CN114075548B - AXL-targeted CAR-T cell, and preparation method and application thereof - Google Patents

Abstract

The invention discloses an application of a CAR-T cell targeting AXL, which utilizes scFv protein targeting AXL to prepare a third-generation CAR-T cell targeting AXL and is applied to tumor treatment, so that the problem that a CAR-T treatment solid tumor is difficult to find a tumor specific target is solved, the off-target is effectively avoided, the probability of side effect is reduced, and the treatment effect of the solid tumor is improved. Based on the high expression of the AXL in lung cancer, the method can specifically kill lung cancer tissues positively expressed by the AXL and realize the treatment of lung cancer; especially, the composition acts on lung cancer cells which are resistant to EGFR-TKI caused by up-regulation of AXL, and the synergistic EGFR-TKI medicine can obviously improve the treatment effect of lung cancer when being used for treating lung cancer, and overcomes the defect of poor treatment effect caused by drug resistance.

Description

AXL-targeted CAR-T cell, and preparation method and application thereof

Technical Field

The invention relates to the field of immunology, in particular to a CAR-T cell targeting AXL, a preparation method and application thereof.

Background

Lung cancer is one of the most common malignant tumors worldwide. According to statistics of the national tumor registration center (National Central Cancer Registry of China, NCCR), the incidence and mortality of lung cancer are the first in China. In recent years, although methods for treating lung cancer have been developed, such as surgery, radiotherapy, chemotherapy, targeted therapy, and the like. However, because many patients have local or extensive metastasis when they find lung cancer, the overall efficacy of lung cancer is still very limited, with survival rates of only about 15% in 5 years. Therefore, a new therapeutic strategy or drug is needed to improve the therapeutic effect and survival rate of lung cancer patients.

CAR-T therapy (Chimeric Antigen Receptor T-Cell Immunotherapy), i.e. chimeric antigen receptor T cell immunotherapy, is a novel cell therapy. T cells of a tumor patient are artificially modified through a genetic engineering technology, a large number of tumor-specific CAR-T cells are generated after in vitro culture, and then the CAR-T cells are infused back into the patient for attacking cancer cells. The CAR-T cell therapy in the prior art can produce significant therapeutic effects in hematological tumors, for example, the application of CD 19-targeted CAR-T cell immunotherapy to B-cell derived leukemia or lymphoma has been revolutionarily successful, with even complete cure in some of the most advanced cases. However, there are a number of problems in the treatment of solid tumors with CAR-T therapy, including: it is difficult to find tumor targets as specific as CD19 in treating hematological tumors, off-target effects are likely to occur and bring about side effects; the heterogeneity of solid tumors is liable to lead to conditions such as drug resistance due to antigen loss; the effect of treating the solid tumor is obviously inferior to that of treating the blood tumor. However, if a suitable target can be found, the CAR-T cell therapy may be enabled to significantly improve the effect of treating the corresponding solid tumor (such as lung cancer), thereby providing a new therapeutic direction.

AXL was originally found in chronic myelogenous leukemia patients, and as with the other two kinases Tyros and MERs, AXL belongs to the TAM (Tyros, AXL, MER) RTK (receptor tyrosine kinase) family. Research shows that the AXL is highly expressed in lung cancer and plays an important role in the occurrence and development of lung cancer and in drug resistance. For example, the positive expression rate of AXL was 59.8% in 112 lung cancer tissues detected by immunohistochemistry by Japanese scholars Iida and the like. AXL may be able to serve as a therapeutic target for lung cancer or may provide a stable target cell surface antigen for immunotherapy, thereby providing a new lung cancer therapeutic strategy or drug in combination with CAR-T cell immunotherapy to improve lung cancer therapeutic efficacy, improving survival.

Disclosure of Invention

The invention aims to overcome at least one defect of the prior art, provides an AXL-targeted CAR-T cell, a preparation method and application thereof, utilizes an AXL-targeted scFv protein to prepare the AXL-targeted CAR-T cell, and helps to effectively treat lung cancer to improve survival rate of patients by applying the AXL-targeted CAR-T cell to lung cancer, breaks through the bottleneck of poor effect of the prior art CAR-T on treating solid tumors, and provides a new strategy and direction for treating lung cancer.

One of the objects of the present invention is to provide the use of AXL-targeting CAR-T cells for the preparation of a product for the treatment of AXL-expressing lung cancer; the CAR-T cells targeting the AXL can be directly and specifically identified and combined with tumor cells expressing the AXL to be activated, the tumor cells are directly killed by releasing perforin, granzyme B and the like, and meanwhile, the human endogenous immune cells are recruited to kill the tumor cells by releasing cytokines, so that the CAR-T cells targeting the AXL are used for treating tumors, and have good targeting, killing performance and durability. In one embodiment of the invention, AXL-targeted CAR-T cells have a significant cancer inhibiting effect on AXL-expressing lung cancer, a significant killing effect on positive AXL-expressing lung cancer cells, and significant cytokine secretion; the method shows that the CAR-T cells targeting the AXL can have an effective treatment effect on lung cancer, and the CAR-T cells targeting the AXL are applied to the market for preparing products for treating the lung cancer expressing the AXL, so that the immune system of a patient can be utilized to kill tumors, and immune memory T cells can be formed to obtain a specific anti-tumor long-acting mechanism, thereby being beneficial to further improving the treatment effect and the survival rate of the patient.

Further, the lung cancer is EGFR-TKI resistant lung cancer. Some studies have found that in non-small cell lung cancer (NSCLC) with Epidermal Growth Factor Receptor (EGFR) mutations, activation of AXL can lead to acquired resistance of first-generation EGFR-TKI drugs (e.g., erlotinib), and thus treatment targeting AXL may prevent or overcome TKIs resistance in EGFR mutant lung cancer patients. In the embodiment of the invention, the AXL is proved to be more obviously expressed in EGFR-TKI drug-resistant lung cancer besides being highly expressed in lung cancer tissues, and the CAR-T cell targeting the AXL can effectively act on EGFR-TKI drug-resistant lung cancer to play a role in inhibiting cancer; therefore, the CAR-T cells based on the targeted AXL are helpful for providing more remarkable lung cancer treatment effect by combining EGFR-TKI drugs, and the poor treatment effect caused by drug resistance is overcome. Provides a new selection and application strategy for the treatment of EGFR-TKI drug-resistant patients.

Further, the CAR-T cells carry protein sequences and/or proteins that down-regulate AXL expression, and the products include EGFR-TKI drugs. Namely, the CAR-T cells can express proteins which can down regulate the expression of the AXL, or directly carry the proteins which act on the AXL to down regulate the expression, and the drug resistance of EGFR-TKI drug-resistant lung cancer can be reduced by down regulating the expression of the AXL, so that the EGFR-TKI drug can be conveniently combined to realize combined treatment, and the treatment effect is improved. Still further, the EGFR-TKI drug includes Erlotinib.

Further, the lung cancer is non-small cell lung cancer. In the embodiment of the invention, the AXL-CAR T cells have good tumor inhibiting effect on the NSCLC cells positive to the AXL in vivo and in vitro, so that the CAR-T cells targeting the AXL can realize effective treatment on non-small cell cancers.

Further, the AXL-targeting CAR-T cells are third generation CAR-T cells. The third generation CAR-T cells comprising both CD28 and CD137 have better persistence and killing function in vivo compared to the second generation CAR-T cells. It combines the advantages of CD28 and CD137, and enables CAR-T cells to exhibit greater cytotoxicity, persistence, and capacity of memory T cell differentiation.

Further, the AXL-targeting CAR-T cell scFV protein is selected from one or more of 3E8, 20G7D9 or YW327.6S2. In the embodiment of the invention, CAR T cells constructed based on the three scFvs have stronger killing capacity to the NSCLC cells positive to the AXL, the killing efficiency of the three AXL-CAR T cells is increased along with the increase of E:T, and the killing capacity of the three CAR T cells is strongest by YW327.6S2-CAR-T cells. Furthermore, the scFv protein adopts YW327.6S2, which is a fully humanized antibody, and the generation of potential human anti-mouse antibodies of a human body can be reduced by constructing the CAR T by using the antibody, so that the CAR-T cells are prevented from being cleared by the human body.

It is a further object of the present invention to provide a product for the treatment of AXL-expressing lung cancer, characterized in that the product comprises AXL-targeted CAR-T cells and EGFR-TKI drugs as described above.

It is a further object of the present invention to provide a method of preparing AXL-targeted CAR-T cells comprising the steps of:

s1, constructing a lentiviral vector containing a CAR molecular sequence, wherein the CAR molecular sequence comprises an AXL scFv sequence, a Hinge region-finger sequence, a transmembrane region sequence, a costimulatory domain sequence and a CD3 zeta sequence, and the AXL scFv sequence expresses a scFv protein targeting AXL;

s2, carrying out slow virus packaging on the slow virus vector obtained in the step S1 to obtain slow virus containing the slow virus vector;

s3, separating human Peripheral Blood Mononuclear Cells (PBMCs), separating T cells from the separated PBMCs, and stimulating the T cells;

s4, infecting the T cells stimulated in the step S3 by using the slow virus obtained in the step S2 to obtain the AXL-targeted CAR-T cells.

The method can quickly and simply construct the CAR-T cell targeting the AXL, is convenient for practical production to prepare the medicine for treating the lung cancer based on the CAR-T cell targeting the AXL, thereby improving the lung cancer treatment effect and providing a new choice for the lung cancer treatment in the prior art.

Further, the scFv sequence targeting AXL is a gene sequence expressing 3E8, YW327.6S2 or 20G7D9 protein.

