CN114560948A

CN114560948A - Chimeric antigen receptor, CAR-T cell and application thereof

Info

Publication number: CN114560948A
Application number: CN202210214436.5A
Authority: CN
Inventors: 杨安钢; 阎博; 王鹏举; 王依依; 张仪婷; 贾林涛; 赵晶
Original assignee: Air Force Medical University of PLA
Current assignee: Air Force Medical University of PLA
Priority date: 2022-03-07
Filing date: 2022-03-07
Publication date: 2022-05-31
Anticipated expiration: 2042-03-07
Also published as: CN114560948B

Abstract

The invention discloses a chimeric antigen receptor with a CD3 gamma intracellular signal region, belonging to the technical field of biological medicines. The invention also discloses a CAR-T cell capable of expressing a chimeric antigen receptor having a CD3 γ intracellular signaling region, which may further include a CD8 signal peptide, a HER2 single chain antibody, a CD8hinge region, a CD8 transmembrane region, and a CD28 intracellular signaling region. The CAR-T cell provided by the invention has good targeting property, can accurately and specifically recognize and kill HER2 positive tumor cells, and simultaneously reduces the risk of inducing cytokine storm.

Description

Chimeric antigen receptor, CAR-T cell and application thereof

Technical Field

The invention relates to the technical field of biological medicines, and particularly relates to a chimeric antigen receptor, CAR-T cells and application thereof.

Background

CAR-T cell therapy has shown encouraging efficacy in treating hematological malignancies, however for solid tumors the complex tumor microenvironment limits the efficacy of CAR-T cells, preventing widespread use of CAR-T cell therapy. The obstacles facing CAR-T cell immunotherapy mainly include problems of easy CAR-T cell depletion, poor chemotactic ability and infiltration to tumor sites, tumor antigen escape and heterogeneity, and complex immunosuppressive tumor microenvironment. To overcome these limitations and expand the use of CAR-T cells to a wider range of malignancies, it is necessary to design entirely new "super" CARs from traditional CARs over conventional structures, giving CAR-T cells a more powerful and sophisticated "weaponry".

The classical CAR structure consists of a tumor antigen binding region, a hinge region, a transmembrane region, and an intracellular signal region. The extracellular antigen-binding region is typically a single chain antibody (scFv) from a monoclonal antibody that specifically binds to a tumor antigen in a Major Histocompatibility Complex (MHC) -independent manner. The intracellular signaling region consists of a signaling domain that triggers T cell activation. The first generation CAR had only one CD3 zeta domain; the second generation comprises a CD3 zeta domain and one costimulatory domain (CD28 or 4-1 BB); the third generation comprises the CD3 zeta domain and two costimulatory domains (CD28+ OX40 or CD28+4-1 BB); fourth generation CARs add pro-inflammatory cytokines and co-stimulatory ligands such as IL-7, IL-12, etc. on a second generation basis; fifth generation CARs were also based on second generation CARs with an additional insertion between the CD3 ζ and CD28 signaling domains of a STAT3/5 (transcription factor) sequence derived from a truncated cytoplasmic domain of the IL-2 receptor beta chain (IL-2R β) and fused at the C-terminus of CD3 ζ for binding to the tyrosine-XX-glutamine motif (YXXQ).

When CAR-T cells specifically recognize and bind to tumor antigens, tyrosine residues on immunoreceptor tyrosine-based activation motifs (ITAMs) can be phosphorylated by recruitable tyrosine kinases (lcks), and phosphorylation of ITAMs subsequently leads to recruitment and activation of ZAP-70 tyrosine kinases, which in turn leads to activation of downstream pathway signaling molecules such as LAT, SLP-76, Ras-MAP, etc., causing CAR-T cells to activate and kill tumor cells. Therefore, ITAMs play a key role in CAR-T cell signaling. Most of the CAR structures reported so far are based on the first generation CAR structure, using CD3 ζ containing 3 ITAMs as the first activation signal. Although all are engineered to combine different signal molecule domains, they are still limited to modifications based on the original domain. For example, using the native sequence of the CD3 zeta chain signaling motif (containing 3 ITAMs), or using the original sequence of the intracellular signaling domain of CD28, 4-1BB, the domain itself was not engineered at all. In particular, the most important CD3 ζ first signaling domain in CAR design is almost all intracellular full-length sequences containing 3 ITAMs.

CD3 is a transmembrane protein found on T cells and has four isoforms, CD3 δ, CD3 e, CD3 γ and CD3 ζ, where CD3 δ/CD3 e, CD3 γ/CD3 e constitute heterodimers. All CD3 chains contain ITAMs in their cytoplasmic domains. One CD3 zeta chain contained 3 ITAMs, while one CD3 delta/CD 3 epsilon/CD 3 gamma chain contained 1 ITAM each. Among them, CD3 γ not only plays a role in signal transduction during T cell activation, but also dynamically regulates the expression of TCR on the cell membrane surface.

Disclosure of Invention

Accordingly, the present invention is directed to a chimeric antigen receptor having intracellular signaling domains of CD28 and CD3 γ, which can perform targeted recognition and signal transduction during T cell activation.

The invention also aims to provide the CAR-T cell which can express the chimeric antigen receptor with the intracellular signal regions of CD28 and CD3 gamma, can obviously inhibit the growth of tumors and prolong the survival period of mice.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a chimeric antigen receptor which has intracellular signal regions of CD28 and CD3 gamma, wherein the nucleotide sequence of the CD3 gamma is shown as SEQ ID NO. 1.

Preferably, the amino acid sequence of the CD3 gamma is shown as SEQ ID NO. 2.

