CAR-gammadelta t cells targeting axl and uses thereof

Abstract

The application relates to the technical field of medical treatment and provides a CAR-gammadelta T cell targeting AXL and application thereof, the CAR-gammadelta T cell has a CAR molecule, the CAR molecule comprises the following structure: signal peptide-antiAXL scFv-extracellular hinge region-transmembrane region-co-stimulating factor-CD3 zeta intracellular region, the "-" is independently a connecting peptide or a peptide bond; the co-stimulating factor comprises a CD28 intracellular region and a 41BB intracellular region. Advantages: the CAR-gammadelta T cell targeting AXL integrates a third-generation CAR structure (CD28+4-1BB double co-stimulating domain) and a gammadelta T cell, realizes high targeting precision and killing efficiency. The in-vitro killing rate of the CAR-gammadelta T cell targeting AXL to AXL positive lung cancer cells (A549 and HCC827-ER3) is >90% (effector to target ratio 10:1), is obviously improved compared with common gammadelta T cells, and the tumor volume is obviously reduced in a PDX mouse model, which proves excellent treatment effect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a CAR-γδT cell targeting AXL and its application. Background Technology

[0002] Lung cancer has the highest mortality rate among malignant tumors worldwide, and its incidence rate has been on the rise. my country accounts for more than 40% of the global cases, resulting in a heavy disease burden. Due to its insidious onset, rapid progression, and tendency to metastasize early, more than 60% of patients are diagnosed at an advanced stage, making radical surgery impossible, and current treatment methods are not ideal.

[0003] AXL is overexpressed in various cancers such as lung cancer and breast cancer. Its high expression often indicates a poor prognosis for patients. Moreover, its activation is not only a necessary step in the epithelial-mesenchymal transition, but also promotes erlotinib resistance in EGFR-mutant non-small cell lung cancer. Although AXL also has physiological functions in normal cells such as neurons and immune cells, which may bring potential treatment side effects, small molecule inhibitors, monoclonal antibodies and CAR-T cells targeting AXL have shown anti-cancer activity in some tumor models.

[0004] Chimeric antigen receptor T-cell (CAR-T) therapy involves genetically engineering T cells to express chimeric receptors that specifically recognize tumor antigens, thereby achieving targeted killing of tumor cells. It is a type of immunotherapy. Currently, CD19-targeted CAR-T therapy has shown good efficacy in treating hematologic malignancies such as leukemia and lymphoma, but its application in solid tumors presents several challenges: First, it is prone to off-target toxicity, cytokine release syndrome, neurotoxicity, or graft-versus-host disease. Second, the immunosuppressive microenvironment of solid tumors significantly reduces the infiltration capacity and survival time of effector cells, making CAR-T therapy far less effective for solid tumors than for hematologic malignancies. Furthermore, traditional CAR-T therapy primarily uses αβ-T cells as carriers. These cells rely on the presentation of the major histocompatibility complex (MHC) for antigen recognition, and graft-versus-host disease is easily induced during allogeneic transplantation, thus limiting its clinical application.

[0005] Therefore, the development of novel, highly efficient, and low-toxicity CAR-modified effector cells has significant clinical implications and practical application value. Summary of the Invention

[0006] The present invention aims to overcome at least one of the shortcomings of the prior art and provide a CAR-γδT cell targeting AXL and its application, achieving high targeting precision and killing efficiency, so as to break through the bottleneck of existing solid tumor immunotherapy and provide a new treatment strategy for patients with advanced lung cancer.

[0007] One object of the present invention is to provide a CAR-γδT cell targeting AXL, the CAR-γδT cell having a CAR molecule, the CAR molecule comprising the following structure: signal peptide - antiAXLscFv - extracellular hinge region - transmembrane region - costimulatory factor - CD3ζ intracellular region, wherein the "-" is independently a linking peptide or peptide bond; The co-stimulatory factors include the CD28 intracellular region and the 41BB intracellular region.

[0008] Preferably, the signal peptide is a CD8 signal peptide, and the amino acid sequence of the CD8 signal peptide is shown in SEQ ID NO:1; The amino acid sequence of the antiAXL scFv is shown in SEQ ID NO:2 and 3; The hinge region is a CD8 hinge region, and the amino acid sequence of the CD8 hinge region is shown in SEQ ID NO:4; The transmembrane region is the CD28 transmembrane region, and the amino acid sequences of the CD28 transmembrane region and the CD28 intracellular region are shown in SEQ ID NO:5; The amino acid sequence of the intracellular region of the 41BB is shown in SEQ ID NO:6; The amino acid sequence of the CD3ζ intracellular region is shown in SEQ ID NO:7.

