III cells ( Fig. 7A). In addition, the CBA method was used to analyze the contents of IL-2, TNF-α, IFN-γ and granzyme B in the supernatant of each group in the above cytotoxicity test. The results showed that after co-culture with B7-H3 positive tumor cells, the IL-2, TNF-α, IFN-γ and granzyme B secretion ability of αhB7-H3CAR-T cells was significantly stronger than that of T cells, but after co-culture with B7-H3 negative KOTA-III cells, there was no significant difference in cytokine secretion between NT and B7-H3 CAR-T cells ( Figure 7B ).

[0395] The RTCA method was also used to evaluate the in vitro antitumor activity of αhB7-H3 CAR-T cells. The results showed that αhB7-H3 CAR-T cells prepared from three PBMC samples could effectively inhibit the growth of HGC-27 and AGS cells ( Fig. 12A , only one set of data is shown). At the same time, the results of supernatant cytokine detection also showed that αhB7-H3 CAR-T cells have significantly enhanced cytokine secretion ability ( Fig. 12B )

[0396] In the in vivo experiment, HGC-27 cells were used to establish a xenograft tumor NSG mouse model. The experimental process is as follows Fig. 8A As shown. 6 HGC-27 cells were injected subcutaneously into 7-week-old female NSG mice. Tumor size and weight were measured twice a week. When the average tumor volume reached 100 mm 3 The mice were randomly divided into 3 groups: PBS group, T cell group, and CAR-T cell group, with 5 mice in each group. The same volume of PBS, 1×10 7 T cells and 1×10 7 CAR-T cells.

[0397] The results showed that compared with the PBS group and the T cell group, the tumor growth of mice in the αhB7-H3 CAR-T cell group was significantly inhibited ( Figure 8B -D). During the treatment, there was no significant change in the body weight of mice in the αhB7-H3 CAR-T cell group ( Fig. 8E ), and the coefficients of other organs except the liver were not significantly different from those of other groups of mice ( Fig.8F In addition, the results of the blood routine test of mice showed that there was no significant difference in the blood routine parameters of each group of mice ( Figure 8G ).

[0398] The above experimental results show that αhB7-H3 CAR-T cells have good safety and effectiveness in treating gastric cancer cell model mice.

[0399] Example 8 Treatment of triple-negative breast cancer with αB7-H3 CAR-T cells

[0400] B7-H3 antigen is highly expressed in metastatic and malignant breast cancer tissues, so the anti-tumor activity of αhB7-H3 CAR-T cells against triple-negative breast cancer was further evaluated.

[0401] Two breast cancer cell lines, T47D and MDA-MB-468, were selected as target cells. MDA-MB-468 is a triple-negative breast cancer cell. First, the expression of B7-H3 on the surface of these two breast cancer cell lines was detected. The flow cytometry results showed that both cell lines highly expressed B7-H3 antigen ( Fig. 9A ). Then, the RTCA method was used to verify the inhibitory effect of αhB7-H3 CAR-T cells on the growth of two breast cancer cells. The results showed that compared with T cells, the effector-target ratio of 1:1 and 5:1 could significantly inhibit the growth of two breast cancer cells ( Fig. 9B ). At the same time, the results of the test on cytokine content in the supernatant of this experiment showed that co-incubation with two breast cancer cells could significantly enhance the secretion of IL-2, TNF-α, IFN-γ and granzyme B ( Fig. 9C ). The above experimental results show that αhB7-H3 CAR-T cells have significant specific cytotoxic effects on breast cancer cells in vitro.

[0402] Example 9 B7-H3 CAR-T cells for the treatment of osteosarcoma

[0403] The B7-H3 molecule is highly expressed in the tumor tissues of more than 90% of osteosarcoma patients. At the same time, the expression of B7-H3 is closely related to the recurrence of osteosarcoma and the survival of patients. Therefore, the therapeutic effect of αhB7-H3 CAR-T cells on osteosarcoma was further evaluated.

