CN114075548A - AXL-targeted CAR-T cell and preparation method and application thereof - Google Patents

Abstract

The invention discloses an AXL-targeted CAR-T cell application, wherein an AXL-targeted scFv protein is used for preparing an AXL-targeted third-generation CAR-T cell, and the AXL-targeted third-generation CAR-T cell application is applied to tumor treatment, so that the problem that a tumor specific target is difficult to find in CAR-T treatment of solid tumors is solved, the off-target is effectively avoided, the probability of side effects is reduced, and the solid tumor treatment effect is improved. Based on high expression of AXL in lung cancer, the compound has specific killing effect on lung cancer tissues positively expressed by AXL, and realizes treatment of the lung cancer; particularly acting on lung cancer cells which are resistant to EGFR-TKI due to AXL up-regulation, can remarkably improve the treatment effect of the lung cancer by cooperating with EGFR-TKI drugs to treat the 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, and particularly relates to an AXL-targeted CAR-T cell and a preparation method and application thereof.

Background

Lung cancer is one of the most common malignancies worldwide. According to the statistics of the National center for Cancer registration of China (NCCR), the incidence and mortality of lung Cancer are ranked the first in China. In recent years, lung cancer treatment methods such as surgery, radiotherapy, chemotherapy, and targeted therapy have been developed. However, since many patients have local or extensive metastases at the time of lung cancer discovery, the overall efficacy of lung cancer remains quite limited, with a 5-year survival rate of only about 15%. Therefore, a new therapeutic strategy or drug is urgently needed to improve the therapeutic effect and survival rate of lung cancer patients.

CAR-T therapy (Chimeric Antigen Receptor T-Cell Immunotherapy), a Chimeric Antigen Receptor T-Cell Immunotherapy, is a novel Cell therapy. The T cells of a tumor patient are artificially modified by a genetic engineering technology, and are cultured in vitro in a large amount to generate tumor-specific CAR-T cells which are then infused back into the patient body to attack cancer cells. CAR-T cell therapy of the prior art can produce significant therapeutic effects in hematological tumors, e.g., CD 19-targeted CAR-T cell immunotherapy has revolutionized success in B cell-derived leukemias or lymphomas, with partial or complete cure in the latest cases. However, when CAR-T therapy is used to treat solid tumors, there are a number of problems, including: tumor targets that are as specific as CD19 in treating hematological tumors are difficult to find, off-target effects easily occur and side effects are brought; heterogeneity of solid tumors is likely to lead to conditions such as drug resistance due to antigen loss; the effect of treating solid tumor is obviously not as obvious as that of treating blood tumor. However, if a more suitable target can be found, the CAR-T cell therapy can be enabled to significantly improve the effect of treating corresponding solid tumors (such as lung cancer), thereby providing a new therapeutic direction.

AXL was originally found in chronic myeloid leukemia patients and, like the other two kinases Tyros and MER, AXL belongs to the TAM (Tyros, AXL, MER) RTK (receptor tyrosine kinase) family. Research shows that AXL is highly expressed in lung cancer and plays an important role in the aspects of occurrence, development and drug resistance of lung cancer. For example, Iida et al in Japan examined 112 lung cancer tissues by immunohistochemistry found that the positive expression rate of AXL reached 59.8%. AXL may serve as a therapeutic target for lung cancer, and may provide a stable target cell surface antigen for immunotherapy, so that a new lung cancer treatment strategy or drug is provided in combination with CAR-T cell immunotherapy to improve the lung cancer treatment effect and survival rate.

Disclosure of Invention

The invention aims to overcome at least one defect of the prior art and provides an AXL-targeted CAR-T cell and a preparation method and application thereof, the AXL-targeted CAR-T cell is prepared by utilizing an AXL-targeted scFv protein, and the AXL-targeted CAR-T cell is applied to lung cancer, so that the AXL-targeted CAR-T cell is beneficial to effectively treating the lung cancer to improve the survival rate of patients, the bottleneck that the CAR-T in the prior art has poor effect on treating solid tumors is broken through, and a new strategy and direction are provided for treating the lung cancer.

One of the objects of the present invention is to provide the use of AXL-targeted CAR-T cells for the preparation of a product for the treatment of AXL expressing lung cancer; the CAR-T cell targeting AXL can directly and specifically recognize and combine with tumor cells expressing AXL to be activated, directly kills the tumor cells by releasing perforin, granzyme B and the like, and simultaneously recruits human endogenous immune cells to kill the tumor cells by releasing cytokines, so that the tumor is treated, and the CAR-T cell targeting AXL has good targeting property, killing property and durability. In one embodiment of the invention, AXL-targeted CAR-T cells have significant cancer suppression, killing and cytokine secretion on AXL-expressing lung cancer cells positively expressing AXL; the AXL-targeted CAR-T cell can play an effective role in treating lung cancer, and the AXL-targeted CAR-T cell is applied to the preparation of products for treating the AXL-expressed lung cancer, so that the tumor can be killed by utilizing the immune system of a patient, and an immune memory T cell 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. In some studies, activation of AXL in non-small cell lung cancer (NSCLC) with Epidermal Growth Factor Receptor (EGFR) mutation may 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 highly expressed in lung cancer tissues and more obviously expressed in EGFR-TKI drug-resistant lung cancer, and the CAR-T cell targeting the AXL can effectively act on the EGFR-TKI drug-resistant lung cancer to play a role in inhibiting cancers; therefore, based on the AXL-targeted CAR-T cells, the combined EGFR-TKI medicine is helpful for providing more remarkable treatment effect on lung cancer, and the defect of poor treatment effect caused by drug resistance is overcome. Provides a new selection and application strategy for the treatment of EGFR-TKI 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. The CAR-T cell can express a protein which can reduce the expression of AXL, or directly carry a protein which acts on AXL to reduce the expression, and the AXL expression is reduced to reduce the drug resistance of EGFR-TKI drug-resistant lung cancer, so that the combined treatment can be realized by combining EGFR-TKI drugs, and the treatment effect is improved. 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 cell has good tumor inhibition effect on AXL positive NSCLC cells in vivo and in vitro, so the CAR-T cell targeting AXL can realize effective treatment on non-small cell cancer.

Further, the AXL-targeted CAR-T cell is a third generation CAR-T cell. Third generation CAR-T cells containing both CD28 and CD137 had better in vivo persistence and killing function compared to second generation CAR-T cells. It combines the advantages of CD28 and CD137, enabling CAR-T cells to exhibit greater cytotoxicity, persistence, and differentiation capacity of memory T cells.

Further, the AXL-targeted CAR-T cell scFV protein is selected from one or more of 3E8, 20G7D9, or YW327.6S2. In the embodiment of the invention, the CAR T cells constructed based on the three scFv all have stronger killing capacity on the AXL positive NSCLC cells, the killing efficiency of the three AXL-CAR T cells is increased along with the increase of E: T, and YW327.6S2-CAR-T cells have the strongest killing capacity among the three. Further, the scFv protein used YW327.6S2, which is a fully humanized antibody, used to construct CAR T can mitigate the production of potential human anti-mouse antibodies in humans, preventing CAR-T cells from being cleared by the human body.

Still another object of the present invention is to provide a product for treating AXL-expressing lung cancer, wherein the product comprises the AXL-targeted CAR-T cell and EGFR-TKI drug described above.

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

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

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

s3, separating PBMCs (human peripheral blood mononuclear cells), sorting T cells from the separated PBMCs, and stimulating the T cells;

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

The method can quickly and simply construct the CAR-T cell targeting the AXL, and is convenient for practical production, so that the CAR-T cell targeting the AXL can be used for preparing a medicament for treating the lung cancer, the treatment effect of the lung cancer is improved, and a new choice is provided for the treatment of the lung cancer in the prior art.

Further, the AXL-targeting scFv sequence 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, 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 AXL-targeted CAR-T cell, wherein the AXL CAR-T cell can specifically bind to cancer cells with high expression of AXL, so that the killing property of the AXL CAR-T cell on the cancer cells is activated, the tumor growth is inhibited, and the effect of treating tumors is achieved. And the drug resistance caused by the AXL can be inhibited by targeting the AXL, so that the effect of treating tumors is improved by further combining other drugs.

