Preparation and application of chimeric antigen receptor immune cells constructed based on GAS6

Abstract

The present invention provides a chimeric antigen receptor immune cell preparation and application thereof constructed based on growth arrest-specific protein 6 (GAS6, AXSF or AXLLG). Specifically, the present invention provides a chimeric antigen receptor (CAR) based on GAS6 modification, wherein the CAR contains an extracellular binding domain, and the extracellular binding domain can specifically target the GAS6 receptor. The CAR immune cell of the present invention has strong specificity and target affinity, so it has strong killing ability to target cells and high safety.

Description

Technical Field

[0001] The present invention belongs to the field of immune cell therapy, and specifically relates to the preparation and application of chimeric antigen receptor immune cells constructed based on GAS6. Background Art

[0002] Cancer is the second-leading threat to human health. In 2018, there were 18.1 million new cancer cases and 9.5 million cancer-related deaths worldwide. It is estimated that by 2040, there will be 29.5 million new cancer cases and 16.4 million cancer deaths annually. While traditional cancer treatments such as radiotherapy, chemotherapy, and surgical resection can prolong survival, they are limited by decreased quality of life and high recurrence rates.

[0003] Biological immunotherapy has become the fourth means of treating tumors after surgery, radiotherapy, and chemotherapy, and will become an essential means of treating tumors in the future. Chimeric Antigen Receptor-T cell (CAR-T) refers to T cells that have been genetically modified to recognize specific target antigens in an MHC-unrestricted manner and to continuously activate and proliferate. The structure of CAR includes a tumor-associated antigen binding region, an extracellular hinge region, a transmembrane region, and an intracellular signaling region. Currently, CAR-T therapy has shown good therapeutic effects in hematological malignancies, but the application of CAR-T therapy in solid tumors is limited by the presence of tumor heterogeneity, lack of tumor-specific antigens, and tumor immunosuppressive microenvironment.

[0004] Numerous studies have shown that AXL, a member of the TAM family of receptor tyrosine kinases (RTKs), is involved in a variety of cellular processes, including cell growth, proliferation, survival, apoptosis, and adhesion. AXL is highly expressed in various malignancies, from solid tumors to hematological tumors, including lung cancer, pancreatic cancer, gliomas, prostate cancer, breast cancer, liver cancer, colon cancer, ovarian cancer, gastric cancer, and nasopharyngeal cancer; and in hematological tumors such as acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), and diffuse large B-cell lymphoma (DLBCL), promoting tumor formation. At the same time, studies have found that AXL is upregulated in macrophages in inflammatory diseases or in macrophages associated with the tumor microenvironment. Therefore, targeting AXL has the potential to inhibit inflammation or have anti-tumor effects. Compared with normal tissues, AXL is highly expressed in primary tumors and metastases, which is associated with poor tumor prognosis and chemoresistance in various cancers. AXL's novel role in biological processes has made it a tumor biomarker and tumorigenesis marker molecule.

[0005] Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer and is projected to become one of the leading causes of cancer death in the United States by 2030. Despite advances in systemic chemotherapy for metastatic PDAC, overall survival remains low. Key characteristics of pancreatic cancer are metastasis, treatment resistance, and immunosuppression. AXL, a member of the TAM family of receptor tyrosine kinases, has been implicated in pancreatic cancer cell plasticity, chemoresistance, immunosuppression, and metastasis. Studies have shown that AXL expression is significantly elevated in tumor tissues of approximately 70% of pancreatic cancer patients, while expression is virtually absent in normal pancreatic ductal epithelium. In cancer patients, AXL overexpression is positively correlated with lymph node metastasis, tumor resistance, distant metastasis, disease progression, poor overall survival, and recurrence rates. Studies have shown that knocking down AXL expression increases the sensitivity of pancreatic cancer cells to gemcitabine.

[0006] The above studies show that AXL is an important target for tumor treatment; targeting AXL can effectively inhibit tumor growth, invasion, metastasis and drug resistance, thereby prolonging patient survival.

[0007] Therefore, there is an urgent need in the art to develop a chimeric antigen receptor immune cell targeting AXL and a therapeutic method thereof. Summary of the Invention

[0008] The purpose of the present invention is to provide a chimeric antigen receptor immune cell targeting the GAS6 receptor and a preparation and application method thereof.

[0009] In the first aspect of the present invention, a chimeric antigen receptor (CAR) is provided, characterized in that the CAR contains an extracellular binding domain, the extracellular binding domain has a structure based on the GAS6 protein or a fragment thereof based on the amino acid sequence shown in SEQ ID NO: 1, and the extracellular binding domain can specifically bind to the GAS6 receptor.

[0010] In another preferred embodiment, the extracellular binding domain has an amino acid sequence derived from GAS6.

[0011] In another preferred embodiment, the extracellular binding domain comprises GAS6 protein or a fragment thereof.

[0012] In another preferred embodiment, the extracellular binding domain comprises the extracellular region of GAS6 protein.

[0013] In another preferred embodiment, the extracellular binding domain contains the laminin G-like (LG) domain of the GAS6 protein.

[0014] In another preferred embodiment, the LG domain includes LG1 and LG2 domains.

[0015] In another preferred embodiment, the LG domain includes wild-type and mutant LG domains.

[0016] In another preferred embodiment, the amino acid sequence of the LG domain is as shown in positions X to 678 of the sequence shown in SEQ ID NO: 1, wherein X is any positive integer from 250 to 285, preferably X is a positive integer from 260 to 279, and most preferably is 261.

[0017] In another preferred example, the amino acid sequence of the LG domain is as shown in positions 279-678 of the sequence shown in SEQ ID NO: 1.

[0018] In another preferred embodiment, the GAS6 protein or its fragment includes the laminin G-like (LG) region of the GAS6 protein, and the amino acid sequence of the LG domain corresponds to positions 279-678 of the sequence shown in SEQ ID NO:1.

[0019] In another preferred embodiment, the GAS6 protein or its fragment further includes a partial fragment of the epidermal growth factor (EGF)-like domain of the GAS6 protein, and the amino acid sequence of the EGF-like domain is positions 118-278 of the sequence shown in SEQ ID NO:1.

[0020] In another preferred embodiment, the amino acid sequence of the partial fragment of the EGF-like domain is n-278 of the sequence shown in SEQ ID NO: 1,

[0021] wherein n is a positive integer from 118 to 275, preferably a positive integer from 250 to 270, and most preferably 261.

[0022] In another preferred embodiment, the GAS6 protein or its fragment includes the extracellular domain of the GAS6 protein, and the amino acid sequence of the extracellular domain of the GAS6 protein corresponds to positions 31-678 of the sequence shown in SEQ ID NO:1.

[0023] In another preferred embodiment, the extracellular binding domain of the CAR contains, in addition to the first extracellular domain targeting the GAS6 receptor, a second extracellular domain targeting an additional target.

[0024] In another preferred embodiment, the additional target is a tumor-specific target.

[0025] In another preferred embodiment, the GAS6 protein or its fragment specifically binds to GAS6 receptors including AXL protein.

[0026] In another preferred embodiment, the GAS6 receptor is a receptor tyrosine kinase.

[0027] In another preferred embodiment, the receptor tyrosine kinase is selected from the group consisting of AXL, Tyro3, ​​MERTK, or a combination thereof.

[0028] In another preferred embodiment, the GAS6 receptor is selected from the following group: AXL, Tyro3, ​​and MERTK.

[0029] In another preferred embodiment, the receptor tyrosine kinase AXL is located on the cell membrane (or transmembrane, cytoplasm).

[0030] In another preferred embodiment, the receptor tyrosine kinase is AXL.

[0031] In another preferred embodiment, the receptor tyrosine kinase AXL is derived from human or non-human mammals.

[0032] In another preferred embodiment, the non-human mammals include: rodents (such as rats and mice), primates (such as monkeys), preferably primates.

[0033] In another preferred embodiment, the receptor tyrosine kinase AXL is of human or monkey origin.

[0034] In another preferred embodiment, the AXL is of human origin.

[0035] In another preferred example, the GAS6 protein or a fragment thereof has an amino acid sequence as shown in SEQ ID NO: 1, or has an amino acid sequence from positions 1 to 678 (preferably from positions 31 to 678, more preferably from positions 118 to 678, and more preferably from positions 261 to 678) of the sequence shown in SEQ ID NO: 1.

[0036] In another preferred embodiment, the amino acid sequence of the GAS6 protein or its fragment is selected from the following group:

[0037] (i) the sequence shown in positions 31 to 678 of the sequence shown in SEQ ID NO: 1; and

[0038] (ii) an amino acid sequence obtained by replacing, deleting, changing or inserting one or more amino acid residues, or adding 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues, at the N-terminus or C-terminus of the sequence shown in SEQ ID NO: 1; and the obtained amino acid sequence has a sequence identity of ≥85% (preferably ≥90%, more preferably ≥95%, for example ≥96%, ≥97%, ≥98% or ≥99%) with the sequence shown in SEQ ID NO: 1 at positions 261 to 678; and the obtained amino acid sequence has the same or similar function as the sequence shown in (i).

[0039] In another preferred embodiment, the structure of the chimeric antigen receptor is shown in Formula I below:

[0040] L-EB-H-TM-C-CD3ζ-RP (I)

[0041] Where,

[0042] Each "-" is independently a connecting peptide or a peptide bond;

[0043] L is none or a signal peptide sequence;

[0044] EB is the extracellular binding domain;

[0045] H is the absence or hinge region;

[0046] TM is the transmembrane domain;

[0047] C is none or a costimulatory signaling molecule;

[0048] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ;

[0049] RP is none or reporter protein.

[0050] In another preferred embodiment, the reporter protein RP further comprises a self-cleavage recognition site at its N-terminus, preferably a T2A sequence.

[0051] In another preferred embodiment, the reporter protein RP is a fluorescent protein.

[0052] In another preferred embodiment, the reporter protein RP is mKate2 red fluorescent protein.

[0053] In another preferred embodiment, the amino acid sequence of the mKate2 red fluorescent protein is shown in SEQ ID NO: 2.

[0054] In another preferred embodiment, the L is a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

[0055] In another preferred embodiment, the L is a signal peptide derived from CD8.

[0056] In another preferred embodiment, the amino acid sequence of L is shown in SEQ ID NO: 3.

[0057] In another preferred embodiment, the H is the hinge region of a protein selected from the group consisting of CD8, CD28, CD137, or a combination thereof.

[0058] In another preferred embodiment, the H is a hinge region derived from CD8.

[0059] In another preferred embodiment, the amino acid sequence of H is shown in SEQ ID NO: 4.

