CN114163538B - Chimeric antigen receptor and chimeric antigen receptor T cell simultaneously targeting GPC3 and CD276, and preparation methods and applications thereof - Google Patents

Abstract

The invention discloses a chimeric antigen receptor and a chimeric antigen receptor T cell which simultaneously target GPC3 and CD276, and a preparation method and application thereof. Experiments show that chimeric antigen receptor T cells which simultaneously target GPC3 and CD276 have strong killing activity on tumor cell lines which simultaneously express GPC3 and CD276, and have strong killing activity on tumor cell lines which are negative to GPC3 and positive to CD 276. Therefore, the chimeric antigen receptor T cell has a great application prospect in the treatment of hepatocellular carcinoma and the like.

Description

Chimeric antigen receptor, chimeric antigen receptor T cells targeting both GPC3 and CD276 Preparation methods and applications thereof

Technical field

The present invention relates to biological products, specifically to chimeric antigen receptors targeting GPC3 and CD276 simultaneously, chimeric antigen receptor T cells and their preparation methods and applications.

Background technique

Hepatocellular carcinoma is a common malignant tumor, accounting for 90% of all primary liver cancers and the second most lethal cancer in the world. Most patients with hepatocellular carcinoma receive local or systemic therapies, such as chemotherapy, radiotherapy, liver resection, skin ethanol injection, radiofrequency ablation, various embolization therapies, antibody therapies such as immune checkpoint inhibitors, vascular endothelial growth factor receptor Inhibitors and targeted therapies such as sorafenib, however, the efficacy of these therapies are insufficient (Lee, Y.H., et al., Combined Immunotherapy for Hepatocellular Carcinoma: Radiotherapy, ImmuneCheckpoint Blockade and Beyond. Frontiers in Immunology, 2020.11), Irreparable harm is done to patients and their families. There is therefore an urgent need for new drugs to treat hepatocellular carcinoma.

CAR-T (chimeric antigen receptor T cell) technology is a cellular immunotherapy that has been developing rapidly in recent years. It uses genetic engineering means (such as transduction based on recombinant viral vectors) to express chimeric antigen receptors on the surface of T cells. By preparing and amplifying chimeric antigen receptor T cells in vitro, and then infusing them back into the human body, they activate the autoimmune system and exert efficient killing of tumor cells (Rafiq, S., C.S. Hackett, and R.J. Brentjens, Engineering strategies to overcome the current roadblocks in CAR T cell therapy.Nature Reviews ClinicalOncology,2020.17(3):p.147-167.Benmebarek,M.R.,et al.,Killing Mechanisms ofChimeric Antigen Receptor(CAR)T Cells.International Journal of MolecularSciences,2019.20(6 ).). Chimeric antigen receptor T cell technology is considered the most likely therapy to conquer cancer, and CAR-T cells targeting CD19 have produced good results in blood tumors. Currently, six CAR-T drugs have been approved for marketing around the world, demonstrating the huge application prospects of CAR-T cells in the treatment of malignant tumors.

However, CAR-T cell therapy faces the problems of heterogeneous expression of tumor antigens and antigen loss. First, in acute lymphoblastic leukemia, the CD19-negative cell population can evade recognition by T cells targeting the CD19 chimeric antigen receptor, leading to overgrowth of the cell population and relapse of the disease. In addition, in preclinical studies of pancreatic cancer, prostate cancer, and neuroblastoma, single-antigen-targeted chimeric antigen receptor T cells failed to eradicate tumors because the chimeric antigen receptor T cells were unable to recognize these tumor cells. Expressed low-density target antigens (Anurathapan, U.; Chan, R.C.; Hindi, H.F.; Mucharla, R.; Bajgain, P.; Hayes, B.C.; Fisher, W.E.; Heslop, H.E.; Rooney, C.M.; Brenner, M.K. ; Morrissette, J.J.D.; Martinez-Lage, M.; Brem, S.; Maloney, E.; Shen, A.; et al. A single dose of peripherally infused egfrvii-directed car t cells mediates antigen loss and induces adaptiveresistance in patients with recurrent glioblastoma.Sci.Transl.Med.2017,9,eaaa0984). In hepatocellular carcinoma, the elevated levels of GPC3 in serum inhibit the binding of GPC3-targeted chimeric antigen receptor T cells to cell surface GPC3, hindering the efficacy of chimeric antigen receptor T cells (Sun, L., et al., Shed antigen-induced blocking effect on CAR-T cells targeting Glypican-3 in HepatocellularCarcinoma. Journal for Immunotherapy of Cancer, 2021.9(4)).