Compared with the prior art, the invention has the beneficial effects that: the CAR-T cell targeted to the AXL gene can be constructed, so that the problem that the tumor specific target is difficult to find in the prior art when the CAR-T is used for treating the solid tumor is solved, off-target is avoided, the probability of side effects is reduced, the treatment effect of the solid tumor is improved, the tumor treatment application range of the CAR-T is improved, and a new strategy and direction are provided for treating cancer. And based on high expression of AXL in lung cancer, the compound has a specific killing effect on lung cancer tissues positively expressed by AXL, so that the treatment of the lung cancer is realized, and the development of a lung cancer treatment technology is facilitated. Meanwhile, the CAR-T cell targeting the AXL can also act on lung cancer cells resistant to EGFR-TKI, and is beneficial to synergistically treating the lung cancer by combining with the EGFR-TKI, so that the treatment effect of the lung cancer is remarkably improved, and the defect of poor treatment effect caused by drug resistance is overcome. The CAR-T cell targeting AXL can realize effective lung cancer treatment, improve the survival rate of patients and overcome the defects of the treatment medicines and technologies in the prior art.

Drawings

FIG. 1 shows the result of immunohistochemical detection of AXL in human normal tissues in accordance with an embodiment of the present invention.

FIG. 2 shows the results of immunohistochemical detection of AXL in lung cancer tissues in accordance with an embodiment of the present invention.

FIG. 3 shows that WB of the invention detects the AXL expression of lung cancer tissues and tissues beside the lung cancer tissues of EGFR-TKI drug resistant patients.

FIG. 4 shows the expression of WB and AXL by flow-detection in lung cancer cell lines A549, HCC827 ER3 and HCC 827.

FIG. 5 shows the results of the validation of the affinity of AXL scFv in accordance with an embodiment of the present invention.

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

FIG. 7 shows the construction results of the CAR molecule lentiviral vector of the present embodiment.

FIG. 8 shows the construction of CAR T cells and the results of their identification in accordance with an embodiment of the invention.

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

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

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

FIG. 12 shows the results of phenotypic analysis of T cells of example of the present Invention (IV).

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

FIG. 14 shows the results of in vitro killing of AXL-CAR T, CD19-CAR T, and T cells according to an embodiment of the invention.

FIG. 15 shows the result of the in vitro killing experiment cytokine secretion detected by ELISA method according to the embodiment of the present invention.

FIG. 16 shows the results of the A549 cell subcutaneous tumor model treatment with example YW327.6S2-CAR T cells of the invention.

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

FIG. 18 shows the results of YW327.6S2-CAR T combined with Erlotinib in the model of HCC827 ER3 GL subcutaneous tumors.

Detailed Description

The drawings are only for purposes of illustration and are not to be construed as limiting the invention. For a better understanding of the following embodiments, certain features of the drawings may be omitted, enlarged or reduced, and do not represent the size of an actual product; it will be understood 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 (cell culture, cryopreservation and recovery)

First, cell culture

(1) All cells were incubated at 37 ℃ in 5% CO₂And (5) culturing in a constant-temperature incubator. (2) HCC827, HCC827 ER 3: the culture was carried out using 10% RPMI-1640 complete medium. HCC827 cells are passaged according to the ratio of 1:2-1:3, and are subjected to liquid change, passage or frozen storage for 2-3 days. HCC827 ER3 cells were passaged at 1:2 for 2-3 days, and then subjected to liquid change, passaging or cryopreservation. (3) A549: the culture was carried out using 10% DMEM/F12 complete medium. Carrying out passage according to the ratio of 1:3-1:6, and carrying out liquid change, passage or frozen storage for 1-2 days. (4) 293T: the culture was performed using 10% DMEM complete medium. Carrying out passage according to the ratio of 1:3-1:6, and carrying out liquid change and passage for 1-2 daysOr freezing and storing. 293T cells are semi-adherent cells, the adherence is not firm, violent whipping or violent shaking is avoided in the culture process, and the digestion time is less than 1 min. (5) 293F: for AXL scFv protein purification. Carrying out passage according to the ratio of 1:2-1:3, and changing liquid, passage or freezing and storing for 2-3 days. 293F is suspension cells, and a half-amount liquid changing method is adopted or liquid changing or passage is carried out after all culture media are collected and centrifuged. (6) T cell: the T cell culture medium was prepared in a gentle and complete medium (IL-2 was added by itself). And the T cells grow in a suspension way, and liquid exchange or passage is carried out by adopting a half liquid exchange method or a centrifugal method. Because of the limited T cell expansion capacity, T cells are generally not cultured for more than 3 weeks after sorting.

Secondly, freezing and storing cells

(1) Cell cryopreservation solution: 90% serum + 10% DMSO, should be prepared in advance, and stored at 4 ℃ under refrigeration. (2) The newly recovered cells can be frozen and stored after passage for 3-4 times, and generally, the cells with better state are selected for seed preservation, so that higher recovery success rate can be ensured. (3) Count adherent cells after being digested by pancreatin, 300g and centrifuge for 5 minutes; suspension cells were not digested and were counted directly and centrifuged. In order to ensure the recovery success rate, the number of the frozen cells is generally over 100 ten thousand. (4) The supernatant of the centrifuge tube was aspirated, the cells were removed, 1mL of pre-cooled cell freezing medium was added to resuspend the cells, and the cells were aspirated into the tube. (5) And (4) placing the freezing tube into a programmed cooling freezing box (the freezing box is placed at the normal temperature firstly). After the frozen storage box is placed in a refrigerator at minus 80 ℃ for 48 hours, the cells are transferred into liquid nitrogen for long-term storage.

Third, cell recovery

(1) The frozen cells were carefully removed from the liquid nitrogen tank and quickly placed in a 37 ℃ water bath. (2) Shake rapidly (about 200 times) until completely thawed, preventing ice crystals from damaging the cells. (3) The 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, the pre-warmed medium was added for resuspension, and the cells were transferred to the corresponding flask or dish depending on the number of cells. (5) The next day, the cell status determines whether a change of medium or a further culture is required.

Example 2

Detection of AXL expression in Lung cancer and Normal organs or tissues of human body

In order to verify that AXL is an ideal target for treating lung cancer and avoid potential off-target phenomena, the present example uses immunohistochemistry to detect the expression of AXL in 90 cases of lung cancer (including 40 cases of EGFR-TKI resistant patients) and 19 normal human organs or tissues. Meanwhile, Western-Blot is used for detecting the expression condition of the AXL in 8 EGFR-TKI drug-resistant lung cancer and the corresponding paracancer lung tissue, so that the specific expression relation of the AXL is further obtained.

Specifically, the immunohistochemical operation step in this embodiment includes: (1) baking slices: placing the tissue slices in a 65 ℃ oven for baking for 1-2 hours; (2) dewaxing: placing the grill in xylene to dewax for 2 times, each time for 10 minutes; (3) hydration: orderly hydrating in 100%, 95%, 75% and 50% ethanol for 5 minutes per cylinder; then washed 3 times in TBST for 5 minutes each; (4) antigen retrieval: placing the slices in a sodium citrate repairing solution (pH is 6.0) for microwave antigen repairing, and cooling at room temperature for 20-30 minutes after repairing; washing in TBST for 5 min 3 times; (5) blocking endoperoxidase activity: acting with 3% H2O2 at room temperature for 10 min; washing in TBST for 5 min 3 times; (6) and (3) sealing: blocking with 5% BSA at room temperature for 2 hours; (7) incubating the primary antibody: rabbits were incubated 1:500 rabbit anti-human AXL primary antibody overnight at room temperature. Primary antibody was recovered and then washed 3 times in TBST for 5 minutes each; (8) incubation of secondary antibody: incubate the secondary antibody solution at room temperature for 30 minutes, wash 3 times in TBST, 5 minutes each; (9) DAB color development: incubating for 3 minutes by using a DAB chromogenic substrate or observing under a mirror until the section turns yellow to stop chromogenic; washing in TBST for 5 min 3 times; (10) hematoxylin counterstaining: hematoxylin staining is carried out for 2 minutes, hydrochloric acid alcohol differentiation is carried out for 15 seconds, and blue returning reagent (PBS) staining is carried out for 15 minutes; (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 including liver, spleen, lung, kidney, brain, esophagus, stomach, colon, rectum, appendix, pancreas, gallbladder, tonsil, thyroid, prostate, testis, mammary gland, uterus and skin, and more than 3 tissues are collected from each tissue, and the detection results are shown in FIG. 1; and a549 was used as AXL positive control and HCC827 was used as negative control (400 ×) and scored using a four-point scoring standard, with the statistical results of the four-point scoring shown in table 1 below:

TABLE 1 human Normal tissue AXL expression

The results of immunohistochemical detection of normal tissues show that only 1 case of each colorectal tissue shows 1+ expression, and the other normal tissues show no AXL expression.