Preferably, the chimeric antigen receptor consists of a CD8 signal peptide, a HER2 single chain antibody, a CD8hinge region, a CD8 transmembrane domain, CD28, and an intracellular signal region of CD3 γ in tandem.

Preferably, the nucleotide sequence of the chimeric antigen receptor is shown as SEQ ID NO. 3.

Preferably, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO. 4.

The invention also provides a CAR-T cell expressing the chimeric antigen receptor described above.

The invention also provides a lentivirus expression vector, which comprises the nucleotide sequence of the chimeric antigen receptor.

The invention also provides a recombinant lentivirus which comprises the lentivirus expression vector.

Preferably, the recombinant lentivirus is prepared by cotransfecting mammalian cells with the lentivirus expression vector and a lentivirus packaging plasmid.

The invention also provides application of the CAR-T cell in preparing a medicine for treating tumors.

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

the chimeric antigen receptor prepared by the invention has intracellular signal regions of CD28 and CD3 gamma, can play the roles of target recognition and signal transduction in the process of activating T cells, and can dynamically regulate the expression of TCR on the surface of a cell membrane.

The Zeta-CAR-T, G-CAR-T cells prepared by the invention can obviously inhibit the growth of tumors and kill tumor cells specifically. The tumor inhibition effect of the modified G-CAR-T is more obvious compared with that of the traditional Zeta-CAR-T, the cytokine production level of the G-CAR-T is obviously reduced, the risk of inducing cytokine storm is greatly reduced, the perforin level of the G-CAR-T cell is slightly higher than that of the Zeta-CAR-T cell, and the survival period of the G-CAR-T treated mouse is longest.

Drawings

FIG. 1: the CAR structure of Zeta-CAR and G-CAR.

FIG. 2 is a schematic diagram: detecting the sorting effect of the T cells by flow cytometry; a: control; b: not sorting; c: and (6) after sorting.

FIG. 3: and detecting the CAR-T cell preparation effect by flow cytometry.

FIG. 4: the killing effect of different structures of CAR-T cells on target cells was observed by fluorescence microscopy (scale: 100 μm).

FIG. 5: the luciferase activity method is used for detecting the killing effect of the CAR-T cells in vitro.

FIG. 6: cytokine secretion levels after co-incubation of Zeta/G-CAR-T cells with target cells.

FIG. 7: granzyme B and perforin levels of Zeta/G-CAR-T cells.

FIG. 8: therapeutic profile of Zeta/G-CAR-T cells on mouse tumors.

FIG. 9: survival of NSG-bearing mice in the Zeta/G-CAR-T cell treatment group.

Detailed Description

The invention provides a chimeric antigen receptor, which has intracellular signal regions of CD28 and CD3 gamma, wherein the nucleotide sequence of the CD3 gamma is shown as SEQ ID NO. 1; the amino acid sequence of the CD3 gamma is shown as SEQ ID NO. 2.

The invention takes the chimeric antigen receptor containing the second generation CAR structure as the basis, selects CD3 gamma only containing one ITAM motif as a first activation signal, and carries out engineering transformation on the structural domain of the intracellular signal region of the chimeric antigen receptor to prepare the chimeric antigen receptor with the intracellular signal region of CD3 gamma, which can play the roles of target recognition and signal transduction in the T cell activation process.

The chimeric antigen receptor of the invention consists of a CD8 signal peptide, a HER2 single-chain antibody, a CD8hinge region, a CD8 transmembrane structural region, a CD28 and a CD3 gamma cellThe signal regions are formed in series, as shown in detail in FIG. 1. P in FIG. 1 of the present invention_EF1αFor promoter, CD8 Leader is signal peptide, P1h2 is HER2 single chain antibody, CD8Hinge + TM is CD8Hinge region + transmembrane region, CD28 is intracellular signal region, CD3 γ is intracellular signal region, wherein CD28 and CD3 γ belong to signaling region.

The invention changes a CD3 zeta molecule containing 3 ITAM motifs into a CD3 gamma sequence only containing 1 ITAM on the basis of a classical second-generation CD28-CD3 zeta-CAR structure, prepares a CAR structure containing different intracellular signal strengths, and can be used for preparing CAR-T cells with a novel structure.

The nucleotide sequence of the chimeric antigen receptor is shown as SEQ ID NO. 3. In the nucleotide sequence, the 1 st to 66 th positions are CD8 signal peptide sequences, the 67 th to 783 th positions are HER2 single-chain antibody sequences, the 784 th to 1032 th positions are CD8hinge regions and transmembrane regions, the 1033 th to 1155 th positions are CD28 intracellular signal regions, and the 1162 th to 1296 th positions are CD3 gamma intracellular signal regions.

The amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 4. In the amino acid sequence, the 1 st to 22 th sites are CD8 signal peptide sequences, the 23 th to 261 th sites are HER2 single chain antibody sequences, the 262 th to 344 th sites are CD8hinge region and transmembrane region, the 345 th to 385 th sites are CD28 intracellular signal region, and the 388 th to 432 th sites are CD3 gamma intracellular signal region.

The invention takes a humanized single-chain antibody P1h2(ZL 201510622152.X) targeting HER2 as an extracellular antigen binding region element of a CAR molecule, fuses a CD3 gamma single ITAM sequence at the end of an intracellular signal CD28, clones the single ITAM sequence into a lentiviral expression vector pLVX-EF1 alpha-IRES-Puro, and prepares a CAR molecule lentiviral expression plasmid containing a CD3 gamma first signal, which is named as G-CAR. After lentivirus packaging and concentration, infecting activated human primary T cells to successfully obtain G-CAR-T cells, namely the CAR-T cells expressing the chimeric antigen receptor.

