CN111057158A

CN111057158A - Chimeric antigen receptor targeting human GPC3 and application thereof

Info

Publication number: CN111057158A
Application number: CN202010179722.3A
Authority: CN
Inventors: 李俊; 郭志刚
Original assignee: Nanjing Landun Biotechnology Co Ltd
Current assignee: Nanjing Landun Biotechnology Co Ltd
Priority date: 2020-03-16
Filing date: 2020-03-16
Publication date: 2020-04-24

Abstract

The invention discloses a chimeric antigen receptor targeting human GPC3, which comprises an extracellular antigen binding region, a transmembrane region and an intracellular signal domain which are sequentially connected in series and are used for binding GPC3 antigen, wherein the extracellular antigen binding region comprises anti-GPC3scFv used for binding GPC3 antigen, the heavy chain amino acid sequence of the chimeric antigen receptor is shown as SEQ ID No.1, and the light chain amino acid sequence of the chimeric antigen receptor is shown as SEQ ID No. 2. Compared with the prior art, the invention takes human GPC3 as a target point to construct a chimeric antigen receptor, particularly carries out mutation on amino acids in ITAM2 and ITAM3 regions of CD3zeta, and the constructed T cells modified by the chimeric antigen receptor have stronger killing activity on tumor cells, have more central memory T cells, target-kill GPC3 positive tumor cells, effectively reduce the recurrence rate of tumors after treatment and prolong the life cycle of patients.

Description

Chimeric antigen receptor targeting human GPC3 and application thereof

Technical Field

The invention relates to a chimeric antigen receptor targeting human GPC3 and application thereof, belonging to the technical field of chimeric antigen receptors.

Background

Chimeric Antigen Receptors (CARs) are artificial receptors that mimic the function of T cell receptors, fusing the recognition and binding specificity of antigen and antibody or ligand and receptor and the killing ability of effector T cells to recognized tumor cells. The CAR is formed by sequentially connecting a CD8a leader peptide, an antigen recognition region (ligand or single chain antibody or Fab fragment), a transmembrane region, and a series of signal transduction regions of T cells (CD28, CD3, CD137 intracellular signaling domains). After the T cells are modified, the surface-expressed CAR is firstly combined with the surface antigen of the tumor cells through an antigen recognition area, and then an activation signal is transmitted into the cells through a signal transduction area of the CAR, so that the killing activity of the T cells on the tumor cells is activated in a targeted mode. The DNA sequence expressing the CAR is cloned into a lentivirus expression vector, T cells separated from blood of a patient are infected, the corresponding CAR is expressed on the surface of the T cells, the modified T cells are infused back into the body of the patient, and the modified T cells can kill tumor cells expressing related antigens in a targeted manner in the body of the patient, so that the effect of removing the tumor cells is achieved.

Liver cancer is one of the common malignant tumors, about 50 ten thousand cases of the common malignant tumors occur worldwide each year, the fatality rate is 3 rd in the malignant tumors, and the second is only lung cancer and gastric cancer. The characteristics of latent onset, high malignancy and high mortality of most patients cause the symptoms of the patients to be in the middle and late stages and lose the best time for surgical treatment. Even when tumors are resected, high postoperative recurrence and metastasis lead to poor therapeutic efficacy. Therefore, it is important to research and explore new therapeutic strategies.

Phosphatidylinositol proteoglycan 3(Glypican3, GPC3) is a membranous heparan sulfate proteoglycan protein. High expression in fetal liver, no expression in adult normal liver, but reactivation in liver cancer. The positive expression of GPC3 in primary liver cancer is about 80%, and the specificity is about 90%, which indicates that GPC3 can be used as a biomarker for specific diagnosis and prognosis evaluation of liver cancer, and is expected to become a new target point of targeted therapy of liver cancer.

At present, the application of CAR-T technology is limited to blood system tumors such as leukemia, myeloma and lymphoma, the CAR-T technology has less application in solid tumors such as liver cancer, and the CAR-T cell has greatly reduced recognition and killing capacity on tumor cells in a complex tumor microenvironment, so that the clinical application of the CAR-T technology is limited.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention discloses a chimeric antigen receptor targeting human GPC3 and application thereof.