CD276, also known as B7-H3, is a type of transmembrane protein belonging to the B7 family. CD276 is highly expressed in a variety of cancers, including lung adenocarcinoma, glioma, neuroblastoma, pancreatic cancer, and ovarian cancer, and is barely expressed in normal tissues. Monoclonal antibodies that specifically recognize CD276 mediate effective tumor clearance in multiple tumor models. CD276 is highly expressed in hepatocellular carcinoma and is associated with the progression of liver cancer and poor prognosis of tumor patients. CAR-T cells targeting CD276 can effectively kill hepatocellular carcinoma cell lines in vitro. However, single-target CAR-T cell therapy for solid tumors (such as hepatocellular carcinoma) faces the obstacle of heterogeneous expression of antigens.

Contents of the invention

In order to solve the above problems, the present invention provides a chimeric antigen receptor that simultaneously targets GPC3 and CD276, a chimeric antigen receptor T cell and its preparation method and application.

In order to achieve the above objects, the present invention adopts the following technical solutions:

In the first aspect, the present invention provides a chimeric antigen receptor that simultaneously targets GPC3 and CD276, and the chimeric antigen receptor is named GPC3-CD276 TanCAR.

Preferably, the chimeric antigen receptor includes a single-chain antibody targeting GPC3, a linker, a single-chain antibody targeting CD276, an extracellular hinge region, and a transmembrane region arranged in order from the amino terminus to the carboxyl terminus. , costimulatory signal area and CD3ζ intracellular area.

Preferably, the amino acid sequence of the single-chain antibody targeting GPC3 is shown in SEQ.ID.NO.3.

Preferably, the nucleotide sequence of the gene encoding the single-chain antibody targeting GPC3 is shown in SEQ.ID.NO.4.

Preferably, the linker includes repeated tandem G4S, such as (G4S)3 whose amino acid sequence is shown in SEQ.ID.NO.5.

Preferably, the linker, for example, the nucleotide sequence of the gene encoding (G4S)3 is shown in SEQ.ID.NO.6.

Preferably, the amino acid sequence of the single-chain antibody targeting CD276 is shown in SEQ.ID.NO.7.

Preferably, the nucleotide sequence of the gene encoding the single-chain antibody targeting CD276 is shown in SEQ.ID.NO.8.

Preferably, the extracellular hinge region is selected from the group consisting of CD8α hinge region, CD28 hinge region, IgG1 hinge region or IgG4 hinge region.

Preferably, the amino acid sequence of the CD8α hinge region is shown in SEQ.ID.NO.9.

Preferably, the nucleotide sequence of the gene encoding the CD8α hinge region is shown in SEQ.ID.NO.10.

Preferably, the transmembrane region is selected from the group consisting of CD8α transmembrane region, CD4 transmembrane region, CD28 transmembrane region, CD3ζ transmembrane region or ICOS transmembrane region.

Preferably, the amino acid sequence of the CD8α transmembrane region is shown in SEQ.ID.NO.11.

Preferably, the nucleotide sequence of the gene encoding the CD8α transmembrane region is shown in SEQ.ID.NO.12.

Preferably, the costimulatory signal region is selected from the group consisting of 4-1BB intracellular region, CD28 intracellular region, OX40 intracellular region, ICOS intracellular region or CD27 intracellular region.

Preferably, the amino acid sequence of the 4-1BB intracellular region is shown in SEQ.ID.NO.13.

Preferably, the nucleotide sequence of the gene encoding the 4-1BB intracellular region is shown in SEQ.ID.NO.14.

Preferably, the amino acid sequence of the CD3ζ intracellular region is shown in SEQ.ID.NO.15.

Preferably, the nucleotide sequence of the gene encoding the intracellular region of CD3ζ is shown in SEQ.ID.NO.16.

Preferably, the chimeric antigen receptor includes the amino acid sequence shown in SEQ.ID.NO.1.

Preferably, the gene sequence of the chimeric antigen receptor includes the nucleotide sequence shown in SEQ.ID.NO.2.

In a second aspect, the present invention provides a chimeric antigen receptor T cell that simultaneously targets GPC3 and CD276. The T cell includes the above chimeric antigen receptor that simultaneously targets GPC3 and CD276 (that is, the T cell surface has simultaneous targets. chimeric antigen receptor to GPC3 and CD276).

In a third aspect, the present invention provides a recombinant virus vector and a recombinant virus. The recombinant virus vector includes the encoding gene of the above-mentioned chimeric antigen receptor. The recombinant virus is packaged by lentivirus using the recombinant virus vector.