The invention also provides a lentivirus expression vector, which comprises the nucleotide sequence (SEQ ID NO:3) of the chimeric antigen receptor. The lentivirus expression vector is preferably a lentivirus expression vector pLVX-EF1 alpha-IRES-Puro.

The plasmid pLVX-P1h2-CD28-CD3 gamma containing the chimeric antigen receptor can be obtained by carrying out double enzyme digestion and enzyme digestion product connection on the base sequence of the G-CAR and a lentivirus expression vector pLVX-EF1 alpha-IRES-Puro, and is named as G-CAR.

The invention also provides a recombinant lentivirus which comprises the lentivirus expression vector. The recombinant lentivirus is prepared by co-transfecting a mammalian cell with the lentivirus expression vector and a lentivirus packaging plasmid, a three-plasmid lentivirus packaging system is preferably adopted in the invention, the lentivirus expression vector is preferably pLVX-EF1 alpha-IRES-Puro, and the packaging plasmid is preferably pSPAX2 and pMD2. G.

The invention further provides a method for preparing the G-CAR-T cell by using the recombinant lentivirus, wherein the preparation method comprises the following steps:

(1) amplifying the G-CAR gene to construct a lentiviral expression vector;

(2) using a lentivirus expression vector to carry out virus packaging preparation;

(3) sorting and activating T cells;

(4) lentiviruses infect activated T cells to give G-CAR-T cells.

The invention also provides the application of the CAR-T cell in preparing a medicament for treating tumors, preferably, the tumors are HER2 positive.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In embodiments of the invention, HER2⁺Fluc⁺And HER2^-Fluc⁺PC9 (human lung cancer cell line) cell is stored in laboratory of China civil liberation army military medical university, and is usedRPMI 1640 culture medium containing 10% fetal bovine serum; HEK 293T (human renal epithelial cells) was purchased from shanghai cell bank of chinese academy of sciences using DMEM medium containing 10% fetal bovine serum; primary PBMC and T cells were isolated from peripheral blood of healthy volunteers and cultured in X-VIVO15 medium containing 5% human serum and 100IU/mL IL-2.

Example 1

This example describes the construction and identification of a Zeta/G-CAR plasmid.

1. Design of primers and PCR

(1) Based on the base sequences of the Zeta-CAR and the G-CAR, the Zeta-CAR and the G-CAR genes were amplified by primers shown in Table 1 using the base sequences as templates in the entire gene synthesis by the Oncomen Biotechnology (Beijing) Ltd.

The base sequence of the G-CAR is shown as SEQ ID NO. 3, the base sequence of the Zeta-CAR is shown as SEQ ID NO. 5, and the amino acid sequence of the Zeta-CAR is shown as SEQ ID NO. 6.

TABLE 1 amplification primers

The Zeta-F sequence in Table 1 is shown in SEQ ID NO 7; the Zeta-R sequence is shown as SEQ ID NO 8; the sequence of G-F is shown in SEQ ID NO. 9; the sequence of G-R is shown in SEQ ID NO 10.

PCR amplification was performed according to the instructions of Takara PrimeSTAR Max DNA Polymerase, see Table 2:

TABLE 2 PCR amplification reaction System

The PCR reaction conditions were as follows: pre-denaturation at 98 ℃ for 5 min; the denaturation reaction temperature is 98 ℃, and the time is 10 s; the annealing reaction temperature is 56 ℃, and the time is 5 s; the extension reaction temperature is 72 ℃ and the time is 20 s; the denaturation-annealing-extension process was carried out for 25 cycles. After the last cycle, the extension was carried out at 72 ℃ for another 10min to fully amplify the reaction.

(2) Agarose gel with concentration of 1% is prepared and put into an electrophoresis tank added with 0.5 × TBE electrophoresis buffer. After the PCR reaction is finished, adding 4 mu L of 6 × loading buffer into each tube of reaction product, uniformly mixing, adding into gel pores, and setting the constant current at 150mA and the electrophoresis time at 30min during electrophoresis. After the electrophoresis is finished, analyzing the electrophoresis result by using a gel imager, cutting off a gel fragment with a size suitable for a target strip, and recovering the gel by using a gel recovery kit of Beijing Optimalaceae New Biotechnology Limited.

The glue recovery steps are as follows:

a. adding 250 μ L Buffer BL into the adsorption column, centrifuging at 12000g for 1min, and activating silica gel membrane;

b. cutting a target DNA fragment from the agarose gel, cutting the gel without DNA as much as possible, and putting the gel into a 1.5mL EP tube when the obtained gel volume is smaller and better;

c. adding 500 μ L Buffer GL into the tube, placing the EP tube into a 65 deg.C water bath for 4-6min, and mixing once every 2-3min until the gel is completely melted and the solution is light yellow;

d. transferring the solution into an adsorption column EC, centrifuging at 12000g for 1min, discarding waste liquid, and returning the adsorption column EC into a collection tube;

e. adding 700 mu L of rinsing liquid Buffer W2 into the adsorption column for rinsing, centrifuging at 12000g for 1min, and discarding the waste liquid; repeating the steps once;

f. the adsorption column is put back into an empty collection tube and is centrifuged for 2min at 12000 g;

g. the column was taken out and put into a clean 1.5mL EP tube, and 50. mu.L of preheated ddH was added to the middle part of the adsorption film₂And O, standing at room temperature for 2min, centrifuging at 12000g for 2min, and collecting the liquid in the centrifuge tube after elution.