[0009] Preferably, the CAR molecule further includes a linker, the linker being a self-cleaving peptide, and the CAR molecule includes the following structure: signal peptide - antiAXLscFv - extracellular hinge region - transmembrane region - co-stimulatory factor - CD3ζ intracellular region - self-cleaving peptide, wherein the "-" is independently a linker peptide or a peptide bond.

[0010] Preferably, CAR molecule variants that have at least 90% sequence identity with the CAR molecule of this application can also produce similar effects to this application.

[0011] One object of the present invention is to provide the use of the above-mentioned AXL-targeting CAR-γδT cells in the preparation of products for treating lung cancer expressing AXL.

[0012] One object of the present invention is to provide the use of an immune checkpoint inhibitor in combination with the above-mentioned CAR-γδT cells targeting AXL in the preparation of a product for treating lung cancer expressing AXL.

[0013] One object of the present invention is to provide a pharmaceutical composition comprising an immune checkpoint inhibitor and the above-described CAR-γδT cells.

[0014] Preferably, the immune checkpoint inhibitor is at least one of PD-1 antibody and CTLA-4 antibody.

[0015] Preferably, the PD-1 antibody is pembrolizumab; and the CTLA-4 antibody is tesimazine.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention provides a CAR-γδT cell targeting AXL, which integrates a third-generation CAR structure (CD28+4-1BB dual co-stimulatory domain) with γδT cells, achieving high targeting precision and killing efficiency. Its in vitro killing rate against AXL-positive lung cancer cells (A549 and HCC827-ER3) is >90% (effect-to-target ratio 10:1), significantly improved compared to ordinary γδT cells, and it significantly reduces tumor volume in a PDX mouse model, demonstrating its excellent therapeutic effect.

[0017] 2. This invention achieves efficient in vitro expansion of γδT cells based on ZOL (zoledronic acid), and further evaluates the effects of this method on the purity, expansion fold, and cytotoxicity of γδT cells, thus confirming the high efficiency and reliability of the method.

[0018] 3. In this invention, it is demonstrated that intratumoral injection significantly improves efficacy compared to tail vein injection, and this local administration avoids systemic CRS. Attached Figure Description

[0019] Figure 1 The results showed that ZOL was used to stimulate and expand γδ T cells in vitro, and the effect of ZOL at a concentration of 10 μM was the best.

[0020] Figure 2 Display: Basic structural design of CAR molecules targeting AXL.

[0021] Figure 3 The results show the positive rate of CAR-γδ T detected by flow cytometry.

[0022] Figure 4 The results show that after co-culturing human PBM with ZOL, the expression level of PD-1 in γδ T cells was detected by flow cytometry on days 3, 5 and 7.

[0023] Figure 5 The results show the flow cytometry findings of PD-1 expression levels in γδ T cells after co-culturing γδ T cells and A549 cells at a 5:1 effector-target ratio for different durations.

[0024] Figure 6 Display: Flowchart of the construction and experiment of the A549 subcutaneous tumor model with high AXL expression.

[0025] Figure 7 Display: Growth curves showing the change in subcutaneous tumor volume over time in each group of mice.

[0026] Figure 8 The results show the volume and weight of subcutaneous tumors in each group of mice at the end of day 22 of the experiment.

[0027] Figure 9 Display: H&E staining of organs (heart, lungs, liver, spleen and kidney tissues) of mice in each group. Detailed Implementation

[0028] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0029] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0030] One object of the present invention is to provide a CAR-γδT cell targeting AXL, the CAR-γδT cell having a CAR molecule, the CAR molecule comprising the following structure: signal peptide - antiAXLscFv - extracellular hinge region - transmembrane region - costimulatory factor - CD3ζ intracellular region, wherein the "-" is independently a linking peptide or peptide bond; The co-stimulatory factors include the CD28 intracellular region and the 41BB intracellular region.

[0031] The 41BB intracellular region is the 4-1BB intracellular region. γδT cells possess unique anti-tumor characteristics: they do not rely on MHC to recognize antigens, can directly target various tumor-associated antigens, and are less likely to induce graft-versus-host disease during xenograft transplantation, exhibiting better safety and tolerability. These characteristics make γδT cells a highly promising carrier for solid tumor immunotherapy. The CAR molecule of this invention has dual co-stimulatory domains of CD28 and 4-1BB, making it a third-generation CAR molecule. This invention integrates a third-generation CAR structure with γδT cells, achieving high targeting precision and killing efficiency.

[0032] In one or more embodiments of the present invention, the CAR molecule further includes a fluorescent protein sequence for detection. It can be understood that the CAR molecule includes the following structure: signal peptide-antiAXLscFv-extracellular hinge region-transmembrane region-CD28 intracellular region-41BB intracellular region-CD3ζ intracellular region-fluorescent protein sequence.