Further, the Hinge region-Hinge is CD8 Hinge, the transmembrane region is CD28 transmembrane region, and the costimulatory domain is CD28 and/or 4-1BB costimulatory domain.

The invention also aims to provide the CAR-T cell obtained by the method for preparing the CAR-T cell targeting the AXL, and the CAR-T cell of the AXL can specifically bind to a cancer cell which highly expresses the AXL so as to activate the killing property of the cancer cell, inhibit the growth of tumors and achieve the effect of treating tumors. And the drug resistance caused by AXL can be inhibited by targeting on the AXL, so that the effect of tumor treatment is further improved by combining other drugs.

Compared with the prior art, the invention has the beneficial effects that: the CAR-T cell targeting the AXL gene can be constructed, so that the problem that the CAR-T is difficult to find a tumor specific target point in the prior art for treating solid tumors is solved, the occurrence of off-target is avoided, the probability of side effects is reduced, the treatment effect of the solid tumors is improved, the application range of the CAR-T tumor treatment is perfected, and a new strategy and direction are provided for treating cancers. And based on the high expression of the AXL in lung cancer, the method can specifically kill lung cancer tissues positively expressed by the AXL, thereby realizing the treatment of lung cancer and being beneficial to the development of lung cancer treatment technology. Meanwhile, the CAR-T cells targeting the AXL can also act on lung cancer cells resistant to EGFR-TKI, and the combination of the CAR-T cells and the EGFR-TKI is helpful for cooperatively treating lung cancer, so that the lung cancer treatment effect is obviously improved, and the poor treatment effect caused by drug resistance is overcome. The CAR-T cell targeted to the AXL can realize effective lung cancer treatment, improve survival rate of patients and overcome the defects of the prior art therapeutic drugs and techniques.

Drawings

FIG. 1 shows the results of immunohistochemical detection of human normal tissue by AXL according to the embodiment of the invention.

FIG. 2 shows the result of immunohistochemical detection of AXL in lung cancer tissue according to the embodiment of the present invention.

FIG. 3 shows the detection of the AXL expression of lung cancer tissues and paracancerous tissues of EGFR-TKI drug resistant patients in accordance with the embodiment of the present invention.

FIG. 4 shows the expression of WB and flow assay AXL according to the examples of the present invention in lung cancer cell lines A549, HCC827 ER3 and HCC 827.

FIG. 5 shows the results of the verification of affinity of the AXL scFv of the example of the invention.

FIG. 6 is a schematic diagram of the molecular structure of a CAR according to an embodiment of the present invention.

FIG. 7 shows the results of construction of a CAR molecule lentiviral vector of an embodiment of the invention.

FIG. 8 shows the construction of CAR T cells and their identification results according to the examples of the present invention.

FIG. 9 shows the phenotypic analysis results (one) of T cells according to the example of the present invention.

FIG. 10 shows the phenotypic analysis results (II) of T cells according to the example of the present invention.

FIG. 11 shows the phenotypic analysis results (III) of T cells according to the example of the present invention.

FIG. 12 shows the results of phenotyping of T cells according to the example of the Invention (IV).

FIG. 13 shows the construction results of GL cell lines of the present invention.

FIG. 14 shows the results of in vitro killing of example AXL-CAR T, CD19-CAR T and T cells of the invention.

FIG. 15 shows the results of in vitro killing assay cytokine secretion by ELISA method of an embodiment of the invention.

Figure 16 shows the results of a T cell therapy a549 cell subcutaneous tumor model of example YW327.6S2-CAR of the invention.

FIG. 17 shows the results of the example YW327.6S2-CAR T cell therapy of HCC827 ER3 cell subcutaneous tumor model of the present invention.

FIG. 18 shows the results of the example YW327.6S2-CAR T combined with Erlotinib treatment of HCC827 ER3 GL cell subcutaneous tumor model of the present invention.

Detailed Description

The drawings are for illustrative purposes only and are not to be construed as limiting the invention. For better illustration of the following embodiments, some parts of the drawings may be omitted, enlarged or reduced, and do not represent the actual product dimensions; it will be appreciated by those skilled in the art that certain well-known structures in the drawings and descriptions thereof may be omitted.

Example 1

Preparation of subsequent examples off-work (cell culture, cryopreservation and resuscitation)

1. Cell culture

(1) All cells were exposed to 5% CO at 37℃ ₂ Culturing in a constant temperature incubator. (2) HCC827, HCC827 ER3: the culture was performed using 10% RPMI-1640 complete medium. HCC827 cells were passaged 1:2-1:3, and 2-3 days for replacement, passaging, or cryopreservation. HCC827 ER3 cells were passaged 1:2, and 2-3 days with liquid changes, passaged or frozen. (3) a549: the culture was performed using 10% DMEM/F12 complete medium. Passaging is carried out according to the ratio of 1:3-1:6, and liquid exchange, passaging or freezing storage are carried out for 1-2 days. (4) 293T: the culture was performed using 10% dmem complete medium. Passaging is carried out according to the ratio of 1:3-1:6, and liquid exchange, passaging or freezing storage are carried out for 1-2 days. 293T cells are semi-adherent cells, the adherence is not firm, the culture process is not limited to violent blowing or violent shaking, and the digestion time is less than 1 min. (5) 293F: for AXL scFv protein purification. Passaging is carried out according to the ratio of 1:2-1:3, and liquid exchange, passaging or freezing storage are carried out for 2-3 days. 293F is suspension cells, and the suspension cells are subjected to liquid exchange or passage after the liquid exchange method is adopted or all culture media are collected and centrifuged. (6) T cells: t cell culture medium was complete medium (self-addition IL-2) prepared by Methaemamectin benzoate, germany. T cells grow in a suspension holding mode, and liquid exchange or passage adopts a half-quantity liquid exchange method or a centrifugal method for liquid exchange and passage. Because of the limited expansion capacity of T cells, T cells are generally not cultured for more than 3 weeks after sorting.

2. Cell cryopreservation

(1) Cell cryopreservation solution: 90% serum +10% DMSO should be prepared in advance and stored in cold storage at 4deg.C. (2) The newly recovered cells can be frozen after 3-4 passages, and the higher recovery success rate can be ensured by generally selecting the cells with better states for seed preservation. (3) The adherent cells are counted after being digested by pancreatin, 300g and centrifuged for 5 minutes; the suspended cells do not need to be digested, and can be directly counted and centrifuged. In order to ensure the success rate of recovery, the number of the frozen cells is generally more than 100 ten thousand. (4) The supernatant of the centrifuge tube was pipetted off, the cells were not aspirated, 1mL of pre-chilled cell cryopreservation solution was added to resuspend the cells, and the tube was aspirated. (5) The freezing tube is placed in a program cooling freezing box (the freezing box is firstly placed at normal temperature). After the freezing box is placed in a refrigerator at the temperature of minus 80 ℃ for 48 hours, the cells are transferred into liquid nitrogen for long-term storage.

3. Cell resuscitation

(1) The frozen cells were carefully removed from the liquid nitrogen tank and rapidly placed in a 37 ℃ water bath. (2) Rapidly shaking (about 200 times) until completely thawing, preventing ice crystals from damaging the cells. (3) Cells were carefully transferred to a 15mL centrifuge tube, and more than 5 volumes of pre-warmed medium were added prior to centrifugation to reduce cell damage, 300g, and centrifuged for 5 minutes. (4) The supernatant was discarded, resuspended in pre-warmed medium, and the cells were transferred to corresponding flasks or dishes depending on how much cells were. (5) The next day, depending on the cell status, it is determined whether a change of fluid is required or culture is continued.

Example 2

Detecting expression of human normal organs or tissues AXL of lung cancer

In order to verify that AXL is an ideal target for treating lung cancer and to avoid potential "off-target" phenomena, the present example uses immunohistochemical methods to detect AXL expression in 90 lung cancer (including 40 EGFR-TKI resistant patients) tissues and 19 normal organs or tissues of the human body. Meanwhile, the expression condition of the AXL in 8 pairs of EGFR-TKI drug-resistant lung cancer and lung tissues beside the corresponding cancer is detected by using Western-Blot, so that the specific expression relation of the AXL is further obtained.

Specifically, the immunohistochemical operation steps in this embodiment include: (1) baking slices: placing the tissue slices in a 65 ℃ oven to bake the slices for 1-2 hours; (2) dewaxing: placing the grill into xylene for dewaxing for 2 times and 10 minutes each time; (3) hydration: sequentially hydrating in 100%,95%,75% and 50% ethanol for 5 min each; then washed 3 times in TBST for 5 minutes each; (4) antigen retrieval: placing the slice in sodium citrate repairing solution (pH=6.0) for microwave antigen repairing, and cooling for 20-30 minutes at room temperature after repairing; washing 3 times in TBST for 5 minutes each; (5) blocking endoperoxidase activity: the reaction was performed at room temperature using 3% H2O2 for 10 min; washing 3 times in TBST for 5 minutes each; (6) closing: blocking with 5% BSA for 2 hours at room temperature; (7) incubating the primary antibody: the rabbit anti-human AXL primary antibody was incubated at 1:500 at room temperature overnight. Recovering the primary antibody, and then washing 3 times in TBST for 5 minutes each time; (8) incubating a secondary antibody: incubating the secondary antibody solution for 30 min at room temperature, washing 3 times in TBST for 5 min each; (9) DAB color development: incubation with DAB chromogenic chromogen substrate for 3 min or microscopic observation of slice yellowing to terminate development; washing 3 times in TBST for 5 minutes each; (10) hematoxylin counterstain: hematoxylin staining for 2 min, hydrochloric acid alcohol differentiation for 15s, and blue-returning reagent (PBS) staining for 15 min; (11) Dehydrating, sealing, air drying, and observing under microscope.

