CN110684120A - A chimeric antigen receptor targeting GPC3 and its application - Google Patents

Abstract

The invention discloses a chimeric antigen receptor targeting GPC3 and its application, belonging to the field of tumor immunotherapy. The chimeric antigen receptor includes: signal peptide linked in sequence, single chain antibody targeting GPC3, Myc tag, CD8α hinge region, CD28 transmembrane region, costimulatory factor, intracellular signal domain, P2A linker peptide and CCL19 secretion Area. The chimeric antigen receptor can recognize the GPC3 antigen, and the CAR-T cells constructed with the new chimeric antigen receptor have good killing ability to the GPC3 target protein. At the same time, the chimeric antigen receptor targeting GPC3 can also secrete chemotaxis The chemokine CCL19, while not changing the anti-tumor activity, is expected to promote the infiltration of immune cells into the tumor microenvironment, inhibit the growth rate of the tumor, and reduce the size of the tumor, making the GPC3-targeting chimeric antigen receptor. It has stronger anti-solid tumor tumor activity, thus showing a good application prospect.

Description

A chimeric antigen receptor targeting GPC3 and its application

technical field

The invention relates to the field of tumor immunotherapy, in particular to a chimeric antigen receptor targeting GPC3 and its application.

Background technique

Chimeric antigen receptor T cells (Chimeric Antigen Receptor T-Cell, CAR-T) are constructed by introducing the fusion of CAR gene into the host T lymphocyte genome in the form of nucleic acid. The application of CAR-T in hematological tumors has shown promising therapeutic effects, especially for patients with relapsed and refractory B lymphocytic leukemia, with a remission rate of over 90%. However, in the application of solid tumors, due to the lack of tumor-specific antigens and other reasons, CAR-T has not yet achieved good clinical efficacy in the treatment of solid tumors.

SUMMARY OF THE INVENTION

In order to solve the problems in the prior art, the embodiments of the present invention provide a chimeric antigen receptor targeting GPC3 and its application. The technical solution is as follows:

In one aspect, an embodiment of the present invention provides a chimeric antigen receptor targeting GPC3, the chimeric antigen receptor comprising: a signal peptide connected in sequence, a single-chain antibody targeting GPC3, a Myc tag, and a CD8α hinge region , CD28 transmembrane region, costimulatory factor, intracellular signal domain, P2A connecting peptide and CCL19 secretory region, the nucleotide sequence of the signal peptide is shown in SEQ ID NO: 1 in the sequence listing, the single targeting GPC3 The nucleotide sequence of the chain antibody is shown in SEQ ID NO: 2 in the sequence listing, the nucleotide sequence of the Myc tag is shown in SEQ ID NO: 3 in the sequence listing, and the nucleotide sequence of the CD8α hinge region As shown in SEQ ID NO: 4 in the sequence listing, the nucleotide sequence of the CD28 transmembrane region is shown in SEQ ID NO: 5 in the sequence listing, and the nucleotide sequence of the intracellular signal domain is shown in the sequence listing As shown in SEQ ID NO: 6, the nucleotide sequence of the P2A connecting peptide is shown in SEQ ID NO: 7 in the sequence listing, and the nucleotide sequence of the CCL19 secretory region is shown in SEQ ID NO: 8 in the sequence listing. Show.

Further, the chimeric antigen receptor targeting GPC3 also includes a costimulatory factor, one end of the costimulatory factor is connected to the other end of the CD28 transmembrane region, and the costimulatory factor is CD27, CD28, 4 - At least one of 1BB, OX40, CD30, CD40, ICOS, NKG2D and B7-H3.

Further, the costimulatory factor is CD28, and the nucleotide sequence of the costimulatory factor is shown in SEQ ID NO: 9 in the sequence listing.

On the other hand, an embodiment of the present invention provides an application of the above-mentioned GPC3-targeting chimeric antigen receptor, the application comprising: using the GPC3-targeting chimeric antigen receptor as an antitumor drug.

Further, the tumors include liver cancer, lung cancer, gastric cancer, breast cancer, melanoma, ovarian cancer, yolk sac tumor and neuroblastoma.

Preferably, the tumor is liver cancer.