In the example, 90 cases of lung cancer were detected in the immunohistochemical detection of lung cancer tissues, and 40 cases of lung cancer were EGFR-TKI drug-resistant patients (400X). The AXL expression was evaluated using a four-level score scale, with four levels of immunohistochemical results as shown in fig. 2 and four-level score statistics as shown in table 2 below, wherein 0 level indicates negative expression and 1 to 3 levels indicate weak to strong expression, respectively.

TABLE 2 Lung cancer tissue AXL expression intensity Classification

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 AXL in 8 EGFR-TKI drug-resistant lung cancer and the paracarcinoma lung tissue corresponding to the EGFR-TKI drug-resistant lung cancer, wherein the Western-Blot detection step comprises the following steps: (1) extracting tissue protein: transporting the human lung cancer tissue and the tissue beside the lung cancer in an ice box, washing the tissue with PBS for 3 times on ice, washing blood, sucking the PBS dry, shearing the mung bean tissue with tissue scissors, adding RIPA lysate to crack the tissue on ice for 15 minutes. After the lysis is completed, the tissue is thoroughly lysed using an ultrasonic crusher. Removing connective tissue, centrifuging, and collecting supernatant; (2) protein quantification is carried out by using a BCA protein quantification kit, and the protein concentration is calculated after reading by an enzyme-labeling instrument. Adding 5 xSDS-PAGE protein Loading Buffer (5 xLoading Buffer) into the volume of the protein sample according to the proportion of 4:1, fully and uniformly mixing, carrying out metal bath at 95 ℃ for 5-10 minutes to denature the protein, and preparing a sample; (3) electrophoresis: 8% glue concentration was prepared according to the formulation of Biyuntian SDS-PAGE gel preparation kit. After coagulation, loading, and adding 40 mu g of protein into each hole; the excess wells were trimmed with 1 × Loading Buffer. Adding an electrophoresis solution, and performing electrophoresis at constant voltage of 90V until a bromophenol blue indicator line runs to the bottom of the gel; (4) film transfer: preparing a film transfer liquid in advance, and precooling at 4 ℃ for later use. And sequentially clamping a cathode carbon plate, a sponge, filter paper, gel, a PVDF membrane, filter paper, a sponge and an anode carbon plate. Rotating the film for 90 minutes in an ice box at a constant current (250 mA); (5) and (3) sealing: sealing with 5% skimmed milk for 2 hr at room temperature on a shaking table; (6) incubating the primary antibody: diluting AXL antibody (CST) with primary antibody diluent at a ratio of 1:1000, and shaking overnight at 4 deg.C; (7) incubation of secondary antibody: after recovery of the primary antibody, the antibody was washed 3 times with TBST for 5 minutes each. Secondary antibody (rabbit antibody) was then added. The secondary antibody is prepared by adopting 5% skimmed milk or BSA according to the dilution ratio of 1: 5000; (8) protein detection (exposure): the washing was performed 3 times for 5 minutes each with TBST. Developing on a developing instrument after preparing the hypersensitive luminous liquid; (9) and (5) image analysis.

The WB detection result of AXL expression in lung cancer tissues and tissues beside the lung cancer of EGFR-TKI drug resistant patients is shown in FIG. 3A (wherein T represents tumor tissues and N represents tissues beside the lung cancer), and it can be seen from the graph that the lung cancer tissues of EGFR-TKI drug resistant patients highly express AXL, while the tissues beside the lung cancer do not substantially express AXL, and FIG. 3B/C is a WB detection gray scale analysis result graph.

The result that the tumor tissue is highly expressed and the normal tissue is not substantially expressed shows that the AXL is very likely to be an ideal therapeutic target for patients with lung cancer, particularly drug-resistant patients with lung cancer EGFR-TKI, and the AXL-targeted immunotherapy has good safety and is helpful for providing new strategies and medicines for treating lung cancer based on the AXL-targeted therapy.

Example 3

Lung cancer cell line AXL phenotype assay

Western-Blot detection of lung cancer cell line AXL expression

A549, HCC827 and HCC827 ER3 cells were cultured in 10cm dishes and total protein was extracted on ice after overnight serum-free. The following steps were as above, and the WB detection step in example 1 was used.

Human lung adenocarcinoma a549 and HCC827 cells were purchased from ATCC in this example and the following examples. HCC827 ER3 is stored in the subject group laboratory, and can be seen in Activation of the axL kinase restriction to EGFR-targeted therapy in lung cancer, and HCC827 ER3 is Erotinib drug-resistant cell model of HCC 827.

WB assay as shown in fig. 4A, HCC827 cell line does not express AXL. As in the previous studies of the subject group of the present inventors, the details are described in the literature of Activation of the AXL kinases resistance to EGFR-targeted therapy in the lung cancer.

Secondly, detecting the AXL phenotype of the lung cancer cell line by a flow cytometer

(1) A549, HCC827 and HCC827 ER3 cells were cultured, 30 to 50 ten thousand cells each were collected, washed with PBS and then resuspended in 200. mu.L of PBS containing 2% FBS. (2) Add 1. mu.L of PE-AXL flow monoclonal antibody and incubate for 30 min on ice in the dark. (3) 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 signals.

FIG. 4B shows the results of flow analysis, indicated by isotype in dotted lines and lung cancer cells in solid lines.

The previous research proves that HCC827 is a lung cancer cell line which does not express AXL, and on the basis, the inventor constructs an Erotinib drug-resistant cell model HCC827 ER3 of HCC827 at the early stage and reveals that the expression up-regulation of AXL is an important mechanism of drug resistance. The result that the HCC827 does not express AXL but HCC827 ER3 highly expresses AXL can be repeated by Western-Blot in the experiment; in addition, the AXL expression of the lung cancer cell line a549 was also detected in this study, and the results showed that a549 also highly expressed AXL (fig. 4A). The flow results showed that AXL expression reached 97.7% and 94.7% in a549 and HCC827 ER3, respectively, while it was only 1.86% in HCC827 (fig. 4B). These results again indicate that AXL is highly expressed in various lung cancer cell lines and thus AXL may be an ideal target for lung cancer treatment. In subsequent cell and animal experiments, i.e. in subsequent examples, a549 and HCC827 ER3 were selected as AXL positive expressing cell lines in this study, while HCC827 served as negative controls. To verify the targeting and killing properties of AXL-CAR T cells.

Example 4

Validation of three AXL scFv affinities

Purification of AXL scFv recombinant protein

AXL scFv expression plasmid transfection of 293F cells (Large Scale culture/1L Shake flask)

The expression plasmids pComb3-3E3E8, pComb3-YW327.6S2 and pComb3-20G7D9 of the three AXL scFv are successfully constructed, then plasmid extraction is carried out, and then enzyme digestion and gene sequencing identification are carried out. Post-transfection 293F cells were identified for AXL scFv protein expression and purification. The method comprises the following specific steps: (1) 300mL of medium (containing 0.5X 106 293F cells) was added to a 1L shake flask; (2) after culturing in a constant temperature shaking incubator (37 ℃, 120rpm, 5% CO2) for 24 hours, the cells were counted so that the number of cells was controlled to 1X 106. (typically the number of cells doubled after 24h of culture); (3) aspirate 300 μ g of plasmid filtered through 0.22 μm filter into 30mL PBS and mix well, taking care not to vigorously blow or shake to prevent plasmid DNA fragmentation; (4) adding 1.2mL of PEI (0.5mg/mL) subjected to filtration sterilization into the PBS/plasmid mixed solution, performing vortex oscillation for 3 seconds, thoroughly mixing, and standing 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 ℃, 120rpm, in a constant temperature shaking incubator with 5% carbon dioxide concentration; (7)3000g, 5 min centrifugation to harvest all cell pellets, storage at-80 ℃ or direct purification of the protein.

In this example, 293F cells were used for purification of AXL scFv protein as supplied by Zhao Qi professor of Australian university. Three expression plasmids (pComb3-3E3E8, pComb3-YW327.6S2, pComb3-20G7D9) for AXL scFv, each of which has His and Flag tags for protein purification, were constructed with the assistance of the task group of Qi professor, university of Macao Zhao.