Preferably, the gene encoding the chimeric antigen receptor includes a gene encoding a signal peptide connected in sequence from the 5' end to the 3' end, a gene encoding a single chain antibody targeting GPC3, and a linker (such as (G4S)3 ), genes encoding single-chain antibodies targeting CD276, genes encoding extracellular hinge regions (such as the CD8α extracellular hinge region), genes encoding transmembrane regions (such as the CD8α transmembrane region), and costimulatory signal regions (For example, genes encoding the intracellular region of 4-1BB) and genes encoding the intracellular region of CD3ζ.

Preferably, the signal peptide is a CD8α signal peptide, and the nucleotide sequence of the gene encoding the CD8α signal peptide is shown in SEQ.ID.NO.19.

Preferably, the amino acid sequence of the CD8α signal peptide is shown in SEQ.ID.NO.20.

Preferably, in the recombinant virus vector and the recombinant virus, the gene sequence of the chimeric antigen receptor that simultaneously targets GPC3 and CD276 is shown in SEQ.ID.NO.17, and the gene sequence includes the chimeric antigen receptor. The nucleotide sequence of the coding gene. Compared with the nucleotide sequence shown in SEQ.ID.NO.2 in the first aspect, the nucleotide sequence shown in SEQ.ID.NO.17 has an added gene encoding the CD8α signal peptide. The gene encoding the signal peptide can better direct the expression of the chimeric antigen receptor to the cell surface.

Preferably, the amino acid sequence of the chimeric antigen receptor is shown in SEQ.ID.NO.18, specifically as follows: MALPVTALLLPLALLLHAARPDVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKRGGGGSGGGGSGGGGSQVQLVQ SGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRD EDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQLTQSPSFLSSVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Preferably, the recombinant viral vector is a pWPXLd lentiviral vector.

In a fourth aspect, the present invention provides a method for preparing chimeric antigen receptor T cells that simultaneously target GPC3 and CD276, including the following steps:

1) The gene sequence of the chimeric antigen receptor GPC3-CD276 TanCAR targeting both GPC3 and CD276 is constructed or artificially synthesized through gene cloning. The gene sequence of the GPC3-CD276 TanCAR includes the core as shown in SEQ.ID.NO.2 The nucleotide sequence (for example, the gene sequence of GPC3-CD276 TanCAR is shown in SEQ.ID.NO.17);

2) Insert the gene sequence of the GPC3-CD276 TanCAR into the pWPXLd vector to obtain the recombinant plasmid pWPXLd-GPC3-CD276 TanCAR;

3) Co-transfect host cells with the recombinant plasmid pWPXLd-GPC3-CD276 TanCAR, envelope plasmid and packaging plasmid to obtain recombinant lentivirus;

4) The recombinant lentivirus is transfected into CD3-positive T lymphocytes (CD3-positive T cells for short), and chimeric antigen receptor T cells targeting both GPC3 and CD276 are obtained after isolation.

Preferably, the envelope plasmid is PMD2G, the packaging plasmid is psPAX2, and the host cell is HEK293T cells.

Preferably, in step 4, CD3-positive T lymphocytes are isolated from human peripheral blood mononuclear cells.

Preferably, the human peripheral blood mononuclear cells are derived from autologous venous blood, autologous bone marrow, umbilical cord blood or placental blood.

In a fifth aspect, the present invention provides a drug for treating hepatocellular carcinoma, which includes the above chimeric antigen receptor T cells targeting both GPC3 and CD276.

The beneficial effects of the present invention are reflected in:

The chimeric antigen receptor T cells proposed by the present invention that simultaneously target GPC3 and CD276 not only have a killing effect on tumor cells expressing GPC3 and CD276, but also have killing activity on GPC3-negative CD276-positive tumor cells, solving the problem of antigen heterogeneity. The problem of poor efficacy of chimeric antigen receptor T cells in the treatment of solid tumors such as hepatocellular carcinoma caused by sexual expression or antigen loss.

Description of the drawings

Figure 1 is a schematic structural diagram of the gene encoding GPC3-CD276 TanCAR.

Figure 2 shows the flow cytometric analysis results of GPC3-CD276 TanCAR expression on T cells; the UTD group is CD3-positive T cells that have not been transduced with the virus, and the GPC3-CD276 TanCAR-T group is chimeric that targets both GPC3 and CD276. Antigen receptor T cells.

Figure 3 shows the analysis results of the killing activity of chimeric antigen receptor T cells targeting both GPC3 and CD276 against the Huh7 cell line; among them: the UTD group is CD3-positive T cells that have not been transduced with the virus, and the GPC3-CD276 TanCAR-T group is Chimeric antigen receptor T cells targeting both GPC3 and CD276.