Wherein, the detection tissues for immunohistochemical detection of normal tissues of human body comprise 19 tissues of liver, spleen, lung, kidney, brain, esophagus, stomach, colon, rectum, appendix, pancreas, gall bladder, tonsil, thyroid, prostate, testis, breast, uterus and skin, and more than 3 tissues are collected for each tissue, and the detection result is shown in figure 1; and a549 was used as AXL positive control and HCC827 was used as negative control (400×), scored using a four-level scoring criteria, the four-level scoring statistics being shown in table 1 below:

TABLE 1 expression of human normal tissue AXL

The results of immunohistochemical detection of normal tissues showed that only 1 colorectal case showed 1+ expression, and none of the other normal tissues showed AXL expression.

In this example, a total of 90 lung cancers were detected by immunohistochemical detection of lung cancer tissues, 40 of which were EGFR-TKI drug resistant patients (400X). The AXL expression was assessed using a four-level scoring criteria, the four-level corresponding immunohistochemical detection results are shown in fig. 2, the four-level scoring statistics are shown in table 2 below, wherein 0 level indicates negative expression and 1 to 3 levels indicate weak to strong expression, respectively.

TABLE 2 fractionation of the expression intensity of AXL in lung cancer tissue

The result of immunohistochemical detection of lung cancer tissues shows that AXL is highly expressed in the lung cancer tissues, and the total expression rate reaches 69%.

Specifically, the invention also adopts Western-Blot to detect the expression condition of the AXL in 8 pairs of EGFR-TKI drug-resistant lung cancer and lung tissues beside the corresponding cancer, wherein the Western-Blot detection steps comprise: (1) extraction of tissue proteins: transporting human lung cancer tissues and tissues beside the lung cancer in an ice box, cleaning the tissues on ice for 3 times by using PBS, cleaning blood, sucking the PBS, cutting mung bean size tissues by using tissue scissors, adding RIPA lysate, and performing ice lysis for 15 minutes. After completion of the lysis, the tissue was thoroughly lysed using an ultrasonic pulverizer. Removing connective tissue, centrifuging, and collecting supernatant; (2) Protein quantification was performed using BCA protein quantification kit and protein concentration was calculated after microplate reader reading. Adding the protein sample volume into 5 XSDS-PAGE protein Loading Buffer (5 XSDS-PAGE Buffer) according to the ratio of 4:1, fully and uniformly mixing, and carrying out metal bath at 95 ℃ for 5-10 minutes to denature the protein, so as to prepare a sample; (3) electrophoresis: the 8% concentration gel was prepared according to the formulation of the Biyun SDS-PAGE gel preparation kit. Loading after solidification, and adding 40 mug protein into each hole; the extra holes are balanced by adding 1×loading Buffer. Adding an electrophoresis solution, and performing constant-pressure electrophoresis at 90V until a bromophenol blue indication line runs to the bottom of the gel; (4) film transfer: preparing membrane liquid in advance, and pre-cooling at 4 ℃ for standby. The cathode carbon plate-sponge-filter paper-gel-PVDF membrane-filter paper-sponge-anode carbon plate are sequentially clamped. Film transfer for 90 minutes with constant current (250 mA) in the ice box; (5) closing: sealing with 5% skimmed milk, and sealing on a shaker at room temperature for 2 hr; (6) incubating the primary antibody: diluting AXL antibody (CST) with primary anti-diluent at a ratio of 1:1000, shaking overnight at 4 ℃; (7) incubating a secondary antibody: after recovering the primary antibody, the primary antibody was washed 3 times for 5 minutes with TBST. Secondary antibodies (rabbit antibodies) were then added. The secondary antibody is prepared by adopting 5% skimmed milk or BSA according to a dilution ratio of 1:5000; (8) protein detection (exposure): the washing was performed 3 times for 5 minutes with TBST. After preparing the hypersensitive luminous solution, developing on a developing instrument; (9) image analysis.

The results of the detection of the AXL expression of lung cancer tissues and other tissues of EGFR-TKI drug resistant patients are shown in fig. 3A (wherein T represents tumor tissues and N represents other tissues), and as can be seen from the figure, the lung cancer tissues of EGFR-TKI drug resistant patients highly express AXL, but other tissues do not substantially express AXL, and the results of the detection of the gray scale analysis of the WB are shown in fig. 3B/C.

The results of the high expression of the tumor tissues and the substantial non-expression of the normal tissues show that the AXL is very likely to be an ideal therapeutic target for lung cancer patients, especially drug resistant patients of lung cancer EGFR-TKI, and the immune therapy targeting the AXL can have good safety and is helpful for providing novel strategies and drugs for treating lung cancer based on the targeting of the AXL.

Example 3

Lung cancer cell line AXL phenotype assay

1. Western-Blot detection of lung cancer cell line AXL expression

A549, HCC827 and HCC827 ER3 cells were cultured in a 10cm dish, and total protein was extracted on ice after overnight serum-free. The subsequent steps are as described above, using the WB detection procedure of example 1.

In this example, human lung adenocarcinoma A549, HCC827 cells were purchased from ATCC in the following examples. HCC827 ER3 was stored by the subject group laboratory and see Activation of the AXL kinase causes resistance to EGFR-targeted therapy in lung cancer, HCC827 ER3 being an Erotinib resistant cell model of HCC 827.

WB detection as shown in fig. 4A, HCC827 cell line did not express AXL. As in the previous studies of the present inventors' task group, the details are described in documents Activation of the AXL kinase causes resistance to EGFR to targeted therapy in lung cancer.

2. Flow cytometry detection of lung cancer cell line AXL phenotype

(1) A549, HCC827 and HCC827 ER3 cells were cultured, 30-50 ten thousand cells were collected each, washed with PBS, and resuspended in 200 μl of PBS containing 2% fbs. (2) mu.L of PE-AXL flow-through monoclonal antibody was added and incubated for 30 min on ice protected from light. (3) The PBS was washed two to three times, resuspended in 200. Mu.L of PBS containing 2% FBS, and prepared for flow-on-machine.

(4) And collecting and analyzing the signals.

FIG. 4B shows the results of a flow assay, with the dotted line showing isotype and the solid line showing lung cancer cells.

Previous studies demonstrated that HCC827 is a lung cancer cell line that does not express AXL, on the basis of which the inventors previously constructed an Erotinib resistant cell model HCC827 ER3 for HCC827 and revealed that upregulation of AXL expression is an important mechanism for its resistance. The Western-Blot of this experiment can repeat the results of the above HCC827 without AXL expression, but HCC827 ER3 with high AXL expression; in addition, the present study also examined AXL expression of lung cancer cell line a549, which showed that a549 also highly expressed AXL (fig. 4A). The flow results showed that AXL reached 97.7% and 94.7% expression in a549 and HCC827 ER3, respectively, while only 1.86% was found in HCC827 (fig. 4B). These results again demonstrate that AXL is highly expressed in a variety of lung cancer cell lines, and therefore AXL can be an ideal target for lung cancer treatment. In subsequent cell and animal experiments, i.e. in the subsequent examples, the study selected a549 and HCC827 ER3 as AXL positive expression cell lines and HCC827 as negative control. To verify the targeting and killing of AXL-CAR T cells.

Example 4

Verification of affinity of three AXL scFv

1. Purification of AXL scFv recombinant proteins

Transfection of AXL scFv expression plasmid into 293F cells (Large Scale culture/1L shake flask)

The expression plasmids pComb3-3E 8, pComb3-YW327.6S2 and pComb3-20G7D9 of the three AXL scFv are successfully constructed, and then plasmid extraction is carried out, and enzyme digestion and gene sequencing identification are carried out. After identification, 293F cells were transfected for expression and purification of the AXL scFv protein. The method comprises the following specific steps: (1) 300mL of medium (containing 0.5X106 293F cells) was added to a 1L flask; (2) After culturing in a constant temperature shaking incubator (37 ℃,120rpm,5% CO 2) for 24 hours, the cells are counted so that the number of cells is controlled to 1X 106. (typically, the number of cells doubles after 24 hours of culture); (3) Mu.g of plasmid filtered with 0.22 μm filter was pipetted into 30mL of PBS and thoroughly mixed, taking care not to vigorously blow or shake, to prevent plasmid DNA fragmentation; (4) 1.2mL of PEI (0.5 mg/mL) after filtration and sterilization is added into the mixed solution of PBS/plasmid, vortex shaking is carried out for 3 seconds to thoroughly mix, and the mixture is kept stand at room temperature for 20 minutes; (5) After 20 minutes the plasmid/PEI mixture was added to 293F cells; (6) After co-transfection, incubation was carried out for 48h at 37℃in a shaking incubator with a constant temperature of 120rpm and a carbon dioxide concentration of 5%; (7) 3000g,5 min centrifugation to harvest whole cell pellet, storage at-80℃or direct purification of protein.

293F cells in this example were given benefit from the university of Australian Zhao Qi for purification of the AXL scFv protein. Expression plasmids for three AXL scFv (pComb 3-3e 8, pComb3-YW327.6S2, pComb3-20G7D 9) were constructed with the teaching task group of university of australia Zhao Qi, all three scFv bearing His and Flag tags to facilitate protein purification.

