Chimeric antigen receptors and their derivatives and applications

Abstract

In one embodiment of the present invention, a chimeric antigen receptor having a light chain variable region sequence set forth in SEQ ID NO: 1 and a heavy chain variable region sequence set forth in SEQ ID NO: 2 is provided, which can have a killing effect on tumor cells, particularly tumor cells expressing Axl. Embodiments of the present invention also provide derivatives of the chimeric antigen receptor and their applications.

Description

[Technical Field]

[0001] The present invention belongs to the field of genetic engineering, and particularly relates to chimeric antigen receptors and their derivatives and applications. [Background technology]

[0002] Axl is a receptor tyrosine kinase that is highly expressed in many types of cancer, including granular cell leukemia, pseudopyrethrum ester leukemia, megakaryocytic leukemia, endometrial cancer, gastric cancer, colon cancer, prostate cancer, thyroid cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, renal cell carcinoma, gliomastoid tumor, melanoma, and osteosarcoma, and is a potential anticancer drug target.

[0003] Therefore, there is a need to provide new Axl-specific chimeric antigen receptors. Summary of the Invention

[0004] The present invention provides a chimeric antigen receptor that has killing activity against tumor cells, particularly tumor cells that express Axl.

[0005] The chimeric antigen receptors of the present invention include Axl-specific antibodies, which can be single-chain antibodies (scFV), monoclonal antibodies, domain antibodies, Fab fragments, Fd fragments, Fv fragments, F(ab')2 fragments, and derivatives thereof, or other forms of antibodies.

[0006] Preferably, the Axl-specific antibody is a humanized antibody. The humanized antibody is a full-length antibody and is of any of the subtypes hIgG1, hIgG2, hIgG3, and hIgG4.

[0007] Preferably, the sequence of the light chain variable region is as shown in SEQ ID NO:1 and the sequence of the heavy chain variable region is as shown in SEQ ID NO:2.

[0008] Preferably, the Axl-specific antibody further comprises a binding region located between the light chain variable region and the heavy chain variable region, as shown in SEQ ID NO:3.

[0009] Preferably, the Axl-specific antibody comprises the light chain variable region, the linking region, and the heavy chain variable region linked in that order.

[0010] Preferably, the chimeric antigen receptor further comprises a hinge region connecting the C-terminus of the heavy chain variable region of the Axl-specific antibody, and a transmembrane domain connecting the hinge regions.

[0011] Preferably, the sequence of the hinge region is derived from at least one of CD8α, CD28, 4-1 BB, ICOS, OX40, CD40, CD80 and IgG.

[0012] Preferably, the sequence of the transmembrane domain is derived from at least one of CD2, CD27, LFA-1, CD8α, CD28, 4-1 BB, ICOS, OX40, CD40, CD80, CD3ζ, and CD3ε.

[0013] Preferably, the sequence of the hinge region is shown in SEQ ID NO:4 and the sequence of the transmembrane domain is shown in SEQ ID NO:5.

[0014] Preferably, the chimeric antigen receptor further comprises a signal peptide having an expression driving region and a light chain variable region that links the expression driving region to the AXL-specific antibody.

[0015] The expression driver region of the present invention can tag and encode promoters for CAR cell amplification and suicide.

[0016] Preferably, the sequence of the expression driver region is derived from at least one of EF 1α, CMV, PGK, MPSV, MMLV, and SFV.

[0017] Preferably, the sequence of the expression driver region is shown in SEQ ID NO:6.

[0018] Preferably, the sequence of the signal peptide is shown in SEQ ID NO:7.

[0019] Preferably, the sequence of the chimeric antigen receptor is shown in SEQ ID NO:8.

[0020] The present invention also provides a nucleic acid encoding at least a partial fragment of a chimeric antigen receptor, and a vector comprising the nucleic acid.

[0021] Preferably, the vector may be an adenoviral vector, a slow viral vector, or a reverse transcription viral vector.

[0022] The invention also provides a vector containing the nucleic acid, and a host cell containing the vector.

[0023] Preferably, the host cell may be a recombinant microbial host cell or a mammalian cell.

[0024] Preferably, the host cell is capable of expressing an Axl-specific antibody.

[0025] The present invention also provides the application of said chimeric antigen receptor in the preparation of tumor-related drugs and tumor treatment.