[0033] The fluorescent protein includes enhanced green fluorescent protein, but other types of fluorescent proteins or markers may also be used for easy observation and detection. In some embodiments of the present invention, the amino acid sequence of the fluorescent protein is shown in SEQ ID NO:15.

[0034] Preferably, the signal peptide is a CD8 signal peptide, and the amino acid sequence of the CD8 signal peptide is shown in SEQ ID NO:1; The amino acid sequence of the antiAXL scFv is shown in SEQ ID NO:2 and 3 [W1.1]; The hinge region is a CD8 hinge region, and the amino acid sequence of the CD8 hinge region is shown in SEQ ID NO:4; The transmembrane region is the CD28 transmembrane region, and the amino acid sequences of the CD28 transmembrane region and the CD28 intracellular region are shown in SEQ ID NO:5; The amino acid sequence of the intracellular region of the 41BB is shown in SEQ ID NO:6; The amino acid sequence of the CD3ζ intracellular region is shown in SEQ ID NO:7.

[0035] Preferably, the CAR molecule further includes a linker, the linker being a self-cleaving peptide, and the CAR molecule includes the following structure: signal peptide - antiAXLscFv - extracellular hinge region - transmembrane region - co-stimulatory factor - CD3ζ intracellular region - self-cleaving peptide, wherein the "-" is independently a linker peptide or a peptide bond.

[0036] In one or more embodiments of the present invention, the self-cleaving peptide is a 2A peptide, as shown in SEQ ID NO:8. In one or more embodiments of the present invention, the CAR molecule comprises the following structure: CD8 signal peptide (CD8SP) - Single-chain antibody variable region (scFv) of AXL antibody - CD8 hinge region (CD8 Hinge) - CD28 transmembrane region (CD28TM) - Intracellular region (CD28 IC) - 41BB - CD3ζ signal domain - 2A peptide (2A peptide) - Enhanced green fluorescent protein (eGFP).

[0037] In one or more embodiments of the present invention, the CAR molecule may further contain chemically modified or non-functional groups. The chemical modifications include: phosphorylation, acetylation, methylation, ubiquitination or ubiquitination-like processes, glycosylation, esterification, cyclization, enzymatic cleavage, disulfide bond formation, and hydroxylation. The non-functional groups include, but are not limited to, tag sequences, targeting peptides, dyes, biotin, and affinity ligands. The tag sequences include, but are not limited to: His-tag, GST-tag, maltose-binding protein tag, Strep-tag, c-Myc tag, HA tag, FLAG tag, V5 tag, AviTag, SNAP-tag, and SUMO tag. The method of obtaining the CAR molecule is not limited and can be obtained using any conventional method in the art, including, but not limited to, solid-phase synthesis and biosynthesis.

[0038] In this invention, the term "sequence identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific peptide or polypeptide sequence, provided that the sequences are aligned and (if necessary) vacancies are introduced to achieve maximum sequence identity, and no conservative substitutions are considered part of the sequence identity. Sequence alignment used to determine the percentage of amino acid sequence identity can be performed by various methods known to those skilled in the art, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR). Those skilled in the art can determine appropriate parameters for determining the alignment, including any algorithms required to achieve maximum alignment across the full length of the compared sequences.

[0039] In one or more embodiments of the present invention, the amino acid molecules of CD8 signal peptide (CD8SP), single-chain antibody variable region (scFv) (VH) of AXL antibody, single-chain antibody variable region (scFv) (VL) of AXL antibody, CD8 hinge region (CD8 Hinge), CD28 transmembrane region (CD28 TM) and intracellular region (CD28 IC), 4-1BB, CD3ζ signal domain, 2A peptide (2A peptide), and enhanced green fluorescent protein (eGFP) for detection are sequentially shown as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8 and 9.

[0040] Another object of the present invention is to provide an expression vector for the above-mentioned nucleic acid molecules, said expression vector comprising plasmids.

[0041] In one or more embodiments of the present invention, the expression vector is the lentiviral expression plasmid pWPXLd. In one or more embodiments of the present invention, a three-plasmid packaging system is used for lentiviral packaging, wherein the helper plasmids are commercially available helper plasmids. Exemplarily, the helper plasmids are pSPAX2 and pMD2.G.

[0042] The expression vector of the present invention can be any suitable expression vector capable of being used to transform or transfect one or more genes or sequences of interest into any suitable host cell and preferably to express the genes or sequences in the host cell. Suitable vectors include those designed for amplification and expansion or for expression or both of the above, and examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0043] Preferably, the use of any of the above-mentioned AXL-targeting CAR-γδT cells in the preparation of products for treating lung cancer expressing AXL.