2. Extraction of AXL scFv purified proteins from 293F cells

The method comprises the following specific steps: (1) Cells were lysed with pre-chilled cell lysate (40 mL lysate/1000 mL medium) on ice; (2) Blowing the cells to allow them to lyse sufficiently, taking care not to swirl or foam; (3) the glass homogenate resuspended cells. The cells were thoroughly lysed by an ultrasonic pulverizer (3 cycles), 30000g, centrifuged at 4℃for 25 min, the pellet was discarded, and the supernatant was left; (4) 1.25mL agarose gel resin containing Flag tag is added to each 1000mL cell culture medium, and the mixture is washed three times by resin balance buffer; (5) Incubating the supernatant obtained in the step (3) with Flag-tagged affinity resin, centrifuging the tube by 50mL, and shaking the tube at 4 ℃ for 30-120 minutes; (6) centrifuging at 4 ℃ for one minute with 3000g, and discarding the supernatant; (7) The affinity resin was washed with 45mL of pre-chilled buffer (100 mM potassium acetate, 50mM Tris pH 7.5,5% glycerol, 0.3% Triton X100), 3000g, centrifuged for one minute and the supernatant discarded; (8) Repeating step (7) with high salt buffer (300 mM potassium acetate, 50mM Tris pH 7.5,5% glycerol), low salt buffer (50 mM potassium acetate, 50mM Tris pH 7.5,5% glycerol) and TEV cleavage buffer (50 mM potassium acetate, 50mM Tris pH 7.5,0.5mM TCEP), respectively; (9) Collecting 10 mu L of affinity resin sample by using non-denaturing gel for dyeing with carbamaze brilliant blue; (10) The affinity resin was resuspended using 8-10mL pre-chilled TEV cleavage buffer and then transferred to a 15mL centrifuge tube; (11) About 40ug of TEV protease (1 mg/mL) was added and the mixture was homogenized; (12) The gas in the 15mL centrifuge tube is changed into 100% N2, so that the oxidation of protein is prevented; (13) shaking table at 4℃overnight; (14) 3000g, centrifuged for 10 minutes, and the supernatant is transferred to an ultracentrifuge filter with an appropriate molecular weight cut-off and concentrated to 500. Mu.L; (15) Using non-denaturing gel, taking 10 μl sample of concentrated protein for coomassie brilliant blue detection, using TEV eluate as control; (16) Using non-denaturing gel, taking 10 μl of resin sample as coomassie brilliant blue detection, and using TEV positive resin as control; (17) The column was run with an equal amount of gel filtration buffer (50 mM potassium acetate, 50mM Tris pH 7.5,0.5mM TCEP); (18) Filtering the protein with a 0.22 mu m filter membrane, loading the sample to a chromatographic column, collecting the sample, and detecting whether the target protein is collected by dyeing with the Brilliant blue of Lecoomassie; (19) Sampling from the steps (9), (15), (16) and (18) to perform the examination. And the examination of steps (9), (15) and (16) is needed before gel filtration to ensure the expression of the target protein.

2. Affinity of indirect ELISA for detecting AXL scFv

After protein collection was completed, the affinity of the AXL three scFv binding to AXL antigen was detected by indirect ELISA. The method comprises the following specific steps: (1) coating AXL antigen: 100ng/well (control group is needed to be arranged, each group comprises 2-3 compound holes), 100 mu L of volume is added to each hole, and the temperature is 4 ℃ overnight; (2) Discarding the coating liquid, washing 3 times with TBST, sealing with 5% skimmed milk (filling up each hole), and standing at room temperature for 1 hr; (3) Discarding the blocking solution, washing 3 times by TBST, respectively adding three AXL scFv to be detected with different concentrations and a control antibody of 100 mu L/well, and carrying out room temperature for 1h; (4) Discarding liquid, washing with TBST for 3 times, adding corresponding enzyme-labeled secondary antibody 100 μl/well, covering in dark place, and incubating at 37deg.C for 1 hr; (5) Discarding liquid, washing 3 times by TBST, adding 100 mu L/well TMB color development liquid, and developing color in dark place for 5-10 minutes; (6) Adding stop solution 50 mu L/well, measuring the absorbance value of each hole at 450nm by an enzyme-labeled instrument, and drawing a curve.

scFv is a structure belonging to the extracellular domain of a CAR molecule, whose affinity has a decisive role for CAR T cells to recognize tumor antigens. Thus, to screen for optimized scFv, this example demonstrates the affinities of three AXL scFv. The amino acid or nucleotide sequences of the three scfvs are all from patents, and the affinities of the three scfvs are respectively: 3E (E) 3E8(1.6×10 ^-9 M)，YW327.6S2(1×10 ^-9 M)，20G7D9(5.3×10 ^-8 M)。

To verify its affinity, this example constructed three expression vectors for AXL scFv: pComb3-3E 8, pComb3-YW327.6S2, pComb3-20G7D9, results of digestion and sequencing showed successful vector construction, as shown in FIG. 5A. These three scfvs were then expressed and purified using 293F cells and their affinities were confirmed by ELISA from strong to weak as YW327.6S2,3E 8 and 20G7D9, respectively, as shown in fig. 5B, which matches the known affinity trends of the patent (see example 5). Thus, the present study constructed 3E3E8-CAR-T, YW327.6S2-CAR-T,20G7D9-CAR-T cells with these three scFvs, respectively, in the cell killing experiments of the subsequent examples, and explored whether scFvs of different affinities had an effect on the killing efficiency of the CAR-T cells. Specifically, the agarose gel electrophoresis result of FIG. 5A shows that the expression vectors of pComb3-3E 8, pComb3-YW327.6S2 and pComb3-20G7D9 are successfully constructed; FIG. 5B shows the results of an indirect ELISA assay for the binding capacity of three scFvs to the AXL antigen. The results of this example were obtained from three independent experiments, and the data were expressed as mean ± standard error.

Example 5

Construction of AXL-CAR T cells

1. Construction of three pWPXLd-CAR-AXL-GFP lentiviral vectors

The three AXL scFv sequences were from patents, with the patent numbers: WO2012/175692A1 (3E 8), US8853369B2 (YW327.6S2), US9249228B2 (20G 7D 9). The method comprises the following specific steps:

(1) Designing a CAR molecule gene sequence: including the AXL scFv sequence, CD8 hinge, CD28 transmembrane region, CD28 and CD137 costimulatory domain, cd3ζ intracellular domain. Respectively adding enzyme cutting sites PmeI and SpeI before and after so as to facilitate the enzyme cutting of the cloning vector;

(2) The corresponding CAR molecule sequences were synthesized by the company nanjing, and cloned into pUC57 vectors (cloning plasmids pUC57-3E 8, pUC57-YW327.6S2, pUC57-20G7D9 for three AXL scFv, synthesized by the company nanjing), respectively, to obtain: pUC57-3E3E8-CAR, pUC57-YW327.6S2-CAR and pUC57-20G7D9-CAR; pUC57-CD19-CAR cloning vector was also synthesized as experimental control in this example;

(3) Double-enzyme cutting pUC57-3E3E8-CAR, pUC57-YW327.6S2-CAR and pUC57-20G7D9-CAR by using PmeI and SpeI respectively, cutting and recovering the AXL CAR molecular gene fragments, and simultaneously double-enzyme cutting pWPXLd-GFP framework plasmids by using PmeI and SpeI to recover a large fragment framework; (pWPXLd-GFP was offered by the Li Peng professor group, and contained GFP and plasmid backbone, used to clone the CAR molecule lentiviral vector, GFP could be used as a marker for subsequent detection of lentivirus packaging success and T cell positive rate of transfection.)

(4) Three AXL CARs were cloned into a ppplxld-GFP backbone plasmid, respectively, using T4 ligase at 16 ℃ for 1 hour, constructed as a ppplxld-3E 8-CAR-GFP, a ppplxld-YW327.6S2-CAR-GFP and a ppplxld-20G 7D9-CAR-GFP lentiviral vector, respectively;

(5) Transforming TOP10 competent cells; in this example competent cell TOP10, purchased from Nanjinopran, inc., was used for plasmid extraction and vector construction.

(6) 3-5 monoclone are selected, and the fungus is shaken overnight;

(7) Extracting plasmid with plasmid small extraction kit, including the steps of: a) The bacterial liquid was centrifuged (3500 g,10 minutes) using a centrifuge; b) The old culture broth was pipetted off and 500. Mu.L of Solution I/RNase A (lysis, 4 ℃) was added to the culture tube. Resuspension of cells; c) The cell suspension was transferred to a new 2mL microcentrifuge tube. Add 500. Mu.L Solution II (release ribozyme) and gently mix back and forth (7-10 times) to thoroughly lyse. The mixture was left at room temperature for 2 minutes. Severe vibration was avoided as this would lead to disruption of chromosomal DNA and reduced plasmid purity (Solution II should be sealed); d) mu.L of frozen Buffer N3 was added to the cell suspension and gently and thoroughly shaken until white flocculent precipitate appeared. Centrifugation at room temperature (preferably 4 ℃) is not less than 12000g for 10 minutes. And (5) warm prompting: the Buffers must be thoroughly shaken. If sticky, brown and spherical precipitates appear in the shaking process, further mixing and neutralization are needed, and the mixing is a key for obtaining a better result; e) The supernatant was carefully transferred to a 1.5mL centrifuge tube. An equal amount of ETR Binding Buffer was added to the lysate. Shaking the centrifuge tube back and forth for 7-10 times; f) 700. Mu.L of the above mixture was added to a clean 2mL HiBindTM collection tube. 10000g at room temperature, for 1 minute. The solution was filtered with Column. Discarding the flow-through and reusing the collection tube; g) Repeating step 6 until all the clear lysate passes through the microtubes; h) Adding 500 mu L ETR Wash Buffer I to mini column,10000g, centrifuging for 1 min, discarding flow-through, and recycling collection tube; i) Add 500. Mu.L Buffer EHB to mini column.10000g, centrifuged for 1 min. Discarding flow-through, and recycling collection tube; j) Ethanol was diluted by adding 700 mu L DNA Wash Buffer. 10000g, centrifuged for 1 min. Discarding flow-through, and recycling collection tube; k) Adding 700 mu L DNA Wash Buffer, and repeating the step 10 once; l) discard the liquid in the centrifuge tube, centrifuge the empty column for 3 minutes at maximum speed (. Gtoreq.13000 g) and dry column Matrix. This step is critical to eliminating ethanol within column; m) column was placed in a new 1.5mL microcentrifuge tube. 60-100. Mu.L (depending on the target final concentration) of Endotoxin-Free Elution Buffer was added to the column matrix and left standing at room temperature for 2 minutes. The DNA was eluted by centrifugation at 13000g for 1 min. At this time, about 70-85% of DNA remains, and almost most of DNA can be eluted by washing again; n) measuring plasmid concentration and purity;