The beneficial effects brought by the technical solutions provided in the embodiments of the present invention are as follows: the embodiments of the present invention provide a chimeric antigen receptor targeting GPC3, and the GPC3 antigen is specifically highly expressed in primary liver cancer HCC, and is Tumors can be detected at the early stage, especially in patients with small liver cancer and alpha-fetoprotein AFP-negative HCC patients, and the positive rate is very high, and the level of GPC3 in liver cancer tissue is significantly increased. The GPC3 antigen is used as the target and can recognize the GPC3 antigen, which makes the CAR-T cells constructed with the new chimeric antigen receptor have good killing ability to the GPC3 target protein, and can exert stronger anti-tumor effect. The chimeric antigen receptor targeting GPC3 can also secrete the cellular chemokine CCL19. The chemokine produced is expected to promote the infiltration of immune cells into the tumor microenvironment, inhibit the growth rate of the tumor, and shrink the tumor without changing the anti-tumor activity. The volume of chimeric antigen receptor targeting GPC3 has stronger anti-solid tumor tumor activity, thus showing a good application prospect.

Description of drawings

In order to illustrate the technical solutions in the embodiments of the present invention more clearly, the following briefly introduces the accompanying drawings used in the description of the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained from these drawings without creative effort.

Fig. 1 is the structural schematic diagram of the chimeric antigen receptor provided in the embodiment of the present invention;

Fig. 2 is the agarose gel electrophoresis figure that the embodiment of the present invention provides;

Fig. 3 is the CAR19 lentivirus transfection efficiency diagram provided in the embodiment of the present invention;

Fig. 4 is the contrast diagram of IL-2 secretion amount provided in the embodiment of the present invention;

Fig. 5 is the comparison chart of the secretion amount of IFNγ provided in the embodiment of the present invention;

Figure 6 is a comparison chart of the cell killing efficiency provided by the embodiment of the present invention. In the figure, A is the killing ability of CAR-T cells to HepG2 cells, A is the killing ability of control cells to HepG2 cells, and the abscissa is effector cells and target cells. The number ratio, the ordinate is the cell killing efficiency, the unit is %.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

Example

The embodiment of the present invention provides a chimeric antigen receptor targeting GPC3, the chimeric antigen receptor comprising: a signal peptide connected in sequence, a single-chain antibody targeting GPC3 (Glypican-3), a Myc tag, a CD8α hinge region, CD28 transmembrane region, costimulatory factor, intracellular signal domain, P2A connecting peptide and CCL19 secretion region, the nucleotide sequence of the signal peptide is shown in SEQ ID NO: 1 in the sequence listing, and the targeting GPC3 The nucleotide sequence of the single-chain antibody is shown in SEQ ID NO: 2 in the sequence listing, the nucleotide sequence of the Myc tag is shown in SEQ ID NO: 3 in the sequence listing, and the nucleoside of the CD8α hinge region The acid sequence is shown in SEQ ID NO: 4 in the sequence listing, the nucleotide sequence of the CD28 transmembrane region is shown in SEQ ID NO: 5 in the sequence listing, and the nucleotide sequence of the intracellular signal domain is shown in the sequence SEQ ID NO: 6 in the list, the nucleotide sequence of the P2A linking peptide is shown in SEQ ID NO: 7 in the sequence table, and the nucleotide sequence of the CCL19 secretory region is shown in the sequence table SEQ ID NO: 8 shown. When implemented, the single-chain antibody targeting GPC3 includes three parts of the light chain _VL variable region, the Inter-Linker and the heavy chain _VH variable region of the GPC3 single-chain antibody.

Further, one end of the costimulatory factor is connected to the other end of the CD28 transmembrane region, and the costimulatory factor is at least one of CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, NKG2D and B7-H3. In implementation, the costimulatory factor can be a combination of two or more of the above-mentioned costimulatory factors. In this case, two or more costimulatory factors are connected end-to-end in sequence, and then the two ends are respectively connected to the CD8α transmembrane domain and the intracellular signal domain.

Further, the costimulatory factor is CD28, and the nucleotide sequence of the costimulatory factor is shown in SEQ ID NO: 6 in the sequence listing.