[0060] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0061] In another preferred embodiment, the TM is a transmembrane region derived from CD8.

[0062] In another preferred embodiment, the amino acid sequence of the TM is shown in SEQ ID NO: 5.

[0063] In another preferred embodiment, the 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.

[0064] In another preferred embodiment, the C is a co-stimulatory signal molecule derived from 4-1BB.

[0065] In another preferred embodiment, the amino acid sequence of C is shown in SEQ ID NO:6.

[0066] In another preferred embodiment, the amino acid sequence of the cytoplasmic signaling sequence derived from CD3ζ is shown as SEQ ID NO: 7.

[0067] In another preferred embodiment, the amino acid sequence of the chimeric antigen receptor CAR is shown in SEQ ID NO: 8.

[0068] In a second aspect of the present invention, a nucleic acid molecule is provided, wherein the nucleic acid molecule encodes the chimeric antigen receptor according to the first aspect of the present invention.

[0069] In the third aspect of the present invention, a vector is provided, characterized in that the vector contains the nucleic acid molecule as described in the second aspect of the present invention.

[0070] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, or a combination thereof.

[0071] In another preferred embodiment, the vector is a lentiviral vector.

[0072] In another preferred embodiment, the vector is selected from the following group: pTomo lentiviral vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, pLV, etc.

[0073] In another preferred embodiment, the vector is a pTomo lentiviral vector.

[0074] In another preferred embodiment, the vector further comprises a member selected from the group consisting of: a promoter, a transcription enhancing element WPRE, a long terminal repeat sequence LTR, and the like.

[0075] In another preferred example, the vector comprises the nucleotide sequence shown in SEQ ID NO:9.

[0076] In the fourth aspect of the present invention, a host cell is provided, wherein the host cell contains the vector as described in the third aspect of the present invention or the chromosome into which the exogenous nucleic acid molecule as described in the second aspect of the present invention is integrated or expresses the chimeric antigen receptor as described in the first aspect of the present invention.

[0077] In the fifth aspect of the present invention, an engineered immune cell is provided, wherein the immune cell contains the vector or chromosome as described in the third aspect of the present invention, in which an exogenous nucleic acid molecule as described in the second aspect of the present invention is integrated, or expresses the chimeric antigen receptor as described in the first aspect of the present invention.

[0078] In another preferred embodiment, the engineered immune cells are selected from the group consisting of T cells, NK cells, NKT cells, or macrophages.

[0079] In another preferred embodiment, the engineered immune cells are chimeric antigen receptor T cells (CAR-T cells) or chimeric antigen receptor NK cells (CAR-NK cells).

[0080] In another preferred embodiment, the engineered immune cells are CAR-T cells.

[0081] In the sixth aspect of the present invention, a method for preparing the engineered immune cells as described in the fifth aspect of the present invention is provided, comprising the following steps: transducing the nucleic acid molecule as described in the second aspect of the present invention or the vector as described in the third aspect of the present invention into the immune cells, thereby obtaining the engineered immune cells.

[0082] In another preferred embodiment, the method further comprises the step of performing functional and effectiveness testing on the obtained engineered immune cells.

[0083] In the seventh aspect of the present invention, a pharmaceutical composition is provided, which contains the chimeric antigen receptor as described in the first aspect of the present invention, the nucleic acid molecule as described in the second aspect of the present invention, the vector as described in the third aspect of the present invention, the host cell as described in the fourth aspect of the present invention, and / or the engineered immune cell as described in the fifth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0084] In another preferred embodiment, the preparation is a liquid preparation.

[0085] In another preferred embodiment, the preparation is in the form of an injection.

[0086] In another preferred embodiment, the concentration of the engineered immune cells in the preparation is 1×10 3 -1×10 8 cells / ml, preferably 1×10 4 -1×10 7 cells / ml.

[0087] In the eighth aspect of the present invention, a chimeric antigen receptor as described in the first aspect of the present invention, a nucleic acid molecule as described in the second aspect of the present invention, a vector as described in the third aspect of the present invention, or a host cell as described in the fourth aspect of the present invention, and / or an engineered immune cell as described in the fifth aspect of the present invention is provided for the preparation of a drug or preparation for preventing and / or treating diseases related to abnormal expression of the GAS6 receptor.

[0088] In another preferred embodiment, the GAS6 receptor includes but is not limited to Tyro3, ​​AXL, MERTK and the like.

[0089] In another preferred embodiment, the abnormal expression of the GAS6 receptor refers to overexpression of the GAS6 receptor.

[0090] In another preferred embodiment, the excessive expression of the GAS6 receptor means that the expression level of the GAS6 receptor is ≥1.5 times, preferably ≥2 times, and more preferably ≥2.5 times the expression level under normal physiological conditions.

[0091] In another preferred embodiment, the diseases associated with abnormal expression of GAS6 receptor include but are not limited to tumors, venous thromboembolic diseases, systemic lupus erythematosus, chronic renal failure and preeclampsia.

[0092] In another preferred embodiment, the diseases related to abnormal expression of GAS6 receptor include: drugs or preparations for diseases related to abnormal expression of receptor tyrosine kinase AXL.

[0093] In another preferred embodiment, the diseases associated with abnormal AXL expression include: tumors, venous thromboembolic diseases, systemic lupus erythematosus, chronic renal failure and preeclampsia.

[0094] In another preferred embodiment, the disease is a malignant tumor with high expression of AXL.

[0095] In another preferred embodiment, the tumor includes blood tumors and solid tumors.

[0096] 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.

[0097] In another preferred embodiment, the solid tumor is selected from the group consisting of pancreatic cancer, breast cancer, gastric cancer, hepatobiliary cancer, colorectal cancer, bladder cancer, non-small cell lung cancer, ovarian cancer and esophageal cancer, glioma, lung cancer, prostate cancer, nasopharyngeal cancer or a combination thereof.

[0098] In another preferred embodiment, the tumor is selected from the group consisting of pancreatic cancer, liver cancer, glioma, gastric cancer, and prostate cancer.

[0099] In the ninth aspect of the present invention, there is provided a use of the engineered immune cells as described in the fifth aspect of the present invention, or the pharmaceutical composition as described in the seventh aspect of the present invention, for preventing and / or treating cancer or tumors.

[0100] In the tenth aspect of the present invention, a method for treating a disease is provided, comprising administering an effective amount of the engineered immune cells as described in the fifth aspect of the present invention, or the pharmaceutical composition as described in the seventh aspect of the present invention to a subject in need of treatment.

[0101] In another preferred embodiment, the disease is a disease associated with abnormal expression of GAS6 receptor.

[0102] In another preferred embodiment, the disease is cancer or tumor.

[0103] In another preferred embodiment, the engineered immune cells or CAR immune cells contained in the pharmaceutical composition are cells derived from the subject (autologous cells).

[0104] In another preferred embodiment, the engineered immune cells or CAR immune cells contained in the pharmaceutical composition are cells derived from healthy individuals (allogeneic cells).

[0105] In another preferred embodiment, the method can be used in combination with other treatment methods.

[0106] In another preferred embodiment, the other treatment methods include chemotherapy, radiotherapy, targeted therapy and the like.

[0107] 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 described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0108] Figure 1 A schematic diagram of the GAS6-CAR vector construction is shown.

[0109] Among them, A is a schematic diagram of the GAS6 sequence, in which 1-30AA is the signal peptide, 31-678AA is the extracellular domain, 49-90AA is the γ-carboxyglutamic acid (Gla) domain, 91-117AA is the disulfide bridge, 118-278AA is the epidermal growth factor (EGF)-like domain, and 279-678AA are two laminin G-like (LG) domains; B is a schematic diagram of the control group plasmid Mock-CAR and GAS6-CAR structure, in which the signal peptide, hinge region, and transmembrane region are all derived from the human CD8 molecule, 4-1BB is derived from human CD137, CD3ζ is derived from human CD3, and mKate2 is a fluorescent marker for detecting CAR expression; C is HindIII enzyme digestion identification of the pTomo-GAS6-CAR vector.

[0110] Figure 2 The results of CAR transfection efficiency detection are shown.

[0111] Among them, A is the cell fluorescence expression result after T cells were infected with Mock-CAR and GAS6-CAR for 72 hours, where BF is bright field and mKate2 is CAR fluorescence expression; B is the fluorescence expression result of flow cytometry detection.

[0112] Figure 3 The killing effect of GAS6-CAR on different tumor cell lines was shown.

[0113] Among them, A, pancreatic cancer. B, glioma. C, non-small cell lung cancer. D, prostate cancer. E, liver cancer. F, breast cancer. G, gastric cancer.

[0114] Figure 4 Shown are the results of AXL expression detection in different pancreatic cancer cell lines.

[0115] A shows Western blotting to detect AXL protein expression. B shows qPCR to detect AXL mRNA levels. C shows flow cytometry to detect AXL expression. D shows immunofluorescence to detect AXL expression on the cell membrane.

[0116] Figure 5 The results of gradient killing of different pancreatic cancer cell lines by GAS6-CAR are shown.

[0117] Figure 6 The results of IFNγ release after GAS6-CAR killed different pancreatic cancer cell lines were shown.

[0118] Figure 7 Overexpression of AXL in the pancreatic cancer cell line BXPC3 enhanced the killing effect of GAS6-CAR.

[0119] A shows Western blotting to detect AXL protein expression. B shows qPCR to detect AXL mRNA levels. C shows flow cytometry to detect AXL expression. D shows immunofluorescence to detect AXL expression on the cell membrane. E shows the cytotoxicity of GAS6-CAR against BXPC3-AXL. F shows the results of IFNγ release assay.

[0120] Figure 8 It was shown that knockdown of AXL expression in the pancreatic cancer cell line PANC1 reduced the killing effect of GAS6-CAR.

[0121] A shows Western blotting to detect AXL protein expression. B shows qPCR to detect AXL mRNA levels. C shows flow cytometry to detect AXL expression. D shows immunofluorescence to detect AXL expression on the cell membrane. E shows the cytotoxicity of GAS6-CAR against PANC1-shAXL. F shows the results of IFNγ release.

[0122] Figure 9 The results show the killing effect of GAS6-CAR on the normal cell line HEK-293T.

[0123] A shows Western blotting to detect AXL protein expression. B shows qPCR to detect AXL mRNA levels. C shows flow cytometry to detect AXL expression. D shows immunofluorescence to detect AXL expression on the cell membrane. E shows GAS6-CAR killing of HEK-293T cells. F shows IFNγ release. DETAILED DESCRIPTION

[0124] After extensive and in-depth research and extensive screening, the inventors have developed, for the first time, a chimeric antigen receptor immune cell preparation and application based on GAS6. Experimental results demonstrate that the GAS6 receptor-targeting CAR-T cell of the present invention has a significant killing effect on target cells and a specific anti-tumor cell effect. This is the basis for the completion of the present invention.