[0026] Preferably, the tumor is an Axl-high expressing tumor. [Brief explanation of the drawings]

[0027] [Figure 1] 1 shows a plasmid pattern of a shuttle plasmid according to an embodiment of the present invention. [Figure 2] FIG. 10 is a comparative diagram of flow analysis results between a transfection control group and a transfection experimental group in an example of the present invention. [Figure 3] FIG. 10 is a diagram comparing the relative expression levels of anti-Axl-scFv mRNA between a transfection control group and an experimental transfection group in an example of the present invention. [Figure 4]FIG. 10 is a comparative diagram showing the change trends of LCLC-103 H proliferation rates at different co-culture times between the LCLC-103 H blank group and the corresponding control group at E:T=4:1 and the experimental group according to an embodiment of the present invention. [Figure 5] FIG. 1 is a comparative diagram showing the change trends of MDA-MB-231 proliferation rates under different co-culture times in the MDA-MB-231 blank group, the corresponding control group under different E:T, and the corresponding experimental group under different E:T in an embodiment of the present invention. [Figure 6] FIG. 10 is a graph showing the growth rate trends of the corresponding control group of LCLC-103 H and the experimental group at different E:T according to an embodiment of the present invention, and a comparison of the growth rate trends of the corresponding control group and the experimental group of H460 at different E:T. [Figure 7] This is a comparison diagram of the proliferation rate trends of the matched control group of MDA-MB-231 and the experimental group at different E:T in an embodiment of the present invention, and the proliferation rate trends of the matched control group of MDA-MB-453 at different E:T and the experimental group at different E:T. [Figure 8] FIG. 1 is a graph comparing the relative expression levels of IFNγ in blank groups, control groups, and experimental groups corresponding to MV 4-11 in an example of the present invention. [Figure 9] FIG. 1 is a graph comparing the relative expression levels of IFNγ in blank groups, control groups, and experimental groups corresponding to HL 60 according to an embodiment of the present invention. [Figure 10] FIG. 1 is a graph comparing the relative expression levels of IFNγ in blank groups, control groups, and experimental groups corresponding to MDA-MB-231 in an example of the present invention. [Figure 11] 1 shows photographs comparing the tumor metastasis status of lung tissue samples from the PBS group, the control animal group, and the treatment animal group in an example of the present invention. [Figure 12] FIG. 12 is a graph comparing the pulmonary metastatic area occupancy rates of lung tissue samples in each group, statistically calculated based on the photographs shown in FIG. 11. [Figure 13] FIG. 1 is a comparative diagram of immunostained photographs of in situ tumor tissue samples from a PBS group, an animal control group, and an animal treatment group according to an embodiment of the present invention. [Figure 14]FIG. 1 is a comparative diagram of the positive area occupancy ratios of CD3 and CD8 in in situ tumor tissue samples from the PBS group, animal control group, and animal treatment group, which are examples of the present invention. [Figure 15] FIG. 1 is a comparative diagram of the expression levels of CD3, CD8A, CD4, IFNG, GZMB, and TNFA in the PBS group, animal control group, and animal treatment group of an example of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0028] In order to clarify the objectives, technical solutions and advantages of the embodiments of the present invention, the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present invention, and not all of the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative work fall within the scope of protection of the present invention. Unless otherwise defined, technical or scientific terms used herein should have the ordinary meanings understood by those with ordinary skills in the field to which the present invention belongs. As used herein, similar words such as "comprise" mean that the element or object appearing before the word covers the elements or objects listed after the word and their equivalents, without excluding other elements or objects.

[0029] The scientific and technical terms used in the embodiments of the present invention have the same or similar meanings as those commonly understood by those skilled in the art. To facilitate understanding of the present invention, some terms are defined as follows: "Antibody" in embodiments of the present invention refers to an antigen-binding protein of the immune system, including a complete, full-length antibody having an antigen-binding region, and any fragment that retains the "antigen-binding portion" or "antigen-binding region," or a single chain, such as a single-chain variable fragment (scFv). "Antibody" also includes all recombinant forms of antibodies (particularly those described herein), such as antibodies expressed in prokaryotic cells, non-glycosylated antibodies, and antigen-binding antibody fragments and derivatives.

[0030] "CD3ζ" is defined as the protein provided by GenBank Accession No. BAG 36664.1, or equivalent residues from non-human species such as mouse, rodent, monkey, or ape. A "CD3 zeta domain" is defined as the amino acid residues from the cytoplasmic domain of the zeta chain sufficient to functionally transmit the initial signal required for T cell activation. In one embodiment, the zeta cytoplasmic domain comprises residues 52-163 of GenBank Accession No. BAG 36664.1, or equivalent residues from non-human species such as mouse, rodent, monkey, or ape.