The technical scheme is as follows: in order to achieve the purpose, the invention adopts the following technical scheme:

a chimeric antigen receptor targeting human GPC3, comprising an extracellular antigen-binding region, a transmembrane region and an intracellular signaling domain which are sequentially connected in series and bind GPC3 antigen, wherein the extracellular antigen-binding region comprises anti-GPC3scFv for binding GPC3 antigen, and the heavy chain amino acid sequence of the chimeric antigen receptor is shown as SEQ ID No.1, and the light chain amino acid sequence of the chimeric antigen receptor is shown as SEQ ID No. 2.

The chimeric antigen receptor can also comprise an extracellular signal peptide structure, and as an option, the extracellular signal peptide structure is CD8a signal peptide, and the amino acid sequence of the extracellular signal peptide structure is shown as SEQ ID number 4.

The chimeric antigen receptor may also include a hinge region structure connecting the extracytoplasmic antigen binding region and the transmembrane region. Alternatively, the hinge structure is selected from a CD8a hinge peptide segment, and the amino acid sequence of the hinge peptide segment is shown as SEQ ID NO. 5.

The transmembrane region may be selected from the CD4, CD8a, CD28 or 4-1BB transmembrane regions.

As an embodiment of the invention, the transmembrane region is selected from the CD8a transmembrane region, and the amino acid sequence thereof is shown as SEQ ID NO. 6.

The intracellular signal domain can be selected from one or more of CD27, CD28, 4-1BB (also named CD 137), OX40, intracellular domain peptide segments of CD3zeta or intracellular domain peptide segments of mutants thereof.

As an embodiment of the present invention, the intracellular signaling domain is an intracellular domain peptide segment of a CD3zeta mutant, wherein the amino acids of an Immunoreceptor Tyrosine Activation Motif (ITAM) 2 region and an Immunoreceptor Tyrosine Activation Motif (ITAM) 3 region are mutated, and the amino acid sequence is shown as SEQ ID NO. 3.

The intracellular signaling domain further comprises the amino acid sequence of the 4-1BB intracellular domain peptide fragment shown in SEQ ID NO. 7.

As a specific embodiment of the invention, the amino acid sequence of the chimeric antigen receptor targeting human GPC3 is shown in SEQ ID NO. 8.

The invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding the chimeric antigen receptor targeting human GPC 3. The nucleic acid molecule comprises a nucleotide sequence that can encode any of the extracellular membrane-binding regions, transmembrane regions, intracellular signaling domains, extracellular signal peptide structures, or hinge-containing structures described herein.

As a specific embodiment of the invention, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.9, and the coded amino acid sequence thereof is shown as SEQ ID NO. 8.

The invention also provides a vector containing the nucleic acid molecule.

The invention also provides a host cell containing the nucleic acid molecule or the vector.

The invention finally provides the application of the chimeric antigen receptor, the nucleic acid molecule, the vector and the host cell in preparing medicaments for preventing or treating tumors.

Has the advantages that: compared with the prior art, the invention takes human GPC3 as a target point to construct a chimeric antigen receptor, particularly carries out mutation on amino acids in ITAM2 and ITAM3 regions of CD3zeta, and the constructed T cells modified by the chimeric antigen receptor have stronger killing activity on tumor cells, have more central memory T cells, target-kill GPC3 positive tumor cells, effectively reduce the recurrence rate of tumors after treatment and prolong the life cycle of patients.

Drawings

FIG. 1 is a schematic diagram of a chimeric antigen receptor according to the present invention.

FIG. 2 is a graph of the flow results of GPC3-CAR-T cell lentivirus transfection efficiency.

FIG. 3 is a graph comparing the lytic killing ability of GPC3-CAR-T cells against HEPG2, a target cell.

FIG. 4 is a graph comparing the lytic killing ability of GPC3-CAR-T cells against Hu7 as a target cell.

FIG. 5 is a graph comparing the IFN- γ secretion capacity of GPC3-CAR-T cells.

FIG. 6 is a graph comparing the ability of GPC3-CAR-T cells to secrete IL-2.

Detailed Description

The present invention will be further illustrated with reference to the following specific examples.

Example 1 preparation of chimeric antigen receptor of the invention targeting human GPC 3.