[0125] the term

[0126] In order to make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings generally understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can change. It should also be understood that the terms used herein are intended only to describe specific embodiments and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0127] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0128] In addition, any positive integer of X1-X2 represents any positive integer between X1 and X2 (including the endpoints), for example, the positive integers of 1-5 include 1, 2, 3, 4 and 5, and so on.

[0129] As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance may occur but need not.

[0130] As used herein, the term "comprising" or "including" may be open, semi-closed, or closed. In other words, the term also includes "consisting essentially of" or "consisting of."

[0131] "Transduction", "transfection", "transformation" or other terms used in this article refer to the process of transferring exogenous polynucleotides into host cells, transcribing and translating them to produce polypeptide products, including the use of plasmid molecules to introduce exogenous polynucleotides into host cells (such as Escherichia coli).

[0132] "Gene expression" or "expression" refers to the process of gene transcription, translation, and post-translational modification to produce the RNA or protein product of a gene.

[0133] "Polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxynucleotides (DNA), ribonucleotides (RNA), hybrid sequences thereof, and analogs thereof. A polynucleotide may include modified nucleotides, such as methylated or capped nucleotides or nucleotide analogs. As used herein, the term polynucleotide refers interchangeably to single-stranded and double-stranded molecules. Unless otherwise indicated, any polynucleotide described herein includes a double-stranded form and two complementary single strands that are known or predicted to constitute a double-stranded form.

[0134] Conservative amino acid substitutions are known in the art. In some embodiments, potential substituting amino acids are within one or more of the following groups: glycine, alanine; and valine, isoleucine, leucine, and proline; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine, lysine, arginine, and histidine; and / or phenylalanine, tryptophan, and tyrosine; methionine, and cysteine. In addition, the present invention also provides non-conservative amino acid substitutions that allow substitution of amino acids from different groups.

[0135] Those skilled in the art will readily understand the meaning of all parameters, dimensions, materials, and configurations described herein. Actual parameters, dimensions, materials, and / or configurations will depend on the specific application for which the invention is to be used. Those skilled in the art will appreciate that the embodiments or claims are given by way of example only, and that, within the scope of equivalents or claims, the scope of the embodiments of the invention is not limited to the scope specifically described and claimed.

[0136] Definitions and all definitions used herein should be understood to control over dictionary definitions or definitions in documents incorporated by reference.

[0137] All references, patents, and patent applications cited herein are incorporated by reference with respect to the subject matter in which they are cited, in some cases in their entirety.

[0138] It should be understood that for any method described herein comprising more than one step, the order of the steps is not necessarily limited to the order described in the examples.

[0139] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0140] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0141] The term "administering" refers to the physical introduction of the product of the invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion.

[0142] TAM family

[0143] The TAM family of receptor tyrosine kinases (RTKs), including AXL, Tyro3, ​​and MERTK, plays a crucial role in tumorigenesis and progression. The AXL gene, located on the long arm of chromosome 19 and consisting of 20 exons, was first discovered in patients with chronic myeloid leukemia. The protein is encoded by the AXL gene (or UFO, ARK, Tyro7, or JTK11). The full-length AXL protein consists of 894 amino acids and has a molecular weight of 98 kDa; however, due to post-translational glycosylation, the actual AXL protein is 120 kDa (partially glycosylated) or 140 kDa (fully glycosylated).

[0144] AXL is composed of extracellular, transmembrane, and intracellular domains. The extracellular domain is composed of two immunoglobulin (IgL)-like repeats and two fibronectin type III (Fro III)-like repeats. The Fro III domain is responsible for binding of AXL to its ligand, GAS6. The intracellular domain of AXL plays an important role in autophosphorylation and downstream kinase activity.

[0145] Studies have shown that AXL is highly expressed in a variety of clinical tumor tissues (such as lung cancer, pancreatic cancer, glioma, prostate cancer, breast cancer, liver cancer, colon cancer, ovarian cancer, gastric cancer, etc.), including protein and RNA levels; and is highly expressed in corresponding tumor cell lines; at the same time, studies have shown that AXL is highly expressed in macrophages associated with inflammatory diseases and the tumor microenvironment. High expression of AXL is closely related to the survival, proliferation, metastasis, invasion, epithelial-mesenchymal transition and stemness of cancer cells, and is also related to the patient's recovery and survival rate; AXL expression even affects the tumor microenvironment to promote tumor immunosuppression. Reducing AXL expression by gene knockdown can significantly inhibit the invasion and metastasis of tumor cells and improve prognosis.

[0146] In the field of tumor immunotherapy, T cells can exert anti-tumor effects (such as in triple-negative breast cancer) through AXL. Compared with AXL-negative tumor cells, tumor cells with high AXL expression are more easily killed by AXL-CAR T cells. These results indicate that AXL is an important target molecule for tumor treatment.

[0147] Growth arrest-specific protein 6 (GAS6 or AXSF, AXLLG)

[0148] GAS6 is a vitamin K-dependent, multidomain protein with a molecular weight of 75 kDa. GAS6 comprises 678 amino acids, including an N-terminal γ-carboxyglutamate (Gla) domain (amino acids 49-90), which confers GAS6 the ability to bind to anionic phospholipids on the cell surface and is associated with stimulation of cell proliferation; four epidermal growth factor (EGF)-like domains (amino acids 118-278); and two laminin G-like (LG) domains (amino acids 279–678). Crystalline complexes of AXL and GAS6 reveal that the C-terminal LG domain of GAS6 binds to the extracellular domains of AXL, IgL-1 and IgL-2.

[0149] The GAS6 gene is highly expressed in various cancers and has been considered a poor prognostic marker. Elevated levels of its protein are also associated with various disease states, including venous thromboembolic disease, systemic lupus erythematosus, chronic renal failure, and preeclampsia.

[0150] GAS6 is a ligand for the TAM family (including Tyro3, ​​AXL, and MERTK). In addition to AXL, it can also bind to other TAM family members. GAS6 has the highest binding affinity to AXL and is the only activating ligand for AXL discovered to date. When AXL binds with high affinity to its ligand, GAS6, the intracellular kinase domain of the AXL receptor undergoes homodimerization and trans-autophosphorylation, recruiting adaptor molecules and effector proteins containing Src homology 2 (SH2) or other phosphotyrosine binding domains (PTBs), thereby activating downstream signaling pathways such as RAS, RAF, MAPK, ERK1 / 2, PI3K, and TGF-β1 / 2, ultimately exerting its effects. The GAS6 / AXL signaling pathway is closely associated with tumor cell growth, metastasis, invasion, epithelial-mesenchymal transition (EMT), angiogenesis, drug resistance, immune regulation, and stem cell maintenance.

[0151] Based on this, the present invention integrates a specific fragment of GAS6 into a CAR vector for the first time through genetic engineering, and modifies related immune cells, thereby achieving specific killing of cells positive for GAS6 binding protein, which can be used to treat related diseases.

[0152] Chimeric antigen receptor (CAR) of the present invention

[0153] Chimeric antigen receptor (CAR) is composed of an extracellular antigen recognition region, a transmembrane region, and an intracellular co-stimulatory signaling region.

[0154] The design of CARs has evolved through the following process: First-generation CARs contain only a single intracellular signaling component, CD3ζ or FcγRI. Because they only have a single activation domain, they can only induce transient T cell proliferation and minimal cytokine secretion, but cannot provide long-term T cell proliferation signals or sustained in vivo anti-tumor effects, resulting in limited clinical efficacy. Second-generation CARs incorporate a co-stimulatory molecule, such as CD28, 4-1BB, OX40, or ICOS, into the existing structure. This significantly improves the function of first-generation CARs, further enhancing the persistence of CAR-T cells and their ability to kill tumor cells. New immune co-stimulatory molecules, such as CD27 and CD134, are added in tandem to the second-generation CARs, resulting in the development of third- and fourth-generation CARs.

[0155] The extracellular segment of CAR can recognize a specific antigen, and then transduce the signal through the intracellular domain, causing cell activation and proliferation, cytolytic toxicity and secretion of cytokines, thereby eliminating the target cells. First, the patient's autologous cells (or allogeneic donors) are isolated, activated and genetically modified to produce CAR immune cells, and then injected into the same patient. This method has a very low probability of developing graft-versus-host disease, and the antigen is recognized by the immune cells in a non-MHC restricted manner.

[0156] CAR-immune cell therapy has achieved a very high clinical response rate in the treatment of hematological malignancies, a high response rate that has not been achieved by any previous treatment method, and has triggered a wave of clinical research around the world.

[0157] Specifically, the chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain.

[0158] The extracellular domain includes a target-specific binding element. The extracellular domain can be an ScFv of an antibody based on antigen-antibody specific binding, or a natural sequence or derivative thereof based on ligand-receptor specific binding.

[0159] In the present invention, the extracellular domain of the chimeric antigen receptor is a GAS6 protein or a fragment thereof that can specifically bind to the AXL target of the CAR of the present invention. More preferably, the extracellular binding domain of the chimeric antigen receptor of the present invention has the amino acid sequence of positions 261 to 678 of the sequence shown in SEQ ID NO: 1.

[0160] The intracellular domain includes a costimulatory signaling region and a zeta chain portion. The costimulatory signaling region refers to a portion of the intracellular domain that includes costimulatory molecules. Costimulatory molecules are cell surface molecules required for lymphocytes to effectively respond to antigens, rather than antigen receptors or their ligands.

[0161] Between the extracellular domain and the transmembrane domain of CAR, or between the cytoplasmic domain and the transmembrane domain of CAR, a linker can be incorporated. As used herein, the term "linker" 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 linker may include 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

[0162] The CAR of the present invention, when expressed in T cells, can recognize antigens based on antigen binding specificity. When it binds to its associated antigen, it affects tumor cells, causing them to not grow, be caused to die or be affected in other ways, and causing the patient's tumor burden to shrink or be eliminated. The antigen binding domain is preferably fused with the intracellular domain of one or more of the co-stimulatory molecules and the ζ chain. Preferably, the antigen binding domain is fused with the intracellular domain of the CD8 signaling domain and the CD3ζ signaling domain combination.

[0163] In the present invention, the extracellular binding domain of the CAR of the present invention also includes sequence-based conservative variants, which means that compared with the amino acid sequence of positions 261 to 678 (or positions 279-678) of SEQ ID NO: 1, up to 10, preferably up to 8, and more preferably up to 5 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.