Figure 4 shows the results of the analysis of the killing activity of chimeric antigen receptor T cells targeting both GPC3 and CD276 against the SK-HEP-1-CD276 cell line; among them: the UTD group is CD3-positive T cells that have not been transduced with the virus, GPC3 CAR- The T group is a chimeric antigen receptor T cell that targets GPC3 alone, and the GPC3-CD276 TanCAR-T group is a chimeric antigen receptor T cell that targets both GPC3 and CD276; * indicates a significant difference.

Detailed ways

The present invention will be further described in detail below with reference to the accompanying drawings and examples. The embodiments are only used to explain the present invention, but not to limit the scope of protection of the present invention.

(1) Preparation of chimeric antigen receptor T cells targeting GPC3 and CD276 simultaneously

(1) Preparation of the gene sequence of the chimeric antigen receptor GPC3-CD276 TanCAR targeting both GPC3 and CD276

Referring to Figure 1, the gene sequence of the chimeric antigen receptor GPC3-CD276 TanCAR, which targets both GPC3 and CD276, includes the coding genes for the following elements from the 5' end to the 3' end: the coding gene for CD8α signal peptide (CD8αSP) , the coding gene of the single-chain antibody targeting GPC3 (GPC3-scFv), the coding gene of the linker "(G4S)3", the coding gene of the single-chain antibody targeting CD276 (CD276-scFv), the CD8α hinge region (CD8αhinge ), the coding gene for the CD8α transmembrane region (CD8αTM), the coding gene for the 4-1BB intracellular region (4-1BB), and the coding gene for the CD3ζ intracellular region (CD3-zeta).

The nucleotide sequence of the gene encoding the CD8α signal peptide is shown in SEQ.ID.NO.19, and the nucleotide sequence of the gene encoding the single-chain antibody targeting GPC3 is shown in SEQ.ID.NO.4. The nucleotide sequence of the gene encoding (G4S)3 is shown in SEQ.ID.NO.6. The nucleotide sequence of the gene encoding the single-chain antibody targeting CD276 is shown in SEQ.ID.NO.8. CD8α The nucleotide sequence of the gene encoding the hinge region is shown in SEQ.ID.NO.10, the nucleotide sequence of the gene encoding the CD8α transmembrane region is shown in SEQ.ID.NO.12, and the 4-1BB intracellular region The nucleotide sequence of the encoding gene is shown in SEQ.ID.NO.14, and the nucleotide sequence of the encoding gene of the CD3ζ intracellular region is shown in SEQ.ID.NO.16. By connecting the stop codon at the 3' end of the gene encoding the CD3ζ intracellular region, the gene sequence of the chimeric antigen receptor GPC3-CD276 TanCAR is obtained, as shown in SEQ.ID.NO.17.

The gene sequence of the chimeric antigen receptor GPC3-CD276 TanCAR was gene synthesized by Jiangsu Jinweizhi Biotechnology Co., Ltd.

(2) Construction of recombinant plasmid pWPXLd-GPC3-CD276 TanCAR

The gene sequence of the synthesized GPC3-CD276 TanCAR was inserted between the BamH1 and EcoR1 restriction sites of the pWPXLd vector, and then transferred into E. coli competent cells DH5α for positive clone PCR identification and sequencing identification. The verified correct plasmid was labeled as the recombinant plasmid pWPXLd-GPC3-CD276 TanCAR for subsequent experiments.

(3) Construction of recombinant lentivirus

The recombinant plasmid pWPXLd-GPC3-CD276 TanCAR, packaging plasmid psPAX2 and envelope plasmid pMD2G were co-transfected into cultured HEK293T cells using lipofectamine3000 transfection reagent. Collect the virus-containing supernatant at 48 hours: First, centrifuge the culture system at room temperature at 2000 rpm for 5 minutes, take the supernatant, and then filter it through a 0.45 μm filter membrane. The obtained recombinant lentivirus supernatant is used for T cell infection.

(4) Preparation of chimeric antigen receptor T cells

a) Isolation of PBMC (Peripheral Blood Mononuclear Cells)

Source of PBMC: autologous venous blood from healthy volunteers.

The operation process of isolating PBMC is as follows: extract the blood of the healthy volunteers, use Ficoll to collect peripheral blood mononuclear cells, centrifuge and separate the middle layer cells, and obtain PBMC after washing with PBS and counting.

b) Isolation of antigen-specific T lymphocytes using immunomagnetic beads

Take the above PBMC, add KBM581 culture medium containing 10% serum and an appropriate amount of IL-2 to form a cell suspension; according to the ratio of the number of magnetic beads to cells is 1:1, add CD3/CD28 immunomagnetic beads and shake at room temperature. The bed was incubated at a speed of 20 rpm for 45 minutes; a magnet was used to screen the cells incubating the magnetic beads. After removing the unadsorbed cell suspension, the above-mentioned KBM581 medium was added to resuspend the magnetic bead-cell mixture to obtain CD3-positive T lymphocytes. Continue After 24 hours of culture, the cells were used for lentiviral infection.

c) Preparation of antigen-specific T lymphocytes by viral transfection

The CD3-positive T lymphocytes separated by the immunomagnetic bead method are taken, and the recombinant lentivirus corresponding to the number of CD3-positive T lymphocytes is added for culture.