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

The method comprises the following specific steps: (1) lysing the cells with pre-cooled cell lysate (40mL lysate/1000 mL medium) on ice; (2) blowing the cells to lyse them sufficiently, taking care not to vortex or foam; (3) the glass homogenizer resuspended cells. The cells were lysed thoroughly with an ultrasonic disintegrator (3 cycles), 30000g, centrifuged at 4 ℃ for 25 minutes, the precipitate was discarded, and the supernatant was retained; (4) adding 1.25mL of Flag-tag-containing agarose gel resin into every 1000mL of cell culture medium, and washing three times with resin equilibrium buffer; (5) incubating the supernatant obtained in the step (3) with Flag-labeled affinity resin, centrifuging a 50mL centrifuge tube, and shaking the tube for 30-120 minutes at 4 ℃; (6) centrifuging at 4 deg.C for one minute at 3000g, and discarding the supernatant; (7) the affinity resin was washed with 45mL of pre-chilled buffer (containing 100mM potassium acetate, 50mM Tris pH 7.5, 5% glycerol, 0.3% Triton. times.100), 3000g, centrifuged for one minute, and the supernatant discarded; (8) repeating step (7) with high salt buffer (300mM potassium acetate, 50mM Tris pH 7.5, 5% glycerol), low salt buffer (50mM potassium acetate, 50mM Tris pH 7.5, 5% glycerol) and TEV digestion buffer (50mM potassium acetate, 50mM Tris pH 7.5, 0.5mM TCEP), respectively; (9) using non-denatured glue, collecting 10 μ L of affinity resin sample for Kamasie brilliant blue staining; (10) resuspending the affinity resin using 8-10mL of pre-cooled TEV enzyme digestion buffer, and then transferring the affinity resin to a 15mL centrifuge tube; (11) adding about 40ug TEV protease (1mg/mL), and blowing uniformly; (12) the gas in a 15mL centrifugal tube is changed into 100% N2 to prevent the oxidation of protein; (13) shaking overnight at 4 ℃; (14)3000g, centrifuged for 10 min and the supernatant was transferred to an ultracentrifuge filter with appropriate molecular weight cut-off and concentrated to 500 μ L; (15) using non-denatured gel, taking 10 μ L of the protein concentrate sample for Coomassie brilliant blue detection, and using TEV eluate as a control; (16) using non-denatured glue, taking 10 mu L of resin sample for Coomassie brilliant blue detection, and using TEV positive resin for comparison; (17) exclusion chromatography column with equal amount of gel filtration buffer (50mM potassium acetate, 50mM Tris pH 7.5, 0.5mM TCEP); (18) filtering the protein by using a filter membrane of 0.22 mu m, loading the sample to a chromatographic column, collecting the sample, and carrying out Coomassie brilliant blue staining to detect whether the target protein is collected or not; (19) sampling from the steps (9), (15), (16) and (18) for examination. And before gel filtration, the examination of the counterstain in steps (9), (15) and (16) is required to ensure the expression of the target protein.

Two, indirect ELISA method for detecting AXL scFv affinity

After protein collection, the affinity of the three scFv of AXL for binding the AXL antigen was detected by indirect ELISA. The method comprises the following specific steps: (1) envelope AXL antigen: 100ng/well (a control group needs to be arranged, each group has 2-3 multiple wells), each well is added with 100 mu L of volume, and the mixture stays overnight at 4 ℃; (2) discarding the coating solution, washing with TBST for 3 times, sealing with 5% skimmed milk (filling each well), and standing at room temperature for 1 h; (3) removing the sealing liquid, washing for 3 times by TBST, respectively adding three kinds of AXL scFv to be detected with different concentrations and a control antibody of 100 muL/well, and keeping the temperature at 1 h; (4) discarding the liquid, washing for 3 times by TBST, adding corresponding enzyme-labeled secondary antibody 100 mu L/well, covering and protecting from light, and incubating for 1h at 37 ℃; (5) discarding the liquid, washing for 3 times by TBST, adding 100 mu L/well TMB color development liquid, and developing for 5-10 minutes in a dark place; (6) adding 50 mu L/well of stop solution, measuring the light absorption value of each hole with the wavelength of 450nm by using an enzyme-labeling instrument, and drawing a curve.

scFv is a structure belonging to the extracellular region of the CAR molecule, the affinity of which is crucial for CAR T cells to recognize tumor antigens. Thus, to screen for optimized scfvs, this example demonstrates the affinity of three AXL scfvs. The amino acid or nucleotide sequences of all three scfvs are derived from patents with affinity: 3E3E8 (1.6X 10)^-9M)，YW327.6S2(1×10^-9M)，20G7D9(5.3×10^-8M)。

In order to verify the strength of the affinity, three expression vectors of AXL scFv were constructed in this example: pComb3-3E3E8, pComb3-YW327.6S2 and pComb3-20G7D9, and the enzyme digestion and sequencing results show that the vector construction is successful, as shown in FIG. 5A. The three scfvs were then expressed and purified using 293F cells and confirmed by ELISA to have affinity of YW327.6S2, 3E8 and 20G7D9, respectively, from strong to weak, as shown in figure 5B, which is consistent with the known affinity trends of the patents (see example 5). Therefore, this study constructed 3E3E8-CAR-T, YW327.6S2-CAR-T, 20G7D9-CAR-T cells with these three scFv in cell killing experiments in subsequent examples, respectively, and explored whether scFv of different affinities had an effect on killing efficiency of CAR-T cells. Specifically, the agarose gel electrophoresis result of FIG. 5A shows that the expression vectors of pComb3-3E3E8, pComb3-YW327.6S2 and pComb3-20G7D9 are successfully constructed; FIG. 5B shows the results of indirect ELISA testing of the binding ability of three scFv to AXL antigen. The results of this example were obtained from three independent experiments, and the data are expressed as mean ± sem.

Example 5

Construction of AXL-CAR T cells

Construction of one and three pWPXld-CAR-AXL-GFP lentiviral vectors

The three AXL scFv sequences are derived from patents with patent numbers: WO2012/175692a1(3E 8), US8853369B2(YW327.6S2), US9249228B2(20G7D 9). The method comprises the following specific steps:

(1) design CAR molecule gene sequence: including the AXL scFv sequence, CD8 hinge, CD28 transmembrane region, CD28 and CD137 costimulatory domain, CD3 ζ intracellular domain. Adding enzyme cutting sites PmeI and SpeI respectively before and after the cloning vector is cut by enzyme;

(2) the corresponding CAR molecular sequences were synthesized by Nanjing Kinsley and cloned into pUC57 vector (three AXL scFv cloning plasmids pUC57-3E3E8, pUC57-YW327.6S2, pUC57-20G7D9, synthesized by Nanjing Kinsley) respectively to obtain: pUC57-3E3E8-CAR, pUC57-YW327.6S2-CAR and pUC57-20G7D 9-CAR; pUC57-CD19-CAR cloning vector was also synthesized in this example as an experimental control;

(3) carrying out double enzyme digestion on the AXL CAR molecular gene fragments of pUC57-3E3E8-CAR, pUC57-YW327.6S2-CAR and pUC57-20G7D9-CAR by using PmeI and SpeI respectively, cutting the gel and recovering, simultaneously carrying out double enzyme digestion on pWPXld-GFP framework plasmid by using PmeI and SpeI, and recovering a large-fragment framework; (pWPXld-GFP, a good gift from the professor Lepeng, contains GFP and plasmid backbone, and is used to clone CAR molecule lentiviral vector, GFP can be used as a marker for the subsequent detection of whether the lentivirus is successfully packaged and the positive rate of transfected T cells.)

(4) Connecting for 1 hour at 16 ℃ by using T4 ligase, respectively cloning three AXL CARs to pWPXld-GFP framework plasmids to respectively construct pWPXld-3E3E8-CAR-GFP, pWPXld-YW327.6S2-CAR-GFP and pWPXld-20G7D9-CAR-GFP lentiviral vectors;

(5) transforming TOP10 competent cells; competent cell TOP10, purchased from Nanjing Novodka, was used for plasmid extraction and vector construction in this example.