[0164] In the present invention, the number of added, deleted, modified and / or substituted amino acids is preferably no more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably no more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, more preferably 15-20%.

[0165] In the present invention, the number of amino acids added, deleted, modified and / or substituted is usually 1, 2, 3, 4 or 5, preferably 1-3, more preferably 1-2, and most preferably 1.

[0166] 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 such a domain being bound to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complex.

[0167] The intracellular domain in the CAR of the present invention includes a 4-1BB costimulatory domain and a CD3ζ signaling domain.

[0168] In one embodiment of the present invention, the CAR is a CAR that can specifically target AXL.

[0169] Chimeric antigen receptor immune cells (CAR-immune cells)

[0170] The present invention provides a chimeric antigen receptor immune cell, which comprises the chimeric antigen receptor of the present invention that specifically targets the GAS6 receptor (preferably AXL).

[0171] The chimeric antigen receptor immune cell of the present invention can be a CAR-T cell, or a CAR-NK cell, or a CAR-macrophage. Preferably, the chimeric antigen receptor immune cell of the present invention is a CAR-T cell.

[0172] As used herein, the terms "CAR-T cells", "CAR-T", and "CAR-T cells of the present invention" all refer to the CAR-T cells described in the fifth aspect of the present invention.

[0173] CAR-T cells have the following advantages over other T-cell-based treatments: (1) The action of CAR-T cells is not restricted by MHC; (2) Since many tumor cells express the same tumor markers, once the CAR gene construction for a certain tumor marker is completed, it can be widely used; (3) CAR can use both tumor protein markers and glycolipid non-protein markers, expanding the target range of tumor markers; (4) The use of the patient's own cells reduces the risk of rejection; (5) CAR-T cells have immune memory function and can survive in the body for a long time.

[0174] As used herein, the terms "CAR-NK cell," "CAR-NK," and "CAR-NK cell of the present invention" all refer to the CAR-NK cell of the fifth aspect of the present invention. The CAR-NK cell of the present invention can be used for tumors that highly express the AXL receptor (preferably AXL).

[0175] Natural killer (NK) cells are a major type of immune effector cell that protects the body from viral infection and tumor cell invasion through non-antigen-specific pathways. Engineered (genetically modified) NK cells may acquire new functions, including the ability to specifically recognize tumor antigens and possess enhanced anti-tumor cytotoxicity.

[0176] Compared with CAR-T cells, CAR-NK cells have the following advantages, such as: (1) they directly kill tumor cells by releasing perforin and granzymes, but have no killing effect on normal cells in the body; (2) they release very small amounts of cytokines, thereby reducing the risk of cytokine storms; (3) they are extremely easy to expand in vitro and develop into "ready-made" products. Other than that, it is similar to CAR-T cell therapy.

[0177] As used herein, the terms "CAR-macrophage (M) cell", "CAR-macrophage (M)", and "CAR-macrophage (M) cell of the present invention" all refer to the CAR-macrophage described in the fifth aspect of the present invention. The CAR-macrophage of the present invention can be used for inflammatory diseases and tumors with high expression of AXL receptors (preferably AXL).

[0178] Macrophages are phagocytic cells derived from monocytes and play an important role in the body's innate and cellular immunity. Engineered (genetically modified) macrophages can enhance their ability to specifically recognize tumor antigens and possess enhanced anti-tumor cytotoxicity.

[0179] carrier

[0180] The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by directly isolating from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically.

[0181] The present invention also provides vectors comprising the nucleic acid molecules of the present invention. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for 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.

[0182] In brief summary, the expression cassette or nucleic acid sequence of the present invention is generally operably linked to a promoter and incorporated into an expression vector. Such vectors are suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that can be used to regulate expression of the desired nucleic acid sequence.

[0183] 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, and 5,589,466, which are incorporated herein by reference in their entireties. In another embodiment, the present invention provides gene therapy vectors.

[0184] 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.

[0185] Further, expression vector can be provided to cell in the form of viral vector.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. The virus that can be used as a vector includes but is not limited to retrovirus, adenovirus, adeno-associated virus, herpes virus and slow virus. Generally, suitable vectors are included in at least one organism and work in the origin of replication, promoter sequence, convenient restriction enzyme site and one or more selectable markers (for example, WO01 / 96584; WO01 / 29058; and U.S. Patent number 6,326,193).

[0186] 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 the 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.

[0187] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. Typically, these are located in the 30-110 bp 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, promoter function is maintained. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50 bp before activity begins to decline. Depending on the promoter, it has been shown that individual elements can work together or independently to initiate transcription.

[0188] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive any polynucleotide sequence operably connected thereto for high-level expression. 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 expression of a polynucleotide sequence operably linked to the 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, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0189] In order to evaluate the expression of the CAR polypeptide or its portion, 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 that is sought to be transfected or infected by a viral vector. In other aspects, selectable markers can 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 to enable expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

[0190] Reporter genes are used to identify cells of potential transfection and to evaluate the functionality of regulatory sequences. Typically, reporter genes are genes that are not present in or expressed by recipient organisms or tissues, and that encode polypeptides whose expression is clearly indicated by some easily detectable properties, such as enzymatic activity. After DNA has been introduced into recipient cells, the expression of the reporter gene is measured at an appropriate time. Suitable reporter genes can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). In one embodiment of the invention, the reporter gene is a gene encoding mKate2 red fluorescent protein. Suitable expression systems are well known and can be prepared or commercially available using known techniques. Typically, constructs with at least 5 flanking regions that show the highest level of reporter gene expression are identified as promoters. Such promoter regions can be connected to reporter genes and used to evaluate the ability of reagents to regulate promoter-driven transcription.

[0191] Methods for introducing genes into cells and expressing genes in cells are known in the art. In the context of expression vectors, the vectors can be readily 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.

[0192] 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.

[0193] 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 method for inserting genes into mammalian, for example, human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0194] 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).

[0195] In the case of using non-viral delivery system, exemplary delivery means is liposome. Consider using lipid preparation, to introduce nucleic acid into host cell (in vitro, in vitro (ex vivo) or in vivo). On the other hand, this nucleic acid can be associated with lipid. The nucleic acid associated with lipid can be encapsulated in the aqueous interior of liposome, dispersed in the lipid bilayer of liposome, attached to liposome through the connecting molecule associated with liposome and oligonucleotide, trapped in liposome, compounded with liposome, dispersed in the solution comprising lipid, mixed with lipid, united with lipid, included in lipid as suspension, included in micelle or with micelle compound, or otherwise associated with lipid. The lipid, lipid / DNA or lipid / expression vector associated with composition are not limited to any specific structure in solution. For example, they can be present in bilayer structure, as micelle or have " collapsed (collapsed) " structure. They can also be simply dispersed in solution, 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.

[0196] In a preferred embodiment of the present invention, the vector is a lentiviral vector.

[0197] preparation

[0198] The present invention provides a preparation containing the chimeric antigen receptor CAR described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the vector described in the third aspect of the present invention, or the host cell described in the fourth aspect of the present invention or the engineered immune cell described in the fifth aspect 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.

[0199] In one embodiment, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, or the like; a carbohydrate such as glucose, mannose, sucrose, or dextran, mannitol; a protein; a polypeptide or 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.

[0200] Therapeutic applications

[0201] 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 a receptor tyrosine kinase (preferably AXL), a marker of tumor cells, synergistically activate T cells, elicit an immune response from immune cells, and significantly enhance their tumor cell-killing efficiency.

[0202] 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 steps of administering the CAR-T cells of the present invention to the mammal.

[0203] In one embodiment, the present invention comprises a type of cell therapy in which a patient's own T cells (or those of an allogeneic donor) are isolated, activated, and genetically modified to produce CAR-T cells, which are then infused back into the same patient. This approach significantly reduces the risk of graft-versus-host disease, as antigens are recognized by T cells in an MHC-free manner. Furthermore, a single CAR-T cell can treat all cancers expressing that antigen. Unlike antibody therapies, CAR-cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

[0204] In one embodiment, the CAR-T cells of the present invention can undergo a stable in vivo T cell expansion and a sustainable amount of time. In addition, the immune response mediated by CAR can be part of an adoptive immunotherapy step, wherein CAR-modified T cells induce an immune response specific to the antigen binding domain in CAR. For example, CAR-T cells of receptor tyrosine kinase (preferably AXL) cause a specific immune response of receptor tyrosine kinase expressing (preferably AXL) cells.

[0205] Although the data disclosed herein specifically disclose lentiviral vectors comprising a GAS6 protein or fragment thereof, hinge and transmembrane regions, and 4-1BB and CD3ζ signaling domains, the invention should be construed to encompass any number of variations to each of the construct components.

[0206] Treatable cancers include tumors that are not vascularized or substantially not yet 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 lymphoma, benign and malignant tumors and malignant tumors, such as sarcoma, cancer and melanoma. Also included are adult tumors / cancers and childhood tumors / cancers.

[0207] 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, granulocytic-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 lymphoma (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0208] Solid tumors are abnormal masses of tissue that do not usually include cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell types 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, breast cancer, gastric cancer, hepatobiliary cancer, colorectal cancer, bladder cancer, non-small cell lung cancer, ovarian cancer, and esophageal cancer, glioblastoma, lung cancer, prostate cancer, nasopharyngeal carcinoma, etc.

[0209] The CAR-modified T cells of the present invention can also be used as a vaccine type for ex vivo immunization and / or in vivo therapy of mammals. Preferably, the mammal is a human.

[0210] 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.

[0211] In vitro 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 a vector 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.

[0212] 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.

[0213] The present invention provides a method for treating tumors, comprising administering a therapeutically effective amount of CAR-modified T cells of the present invention to a subject in need thereof.

[0214] The CAR-modified T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as IL-2, IL-17 or other cytokines or cell groups. Briefly, the pharmaceutical composition of the present invention may include a target cell group as described herein, in combination 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 compositions of the present invention are preferably formulated for intravenous administration.

[0215] The pharmaceutical compositions of the present invention can be administered in a manner appropriate to 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 appropriate dosages can be determined by clinical trials.

[0216] When an "effective amount," "immunologically effective amount," "anti-tumor effective amount," "tumor-inhibitory effective amount," or "therapeutic amount" is indicated, the precise 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 a pharmaceutical composition comprising the T cells described herein can be administered in an amount of 10 4 to 10 9 The dose of cells / kg body weight is preferably 10 5 to 10 6The T cell composition can be administered at a dose of 10 cells / kg body weight (including all integer values ​​within those ranges). The T cell composition can also be administered multiple times at these doses. The cells can be administered using infusion techniques well known 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 person skilled in the art of medicine by monitoring the patient's disease signs and adjusting the treatment accordingly.