(8) The extracted plasmid was identified by digestion with PmeI and SpeI and the correct clone was stored in a-80℃freezer with glycerol. In order to ensure the correctness of the cloned sequence, the CAR molecular vector sequence is identified by line gene sequencing;

(9) The correct pWPXLd-3E3E8-CAR-GFP, pWPXLd-YW327.6S2-CAR-GFP, pWPXLd-20G7D9-CAR-GFP and pWPXLd-CD19-CAR-GFP lentiviral vector plasmids were then subjected to a large extraction using a plasmid large extraction kit, while lentiviral-packaged helper plasmids (pSPAX 2 and pMD2. G) were extracted in large quantities. Re-sequencing ensures the correctness of each gene sequence. Preserving at-20 ℃ for subsequent lentiviral packaging.

To construct AXL-CAR-T cells, this example first constructed three CAR molecule lentiviral vectors comprising AXL scFv (3e3e8, yw327.6s2, 20G7D 9) and CD19-CAR molecule lentiviral vectors (for experimental control); in this example, the schematic structure of the CAR molecule is shown in fig. 6, VH and VL are scFv heavy and light chains, TM is the transmembrane region, CD28, 4-1BB and CD3 are the intracellular regions, and eGFP is an enhanced green fluorescent protein. The cleavage and sequencing results of fig. 7 indicate that the CAR molecule lentiviral vector was successfully constructed.

2. CAR molecule lentiviral packaging

The method comprises the following steps: (1) Lentiviral packaging was performed using 293T cells (purchased from ATCC): 293T cells were seeded onto 10cm dishes at a density of about 40-50% on the first day, ensuring that the cell density was as high as about 80-90% after overnight; (2) The medium was removed and starved with DMEM medium containing 1% fbs for 2 hours; (3) The plasmid of interest, pSPAX2 and pMD2.G, were added in a 3:4:1 ratio to a 15mL centrifuge tube containing Opti-MEM medium, labeled A; adding PEI which absorbs three times of the total plasmid amount into another 15mL centrifuge tube, and marking as B; after standing for 5 minutes, uniformly mixing the A and the B, and standing for 20 minutes at room temperature; (4) Adding the A+B mixed solution into 293T cells, adding 1mL of the mixed solution into each dish, and infecting the mixed solution for 6 to 10 hours at a temperature of 37 ℃; (5) Removing the A+B mixed solution after infection, adding a DMEM fresh medium containing 1% FBS, and continuing to culture; (6) the infection efficiency can be observed under a fluorescence microscope; (7) The virus supernatant was collected once every 48 and 72 hours and filtered through a 0.22 μm filter to directly infect the target cells. Can be stored at 4deg.C for one week. The long-term preservation is carried out at-80 ℃;

3. Lentivirus titer measurement

The method comprises the following steps: (1) On day 1, 96-well plates were plated with 293T cells, 5000-10000 cells per well, 100. Mu.L/well. Culturing in a cell incubator to enable the cells to grow to 30-50% of density in the next day; (2) On day 2, a multiple dilution of slow virus solution was prepared with 10 EP tubes. Add 90. Mu.L of 10% DMEM medium per tube, 10. Mu.L of lentivirus stock solution to be tested in the first tube, 10. Mu.L from the first tube into the second tube, and so on until the last tube, the first tube has 10 ^-3 The final tube had 10 of mL virus stock ^-12 mL virus stock; (3) Sequentially adding the 10 dilution gradient virus dilutions (90 mu L) into a 96-well plate for slow virus infection; (4) Day 3 (or 12-24 hours after infection), determining whether to change the liquid according to the cell state; (5) On day 4, the number of fluorescent cells in each well was observed under a fluorescent microscope, and the viral titer was the number of cells expressing fluorescence divided by the corresponding dilution. For example, expression was observed in the fifth wellThe number of GFP cells was 10, and the corresponding viral titer was 10 TU/(10) ^-7 mL)＝1×10 ⁸ /mL; (6) Repeated freeze thawing reduces virus titer (each freeze thawing reduces virus titer by 10% -50%); the repeated freezing and thawing should be avoided as much as possible during the use of the virus, and the virus can be packaged (200 mu L/tube) and directly placed at-80deg.C for preservation; (8) If the virus is stored for more than 6 months, the virus titer is re-determined before use.

After the lentiviral vector was successfully constructed, the present example packaged lentiviruses with 293T cells and tested for lentiviral titres. Four lentiviral titers are shown in table 3 below, TU: viral titer units.

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Table 3CAR molecule lentiviral titres

4. Sorting and stimulation of human Peripheral Blood Mononuclear Cells (PBMCs) (exemplified by Meitian and Gentle yang selection kit)

The method comprises the following steps: (1) Mixing lymphocyte separating liquid and peripheral blood according to a ratio of 1:1, taking attention that the separating liquid is firstly added, then dropwise and slowly adding blood, wherein the upper layer of blood can be seen, the separating liquid is arranged at the lower layer, and the boundary between the separating liquid and the peripheral blood is obvious; (2) Then it was centrifuged (raise 4 to 0, 25 ℃,800g,25-30 minutes) with a flat angle centrifuge; (3) The inside of the tube is divided into three layers after centrifugation, the upper layer is blood plasma and PBS, the lower layer is mainly red blood cells and granulocytes, the middle layer is Ficoll, a white cloud layer narrow band (white film layer) is arranged at the interface between the upper layer and the middle layer, and the white film layer is carefully sucked into a 15mL centrifuge tube; (4) Gently beating the cells into single cell suspension with 10mL PBS, and filtering the cells with a 30 μm filter membrane to prevent blood clots from blocking the sorting column, 300g, for 10 minutes; (5) Discarding the supernatant, adding 4-6mL of erythrocyte lysate to resuspend, and lysing the erythrocytes (5 minutes); (6) washing the erythrocyte lysate by adding 10mL of PBS, and centrifuging. If the lysis is incomplete, adding red blood cell lysate again for lysis; (7) Discard supernatant PBMC were resuspended in 10mL MACS PBS and 10. Mu.L of the cell suspension was counted. 500g,5 min centrifugation of cells; (8) The supernatant was discarded, and Methaemaphyla-and-Ishizome MACS buffer resuspended cells (40. Mu.L buffer/10) ⁷ Cells), adding biotin-labeled CD3 magnetic bead antibody (20. Mu.L/10) ⁷ Cells). Incubate on ice or in a refrigerator at 4℃for 15 minutes. Vortex shaking once every 1-2 minutes; (9) Meanwhile, installing and preprocessing a Meitian and gentle sorting column, and adding a MACS buffer to enable the MACS buffer to flow out under the action of gravity; (10) After the cell incubation was completed, it was resuspended with MACS buffer, 500g,5 min; (11) The MACS buffer was removed, resuspended with 3-5mL MACS buffer again, and the cells were added to the separation column to drain off under the force of gravity and the column-passed liquid was discarded. The kit is a positive selection kit, so that T cells can remain on the tube wall; (12) After MACS buffer is drained, adding 5mL of MACS buffer, eluting T cells in the column; (13) repeating the previous step; (14) Centrifuging the T cells obtained by separation, washing once with PBS, and counting; (15) Every 500 ten thousand T cells were activated with 25. Mu.L of a merday gentle CD3/CD28 magnetic bead antibody. The T cell culture medium is added to inoculate a 48-well plate or a T25 culture flask for culture. Costimulation for 24-48 hours; (16) A small amount of T cells were taken for flow detection of CD3/4/8 phenotype.

Peripheral blood source in this example: the aseptic blood collection room collects peripheral blood of healthy donors of the inventor and members of the same experimental group, about 10-20mL each time, and the peripheral blood is collected in a blood collection tube or a blood collection bag for storage at 4 ℃. And Mononuclear Cells (PBMCs) were isolated as soon as possible after completion of blood collection.

5. Lentiviral infection of T cells

The method comprises the following steps: (1) On day 1, after T cells are stimulated, the magnetic bead antibodies and the old culture medium are removed by centrifugation, and the count is carried out; (2) Calculating the required lentivirus amount according to the number of T cells and the infection complex number (MOI value), adding 6-8 mug of a transfection-assisting reagent Polybrene into each mL of culture medium, and carrying out infection for 12-24 hours; (3) On day 2, after T cell infection, removing virus infection liquid, and continuously adding a T cell culture medium for culture; (4) On days 3-5, infection can be observed under a fluorescence microscope, and the transfection efficiency can be determined by detecting GFP positive rate through a flow; (5) CD19-CAR-T cells were constructed as experimental controls in the same manner as described above.