On the 3rd day of culture, a certain number of CD3-positive T lymphocytes infected with the recombinant lentivirus were collected, and the expression of CAR on the cell surface was analyzed by flow cytometry. The results are shown in Figure 2. Compared with CD3-positive T lymphocytes that were not transduced with the virus, Compared with the cells, T cells infected with the recombinant lentivirus had approximately 14% surface expression of GPC3-CD276 TanCAR, indicating that chimeric antigen receptor T cells targeting both GPC3 and CD276 were successfully prepared. Continue to culture for 48 hours, collect chimeric antigen receptor T cells targeting both GPC3 and CD276 for killing experiment analysis, or store them in cell cryopreservation solution and place them in a programmed cooling box at -80°C for 24 hours before transfer. to liquid nitrogen tank for long-term storage.

(2) Use the RTCA system to analyze the killing activity of chimeric antigen receptor T cells that simultaneously target GPC3 and CD276 against liver cancer cell lines.

2.1 Experiment 1 Control and Effector Cell Grouping

According to the effector cells added to the target cell Huh7 (a liver cancer cell line expressing both GPC3 and CD276), it was divided into: UTD group (adding CD3-positive T lymphocytes without virus transduction) and GPC3-CD276 TanCAR-T group (simultaneously Chimeric antigen receptor T cells targeting GPC3 and CD276), and a control group (control, culture medium group) was set up.

2.2 Experiment 2 Control and Effector Cell Grouping

According to the effector cells added to the target cell SK-HEP-1-CD276 (a liver cancer cell line that overexpresses CD276 but does not express GPC3), it is divided into: UTD group (added CD3-positive T lymphocytes without virus transduction), GPC3 CAR-T group (the gene sequence corresponding to the chimeric antigen receptor GPC3 CAR lacks linker-CD276-scFv compared with the GPC3-CD276Tan CAR; the CAR-T cell production method is consistent, and the positive rate of GPC3 CAR expression is approximately 30%), GPC3 -CD276 TanCAR-T group (chimeric antigen receptor T cells targeting both GPC3 and CD276), and a control group (control, medium group).

2.3 Experimental operations

First, use 50 μL DMEM or RPMI1640 culture medium to balance the RTCA single plate, and then collect the cultured target cells Huh7 or SK-HEP-1-CD276. After counting the cells, add 50 μL cell suspension containing 5000 target cells to each well. solution, place it in a 37°C incubator for 15 minutes, and then place it in an RTCA resistance system. After 24 hours, according to the ratio of different effector cells (such as collected GPC3-CD276 TanCAR-T cells) to target cells (E:T) 20 :1, 10:1, 5:1, add 100μL of the effector cell suspension of the corresponding number of cells to the target cells, set at least 2 duplicate wells for each group, and then put them into the RTCA resistance system to kill them after a certain period of time. Activity analysis.

2.4 Result analysis

Effector cell killing rate analysis was performed using cell index at specified time points.

The formula for calculating the killing rate of the UTD group:

UTD killing rate=(control _{cell index} -UTD _{cell index} )/control _{cell index} ×100

The calculation formula for the killing rate of the GPC3-CD276 TanCAR-T group and GPC3 CAR-T group:

CAR-T killing rate = (control _{cell index} -CAR-T _{cell index} )/control _{cell index} ×100

Experimental results show (Figure 3) that compared with UTD (killing for 24 hours), GPC3-CD276 TanCAR-T, chimeric antigen receptor T cells targeting both GPC3 and CD276 have a stronger effect on the GPC3-positive CD276-positive liver cancer cell line Huh7 The killing activity is better when the effect-to-target ratio (E:T) is 10:1 or 20:1.

In addition, the experimental results also show (Figure 4) that when the effect-to-target ratio is 20:1, compared with GPC3 CAR-T (killing for 20 hours), GPC3-CD276 TanCAR-T targets the chimeric antigen receptor of GPC3 and CD276 simultaneously. T cells have a stronger killing effect on the GPC3-negative CD276-positive liver cancer cell line SK-HEP-1-CD276.