[0217] 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.

[0218] In certain embodiments of the present invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in combination with (e.g., before, simultaneously, or after) any number of relevant treatment modalities, including, but not limited to, treatment with 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 specific tumor patients. In further embodiments, the T cells of the present invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell compositions of the present invention are administered to a patient in combination with (e.g., before, simultaneously, or after) bone marrow transplantation, chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide. For example, in one embodiment, a subject may undergo standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. 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 before or after surgery.

[0219] 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. Dosage ratios for human administration may be implemented according to practices accepted in the art. Typically, 1×10 6 to 1×10 10The CAR-T cells of the present invention are administered to the patient, for example, by intravenous infusion.

[0220] The main advantages of the present invention include:

[0221] 1) Target specificity: AXL is basically not expressed on the cell membrane of normal cells, but is highly expressed on the cell membrane of tumor tissues and macrophages. Therefore, this CAR specifically kills tumor cells and macrophages that highly express AXL on their membranes, while having no obvious killing effect on normal cells.

[0222] 2) This invention utilizes a ligand-receptor binding mechanism, rather than the traditional scFv. The conservative nature of receptor-ligand interactions means that safety studies in animals, particularly primates, are more reflective of safety in humans.

[0223] 3) GAS6 can also bind to other members of the TAM family, and multi-target effects can prevent tumor cells from immune escape.

[0224] 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 and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0225] Unless otherwise stated, the reagents, plasmids, and cells in the examples of this application are commercially available.

[0226] Table A Sequence

[0227]

[0228]

[0229] cell lines

[0230] cell lines type PANC1 pancreatic cancer cells BXPC3 pancreatic cancer cells ASPC1 pancreatic cancer cells U251 glioma cells U87 glioma cells H1299 Non-small cell lung cancer cells A549 Non-small cell lung cancer cells PC3 prostate cancer cells MHCC-97H liver cancer cells HepG2 liver cancer cells SMMC-7721 liver cancer cells MDA-MB468 breast cancer cells MDA-MB231 breast cancer cells NUGC4 Gastric cancer cells MKN28 Gastric cancer cells AGS Gastric cancer cells

[0231] Example 1: Preparation of GAS6-CAR vector

[0232] Based on the nucleotide sequence of GAS6 (NM_000820.4), human CD8 signal peptide, human CD8α hinge region, human CD8 transmembrane region, human 4-1BB intracellular region, and human CD3ζ intracellular region, the corresponding nucleotide sequences were obtained by synthetic methods or PCR. The CD8 signal peptide and GAS6 extracellular region were synthesized (to ensure the integrity of the fibronectin G-like structure of the GAS6 extracellular domain, 18 amino acids of the epidermal growth factor-like domain were retained at the N-terminus). The nucleotide sequence of the CAR molecule was digested with AgeI (Thermo Fisher Scientific) and NheI (Thermo Fisher Scientific) and ligated with T4 DNA ligase (NEB) into the lentiviral vector pTomo, which already contained the CD8 transmembrane region, 4-1BB costimulatory domain, and CD3ζ signaling region. The cells were then transformed into competent Escherichia coli (Stbl3).

[0233] The recombinant plasmid was sequenced and the sequencing results were compared to confirm whether the plasmid was correct. The sequencing primer was a universal sequencing primer. The sequencing and enzyme digestion identification results showed that the CAR coding sequence was correctly inserted into the predetermined position of the plasmid ( Figure 1 C).

[0234] All plasmids were extracted using QIAGEN's endotoxin-free extraction kit, and the purified plasmids were transfected into HEK-293T cells using Beyotime Lipo6000 for lentiviral packaging.

[0235] Example 2: Virus packaging

[0236] HEK-293T cells were cultured in 15cm dishes for viral packaging. Transfection was performed when the HEK-293T cells reached approximately 80%-90% confluency. A plasmid mixture (20 μg of core plasmid, 10 μg of pCMVΔR8.9, and 4 μg of PMD2.G) was prepared by dissolving the plasmids in 2 ml of OPTIMEM. In a separate centrifuge tube, 2 ml of OPTIMEM and 68 μl of Lipo 6000 were added. After incubation at room temperature for 5 minutes, the plasmid complex was added to the liposome complex and allowed to incubate at room temperature for 20 minutes. The above mixture was added dropwise to the HEK-293T cells and incubated at 37°C for 6 hours, after which the culture medium was removed. Prewarmed complete culture medium was added. Viral supernatants were collected at 48 and 72 hours and centrifuged at 3000 rpm for 20 minutes at 4°C. After filtration through a 0.45 μm filter, the virus was concentrated by centrifugation at 25,000 rpm at 4°C for 2.5 hours. The concentrated virus was dissolved in 30 μl of virus lysis buffer overnight, and the virus titer was detected by QPCR. The results showed that the virus titer met the requirements.

[0237] Example 3: CAR-T cell preparation

[0238] Mononuclear cells were isolated from human peripheral blood using Ficool separation medium, and purified CD3+ T cells were obtained using RosetteSep Human T Cell Enrichment Cocktail (Stemcell Technologies). T cells were activated using CD3 / CD28 magnetic beads (Life Technologies) and then stimulated with 200 U / ml of IL-2 (PeproTech). After 48 hours of stimulation, viral infection was performed. CAR-T cells were generated by infecting T cells with lentivirus at an MOI of 100 in the presence of lentiboost. The culture medium was changed one day after infection.

[0239] Example 4: Detection of the positive rate of infected CAR-T cells by flow cytometry

[0240] CAR-T cells and NTD cells (control group) infected with the virus for 72 hours were collected by centrifugation, washed once with PBS, and the supernatant was discarded. The cells were resuspended in PBS containing 2% FBS, and the positive rate was detected by flow cytometry.

[0241] The results of transfection efficiency are as follows Figure 2 shown.

[0242] like Figure 2 As shown in A, after the CAR-T2A-mKate2 fusion protein expressed by CAR-T cells is cleaved, the resulting mKate2 protein exhibits red fluorescence in the cell.

[0243] Figure 2 B shows that flow cytometry was used to detect the positive expression rate of CAR or mKate2CAR-T, which was approximately 30%.

[0244] Example 5: Detection of AXL expression in each target cell

[0245] (1) Cell immunofluorescence: Target cells were plated on discs in a 24-well plate. After 24 hours, the cells were fixed with 4% paraformaldehyde (PFA) for 20 minutes and washed three times with PBST for 5 minutes each. The cells were blocked with 10% goat serum at room temperature for 1 hour and incubated with an antibody that specifically recognizes AXL at four degrees Celsius overnight. The next day, the cells were washed three times with PBST for 5 minutes each. The cells were incubated with a CY3-labeled secondary antibody that specifically recognizes the primary antibody for 1 hour at room temperature. After washing three times with PBS, the nuclei were stained with DAPI. Confocal microscopy was used for imaging.

[0246] (2) Flow cytometry: Collect 1 million cells, fix the cells with 4% PFA at room temperature for 15 minutes, and wash them by centrifugation in 1X PBS; permeabilize the cells with 100% methanol on ice for 15 minutes, and wash them by centrifugation in 1X PBS; resuspend the cells in 100 μl of diluted primary antibody (1:300), incubate them at room temperature for 1 hour, and wash them by centrifugation in 1X PBS. Discard the supernatant. Repeat the operation. Resuspend the cells with 100 μl of diluted fluorescent substance-conjugated secondary antibody (FITC-488 anti-rabbit), incubate them at room temperature in the dark for 30 minutes, and wash them by centrifugation in 1X PBS. Discard the supernatant. Repeat the operation. Resuspend the cells in 300 μl of 1X PBS and analyze them by flow cytometry.

[0247] (3) Immunoblotting: Collect cells from a 6 cm culture dish and centrifuge at 5500 rpm at 4°C for 5 min. After removing the supernatant, add RIPA cell lysis buffer containing the protease inhibitor PMSF according to the number of cells, lyse on ice for 20 min, centrifuge at 14,000 rpm at 4°C for 30 min, collect the supernatant solution and measure the concentration. Load 50 μg of protein sample and perform protein electrophoresis to detect the expression of AXL in target cells.

[0248] (4) qPCR: Collect cells from 6-well plates, remove the culture medium, add 1 ml of Trizol to lyse the cells, let stand at room temperature for 5 min, add 200 μl / ml Trizol in chloroform, mix by inversion 6-8 times, let stand at room temperature for 5 min; centrifuge at 12000 g, 4°C for 15 min, and transfer the supernatant to another centrifuge tube; add an equal volume of isopropanol, mix by inversion, and let stand at room temperature for 10 min; centrifuge at 12000 g, 4°C for 10 min, and discard the supernatant; add 1 ml of 70% ethanol (prepared with RNase-free H2O) to wash, centrifuge at 7500 g, room temperature for 5 min; discard the supernatant, let stand at room temperature for 10 min to dry RNA, and add 30 μl of RNase-free water to dissolve RNA; determine RNA concentration by Nandrop 2000, and check RNA integrity and quantitative accuracy by 1% agarose gel electrophoresis. cDNA was synthesized according to the instructions of the RevertAid™ First Strand cDNA Synthesis Kit (Thermo Scientific), and mRNA levels were detected.

[0249] The results of AXL expression detection in each cell line are shown in Figure 4 By WB ( Figure 4 A), qPCR ( Figure 4 B) Flow cytometry ( Figure 4C) Detection of AXL expression in target cells revealed consistent results indicating that PANC1 and ASPC1 expressed AXL highly, while BXPC3 expressed AXL lowly. Immunofluorescence localization confirmed that AXL was highly expressed on the cell membranes of PANC1 and ASPC1, and AXL was highly expressed on the cell membranes of PANC1 and BXPC3 ( Figure 4 D).

[0250] Example 6: Construction of target cells carrying luciferase

[0251] The pTomo-CMV-Luciferase-IRES-Puro lentivirus packaging steps were the same as those in Example 2.

[0252] After virus infection of PANC1, BXPC3, ASPC1, U251, U87, H1299, A549, PC3, MHCC-97H, HepG2, SMMC-7721, MDA-MB468, MDA-MB231, NUGC4AGS, MKN28, and HEK-293T cells, they were screened with Puromycin (1ug / ml) for 2 weeks, and PANC1, BXPC3, ASPC1, U251, U87, H1299, A549, PC3, MHCC-97H, HepG2, SMMC-7721, MDA-MB468, MDA-MB231, NUGC4AGS, MKN28, and HEK-293T cells were successfully obtained.