After CAR molecule lentiviruses are packaged, CAR molecule lentiviruses of T cells are subsequently infected. The expression of green fluorescence of T cells was seen under a fluorescence microscope 3-5 days after infection. To further clarify the expression of CAR molecules, a series of experiments were performed in this study.

After CAR molecule lentiviruses are packaged, CAR molecule lentiviruses of T cells are subsequently infected. The expression of green fluorescence of T cells was seen under a fluorescence microscope 3-5 days after infection.

6. Detection of CAR molecule expression

1. Extraction of CAR-T cell RNA

The method comprises the following steps:

(1) Extracting total RNA of three types of AXL-CAR T cells, CD19-CAR T cells and fresh T cells by adopting a TRIZOL method;

(2) According toInstructions for Q RT SuperMix for qPCR (+gDNA wind) reverse transcription kit reverse the total RNA extracted into cDNA.

a) The DNA in the total RNA is removed, and the reaction system is as follows:

b) After mixing and centrifuging, the mixture was reacted at 42℃for 2 minutes.

c) Reverse transcription, the system is as follows:

d) The reaction procedure: 15 minutes at 50℃to 2 minutes at 85 ℃.

7. qPCR detection of expression of CAR molecules

To further quantify the expression of the CAR molecule, the present example follows the cDNA inverted in step six qPCR/>qPCR experiments were performed using the instructions provided in the Green Master Mix (High ROX Premixed) kit.

(1) Primer synthesis: the primers used in the qPCR reaction of the experiment are designed and synthesized by Shanghai JieRui bioengineering Limited company, and the relative expression quantity of all target genes takes GAPDH as an internal reference gene. The primer sequences are shown in Table 4:

TABLE 4qPCR primer sequences

(2) The qPCR reaction system is shown below:

(3) The qPCR reaction conditions are as follows:

(4) Fluorescence signals were collected using an ABI Quantum studio 7Flex reactor to obtain corresponding Ct values, and all genes of interest were GAPDH as reference gene according to 2 ^-△△Ct The relative expression level of the related gene mRNA was calculated by the method.

(5) Statistical analysis was performed using SPSS 22.0 statistical software, P <0.05 indicating that the difference was statistically significant.

First, total RNA of CAR T cells was extracted, and qPCR experiments were performed after reverse transcription. qPCR results showed that the relative amount of CAR molecules expressed by CAR T cell groups was significantly higher than that of T cell groups, and the results were statistically different, as shown in fig. 8A.

8. Western-Blot detection of expression of CAR molecules

(1) Three AXL-CAR T cells, CD19-CAR T cells and fresh T cell protein samples were extracted on ice after overnight serum-free. Protein quantification was performed using BCA protein quantification kit and protein concentration was calculated after microplate reader reading. The protein sample volume is added into 5 XSDS-PAGE protein Loading Buffer (5 XSDS Buffer) according to the ratio of 4:1, and the mixture is fully mixed, and the protein is denatured by a metal bath at 95 ℃ for 5-10 minutes, so as to prepare a sample. (2) electrophoresis: 12% concentration gel was prepared according to the Biyun SDS-PAGE gel formulation. After clotting, 40ug protein was added to each well. The extra holes are balanced by adding 1×loading Buffer. And adding an electrophoresis solution, and carrying out constant-pressure electrophoresis at 90V until a bromophenol blue indication line runs to the bottom of the gel. (3) film transfer: preparing membrane liquid in advance, and pre-cooling at 4 ℃ for standby. The cathode carbon plate-sponge-filter paper-gel-PVDF membrane-filter paper-sponge-anode carbon plate are sequentially clamped. Film transfer was performed for 90 minutes with constant current (200 mA) in the ice box. (4) closing: the block was performed with 5% skimmed milk and on a shaker at room temperature for 2 hours. (5) incubating the primary antibody: the CD3 antibody was diluted with primary anti-dilution at a ratio of 1:500 and shaken overnight at 4 ℃. (6) incubating the secondary antibody: after recovering the primary antibody, the primary antibody was washed 3 times for 5 minutes with TBST. Secondary antibodies (rabbit antibodies) were then added. The secondary antibody is prepared by 5% skimmed milk according to a dilution ratio of 1:4000. (7) protein detection (exposure): the washing was performed 3 times for 5 minutes with TBST. After the prepared hypersensitive luminous solution (1:1 preparation), developing on a developing instrument. And (8) image analysis.

In this example, the total protein of CAR-T cells and uninfected T cells (control) was also extracted, and Western-Blot detected the expression of intrinsic endogenous CD3 protein in T cells and CAR-T cells, while CAR-T cells detected the expression of the introduced exogenous CD3 gene in addition to endogenous CD3 protein. Indicating successful introduction of the CAR molecule into T cells, as shown in fig. 8B.

9. Detection of expression of CAR molecules by flow cytometry

(1) AXL-CAR T, CD19-CAR T and fresh T cells were collected by centrifugation. (2) The cells were washed twice with PBS containing 2% FBS, and finally resuspended with 200. Mu.L of PBS containing 2% FBS, and prepared for flow-on-machine. (3) And detecting the GFP positive rate of the cells by using a flow cytometer, wherein the GFP positive rate is the infection efficiency of the CAR molecule lentivirus.

In this example, the expression of CAR molecules was also examined by flow cytometry, and the results are shown in fig. 8C (data are expressed as mean ± standard deviation), and the positive rates of CAR-T cells at total T cells are respectively: 3E3E 8-CAR-T25.3%, YW327.6S2-CAR-T29.1%, 20G7D 9-CAR-T30.5%, CD 19-CAR-T35.8%.

The above results all demonstrate that the study successfully constructed CAR T cells.

10. CAR T cell phenotype detection

The CAR-T cells were subjected to flow tests on the T cell phenotype on day 0 and 14 after activation, respectively, after successful construction to determine activation and differentiation.

(1) Fresh T cells sorted on day 0 were collected separately and AXL-CAR T cells were cultured for 14 days after activation. (2) Different combinations of flow monoclonal antibodies or nonspecific isotype monoclonal antibodies (1 μl) such as CD3, CD4, CD8, CD45RO, CD62L, PD-1, CTLA-4, TIM-3 were added respectively and incubated for 30 min on ice or at 4 ℃ protected from light. (3) The PBS was washed two to three times, resuspended in 200. Mu.L of PBS containing 2% FBS, and then prepared for flow-on-machine. (4) Setting corresponding fluorescence channels, gating by taking non-specific homotype monoclonal antibodies as a control group, and detecting the fluorescent expression condition of cells of an experimental group.

After T cells were sorted from peripheral blood of three healthy volunteers, the study performed a flow-through identification of T cell phenotypes such as CD3, CD4, CD8, etc. The results showed that three healthy donors had CD3 positives of 96.8%,98.7% and 99.2%, respectively, which could be used for CAR-T construction, as shown in figure 9.

The memory cell phenotypes CD45RO and CD62L were significantly up-regulated for both unmodified and modified CAR T cells, suggesting that CAR T cells differentiated towards memory T cells, and that CAR T cells were not significantly different from unmodified T cells after modification, as shown in figures 10, 11. More importantly, the inhibitory related molecules PD-1, ctla-4 and TIM3 were upregulated with increasing in vitro culture time of T cells, suggesting T cell depletion, as shown in fig. 12.

Fig. 9: flow-testing fresh T cell CD3/4/8 phenotype; fig. 10 and 11: detecting CD45RO and CD62L phenotype changes of uninfected T cells and CAR T cells on day 0 and day 14, respectively; fig. 12: phenotypic changes of TIM-3, ctla4 and PD1 on day 0 and day 14 were examined for uninfected T cells and CAR T cells, respectively. The constructed CAR T cells can be activated and proliferated in a large quantity, and can show migration, cell killing toxicity, memory and failure cell phenotype.

Example 6

In vitro killing experiments of AXL-CAR-T cells

1. Establishment of GFP and Luciferase double positive (GL) cell lines

(1) A549, HCC827 ER3 were plated on 6-well plates on the first day, with specific plating densities up to about 50% on the second day.

(2) The next day, the amount of virus required was calculated from (cell MOI value X number of cells)/virus titer, and LV5-LUC-GFP-Puro lentivirus and Polybrene (6-8 ug/mL medium) were added. The lentiviral plasmid containing Luciferase (LV 5-LUC-GFP-Puro) in this example was purchased from Shanghai Ji Ma pharmaceutical technologies Inc., and contains Luciferase and GFP to construct a Luciferase-containing lung cancer cell line, and Puromycin was used to screen positively infected cells.

(3) On the third day, it is determined whether to change the liquid and continue the infection according to the cell state, and the infection time is generally not more than 24 hours.

(4) After infection, puromycin (2 ug/medium) was used for 7-14 days.

(5) After screening, GFP positive rate can be detected in a flow mode, if the positive rate is low or the infection intensity is uneven, the infection can be carried out again or flow sorting can be carried out (GFP is used as a sorting index).

(5) After GL cells are successfully constructed, in vivo and in vitro experiments can be directly carried out.

In order to perform in vitro killing experiments by using a Luciferase method, a lung cancer GL cell line expressing GFP and Luciferase (Luciferase) is constructed in the embodiment, and flow results show that GL expression ratios of three lung cancer cells are all above 97.9%, as shown in FIG. 13, GFP is a detection marker. Luciferases can oxidize sodium salts of the substrate fluorescein, which can fluoresce during oxidation. Only the living cell luciferase can oxidize the luciferin, and the ratio of living cells can be approximately reflected by the fluorescence intensity, so that the killing ratio of the cell experiment can be calculated.

