Chimeric antigen receptor modified based on receptor tyrosine kinase family AXL and application thereof

Abstract

The invention relates to the field of immune cell therapy, and provides a chimeric antigen receptor modified based on receptor tyrosine kinase family AXL and application thereof.The chimeric antigen receptor contains an extracellular binding domain, and the extracellular binding domain can be specifically bound with an AXL ligand; the amino acid sequence of the AXL is as shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5. Target specificity: GAS6 is low in expression on cell membranes of normal cells, but is high in expression on cell membranes of tumor tissues, so that the CAR provided by the invention can specifically kill tumor cells with high expression of GAS6 on the membranes, and has no obvious toxic effect on the normal cells. According to the invention, an action mode of combining a ligand and a receptor is utilized instead of scFv in a traditional sense. The conservative property of the receptor-ligand interaction determines the safety test in animals, especially primates, and can reflect the safety of the receptor-ligand in human bodies.

Description

Technical Field

[0001] The present application belongs to the field of immune cell therapy, and in particular relates to a chimeric antigen receptor based on the modification of the receptor tyrosine kinase family AXL and its application. Background Art

[0002] CAR-T cell therapy is a new immunotherapy method that has not only made significant research progress in blood tumors, but also demonstrated considerable application prospects in solid tumors. However, CAR-T cell therapy still faces the following challenges: antigen escape, CAR-T infiltration, immunosuppressive microenvironment, non-tumor targeted toxicity and CAR-T safety.

[0003] Traditional CAR-T uses an antigen-specific scFv design, and the high affinity between the antigen and the antibody increases the immune response, such as "non-tumor targeted toxicity", resulting in reduced safety of traditional CAR-T. Summary of the invention

[0004] The purpose of this application is to provide a chimeric antigen receptor based on the modification of the receptor tyrosine kinase family AXL, aiming to solve the safety issues of traditional CAR-T.

[0005] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0006] In a first aspect, the present application provides a chimeric antigen receptor (CAR) modified based on the receptor tyrosine kinase family AXL, wherein the chimeric antigen receptor contains an extracellular binding domain, and the extracellular binding domain can specifically bind to the AXL ligand.

[0007] A possible design is a chimeric antigen receptor (CAR) based on the modification of the receptor tyrosine kinase family AXL, characterized in that the chimeric antigen receptor contains an extracellular binding domain, and the extracellular binding domain can specifically bind to the AXL ligand;

[0008] The amino acid sequence of AXL is shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5.

[0009] In a possible design, the structure of the chimeric antigen receptor is shown in Formula I below:

[0010] L-EF1α-EB-H-TM-C-CD3ζ-RP(I)

[0011] In the formula,

[0012] Each "-" is independently a connecting peptide or a peptide bond;

[0013] L is none or a signal peptide sequence;

[0014] EF1α is the promoter element sequence;

[0015] EB is the extracellular binding domain;

[0016] H is the hinge region;

[0017] TM is the transmembrane domain;

[0018] C is no or co-stimulatory signal molecule;

[0019] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ;

[0020] RP is none or reporter protein.

[0021] The amino acid sequence of the signal peptide sequence is shown in SEQ ID NO:9, the amino acid sequence of the hinge region is shown in SEQ ID NO:11, and the amino acid sequence of the transmembrane domain is shown in SEQ ID NO:13.

[0022] The C is selected from the co-stimulatory signal molecules of the following histones: CD28, 4-1BB or a combination thereof, and the amino acid sequence of the 4-1BB is shown in SEQ ID NO:15.

[0023] The amino acid sequence of CD3ζ is shown in SEQ ID NO: 17;

[0024] The reporter protein is a red fluorescent protein mKate2, and the amino acid sequence of the red fluorescent protein mKate2 is shown in SEQ ID NO:7.

[0025] In a second aspect, the present invention provides a nucleic acid molecule encoding the aforementioned chimeric antigen receptor.

[0026] In a third aspect, the present invention provides a vector, wherein the vector contains the aforementioned nucleic acid molecule.

[0027] In a fourth aspect, the present invention provides an engineered immune cell, wherein the engineered immune cell contains the aforementioned vector or chromosome in which the aforementioned exogenous nucleic acid molecule is integrated or expresses the aforementioned chimeric antigen receptor.