[0044] In one or more embodiments of the present invention, the lung cancer expressing AXL is A549 or HCC827-ER3.

[0045] 7. The use of an immune checkpoint inhibitor in combination with CAR-γδT cells targeting AXL as described in claims 1-5 in the preparation of a product for treating lung cancer expressing AXL.

[0046] Another object of the present invention is to provide a pharmaceutical composition comprising an immune checkpoint inhibitor and any of the above-mentioned CAR-γδT cells.

[0047] In one or more embodiments of the present invention, the pharmaceutical composition further includes optional pharmaceutically acceptable excipients. The pharmaceutically acceptable excipients are rationally selected based on factors such as the product form, intended use, and route of administration of the pharmaceutical composition, and include, but are not limited to: buffers, coenzymes, enzyme protectants, metal ions, catalysts, antifoaming agents, diluents (such as starch, dextrin, sucrose, lactose, mannitol, etc.), absorbents (such as calcium sulfate, calcium hydrogen phosphate, etc.), wetting agents (such as ethanol), binders (such as hydroxypropyl methylcellulose, povidone, etc.), solvents, pH adjusters, antibacterial agents (such as sodium sulfite, sodium thiosulfate, etc.), isotonic adjusters (such as glucose, sodium chloride, etc.), chelating agents (such as disodium EDTA), etc.

[0048] In one or more embodiments of the present invention, the antitumor drug can be administered via intratumoral injection and tail vein injection.

[0049] In one or more embodiments of the present invention, the antitumor drug is administered via intratumoral injection.

[0050] Preferably, the immune checkpoint inhibitor is at least one of PD-1 antibody and CTLA-4 antibody.

[0051] Preferably, the PD-1 antibody is pembrolizumab; and the CTLA-4 antibody is tesimazine.

[0052] The sequence information involved in this invention is shown in the table below: Table 1: Sequence Description

[0053] The present invention will now be further illustrated with specific examples. The following embodiments are only for explaining the present invention and do not constitute a limitation thereof. The test samples and test procedures used in the following embodiments include the following (if the specific experimental conditions are not specified in the embodiments, they are usually performed according to conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following embodiments can be obtained commercially unless otherwise specified).

[0054] Example 1: Obtaining and Expanding γδT Cells The specific steps for obtaining and expanding γδT cells include: (1) Extraction of PBMCs from healthy donors: Take an appropriate amount of peripheral blood sample from a healthy donor and dilute it with an equal volume of PBS to reduce blood viscosity and facilitate better separation with the lymphocyte separation medium. Add a certain amount of Ficoll lymphocyte separation medium (purchased from TBD) to a new centrifuge tube. The amount of Ficoll lymphocyte separation medium added should be approximately half the volume of the diluted blood, i.e., peripheral blood sample volume: PBS volume: Ficoll lymphocyte separation medium volume = 1:1:1. Slowly add the diluted peripheral blood sample along the wall of the centrifuge tube onto the surface of the Ficoll lymphocyte separation medium using a pipette. The addition should be slow to avoid disrupting the separation interface. Centrifuge. Centrifugation parameters can be adjusted appropriately based on centrifuge machine differences. In this example, the centrifugation force is 800 g, 4°C for the initial acceleration and 0°C for the final acceleration, and centrifugation for 30 minutes. After centrifugation, the centrifuge tube will roughly separate into a red blood cell layer, a separation medium layer, a PBMC layer, and a plasma layer from bottom to top. Carefully aspirate the PBMC layer using a pipette, avoiding the red blood cell layer as much as possible. Transfer the aspirated PBMC layer to a new centrifuge tube, add an appropriate amount of PBS, and centrifuge again (300 g, 10 min). Discard the supernatant. Repeat this step twice to remove residual lymphocyte separation fluid and other impurities. Add an appropriate amount of erythrocyte lysis buffer (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) according to the cell volume. Gently mix with a pipette and let stand at room temperature for 5-10 minutes. Add an appropriate amount of PBS to terminate the lysis reaction, where the volume of PBS added is approximately equal to the volume of erythrocyte lysis buffer added. Centrifuge again (300 g, 10 min). Discard the supernatant and resuspend the PBMC pellet in T cell expansion medium (purchased from Stemcell) + ZOL (zoledronic acid, final concentration 10 μM) + IL-2 (final concentration 500 IU / mL). ZOL (zoledronic acid) can effectively stimulate γδT cells in PBMCs, promoting their activation and expansion.

[0055] (2) Sorting of γδT cells: Using the TCR γδT cell isolation kit (purchased from Miltenyi Biotec), γδT cells were isolated from the PBMC cell suspension obtained in the above steps according to the instructions for use.