2. In vitro killing efficiency of detecting AXL-CAR T cells by luciferase method

(1) AXL-, CD19-CAR T cells and control T cells are prepared, and the actual CAR T cell number of each batch of CAR T cells is calculated according to the GFP positive rate of flow detection, so that effector cells needing to be added are obtained.

(2) According to the pre-planned effective target ratio (E: T), effector cells (CAR T cells or T cells) and target cells (GL tumor cells) are sequentially added, for example 10000 target cells, the effective target ratio is 8:1,4:1 …, then the effector cells need to be added, 80000 and 40000 … are pushed in this way, the above cells are paved into a 96-well plate with a round bottom and no light transmission, and three compound wells are arranged.

(3) The co-cultured cells were placed in a cell incubator for 24 hours.

(4) After the co-culture is completed, 100 mu L of supernatant is gently sucked into a new 96-well plate by a row gun, and the detection of cytokines can be realized.

(5) Sodium fluorescein salt was added to the 96-well plate, 100. Mu.L of Luciferin substrate solution (150 ug/mL) was added to each well, and after 5-10 minutes of reaction, fluorescence was measured in a fluorometer.

(6) Calculating killing efficiency: the ratio of decrease in fluorescence values was determined as percent killing = (control Kong Yingguang value-target Kong Yingguang value)/control wells×100% (fluorescence values of wells without GL target cells are negligible) with wells without T cells as control wells.

To verify the targeting of AXL-CAR T cells, this example groups 3E8-, YW327.6S2-, 20G7D9-CAR T with CD19-CAR T and uninfected T cells, respectively, for their ability to kill target cells. The present example continues to employ different potency target ratios (8:1, 4:1,2:1, 1:1) and killing experiments were performed with A549, HCC827 ER3, and HCC827 as target cells. The result shows that YW327.6S2-CAR T has obvious killing effect on AXL positive expressed A549 and HCC827 ER3, and the killing effect is obviously increased along with the increase of the effective target ratio; YW327.6S2-CAR T, however, had substantially no killing effect on AXL negative-expressing HCC827 cells. Furthermore, both CD 19-targeted CAR T cells and uninfected T cells had no killing effect on the target cells, as shown in fig. 14. These results demonstrate that three AXL-CAR T have high specificity (targeting) and strong killing capacity for AXL-positive expressing lung cancer cells, with the CAR T cells constructed with YW327.6S2 as scFv having the strongest killing capacity in vitro and statistically different killing ratios than the other two AXL-CAR-T cells. This is probably due to the strongest affinity of YW327.6S2 for AXL among the three scFv, which is consistent with the trend of affinity of the three scFv for AXL as verified by ELISA in this study. And considering YW327.6S2 to be a fully humanized antibody, it may be less immunogenic in human applications, avoiding the generation of human anti-murine antibodies, thereby reducing immune rejection of CAR T cells by the host. Therefore, CAR T (YW327.6S2-CAR T) cells constructed with YW327.6S2 as scFv were used as effector cells in the subsequent examples for further cell killing and in vivo experiments.

The results were obtained from three independent experiments, where data are expressed as mean ± standard deviation, p <0.05, p <0.01, p <0.001.

3. Detection of cytokines by enzyme-linked immunosorbent assay (ELISA)

(1) At the end of the killing experiment, supernatants from wells 1:1 at E: T in 96-well plates were collected and immediately detected or stored at-20 ℃. (2) centrifuging the supernatant, 1000g,15 minutes. (3) Wash Buffer, substrate Solution and cytokine standards were prepared at room temperature according to the instructions prior to the experiment. (4) Excess microporous strips are removed from the panel frame, placed back into the aluminum foil bag containing the desiccant package, and then resealed. (5) 100. Mu.L of the diluting reagent RD1W was added to the detection well. (6) 100 μl of standard, control or sample was added to each well and covered against light. Incubate for 2 hours at room temperature. The standard and the sample wells measured are labeled. (7) The liquid in each well was aspirated and 3 washes were performed using wash buffer (400. Mu.L/well). Thorough removal of the liquid in each step is critical to achieving good detection results. After the last wash, all remaining wash buffer in the wells is removed by aspiration or decanting. The plate was inverted and blotted dry with a clean paper towel. (8) 200 μl of the corresponding human cytokine conjugate was added to each well. Covering from light. Incubate for 2 hours at room temperature. (9) washing the well plate according to the step (7) after the incubation is finished. (10) 200 mu L Substrate Solution was added to each well and incubated at room temperature for 20 minutes in the absence of light. (11) 50. Mu.L of Stop Solution was added to each well, and the color in the well after addition should change from blue to yellow. If the color in the wells is green or the color change is not uniform, the plate is gently tapped to ensure adequate mixing. (12) The OD of each well was read by a microplate reader at a wavelength of 450nm over 30 minutes. If wavelength correction is possible, the wavelength is set to 540nm or 570nm for correction. If wavelength correction is not possible, the reading at 450nm is subtracted by the reading at 540nm or 570 nm. This method can correct optical defects in the board. The read value at the wavelength of 450nm directly without correction may be so high that the accuracy is lower. (13) And (5) making a standard curve according to the OD value, and calculating the concentration of the sample. If the sample is diluted, the final result is multiplied by the dilution factor.

T cells release a large amount of cytokines during the process of killing tumor cells. To further verify the killing function of T cells, this study examined cytokine secretion when T cells were co-cultured with target cells. Three effector cells, YW327.6S2-CAR-T, CD19-CAR T and T cells, were co-cultured with three target cells, A549, HCC827 ER3 and HCC827, respectively, at a target ratio of 1:1 for 24 hours, and the supernatants were assayed for cytokine secretion using ELISA kits. The results showed that the secretion of six cytokines IL-2, TNF- α, IFN- γ, GM-CSF, perforin and Granzyme B was significantly increased and the differences were statistically significant when the YW327.6S2-CAR T was co-cultured with the AXL-positive expression cell line A549, HCC827 ER3, as compared to the AXL-negative expression cell line. In addition, neither CD19-CAR T nor T cells seen significant cytokine secretion after co-culture with target cells, as shown in figure 15. The above results demonstrate from the side the specific recognition and killing ability of YW327.6S2-CAR T on AXL positive target cells.

The results were obtained in three independent experiments, p <0.05, < p <0.01, < p <0.001 in fig. 15.

In the above examples, the experimental results are expressed as mean.+ -. SD or SEM. Group differences were detected by one-way ANOVA method. The non-normal distribution data was examined using Kruskal-Wallis. All statistical analyses were performed using Graph-pad Prism 7.0. * p <0.05, p <0.01 and p <0.001 are considered statistically significant.

Example 7

In vivo killing experiments of YW327.6S2-CAR T cells on non-small cell lung cancer

The experimental mice were female 3-4 week NSG mice purchased from Beijing Bai Osai Corp (NOD-Prkdc) ^scid IL2rg ^tm1 Bcgen, BIOCYTOGEN), which is a three-line knockout mouse, mature T/B/NK cells are not detected in vivo, and thus there is no interference of endogenous T/B/NK cells on CAR T cells. All mice were kept in an SPF environment, provided autoclaved food and water for free feeding, 12 hours each for light and dark times each day.

The experimental materials used in this example include: 4% paraformaldehyde fixative (Nanjinouzan Biotechnology Co., ltd.), ki-67 monoclonal antibody (US CST Co.), small animal living body optical imaging system (Perkinelmer IVIS, USA), dissecting instrument (Shanghai Jade research science instruments Co., ltd.), vernier caliper (Beijing space peak optoelectronics Co., ltd.), the rest of the required experimental reagents, instruments and consumables are as described in the above examples.

1. Construction of NSCLC subcutaneous tumor model and tumor biological monitoring

(1) Culture of NSCLC cells positive for both AXL-expressed and HCC827 ER3, 2X 10 cells were prepared per NSG mouse (6-8 weeks) ⁶ And (3) tumor cells.

(2) The two cells, A549 and HCC827 ER3, were digested with 0.25% pancreatin every 2X 10 ⁶ Individual tumor cells were resuspended in 100 μl PBS and prepared for inoculation.

(3) After disinfecting the skin in the groin of the left side of the mice, tumor cells were aspirated with a 1mL syringe, gently lifted up to the skin, and inoculated, and local skin doming was seen as a small packet. Care should be taken not to inject into the abdominal cavity or muscle to avoid tumor adhesion. A549 and HCC827 ER3 independent packets were tested and divided into: YW327.6S2-CAR T cell group, CD19-CAR T cell group and uninfected T cell group, 6 mice per group.

(4) The body weight change of the mice was measured every three days, and the growth of the tumor was observed, and the size of the tumor was measured with a vernier caliper. The tumor volume calculation formula is: v=ab ² V is the volume, a is the tumor major diameter, and b is the tumor minor diameter.

2. CAR T cell therapy for NSCLC subcutaneous tumor model

(1) After CAR T cells were constructed using the method set forth in the first section, they were cultured for 7-14 days and used for in vivo experiments.

(2) To the extent that the tumor volume is about 50mm on average ³ At this time, mice were randomly divided into 3 groups: YW327.6S2-CAR T cell group, CD19-CAR T cell group and uninfected T cell group, 6 mice per group. Tail vein infusions were performed separately, 1X 10 infusions per mouse ⁷ Individual effector cells (YW327.6S2-CAR T, CD19-CAR T or uninfected T cells).