[0404] Three osteosarcoma cell lines, HOS, MG63 and SJSA-1, were selected for the study. The results of flow cytometry detection of the B7-H3 antigen expression of the three cell lines showed that the three osteosarcoma cell lines had a B7-H3 antigen expression level of nearly 100% ( Fig. 10A ). The results of RTCA assay to detect the growth inhibition of αhB7-H3 CAR-T cells on HOS cells, MG63 cells and SJSA-1 cells showed that under different effector-target ratios, αhB7-H3 CAR-T cells showed specific inhibition of the growth of these three cell lines ( Fig. 10B). At the same time, the results of the test on cytokine content in the supernatant of this experiment also showed that after co-incubation with HOS cells, MG63 cells and SJSA-1 cells, the secretion of IL-2, IFN-γ and granzyme B of αhB7-H3 CAR-T cells was significantly increased compared with that of T cells ( Fig. 10C ). The above experimental results show that αhB7-H3 CAR-T cells have significant anti-tumor activity against osteosarcoma in vitro.

[0405] In the in vivo experiments, MG63 cells were used to establish the xenograft tumor NSG mouse model. Fig.11A On day 0, 5×10 6 MG63 cells mixed with matrix gel were subcutaneously injected into the right back of NSG mice, and the tumor size and body weight of the mice were measured twice a week. When the average tumor volume reached 100 mm3, they were randomly divided into 3 groups: PBS group, T cell group, and αhB7-H3 CAR-T cell group, with 5 mice in each group. Each group of mice was injected intratumorally with PBS equal to the volume of effector cell suspension, 1×10 7 T cells and 1×10 7 αhB7-H3 CAR-T cells.

[0406] The results showed that compared with PBS and T cells, αhB7-H3 CAR-T cells can effectively inhibit the growth of osteosarcoma cells ( Fig. 11B ), and the weight of mice in the αhB7-H3 CAR-T cell group did not change significantly during the treatment. However, the weight of mice in the T cell group decreased during the experiment, which may be due to the same reason as the weight loss of mice in the triple-negative breast cancer treatment experiment ( Fig. 11C ).

[0407] The above experimental results show that αhB7-H3 CAR-T cells can effectively inhibit the growth of osteosarcoma cells both in vivo and in vitro, and have good safety.

[0408] Example 10 In vivo anti-tumor effect of αhB7-H3 CAR-T on neuroblastoma cell lines

[0409] The subcutaneous tumor model of NSG mice was established using the neuroblastoma cell line SK-N-AS (B7-H3 positive cell line). Fig.13A Each mouse was inoculated with 1.5×10 6 SK-N-AS cells were inoculated into 10 mice in total and divided into 2 groups, with 5 mice in each group. Then, 2×10 7CAR-T cells (αhB7-H3 CAR-T) or T cells (Mock-T), and then the tumor size and mouse body weight were measured regularly.

[0410] The results showed that compared with the control group, αhB7-H3 CAR-T cells could significantly inhibit the growth and proliferation of tumor cells in mice (such as Fig. 13B , 13C). In addition, there was no significant change in the body weight of mice compared with the control group (e.g. Fig.13D ).

[0411] The above results indicate that the αhB7-H3 CAR-T cells constructed in the present invention have significant in vivo anti-tumor effects on the neuroblastoma cell line SK-N-AS.

[0412] Example 11 Antitumor effects of B7-H3 CAR-T on colon cancer cell lines in vitro and in vivo

[0413] The third-generation B7-H3-CAR vector containing both ICOS and 41BB co-stimulatory molecules was constructed using the B7-H3 humanized antibody sequence T1H1-T1L1. The vector structure is shown in the figure below. Fig.14 As shown, the amino acid sequence of the third-generation B7-H3-CAR is shown in SEQ ID NO: 19.