[0056] The specific steps are as follows: Resuspend the PBMC cell suspension in PBS buffer containing 2% FBS, adjusting the PBMC cell suspension volume appropriately based on the PBMC cell concentration. When the cell concentration is approximately 1×10⁻⁶ cells / mL... 8 When the cell concentration is <1×10⁶ cells / mL, the cell suspension volume is 100 µL - 2.5 mL; when the cell concentration is <1×10⁶ cells / mL, the cell suspension volume is 100 µL - 2.5 mL. 7 The cell suspension volume was 0.1 mL. If the PBMC cell content was less than 2%, the concentration was adjusted to 2 × 10⁻⁶ cells / mL. 8cells / mL.

[0057] Add Anti-Human CD32 (Fc gamma RII) Blocker to the PBMC cell suspension at a ratio of 100 µL / mL (i.e., 100 µL of Anti-Human CD32 (Fc gamma RII) Blocker to 1 mL of cell suspension) and mix well. Then add PE anti-human TCRγ / δ (400 µg / mL) at a ratio of 0.3–3 µg / mL (relative to the cell suspension), mix well, and incubate at room temperature for 15 minutes. After incubation, add an excess of 10 volumes of buffer (PBS + 2% FBS + 1 mM EDTA), centrifuge at 300×g for 10 minutes at room temperature, centrifuge at 4°C and 0°C, carefully remove the supernatant, and resuspend to the initial volume. Add EasySep™ PE Selection Cocktail at a ratio of 100 µL / mL (i.e., 100 µL of Cocktail to 1 mL of cell suspension), mix well, and incubate at room temperature for 15 minutes. Vortex the magnetic beads (EasySep™ DextranRapidSpheres™) for 30 seconds to ensure uniform dispersion. Add the dispersed magnetic beads at a ratio of 50 µL / mL, mix thoroughly, and incubate at room temperature for 10 minutes. Adjust the volume to 2.5 mL using the above buffer solution and gently mix with a pipette. Place the flow cytometry tube (without the cap) into the magnet (inserting the tube to the bottom) and incubate at room temperature for 5 minutes. While maintaining the flow cytometry tube position, tilt the entire magnet and flow cytometry tube, holding this tilted position for 2-3 seconds to allow the liquid to flow naturally. Do not shake or aspirate with a pipette to preserve the target cells. Remove the flow cytometry tube from the magnet. Repeat twice to obtain the target cells. Resuspend the cells in the above T cell expansion medium to obtain purified TCR γδ+ cells. Continue culturing using T cell expansion medium.

[0058] The growth of TCR γδ+ cells was detected using the CCK-8 reagent. The specific steps were as follows: Prepare five 96-well plates, and precisely add 100 μL of purified γδT cell suspension (approximately 2000 cells / well) to each well. After seeding, place all five 96-well plates together in a cell culture incubator. On days 1, 3, 5, 7, and 9 of culture, remove one well from the incubator for CCK-8 assay. The CCK-8 assay must be performed in the dark. Specifically, slowly add 10 μL of CCK-8 solution to each well. Avoid introducing air bubbles into the wells during addition to prevent interference with absorbance (OD) measurement. Then return the plate to the incubator and continue incubation for 2.5 hours to ensure sufficient reaction between CCK-8 and the cells. After incubation, remove the plate from the incubator. Before reading the absorbance, gently place the plate on a shaker to mix and ensure uniform cell distribution. The absorbance of each well at a wavelength of 450 nm was measured using an ELISA reader.

[0059] Example 2: Construction of a lung cancer cell line with double GFP and Luciferase (GL) positivity On day 1 of the experiment, A549 cells were seeded into 6-well plates. The seeding density was adjusted according to the cell growth rate, with the standard being that the cells could grow to approximately 50% confluence by the next day. The following day, the required viral load for each cell line was calculated based on the cell count and the multiplicity of infection (MOI). The corresponding amounts of luciferase-containing lentiviral plasmid (LV5-LUC-GFP-Puro) (purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd.) and polybrene (final concentration 10 μg / mL) were added to the corresponding wells for cell transfection. Twelve hours after transfection, the cell status was observed under a microscope. The transfection time was extended appropriately based on the cell status, but generally, the total infection time was controlled within 24 hours. After transfection, the culture medium was replaced with fresh medium containing 2 μg / mL puromycin and cultured for 7 days to complete the selection. On day 7 of culture, cells were digested with trypsin, washed once with PBS, the supernatant was removed, and the cell pellet was resuspended in PBS to obtain GFP and Luciferase double-positive (GL) lung cancer cell lines. GFP expression rates in each cell line were detected by flow cytometry. If the results showed low expression rates, the above procedure could be repeated to obtain a cell population with a high positivity rate.