(6) Selecting 3-5 monoclonals, and shaking the bacteria overnight;

(7) extracting plasmids by using a plasmid miniprep kit, comprising the following steps: a) centrifuging the bacterial liquid by using a centrifuge (3500g, 10 minutes); b) the old culture was aspirated off, and 500. mu.L of Solution I/RNase A (lysis, 4 ℃) was added to the culture tube. Resuspending the cells; c) the cell suspension was transferred to a new 2mL microcentrifuge tube. Add 500. mu.L Solution II (release ribozyme) and mix gently back and forth (7-10 times) to allow thorough cleavage. The mixture was left at room temperature for 2 minutes. Avoid violent vibration, because this will lead to the chromosome DNA rupture and reduce plasmid purity (Solution II should seal); d) mu.L of ice-frozen Buffer N3 was added to the cell suspension and gently and thoroughly shaken until a white flocculent precipitate appeared. Centrifugation is carried out at room temperature (preferably 4 ℃) for 10 minutes at 12000g or more. Warm prompt: buffers must be thoroughly shaken. If viscous, brown and spherical precipitates appear in the shaking process, further uniform mixing and neutralization are needed, and the key point for obtaining better results is that the uniform mixing is carried out; e) carefully transfer the supernatant 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) add 700. mu.L of the above mixture to a clean 2mL HiBindTM collection tube. 10000g at room temperature, and centrifugation for 1 minute. The solution was filtered through Column. Discard flow-through and reuse collection tube; g) repeating step 6 until all the clear lysate passes through the microtubes; h) adding 500 μ L ETR Wash Buffer I to mini column, 10000g, centrifuging for 1min, discarding flow-through, and recycling the collection tube; i) add 500. mu.L Buffer EHB into mini column. 10000g, centrifuged for 1 min. Discarding flow-through, and recycling the collection tube; j) add 700. mu.L of DNA Wash Buffer to dilute the ethanol. 10000g, centrifuged for 1 min. Discarding flow-through, and recycling the collection tube; k) adding 700 mu L of DNA Wash Buffer, and repeating the step 10 once; l) discard the liquid in the centrifuge tube, centrifuge empty column at maximum speed (. gtoreq.13000 g) for 3 minutes, oven dry column Matrix. This step is critical to eliminating ethanol in the column; m) column was placed into a new 1.5mL microcentrifuge tube. 60-100. mu.L (in terms of target final concentration) of Endotoxin-Free Elution Buffer was added to the column matrix and left standing at room temperature for 2 minutes. 13000g for 1 minute to elute DNA. At this time, about 70-85% of DNA remains, and almost most of DNA can be eluted by washing once again; n) measuring the concentration and purity of the plasmid;

(8) the extracted plasmid was digested 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 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 mass extracted with a plasmid Mass extraction kit, along with the lentiviral packaging helper plasmids (pSPAX2 and pMD2. G). Re-sequencing ensures the correctness of each gene sequence. Stored at-20 ℃ for subsequent lentiviral packaging.

To construct AXL-CAR-T cells, three CAR molecule lentiviral vectors comprising AXL scFv (3E 8, YW327.6S2, 20G7D9) and CD19-CAR molecule lentiviral vectors (for experimental controls) were constructed; FIG. 6 shows the schematic structural diagram of the CAR molecule in this example, VH and VL are scFv heavy and light chains, TM is the transmembrane region, CD28, 4-1BB and CD3 are intracellular regions, and eGFP is enhanced green fluorescent protein. The enzyme cutting and sequencing results of FIG. 7 show that the CAR molecule lentiviral vector is successfully constructed.

II, CAR molecular lentivirus packaging

The method comprises the following steps: (1) lentivirus packaging was performed using 293T cells (purchased from ATCC): the first day 293T cells were seeded into 10cm dishes at a density of about 40-50% to ensure that the cell density grew to about 80-90% overnight; (2) removing the culture medium, and starving with DMEM medium containing 1% FBS for 2 hours; (3) the target plasmid, pSPAX2 and pMD2.G, were added to a 15mL centrifuge tube containing Opti-MEM medium at a ratio of 3:4:1, labeled A; drawing PEI with three times of total plasmid amount, adding the PEI into another 15mL centrifuge tube, and marking as B; 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 A + B mixed solution into each dish, and infecting the mixture for 6-10 hours in an incubator at 37 ℃; (5) removing the A + B mixed solution after infection, adding 1% FBS DMEM fresh culture medium, and continuing to culture; (6) the infection efficiency can be observed under a fluorescence microscope; (7) the virus supernatants were collected at 48 and 72 hours, and filtered through 0.22 μm filters to directly infect the desired cells. Can be stored at 4 deg.C for one week. The long-term preservation needs to be carried out at-80 ℃;

third, lentivirus titer measurement

The method comprises the following steps: (1) day 1, 96-well plates were plated with 293T cells, and 5000-10000 cells/well were added to each well at 100. mu.L/well. Culturing in a cell culture box to allow the cells to grow to 30-50% of density the next day; (2) on day 2, 10 EP tubes were used to prepare a multiple dilution of slow virus fluid. Adding 90 μ L of 10% DMEM medium into each tube, adding 10 μ L of lentivirus stock solution to be detected into the first tube, taking 10 μ L from the first tube, adding into the second tube, and so on until the last tube, and the first tube has 10 tubes^-3mL virus stock solution, 10 in the last tube^-12mL of a virus stock solution; (3) sequentially adding the virus diluents (90 mu L) with the 10 dilution gradients into a 96-well plate for lentivirus infection; (4) on day 3 (or 12-24 hours after infection), whether to change the fluid is determined 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 virus titer was the number of cells expressing fluorescence divided by the corresponding dilution factor. For example, if 10 GFP expressing cells were observed in the fifth well, the corresponding virus titer was 10 TU/(10. sup. th well)^-7mL)＝1×10⁸Per mL; (6) repeated freeze thawing will reduce virus titer (each freeze thaw will reduce virus titer by 10% -50%); repeated freeze thawing should be avoided as much as possible during the use of the virus, the virus can be subpackaged (200 μ L/tube), and the virus can be directly stored at-80 ℃; (8) if the virus is stored for more than 6 months, the virus titer needs to be re-determined before use.

After the lentiviral vector was successfully constructed, this example was packaged with 293T cells and the titer of the lentivirus was tested. The four lentivirus titers are shown in table 3 below, TU: viral titer units.

TABLE 3CAR molecule lentivirus Titers

Sorting and stimulation of human Peripheral Blood Mononuclear Cells (PBMCs) (taking the kit of American day and whirlpool selection as an example)

The method comprises the following steps: (1) mixing lymphocyte separating medium and peripheral blood according to a ratio of 1:1, adding the separating medium, and then adding blood drop by drop and slowly, wherein the upper layer of the blood is visible, the lower layer of the separating medium is visible, and the boundary between the two layers is obvious; (2) then centrifuging the mixture by using a flat angle centrifuge (rising 4 and falling 0, 25 ℃,800 g, 25-30 minutes); (3) after centrifugation, the tube is divided into three layers, wherein the upper layer is 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 of the upper layer and the middle layer, and the white film layer is carefully sucked into a 15mL centrifuge tube; (4) gently blowing and beating the mixture into single cell suspension by using 10mL PBS, and filtering the cells by using a 30-micron filter membrane to prevent blood clots from blocking the sorting column for 300g for 10 minutes; (5) discarding the supernatant, adding 4-6mL of erythrocyte lysate for resuspension, and lysing erythrocytes (5 minutes); (6) the erythrocyte lysate was washed with 10mL of PBS and centrifuged. If the lysis is not complete, the erythrocyte lysate can be added again for lysis; (7) the supernatant was discarded, 10mL of MACS PBS was added to resuspend the PBMCs, and 10. mu.L of the cell suspension was counted. Centrifuging the cells at 500g for 5 min; (8) discard the supernatant and add a gentle and gentle MACS buffer to resuspend the cells (40. mu.L buffer/10)⁷Cells), biotin-labeled CD3 magnetic bead antibody (20 μ L/10) was added⁷A cell). Incubate for 15 minutes on ice or in a refrigerator at 4 ℃. Vortex every 1-2 minutes; (9) simultaneously, mounting and pretreating a separation column which is beautiful, gentle and whirly, and adding a MACS buffer to ensure that the separation column completely flows out under the action of gravity; (10) after the cell incubation was complete, it was resuspended in MACS buffer, 500g, 5 min; (11) the MACS buffer was removed, resuspended in 3-5mL of MACS buffer, the cells were loaded into the sorting column, allowed to drain by 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 the MACS buffer is drained, adding 5mL of MACS buffer to elute the T cells in the column; (13) repeating the previous step; (14) centrifuging the sorted T cells, washing the T cells once by PBS, and counting; (15) each 500 ten thousand T cells with 25 u L, Meitian and whirlwind CD3/CD28 magnetic bead antibody for activation. T cell-adding cultureThe medium was inoculated in 48-well plates or T25 flasks for culture. Co-stimulating for 24-48 hours; (16) a small number of T cells were taken for flow testing for the CD3/4/8 phenotype.