[0253] Example 7: CAR-T cell killing

[0254] In this example, the cytotoxicity of the CAR-T cells of the present invention against different target cells was tested. The target cells used included: AXL-high-expressing target cells: PANC1, ASPC1, U251, U87, H1299, PC3, HepG2, MDA-MB231, AGS; and AXL-low-expressing target cells: BXPC3, A549, MHCC-97H, SMMC-7721, MDA-MB468, NUGC4, and MKN28.

[0255] After digestion and counting of PANC1, BXPC3, ASPC1, U251, U87, H1299, A549, PC3, MHCC-97H, HepG2, SMMC-7721, MDA-MB468, MDA-MB231, NUGC4AGS, and MKN28-luciferase cells, the cell density was adjusted to 2×10 4 / ml. 100ul luciferase cells were inoculated into a 96-well plate, and the cell density of CAR-T and NT cells was adjusted to 1×10 5 / ml, inoculated into a black 96-well plate at an E:T ratio of 5:1, with 100 μl per well. The target cells and T cells were mixed and incubated in an incubator for 24 hours.

[0256] After digestion and counting of PANC1, BXPC3, and ASPC1-luciferase cells, the cell density was adjusted to 2×10 4 / ml. 100ul PANC1, BXPC3, and ASPC1-luciferase cells were seeded into a 96-well plate, and the cell density of CAR-T and NT cells was adjusted to 1×10 5 / ml, and inoculated into a black 96-well plate at E:T ratios of 0.5:1, 1:1, 2:1, and 4:1, with 100 μl per well. The target cells and T cells were mixed and incubated in an incubator for 24 hours.

[0257] The cell supernatant was collected and frozen at -80°C to detect the amount of IFNγ released (see Example 8). Cell killing was detected using a Promega fluorescence detection kit. First, the cells were treated with 20ul 1*PLB lysis buffer for 20 minutes, and 100ul substrate was added to each well and immediately detected using a BioTek microplate reader.

[0258] Cytotoxic killing cell % = (1-fluorescence value of target cells with effector cells / fluorescence value of target cells without effector cells) × 100%

[0259] The results of GAS6-CAR's killing effect on different tumor cell lines are as follows Figure 3 As shown. Pancreatic cancer cells (A); glioma cells (B); non-small cell lung cancer cells (C); prostate cancer cells (D); liver cancer cells (E); breast cancer cells (F); gastric cancer cells (G). The results showed that GAS6-CAR has a good killing effect on multiple tumor cell lines.

[0260] The results of the gradient killing of different pancreatic cancer cell lines by GAS6-CAR are as follows Figure 5 Figure 2. (A) Gradient killing effect of GAS6-CAR on PANC1; (B) Gradient killing effect of GAS6-CAR on BXPC3; (C) Gradient killing effect of GAS6-CAR on ASPC1. The results showed that the killing effect of GAS6 CAR-T cells on AXL-overexpressing tumor cells gradually increased with increasing effector-target ratio (E:T).

[0261] Example 8: IFNγ cytokine release

[0262] In this example, the release of cytokines when the CAR-T cells of the present invention were co-incubated with target cells was detected using the supernatant of the co-incubated cells in a cell killing experiment.

[0263] The method is as follows: the cell supernatant obtained by co-incubating the CAR-T cells of the present invention with PANC1, BXPC3, ASPC1, and HEK-293T target cells (E:T ratio of 4:1) in Example 7 was used to detect IFNγ using the IFN gamma Human ELISA Kit (life technology).

[0264] Dissolve the standard in Standard Dilution Buffer and perform serial dilutions to obtain standards at concentrations of 1000 pg / ml, 500 pg / ml, 250 pg / ml, 125 pg / ml, 62.5 pg / ml, 31.2 pg / ml, 15.6 pg / ml, and 0 pg / ml.

[0265] Add 50ul of Incubation buffer, 50ul of test sample, and 50ul of IFNγbiotin conjugated solution to each well, mix well, and let stand at room temperature for 90 minutes.

[0266] Then follow these steps in order:

[0267] (1) Wash the wells 4 times with 1*Wash Buffer, each time for 1 minute.

[0268] (2) Add 100 μl of 1*Streptavidin-HRP solution to each well and let it stand at room temperature for 45 minutes.

[0269] (3) Wash the wells 4 times with 1*Wash Buffer, each time for 1 minute.

[0270] (4) Add 100 μl of stabilized chromogen and let stand at room temperature for 30 minutes.

[0271] (5) Add 100 μl of Stop solution to each well and mix thoroughly.

[0272] (6) Detect the absorbance at 450 nm.

[0273] The results are as follows Figure 6 As shown in the figure, cytokine levels increased significantly after GAS6-CAR-T cells killed PANC1 and ASPC1, while BXPC3 showed no significant changes. These results indicate that the killing effect of GAS6-CAR-T cells on tumor cells is accompanied by IFNγ release, suggesting that this killing effect is related to IFNγ release.

[0274] Example 9: Effect of AXL overexpression on the killing effect of GAS6-CAR-T

[0275] According to the CDS region sequence of AXL, an AXL overexpression plasmid (EX-Z7835-Lv105-B, ORF lentiviral expression clone) was purchased from Yijin Biotechnology and transiently transfected with the AXL overexpression plasmid.

[0276] One day before transfection, 500,000 target cells were seeded into a six-well plate for culture. Before transfection, each well of the six-well plate was replaced with 2 ml of fresh culture medium (containing serum but not antibiotics); two clean sterile centrifuge tubes were taken and 125 μl was added to each well. Then add 2.5 μg of plasmid DNA to one tube and mix it by gently blowing with a gun; add 5 μl of Lipo6000 to the other tube. TM After standing at room temperature for 5 minutes, add the culture medium containing DNA to the transfection reagent containing Lipo6000. TM In the culture medium of the transfection reagent, gently invert the centrifuge tube or gently blow with a gun to mix. After standing at room temperature for 5 minutes, add it to the 6-well plate and mix it. After 48 hours, detect the overexpression efficiency and perform GAS6-CAR-T killing test.

[0277] The cells of different pancreatic cancer cell lines BXPC3-Vector and BXPC3-AXL-luciferase were digested and counted, and the cell density was adjusted to 2×10 4 / ml. 100ul luciferase cells were inoculated into a 96-well plate, and the cell density of CAR-T / NT cells was adjusted to 1×10 5 Target cells and T cells were mixed and incubated in a black 96-well plate at an E:T ratio of 4:1, with 100 μl per well.

[0278] The killing effect of GAS6-CAR-T on pancreatic cancer cell line BXPC3 after overexpression of AXL is shown in the figure. Figure 7 shown. Figure 7 -A is WB detection of AXL protein expression. Figure 7 -B: qPCR detection of AXL mRNA levels. Figure 7 -C is flow cytometry detection of AXL expression. Figure 7 -D is the expression level of AXL on the cell membrane detected by immunofluorescence. Figure 7 -E represents the killing effect of GAS6-CAR on BXPC3 after overexpression of AXL. Figure 7-F represents the IFNγ release after GAS6-CAR-T cells kill BXPC3 cells overexpressing AXL. The results showed that compared with the control group (BXPC3-Vector), the killing rate and IFNγ release of GAS6-CAR-T cells against BXPC3-AXL cells overexpressing AXL were significantly increased. This result indicates that GAS6-CAR-T cells have a significantly enhanced killing effect on AXL-overexpressing tumor cells.

[0279] Example 10: Effect of specific knockdown of AXL on the killing effect of GAS6-CAR-T

[0280] Based on the shRNA sequence library provided by Sigma, MISSION shRNA Lentiviral Transduction Particles were selected, shRNAs with validated CDS regions were selected, and each selected shRNA was BLASTed on NCBI to ensure target specificity.

[0281] One day before transfection, seed 1 million HEK-293T cells per dish into a 6 cm dish for culture. Before transfection, replace the 6 cm dish with 5 ml of fresh culture medium (containing serum but without antibiotics); take two clean sterile centrifuge tubes and add 250 μl of each. Then add 5 μg of plasmid DNA to one tube and mix it by gently blowing with a gun; add 10 μl of Lipo6000 to the other tube. TM After standing at room temperature for 5 minutes, add the culture medium containing DNA to the transfection reagent containing Lipo6000. TM In the culture medium containing the transfection reagent, gently invert the centrifuge tube or gently blow it with a gun to mix. After standing at room temperature for 20 minutes, add it to a 6 cm dish and mix it. Collect the supernatant after 48 hours and 72 hours respectively, filter it, and centrifuge it at 3000r / min and 4℃ for 15 minutes to obtain the virus-containing supernatant.

[0282] The day before infection, 500,000 target cells were seeded into six-well plates and cultured. Before infection, each well of the six-well plate was replaced with 1 ml of fresh culture medium (containing serum but without antibiotics), followed by 1 ml of viral supernatant and 2 μl of polybrane (10 mg / ml). After 24 hours, complete culture medium was replaced. After 72 hours, shRNA knockdown efficiency was tested and GAS6-CAR-T cell killing was performed. In vitro killing was performed as described above, and the killing of PANC1 and PANC1-shAXL by GAS6-CAR was detected by fluorescence value changes.

[0283] After digestion and counting of PANC1-shCOO2, PANC1-shAXL#1, and PANC1-shAXL#2-luciferase cells, the cell density was adjusted to 2×10 4 / ml. 100ul luciferase cells were inoculated into a 96-well plate, and the cell density of CAR-T / NT cells was adjusted to 1×10 5 100 μl of T cells were seeded into each well of a black 96-well plate at an E:T ratio of 4:1. The target cells and T cells were mixed and incubated in an incubator for 24 hours before the killing effect was measured.

[0284] The results are as follows Figure 8 shown. Figure 8 -A is WB detection of AXL protein expression. Figure 8 -B: qPCR detection of AXL mRNA levels. Figure 8 -C is flow cytometry detection of AXL expression. Figure 8 -D is the expression level of AXL on the cell membrane detected by immunofluorescence. Figure 8 -E represents the killing effect of GAS6-CAR on AXL after PANC1 knockdown. Figure 8 -F represents the IFNγ release after GAS6-CAR silenced PANC1 AXL. The results showed that compared with the control PANC1-shCOO2 cells, the killing rate and IFNγ release of GAS6-CAR-T cells against AXL knockdown PANC1-shAXL#1 and PANC1-shAXL#1 cells were significantly reduced. This result indicates that knockdown of AXL on the cell membrane reduces the killing effect of GAS6-CAR-T.