In summary, the chimeric antigen receptor T cells constructed in the present invention targeting both GPC3 and CD276 have strong killing activity against GPC3 and CD276-positive tumor cell lines, and compared with GPC3 CAR-T, they are more effective against GPC3-negative CD276-positive tumors. The cell line has a stronger killing effect and overcomes the problem of tumor antigen escape caused by heterogeneous expression of GPC3. Therefore, the chimeric antigen receptor T cells that simultaneously target GPC3 and CD276 have great application prospects in the treatment of solid tumors such as hepatocellular carcinoma.

<110> Shenzhen Institute of Advanced Technology; Shenzhen University of Technology, Chinese Academy of Sciences (in preparation)

<120> Chimeric antigen receptors and chimeric antigen receptor T cells targeting GPC3 and CD276 simultaneously and their preparation methods and applications

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gggggtggag gatctggagg tgggggctct gggggtgggg gatctgacat tcagctgaca 1200

cagagcccta gcttcctgtc tgcctctgtg ggggacagag tgaccatcac ctgcaaggct 1260

tctcagaatg tggacaccaa tgtggcctgg tatcagcaga agcctggcaa ggcccccaag 1320

gccctgatct actctgctag ctacagatac tctggggtgc ctagcagatt ctctggctct 1380

ggctctggca cagacttcac cctgaccatc agcagcctgc agcctgagga ctttgccacc 1440

tactactgtc agcagtacaa caactacccc ttcacctttg gccaaggcac taagctggaa 1500

atcaagacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 1560

cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 1620

gggctggact tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 1680

cttctcctgt cactggttat caccctttac tgcaaacggg gcagaaagaa actcctgtat 1740

atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1800

tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 1860

gcagacgccc ccgcgtacaa gcagggccag aaccagctct ataacgagct caatctagga 1920

cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1980

aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 2040

gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 2100

ggcctttacc agggtctcag tacagccacc aaggacacct acgacgcct tcacatgcag 2160

gccctgccccctcgctga 2178

<210> 3

<211> 243

<212> PRT

<213> GPC3-scFv

<400> 3

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Ala Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn

85 90 95

Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe

180 185 190

Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

195 200 205

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

210 215 220

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

225 230 235 240

Val Ser Ser

243

<210> 4

<211> 729

<212> DNA

<213> GPC3-scFv

<400> 4

gatgtggtta tgacccaaag ccccctgagc ctgcctgtga ctcctgggga gcctgctagc 60

atcagctgca gaagctctca gagcctggtg cacagcaatg ccaacacccta cctgcactgg 120

tacctgcaga agcctgggca gagccctcag ctgctgatct acaaggtgag caatagattt 180

tctggggtgc ctgacagatt ttctggctct ggctctggca cagacttcac cctgaagatc 240

agcagagtgg aggctgagga tgtgggggtg tactactgct ctcagaacac ccatgtgccc 300

cccacctttg gccaaggcac aaagctggag atcaagagag gtggcggtgg ctcgggcggt 360

ggtgggtcgg gtggcggcgg atctcaagtg cagctggtgc agtctggggc tgaggtgaag 420

aagcctgggg cctctgtgaa ggtgagctgc aaggcctctg gctacacctt cacagactat 480

gagatgcact gggtcagaca agcccctggc caaggcctag aatggatggg ggccctggac 540

cccaagactg gggacacagc ctactctcag aagttcaagg gcagagtgac cctgacagct 600

gatgagagca caagcacagc ctacatggag ctgagcagcc tgagatctga ggacacagct 660

gtgtactact gcactagatt ctacagctac acctactggg gccaaggcac cctggtgaca 720

gtgagcagc 729

<210> 5

<211> 15

<212> PRT

<213> (G4S) 3

<400> 5

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 6

<211> 45

<212> DNA

<213> (G4S) 3

<400> 6

ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatct 45

<210> 7

<211> 244

<212> PRT

<213> CD276-scFv

<400> 7

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Asp Ser Ser Ala Ile Tyr Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Gly Arg Gly Arg Glu Asn Ile Tyr Tyr Gly Ser Arg Leu Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser

130 135 140

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

145 150 155 160

Lys Ala Ser Gln Asn Val Asp Thr Asn Val Ala Trp Tyr Gln Gln Lys

165 170 175

Pro Gly Lys Ala Pro Lys Ala Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr

180 185 190

Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

195 200 205

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr

210 215 220

Cys Gln Gln Tyr Asn Asn Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys

225 230 235 240

Leu Glu Ile Lys

<210> 8

<211> 732

<212> DNA

<213> CD276-scFv

<400> 8

gaggtacaac tggtggagtc tggggggggc ctggttcagc ctgggggctc tctgagactg 60

agctgtgctg cctctggctt caccttcagc agctttggca tgcactgggt gagacaagcc 120

cctggcaagg gcctggagtg ggtggcctac atcagctctg acagctctgc catctactat 180

gctgacacag tgaagggcag attcaccatc agcagagaca atgccaagaa cagcctgtac 240

ctgcagatga acagcctgag agatgaggac acagctgtgt actactgtgg cagaggcaga 300

gagaacatct actatggcag cagactggac tattggggcc aaggcacaac agtgacagtc 360

agctctgggg gtggaggatc tggaggtggg ggctctgggg gtgggggatc tgacattcag 420

ctgacacaga gccctagctt cctgtctgcc tctgtggggg acagagtgac catcacctgc 480

aaggcttctc agaatgtgga caccaatgtg gcctggtatc agcagaagcc tggcaaggcc 540

cccaaggccc tgatctactc tgctagctac agatactctg gggtgcctag cagattctct 600

ggctctggct ctggcacaga cttcaccctg accatcagca gcctgcagcc tgaggacttt 660

gccacctact actgtcagca gtacaacaac taccccttca cctttggcca aggcactaag 720

ctggaaatca ag 732

<210> 9

<211> 45

<212> PRT

<213> CD8α hinge

<400> 9

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

1 5 10 15

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

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 10

<211> 135

<212> DNA

<213> CD8α hinge

<400> 10

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 11

<211> 24

<212> PRT

<213> CD8αTM

<400> 11

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 12

<211> 72

<212> DNA

<213> CD8αTM

<400> 12

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210> 13

<211> 42

<212> PRT

<213> 4-1BB

<400> 13

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 14

<211> 126

<212> DNA

<213> 4-1BB

<400> 14

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 15

<211> 112

<212> PRT

<213> CD3-zeta

<400> 15

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 16

<211> 336

<212> DNA

<213> CD3-zeta

<400> 16

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttggggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 17

<211> 2241

<212> DNA

<213> Chimeric Antigen Receptor (containing CD8α SP)

<400> 17

60

cctgatgtgg ttatgaccca aagccccctg agcctgcctg tgactcctgg ggagcctgct 120

agcatcagct gcagaagctc tcagagcctg gtgcacagca atgccaacac ctacctgcac 180

tggtacctgc agaagcctgg gcagagccct cagctgctga tctacaaggt gagcaataga 240

ttttctgggg tgcctgacag attttctggc tctggctctg gcacagactt caccctgaag 300

atcagcagag tggaggctga ggatgtgggg gtgtactact gctctcagaa cacccatgtg 360

ccccccacct ttggccaagg cacaaagctg gagatcaaga gaggtggcgg tggctcgggc 420

ggtggtgggt cgggtggcgg cggatctcaa gtgcagctgg tgcagtctgg ggctgaggtg 480

aagaagcctg gggcctctgt gaaggtgagc tgcaaggcct ctggctacac cttcacagac 540

tatgagatgc actgggtcag acaagcccct ggccaaggcc tagaatggat gggggccctg 600

gaccccaaga ctggggacac agcctactct cagaagttca agggcagagt gaccctgaca 660

gctgatgaga gcacaagcac agcctacatg gagctgagca gcctgagatc tgaggacaca 720

gctgtgtact actgcactag attctacagc tacacctact ggggccaagg caccctggtg 780

acagtgagca gcggtggcgg tggctcgggc ggtggtgggt cgggtggcgg cggatctgag 840

gtacaactgg tggagtctgg ggggggcctg gttcagcctg ggggctctct gagactgagc 900

tgtgctgcct ctggcttcac cttcagcagc tttggcatgc actgggtgag acaagcccct 960

ggcaagggcc tggagtgggt ggcctacatc agctctgaca gctctgccat ctactatgct 1020

gacacagtga agggcagatt caccatcagc agagacaatg ccaagaacag cctgtacctg 1080

cagatgaaca gcctgagaga tgaggacaca gctgtgtact actgtggcag aggcagagag 1140

aacatctact atggcagcag actggactat tggggccaag gcacaacagt gacagtcagc 1200

tctgggggtg gaggatctgg aggtgggggc tctgggggtg ggggatctga cattcagctg 1260

acacaggcc ctagcttcct gtctgcctct gtgggggaca gagtgaccat cacctgcaag 1320

gcttctcaga atgtggacac caatgtggcc tggtatcagc agaagcctgg caaggccccc 1380

aaggccctga tctactctgc tagctacaga tactctgggg tgcctagcag attctctggc 1440

tctggctctg gcacagactt caccctgacc atcagcagcc tgcagcctga ggactttgcc 1500

acctactact gtcagcagta caacaactac cccttcacctttggccaagg cactaagctg 1560

gaaatcaaga ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 1620

cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 1680

agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 1740

gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 1800

tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1860

agctgccgatttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1920

agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta 1980

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 2040

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 2100

atggcggagg cctacagtga gattggggatg aaaggcgagc gccggagggg caaggggcac 2160

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 2220

caggccctgccccctcgctg a 2241

<210> 18

<211> 746

<212> PRT

<213> Chimeric Antigen Receptor (containing CD8α SP)

