Chimeric antigen receptors based on receptor tyrosine kinase family AXL and their applications

Abstract

The present application relates to the field of immune cell therapy, and provides a chimeric antigen receptor based on the modification of the receptor tyrosine kinase family AXL and its application. 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 the AXL is shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5. Target specificity: GAS6 is lowly expressed on the cell membrane of normal cells, but highly expressed on the cell membrane of tumor tissues, so that 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. The present invention utilizes the mode of action of ligand binding to receptor, rather than scFv in the traditional sense. The conservation of receptor-ligand interaction determines the safety test in animals, especially primates, which can better reflect its safety in humans.

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, a new immunotherapy approach, has not only achieved significant research progress in hematologic malignancies but also demonstrated promising application prospects in solid tumors. However, CAR-T cell therapy still faces several challenges: antigen escape, CAR-T infiltration, an immunosuppressive microenvironment, non-tumor-targeted toxicity, and CAR-T safety.

[0003] Traditional CAR-T uses 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 receptor tyrosine kinase family AXL, aiming to solve the safety issues of traditional CAR-T.

[0005] To achieve the above application objectives, the technical solutions adopted in this application are 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 that can specifically bind to the AXL ligand.

[0007] One possible design is 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 that 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 one 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] Where,

[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 none or a costimulatory signaling 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 the 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 comprising 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 the aforementioned exogenous nucleic acid molecule integrated into the chromosome 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) This invention utilizes the binding of ligands to receptors, rather than the traditional scFv. The conservative nature of receptor-ligand interactions dictates safety testing in animals, particularly primates, which better reflects 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 following briefly introduces the drawings required for use in the embodiments or descriptions of the prior art. 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 any creative work.

[0033] Figure 1 This is a diagram showing the characteristics 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 this application. DETAILED DESCRIPTION

[0036] In order to make the technical problems, technical solutions and beneficial effects to be solved by this 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 this application and are not intended to limit this application.

[0037] In this application, the term "and / or" describes the relationship between associated objects, indicating that three possible relationships exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, 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 "more than one" 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, and c can be single or multiple.

[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. 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 for the purpose of describing specific embodiments only and are not intended to limit the present application. The singular forms "a", "an", "the" 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 otherwise.

[0041] The weights of the relevant components mentioned in the examples of this application may not only refer to the specific content of each component, but also represent the weight ratio between the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the examples of this application, it is within the scope disclosed in the examples of this application. Specifically, the mass described in the examples of this application may 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), the human CD8 signal peptide (amino acid sequence as shown in SEQ ID NO: 9, nucleotide sequence as shown in SEQ ID NO: 10), the human CD8α hinge region (amino acid sequence as shown in SEQ ID NO: 11, nucleotide sequence as shown in SEQ ID NO: 12), the human CD8 transmembrane region (amino acid sequence as shown in SEQ ID NO: 13, nucleotide sequence as shown in SEQ ID NO: 14), the 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 the 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 was obtained by artificial synthesis or PCR. Artificial synthesis or PCR methods 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, and 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 via XbaI. The synthesis and extraction of all plasmids are conventional 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 the extracellular binding domain that specifically binds to the GAS6 ligand, and its amino acid sequences are 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 costimulatory signal molecule, selected from the group consisting of the following costimulatory signal molecules: 4-1BB;

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

[0057] RP is null or reporter protein, wherein the reporter protein is the red fluorescent protein mKate2.

[0058] Example 2

[0059] Lentivirus preparation

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

[0061] (2) When the cell density reaches about 80%, transfection is performed. 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 per 15 cm dish. At the same time, 68 μL of lipo8000 is added, mixed, and added to the culture 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 h after transfection and replace with 15 mL of fresh culture medium; and collect the culture supernatant 72 h 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 aliquoted and stored at -80°C.

[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 the Human T Cell Enrichment Cocktail kit as follows:

[0069] (a) 10 mL of peripheral blood was added to 500 μL of RosetteSep™ Cocktail, mixed well, allowed to stand for 10 min, and diluted 1-fold with PBS (containing 2% FBS);

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

[0071] (c) The supernatant was transferred to a 50 mL centrifuge tube, and an equal volume of PBS (containing 2% FBS) was added. The tube was then 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 CD3 expression 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 72 h;

[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 were infected with 1 μL lentiBoost (1 μg / mL) and CAR lentiviral particles (MOI = 100). After 24 hours of infection, the cells were centrifuged at 500 g for 5 minutes, the supernatant was discarded, and the cells were transferred to a 24-well plate. On the third day after CAR-T cell infection, the infection efficiency of CAR-T was monitored by flow cytometry for subsequent experimental studies.