[0028] In a fifth aspect, the present invention provides a pharmaceutical composition comprising the aforementioned chimeric antigen receptor, the aforementioned nucleic acid molecule, the aforementioned vector, and / or the aforementioned engineered immune cell, and a pharmaceutically acceptable carrier, diluent or excipient.

[0029] The beneficial effects of the present invention are:

[0030] 1) Target specificity: GAS6 is lowly expressed on the cell membrane of normal cells, but highly expressed on the cell membrane of tumor tissues. Therefore, the CAR provided by the present invention can specifically kill tumor cells with high expression of GAS6 on the membrane, without obvious toxic effects on normal cells.

[0031] 2) The present invention utilizes the mode of action of ligand binding to receptor, rather than the traditional scFv. The conservative nature of receptor-ligand interaction determines the safety test in animals, especially primates, which can better reflect its safety in humans. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 It is a characteristic presentation diagram of AXL-CAR in the embodiment of the present application;

[0034] Figure 2 This is a graph showing the expression of GAS6 in different tumor cells in the examples of this application;

[0035] Figure 3 This is a graph showing the results of AXL-CAR-T cells effectively killing GAS6-positive tumor cell lines in the examples of the present application. DETAILED DESCRIPTION

[0036] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0037] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0038] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0039] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0040] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0041] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as μg, mg, g, kg, etc.

[0042] The term "PET" is the abbreviation of "Polyethylene terephthalate", which means polyethylene terephthalate; the term "PU" is the abbreviation of "polyurethane", which means polyurethane material and is the abbreviation of polyurethane.

[0043] Example 1

[0044] Construction of AXL-CAR vector

[0045] Based on the partial nucleotide sequence of AXL (as shown in SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6), human CD8 signal peptide (amino acid sequence as shown in SEQ ID NO: 9, nucleotide sequence as shown in SEQ ID NO: 10), human CD8α hinge region (amino acid sequence as shown in SEQ ID NO: 11, nucleotide sequence as shown in SEQ ID NO: 12), human CD8 transmembrane region (amino acid sequence as shown in SEQ ID NO: 13, nucleotide sequence as shown in SEQ ID NO: 14), human 4-1BB intracellular region (amino acid sequence as shown in SEQ ID NO: 15, nucleotide sequence as shown in SEQ ID NO: 16) and human CD3ζ intracellular region gene sequence (amino acid sequence as shown in SEQ ID NO: 17, nucleotide sequence as shown in SEQ ID NO: 18), the corresponding nucleotide sequence is obtained by artificial synthesis method or PCR method. Artificial synthesis method or PCR method are both existing technologies and will not be described in detail here.

[0046] The CD8 signal peptide, AXL extracellular region, CD8 transmembrane region, 4-1BB costimulatory domain, CD3ζ signaling region, T2A-mKate2 fluorescent protein (amino acid sequence as shown in SEQ ID NO: 7, nucleotide sequence as shown in SEQ ID NO: 8) were seamlessly cloned into the lentiviral vector PGC-EF1α-P2A-EGFP by XbaI. The synthesis and extraction of all plasmids are prior art and will not be described in detail here.

[0047] The structure of the AXL-CAR vector in this example is as follows:

[0048] L-EF1α-EB-H-TM-C-CD3ζ-RP(I)

[0049] Each "-" is independently a connecting peptide or a peptide bond;

[0050] L is none or a signal peptide sequence, selected from the signal peptides of the following histones: CD8;

[0051] EF1α is the promoter element sequence;

[0052] EB is an extracellular binding domain that specifically binds to the GAS6 ligand, and its amino acid sequence is shown in SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6;

[0053] H is a hinge region, selected from the hinge region of a protein of the following group: CD8;

[0054] TM is a transmembrane domain, selected from the transmembrane region of a protein of the following group: CD8;

[0055] C is no or a co-stimulatory signal molecule, selected from the group consisting of the co-stimulatory signal molecules of the following proteins: 4-1BB;

[0056] CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ, and its amino acid sequence is shown in SEQ ID NO: 17;

[0057] RP is None or Reporter protein, where the reporter protein is the red fluorescent protein mKate2.

[0058] Example 2

[0059] Lentivirus preparation

[0060] (1) HEK-293T cells were revived and placed in a 15 cm culture dish. When the cell density reached 90%, they were trypsinized and passaged at a ratio of 1:2.