The application of the GPC3-targeting chimeric antigen receptor provided in the embodiments of the present invention includes: using the GPC3-targeting chimeric antigen receptor as an anti-tumor drug. Tumors include liver cancer, lung cancer, gastric cancer, breast cancer, melanoma, ovarian cancer, yolk sac tumor and neuroblastoma, preferably, the tumor is liver cancer.

At the time of implementation, the NCBI website database was searched for: human CD8α signal region, human CD28 hinge region, CD28 intracellular region, human CD3ζ intracellular signal domain, human CCL19 gene sequence information, anti-GPC3 monoclonal antibody Sequence information of the heavy chain and light chain variable regions of anti-GPC3 monoclonal antibodies. The above sequences are codon-optimized on the website http://sg.idtdna.com/site to ensure that the encoded amino acid sequences remain unchanged. more suitable for human cell expression. Fully synthesized chimeric antigen receptor gene sequence, its structure is: signal peptide-single-chain antibody targeting GPC3-Myc tag-CD8α hinge region-CD28 transmembrane region-costimulator CD28-intracellular signal domain CD3ζ-connection Peptide P2A-CCL19 secretory region, specifically: CD8α leader-GPC3 scFv-Myc tag-CD8α hinge-CD28-CD3ζ-P2A-CCL19, labeled as CAR19, the structure of the chimeric antigen receptor is shown in Figure 1, the chimeric antigen receptor is shown in Figure 1. The nucleotide sequence of the chimeric antigen receptor is shown in SEQ ID NO: 10 in the sequence listing, and correspondingly, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 11 in the sequence listing.

When the parts of the chimeric antigen receptor are connected, they can be directly connected together, or they can be connected through a linker sequence. The linker sequence may be a flexible sequence known in the art for use in the variable light and heavy chain variable regions of antibodies, eg, a G and S containing ( _{G4S)3 linker} sequence.

To prepare the gene plasmid vector of the chimeric antigen receptor, the specific method is as follows:

Amplify and obtain the CAR19 gene sequence by PCR (Polymerase Chain Reaction, polymerase chain reaction), and add the restriction site Xba I and the restriction site BamH I to the two ends of the CAR19 gene to obtain the product to be digested. It was subjected to Xba I and BamH I double digestion reactions with the lentiviral vector plasmid pCDH-EF1-MCS-T2A-copGFP, respectively, to obtain the restriction fragment containing CAR19 and the restriction fragment containing pCDH-EF1-MCS-T2A-copGFP. The reaction conditions of the digestion were as follows: the digestion temperature was 37°C, and the digestion time was 30 min. The digestion system with a total volume of 50 μL includes: 5 μL of 10×buffer; 5 μg of DNA to be digested; 2 μL of XbaI enzyme; 2 μL of BamH I enzyme; the volume of the digestion system was supplemented to 50 μL with deionized water.

The digested fragment containing CAR19 and the digested fragment containing pCDH-EF1-MCS-T2A-copGFP were electrophoresed on a 1% agarose gel, respectively. The nucleic acid band of the restriction fragment of EF1-MCS-T2A-copGFP was cut out and placed in two clean EP tubes, respectively, and then the DNA in the agarose gel was purified and recovered to obtain the CAR19 restriction product and pCDH- EF1-MCS-T2A-copGFP digestion product.

The DNA fragments of the CAR19 digested product and the pCDH-EF1-MCS-T2A-copGFP digested product were ligated at 16°C overnight to form pCDH-EF1-CAR19-T2A-copGFP. The ligation system with a total volume of 10 μL includes: 1 μL of pCDH-EF1-MCS-T2A-copGFP digestion product, 7 μL of CAR19 digestion product, 1 μL of T4 DNA ligase and 1 μL of 10×T4 DNA ligase Buffer .

The ligation product was transferred into Stbl3 competent cells (purchased from TRANSGEN BIOTECH), the specific method is as follows:

Take out the Stbl3 competent cells stored in the -80°C freezer and thaw them on ice. The ligation product was added to the Stbl3 competent cells, after ice bathing for 30 min, heat shock at 42°C for 45 s, and then ice bathing for 2 min to obtain the transformation product.