[0414] The expression of B7-H3 on the surface of colon cancer cell line SW620 was detected by flow cytometry. The results are as follows Fig.15 The results showed that the positive rate of B7-H3 in SW620 cells was 95.20%, indicating that the colon cancer cell line SW620 also highly expressed B7-H3 cells.

[0415] Then, the third-generation B7-H3-CAR-T cells were used to conduct an in vitro killing experiment on SW620. Fig.16A The results showed that under the conditions of effector-target ratios of 5:1 and 1:1, the third-generation B7-H3-CAR-T cells could significantly inhibit the growth of SW620 cells compared with the control group T cells, and the inhibitory effect on tumors under the condition of effector-target ratio of 5:1 was more obvious than that under the condition of effector-target ratio of 1:1. The above results indicate that the third-generation B7-H3-CAR-T cells have significant specific killing effect on the colon cancer cell line SW620.

[0416] Then, the release of cytokines (IL-2, TNF-α, IFN-γ, Granzyme B) in the supernatant of the above experiment was further analyzed. Fig. 16BThe results showed that compared with the control group, the release of cytokines in the B7-H3-CAR-T group was significantly increased, indicating the release of specific cytokines, further illustrating that the third-generation B7-H3-CAR-T cells constructed by the present invention can be specifically activated by SW620 cells.

[0417] In order to further evaluate the in vivo anti-tumor effect of the third-generation B7-H3-CAR-T cells constructed by the present invention on colon cancer, a tumor model of NCG mice was first constructed using Luc-GFP-SW620 cells (SW620 cell line overexpressing luciferase). The experimental design was as follows: Fig.17A Therefore, on day 0, 1 × 10 6 On the 5th day, the mice were randomly divided into 3 groups: PBS group, T cell group, and CAR-T cell group. Each mouse was infused with 2×10 7 T or CAR-T cells or an equal amount of PBS, 8 mice in each group, 4 for in vivo imaging and survival observation, and the other 4 for other efficacy and safety evaluations after sacrifice and dissection. The results showed that compared with the control group PBS and T cells, the tumor fluorescence signal in mice treated with CAR-T was significantly weakened ( Fig. 17B ,17C), indicating that the third-generation B7-H3-CAR-T cells can significantly inhibit the growth and proliferation of SW620 tumor cells in mice. Mouse survival curve ( Fig.17D ) also showed that the third-generation B7-H3-CAR-T cells significantly prolonged the survival of SW620 tumor-bearing mice.

[0418] The above results indicate that the third-generation B7-H3-CAR-T cells constructed by the present invention have significant in vivo anti-tumor effects on colon cancer cells SW620.

[0419] Example 12 In vivo antitumor effect of low-dose B7-H3 CAR-T cells combined with radiotherapy on pancreatic cancer cells

[0420] First, we detected the expression of B7-H3 on the cell surface of four pancreatic cancer cell lines PANC-1, SW1990, ASPC-1, and PATU8988. Fig.18A As shown, all four pancreatic cancer cell lines highly expressed B7-H3 molecules.

[0421] Then, the in vivo antitumor effect of low-dose third-generation B7-H3 CAR-T cells combined with X-ray irradiation on pancreatic cancer cell line SW1990 was evaluated. 6SW1990 cells were used to subcutaneously implant tumors in 6-8 week-old NCG mice. A total of 12 mice were included. Two tumor-bearing mice were selected as the control group (CONT group). Nine of the mice were subjected to local X-ray irradiation of the tumor site on the 8th day after tumor implantation (such as Fig.18B ), the doses were 2Gy*1 (2Gy irradiation once), 2Gy*4 (2Gy irradiation four times), and 8Gy*1 (8Gy irradiation once). After the irradiation, one mouse was injected with 1×10 6 The remaining unirradiated mouse was injected with 1×10 6 B7-H3-CAR-T cells were added. The size of the tumor in the mouse was then measured using a vernier caliper.