[0285] Example 11: Killing effect of GAS6-CAR-T on non-tumor cells

[0286] HEK-293T cells are a human embryonic kidney cell line and were seeded into black 96-well plates at effector-target ratios of 0.5:1, 1:1, 2:1, and 4:1. GAS6-CAR-T cells were co-incubated with HEK-293T-luciferase cells, and the killing effect of GAS6-CAR-T on HEK-293T cells was detected by changes in fluorescence values.

[0287] The results are as follows Figure 9 shown. Figure 9 -A is WB detection of AXL protein expression. Figure 9 -B: qPCR detection of AXL mRNA levels. Figure 9 -C is flow cytometry detection of AXL expression. Figure 9 -D is the expression level of AXL on the cell membrane detected by immunofluorescence. Figure 9-E represents the killing effect of GAS6-CAR on HEK-293T. Figure 9 -F is the IFNγ release induced by GAS6-CAR killing HEK-293T cells.

[0288] The above results showed that GAS6-CAR-T had no significant killing effect on non-tumor HEK-293T cells.

[0289] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto. Sequence Listing <110> West China Hospital of Sichuan University <120> Preparation and application of chimeric antigen receptor immune cells constructed based on GAS6 <130> P2021-1800 <160> 9 <170> PatentIn version 3.5 <210> 1 <211> 678 <212> PRT <213> Homo sapiens <400> 1 Met Ala Pro Ser Leu Ser Pro Gly Pro Ala Ala Leu Arg Arg Ala Pro 1 5 10 15 Gln Leu Leu Leu Leu Leu Leu Ala Ala Glu Cys Ala Leu Ala Ala Leu 20 25 30 Leu Pro Ala Arg Glu Ala Thr Gln Phe Leu Arg Pro Arg Gln Arg Arg 35 40 45 Ala Phe Gln Val Phe Glu Glu Ala Lys Gln Gly His Leu Glu Arg Glu 50 55 60 Cys Val Glu Glu Leu Cys Ser Arg Glu Glu Ala Arg Glu Val Phe Glu 65 70 75 80 Asn Asp Pro Glu Thr Asp Tyr Phe Tyr Pro Arg Tyr Leu Asp Cys Ile 85 90 95 Asn Lys Tyr Gly Ser Pro Tyr Thr Lys Asn Ser Gly Phe Ala Thr Cys 100 105 110 Val Gln Asn Leu Pro Asp Gln Cys Thr Pro Asn Pro Cys Asp Arg Lys 115 120 125 Gly Thr Gln Ala Cys Gln Asp Leu Met Gly Asn Phe Phe Cys Leu Cys 130 135 140 Lys Ala Gly Trp Gly Gly Arg Leu Cys Asp Lys Asp Val Asn Glu Cys 145 150 155 160 Ser Gln Glu Asn Gly Gly Cys Leu Gln Ile Cys His Asn Lys Pro Gly 165 170 175 Ser Phe His Cys Ser Cys His Ser Gly Phe Glu Leu Ser Ser Asp Gly 180 185 190 Arg Thr Cys Gln Asp Ile Asp Glu Cys Ala Asp Ser Glu Ala Cys Gly 195 200 205 Glu Ala Arg Cys Lys Asn Leu Pro Gly Ser Tyr Ser Cys Leu Cys Asp 210 215 220 Glu Gly Phe Ala Tyr Ser Ser Gln Glu Lys Ala Cys Arg Asp Val Asp 225 230 235 240 Glu Cys Leu Gln Gly Arg Cys Glu Gln Val Cys Val Asn Ser Pro Gly 245 250 255 Ser Tyr Thr Cys His Cys Asp Gly Arg Gly Gly Leu Lys Leu Ser Gln 260 265 270 Asp Met Asp Thr Cys Glu Asp Ile Leu Pro Cys Val Pro Phe Ser Val 275 280 285 Ala Lys Ser Val Lys Ser Leu Tyr Leu Gly Arg Met Phe Ser Gly Thr 290 295 300 Pro Val Ile Arg Leu Arg Phe Lys Arg Leu Gln Pro Thr Arg Leu Val 305 310 315 320 Ala Glu Phe Asp Phe Arg Thr Phe Asp Pro Glu Gly Ile Leu Leu Phe 325 330 335 Ala Gly Gly His Gln Asp Ser Thr Trp Ile Val Leu Ala Leu Arg Ala 340 345 350 Gly Arg Leu Glu Leu Gln Leu Arg Tyr Asn Gly Val Gly Arg Val Thr 355 360 365 Ser Ser Gly Pro Val Ile Asn His Gly Met Trp Gln Thr Ile Ser Val 370 375 380 Glu Glu Leu Ala Arg Asn Leu Val Ile Lys Val Asn Arg Asp Ala Val 385 390 395 400 Met Lys Ile Ala Val Ala Gly Asp Leu Phe Gln Pro Glu Arg Gly Leu 405 410 415 Tyr His Leu Asn Leu Thr Val Gly Gly Ile Pro Phe His Glu Lys Asp 420 425 430 Leu Val Gln Pro Ile Asn Pro Arg Leu Asp Gly Cys Met Arg Ser Trp 435 440 445 Asn Trp Leu Asn Gly Glu Asp Thr Thr Ile Gln Glu Thr Val Lys Val 450 455 460 Asn Thr Arg Met Gln Cys Phe Ser Val Thr Glu Arg Gly Ser Phe Tyr 465 470 475 480 Pro Gly Ser Gly Phe Ala Phe Tyr Ser Leu Asp Tyr Met Arg Thr Pro 485 490 495 Leu Asp Val Gly Thr Glu Ser Thr Trp Glu Val Glu Val Val Ala His 500 505 510 Ile Arg Pro Ala Ala Asp Thr Gly Val Leu Phe Ala Leu Trp Ala Pro 515 520 525 Asp Leu Arg Ala Val Pro Leu Ser Val Ala Leu Val Asp Tyr His Ser 530 535 540 Thr Lys Lys Leu Lys Lys Gln Leu Val Val Leu Ala Val Glu His Thr 545 550 555 560 Ala Leu Ala Leu Met Glu Ile Lys Val Cys Asp Gly Gln Glu His Val 565 570 575 Val Thr Val Ser Leu Arg Asp Gly Glu Ala Thr Leu Glu Val Asp Gly 580 585 590 Thr Arg Gly Gln Ser Glu Val Ser Ala Ala Gln Leu Gln Glu Arg Leu 595 600 605 Ala Val Leu Glu Arg His Leu Arg Ser Pro Val Leu Thr Phe Ala Gly 610 615 620 Gly Leu Pro Asp Val Pro Val Thr Ser Ala Pro Val Thr Ala Phe Tyr 625 630 635 640 Arg Gly Cys Met Thr Leu Glu Val Asn Arg Arg Leu Leu Asp Leu Asp 645 650 655 Glu Ala Ala Tyr Lys His Ser Asp Ile Thr Ala His Ser Cys Pro Pro 660 665 670 Val Glu Pro Ala Ala Ala 675 <210> 2 <211> 233 <212> PRT <213> Artificial Sequence <400> 2 Met Ser Glu Leu Ile Lys Glu Asn Met His Met Lys Leu Tyr Met Glu 1 5 10 15 Gly Thr Val Asn Asn His His Phe Lys Cys Thr Ser Glu Gly Glu Gly 20 25 30 Lys Pro Tyr Glu Gly Thr Gln Thr Met Arg Ile Lys Ala Val Glu Gly 35 40 45 Gly Pro Leu Pro Phe Ala Phe Asp Ile Leu Ala Thr Ser Phe Met Tyr 50 55 60 Gly Ser Lys Thr Phe Ile Asn His Thr Gln Gly Ile Pro Asp Phe Phe 65 70 75 80 Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Val Thr Thr Tyr 85 90 95 Glu Asp Gly Gly Val Leu Thr Ala Thr Gln Asp Thr Ser Leu Gln Asp 100 105 110 Gly Cys Leu Ile Tyr Asn Val Lys Ile Arg Gly Val Asn Phe Pro Ser 115 120 125 Asn Gly Pro Val Met Gln Lys Lys Thr Leu Gly Trp Glu Ala Ser Thr 130 135 140 Glu Thr Leu Tyr Pro Ala Asp Gly Gly Leu Glu Gly Arg Ala Asp Met 145 150 155 160 Ala Leu Lys Leu Val Gly Gly Gly His Leu Ile Cys Asn Leu Lys Thr 165 170 175 Thr Tyr Arg Ser Lys Lys Pro Ala Lys Asn Leu Lys Met Pro Gly Val 180 185 190 Tyr Tyr Val Asp Arg Arg Leu Glu Arg Ile Lys Glu Ala Asp Lys Glu 195 200 205 Thr Tyr Val Glu Gln His Glu Val Ala Val Ala Arg Tyr Cys Asp Leu 210 215 220 Pro Ser Lys Leu Gly His Lys Leu Asn 225 230 <210> 3 <211> 21 <212> PRT <213> Artificial Sequence <400> 3 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 20 <210> 4 <211> 45 <212> PRT <213> Artificial Sequence <400> 4 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 5 <211> 24 <212> PRT <213> Artificial Sequence <400> 5 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys 20 <210> 6 <211> 43 <212> PRT <213> Artificial Sequence <400> 6 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg 35 40 <210> 7 <211> 111 <212> PRT <213> Artificial Sequence <400> 7 Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln 1 5 10 15 Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp 20 25 30 Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro 35 40 45 Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp 50 55 60 Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg 65 70 75 80 Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr 85 90 95 Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 8 <211> 662 <212> PRT <213> Artificial Sequence <400> 8 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 His Cys Asp Gly Arg Gly Gly Leu Lys Leu Ser 20 25 30 Gln Asp Met Asp Thr Cys Glu Asp Ile Leu Pro Cys Val Pro Phe Ser 35 40 45 Val Ala Lys Ser Val Lys Ser Leu Tyr Leu Gly Arg Met Phe Ser Gly 50 55 60 Thr Pro Val Ile Arg Leu Arg Phe Lys Arg Leu Gln Pro Thr Arg Leu 65 70 75 80 Val Ala Glu Phe Asp Phe Arg Thr Phe Asp Pro Glu Gly Ile Leu Leu 85 90 95 Phe Ala Gly Gly His Gln Asp Ser Thr Trp Ile Val Leu Ala Leu Arg 100 105 110 Ala Gly Arg Leu Glu Leu Gln Leu Arg Tyr Asn Gly Val Gly Arg Val 115 120 125 Thr Ser Ser Gly Pro Val Ile Asn His Gly Met Trp Gln Thr Ile Ser 130 135 140 Val Glu Glu Leu Ala Arg Asn Leu Val Ile Lys Val Asn Arg Asp Ala 145 150 155 160 Val Met Lys Ile Ala Val Ala Gly Asp Leu Phe Gln Pro Glu Arg Gly 165 170 175 Leu Tyr His Leu Asn Leu Thr Val Gly Gly Ile Pro Phe His Glu Lys 180 185 190 Asp Leu Val Gln Pro Ile Asn Pro Arg Leu Asp Gly Cys Met Arg Ser 195 200 205 Trp Asn Trp Leu Asn Gly Glu Asp Thr Thr Ile Gln Glu Thr Val Lys 210 215 220 Val Asn Thr Arg Met Gln Cys Phe Ser Val Thr Glu Arg Gly Ser Phe 225 230 235 240 Tyr Pro Gly Ser Gly Phe Ala Phe Tyr Ser Leu Asp Tyr Met Arg Thr 245 250 255 Pro Leu Asp Val Gly Thr Glu Ser Thr Trp Glu Val Glu Val Val Ala 260 265 270 His Ile Arg Pro Ala Ala Asp Thr Gly Val Leu Phe Ala Leu Trp Ala 275 280 285 Pro Asp Leu Arg Ala Val Pro Leu Ser Val Ala Leu Val Asp Tyr His 290 295 300 Ser Thr Lys Lys Leu Lys Lys Gln Leu Val Val Leu Ala Val Glu His 305 310 315 320 Thr Ala Leu Ala Leu Met Glu Ile Lys Val Cys Asp Gly Gln Glu His 325 330 335 Val Val Thr Val Ser Leu Arg Asp Gly Glu Ala Thr Leu Glu Val Asp 340 345 350 Gly Thr Arg Gly Gln Ser Glu Val Ser Ala Ala Gln Leu Gln Glu Arg 355 360 365 Leu Ala Val Leu Glu Arg His Leu Arg Ser Pro Val Leu Thr Phe Ala 370 375 380 Gly Gly Leu Pro Asp Val Pro Val Thr Ser Ala Pro Val Thr Ala Phe 385 390 395 400 Tyr Arg Gly Cys Met Thr Leu Glu Val Asn Arg Arg Leu Leu Asp Leu 405 410 415 Asp Glu Ala Ala Tyr Lys His Ser Asp Ile Thr Ala His Ser Cys Pro 420 425 430 Pro Val Glu Pro Ala Ala Ala Thr Thr Thr Pro Ala Pro Arg Pro Pro 435 440 445 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 450 455 460 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 465 470 475 480 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 485 490 495 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 500 505 510 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 515 520 525 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 530 535 540 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 545 550 555 560 Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 565 570 575 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 580 585 590 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 595 600 605 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 610 615 620 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 625 630 635 640 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 645 650 655 Gln Ala Leu Pro Pro Arg 660 <210> 9 <211> 1986 <212> DNA <213> Artificial Sequence <400> 9 atggccctgc ccgtcaccgc tctgctgctg ccccttgctc tgcttcttca tgcagcaagg 60 ccgcactgtg acgggcgtgg gggcctcaag ctgtcccagg acatggacac ctgtgaggac 120 atcttgccgt gcgtgccctt cagcgtggcc aagagtgtga agtccttgta cctgggccgg 180 atgttcagtg ggacccccgt gatccgactg cgcttcaaga ggctgcagcc caccaggctg 240 gtagctgagt ttgacttccg gacctttgac cccgagggca tcctcctctt tgccggaggc 300 caccaggaca gcacctggat cgtgctggcc ctgagagccg gccggctgga gctgcagctg 360 cgctacaacg gtgtcggccg tgtcaccagc agcggcccgg tcatcaacca tggcatgtgg 420 cagacaatct ctgttgagga gctggcgcgg aatctggtca tcaaggtcaa cagggatgct 480 gtcatgaaaa tcgcggtggc cggggacttg ttccaaccgg agcgaggact gtatcatctg 540 aacctgaccg tgggaggtat tcccttccat gagaaggacc tcgtgcagcc tataaaccct 600 cgtctggatg gctgcatgag gagctggac tggctgacg gagagacac caccatccag gaaacggtga aagtgaacac gaggatgcag tgcttctcgg tgacggagag aggctctttc 720 taccccggga gcggcttcgc cttctacagc ctggactaca tgcggacccc tctggacgtc 780 gggactgaat caacctggga to take gtggctcaca tccgcccagc cgcagacaca 840 ggcgtgctgt ttgcgctctg ggcccccgac ctccgtgccg tgcctctctc tgtggcactg 900 gtagactatc actccacgaa gaaactcaag aagcagctgg tggtcctggc cgtggagcat acggccttgg ccctaatgga gatcaaggtc tgcgacggcc aagagcacgt ggtcaccgtc 1020 tcgctgaggg acggtgaggc caccctggag gtggacggca ccaggggcca gagcgaggtg 1080 agcgccgcgc agctgcagga gaggctggcc gtgctcgaga ggcacctgcg gagccccgtg 1140 ctcacctttg ctggcggcct gccagatgtg ccggtgactt cagcgccagt caccgcgttc 1200 taccgcggct gcatgacact ggaggtcaac cggaggctgc tggacctgga cgaggcggcg 1320. 1320. 1320. 1320. 1320. 1320. 1320. 1320 acgacgccag cgccgcgacc accaacaccg gcgcccacca tcgctagcca gcccctgtcc 1380 ctgcgcccag aggcgtgccg gccagcggcg gggggcgcag tgcacacgag ggggctggac 1440 ttcgcctgtg atatctacat ctgggcgccc ttggccggga cttgtggggt ccttctcctg 1500 tcactggtta tcacccttta ctgcaaacgg ggcaagaaga aactcctgta tatattcaaa 1560 caaccattta tgagaccagt acaaactact caagagaag atggctgtag ctgccgattt 1620 1680 cccgcgtaca agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag 1740 gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga 1800 1860 tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac 1920 cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc 1980 cctcgc 1986