[0031] "CD28" is defined as the protein provided by GenBank Accession No. NP_006130.1, or equivalent residues from non-human species such as mouse, rodent, monkey, or ape. The "CD28 signal region" is defined as the amino acid residues from the cytoplasmic domain of CD28 that transmit the costimulatory signal required for T cell activation, the sequence of which includes residues 180-220 of GenBank Accession No. NP_006130.1, or equivalent residues from non-human species such as mouse, rodent, monkey, or ape. The "CD28 hinge region" is defined as residues 114-152 of GenBank Accession No. NP_006130.1, or equivalent residues from non-human species such as mouse, rodent, monkey, or ape. The "CD28 hinge transmembrane region" includes residues 153-179 of GenBank Accession No. NP_006130.1, its functionally directed homologs, e.g., equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc.

[0032] "CD8" is a leukocyte differentiation antigen molecule, a glycoprotein found on the surface of T cells that helps the T cell receptor (TCR) recognize antigens and participates in the transmission of T cell activation signals. It is also called a co-receptor for TCR. The "CD8 hinge region" is defined as the variable region (V-like region) present in the extracellular portion of the α-chain and β-chain of the CD8 molecule, and the hinge region (connecting peptide) rich in proline, threonine, and serine residues present between the V-like region and the cell membrane portion.

[0033] "EF1α" is defined as a strong mammalian promoter of the elongation factor 1 alpha (EF1A) gene, which can stably drive the constitutive expression of downstream genes in various cell types. The EF1α promoter is suitable for expression in stem cells, primary cells, hematopoietic cells, etc., and is weaker than CMV in commonly used cell lines such as HEK 293 and tumors.

[0034] Axl, also known as Ark, Ufo, or Tyro-7, belongs to the receptor tyrosine kinase TAM family. Its extracellular segment is similar in structure to neural cell adhesion molecule (NCAM) and contains two immunoglobulin-like (Ig) domains and two fibrin III (FN III, FN 3)-like domains. The Ig domains bind to ligands, while the FN III domains regulate the binding process between Axl and its ligands. The intracellular domain of Axl is a tyrosine kinase-like domain and possesses kinase activity.

[0035] All reagents and consumables used in the examples of the present invention can be purchased commercially, and unless otherwise specified, are from Hanheng Bioscience and Technology (Shanghai) Co., Ltd.

[0036] Unless otherwise specified, all cell cultures described in the examples of the present invention were carried out in a carbon dioxide cell culture chamber, with a controlled temperature of 37±0.5°C and a carbon dioxide volume concentration of 5±0.5%.

[0037] The qPCR confidence interval in the examples of the present invention is 95%. For statistical analysis, each group's experiment was repeated at least three times, and the experimental results were statistically analyzed using GraphPad Prism 8.0 software. Comparisons between two sets of data were performed using a two-tailed unpaired t-test to calculate statistical differences, and comparisons between multiple sets of data were performed using ANOVA analysis of variance to calculate statistical differences. p<0.05 was considered to indicate statistical difference. In the specification and drawings: * indicates P<0.05, *represents P<0.001.

[0038] Example 1: Axl-specific chimeric antigen receptor This example provides an Axl-specific chimeric antigen receptor (abbreviated as Axl-CAR) of the sequence as shown in SEQ ID NO:8.

[0039] Example 2: Construction of shuttle plasmids In this example, the nucleotide sequence of Axl-CAR in Example 1 was amplified by PCR, digested with restriction enzymes, and the recovered fragments were inserted into the polycloning site of a shuttle plasmid to obtain a recombinant shuttle plasmid. The plasmid map of the shuttle plasmid is shown in Figure 1. The specific operation procedures are within the ordinary skill in the art.

[0040] In this example, the DH5α strain in E. coli DH5α receptor cell suspension (product number MCC 0010, Frdbio) was used as the receptor cell, and 10 nanograms of the recombinant shuttle plasmid obtained above was added to 100 microliters of E. coli DH5α receptor cell suspension. After mixing, the cells were left in an ice bath for 20-30 minutes and then heat-shocked at 42°C for 90 seconds to transform the recombinant shuttle plasmid into the cells. For specific operating procedures, please refer to the product instructions for the E. coli DH5α receptor cell suspension; a detailed description will not be given here.