[0060] Example 3: Construction of CAR-γδT cells targeting AXL The AXL scFv sequences are all derived from a patent, patent number: WO2012 / 175692A1 (3E3E8). The construction steps include: (1) See Figure 2The CAR gene sequence in this experiment includes the following amino acid molecules in sequence: CD8 signal peptide (CD8SP), single-chain antibody variable region (scFv) (VH) of AXL antibody, single-chain antibody variable region (scFv) (VL) of AXL antibody, CD8 hinge region (CD8 Hinge), CD28 transmembrane region (CD28 TM) and intracellular region (CD28 IC), 4-1BB, CD3ζ signal domain, 2A peptide (2A peptide), and enhanced green fluorescent protein (eGFP) for detection; the amino acid sequences of the CD8 signal peptide, single-chain antibody variable region (scFv) (VH) of AXL antibody, single-chain antibody variable region (scFv) (VL) of AXL antibody, CD8 hinge region (CD8 Hinge), CD28 transmembrane region (CD28 TM) and intracellular region (CD28 IC), 4-1BB, CD3ζ signal domain, and 2A peptide (2A peptide) are shown in SEQ NO: 1, 2, 3, 4, 5, 6, 7, 8, and 9, respectively.

[0061] (2) The lentivirus solution was synthesized by Nanjing Genscript Company with the corresponding CAR molecular sequence and stored at -80℃ for later use.

[0062] (3) Take the lentivirus solution and Polybrene reagent out of the -80℃ freezer and quickly place them on ice to allow them to dissolve slowly.

[0063] (4) Take an appropriate amount of γδ T cell suspension in good condition, transfer it to a centrifuge tube, centrifuge at 800 rpm for 3 minutes, carefully remove the supernatant to avoid removing the precipitate, and then resuspend the cells in fresh culture medium without antibiotics and ZOL.

[0064] (5) Use a pipette to draw 10 μL of cell suspension and 10 μL of trypan blue solution, mix them well, then draw 10 μL of the mixture and add it to a hemocytometer, and count the live cells under a microscope.

[0065] (6) Calculate the total volume of lentivirus required for infection using the appropriate formula based on the multiplicity of infection (MOI) value and cell number. Add the corresponding volume of lentivirus to the cell suspension and add the transfection aid Polybrene. Then, infect the cells with the virus for 12-24 hours.

[0066] (7) On the second day, after the virus infection was completed, the virus fluid was removed by centrifugation, and fresh γδ T cell culture medium was replaced to continue culturing the cells.

[0067] (8) When the cells are cultured to day 3, observe the infection status of the cells under a fluorescence microscope and record it.

[0068] Example 4: After successful construction of GL cells, in vivo and in vitro experiments can be performed directly.

[0069] In vitro killing effect of CAR-γδT cells on lung cancer cell lines The in vitro killing effects of lung cancer cells (GL cells) and CAR-γδT cells were detected using a luciferase assay. Specifically, the corresponding target cells A549 were digested with trypsin and then counted, at a ratio of 1×10⁻⁶. 4 Cells were seeded per well in 96-well plates, with experimental groups (CAR-γδT cells administered) and control groups (no CAR-γδT cells administered). Each group had three replicates to ensure accuracy and reproducibility. Based on the grouping, appropriate amounts of GL cells and CAR-γδT cell suspensions were aspirated, centrifuged at 800 rpm for 3 minutes, the supernatant was discarded, and the cells were resuspended in fresh culture medium and counted. The required cell number for each group was calculated based on different effector-to-target ratios (E:T) (1:1, 2.5:1, 5:1, and 10:1). The 96-well plates were gently rinsed once with PBS, and the prepared cell suspensions were added to the corresponding wells according to the different effector-to-target ratios. The 96-well plates were co-cultured in a cell culture incubator for 6 or 12 hours. The supernatant was discarded, and the 96-well plates were gently rinsed once with PBS. 100 μL of sodium fluorescein at a final concentration of 150 μg / mL was added to each well, and the reaction was carried out for 10 minutes in the dark. The fluorescence value was then detected using a microplate reader. The obtained data were organized and summarized, and the percentage of effector cell killing was calculated according to the formula.

[0070] Percentage of damage = ×100%.

[0071] The control wells are those containing only target cells and no effector cells (i.e., GL cells or CAR-γδT cells). GraphPad Prism software was used for data aggregation, graphing, and statistical analysis.