2. The enzyme digestion is connected to pLVX-EF1 alpha-IRES-Puro vector

(1) Enzyme digestion reaction

The recovered gel product and pLVX-EF1 alpha-IRES-Puro vector were subjected to double digestion with restriction enzymes EcoRI and MluI, respectively, see Table 3:

TABLE 3 cleavage system for target genes or vectors

Reaction conditions are as follows: enzyme digestion is carried out for 1.5h at 37 ℃, and the enzyme digestion product is subjected to glue recovery by using a glue recovery kit of the Scout New Industrial biotechnology (Beijing) Co.

(2) And (3) connection reaction:

the target gene and the restriction enzyme products of pLVX-EF1 alpha-IRES-Puro vector are connected by using Ligation Mix ligase, and the reaction system is shown in Table 4:

TABLE 4 ligation reaction System

Connection conditions are as follows: the reaction temperature was 16 ℃ and the reaction time was 30 min.

3. Identification of bacterial liquid

(1) Transformation of

Stbl3 E.coli competence was taken out from the-80 ℃ freezer and thawed on ice, ligation product was added to 50. mu.L competence, gently mixed and left on ice for 20 min. Then, the mixture was subjected to heat shock in a water bath at 42 ℃ for 90 seconds, rapidly transferred to ice, allowed to stand for 2 minutes, added with 150. mu.L of an antibiotic-free LB medium, placed in a shaker, and shaken at 37 ℃ and 150rpm for 30 minutes. And (3) sucking the bacterial liquid by using a liquid transfer gun, adding the bacterial liquid into an LB agar culture dish containing ampicillin resistance, then uniformly coating the bacterial liquid by using a clean glass coating rod, drying, and inversely placing the dish in a constant-temperature incubator at 37 ℃.

(2) Identification of bacterial liquid

Selecting a monoclonal on an LB culture dish of ampicillin the next day, dissolving the monoclonal in 15mL of LB culture solution containing ampicillin, putting the solution into a shaking table, shaking the solution at 37 ℃ and 220rpm for 12 h; taking 1mL of bacterial liquid sample, submitting the bacterial liquid sample to Beijing Optimalaceae New industry biotechnology limited company for sequencing identification, and extracting plasmids from strains with correct sequencing. The plasmids with correct sequencing results are respectively pLVX-P1h2-CD28-CD3 Zeta and are named as Zeta-CAR; pLVX-P1h2-CD28-CD3 gamma, designated G-CAR.

Example 2

This example prepared CAR-T cells.

1. Cell recovery and subculture

(1) Taking out HEK-293T cells from liquid nitrogen, placing the HEK-293T cells in a 37 ℃ water bath kettle for rapid melting, transferring the cell suspension into a 15mL centrifuge tube in a sterile ultra-clean workbench, adding 4-5 times of volume of DMEM complete culture solution containing 10% fetal calf serum, centrifuging at 800rpm for 5min, removing supernatant, and repeating the steps twice to obtain cell precipitates.

(2) Adding 5mL of complete culture solution into the cell sediment obtained in the step (1), completely resuspending the mixture, transferring the mixture to a 6cm cell culture dish, standing at 37 ℃ and 5% CO₂Culturing in an incubator.

(3) When the cell density reaches more than 80%, 0.05% of pancreatin can be added to digest the cells, and when a small amount of cells fall off from the bottom of the dish, complete culture solution is immediately added to stop digestion. Adjusting the cell state to be optimal can be used for lentiviral packaging.

2. Plasmid extraction

A three-plasmid lentiviral packaging system was used, and the plasmids used included the lentiviral expression vector and the lentiviral packaging plasmids pSPAX2 and pMD2. G. And (3) using an endotoxin-free large-scale extraction kit to extract a large amount of the required plasmid for later use.

3. Lentiviral packaging

(1) Cell preparation: and (3) inoculating the HEK-293T cells in the optimal culture state into a 10cm culture dish, uniformly shaking until the density reaches 80%, and using the HEK-293T cells for slow virus packaging.

(2) Preparing a transfection solution: to a 5mL EP tube, 1.25mL of Opti-MEM culture medium was added, 60. mu.L of Lipofectamine 2000 was added, and the mixture was mixed and allowed to stand for 5 min. To another 5mL EP tube was added an equal volume of culture medium, followed by 10. mu.g of the plasmid of interest, 7.5. mu.g of pSPAX2 and 2.5. mu.g of pMD2.G helper plasmid. Mixing the above mixed solution gently, and standing for 20 min.

(3) Will be provided withThe HEK-293T cell culture supernatant was discarded and 5mL of serum-free DMEM medium was added slowly. And (3) after the mixed solution in the step (2) is completely stood, discarding the culture solution, slowly adding the mixed solution into a dish, supplementing 2.5mL of Opti-MEM culture solution, and slightly shaking the bottom of the dish to ensure that the liquid is uniform. Placing the cells in an incubator at 37 deg.C with 5% CO₂Culturing under the condition for 6 h.

(4) After incubating the cells with the DNA-Lipofectamine 2000 complex for 6h, the liquid was discarded, and 10mL of DMEM was slowly added to complete the culture of the cells. The virus was collected at 48h and 72h after transfection, mixed well and centrifuged at 3000rpm for 5min to remove cell debris from the virus.

4. Lentiviral concentration

(1) The prepared virus solution was collected in a 50mL centrifuge tube and the volume was determined to be 20 mL.

(2) To the virus solution were added ice-cold 4.66mL of 50% PEG-6000 (i.e., final concentration: 8.5%) and 2.74mL of 3M NaCl solution (i.e., final concentration: 0.3M), and the mixture was mixed by inverting it every 20 to 30min and left at 4 ℃ for 1.5 hours.