<400> 18

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 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu

20 25 30

Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln

35 40 45

Ser Leu Val His Ser Asn Ala Asn Thr Tyr Leu His Trp Tyr Leu Gln

50 55 60

Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg

65 70 75 80

Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

85 90 95

Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr

100 105 110

Tyr Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr

115 120 125

Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val

145 150 155 160

Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr

165 170 175

Thr Phe Thr Asp Tyr Glu Met His Trp Val Arg Gln Ala Pro Gly Gln

180 185 190

Gly Leu Glu Trp Met Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala

195 200 205

Tyr Ser Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser

210 215 220

Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr

225 230 235 240

Ala Val Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln

245 250 255

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

260 265 270

Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly

275 280 285

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

290 295 300

Gly Phe Thr Phe Ser Ser Ser Phe Gly Met His Trp Val Arg Gln Ala Pro

305 310 315 320

Gly Lys Gly Leu Glu Trp Val Ala Tyr Ile Ser Ser Asp Ser Ser Ala

325 330 335

Ile Tyr Tyr Ala Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

340 345 350

Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Asp Glu

355 360 365

Asp Thr Ala Val Tyr Tyr Cys Gly Arg Gly Arg Glu Asn Ile Tyr Tyr

370 375 380

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

385 390 395 400

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

405 410 415

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

420 425 430

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Asp Thr Asn

435 440 445

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Ala Leu Ile

450 455 460

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

465 470 475 480

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

485 490 495

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Pro Phe

500 505 510

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala

515 520 525

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

530 535 540

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

545 550 555 560

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

565 570 575

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

580 585 590

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

595 600 605

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

610 615 620

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

625 630 635 640

Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn

645 650 655

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

660 665 670

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

675 680 685

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

690 695 700

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

705 710 715 720

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

725 730 735

Ala Leu His Met Gln Ala Leu Pro Pro Arg

740 745

<210> 19

<211> 63

<212> DNA

<213> CD8α SP

<400> 19

60

cct 63

<210> 20

<211> 21

<212> PRT

<213> CD8α SP

<400> 20

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

20

Claims (6)

1. A chimeric antigen receptor that simultaneously targets GPC3 and CD276, characterized in that: the chimeric antigen receptor comprises a single chain antibody targeting GPC3, a linker, a single chain antibody targeting CD276, an extracellular hinge region, a transmembrane region, a costimulatory signal region, and a cd3ζ intracellular region;

the amino acid sequence of the single-chain antibody targeting GPC3 is shown as SEQ ID No.3, and the amino acid sequence of the single-chain antibody targeting CD276 is shown as SEQ ID No. 7;

the extracellular hinge region is selected from the group consisting of a CD8 a hinge region;

the transmembrane region is selected from the group consisting of a CD8 a transmembrane region;

the costimulatory signal region is selected from the group consisting of the 4-1BB intracellular region;

the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID No. 1.

2. A chimeric antigen receptor T cell that simultaneously targets GPC3 and CD276, characterized in that: the T cell comprising the chimeric antigen receptor of claim 1 that simultaneously targets GPC3 and CD 276.

3. A recombinant viral vector, characterized in that: the recombinant viral vector comprising the corresponding encoding gene of the chimeric antigen receptor of claim 1 that simultaneously targets GPC3 and CD 276; in the recombinant viral vector, the gene sequences of the chimeric antigen receptor which simultaneously targets GPC3 and CD276 are shown in SEQ ID No. 17.

4. A method for preparing chimeric antigen receptor T cells simultaneously targeting GPC3 and CD276, characterized by: the method comprises the following steps:

1) Inserting the corresponding coding genes of the chimeric antigen receptor simultaneously targeting GPC3 and CD276 according to claim 1 into a pwplxld vector to obtain a recombinant plasmid;

2) Co-transfecting the recombinant plasmid with an envelope plasmid and a packaging plasmid to obtain a recombinant lentivirus;

3) The recombinant lentivirus is transfected with CD3 positive T lymphocytes, and chimeric antigen receptor T cells simultaneously targeting GPC3 and CD276 are obtained through separation.

5. A medicament for treating hepatocellular carcinoma, characterized in that: the medicament comprising chimeric antigen receptor T cells of claim 2 that simultaneously target GPC3 and CD 276.

6. Use of a chimeric antigen receptor T cell of claim 2 that simultaneously targets GPC3 and CD276 in the manufacture of an anti-liver cancer medicament.