(3) Mice were monitored every three days after inoculation for changes in body weight and tumor volume.

(4) After the end of the experiment, the mice were euthanized and dissected, and the tumor tissues of the mice were dissected and photographed.

3. YW327.6S2-CAR-T combined Erlotinib treatment

Part of the study shows that AXL up-regulation is an important cause of lung cancer resistance to EGFR-TKI, and YW327.6S2 mab is capable of down-regulating AXL expression. Therefore, the invention also designs an experiment of combining YW327.6S2-CAR-T with Erlotinib treatment;

(1) EGFR-TKI drug resistant cell HCC827 ER3 is cultured, and NSG mice (6-8 weeks) subcutaneous tumor model is constructed.

(2) Tumors grow to about 50mm ³ Afterwards, the experiments were carried out in groups, which were divided into: NC group, erlotinib group, YW327.6S2-CART group, and combination treatment group (erlotinib+ YW327.6S2-CART). Three mice per group.

(1) NC group: no treatment is performed;

(2) erlotinib group: erlotinib lavage, 100 mg/kg/day;

(3) YW327.6S2-CART group: tail vein injection 1 x 10 ⁷ YW327.6S2-CART cells are used for treating HCC827 ER3 subcutaneous tumor;

(4) combination treatment group: tail vein injection 5 x 10 ⁶ YW327.6S2-CART cells were irrigated with Erlotinib (100 mg/kg/day).

(3) Mice were monitored regularly for changes in body weight and tumor volume.

(4) After the experiment, the mice are euthanized, the dissected mice are stripped from the tumor, a part of the dissected mice are fixed, and after paraffin embedding, the dissected mice are subjected to immunohistochemical examination to detect the infiltration of tumor CD3 and the expression of AXL.

Experimental results are expressed as mean ± SD or SEM. Group differences were detected by one-way ANOVA or Bonferroni methods. The non-normal distribution data was examined using Kruskal-Wallis. Survival analysis was performed using Kaplan-Meier. All statistical analyses were performed using Graph-pad Prism 7.0. * p <0.05, p <0.01 and p <0.001 are considered statistically significant.

Experimental results:

to verify whether YW327.6S2-CAR T cells inhibit the growth of NSCLC subcutaneous tumors, the present example constructed subcutaneous tumors with A549 and HCC827 ER3 tumor cells, respectively, when tumors grew to about 50mm ³ The experiments were performed in time groups, divided into three groups of YW327.6S2-CAR T, CD19-CAR T or uninfected T cells, and the tail vein was infused with the corresponding effector cells, respectively. From the tumor growth curves and tumor size of the dissections of fig. 16, 17, YW327.6S2-CAR T cells were effective in inhibiting the growth of a549 and HCC827 ER3 tumors in vivo, whereas CD19-CAR T or uninfected T cells had substantially no tumor inhibiting effect; and the tumor weighing result of YW327.6S2-CAR T group is (A549 0.10g,HCC827 ER3 0.13g), which is obviously lower than that of CD19-CAR T group (A549 0.78,HCC827 ER3 0.60) and uninfected T cell group (A549 0.95,HCC827 ER3 0.68).

Fig. 16 is an experimental result of YW327.6S2-CAR T cell treatment a549 cell subcutaneous tumor model, a is an experimental flow chart: day 0 infusion 2X 10 ⁶ A549 cells, construction of mouse subcutaneous tumor (n=6), tumor growth to about 50mm ³ Time-group experiments were performed with tail vein infusions of YW327.6S2-CAR T, CD19-CAR T or uninfected T cells, respectively, ending the experiment on day 39; b is a tumor stripping batSchematic drawing; c is the tumor growth curve after CAR T infusion; d is the tumor weighing result.

Fig. 17 is a YW327.6S2-CAR T cell therapy HCC827 ER3 cell subcutaneous tumor model, a is an experimental flow chart: day 0 infusion 2X 10 ⁶ HCC827 ER3 cells, mice were constructed for subcutaneous tumors (n=6) up to about 50mm in length ³ Time-group experiments were performed, with tail vein infusions of YW327.6S2-CAR T, CD19-CAR T or uninfected T cells, respectively, at day 18, and the experiment was ended at day 45; b is a schematic drawing of tumor stripping photographing; c is the tumor growth curve after CAR T infusion; d is the tumor weighing result.

FIG. 18 is a flow chart and experimental result diagram of a YW327.6S2-CAR T cell combined Erlotinib treatment experiment, A is an experimental flow chart; day 0 infusion 2X 10 ⁶ Mice were constructed for subcutaneous tumors (n=3) by HCC827 ER3 GL cells. Tumors grow to about 50mm ³ Time-group experiments are carried out, treatment is carried out on the 15 th group, and living body imaging monitoring is carried out on the 22 th day and the 36 th day respectively; b is a living body imaging schematic diagram. The results in fig. 18 demonstrate that the combination treatment group is able to eliminate tumors better than the other groups. YW327.6S2-CAR-T cells at least down regulate the AXL expression of NSCLC cells through YW327.6S2 antibodies and improve the sensitivity of NSCLC cells to Erlotinib, so that the EGFR-TKI resistance of non-small cell lung cancer can be reduced, and the anti-tumor curative effect is improved. Furthermore, for cancer cells which are not resistant to EGFR-TKI, YW327.6S2-CAR-T combined with Erlotinib treatment can be expected to have better treatment effect, because the generation of drug resistance is inhibited on one hand, and on the other hand, the two are subjected to cooperative treatment; after the treatment of the AXL-CAR-T cells, the expression of an HCC827 ER3 tumor model AXL is down-regulated, the sensitivity of the HCC827 ER3 to Erlotinib is recovered, the treatment effect is promoted, and the AXL down-regulation is not generated in both an Erlotinib group and a control group, so that the tumor control effect is poor.

AXL is highly expressed in tissues of primary lung cancer and EGFR-TKI drug-resistant patients, and is basically not expressed in 19 normal tissues of human bodies, which all indicate the importance and feasibility of taking the AXL as a lung cancer treatment target;

Three third generation (CD 28, CD137 costimulatory domain-containing) CAR T cells targeting AXL with different affinities were successfully constructed in this study, respectively: 3E8-CAR T, YW327.6S2-CAR T,20G7D9-CAR T;

3E8-CAR T, YW327.6S2-CAR T,20G7D9-CAR T cells had targeted killing effects on AXL-positive expressing NSCLC cell lines, while there was substantially no killing ability on AXL-negative expressing NSCLC cells. Among these, YW327.6S2-CAR T cells exhibited the strongest killing ability against AXL-positive expressed NSCLC cells;

YW327.6S2-CAR T cells secrete large amounts of cytokines IL-2, TNF- α, IFN- γ, GM-CSF, perforin and Granzyme B while killing NSCLC cells that are positive for AXL expression.

The above examples at least demonstrate: AXL is highly expressed in lung cancer tissues, the positive expression rate reaches 69%, and the AXL is rarely expressed in normal tissues of human bodies; the AXL-CAR T cells have good tumor inhibiting effect on the NSCLC cells positive to the AXL in vivo and in vitro. In addition, the YW327.6S2-CAR T cells of the AXL are used for down regulating the drug resistance of the EGFR-TKI resistant lung cancer, so that EGFR-TKI drugs such as Erlotinib can be conveniently combined, and remarkable treatment effects can be realized on non-drug resistant lung cancer and EGFR-TKI resistant lung cancer.

It should be understood that the foregoing examples of the present invention are merely illustrative of the present invention and are not intended to limit the present invention to the specific embodiments thereof. Any modification, equivalent replacement, improvement, etc. that comes within the spirit and principle of the claims of the present invention should be included in the protection scope of the claims of the present invention.

Claims (7)

1. Use of AXL-targeting CAR-T cells for the preparation of a product for the treatment of AXL-expressing lung cancer, characterized in that the CAR-T cells carry a protein sequence and/or protein that down-regulates AXL expression, i.e. the scFV protein of the AXL-targeting CAR-T cells is selected from one of 3E8, 20G7D9 or YW327.6S2.

2. The use according to claim 1, wherein the lung cancer is EGFR-TKI resistant lung cancer.

3. The use according to claim 1, wherein the lung cancer is non-small cell lung cancer.

4. The use according to claim 1, wherein the product comprises an EGFR-TKI drug.

5. A method of making AXL-targeted CAR-T cells comprising the steps of:

s1, constructing a lentiviral vector containing a CAR molecular sequence, wherein the CAR molecular sequence comprises an AXL scFv sequence, a Hinge region-finger sequence, a transmembrane region sequence, a costimulatory domain sequence and a CD3 zeta sequence, and the AXL scFv sequence expresses a scFv protein targeting AXL; the scFv sequence targeting the AXL is a gene sequence for expressing 3E3E8, YW327.6S2 or 20G7D9 proteins;

S2, carrying out slow virus packaging on the slow virus vector obtained in the step S1 to obtain slow virus containing the slow virus vector;

s3, separating human Peripheral Blood Mononuclear Cells (PBMCs), separating T cells from the separated PBMCs, and stimulating the T cells;

s4, infecting the T cells stimulated in the step S3 by using the slow virus obtained in the step S2 to obtain the AXL-targeted CAR-T cells.

6. The method of claim 5, wherein the Hinge region-Hinge is CD8 Hinge, the transmembrane region is CD28, and the costimulatory domain comprises the CD28 and/or 4-1BB costimulatory domain.

7. A CAR-T cell obtained according to the AXL-targeted CAR-T cell preparation method of claim 5 or 6.