The transformation product was added to 900 μL of liquid LB medium without antibiotics, and the rotating speed of the shaker was 200 rpm at 37° C., and the fermentation was carried out for 45 min to obtain a fermentation broth. The preparation method of the liquid LB medium without antibiotics includes: taking 5g of imported yeast extract, 10g of imported peptone, 10g of anhydrous sodium chloride and 1L of sterile water, mixing them evenly, and sterilizing at 121°C for 20min before use.

The fermentation broth was centrifuged at 4000 rpm for 5 min, the supernatant was discarded, the precipitate was retained, and the precipitate was resuspended in 100 μL of liquid LB medium to obtain a resuspended liquid.

The resuspension was smeared on Amp-resistant solid LB plate medium (purchased from Shanghai CHROMagar Microbial Technology Co., Ltd.), and the solid LB plate medium was placed in a bacterial incubator at 37° C. for overnight culture.

Positive clones were picked on solid LB plate medium.

The positive clones obtained are identified, and the specific methods are as follows:

The obtained positive clone is subjected to Xba I and BamH I double digestion reaction. For the specific operation, refer to the double digestion reaction of the above-mentioned material to be cut by the enzyme. The enzyme digestion product obtained by the positive clone is subjected to agarose gel electrophoresis to identify the target fragment, and obtained. The size of the target fragment is about 2000bp, and the results are shown in Figure 2. It was identified by sequencing that the target sequence was the CAR19 gene.

Plasmid extraction: The positive clones with correct sequencing were prepared into the original bacterial liquid and inoculated into 100 mL of Amp-resistant liquid LB medium, and cultured overnight at 37°C with a shaker speed of 200 rpm to obtain the original bacterial fermentation broth.

The original bacterial fermentation broth was centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the precipitate (cell body) was retained.

The endotoxin-free plasmid extraction kit (purchased from Tiangen Company) was used to extract the bacterial plasmid, and the specific method was carried out according to the instructions of the kit.

CAR19 lentiviral vector packaging: 6h before transfection, 293T cells were seeded into a 10 cm diameter petri dish at about 8.5×10 ⁶ cells per dish. Make sure that the cells are about 80% confluent at the time of transfection and evenly distributed in the dish.

Prepare solution A and solution B, where solution A includes: 4 mL of 2× HEPES buffer (amount packaged together in 8 petri dishes), solution B includes: 72ug plasmid (target plasmid), 37.04ug packaging plasmid PLP1, 34.8 ug packaging plasmid PLP2, 24.08ug packaging plasmid PLP-VSVG and 400 μL of 2.5M calcium ion solution, the total volume of solution B was 4 mL. Fully mix solution B, and while gently vortexing solution A, add solution B dropwise to solution A, and let stand for 3-5 min to obtain a mixed solution. Gently vortex the mixture, add the mixture dropwise to the petri dish containing 293T cells, add 1 mL of the mixture to each petri dish, and gently shake the petri dish back and forth to distribute the mixture evenly on the surface of the petri dish (note that Do not rotate the petri dish when shaking), and place the petri dish in a 37°C incubator. After culturing for 12 h, the medium was replaced with fresh medium, and the culture was continued. After culturing for 48 h, the supernatant of CAR19 lentivirus was collected. After centrifugation at 1500 rpm/min for 5 min, the supernatant was retained, and the supernatant was filtered with a filter membrane with a size of 0.45 μm to obtain a filtrate containing CAR19 lentivirus.

Transfer the CAR19 lentivirus-containing filtrate to an ultracentrifuge tube, and carefully spread a layer of 20% sucrose (1 mL of sucrose per 8 mL of lentivirus-containing filtrate) on the bottom of the ultracentrifuge tube. Equilibrate the ultracentrifuge tube with PBS (phosphate buffersaline, phosphate buffered saline), centrifuge at 27600rpm/min at 4°C for 2h, carefully remove the ultracentrifuge tube, pour off the supernatant, invert the ultracentrifuge tube to remove the residual supernatant, Precipitate is retained. Add 150 μL of PBS to the ultracentrifuge tube, and use a micropipette to gently pipet the bottom of the ultracentrifuge tube several times to dissolve the precipitate in PBS to obtain the concentrated CAR19 lentivirus (gene plasmid vector for chimeric antigen receptor) , when realized, the concentrated CAR19 lentivirus can be divided into centrifuge tubes and stored at -80°C.