[0072] Simultaneously, flow cytometry was used to detect PD-1 expression levels after co-culturing γδT cells and lung cancer cells. The effector-to-target ratio was set at 5:1, and the number of γδT cells was 5 × 10⁶. 4 Cells / well were cultured for 0 hours, 6 hours, and 12 hours, respectively. The antibodies used for flow cytometry were PE-labeled anti-human TCR γδ flow cytometry antibody, FITC-labeled anti-human CD3 flow cytometry antibody, and APC-labeled anti-human PD-1 flow cytometry antibody, respectively.

[0073] Example 5: Treatment of Lung Cell Carcinoma with CAR-γδT Cells Combined with Monoclonal or Bispecific Antibodies CAR-γδT cell therapy combined with monoclonal antibodies (anti-PD-1 antibody) or bispecific antibodies (anti-PD-1 antibody and anti-CTLA-4 antibody) for the treatment of lung cell carcinoma: (1) Construction of a subcutaneous tumor model of lung cancer A549: A549 cells were cultured according to the requirements for animal experiments. When the tumor cells proliferated to the logarithmic growth phase and reached approximately 80% confluence, the supernatant was discarded, and the cells were washed once with PBS. 1 mL of 0.25% trypsin was added to each 100 mm culture dish to digest the cells. The digested cells were then transferred to centrifuge tubes, centrifuged, and washed once with PBS. The supernatant was discarded, and the A549 cell pellet was resuspended in 1 mL of serum-free PBS. The cells were then counted, and the cells were prepared into 2 × 10⁻⁶ cells / mL culture media. 6 One cell per 100 μL of cell suspension. Using a 1 mL syringe, 100 μL of cell suspension was drawn and injected subcutaneously approximately 1 cm below the right axilla of NSG mice. The injection site was visibly punctured, and a small wheal was observed. When the subcutaneous tumor in the mouse reached approximately 50 mm in size... 3 A successful model is considered to have been constructed. Typically, a subcutaneous tumor model can be constructed within 10 days.

[0074] (2) Treatment of lung cancer with CAR-γδT cells combined with monoclonal antibodies (anti-PD-1 antibody) or bispecific antibodies (anti-PD-1 antibody and anti-CTLA-4 antibody): To establish the A549 subcutaneous tumor model of lung cancer, experimental mice were randomly divided into 5 groups, with 5 mice in each group. The specific groupings included: PBS group, γδT cell group, CAR-γδT cell group, CAR-γδT cell combined with anti-PD-1 antibody group, and CAR-γδT cell combined with anti-PD-1 antibody and CTLA-4 antibody group.

[0075] The injection dose of A549 cells was 1×10⁻⁶. 6 50 μL / mouse / dose. The anti-PD-1 antibody was pembrolizumab (injection), administered at a dose of 130 μg / mouse. The CTLA-4 antibody was tesimumab (injection), administered at a dose of 200 μg / mouse. The CAR-γδT cell combination with anti-PD-1 antibody and the CAR-γδT cell combination with both anti-PD-1 and CTLA-4 antibody groups were administered with 1×10... 7 The CAR-γδT cells were mixed to prepare a 50 μL solution for administration. Treatment was administered via injection every 5 days for a total of 3 injections.

[0076] During the experiment, mice in each group were marked, and the longest and shortest diameters of the tumors were measured periodically (every 3 days) using calipers to calculate the volume of the subcutaneous tumors and record the experimental data.

[0077] To further evaluate the potential effects and safety of in vivo CAR-γδ T cell reinfusion on normal organs in mice, mice in each group were euthanized on day 22 after the completion of the entire experimental procedure. Subcutaneous tumors and vital organs such as the heart, lungs, liver, spleen, and kidneys were dissected, isolated, and collected for H&E staining analysis. Microscopic examination of organ sections was used to systematically assess for any tissue damage or pathological changes.

[0078] The following is an analysis of the experimental results.

[0079] 1. In vitro, ZOL can effectively stimulate the proliferation of γδ T cells. In in vitro experiments, ZOL at different concentration gradients was applied directly to freshly extracted, unsorted PBMCs to assess the proliferation of γδ T cells. The results showed that the proliferation rate of γδ T cells exhibited a clear ZOL concentration-dependent characteristic, with the most significant stimulatory effect on γδ T cells observed at a ZOL concentration of 10 μM (Figure 1).

[0080] 2. Construction of CAR-γδ T cells targeting AXL After constructing the CAR molecule, an appropriate lentiviral vector titer was selected, and fresh γδ T cells cultured to day 7 were infected using the aforementioned lentivirus. On day 3 post-infection, the growth status and fluorescence of γδ T cells were observed under a fluorescence microscope. Once the CAR-γδ T cell proliferation reached a certain number, the infection efficiency was detected by flow cytometry. The results showed that the positive rate of CAR-γδ T cells in the total γδ T cell population was 34.1% (…). Figure 3 These results demonstrate the success of constructing CAR-γδ T cells targeting AXL, and these cells can be used in subsequent experiments.