[0422] The results are as follows Fig. 18C As shown, compared with non-irradiated tumor-bearing mice, mice injected with 1×10 6 The tumor size of mice injected with CAR-T cells remained basically unchanged; the tumor size of mice irradiated with X-rays alone tended to decrease compared with the control group, but there was no significant change; and the tumors of mice further injected with low-dose B7-H3-CAR-T cells (2Gy irradiation once, 2Gy irradiation four times, 8Gy irradiation once) showed a significant trend of shrinking, especially in the combined treatment group irradiated once with 8Gy, in which basically no tumor was detected at 30 days, indicating that low-dose B7-H3 CAR-T cells combined with radiotherapy can further enhance the in vivo anti-tumor effect on pancreatic cancer cells SW1990.

[0423] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

[0424] The sequences involved in the sequence table of this application are as follows:

[0425]

[0426] Sequence Listing <110> Boshengji Pharmaceutical Technology (Suzhou) Co., Ltd. <120> B7-H3 chimeric antigen receptor modified T cells and their applications <130> P2020-0881 <160> 19 <170> SIPO Sequence Listing 1.0 <210> 1 <211> 120 <212> PRT <213> Artificial Sequence <400> 1 Glu Val Thr Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Ser Ile Leu Ser Gly Gly Thr Ser Phe Tyr Ala Asp Asn Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Arg Asn Ile Leu Tyr Leu 65 70 75 80 Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Val 85 90 95 Arg Arg Tyr Gly His Asp His Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <400> 2 Gly Phe Thr Phe Ser Thr Tyr 1 5 <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <400> 3 Leu Ser Gly Gly Thr 1 5 <210> 4 <211> 12 <212> PRT <213> Artificial Sequence <400> 4 Arg Tyr Gly His Asp His Tyr Tyr Ala Met Asp Tyr 1 5 10 <210> 5 <211> 108 <212> PRT <213> Artificial Sequence <400> 5 Gln Ile Val Leu Thr Gln Ser Pro Ala Asn Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Met Thr Cys Ala Ala Ser Ser Ser Val Ser Ser Ser 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Gly Arg Phe Asn 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro 85 90 95 Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 6 <211> 12 <212> PRT <213> Artificial Sequence <400> 6 Ala Ala Ser Ser Ser Val Ser Ser Ser Tyr Leu His 1 5 10 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <400> 7 Ser Thr Ser Asn Leu Ala Ser 1 5 <210> 8 <211> 8 <212> PRT <213> Artificial Sequence <400> 8 Gln Tyr His Arg Ser Pro Trp Thr 1 5 <210> 9 <211> 120 <212> PRT <213> Artificial Sequence <400> 9 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Leu Ser Gly Gly Thr Ser Phe Tyr Ala Asp Asn Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Arg Tyr Gly His Asp His Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 10 <211> 108 <212> PRT <213> Artificial Sequence <400> 10 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ala Ala Ser Ser Ser Val Ser Ser Ser 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Leu Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His Arg Ser Pro 85 90 95 Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 11 <211> 120 <212> PRT <213> Artificial Sequence <400> 11 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Leu Ser Gly Gly Thr Ser Phe Tyr Ala Asp Asn Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr His Cys Ala 85 90 95 Arg Arg Tyr Gly His Asp His Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 12 <211> 108 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 12 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ala Ala Ser Ser Ser Val Ser Ser Ser 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr His Arg Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 13 <211> 248 <212> PRT <213> Artificial Sequence <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Leu Ser Gly Gly Thr Ser Phe Tyr Ala Asp Asn Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Arg Tyr Gly His Asp His Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu 130 135 140 Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr 145 150 155 160 Leu Ser Cys Ala Ala Ser Ser Ser Val Ser Ser Ser Tyr Leu His Trp 165 170 175 Tyr Gln Gln Lys Pro Gly Leu Ala Pro Arg Leu Leu Ile Tyr Ser Thr 180 185 190 Ser Asn Leu Ala Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser 195 200 205 Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe 210 215 220 Ala Val Tyr Tyr Cys Gln Gln Tyr His Arg Ser Pro Trp Thr Phe Gly 225 230 235 240 Gly Gly Thr Lys Val Glu Ile Lys 245 <210> 14 <211> 248 <212> PRT <213> Artificial Sequence <400> 14 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Leu Ser Gly Gly Thr Ser Phe Tyr Ala Asp Asn Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr His Cys Ala 85 90 95 Arg Arg Tyr Gly His Asp His Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met 130 135 140 Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly Asp Arg Val Thr 145 150 155 160 Ile Thr Cys Ala Ala Ser Ser Ser Val Ser Ser Ser Tyr Leu His Trp 165 170 175 Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Thr 180 185 190 Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 195 200 205 Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe 210 215 220 Ala Thr Tyr Tyr Cys Gln Gln Tyr His Arg Ser Pro Trp Thr Phe Gly 225 230 235 240 Gln Gly Thr Lys Leu Glu Ile Lys 245 <210> 15 <211> 335 <212> PRT <213> Artificial Sequence <400> 15 Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu 1 5 10 15 Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile 20 25 30 Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe 35 40 45 Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr 50 55 60 Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn 65 70 75 80 Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg 85 90 95 Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile 100 105 110 Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val 115 120 125 Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp 130 135 140 Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn 145 150 155 160 Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu 165 170 175 Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser 180 185 190 Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu 195 200 205 Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln 210 215 220 Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly 225 230 235 240 Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro 245 250 255 