[0041] After transformation, 100 microliters of the resulting cell suspension was added to 300 microliters of fresh, non-antibody LB liquid medium, mixed, and incubated at 37°C for 45-60 minutes with a rocking motion at 230 rpm. The cells were then evenly spread onto 10 cm dishes pre-coated with 20 ml of fresh, non-antibody LB solid medium. The dishes were inverted and incubated at 37°C for 12-16 hours until distinct, non-overlapping single colonies appeared. After color development, white colonies were selected and placed in a fresh, non-antibody LB liquid medium as a bacterial suspension for PCR sequencing. The reaction mixture consisted of 2 microliters of bacterial suspension, 5 microliters of 2x Hieff PCR Master Mix, 2 microliters of ddH2O, 0.5 microliters of upstream primer, and 0.5 microliters of downstream primer. The sequencing results were confirmed to be consistent with the target sequence.

[0042] The upstream primer sequence was 5'-GGTGACCCTGACTTGGAC-3', and the downstream primer sequence was 5'-AGTAGCAGAGAGAGAGCCGGTG-3'.

[0043] The recombinant shuttle plasmid solution was amplified, extracted, and purified using a plasmid extraction kit (Tiangen, DP 117), and the resulting purified recombinant shuttle plasmid was used to transfect cells.

[0044] Example 3: Slow virus packaging In this example, the purified recombinant shuttle plasmid from Example 2 was packaged as a slow virus, and the specific packaging method is as follows: Culture 293 T cells in fresh complete medium DMEM (Thermo, product number 11965118) containing 10% fetal bovine serum to a confluency of 70-80% and then resuspend them to a cell suspension (5 x 10 cells). 6A 20 ml volume of 10 micrograms of pSPAX2 plasmid, 5 micrograms of pMD2G plasmid, 10 micrograms of purified recombinant shuttle plasmid pack, and 75 microliters of transfection reagent (Hanbio Biotechnology, product number HB-TRCF-1000) were mixed and incubated at room temperature for 15 minutes. The mixture was then slowly added dropwise to the cell suspension for transfection. The medium was replaced 16 hours after transfection. 48 hours after transfection, the culture was centrifuged at 2000 g for 10 minutes at 4°C. The viral supernatant was collected and filtered to remove cell debris and impurities. The resulting supernatant was then centrifuged at 82700 g for 120 minutes at 4°C to obtain a slow virus pellet. The slow virus pellet was resuspended in 200 microliters of the same DMEM complete medium to obtain a slow virus resuspension.

[0045] Ten microliters of the slow virus resuspension was taken and added to a 96-well plate previously seeded with HeLa cells. After 24 hours of incubation, microscopic examination revealed that the medium was clear, with no obvious particles in the intercellular spaces, and was free of any bacterial or fungal contamination.

[0046] Ten microliters of the slow virus heavy suspension was taken and incubated in a water bath at 96°C for 15 minutes before PCR reaction. There were no obvious stripes on the PCR gel, indicating that the slow virus was free of mycoplasma contamination.

[0047] The slow virus heavy suspension was subjected to a drop test, and the specific steps were as follows: Day 1: 293 T cells in good proliferative condition were digested and counted to 1 x 10 5 The solution was diluted to 1 / mL and added to a 96-well plate, followed by cell culture for 48 hours.

[0048] The next day: Six 1.5 mL EP tubes were prepared, and 10 μL of the slow virus resuspension was added to the first EP tube, followed by a three-fold gradient dilution to obtain six dilutions of the slow virus resuspension.

[0049] On day 3, for holes requiring promycin screening, first aspirate 100 mL of virus-containing medium and add 100 μL of 10% FBS complete medium containing 1.5 μg / mL promycin.

[0050] On day 5, observations were made under a fluorescence microscope. Six hours before observation, fresh 10% FBS complete medium was replaced, 80 μL of medium was aspirated from the well, and 80 μL of fresh 10% FBS complete medium was added. The well was then cultured at 37°C, 5% CO2 in a medium box. After 6 hours, observations were made under a fluorescence microscope. The percentage of fluorescence was calculated as the viral titer at 10-50% of the wells.

[0051] Titration (TU / mL) = Number of cells × Percentage of positive clones × MOI (1) × Virus dilution × 10 3 TU / mL The viral drop rate is 1 x 10 8 Calculated as TU / ml.