[0081] 3. PD-1 expression levels in γδ T cells gradually increase after activation or co-culture with lung cancer cells. This study used flow cytometry to dynamically analyze the PD-1 expression level in activated γδ T cells. In the experiment, human PBMCs were co-cultured with ZOL (zolium oxychloride), and samples were taken on days 3, 5, and 7 after culture to detect the PD-1 expression level in γδ T cells. The results showed that the PD-1 expression level in γδ T cells activated by ZOL was upregulated, and the PD-1 expression level gradually increased with prolonged culture time. Figure 4 ).

[0082] Furthermore, to further investigate the changes in PD-1 expression in γδ T cells during co-culture with tumor cells, this study used A549 cells as target cells at an effector-to-target ratio of 5:1 and employed flow cytometry to detect PD-1 expression levels after co-culturing γδ T cells with A549 cells for different time periods. The results showed that PD-1 expression levels were upregulated after co-culturing γδ T cells with A549 cells, and these levels continued to increase with prolonged co-culture time. Figure 5 ).

[0083] 4. CAR-γδ T cells targeting AXL have a significant tumor-suppressing effect on A549 subcutaneous tumors, and this effect is further enhanced when combined with anti-PD-1 antibody and CTAL-4 antibody.

[0084] CAR-γδ T cells targeting AXL alone have shown significant tumor-suppressing effects against A549 subcutaneous tumors, and this tumor-suppressing effect is further enhanced by combining anti-PD-1 antibody and CTLA-4 antibody. Figures 6-8 ).

[0085] 5. In vivo infusion of CAR-γδ T cells did not induce GvHD, demonstrating high safety. Experimental results ( Figure 9 The results showed that no significant tissue damage or pathological changes were observed in the heart, lungs, liver, spleen, and kidneys of mice in each group, and no tumor formation was observed. This result also indicates that in vivo infusion of CAR-γδ T cells did not induce GvHD and has a high safety profile.

Claims

1. A CAR-γδT cell targeting AXL, wherein the CAR-γδT cell possesses a CAR molecule, characterized in that, The CAR molecule comprises the following structure: signal peptide-antiAXLscFv-extracellular hinge region-transmembrane region-co-stimulatory factor-CD3ζ intracellular region, wherein the "-" is independently a linking peptide or peptide bond; The co-stimulatory factors include the CD28 intracellular region and the 41BB intracellular region.

2. The CAR-γδT cells targeting AXL according to claim 1, characterized in that, The transmembrane region is the CD28 transmembrane region, and the amino acid sequences of the CD28 transmembrane region and the CD28 intracellular region are shown in SEQ ID NO:

5.

3. The CAR-γδT cells targeting AXL according to claim 1, characterized in that, The amino acid sequence of the intracellular region of the 41BB is shown in SEQ ID NO:

6.

4. The CAR-γδT cells targeting AXL according to claim 1, characterized in that, The signal peptide is a CD8 signal peptide, and the amino acid sequence of the CD8 signal peptide is shown in SEQ ID NO:1; The amino acid sequence of the antiAXLscFv is shown in SEQ ID NO:2 and 3; The hinge region is a CD8 hinge region, and the amino acid sequence of the CD8 hinge region is shown in SEQ ID NO:4; The amino acid sequence of the CD3ζ intracellular region is shown in SEQ ID NO:

7.

5. The CAR-γδT cells targeting AXL according to claim 1, characterized in that, The CAR molecule further includes a linker, which is a self-cleaving peptide. The CAR molecule includes the following structure: signal peptide - antiAXLscFv - extracellular hinge region - transmembrane region - co-stimulatory factor - CD3ζ intracellular region - self-cleaving peptide, wherein the "-" is independently a linker peptide or peptide bond.

6. The use of the CAR-γδT cells targeting AXL as described in any one of claims 1 to 5 in the preparation of products for treating lung cancer expressing AXL.

7. The use of an immune checkpoint inhibitor in combination with CAR-γδT cells targeting AXL as described in any one of claims 1 to 5 in the preparation of a product for treating lung cancer expressing AXL.

8. A pharmaceutical composition, characterized in that, Including immune checkpoint inhibitors and CAR-γδT cells as described in any one of claims 1 to 5.

9. The pharmaceutical composition according to claim 8, characterized in that, The immune checkpoint inhibitor is at least one of PD-1 antibody and CTLA-4 antibody.

10. The pharmaceutical composition according to claim 9, characterized in that, The PD-1 antibody is pembrolizumab; the CTLA-4 antibody is tesimumab.

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