[0061] (2) When the cell density reaches about 80%, transfection is performed. For each 15 cm dish, core plasmid (20 μg): pCMV-dR8.91 (10 μg): pMD2.G (4 μg) = 5:2.5:1 is added to 2 mL of serum-free DMEM medium. At the same time, 68 μL of lipo8000 is added, mixed and added to the dish;

[0062] (3) Add 13 mL of DMEM medium containing 5% FBS to each 15 cm dish and shake gently;

[0063] (4) Collect the cell culture supernatant 48 hours after transfection and replace with 15 mL of fresh culture medium; and collect the culture supernatant 72 hours after transfection;

[0064] (5) Take the supernatant of the culture medium in (4), centrifuge at 3500 rpm, 4°C for 15 min, and filter through a 0.45 μm filter membrane; add 30 mL of supernatant to each ultracentrifuge tube and add 5 mL of 20% sucrose (spread on the bottom of the tube), centrifuge at 25000 rpm, 4°C for 2.5 h; discard the supernatant, invert and dry the precipitate for 5 min, add 50 μL of lentivirus dissolution solution, and dissolve the virus at 4°C overnight;

[0065] (6) The virus solution was packaged and stored at -80℃.

[0066] Example 3

[0067] Preparation of human CAR-T cells

[0068] (1) T cell isolation: T cells were isolated from the peripheral blood of healthy volunteers using RosetteSep gradient centrifugation tubes and Human T Cell Enrichment Cocktail kit as follows:

[0069] (a) 10 mL peripheral blood + 500 μL RosetteSepTM Cocktail, mix well, let stand for 10 min, and dilute 1-fold with PBS (containing 2% FBS);

[0070] (b) Take 15 mL of Ficol Lymphoprep in a RosetteSep™ centrifuge tube, add (a) to (b), and centrifuge at 1200 g for 10 min;

[0071] (c) The supernatant was placed in a 50 mL centrifuge tube, and an equal volume of PBS (containing 2% FBS) was added, and the mixture was centrifuged at 300 g for 10 min.

[0072] (d) The supernatant was discarded, and freezing solution (70% Advanced RPMI 1640 medium + 20% FBS + 10% DMSO) was added for freezing, and the expression of CD3 was detected by flow cytometry.

[0073] (2) Preparation of CAR-T cells, as follows:

[0074] (a) In culture medium (5 mL FBS + 10 μL IL-2 + 500 μL Gluta-MAX + 500 μL P / S + 44 mL Advanced RPMI 1640) + Human T-Activator CD3 / CD28 magnetic beads (25 μL / 1×10 6 T cells) activated T cells for 72h;

[0075] (b) Centrifuge at 500 g for 5 min and count the cells. Infect 96-well plates with CAR lentiviral particles: 1×10 5 T cells + 1 μL lentiBoost (1 μg / mL) + CAR lentiviral particles (MOI = 100), after 24 hours of infection, centrifuge at 500g for 5 minutes, discard the supernatant and transfer to a 24-well plate; and on the third day after CAR-T cell infection, monitor the infection efficiency of CAR-T by flow cytometry for subsequent experimental studies.

[0076] Using the above method, the CAR-T construction model of this embodiment is as shown in the figure Figure 1 As shown, Figure 1 Figure A in the middle is a schematic diagram of the CD19-CAR and AXL-CAR sequences. The vector is PGC-EF1α-P2A-EGFP, in which EF1α is the promoter element, 26-92, 26-128, and 26-222AA are different fragments of the AXL extracellular domain, respectively. The signal peptide, hinge region, and transmembrane region are all derived from human CD8 molecules, 4-1BB comes from human CD137, CD3ζ comes from human CD3, and mKate2 is a fluorescent marker used to detect CAR expression.

[0077] Example 4

[0078] CAR-T cell in vitro toxicity assay

[0079] The Luciferase Assay System was used to detect the killing effect of CAR-T cells on target cells, as follows:

[0080] (1) Target cell digestion and counting: digest the cells with trypsin, centrifuge at 1000 rpm for 3 min, add 1 mL of T cell culture medium, resuspend and count;

[0081] (2) 2×10 per hole 3 Target cells / 100 μL culture medium were added to a 96-well plate;

[0082] (3) CAR-T cell counting: centrifuge at 500 g for 5 min, resuspend and count in 1 mL of T cell culture medium, and add the corresponding number of CAR-T cells / 100 μL / well according to the effector-target ratio;

[0083] (4) Mix (2) + (3), co-culture for 24 hours, collect the supernatant to detect the release of cytokines IFN-γ and TNF-α; add 30 μL / well 1× Cell Culture Lysis, add 30 μL / well Luciferase working solution after 15 minutes, and detect with a multifunctional microplate reader; calculate the killing effect of CAR-T cells on target cells: the ratio of the killing effect of NT cells on target cells.