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

Fifth, Lentiviral infection of T cells

The method comprises the following steps: (1) on day 1, after T cells are stimulated, magnetic bead antibodies and old culture media are removed through centrifugation, and counting is carried out; (2) calculating the required lentivirus amount according to the number of T cells and the infection multiplicity (MOI value), adding 6-8 mu g of transfection assisting reagent Polybrene to each mL of culture medium, and infecting for 12-24 hours; (3) on the 2 nd day, after the T cells are infected, 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 flow detection of GFP positive rate; (5) CD19-CAR-T cells were constructed as experimental controls in the same manner as described above.

After the CAR molecule lentivirus is packaged, a CAR molecule lentivirus infection of T cells follows. Expression of green fluorescence in T cells can be seen under a fluorescence microscope 3-5 days after infection. To further clarify the expression of CAR molecules, this study performed a series of experiments.

After the CAR molecule lentivirus is packaged, a CAR molecule lentivirus infection of T cells follows. Expression of green fluorescence in T cells can be seen under a fluorescence microscope 3-5 days after infection.

Sixth, detection of CAR molecule expression

1. Extraction of CAR-T cell RNA

The method comprises the following steps:

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

(2) according to

Q RT Supermix for qPCR (+ gDNA wiper) reverse transcription kitThe instructions reverse the extracted total RNA to cDNA.

a) Removing DNA in the total RNA, and reacting in the following system:

b) after mixing and centrifugation, the reaction mixture was reacted at 42 ℃ for 2 minutes.

c) Reverse transcription, the system is as follows:

d) reaction procedure: 15 min at 50 ℃ → 2 min at 85 ℃.

Seven, qPCR detection of expression of CAR molecules

To further quantify the expression of the CAR molecule, this example follows the reversal of the cDNA obtained in step six

qPCR

qPCR experiments were performed according to the instructions provided in the Green Master Mix (High ROX Premixed) kit.

(1) Primer synthesis: the primers used in the qPCR reaction are designed and synthesized by Shanghai Czeri bioengineering GmbH, and the relative expression of all target genes takes GAPDH as the 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) collecting fluorescence signals by using an ABI QuantStudio 7Flex reactor to obtain corresponding Ct values, wherein all target genes take GAPDH as an internal reference gene according to the formula 2^-△△CtThe relative expression amount of mRNA of the related gene is calculated by the method.

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

Total RNA of CAR T cells was first extracted and qPCR experiments were performed after reverse transcription. The qPCR results indicated that the relative expression of CAR molecules was significantly higher in the CAR T cell group than in the T cell group, and the results were statistically different, as shown in fig. 8A.

Eighth, Western-Blot detection of expression of CAR molecules

(1) Three AXL-CAR T-cell, CD19-CAR T-cell and fresh T-cell protein samples were taken on ice without serum overnight. Protein quantification is carried out by using a BCA protein quantification kit, and the protein concentration is calculated after reading by an enzyme-labeling instrument. Adding 5 xSDS-PAGE protein Loading Buffer (5 xLoading Buffer) into the protein sample volume according to the proportion of 4:1, fully and uniformly mixing, and performing metal bath at 95 ℃ for 5-10 minutes to denature the protein to prepare the sample. (2) Electrophoresis: the 12% concentration gel was prepared according to the formulation of Biyuntian SDS-PAGE gel preparation kit. After coagulation, samples were loaded and 40ug protein was added per well. The excess wells were trimmed with 1 × Loading Buffer. Add the electrophoresis solution, run at constant 90V until the bromophenol blue indicator line runs to the bottom of the gel. (3) Film transfer: preparing a film transfer liquid in advance, and precooling at 4 ℃ for later use. And sequentially clamping a cathode carbon plate, a sponge, filter paper, gel, a PVDF membrane, filter paper, a sponge and an anode carbon plate. The membrane was rotated in an ice box for 90 minutes at a constant current (200 mA). (4) And (3) sealing: the vessel was sealed with 5% skim milk and kept on a shaker at room temperature for 2 hours. (5) Incubating the primary antibody: the CD3 antibody was diluted with primary anti-diluent at a ratio of 1:500 and shaken overnight at 4 ℃. (6) Incubation of secondary antibody: after recovery of the primary antibody, the antibody was washed 3 times with TBST for 5 minutes each. Secondary antibody (rabbit antibody) was then added. The secondary antibody is prepared by 5% of skimmed milk according to the dilution ratio of 1: 4000. (7) Protein detection (exposure): the washing was performed 3 times for 5 minutes each with TBST. The prepared hypersensitive luminous liquid (1:1 preparation) is developed on a developing instrument. (8) And (5) image analysis.

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

Nine flow cytometry detection of CAR molecule expression

(1) AXL-CAR T, CD19-CAR T and fresh T cells were collected by centrifugation. (2) Flow-on-machine preparation was performed by washing twice with 2% FBS in PBS and finally resuspending the cells in 200. mu.L of 2% FBS in PBS. (3) And detecting the GFP positive rate of the cells by using a flow cytometer, wherein the positive rate is the infection efficiency of the CAR molecule lentivirus.

Flow cytometry was also used in this example to detect CAR molecule expression, and the results are shown in fig. 8C (data expressed as mean ± standard deviation), and the CAR-T cells positive rates in total T cells were: 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 present study successfully constructed CAR T cells.

Ten, CAR T cell phenotype detection

The CAR-T cells were flow tested for T cell phenotype at day 0 and 14 days after activation, respectively, after successful construction to determine their activation and differentiation.

(1) Fresh T cells sorted on day 0 and AXL-CAR T cells cultured for 14 days after activation were collected separately. (2) Different combinations of flow monoclonal antibodies or non-specific isotypic monoclonal antibodies (1. mu.L) such as CD3, CD4, CD8, CD45RO, CD62L, PD-1, CTLA-4, TIM-3, etc. were added, and incubated on ice or at 4 ℃ in the absence of light for 30 minutes. (3) PBS was washed two to three times, resuspended in 200. mu.L of PBS containing 2% FBS, and then ready for flow-on-machine. (4) Setting corresponding fluorescent channel, setting gate with non-specific monoclonal antibody as control group, and detecting the cell fluorescent expression condition of the experiment group.

After sorting T cells from peripheral blood of three healthy volunteers, the study was performed to identify T cell phenotypes such as CD3, CD4, CD8 by flow assay. The results showed that the CD3 positive rates were 96.8%, 98.7% and 99.2% for three healthy donors, respectively, and could be used for CAR-T construction, as shown in FIG. 9.

The memory cell phenotype, CD45RO and CD62L, was significantly upregulated in both the unmodified T cells and the modified CAR T cells, suggesting that the CAR T cells differentiated towards the memory T cells and that the modified phenotype of the CAR T cells was not significantly different from the unmodified T cells, as shown in figures 10 and 11. More importantly, the inhibitory molecules PD-1, CTLA-4 and TIM3 were upregulated with increasing T cell culture time in vitro, suggesting T cell depletion, as shown in figure 12.

FIG. 9: flow assay of fresh T cells for CD3/4/8 phenotype; fig. 10 and 11: flow-detecting changes in CD45RO and CD62L phenotypes of uninfected T cells and CAR T cells at day 0 and day 14, respectively; FIG. 12: flow assay of TIM-3, CTLA4 and PD1 phenotypic changes in uninfected T cells and CAR T cells on days 0 and 14, respectively. The constructed CAR T cells are shown to be capable of activating and proliferating in a large amount and can show migration, cell killing toxicity, memory and exhaustion cell phenotypes.

Example 6

In vitro killing experiment of AXL-CAR-T cells

Establishment of GFP and Luciferase double positive (GL) cell line

(1) On the first day, A549, HCC827, HCC827 ER3 were plated in 6-well plates, with specific plating densities preferably being as high as about 50% on the second day.