(1) Chimeric Antigen Receptor (CAR) lentiviral expression vector construction

The intracellular domain of CD137 and the immune receptor tyrosine activation motif (ITAM) region of CD3Zeta are used as activation signals, and are fused with a single-chain antibody of GPC3 to construct a chimeric antigen receptor expression vector, and the chimeric antigen receptor expression vector is subcloned into a PLVX-EF1a vector (purchased from Clontech), and the combination sequence of each element in the constructed chimeric antigen receptor lentiviral expression vector is shown in FIG. 1:

the amino acid sequences of the elements in the constructed chimeric antigen receptor are respectively (synthesized by Nanjing Kingsrei Biotechnology Co., Ltd.):

signal peptide: SEQ ID NO.4

Extracellular antigen binding region sequence: SEQ NO.1 and SEQ NO.2

CD8a hinge region: SEQ ID NO.5

CD8a transmembrane region: SEQ ID NO.6

CD137 endodomain: SEQ ID NO.7

Intracellular domain of CD3Zeta mutant: SEQ ID NO. 3.

(2) The lentivirus is prepared by the following specific experimental steps:

s1, preparing 15cm dish, inoculating 5X 10⁶293T cells (purchased from ATCC) were cultured in complete medium (DMEM high-sugar, 10% FBS, double antibody) at 37 ℃ in 5% CO₂An incubator for overnight culture;

s2, 100 mu M PEI and lentivirus packaging plasmids (Lenti-EF 1a-CAR, pGP, pVSVG) were taken out of the refrigerator, thawed at room temperature, and then blown up and down by a pipette gun to be mixed completely. Remove PBS or HBSS buffer and warm to room temperature. Taking 2mL PBS to one hole of a 6-hole plate, respectively adding 10 mu g Lenti-EF1a-CAR, 4 mu g pGP and 2 mu g pVSVG, blowing and beating up and down a liquid transfer gun to be fully and uniformly mixed, adding 18 mu L and 100 mu M PEI, immediately blowing and beating up and down by using a liquid transfer machine to be uniformly mixed, and standing for 10 minutes at room temperature;

s3, dropwise adding the DNA/PEI complex into a 15cm culture dish, slightly shaking the culture dish, and fully mixing. Placing the culture dish at 37 ℃ and 5% CO₂An incubator for 6-8 hours, and then containing transfection reagentRemoving the culture medium and replacing the culture medium with a fresh complete culture medium;

s4, after continuous culture for 48 hours, the culture medium supernatant containing the virus in the culture dish was collected, filtered through a 0.45 μm filter, added with 20% by volume of 50% PEG6000 solution, incubated at 4 ℃ for 2 hours, transferred to a centrifuge tube, and after balancing, centrifuged at 3000 Xg 4 ℃ for 0.5 hour. After centrifugation, the liquid in the centrifuge tube was carefully aspirated in a biosafety cabinet, the pellet was resuspended by adding 500. mu.L of PBS buffer, and the virus was stored at-80 ℃.

(3) The separation of primary T cells comprises the following specific experimental steps:

s1, inverting the Lymphoprep reagent several times, and mixing the Lymphoprep reagent thoroughly;

s2, adding 15mL Lymphoprep reagent into a 50mL centrifuge tube (or a 15mL centrifuge tube according to the volume of the separated blood sample) in a biological safety cabinet for later use;

s3, diluting the blood sample with an equal volume of PBS +2% FBS;

s4, carefully adding the diluted blood sample to the upper layer of the separation reagent along the tube wall by using a pipette gun, so as to avoid mixing of the separation reagent and the blood sample;

s5, setting the centrifuge as 800Xg, setting the rotating speed reduction speed as slowest, and centrifuging for 20 minutes at room temperature;

s6, after the centrifugation is finished, collecting the upper layer of light yellow serum to another sterile centrifuge tube, and storing the light yellow serum at-80 ℃;

s7, gently sucking the monocyte layer at the interface of the serum and the separation reagent into a new centrifuge tube, and washing the cells once by using the culture medium;

s8, adjusting the cell density to 1 x 10⁸cells/mL (total volume not more than 2.5 mL), resuspended in 5mL round bottom tube;

s9, adding an antibody cocktail of 100 mu L/mL, fully mixing uniformly, and incubating for 15 minutes at room temperature;

s10, taking out the magnetic beads, blowing and beating the magnetic beads up and down for at least 5 times by using a pipette gun, and fully and uniformly mixing the magnetic beads;

s11, sucking 50 mu L of magnetic beads/mL into the sample, fully mixing the magnetic beads/mL and the sample, and incubating the mixture for 10 minutes at room temperature;

s12, adding a complete culture medium until the total volume in the tube is 2.5mL, inserting the tube (with a cover opened) into the magnetic pole, and standing for 5 minutes at room temperature;

s13, after incubation, keeping the tube in the magnetic pole, slightly inverting, and pouring out the cells in the tube;

s14, resuspend cells in X-vivo 15 medium, and add 10% FBS, 300U/mL IL-2, 5ng/mL IL-15, and 10ng/mL IL-7.