[0084] Experimental Example 1

[0085] The different CAR-T cells prepared in Example 3 were subjected to infection experiments respectively, as follows:

[0086] On the third day after CD19-CAR and AXL-CAR infected T cells, the infection efficiency was analyzed by flow cytometry. Non-transfected T cells were used as controls. The expression of CAR was detected by fusion protein T2A-mKATE2. The infection results are shown in Figure 1 Figure B shows that the infection efficiency of AXL-1-CAR and AXL-2-CAR is about 60%, which is higher than that of AXL-3-CAR.

[0087] Experimental Example 2

[0088] The expression level of GAS6 in tumor cells was Figure 2Flow cytometry analysis of GAS6 protein levels in various pancreatic cancer cell lines (MIA PaCa2, PANC1), liver cancer cell lines (HepG2, SK-Hep1), breast cancer cell lines (MDA-MB231, MDA-MB468, MCF7), and colorectal cancer cell lines (GP2D, HCT116, LS513) was performed. Figure 2 It can be seen that the protein level of GAS6 in SK-Hep1, MDA-MB231 and LS513 reached about 90%.

[0089] Experimental Example 3

[0090] The killing effect of AXL-CAR on tumor cell lines, the results are as follows Figure 3 As shown by Figure 3 It can be seen that AXL-CAR-T cells have no obvious killing effect on HepG2 and MCF7 cell lines with low GAS6 expression, but have significant killing effect on MIA PaCa2, PANC1, SK-Hep1, MDA-MB231, MDA-MB468, GP2D, HCT116, and LS513 with high GAS6 expression. CD19-CAR-T or AXL-CAR-T cells were co-cultured with target cells at an effector-target ratio of 5:1 for 24 hours, and the killing effect of CAR-T cells on target cells was detected by Luciferase Assay System.

[0091] The amino acid or nucleotide sequences involved in this application are as follows:

[0092] 1. AXL series:

[0093] SEQ ID NO:1

[0094] Amino Acids:

[0095] APRGTQAEESPFVGNPGNITGARGLTGTLRCQLQVQGEPPEVHWLRDGQILELADSTQTQVPLGEDE

[0096] SEQ ID NO:2

[0097] Base:

[0098] GCTCCAAGAGGTACACAGGCCGAAGAGTCTCCATTCGTGGGCAATCCAGGCAACATCACCGGAGCAAGAGGCCTGACAGGCACACTGAGATGTCAGCTCCAGGTCCAGGGAGAGCCACCTGAGGTCCATTGGCTGAGAGACGGTCAGATTCTGGAGTTGGCTGACAGCACACAGACACAGGTGCCTTTGGGCGAGGATGAG

[0099] SEQ ID NO:3

[0100] Amino acids:

[0101] APRGTQAEESPFVGNPGNITGARGLTGTLRCQLQVQGEPPEVHWLRDGQILELADSTQTQVPLGEDEQDDWIVVSQLRITSLQLSDTGQYQCLVFLGHQTFVS

[0102] SEQ ID NO:4

[0103] Bases:

[0104] GCACCAAGAGGTACTCAGGCTGAAGAATCTCCATTCGTGGGCAACCCTGGCAACATCACAGGCGCCAGAGGCCTGACCGGCACTCTGAGGTGTCAGCTGCAGGTGCAGGGTGAGCCACCAGAAGTGCATTGGCTGCGCGATGGCCAGATACTTGAGCTGGCTGACTCTACTCAGACACAGGTGCCTCTGGGTGAAGATGAACAAGACGATTGGATCGTGGTGTCTCAGCTCAGAATCACCTCTCTGCAGCTGAGTGACACCGGCCAGTATCAGTGTCTGGTCTTTCTGGGCCATCAGACCTTCGTGAGC

[0105] SEQ ID NO:5

[0106] Amino acids:

[0107] APRGTQAEESPFVGNPGNITGARGLTGTLRCQLQVQGEPPEVHWLRDGQILELADSTQTQVPLGEDEQDDWIVVSQLRITSLQLSDTGQYQCLVFLGHQTFVSQPGYVGLEGLPYFLEEPEDRTVAANTPFNLSCQAQGPPEPVDLLWLQDAVPLATAPGHGPQRSLHVPGLNKTSSFSCEAHNAKGVTTSRTATIT

[0108] SEQ ID NO:6

[0109] Base:

[0110] GCACCTAGAGGCACTCAGGCCGAAGAGTCTCCATTCGTGGGCAATCCAGGCAACATCACCGGAGCTAGAGGTCTGACAGGCACACTGAGGTGTCAGCTGCAGGTGCAGGGAGAGCCTCCAGAGGTGCACTGGCTGAGAGATGGCCAGATCTTGGAACTGGCTGATAGCACACAGACACAGGTACCTCTGGGTGAGGACGAGCAAGACGACTGGATCGTCGTCTCTCAGCTGCGGATCACCAGCCTCCAGCTGAGCGACACTGGACAGTACCAGTGCCTGGTCTTTCTGGGTCACCAGACATTCGTCAGCCAGCCTGGCTACGTGGGCCTGGAAGGTCTGCCTTACTTTCTGGAAGAACCTGAAGATCGGACAGTGGCCGCCAATACTCCATTCAACCTGAGCTGTCAGGCTCAGGGACCACCAGAACCTGTCGATTTGCTGTGGCTGCAAGATGCTGTGCCATTGGCCACCGCACCTGGACACGGACCACAGAGGTCTTTGCACGTTCCAGGTCTGAACAAGACCAGCAGCTTCAGCTGTGAGGCTCACAATGCCAAGGGAGTGACCACAAGTCGCACAGCTACCATCACC

[0111] 2. mKate2 Red Fluorescent Protein

[0112] SEQ ID NO:7

[0113] Amino Acid:

[0114] MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKAVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALKLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYCDLPSKLGHKLN

[0115] SEQ ID NO:8

[0116] Base:

[0117] ATGAGCGAGCTGATTAAGGAGAACATGCACATGAAGCTGTACATGGAGGGCACCGTGAACAACCACCACTTCAAGTGCACATCCGAGGGCGAAGGCAAGCCCTACGAGGGCACCCAGACCATGAGAATCAAGGCGGTCGAGGGCGGCCCTCTCCCCTTCGCCTTCGACATCCTGGCTACCAGCTTCATGTACGGCAGCAAAACCTTCATCAACCACACCCAGGGCATCCCCGACTTCTTTAAGCAGTCCTTCCCCGAGGGCTTCACATGGGAGAGAGTCACCACATACGAAGACGGGGGCGTGCTGACCGCTACCCAGGACACCAGCCTCCAGGACGGCTGCCTCATCTACAACGTCAAGATCAGAGGGGTGAACTTCCCATCCAACGGCCCTGTGATGCAGAAGAAAACACTCGGCTGGGAGGCCTCCACCGAGACCCTGTACCCCGCTGACGGCGGCCTGGAAGGCAGAGCCGACATGGCCCTGAAGCTCGTGGGCGGGGGCCACCTGATCTGCAACTTGAAGACCACATACAGATCCAAGAAACCCGCTAAGAACCTCAAGATGCCCGGCGTCTACTATGTGGACAGAAGACTGGAAAGAATCAAGGAGGCCGACAAAGAGACCTACGTCGAGCAGCACGAGGTGGCTGTGGCCAGATACTGCGACCTCCCTAGCAAACTGGGGCAC

[0118] 3. CD8 signal peptide

[0119] SEQ ID NO:9

[0120] Amino acids: MALPVTALLLPLALLLHAARP

[0121] SEQ ID NO:10

[0122] Bases:

[0123] ATGGCCCTGCCCGTCACCGCTCTGCTGCTGCCCCTTGCTCTGCTTCTTCATGCAGCAAGGCCG

[0124] 4. CD8 hinge region

[0125] SEQ ID NO:11

[0126] Amino Acids:

[0127] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0128] SEQ ID NO:12

[0129] Base:

[0130] ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCTAGCCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT

[0131] 5. CD8 transmembrane region

[0132] SEQ ID NO:13

[0133] Amino Acid: IYIWAPLAGTCGVLLLSLVITLYC

[0134] SEQ ID NO:14

[0135] Base:

[0136] ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC

[0137] 6. Co-stimulatory signaling molecules derived from 4-1BB

[0138] SEQ ID NO:15

[0139] Amino Acids:

[0140] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELR

[0141] SEQ ID NO:16

[0142] Base:

[0143] AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGA

[0144] 7. Cytoplasmic signaling sequence of CD3ζ

[0145] SEQ ID NO:17

[0146] Amino acids:

[0147] VKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0148] SEQ ID NO:18

[0149] Bases:

[0150] GTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

[0151] The full amino acid sequence of AXL is as shown in SEQ ID NO:19.