His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr 260 265 270 Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His 275 280 285 Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro 290 295 300 Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala 305 310 315 320 Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met 325 330 335 <210> 16 <211> 492 <212> PRT <213> Artificial Sequence <400> 16 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu 20 25 30 Ile Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45 Thr Phe Ser Thr Tyr Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys 50 55 60 Gly Leu Glu Trp Val Ser Ser Ile Leu Ser Gly Gly Thr Ser Phe Tyr 65 70 75 80 Ala Asp Asn Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys 85 90 95 Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ala Arg Arg Tyr Gly His Asp His Tyr Tyr Ala Met 115 120 125 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 145 150 155 160 Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro 165 170 175 Gly Glu Arg Ala Thr Leu Ser Cys Ala Ala Ser Ser Ser Val Ser Ser 180 185 190 Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Leu Ala Pro Arg Leu 195 200 205 Leu Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Ile Pro Asp Arg Phe 210 215 220 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu 225 230 235 240 Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His Arg Ser 245 250 255 Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Thr Thr Thr 260 265 270 Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro 275 280 285 Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val 290 295 300 His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro 305 310 315 320 Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu 325 330 335 Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 340 345 350 Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 355 360 365 Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe 370 375 380 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu 385 390 395 400 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 405 410 415 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 420 425 430 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 435 440 445 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 450 455 460 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 465 470 475 480 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <210> 17 <211> 5 <212> PRT <213> Artificial Sequence <400> 17 Gly Gly Gly Gly Ser 1 5 <210> 18 <211> 248 <212> PRT <213> Artificial Sequence <400> 18 Glu Val Thr Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Ser Ile Leu Ser Gly Gly Thr Ser Phe Tyr Ala Asp Asn Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Arg Asn Ile Leu Tyr Leu 65 70 75 80 Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Val 85 90 95 Arg Arg Tyr Gly His Asp His Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Val Leu 130 135 140 Thr Gln Ser Pro Ala Asn Met Ser Ala Ser Leu Gly Glu Arg Val Thr 145 150 155 160 Met Thr Cys Ala Ala Ser Ser Ser Val Ser Ser Ser Tyr Leu His Trp 165 170 175 Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp Ile Tyr Ser Thr 180 185 190 Ser Asn Leu Ala Ser Gly Val Pro Gly Arg Phe Asn Gly Ser Gly Ser 195 200 205 Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala 210 215 220 Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro Trp Thr Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Glu Ile Lys 245 <210> 19 <211> 710 <212> PRT <213> Artificial Sequence <400> 19 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu 20 25 30 Ile Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45 Thr Phe Ser Thr Tyr Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys 50 55 60 Gly Leu Glu Trp Val Ser Ser Ile Leu Ser Gly Gly Thr Ser Phe Tyr 65 70 75 80 Ala Asp Asn Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys 85 90 95 Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ala Arg Arg Tyr Gly His Asp His Tyr Tyr Ala Met 115 120 125 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 145 150 155 160 Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro 165 170 175 Gly Glu Arg Ala Thr Leu Ser Cys Ala Ala Ser Ser Ser Val Ser Ser 180 185 190 Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Leu Ala Pro Arg Leu 195 200 205 Leu Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Ile Pro Asp Arg Phe 210 215 220 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu 225 230 235 240 Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His Arg Ser 245 250 255 Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Glu Ser Lys 260 265 270 Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly 275 280 285 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 290 295 300 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 305 310 315 320 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 325 330 335 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr Tyr Arg 340 345 350 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 355 360 365 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 370 375 380 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 385 390 395 400 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 405 410 415 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 420 425 430 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 435 440 445 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 450 455 460 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 465 470 475 480 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 485 490 495 Gly Lys Trp Leu Pro Ile Gly Cys Ala Ala Phe Val Val Val Cys Ile 500 505 510 Leu Gly Cys Ile Leu Ile Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser 515 520 525 Ser Val His Asp Pro Asn Gly Glu Tyr Met Phe Met Arg Ala Val Asn 530 535 540 Thr Ala Lys Lys Ser Arg Leu Thr Asp Val Thr Leu Lys Arg Gly Arg 545 550 555 560 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 565 570 575 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 580 585 590 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 595 600 605 Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 610 615 620 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 625 630 635 640 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 645 650 655 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 660 665 670 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 675 680 685 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 690 695 700 Gln Ala Leu Pro Pro Arg 705 710