(4) The method comprises the following steps of (1) activating primary T cells and infecting lentiviruses:

s1, adjusting the cell density to 1 x 10⁶Adding cell factor and antibody compound (IL-2 with final concentration of 300U/mL, IL-7 with final concentration of 10ng/mL, IL-15 with final concentration of 5ng/mL, Anti-CD3 (OKT3) with final concentration of 500ng/mL and Anti-CD28 with final concentration of 2 ug/mL) into the cells/mL, and continuously culturing for 48 hours;

s2, calculating the required virus amount according to MOI = 20. The calculation formula is as follows: required amount of virus (mL) = (MOI × number of cells)/viral titer;

s3, taking out the virus from a refrigerator at minus 80 ℃, and quickly melting the virus in a water bath kettle at 37 ℃. Adding the virus amount obtained by the calculation into a six-hole plate, adding polybrene with the final concentration of 6 mug/mL, fully and uniformly mixing, sealing the four sides of the six-hole plate by using a sealing film, and centrifuging at 800Xg for 1 hour;

s4, tearing off the sealing film after the centrifugation is finished, placing the six-hole plate at 37 ℃ and 5% CO₂The incubator (2) is continuously cultured for 24 hours;

s5, 250Xg centrifugation for 10 minutes, remove virus containing culture medium supernatant, fresh medium heavy suspension cell precipitation, cell transfer to new six-well plate, continued to culture for 3-6 days for use.

GPC3-CAR-T cell lentivirus transfection efficiency flow test, the results are shown in FIG. 2.

Example 2 CAR-T cells lyse target cells.

The specific experimental steps are as follows:

s1, adjusting the state of the target cells to logarithmic growth phase, and continuously carrying out passage for 2 times before carrying out the experiment;

s2, digesting and suspending the attached target cells in complete culture medium by pancreatin, and adjusting the cell density to 5 x 10⁵One new 96-well plate was used to inoculate the target cells at a rate of 100. mu.L/well. The unused wells on the periphery of the 96-well plate were filled with 100. mu.L of sterile water per well to prevent evaporation of water from the middle experimental wells. Place the well plate in 5% CO₂Culturing in an incubator at 37 ℃ overnight;

s3, centrifugally collecting the prepared CAR-T cells, and resuspending the cells in a serum-free 1640 culture medium; the 96-well plate was removed from the incubator, the medium in the well was aspirated completely, the cells were washed gently once with sterile PBS, CAR-T cells were added at an E/T ratio of 1:1, 2:1, 4:1, 8:1, and the final volume was made up to 100 μ L/well for 3 replicates; the maximum release pore and the minimum release pore were seeded with the same number of target cells, but without CAR-T cells; place the well plate in 5% CO₂Culturing for 6 hours in an incubator at 37 ℃;

s4, after the culture is finished, taking out the pore plate from the incubator, adding a lysate in the LDH detection kit into the maximum release pore, completely lysing target cells in the lysate, centrifuging the 96 pore plate at the room temperature of 1200Xg for 5 minutes, gently taking out the plate, transferring 50 mu L of the lysate into another new 96 pore plate from each pore, adding an LDH detection reagent, and reading the OD value by using a microplate reader;

target cell lysis percentage calculation formula:

cleavage rate = [ (to-be-measured pore OD value-minimum release pore OD value)/maximum release pore OD value ]. 100%;

s5, the data thus processed are plotted using GraphPad 6.0.