[0152] SEQ ID NO:19

[0153] MAPSLSPGPAALRRAPQLLLLLLAAECALAALLPAREATQFLRPRQRRAFQVFEEAKQGHLERECVEELCSREEAREFFENDPETDYFYPRYLDCINKYGSPYTKNSGFATCVQNLPDQCTPNPCDRKGTQACQDLMGNFFCLCKAGWGGRLCDKDVNECSQENGGCLQ ICHNKPGSFHCSCHSGFELSSDGRTCQDIDECADSEACGEARKNLPGSYSCLCDEGFAYSSQEKACRDVDECLQGRCEQVCVNSPGSYTCHCDGRGGLKLSQDMDTCEDILPCVPFSVAKSVKSLYLGRMFSGTPVIRLRFKRLQPTRLVAEFDFRTFDPEGILLFAG GHQDSTWIVLALRAGRLELQLRYNGVGRVTSSGPVINHGMWQTISVEELARNLVIKVNRDAVMKIAVAGDLFQPERGLYHLNLTVGGIPFHEKDLVQPINPRLDGCMRSWNWLNGEDTTIQETVKVNTRMQCFSVTERGSFYPGSGFAFYSLDYMRTPLDVGTESTWEV EVVAHIRPAADTGVLFALWAPDLRAVPLSVALVDYHSTKKLKKQLVVLAVEHTALALMEIKVCDGQEHVVTVLRDGEATLEVDGTRGQSEVSAAQLQERLAVLERHLRSPVLTFAGGLPDVPVTSAPVTAFYRGCMTLEVNRRLLDLDEAAYKHSDITAHSCPPVEPAAA

[0154] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A chimeric antigen receptor (CAR) based on the receptor tyrosine kinase family AXL, characterized in that: The chimeric antigen receptor contains an extracellular binding domain, and the extracellular binding domain can specifically bind to the AXL ligand; The amino acid sequence of AXL is shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:

5.

2. The chimeric antigen receptor (CAR) according to claim 1, characterized in that The structure of the chimeric antigen receptor is shown in Formula I below: L-EF1α-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; EF1α is the promoter element sequence; EB is the extracellular binding domain; H is the hinge region; TM is the transmembrane domain; C is no or co-stimulatory signal molecule; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ; RP is none or reporter protein.

3. The chimeric antigen receptor (CAR) according to claim 2, characterized in that The amino acid sequence of the signal peptide sequence is shown in SEQ ID NO:9, the amino acid sequence of the hinge region is shown in SEQ ID NO:11, and the amino acid sequence of the transmembrane domain is shown in SEQ ID NO:

13.

4. The chimeric antigen receptor (CAR) according to claim 2, characterized in that The C is selected from the co-stimulatory signal molecules of the following histones: CD28, 4-1BB or a combination thereof, and the amino acid sequence of the 4-1BB is shown in SEQ ID NO:

15.

5. The chimeric antigen receptor (CAR) according to claim 2, characterized in that The amino acid sequence of CD3ζ is shown in SEQ ID NO:

17.

6. The chimeric antigen receptor (CAR) according to claim 2, characterized in that The reporter protein is a red fluorescent protein mKate2, and the amino acid sequence of the red fluorescent protein mKate2 is shown in SEQ ID NO:

7.

7. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the chimeric antigen receptor according to any one of claims 1 to 6.

8. A carrier, characterized in that The vector contains the nucleic acid molecule as described in claim 7.

9. An engineered immune cell, characterized in that: The engineered immune cell contains the vector as claimed in claim 8 or a chromosome in which an exogenous nucleic acid molecule as claimed in claim 7 is integrated or expresses the chimeric antigen receptor as claimed in any one of claims 1-6.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the chimeric antigen receptor according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, the vector according to claim 8, and / or the engineered immune cell according to claim 9, and a pharmaceutically acceptable carrier, diluent or excipient.

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