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

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

(4) After infection is complete, selection is carried out with puromycin (2 ug/medium) for 7-14 days.

(5) After the screening is finished, the GFP positive rate can be detected by flow, and if the positive rate is low or the infection intensity is not uniform, the GFP can be infected again or flow-type sorting can be carried out (GFP is taken as a sorting index).

(5) After the GL cell is successfully constructed, in-vivo and in-vitro experiments can be directly carried out.

In order to perform an in vitro killing experiment by using a Luciferase method, lung cancer GL cell lines expressing GFP and Luciferase (Luciferase) were constructed in this example, and flow-type results showed that the GL expression ratios of the three lung cancer cells were all above 97.9%, as shown in fig. 13, in which GFP was a detection marker. Luciferase can oxidize the substrate sodium luciferin salt, which in the process of oxidation gives off bioluminescence. Only the luciferase of the living cells can oxidize the luciferin, and the proportion of the living cells can be approximately reflected by the fluorescence intensity, so that the killing proportion of the cell experiment can be calculated.

Secondly, detecting the in vitro killing efficiency of the AXL-CAR T cell by a luciferase method

(1) Preparing AXL-, CD19-CAR T cells and control T cells, and calculating the actual CAR T cell number of each batch of CAR T cells according to the GFP positive rate of flow detection, thereby obtaining effector cells to be added.

(2) Effector cells (CAR T cells or T cells) and target cells (GL tumor cells) are added in sequence according to a pre-planned effective target ratio (E: T), for example 10000 target cells are added, the effective target ratio is 8:1, 4:1 …, the effector cells need to be added, 80000 and 40000 … are added, and the like, the above cells are paved into a round bottom opaque 96-well plate, and three multiple wells are arranged.

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

(4) After the co-culture is finished, gently sucking 100. mu.L of supernatant to a new 96-well plate by using a discharge gun, and detecting the cell factor.

(5) Add sodium fluorescein salt to 96-well plate and 100. mu.L Luciferin substrate solution (150ug/mL) per well, react for 5-10 min and then measure fluorescence in a fluorometer.

(6) And (3) calculating killing efficiency: the target cell wells without T cells were used as control wells, the rate of decrease in fluorescence was the percent killing, which was (control well fluorescence-target well fluorescence)/control well × 100% (fluorescence of wells without GL target cells was negligible).

To verify targeting of AXL-CAR T cells, this example grouped 3E8-, YW327.6S2-, 20G7D9-CAR T with CD19-CAR T and uninfected T cells, respectively, for their ability to kill target cells. This example continues with killing experiments using a549, HCC827 ER3 and HCC827 as target cells at different effective target ratios (8:1, 4:1, 2:1, 1: 1). The result shows that YW327.6S2-CAR T has obvious killing effect on AXL positively expressed A549 and HCC827 ER3, and the killing effect is obviously increased along with the increase of the effective target ratio; however, YW327.6S2-CAR T has no substantial killing effect on AXL-negatively expressing HCC827 cells. In addition, both CAR T cells targeting CD19 and uninfected T cells had no killing effect on the target cells, as shown in figure 14. These results show that three AXL-CAR ts all have high specificity (targeting) and strong killing ability against AXL-positively expressed lung cancer cells, wherein the CAR T cell constructed with YW327.6S2 as scFv has the strongest killing ability in vitro, and has statistical difference with the killing ratio exhibited by the other two AXL-CAR-T cells. This is probably due to the strongest affinity of YW327.6S2 for AXL among the three scfvs, which is consistent with the trend in affinity of the three scfvs for AXL as demonstrated by ELISA in this study. And considering YW327.6S2 as a fully humanized antibody that may be less immunogenic for human use, avoiding the production of human anti-mouse antibodies, thereby reducing host immune rejection of CAR T cells. Thus, in the examples that follow, CAR T (YW327.6S2-CAR T) cells constructed with YW327.6S2 as the scFv were used as effector cells for further cell killing and in vivo experiments.

The results were obtained from three independent experiments, and the data in the figure are expressed as mean ± sem, # p <0.05, # p <0.01, # p < 0.001.

Third, enzyme linked immunosorbent assay (ELISA) detection of cytokines

(1) At the end of the killing experiment, supernatants from 96-well plates were collected with an EXT of 1:1 wells and were either detected immediately or stored at-20 ℃. (2) Centrifuge the supernatant, 1000g, 15 min. (3) Before the experiment, the Wash Buffer, the Substrate Solution and the cytokine standard are prepared at room temperature according to the instruction. (4) Excess microporous strips were removed from the panel frame, placed back in an aluminum foil pouch containing desiccant packaging, and then resealed. (5) Add 100. mu.L of dilution reagent RD1W to the wells. (6) Add 100. mu.L of standard, control or sample to each well, and cover with dark. Incubate at room temperature for 2 hours. The standard and the sample well of the assay are labeled. (7) The liquid in each well was aspirated and washed 3 times with wash buffer (400. mu.L/well). Thorough removal of liquid in each step is essential to obtain good detection results. After the last wash, remove all remaining wash buffer from the wells by aspiration or pouring. The plate was inverted and blotted dry with a clean paper towel. (8) To each well was added 200 μ L of the corresponding human cytokine conjugate. Covering and protecting from light. Incubate at room temperature for 2 hours. (9) After incubation, the well plates were washed according to step (7). (10) Add 200. mu.L of Substrate Solution to each well and incubate at room temperature for 20 min in the absence of light. (11) Add 50. mu.L of Stop Solution per well and change the color from blue to yellow in the wells after addition. If the color in the well is green or the color variation is not uniform, please tap the plate gently to ensure adequate mixing. (12) The OD of each well was read in 30 minutes by reading with a microplate reader at a wavelength of 450 nm. 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 from the reading at 540nm or 570 nm. This method allows correction of optical defects in the plate. The uncorrected reading values at a wavelength of 450nm can 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 has a dilution, the final result is multiplied by the dilution factor.

In the process of killing tumor cells, T cells release a large amount of cytokines. To further verify the killing function of T cells, this study examined cytokine secretion when T cells were co-cultured with target cells. YW327.6S2-CAR-T, CD19-CAR T and T cells were cultured for 24 hours in a ratio of 1:1 with A549, HCC827 ER3 and HCC827, respectively, and the supernatants were assayed for cytokine secretion using an ELISA kit. The results show that YW327.6S2-CAR T has significantly increased secretion of six cytokines IL-2, TNF-alpha, IFN-gamma, GM-CSF, Perforin and Granzyme B when co-cultured with the AXL positive expression cell lines A549 and HCC827 ER3, compared to the AXL negative expression cell line, and the differences are statistically significant. In addition, no significant cytokine secretion was seen by both CD19-CAR T and T cells after co-culture with target cells, as shown in figure 15. The above results laterally demonstrate the specific recognition and killing ability of YW327.6S2-CAR T on AXL positive target cells.

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

In the above examples, the experimental results were expressed as mean ± SD or SEM. Differences between groups were detected by the 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 to be statistically significant.

Example 7

YW327.6S2-CAR T cell in vivo killing experiment on non-small cell lung cancer

The experimental mice were female 3-4 week NSG mice purchased from Beijing Baiosaccae (NOD-Prkdc)^scidIL2rg^tm1Bcgen, BIOCYTOGEN), which is a three-line knockout mouse, mature T/B/NK cells cannot be detected in vivo, and thus endogenous T/B/NK cells do not interfere with CAR T cells. All mice were housed in an SPF environment, and were provided with autoclaved food and water for free access to food, 12 hours each per day of light and dark time.

The experimental materials used in this example included: 4% paraformaldehyde fixing solution (Nanjing Nuozhen Biotech Co., Ltd.), Ki-67 monoclonal antibody (CST Co., USA), small animal living body optical imaging system (PerkinElmer IVIS, USA), dissecting instrument (Shanghai Yuyan scientific instruments Co., Ltd.), vernier caliper (Beijing Spaceflight peak optoelectronics technology, Ltd.), and the rest of the required experimental reagents, instruments and consumables as described in the above examples.

Construction of NSCLC subcutaneous tumor model and tumor biological monitoring

(1) Two AXL-positively-expressed NSCLC cells, A549 and HCC827 ER3, were cultured, and 2X 10 cells were prepared per NSG mouse (6-8 weeks)⁶And (4) tumor cells.