[0052] Example 4: Construction of Axl-CAR-T cells In this example, Axl-CAR-T cells were constructed by infecting human peripheral blood mononuclear cells (PBMCs) with the slow virus heavy suspension obtained in Example 3. The specific construction procedure is as follows.

[0053] Ten milliliters of blood from a healthy volunteer was added to 20 milliliters of diluted PBS, then slowly added to 10 ml of Ficoll-Paque PLUS reagent (GE Healthcare). The mixture was centrifuged at 800 g for 30 minutes at room temperature. The serum layer was removed, and the white flocculent cell layer was aspirated into a centrifuge tube. 30 ml of PBS-diluted cell solution was added, followed by centrifugation at 400 g for 15 minutes at room temperature. The supernatant was discarded, and the precipitate was resuspended in 1 ml of PBS. The precipitate was then filtered through a 70 μm filter and centrifuged at 1500 rpm for 5 minutes at room temperature. The supernatant was discarded, and 1 x 10 cells containing 200 U / ml rhIL-2 (Biolegend) were added in RPMI complete medium. 6 After 2 hours of culture, the cells were stimulated using a human T cell activation / proliferation kit (Miltenyi Biotec GmbH), and then cultured for 4–5 days, passaged every 2 days, to obtain the T cells to be transfected.

[0054] The resulting culture containing the transfected T cells was centrifuged at 1500 rpm for 5 min at room temperature, and the supernatant was discarded. The resulting pellet was resuspended in EasySep Buffer, transferred to a 5 mL round-bottom test tube, and a magnet was placed on it for 5 min. The magnet was tilted along with the test tube, and the cell solution was poured out. After centrifugation at 1500 rpm for 5 min at room temperature, the supernatant was discarded and the cells were resuspended in RPMI medium to a concentration of 1 × 10 6 The cells were resuspended at 1000 cells / mL to form the cell suspension to be transfected.

[0055] Virus drip 1 x 10 8 The amount of slow virus used was calculated based on TU / ml and the selected MOI (multiplicity of infection), and 250 microliters of each target cell suspension was added to the transfection system to form the following transfection control and test groups: Transfection control group: 0.5 microliters of condensed amine (final concentration 5 micrograms / milliliter) and 0.5 microliters of IL-2 (final concentration 5 micrograms / milliliter) were added to control MOI = 5. The difference between the added control virus suspension and the slow virus suspension of Example 3 was that the control shuttle plasmid was packaged as a control slow virus according to the method of Example 3, and the control shuttle plasmid was obtained by referring to the manufacturing process of the recombinant shuttle plasmid of Example 2, and the GFP protein sequence was used instead of the Axl-CAR sequence.

[0056] Transfection test group: The difference from the transfection control group is that the virus suspension added was the low-speed virus suspension obtained in Example 3.

[0057] The transfection control and test groups were centrifuged at 200 g for 1 hour at room temperature, then cultured for 6 hours. Next, RPMI complete medium supplemented with 200 U / mL rhIL-2 was added to each group up to a sample volume of 500 microliters, and the cells were cultured for 48 hours.

[0058] In this example, after two days of cell culture, the cells obtained from each group were stained with biotinylated protein L, streptavidin-PE, and anti-CD3-APC, and then detected using flow cell technology. The flow cytometry comparison graph of the transfection control group and the transfection experimental group is shown in Figure 2. The staining process and flow cytometry detection process are conventional techniques used by those skilled in the art. Referring to Figure 2, a comparison between the transfection control group and the transfection experimental group shows that after transfection with the slow virus of Example 3, the T cells successfully expressed Axl-CAR.

[0059] In this example, the cells from each group were cultured for two days, then lysed, total RNA was extracted, and reverse transcribed. qPCR was performed using the resulting cDNA as a template and GAPDH as the reference gene, to obtain the graph comparing the relative expression levels of anti-Axl-scFv mRNA in the transfection control group and the transfection experimental group shown in Figure 3. The steps of total RNA extraction, reverse transcription, and qPCR are common techniques used by those skilled in the art. As shown in Figure 3, the expression level of anti-Axl-scFv mRNA in the transfection experimental group was significantly increased by about three-fold compared to the transfection control group.