(3) The centrifuge precools to 4 ℃ in advance, and the virus liquid is placed in the centrifuge and centrifuged for 30min at the rotating speed of 7000 g.

(4) And (4) discarding the centrifuged virus supernatant, and adding X-VIVO15 serum-free culture solution according to 100 times of concentration volume to resuspend virus precipitates.

5. Isolation of peripheral blood mononuclear cells

(1) Fresh anticoagulated whole blood was taken and diluted with an equal volume of physiological saline.

(2) And (3) adding a certain volume of separation liquid into the centrifugal tube in advance, slowly spreading the diluted blood sample above the liquid level of the separation liquid, and keeping the interface between the two liquid levels clear. The volume of the anticoagulated whole blood, the physiological saline and the separating medium is 1:1: 1.

(3) Centrifuging at room temperature and rotation speed of 800g for 30 min. The centrifuge has a rise damping of 4 and a fall damping of 0.

(4) After centrifugation, a thin, dense white membrane between the plasma and the separation layers was carefully drawn into another centrifuge tube.

(5) Diluting to a certain volume with normal saline, reversing and mixing evenly, leveling, rotating at 250g, centrifuging for 10min, discarding supernatant, and washing repeatedly for 2 times.

(6) The cells were resuspended in physiological saline for use.

6. Isolation of T cells

A defined amount of Human peripheral blood PBMC was collected, resuspended in 100. mu.L of Isolation buffer and transferred to a new tube, per 1X 10 of the instructions of the sorting Kit Mojoport Human CD 3T Cell Isolation Kit (Biolegend catalog: 480022)⁷mu.L of Biotin-Antibody Cocktail was added to each cell, mixed well and incubated on ice for 15 min. Then 10. mu.L of Streptavidin Nanobeads were added, mixed well and incubated on ice for 15 min. Adding the treated cells into 2.5mL of precooled separation buffer solution, placing the mixture in a magnetic frame for adsorption for 5min, taking up the magnetic frame, pouring the liquid into a new 15mL centrifuge tube, and repeating the steps once, wherein the cells in the centrifuge tube are CD3⁺The T cell of (1). Adding 800 μ L sorting buffer solution, centrifuging at 800g for 5min to wash the cell precipitate for 2 times, transferring the cells into X-VIVO15 culture solution containing 5% human serum and 100IU/mL IL-2, and culturing. As a result: flow cytometry detection of CD3 in pre-sorted PBMCs⁺The proportion of T cells was 82.28%, CD3 after sorting⁺The proportion of T cells reaches more than 98 percent, the requirements of subsequent experiments are met, and the sorting result is shown in figure 2.

7. Activation of T cells

Activation of CD3 by CD3/CD28 magnetic bead method⁺T cell: will be 1 × 10⁶The cells were resuspended in 1mL of X-VIVO15 medium containing 5% Human serum, IL-2 solution (100IU/mL) and 1:100 transfection-enhancing agent F108 were added at a ratio of 1:1000, 25. mu.L of magnetic beads (Dynabeads Human T-activator CD3/CD28, Life technologies Catalog:11161D) were added, and after stimulating the cells for 24h, the T cells were infected with lentivirus concentrate.

8. Lentiviral infection of T cells

(1) Lentivirus concentrate was resuspended at 5 × 10 with MOI ═ 50 infection⁵Activated T cells, the cell density was controlled at 1X 10⁶Centrifuge at 800g for 90min per mL.

(2) After centrifugation, the cells were resuspended in 48-well plates and placed at 37 ℃ in 5% CO₂Culturing in a cell culture box.

(3) After the virus infects the cells for 24h, the virus solution is removed and replaced by X-VIVO15 culture solution containing fresh 5% human serum and 100IU/mL IL-2 to maintain cell proliferation.

(4) After the cells are cultured for 5 days, the expression of the CAR molecules on the surface of the T cell membrane is detected by using flow cytometry. Flow detection results show that the virus has good effect of infecting T cells, the infection rate can reach more than 50 percent, namely the Zeta/G-CAR-T cells are successfully prepared, and the results are shown in figure 3.

Example 3

In this example, HER2 positive tumor cells were subjected to an in vitro killing experiment using Zeta/G-CAR-T cells prepared in example 2.

All data were statistically analyzed using GraphPad Prism 8.0, Two comparisons were performed using unpaired t-test, multiple comparisons were performed using Two-way-ANOVA test, when p < 0.05, differences between groups were considered statistically significant, and each experiment was repeated at least three times.

1. Dil staining

(1) HER2⁺PC9 and HER2^-PC9 cells were seeded into 48-well plates at 1X 10 cells/well⁵And (4) cells.

(2) After the cells were cultured overnight, the cell culture fluid was aspirated and the cells were washed 2 times with PBS.

(3) Add 250. mu.L of Dil cell membrane staining solution (10. mu.M concentration) and gently shake to make the dye uniformly cover all cells.

(4) Cells were incubated at 37 ℃ for 20min in the dark.

(5) Cell membrane staining solution was aspirated and washed 3 times with PBS.

(6) The cell culture fluid preheated at 37 ℃ is added to be observed under a fluorescence microscope.

2. Observing the killing condition of effector cells to target cells by using a fluorescence microscope

(1) Effector T cells were compared to Dil-stained target cells in an effective to target ratio of 2: 1 co-incubation, 4 replicate wells per set of experiments.

(2) After 6h, the upper layer of suspended effector T cells was aspirated and the cells were washed 2 times with 200 μ L of 1 × PBS to remove effector and dead target cells.