The experimental results are as follows:

using CAR-T cells as effector cells, using hepatoma cell strains HepG2 and Hu7 naturally expressing GPC3 as target cells, establishing a co-culture system according to different effective target ratios (E/T), namely, 50000 target cells are fixed in each well in a 96-well plate, adding different amounts of CAR-T cells, culturing the co-culture system by using a serum-free culture medium, continuously culturing for 8 hours, taking out the well plate, centrifuging at room temperature of 1200Xg for 10 minutes to precipitate all suspended cells to the bottom of the well plate, taking out 30 mu L of supernatant from each well, and detecting the release amount of LDH in the supernatant of the culture medium to reflect the cracking capability of the CAR-T cells on the target cells, wherein the results are shown in FIGS. 3 and 4, the chimeric antigen receptor T cells targeting human GPC3 are expressed by the effective target ratio of 1:1, the T cells can be efficiently mediated to kill the tumor cells or the recombinant cells; with the increase of the effective target ratio, the killing effect of GPC3-CAR-T cells on target cells HepG2 and Hu7 is increased, and the highest effect is achieved when the effective target ratio is 8: 1.

Example 3 CAR-T cytokine secretion level assay.

The specific experimental steps are as follows:

s2, digesting and suspending the attached target cells in complete culture medium by pancreatin, and adjusting the cell density to 5 x 10⁵One new 96-well plate was used to inoculate the target cells at a rate of 100. mu.L/well. Adding 100 mu L of sterile water into each unused hole on the periphery of the 96-well plate to prevent water in the middle experimental hole from evaporating; place the well plate in 5% CO₂Culturing in an incubator at 37 ℃ overnight;

s3, centrifugally collecting the prepared CAR-T cells, and resuspending the cells in a serum-free 1640 culture medium; the 96-well plate was removed from the incubator, the medium in the wells was aspirated completely, the cells were gently washed once with sterile PBS, and then the incubation was performed as described in 1:1,2: 1,4: 1,8: 1, CAR-T cells were added and the final volume was made up to 100 μ L/well, 3 times in parallel; place the well plate in 5% CO₂Culturing for 6 hours in an incubator at 37 ℃; meanwhile, the T cells obtained in the isolation of primary T cells in example 1 were set as a control group;

s4, after the culture is finished, taking out the pore plate from the incubator, centrifuging the 96 pore plate at 1200Xg room temperature for 5 minutes, gently taking out the plate, transferring 50 mu L of culture medium supernatant from each pore, detecting the expression of IFN-gamma and IL-2 by using an ELISA kit, and reading the OD value by using an enzyme-labeling instrument;

s5, the obtained data are plotted using GraphPad 6.0.

The experimental results are as follows:

using CAR-T cells as effector cells, using a liver cancer cell line Hu7 naturally expressing GPC3 as target cells, establishing a co-culture system according to different effect-target ratios (E/T), namely, in a 96-well plate, the number of target cells fixed in each well is 50000, adding different numbers of CAR-T cells respectively, culturing the co-culture system by using a serum-free culture medium, continuously culturing for 8 hours, taking out the well plate, centrifuging at room temperature of 1200Xg for 10 minutes to ensure that all suspended cells are precipitated to the bottom of the well plate, then taking out 30 mu L of supernatant from each well, and detecting the expression quantities of IFN-gamma and IL-2 secreted after the CAR-T cells are activated by tumor cells in the supernatant of the culture medium by using an ELISA method; as shown in fig. 5 and 6, after the chimeric antigen receptor T cell targeting human GPC3 is combined with the targeted tumor cell, primary T cells can be activated effectively, and the secretory expression of cytokines can be increased; when the effective target ratio is 1:1, after the CAR-T cells are activated by tumor cells, a large amount of IFN-gamma and IL-2 can be secreted, which is obviously higher than that of control T cells; the secretion reached the highest value at an effective target ratio of 4: 1.

Sequence listing

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<120> chimeric antigen receptor targeting human GPC3 and application thereof

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gtgaccagct acgcccagaa gttccagggc cgcgtgaccc tgaccaacga caccagcacc 300

aacaccgtgt acatgcagct gaacagcctg accagcgccg acaccgccgt gtactactgc 360

gcccgctggg ccctgtgggg cgacttcggc atggacgtgt ggggcaaggg caccctggtg 420

accgtgagca gcggcggcgg cggcagcggc ggcggcggca gcggcggcgg cggcagcgac 480

atccagatga cccagagccc cagcaccctg agcgccagca tcggcgaccg cgtgaccatc 540

acctgccgcg ccagcgaggg catctaccac tggctggcct ggtaccagca gaagcccggc 600

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gacttcgcca cctactactg ccagcagtac agcaactacc ccctgacctt cggcggcggc 780