Claims

1. A chimeric antigen receptor (CAR), characterized in that The CAR contains an extracellular binding domain, and the extracellular binding domain can specifically bind to the GAS6 receptor. The structure of the chimeric antigen receptor is shown in the following formula I: L-EB-H-TM-C-CD3ζ-RP(I) In the formula, Each "-" is independently a connecting peptide or a peptide bond; L is none or a signal peptide sequence; EB is an extracellular binding domain, the amino acid sequence of which is shown in positions 261 to 678 of SEQ ID NO: 1; H is no or hinge region; TM is the transmembrane domain; C is no or co-stimulatory signaling molecule; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ; RP is none or reporter protein.

2. The CAR according to claim 1, wherein: The signal peptide is a signal peptide of a protein selected from the group consisting of CD8, CD28, and CD4; The hinge region is a hinge region of a protein selected from the group consisting of CD8, CD28; The transmembrane domain is a transmembrane region of a protein selected from the group consisting of CD28, CD8; The co-stimulatory signal molecule is a co-stimulatory signal molecule of a protein selected from the group consisting of CD28, 4-1BB, or a combination thereof.

3. The chimeric antigen receptor according to claim 1, wherein L is a signal peptide derived from CD8; H is the hinge region of CD8 origin; TM is the transmembrane region of CD8 origin; and C is a co-stimulatory signal molecule derived from 4-1BB.

4. The CAR according to claim 1, wherein: The amino acid sequence of L is shown in SEQ ID NO:

3.

5. The CAR according to claim 1, wherein: The amino acid sequence of H is shown in SEQ ID NO:

4.

6. The CAR according to claim 1, wherein: The amino acid sequence of the TM is shown in SEQ ID NO:

5.

7. The CAR according to claim 1, wherein: The amino acid sequence of C is shown in SEQ ID NO:

6.

8. The CAR according to claim 1, wherein: The amino acid sequence of the cytoplasmic signal transduction sequence derived from CD3ζ is shown in SEQ ID NO:

7.

9. The CAR according to claim 1, wherein: The amino acid sequence of the chimeric antigen receptor CAR is shown in SEQ ID NO:

8.

10. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the chimeric antigen receptor according to claim 1.

11. The nucleic acid molecule according to claim 10, wherein The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

9.

12. A carrier, characterized in that The vector contains the nucleic acid molecule according to claim 10.

13. A host cell, characterized in that The host cell contains the vector as claimed in claim 12 or has the exogenous nucleic acid molecule as claimed in claim 10 integrated into its chromosome or expresses the chimeric antigen receptor as claimed in claim 1.

14. An engineered immune cell, characterized in that: The immune cell contains the vector as claimed in claim 12 or the chromosome into which the exogenous nucleic acid molecule as claimed in claim 10 is integrated or expresses the chimeric antigen receptor as claimed in claim 1.

15. The engineered immune cell according to claim 14, characterized in that The engineered immune cells are chimeric antigen receptor T cells or chimeric antigen receptor NK cells.

16. A method for preparing the engineered immune cell according to claim 14, characterized in that: The following steps are involved: The nucleic acid molecule according to claim 10 or the vector according to claim 12 is transduced into immune cells to obtain the engineered immune cells.

17. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the chimeric antigen receptor as claimed in claim 1, the nucleic acid molecule as claimed in claim 10, the vector as claimed in claim 12, the host cell as claimed in claim 13, and / or the engineered immune cell as claimed in claim 14, and a pharmaceutically acceptable carrier, diluent or excipient.

18. A use of the chimeric antigen receptor according to claim 1, the nucleic acid molecule according to claim 10, the vector according to claim 12, or the host cell according to claim 13, and / or the engineered immune cell according to claim 14, characterized in that: Used for preparing a drug for treating a disease with high expression of the GAS6 receptor AXL, wherein the disease is selected from the following group: pancreatic cancer, breast cancer, gastric cancer, liver cancer, colorectal cancer, bladder cancer, lung cancer, ovarian cancer, esophageal cancer, glioma, prostate cancer, nasopharyngeal carcinoma or a combination thereof.

19. A use of the chimeric antigen receptor according to claim 1, the nucleic acid molecule according to claim 10, the vector according to claim 12, or the host cell according to claim 13, and / or the engineered immune cell according to claim 14, characterized in that: Used for preparing a preparation for treating a disease with high expression of the GAS6 receptor AXL, wherein the disease is selected from the following group: pancreatic cancer, breast cancer, gastric cancer, liver cancer, colorectal cancer, bladder cancer, lung cancer, ovarian cancer, esophageal cancer, glioma, prostate cancer, nasopharyngeal carcinoma or a combination thereof.

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