(3) And adding 100 mu L of 1 XPBS, observing the survival number of the target cells under a fluorescence microscope, randomly selecting 3 fields per hole for photographing, and evaluating the killing condition of effector cells on the target cells.

After Dil-labeled target cells were incubated for 6h with effector cells, the effector cells and dead target cells were removed by washing 2 times with PBS. The killing effect of CAR-T cells was observed under a fluorescent microscope, see figure 4. As can be seen in FIG. 4, both CAR-T cells were able to kill HER2 positive tumor cells and not HER2 negative tumor cells. The CAR-T cells with the two structures have good targeting property, and can accurately and specifically recognize and kill HER2 positive tumor cells.

3. Luciferase Activity assay for killing Activity of two CAR-T cells

(1) Digestion of PC9 (Fluc)⁺HER2⁺) Cell counts were plated in 96-well plates, 2X 10 cells per well⁴Cells were cultured overnight.

(2) Setting 6 groups of effective target ratios which are respectively 1: 1. 1: 2. 1: 4 or 1: effector cells were co-incubated with target cells, 4 replicates per set of experiments.

(3) After 16h, the upper layer of suspended effector T cells was aspirated, and the cells were washed 2 times with 200. mu.L of 1 XPBS to remove as much of the remaining effector and dead cells as possible.

(4) Adding 100 mu L of luciferase substrate working solution, standing for 2min, horizontally placing a 96-well plate on a Xenogen IVIS Lumina II living body for imaging, collecting a fluorescence value of a reaction well, and analyzing data by using software.

Zeta-CAR-T and G-CAR-T cells were incubated with PC9 (Fluc)⁺HER2⁺) Target cells were measured according to an effective target ratio of 1: 1. 1: 2. 1: 4 or 1: 8 incubation for 16h, with no effector cell added sample as control. The results are shown in FIG. 5. As can be seen from FIG. 5, both Zeta-CAR-T and G-CAR-T cells showed very significant killing effect on HER2 positive tumor cells, and with the increase of the effective target ratio, the killing effect of CAR-T cells on tumor cells was better. When the effective target ratio was increased to 1:1, CAR-T cells were able to almost completely lyse the target cells.

4. CAR-T cytokine secretion level detection

HER2⁺PC9 cells were plated in 96-well plates in advance, and Zeta/G-CAR-T effector cells resuspended in cytokine-free supplemented medium at an effective target ratio of 1:1 into a well plate. At 37 ℃ 5% CO₂The cells were incubated in the incubator for 24 hours, and the supernatant was collected by centrifugation at 500g for 10 min. By LEGENDplex^TMAnd (3) detecting the contents of cytokines IL-2, IL-6, IFN-gamma, GM-CSF and TNF-alpha in the supernatant by a multi-factor flow technology.

The results of detecting the secretion expression levels of IL-2, IL-6, IFN-gamma, GM-CSF and TNF-alpha of different CAR-T cells in the process of killing target cells are shown in FIG. 6. From FIG. 6, the levels of IFN- γ, IL-2, IL-6, GM-CSF, TNF- α cytokine production by G-CAR-T cells were significantly lower than that of Zeta-CAR-T (p < 0.0001), indicating that the levels of cytokine production by modified G-CAR-T cells were significantly lower and the risk of inducing cytokine storm was greatly reduced compared to conventional Zeta-CAR-T cells.

5. Flow cytometry detection of CAR-T cell granzyme B and perforin levels

HER2⁺PC9 cells were plated in 12-well plates in advance, at an effective target ratio of 1:1 addition of prepared CAR-T cells and co-incubation with monensin. After 6h of co-incubation, sufficient CAR-T cells were collected and tested for granzyme B and perforin levels by flow cytometry.

The results of the flow cytometry used to detect levels of granzyme B and perforin for both CAR-T cells are shown in figure 7. As can be seen from FIG. 7, granzyme B levels of G-CAR-T cells were comparable to that of Zeta-CAR-T cells, whereas perforin levels of G-CAR-T cells were slightly higher than that of Zeta-CAR-T cells.

Example 4

In this example, NSG tumor-bearing mice were further tested in vivo using Zeta/G-CAR-T cells prepared in example 2.

1. Establishment and treatment of NSG tumor-bearing mouse model

Male healthy NSG mice 5-6 weeks old were purchased from beijing baiosai picture and reared by a specialist. Collecting HER2 in logarithmic growth phase⁺Fluc⁺PC9 cells, perMice 5X 10⁵Individual cells were resuspended in 200 μ L PBS and inoculated subcutaneously in the right dorsal side of NSG mice. After 5 days, mice were randomly divided into 3 groups, and each group was injected via tail vein with 2X 10⁶Individual Zeta-CAR-T, G-CAR-T cells or control untransduced T cells were treated. Thereafter, tumors were imaged twice weekly and evaluated for growth trends.

2. Small animal in vivo imaging technology for monitoring tumor progression

Injecting D-fluorescein sylvite into abdominal cavity of tumor-bearing mouse, anesthetizing the mouse with isoflurane gas after 5-10min, placing the mouse in Xenogen IVIS Lumina II living body imaging equipment, collecting fluorescence, analyzing fluorescence value with software to evaluate tumor size, and imaging twice per week until the mouse dies.

The change in tumor size was recorded by continuous measurement of mouse live imaging as shown in FIG. 8, and it can be seen from FIG. 8 that the group treated with infusion of Zeta/G-CAR-T cells was able to significantly inhibit tumor growth compared to the control non-transduced T cells-treated group. Compared with the traditional Zeta-CAR-T, the G-CAR-T has more remarkable tumor inhibition effect.