[0076] Using the above method, the CAR-T construction model of this embodiment is shown in the figure below: 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 the human CD8 molecule, 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) According to 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 30μL / well 1×Cell Culture Lysis, and after 15 minutes, add 30μL / well Luciferase working solution, 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, as follows:

[0086] On the third day after CD19-CAR and AXL-CAR infection of T cells, the infection efficiency was analyzed by flow cytometry. Non-transfected T cells were used as controls. The expression of CAR was detected using the 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 AXL-CAR-T cells had no significant cytotoxic effect on HepG2 and MCF7 cell lines, which have low GAS6 expression, but showed significant cytotoxic effects on MIA PaCa2, PANC1, SK-Hep1, MDA-MB231, MDA-MB468, GP2D, HCT116, and LS513, which have 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 cytotoxic effect of CAR-T cells on target cells was assessed using the 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] Bases:

[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<无数 <无数

[0106] Amino acids:<无数 <无数

[0107] It should be noted that the tags and

[0106] seem to be incorrect in the original. I've translated them as "无数0000228" and "无数0000229" to maintain their form as you requested. If they are meant to be something else, please clarify. Also, the tag and

[0107] are left as "无数0000230" and "无数0000231" with the same caveat.APRGTQAEESPFVGNPGNITGARGLTGTLRCQLQVQGEPPEVHWLRDGQILELADSTQTQVPLGEDEQDDWIVVSQLRITSLQLSDTGQYQCLVFLGHQTFVSQPGYVGLEGLPYFLEEPEDRTVAANTPFNLSCQAQGPPEPVDLLWLQDAVPLATAPGHGPQRSLHVPGLNKTSSFSCEAHNAKGVTTSRTATIT

[0108] SEQ ID NO:6

[0109] Base:

[0110] GCACCTAGAGGCACTCAGGCCGAAGAGTCTCCATTCGTGGGCAATCCAGGCAACATCACCGGAGCTAGAGGTCTGACAGGCACACTGAGGTGTCAGCTGCAGGTGCAGGGAGAGCCTCCAGAGGTGCACTGGCTGAGAGATGGCCAGATCTTGGAACTGGCTGATAGCACACAGACACAGGTACCTCTGGGTGAGGACGAGCAAGACGACTGGATCGTCGTCTCTCAGCTGCGGATCACCAGCCTCCAGCTGAGCGACACTGGACAGTACCAGTGCCTGGTCTTTCTGGGTCACCAGACATTCGTCAGCCAGCCTGGCTACGTGGGCCTGGAAGGTCTGCCTTACTTTCTGGAAGAACCTGAAGATCGGACAGTGGCCGCCAATACTCCATTCAACCTGAGCTGTCAGGCTCAGGGACCACCAGAACCTGTCGATTTGCTGTGGCTGCAAGATGCTGTGCCATTGGCCACCGCACCTGGACACGGACCACAGAGGTCTTTGCACGTTCCAGGTCTGAACAAGACCAGCAGCTTCAGCTGTGAGGCTCACAATGCCAAGGGAGTGACCACAAGTCGCACAGCTACCATCACC

[0111] 2. mKate2 Red Fluorescent Protein

[0112] SEQ ID NO:7

[0113] [[ID= eighteen]]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] Bases:

[0130] ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCTAGCCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT

[0131] 5. CD8 transmembrane region

[0132] SEQ ID NO:13

[0133] Amino acid: IYIWAPLAGTCGVLLLSLVITLYC

[0134] SEQ ID NO:14

[0135] Bases:

[0136] ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC

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

[0138] SEQ ID NO:15

[0139] Amino acids:

[0140] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELR

[0141] SEQ ID NO:16

[0142] Bases:

[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 entire 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 replacements and improvements made within the spirit and principles of the present application should be included in the scope of protection 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, which 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; The structure of the chimeric antigen receptor is shown in Formula I below: L-EF1α-EB-H-TM-C-CD3ζ-RP (I) Where, 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 costimulatory signaling molecule; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ; RP is none or reporter protein; 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; The nucleotide sequence of the extracellular binding domain is shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6; C is a co-stimulatory signal molecule selected from the group consisting of CD28, 4-1BB, or a combination thereof, wherein the amino acid sequence of 4-1BB is as shown in SEQ ID NO: 15; The amino acid sequence of CD3ζ is shown in SEQ ID NO: 17; 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.

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

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

4. An engineered immune cell, characterized in that: The engineered immune cell contains the vector according to claim 3 or the chromosome into which the exogenous nucleic acid molecule according to claim 2 is integrated or expresses the chimeric antigen receptor according to claim 1.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the chimeric antigen receptor according to claim 1, the nucleic acid molecule according to claim 2, the vector according to claim 3, and / or the engineered immune cell according to claim 4, and a pharmaceutically acceptable carrier.

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