[0060] The qPCR reaction system was prepared using Takara PrimeScript® MRT Master Mix reagent, and the primer pair sequences for the target gene were as follows: AXL-humab 1-F:5'-GGAGAGAGACAAGTCA-3' and AXL-humab 1-R:5'-TCTAAACCTTGGAGAGA-3', CD 3-F:5'-TCCCTCTTCAGTGGCT-3' and CD 3-R:5'-TCCTGCTGAGCAGA-3', CD 8-F:5'-TACTGCACCAGGACG-3' and CD 8-R:5'-AGTATCTCTCTCCCGCC-3', CD 4-F:5'-CCAGCCCTGCATTTC-3' and CD 4-R:5'-TCTTTCCCTGGCTGC-3', IFNG-F:5'-CGTTTGGCTCTTGCT-3' and IFNG-R:5'-TTCTGCTCTCC-3', GZMB-F: 5'-GATCTCCATCCA-3', TNFA-F: 5'-GTAGCCATGTAGCAACC-3' and TNFA-R: 5'-TACTCTCAGCTCACGCA-3'.

[0061] Example 5: In vitro tumor cell killing test by Axl-CAR-T cells In this example, we co-cultured the Axl-CAR-T cells obtained in Example 4 with LCLCC-103 H, HL60, MDA-MB-231, MDA-MB-453, and MV4-11, which express high levels of Axl, and H460, which expresses low levels of Axl, and investigated the killing ability of Axl-CAR-T cells against each tumor cell line.

[0062] LCLC-103 H and H460 are both human large cell lung cancer lines, HL60 is a human leukemia tumor cell, and MV 4-11 is a human acute mononuclear cell leukemia cell, all of which were derived from Nanjing Kebai Bioscience and Technology Co., Ltd.

[0063] Both MDA-MB-231 and MDA-MB-453 are human breast cancer cells and were derived from ATCC.

[0064] Each of the above tumor cells was cultured in RPMI complete medium and passaged at a ratio of 1:3 until corresponding well-growing logarithmic-phase cells were obtained. Then, each tumor cell was resuspended in RPMI complete medium and the concentration was adjusted to obtain each tumor cell suspension. The transfected T cells of Example 4 were resuspended in RPMI complete medium and the concentration was adjusted to obtain a T cell suspension. The Axl-CAR-T cells of Example 4 were resuspended in RPMI complete medium and the concentration was adjusted to obtain an Axl-CAR-T cell suspension.

[0065] A 16-well plate was pre-coated with 50 microliters of RPMI complete medium per well. Tumor cell suspension was added to each well, adjusting the concentration to 5,000 cells / 100 microliters of medium, and then cultured on the tumor cell wall. Then, a T cell suspension was added to obtain a control group, and the T cell to tumor cell ratio (E:T) was adjusted. Axl-CAR-T cell suspension was added to obtain an experimental group, adjusting the E:T ratio, and RPMI complete medium was added to obtain a blank group, adjusting the amount to the same as the control and experimental groups.

[0066] The blank, control, and experimental groups corresponding to each tumor cell line were monitored using real-time label-free cellular analysis (RTCA). The cell index was measured every 15 minutes, and the proliferation rate was calculated as (real-time cell index - cell index at time of T cell addition) / cell index at time of T cell addition.

[0067] For the blank group, control group, and experimental group corresponding to each tumor cell, the number of tumor cells in each pore was 3 × 10 4 After 5 days of co-culture, the relative IFNγ expression levels of the blank group relative to each control group and experimental group were measured using a Human IFNγ ELISPOT Kit (Abcam).

[0068] Figure 4 shows the comparative graph of the change in LCLCC-103 H proliferation rate at different co-culture times between the LCLC-103 H blank group and the control and experimental groups corresponding to E:T = 4:1. The killing effect of Axl-CAR-T cells against LCLC-103 H was stronger than that of T cells in the control group, significantly reducing the proliferation rate of LCLC-103 H. This indicates that Axl-CAR-T cells have a good killing effect on LCLCC-103 H.

[0069] Figure 5 shows the comparative graph of the change in MDA-MB-231 proliferation rate at different co-culture times for the MDA-MB-231 blank group, the corresponding control groups with different E:T ratios, and the corresponding experimental groups with different E:T ratios. At E:T ratios of 1:2 and 1:1, the killing effect of Axl-CAR-T cells on MDA-MB-231 was significantly better than that of the corresponding control groups. This indicates that Axl-CAR-T cells have a good killing effect on MDA-MB-231.