Claims

1. A chimeric antigen receptor (CAR), characterized in that The chimeric antigen receptor comprises a scFv targeting B7-H3, wherein the heavy chain variable region of the scFv comprises the following complementarity determining regions CDR: CDR1 shown in SEQ ID NO:2, CDR2 shown in SEQ ID NO:3, and CDR3 shown in SEQ ID NO:4; And the light chain variable region of the scFv includes the following complementarity determining regions CDR: CDR1' shown in SEQ ID NO:6, CDR2' shown in SEQ ID NO:7, and CDR3' shown in SEQ ID NO:

8.

2. A polynucleotide, characterized in that The polynucleotide encodes the chimeric antigen receptor according to claim 1.

3. A carrier, characterized in that The vector contains the polynucleotide according to claim 2.

4. A genetically engineered immune cell, characterized in that: The immune cell contains the vector according to claim 3 or has the exogenous polynucleotide according to claim 2 integrated into its genome or expresses the chimeric antigen receptor according to claim 1, and the immune cell is a T cell.

5. A method for preparing engineered immune cells, characterized in that: The engineered immune cell expresses the chimeric antigen receptor according to claim 1, comprising the following steps: transducing the polynucleotide according to claim 2 or the vector according to claim 3 into the immune cell, thereby obtaining the engineered immune cell.

6. Use of the immune cell according to claim 4 in the preparation of a drug or preparation for treating cancer, wherein the cancer is gastric cancer, breast cancer, osteosarcoma, neuroblastoma, pancreatic cancer or colon cancer.

7. A pharmaceutical composition comprising the CAR as described in claim 1 or the vector as described in claim 3 or the immune cell as described in claim 4, and a pharmaceutically acceptable carrier, diluent or excipient.

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