(2) Both cells, A549 and HCC827 ER3, were digested with 0.25% pancreatin at 2X 10⁶Each tumor cell was resuspended in 100. mu.L PBS and prepared for inoculation.

(3) After the skin of the left groin of the mouse was disinfected, the tumor cells were aspirated with a 1mL syringe, and the skin was gently lifted and inoculated, with the result that a small lump of local skin was raised. Care was taken not to inject into the abdominal cavity or into the muscles in order to avoid tumor adhesion. Experiments were performed in groups of a549 and HCC827 ER3, which were divided into: YW327.6S2-CAR T cell group, CD19-CAR T cell group and uninfected T cell group, 6 mice per group.

(4) The change in body weight 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 formula for calculating the tumor volume is as follows: ab ═ V²V is volume, a is tumor major diameter, and b is tumor minor diameter.

CAR T cell therapy in NSCLC subcutaneous tumor model

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

(2) The tumor volume is about 50mm³At 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 infusion was performed separately, 1 × 10 per mouse⁷Individual effector cells (YW327.6S2-CAR T, CD19-CAR T, or uninfected T cells).

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

(4) After the experiment, the mice were euthanized, dissected and photographed by stripping the tumor tissue from the mice.

Treatment with YW327.6S2-CAR-T in combination with Erlotinib

Some studies have shown that AXL upregulation is a significant cause of drug resistance of lung cancer to EGFR-TKI, and that YW327.6S2 mab was able to down-regulate AXL expression. Therefore, the invention also designs an YW327.6S2-CAR-T combined Erlotinib treatment experiment;

(1) EGFR-TKI resistant cell HCC827 ER3 was cultured to construct NSG mouse (6-8 weeks) subcutaneous tumor model.

(2) Tumors grow to about 50mm³Thereafter, experiments were performed 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.

NC group: no treatment is carried out;

② Erlotinib group: erlotinib gavage, 100 mg/kg/day;

(iii) YW327.6S2-CART group: tail vein injection of 1X 10⁷YW327.6S2-CART cells were used to treat subcutaneous tumors of HCC827 ER 3;

and (4) a combined treatment group: tail vein injection of 5X 10⁶YW327.6S2-CART cells were gavaged in combination 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, dissected and stripped of tumors, a part of the mice is fixed, sections are subjected to immunohistochemical examination after paraffin embedding, and infiltration of the tumors CD3 and the expression condition of the tumors AXL are detected.

The experimental results are expressed as mean ± SD or SEM. Differences between groups were detected by one-way ANOVA or Bonferroni method. 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 to be statistically significant.

The experimental results are as follows:

to verify that YW327.6S2-CAR T cells inhibited the growth of NSCLC subcutaneous tumorsA549 and HCC827 ER3 tumor cells are used to construct subcutaneous tumor when the tumor grows to about 50mm³Experiments were performed in groups of YW327.6S2-CAR T, CD19-CAR T, or uninfected T cells, and tail vein infusions of the corresponding effector cells, respectively. From the tumor growth curves and the size of the detached tumors in fig. 16 and 17, it was shown that YW327.6S2-CAR T cells could effectively inhibit the growth of a549 and HCC827 ER3 tumors in vivo, while CD19-CAR T or uninfected T cells had essentially no tumor-inhibitory effect; and YW327.6S2-CAR T group was weighed as (A5490.10g, HCC827 ER30.13g) significantly lower than CD19-CAR T group (A5490.78, HCC827 ER 30.60) and uninfected T cell group (A5490.95, HCC827 ER 30.68).

FIG. 16 is the results of an experiment using YW327.6S2-CAR T cells to treat the A549 cell subcutaneous tumor model, A is the experimental flow chart: day 0 infusion of 2X 10⁶A549 cells, mouse subcutaneous tumor (n-6) with tumor length of about 50mm³Experiments were performed in groups, tail vein infusion YW327.6S2-CAR T, CD19-CAR T, or uninfected T cells, respectively, ending the experiment on day 39; b is a schematic diagram of taking a picture of the stripped tumor; c is tumor growth curve after CAR T infusion; d is tumor weighing result.

FIG. 17 is YW327.6S2-CAR T cell therapy HCC827 ER3 subcutaneous tumor model, A is experimental flow chart: day 0 infusion of 2X 10⁶Mouse subcutaneous tumor (n-6) is constructed by HCC827 ER3 cells, and the tumor grows to about 50mm³The experiments were performed in time groups, with tail vein infusion of YW327.6S2-CAR T, CD19-CAR T, or uninfected T cells, respectively, on day 18, and the experiments ended on day 45; b is a schematic diagram of taking a picture by stripping the tumor; c is tumor growth curve after CAR T infusion; d is tumor weighing result.

FIG. 18 is a flow chart and a result chart of YW327.6S2-CAR T cell combined Erlotinib treatment experiment, wherein A is an experiment flow chart; day 0 infusion of 2X 10⁶Mouse subcutaneous tumors (n-3) were constructed from HCC827 ER3 GL cells. Tumors grow to about 50mm³The experiment is carried out in groups, the treatment is carried out in groups on day 15, and the in vivo imaging monitoring is carried out on day 22 and day 36 respectively; b is a living body imaging schematic diagram. The results in fig. 18 show that the combination treatment group is able to destroy tumors better than the other groups. Y isW327.6S2-CAR-T cells at least down-regulate AXL expression of NSCLC cells through YW327.6S2 antibodies and improve the sensitivity of the NSCLC cells to Erlotinib, so that the drug resistance of NSCLC cells to EGFR-TKI can be reduced, and the anti-tumor curative effect is improved. And 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 on one hand, the drug resistance is inhibited, and on the other hand, the drug resistance and the Erlotinib are synergistically treated; c is the expression of an HCC827 ER3 tumor model AXL which is down-regulated after the AXL-CAR-T cell treatment, restores the sensitivity of the HCC827 ER3 to Erlotinib and promotes the treatment effect, and the AXL down-regulation does not occur in the Erlotinib group and the control group, so that the tumor control effect is not good enough.

AXL is highly expressed in tissues of patients with primary lung cancer and EGFR-TKI drug resistance and is not basically expressed in 19 normal tissues of a human body, which all suggest the importance and feasibility of using AXL as a lung cancer treatment target;

this study successfully constructed three AXL-targeting third generation (containing CD28, CD137 costimulatory domain) CAR T cells with different affinities, respectively: 3E8-CAR T, yw327.6s2-CAR T, 20G7D9-CAR T;

3E3E8-CAR T, YW327.6S2-CAR T, 20G7D9-CAR T cells have targeted killing effect on AXL-positively expressed NSCLC cell lines, and have almost no killing ability on AXL-negatively expressed NSCLC cells. Among these, YW327.6S2-CAR T cells showed the strongest killing ability against AXL-positively expressed NSCLC cells;

YW327.6S2-CAR T cells can kill the NSCLC cells positively expressed by AXL and secrete a large amount of cytokines IL-2, TNF-alpha, IFN-gamma, GM-CSF, Perforin and Granzyme B.

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

It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the technical solutions of the present invention, and are not intended to limit the specific embodiments of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention claims should be included in the protection scope of the present invention claims.

Claims (10)

1. Use of AXL-targeted CAR-T cells for the preparation of a product for the treatment of AXL-expressing lung cancer.

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

3. Use according to claim 1, characterized in that CAR-T cells carry protein sequences and/or proteins which down-regulate AXL expression.

4. The use of claim 1, wherein the lung cancer is non-small cell lung cancer.

5. The use of claim 1, wherein the product comprises an EGFR-TKI medicament.

6. The use of any one of claims 1 to 5, wherein the AXL-targeted CAR-T cell scFV protein is selected from one or more of 3E3E8, 20G7D9 or YW327.6S2.

7. A method for preparing CAR-T cells targeted to AXL, comprising the steps of:

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

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

s3, separating PBMCs (human peripheral blood mononuclear cells), sorting T cells from the separated PBMCs, and stimulating the T cells;

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

8. The method of claim 7, wherein the AXL-targeting scFv sequence comprises a gene sequence that expresses 3E3E8, YW327.6S2 or 20G7D9 protein.

9. The method of claim 7, wherein the Hinge region-Hinge is CD8 Hinge, the transmembrane region is CD28 transmembrane, and the costimulatory domain comprises CD28 and/or 4-1BB costimulatory domain.

10. An AXL-targeted CAR-T cell obtained by the method of making a CAR-T cell according to any one of claims 7-9.

Images