[0070] Axl-CAR-T cells are selective in their tumor cell killing effects, specifically: Figure 6 shows the proliferation rate trends of the LCLC-103 H control group and experimental group under different E:Ts, and the proliferation rate trends of the H460 control group and experimental group under different E:Ts. Compared to the LCLCLC-103 H control group under different E:Ts, Axl-CAR-T cells had a better killing effect against LCLC-103 H. At E:Ts of 2:1 and 4:1, the killing effect of Axl-CAR-T cells against H460 was more pronounced than that of the H460 control group. At an E:T of 8:1, the killing effect of Axl-CAR-T cells against H460 was not as pronounced. The killing rate of each LCC-103 H group showed a strong dose-dependence. At E:T ratios of 2:1 and 4:1, the killing effect of Axl-CAR-T cells against LCLC-103 H was more pronounced than that against H460; at E:T ratios of 8:1, the killing effect of Axl-CAR-T cells against LCLC-103 H was less pronounced than that against H460.

[0071] Figure 7 shows the proliferation rate trends of MDA-MB-231 cells under different E:T ratios for the control and experimental groups, and for MDA-MB-453 cells under different E:T ratios for the control and experimental groups. At E:T ratios of 4:1 and 8:1, Axl-CAR-T cells exhibited a significant killing effect against MDA-MB-231 cells relative to the corresponding MDA-MB-231 control group. At E:T ratios of 2:1 and 4:1, Axl-CAR-T cells exhibited a significant killing effect against MDA-MB-453 cells relative to the corresponding MDA-MB-453 control group. At E:T ratios of 2:1, Axl-CAR-T cells exhibited a significant killing effect against MDA-MB-453 cells relative to the corresponding MDA-MB-453 control group. At E:T ratios of 2:1, Axl-CAR-T cells exhibited an insignificant killing effect against MDA-MB-453 cells relative to the corresponding MDA-MB-453 control group. At comparable E:T, Axl-CAR-T cells exhibited stronger killing potential against MDA-MB-453.

[0072] Because IFNγ, a cytokine secreted by immune-activated cells, plays an important immunomodulatory role in inducing antiviral immunity, the IFNγ levels after immune stimulation of the industry-recognized matrix actually reflect the activity of effector cells. Therefore, detecting IFNγ levels is an indirect way of detecting effector cell activity. Figures 8-10 show the relative IFNγ expression levels of the blank, control, and experimental groups corresponding to MV 4-11, HL 60, and MDA-MB-231 tumor cells. Compared to the blank and control groups corresponding to each tumor cell line, the relative IFNγ expression levels of each experimental group tended to be significantly higher. Axl-CAR-T cells demonstrated good killing effects against both MV 4-11, HL 60, and MDA-MB-231 tumor cells.

[0073] Example 6: Experimental study of tumor cell in vivo killing by Axl-CAR-T cells An internal killing experiment was conducted on 10 female NSG mice (Shanghai Nanfang Model Bioscience and Technology Co., Ltd.), aged 6 to 8 weeks. Specifically, 1 × 10 7 MDA-MB-231 cells were transplanted and tumorigenesis occurred 18 days later. After that, interventions were performed to form the following PBS group, animal control group, and animal treatment group: PBS group: 100 microliters of PBS solution was intravenously injected into each tail. Animal control group: Resuspend the transfected T cells of Example 4 in 100 microliters of PBS solution, and the cell number was adjusted to 8 x 10 6 The dose was adjusted individually and injected into the tail vein of each mouse. Animal treatment group: Resuspend the Axl-CAR-T cells from Example 4 in 100 microliters of PBS solution, and administer 8 x 10 cells per tail vein injection to each mouse. 6 individually adjusted.

[0074] Each group received four injections into the tail vein every two days. On day 34 after the start of administration, the mice were euthanized and tissues were collected for analysis.

[0075] In this example, mouse lung tissue samples were immersed in 4% paraformaldehyde for fixation, then paraffin-embedded, sectioned, and stained with Su Mu Jing and Yi Hong (HE) for microscopic examination. Images of the HE stained lung tissue were processed using ImageJ to calculate the area of ​​the tumor within the lung tissue. Photographs comparing the tumor metastasis status of lung tissue samples from the PBS group, control group, and treatment group were obtained as shown in Figure 11 , along with a graph comparing the lung metastasis area percentages of lung tissue samples from each group, based on the photographs shown in Figure 11 . The specific staining, microscopic examination, and image collection and analysis methods are within the skill of those in the art. Referring to Figures 11 and 12 , the lung tumor area percentages in the experimental group were significantly lower than those in the PBS group and control group, even approaching zero, demonstrating that Axl-CAR-T cells can inhibit breast cancer lung metastasis.