3. Survival analysis

NSG tumor-bearing mice were randomly grouped, injected with the same number of Zeta-CAR-T, G-CAR-T cells or control T cells via tail vein, strictly monitored for vital signs, and mice survival curves were plotted, with the results shown in figure 9. As can be seen from FIG. 9, survival was significantly prolonged in Zeta/G-CAR-T cell treated mice and was longest in G-CAR-T treated mice compared to control non-transduced T cell treated mice.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> university of liberated military, air force, military and medical science of Chinese people

<120> chimeric antigen receptor, CAR-T cell and application thereof

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 135

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ggacaggatg gagttcgcca gtcgagagct tcagacaagc agactctgtt gcccaatgac 60

cagctctacc agcccctcaa ggatcgagaa gatgaccagt acagccacct tcaaggaaac 120

cagttgagga ggaat 135

<210> 2

<211> 45

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Gly Gln Asp Gly Val Arg Gln Ser Arg Ala Ser Asp Lys Gln Thr Leu

1 5 10 15

Leu Pro Asn Asp Gln Leu Tyr Gln Pro Leu Lys Asp Arg Glu Asp Asp

20 25 30

Gln Tyr Ser His Leu Gln Gly Asn Gln Leu Arg Arg Asn

35 40 45

<210> 3

<211> 1299

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atggcactgc ccgtcactgc actgctgctg cctctggccc tgctgctgca tgccgctagg 60

cccagcgata ttcagatgac ccagagccca agcagcctga gcgccagcgt tggcgaccgt 120

gtgacgatca cctgccgcgc aacgccgagc gtgagctata tgcattggta tcaacagaag 180

ccgggcaaag cgccaaaact gctgatttat acgaccagca atctggcgag cggcgtgccg 240

agccgtttca gcggtagcgg tagcggcacg gattttaccc tgaccattag cagcctggaa 300

ccggaagatt ttgcgaccta ttattgccag cagtggagcc gtagcccacc gacgttcggc 360

caaggcacca aagtggaaat taagggcggt ggcggtagtg gtggtggcgg ctctggcggt 420

ggtggtagcc aggtgcaact ggttcagagc ggtgcggaag tgaaaaaacc gggtgcgagc 480

gttaaagtga gctgcaaagc cagcggctat acctttaccg gccataccat gaactgggtg 540

cgtcaggcac cgggtcaaca gctggaatgg atgggcctga tcaacccata caatggcgat 600

acgaattaca atcagaagtt taaaggccgc gttacgttta ccgtggacac cagcacgagc 660

accgcgtaca tgcagctgag cagcctgcgt agccaagata ccgcagttta ctattgtgcg 720

cgtcgtgtta ccgattggta ctttgatgtg tggggccagg gcacgctggt tacggtgagc 780

agcttcgtgc ccgtgttcct gcctgccaag cccaccacca cacctgctcc gcggccccca 840

acccccgcac caaccattgc cagccagccc ctgagcctga ggcccgaggc ttgcaggccc 900

gcagctggag gagccgtgca cactagagga ctggacttcg cctgcgacat ctacatctgg 960

gcacccctgg ctgggacctg cggggtcctg ctgctgtccc tggtgattac actgtactgc 1020

aaccacagga atagatccaa aaggtccaga ctgctgcaca gcgactatat gaacatgact 1080

cccaggaggc ctggccccac caggaagcac taccaaccct acgcaccccc cagagacttc 1140

gccgcctaca ggagcactag tggacaggat ggagttcgcc agtcgagagc ttcagacaag 1200

cagactctgt tgcccaatga ccagctctac cagcccctca aggatcgaga agatgaccag 1260

tacagccacc ttcaaggaaa ccagttgagg aggaattga 1299

<210> 4

<211> 432

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Thr

35 40 45

Pro Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala

50 55 60

Pro Lys Leu Leu Ile Tyr Thr Thr Ser Asn Leu Ala Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

85 90 95

Ser Ser Leu Glu Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp

100 105 110

Ser Arg Ser Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser

145 150 155 160

Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly His Thr

165 170 175

Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gln Leu Glu Trp Met Gly

180 185 190

Leu Ile Asn Pro Tyr Asn Gly Asp Thr Asn Tyr Asn Gln Lys Phe Lys

195 200 205

Gly Arg Val Thr Phe Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr Met

210 215 220

Gln Leu Ser Ser Leu Arg Ser Gln Asp Thr Ala Val Tyr Tyr Cys Ala

225 230 235 240

Arg Arg Val Thr Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu

245 250 255

Val Thr Val Ser Ser Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr

260 265 270

Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser

275 280 285

Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly

290 295 300

Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp

305 310 315 320

Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile

325 330 335

Thr Leu Tyr Cys Asn His Arg Asn Arg Ser Lys Arg Ser Arg Leu Leu

340 345 350

His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg

355 360 365

Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg

370 375 380

Ser Thr Ser Gly Gln Asp Gly Val Arg Gln Ser Arg Ala Ser Asp Lys

385 390 395 400

Gln Thr Leu Leu Pro Asn Asp Gln Leu Tyr Gln Pro Leu Lys Asp Arg

405 410 415

Glu Asp Asp Gln Tyr Ser His Leu Gln Gly Asn Gln Leu Arg Arg Asn

420 425 430

<210> 5

<211> 1500

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atggcactgc ccgtcactgc actgctgctg cctctggccc tgctgctgca tgccgctagg 60