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accatcgcca gccagcccct gagcctgcgc cccgaggcct gccgccccgc cgccggcggc 900

gccgtgcaca cccgcggcct ggacttcgcc tgcgacatct acatctgggc ccccctggcc 960

ggcacctgcg gcgtgctgct gctgagcctg gtgatcaccc tgtactgcaa gcgcggccgc 1020

aagaagctgc tgtacatctt caagcagccc ttcatgcgcc ccgtgcagac cacccaggag 1080

gaggacggct gcagctgccg cttccccgag gaggagggcg gctgcgagct gcgcgtgaag 1140

ttcagccgca gcgccgacgc ccccgcctac cagcagggcc agaaccagct gtacaacgag 1200

ctgaacctgg gccgccgcga ggagtacgac gtgctggaca agcgccgcgg ccgcgacccc 1260

gagatgggcg gcaagccccg ccgccagaac ccccaggagg gcctgtacaa cgagctgcag 1320

aaggacaaga tggccgaggc ctacagcgag atcggcatga agggcgagcg ccgccgcggc 1380

cagggccacg acggcctgta ccagggcctg agcaccgcca ccaaggacac ctacgacgcc 1440

ctgcacatgc aggccctgcc cccccgc 1467

Claims

1. A chimeric antigen receptor targeting human GPC3, comprising an extracellular antigen-binding region, a transmembrane region and an intracellular signaling domain in tandem order, which bind GPC3 antigen, wherein the extracellular antigen-binding region comprises anti-GPC3scFv for binding GPC3 antigen, and the heavy chain amino acid sequence is shown as SEQ ID No.1 and the light chain amino acid sequence is shown as SEQ ID No. 2.

2. The chimeric antigen receptor targeting human GPC3 according to claim 1, further comprising an extracellular signal peptide structure, wherein the extracellular signal peptide structure is a CD8a signal peptide, and the amino acid sequence thereof is shown in SEQ ID No. 4.

3. The chimeric antigen receptor targeting human GPC3 according to claim 1, further comprising a hinge region structure selected from a CD8a hinge region peptide fragment, the amino acid sequence of which is shown in SEQ ID No. 5.

4. The chimeric antigen receptor targeting human GPC3 according to claim 1, wherein the transmembrane region is selected from the group consisting of CD4, CD8a, CD28, and 4-1BB transmembrane region.

5. The chimeric antigen receptor targeting human GPC3 according to claim 1, wherein the transmembrane region is selected from the group consisting of the CD8a transmembrane region and has the amino acid sequence shown in SEQ ID No. 6.

6. The chimeric antigen receptor targeting human GPC3 according to claim 1, wherein the intracellular signaling domain is selected from one or more of the intracellular domain peptide fragments of CD27, CD28, 4-1BB, OX40, CD3zeta or mutant intracellular domain peptide fragments thereof.

7. The chimeric antigen receptor targeting human GPC3 according to claim 1, wherein the intracellular signaling domain is selected from the group consisting of intracellular domain peptide fragments of a CD3zeta mutant, the amino acid sequence of which is shown in SEQ ID No. 3.

8. The chimeric antigen receptor targeting human GPC3 according to claim 1, wherein the intracellular signaling domain comprises the amino acid sequence of the 4-1BB intracellular domain peptide segment shown in SEQ ID No. 7.

9. The chimeric antigen receptor targeting human GPC3 according to claim 1, wherein the amino acid sequence is depicted in SEQ ID No. 8.

10. A nucleic acid molecule comprising a nucleotide sequence encoding the chimeric antigen receptor targeted to human GPC3 of claim 1.

11. The nucleic acid molecule of claim 10, wherein the nucleotide sequence of said nucleic acid molecule is set forth in seq id No. 9.

12. A vector comprising the nucleic acid molecule of claim 10 or 11.

13. A host cell comprising the nucleic acid molecule of claim 10 or 11, or the vector of claim 12.

14. Use of the chimeric antigen receptor of any one of claims 1 to 9, the nucleic acid molecule of claim 10 or 11, the vector of claim 12, the host cell of claim 13 for the preparation of a medicament for the prevention or treatment of a tumor.