[0076] In this example, tumor volume was calculated using in situ tumor tissue samples from each mouse as 0.5 × L × W2, where L is length and W is width. The mean tumor volume in the PBS control group was 2800 cubic centimeters, the mean tumor volume in the animal control group was 2430 cubic centimeters, and the mean tumor volume in the animal experimental group was 1650 cubic centimeters. p = 0.0005 compared to the control group.

[0077] In situ tumor tissue samples from each mouse were fixed in 4% polyoxyformaldehyde, embedded in paraffin, and sectioned for immunohistochemistry (IHC). The antibodies used were: anti-CD3 1:100 (Servicebio, GB13014); anti-CD8 1:100 (Servicebio, GB13068); and dual anti-HRP goat anti-rabbit antibody 1:200 (Servicebio, GB 23303). Microscopic examination was then performed, and immunogrouping images were processed using ImageJ. The immunogrouping-positive areas were extracted and calculated using the IHC Tool. Figure 13 shows a comparison of immunostained photographs of the tumor tissue samples from each group, and Figure 14 shows a comparison of the CD3 and CD8 positive area ratios in the tumor tissue samples from each group. Referring to Figures 13 and 14, CD3 and CD8 protein levels were significantly elevated in tumor samples from the experimental animal group compared to the control group.

[0078] Furthermore, in this example, mRNA was extracted from each primary tumor tissue sample and analyzed by qPCR to detect the expression levels of CD3, CD8A, CD4, IFNG, GZMB, and TNFA. Figure 15 shows a comparison of the expression levels of the above factors in each group. Here, PBS refers to the PBS group, control T cells refer to the animal control group, and AXL-CAR T cells refer to the animal experimental group. For specific analysis procedures, see above. As shown in Figure 15, compared with the animal control group, the three T cell marker genes, CD3, CD8A, and CD4, were significantly upregulated in tumor samples from the animal experimental group, and the T cell-secreted cytokine genes, IFNG, GZMB, and TNFA, were also significantly upregulated. The protein levels of CD3 and CD8 in tumor samples from the animal experimental group, as shown in Figures 13 and 14, were also significantly upregulated, all of which demonstrated the stronger tumor infiltration ability of Axl-CAR-T cells compared to the animal control group.

Claims

1. A chimeric antigen receptor comprising a heavy chain variable region and a light chain variable region, wherein the sequence of the light chain variable region is shown in SEQ ID NO: 1 and the sequence of the heavy chain variable region is shown in SEQ ID NO:

2.

2. The chimeric antigen receptor of claim 1, further comprising a connecting region connecting the light chain variable region and the heavy chain variable region, wherein the connecting region sequence is as shown in SEQ ID NO:

3.

3. a transmembrane domain and a hinge region located between the transmembrane domain and the heavy chain variable region of the Axl-specific antibody, the sequence of the hinge region is derived from at least one of CD8, CD8α, CD28, 4-1 BB, ICOS, OX40, CD40, CD80, and IgG; The chimeric antigen receptor according to claim 1, wherein the sequence of the transmembrane domain is derived from at least one of CD2, CD27, LFA-1, CD8α, CD28, 4-1BB, ICOS, OX40, CD40, CD80, CD3ζ, and CD3ε.

4. The chimeric antigen receptor of claim 3, wherein the sequence of the hinge region is set forth in SEQ ID NO: 4 and the sequence of the transmembrane domain is set forth in SEQ ID NO:

5.

5. an expression driver region and a signal peptide located between the expression driver region and the light chain variable region of the Axl-specific antibody, The chimeric antigen receptor of claim 3 , wherein the sequence of the expression driving region is derived from at least one of EF 1α, CMV, PGK, MPSV, MMLV, and SFV.

6. The chimeric antigen receptor of claim 5, wherein the sequence of the expression driver region is shown in SEQ ID NO: 6 and the sequence of the signal peptide is shown in SEQ ID NO:

7.

7. The chimeric antigen receptor according to claim 1, characterized in that the sequence of the chimeric antigen receptor is shown in SEQ ID NO:

8.

8. A nucleic acid encoding at least a portion of the fragment of the chimeric antigen receptor of claim 1.

9. A carrier comprising the nucleic acid according to claim 8.

10. A host cell comprising the carrier according to claim 9.

11. 10. Use of the chimeric antigen receptor of claim 1 in the preparation of a tumor therapeutic drug or in tumor treatment.

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