cccagcgata ttcagatgac ccagagccca agcagcctga gcgccagcgt tggcgaccgt 120

gtgacgatca cctgccgcgc aacgccgagc gtgagctata tgcattggta tcaacagaag 180

ccgggcaaag cgccaaaact gctgatttat acgaccagca atctggcgag cggcgtgccg 240

agccgtttca gcggtagcgg tagcggcacg gattttaccc tgaccattag cagcctggaa 300

ccggaagatt ttgcgaccta ttattgccag cagtggagcc gtagcccacc gacgttcggc 360

caaggcacca aagtggaaat taagggcggt ggcggtagtg gtggtggcgg ctctggcggt 420

ggtggtagcc aggtgcaact ggttcagagc ggtgcggaag tgaaaaaacc gggtgcgagc 480

gttaaagtga gctgcaaagc cagcggctat acctttaccg gccataccat gaactgggtg 540

cgtcaggcac cgggtcaaca gctggaatgg atgggcctga tcaacccata caatggcgat 600

acgaattaca atcagaagtt taaaggccgc gttacgttta ccgtggacac cagcacgagc 660

accgcgtaca tgcagctgag cagcctgcgt agccaagata ccgcagttta ctattgtgcg 720

cgtcgtgtta ccgattggta ctttgatgtg tggggccagg gcacgctggt tacggtgagc 780

agcttcgtgc ccgtgttcct gcctgccaag cccaccacca cacctgctcc gcggccccca 840

acccccgcac caaccattgc cagccagccc ctgagcctga ggcccgaggc ttgcaggccc 900

gcagctggag gagccgtgca cactagagga ctggacttcg cctgcgacat ctacatctgg 960

gcacccctgg ctgggacctg cggggtcctg ctgctgtccc tggtgattac actgtactgc 1020

aaccacagga atagatccaa aaggtccaga ctgctgcaca gcgactatat gaacatgact 1080

cccaggaggc ctggccccac caggaagcac taccaaccct acgcaccccc cagagacttc 1140

gccgcctaca ggagcactag tagggtgaaa ttcagcagaa gcgccgatgc cccagcctac 1200

caacaaggcc agaaccaact gtataacgaa ctgaacctgg gcaggaggga agagtacgac 1260

gtgctggaca aaagaagagg cagggacccc gagatgggcg gcaagcctag aagaaaaaac 1320

cctcaggaag ggctgtacaa cgagctgcag aaggacaaga tggccgaggc ttacagcgag 1380

atcggcatga agggcgagag aagaaggggc aagggccacg acggcctgta tcagggcctg 1440

agcaccgcca ccaaggacac ctacgacgcc ctgcacatgc aagcactgcc tccaaggtga 1500

<210> 6

<211> 499

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Thr

35 40 45

Pro Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala

50 55 60

Pro Lys Leu Leu Ile Tyr Thr Thr Ser Asn Leu Ala Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

85 90 95

Ser Ser Leu Glu Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp

100 105 110

Ser Arg Ser Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser

145 150 155 160

Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly His Thr

165 170 175

Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gln Leu Glu Trp Met Gly

180 185 190

Leu Ile Asn Pro Tyr Asn Gly Asp Thr Asn Tyr Asn Gln Lys Phe Lys

195 200 205

Gly Arg Val Thr Phe Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr Met

210 215 220

Gln Leu Ser Ser Leu Arg Ser Gln Asp Thr Ala Val Tyr Tyr Cys Ala

225 230 235 240

Arg Arg Val Thr Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu

245 250 255

Val Thr Val Ser Ser Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr

260 265 270

Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser

275 280 285

Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly

290 295 300

Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp

305 310 315 320

Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile

325 330 335

Thr Leu Tyr Cys Asn His Arg Asn Arg Ser Lys Arg Ser Arg Leu Leu

340 345 350

His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg

355 360 365

Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg

370 375 380

Ser Thr Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

385 390 395 400

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

405 410 415

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

420 425 430

Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu

435 440 445

Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys

450 455 460

Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu

465 470 475 480

Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu

485 490 495

Pro Pro Arg

<210> 7

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ggtacgaatt cgccaccatg gcactgcccg tc 32

<210> 8

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cgacgcgttc accttggagg cagtgcttgc atg 33

<210> 9

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ggtacgaatt cgccaccatg gcactgcccg tc 32

<210> 10

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cgacgcgttc aattcctcct caactggttt cctt 34

Claims

1. A chimeric antigen receptor is characterized in that the chimeric antigen receptor has intracellular signal regions of CD28 and CD3 gamma, and the nucleotide sequence of the CD3 gamma is shown as SEQ ID NO. 1.

2. The chimeric antigen receptor according to claim 1, wherein the amino acid sequence of CD3 γ is represented by SEQ ID NO 2.

3. The chimeric antigen receptor according to claim 1 or 2, characterized in that it consists of a CD8 signal peptide, a HER2 single chain antibody, a CD8hinge region, a CD8 transmembrane domain, a CD28 and an intracellular signal region of CD3 γ in tandem.

4. The chimeric antigen receptor according to claim 3, wherein the nucleotide sequence of the chimeric antigen receptor is represented by SEQ ID NO. 3.

5. The chimeric antigen receptor according to claim 3, wherein the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO. 4.

6. A CAR-T cell expressing the chimeric antigen receptor of any of claims 1 to 5.

7. A lentiviral expression vector comprising the nucleotide sequence of claim 4.

8. A recombinant lentivirus comprising the vector of claim 7.

9. The recombinant lentivirus of claim 8, wherein the recombinant lentivirus is prepared by co-transfecting a mammalian cell with the lentiviral expression vector of claim 7 and a lentiviral packaging plasmid.

10. Use of a CAR-T cell according to claim 6 in the manufacture of a medicament for the treatment of a tumour.