CAR19 lentivirus titer detection: Take 0.5 μL, 5 μL and 50 μL of concentrated CAR19 lentivirus to infect 293T cells (1×10 ⁵ cells/well) for 24 hours, change the medium after 24 hours, and extract cellular genomic DNA after 72 hours. The concentration is diluted to 5～100ng/μL. TransLv ^™ Lentivirus qPCR Titration Kit (purchased from TransGen) was used, and the specific method was carried out according to its instructions. The detection showed that the titer of the CAR19 lentivirus was 5×10 ⁸ TU/mL.

To prepare chimeric antigen receptor T cells, the specific method is as follows:

Preparation of PBMC (Peripheral Blood Mononuclear Cell, peripheral blood mononuclear cells): Collect 20 mL of peripheral blood from volunteers, add the peripheral blood to a 50 mL centrifuge tube containing heparin, and centrifuge at 2000 rpm for 10 min, and transfer the upper plasma to a new centrifuge tube Freeze. Add an equal volume of pre-warmed physiological saline at 37° C. to the centrifuge tube, mix well, and resuspend the blood cell pellet to obtain a resuspended cell solution. Take another 50mL centrifuge tube and add 20mL of pre-warmed lymphocyte separation solution. 20mL of the resuspended cell solution was slowly added to the upper layer of the lymphocyte separation solution. Centrifuge at 800 rpm for 20 min. The upper layer of plasma was sucked off at a constant speed. When the plasma was 2 to 3 cm away from the buffalo layer, the plasma was stopped, and then the buffalo layer cells were quickly sucked off and transferred to another new 50mL centrifuge tube. 45mL, centrifuged at 1200rpm for 5min, repeated twice, for washing cells. The cell pellet was resuspended in RPMI1640+10% FBS medium, and the number of T cells was counted. In this example, the number of T cells was 1.5×10 ⁷ .

CAR19 lentivirus-transfected human T cells: In this example, the T cell density was adjusted to 1×10 ⁶ /mL, and the T cells were seeded into 1mL/well anti-human 50ng/mL CD3 antibody and 50ng/mL CD28 In the antibody, 200IU/mL of interleukin 2 was added, and the culture was stimulated for 48h. After T cells were activated and cultured for two days, the CAR19 lentivirus was transfected with an infection coefficient of MOI=5, and 8 μg/mL polybrene was added, and cultured in a 37°C incubator. 24h after transfection, the medium was changed, and the cell growth was continuously observed for 8-13 days. Obtain transfected CAR-T cells.

Detection of CAR19 lentivirus transfection efficiency: After transfection, observe the transfected cells regularly under an inverted fluorescence microscope. The transfected CAR-T cells were aspirated, centrifuged at 1000 rpm for 5 min to collect the precipitate, and the precipitate was washed with PBS solution. The proportion of cells expressing GFP fluorescence in transfected CAR-T cells was detected using the FITC channel of flow cytometry. The transfection efficiency is shown in Figure 3.

Chemokine secretion detection of CAR-T cells, as follows:

In order to detect whether the CAR-T cells transfected with CAR19 lentivirus are effectively activated, this example detects the co-culture of CAR-T cells and target cells, and detects the secretion of IFNγ and IL-2 by ELISA kits. Specifically, 1×10 ⁶ CAR-T cells per well and 1×10 ⁵ target cells per well were seeded into 6-well plates, respectively, and cultured at 37°C in a concentration of 5% CO ₂ for 24 h. The cultured supernatant was aspirated, centrifuged at 1000 rpm for 5 min to remove the cell pellet, and the cultured supernatant was harvested. The IFNγ and IL-2 in the culture supernatant were detected according to the instructions of the ELISA kit. As shown in FIG. 4 and FIG. 5 , FIG. 4 is a comparison diagram of the secretion amount of IL-2, and FIG. 5 is a comparison diagram of the secretion amount of IFNγ. Since IFNγ and IL-2 secretion were detected, it can be proved that the CAR-T cells transfected with CAR19 lentivirus have been effectively activated.

In vitro anti-tumor effect: ① Group: 40 wells were taken in a 96-well plate and divided into eight groups, each group was set with five duplicate wells, and only 200 μL of culture medium (referred to as Ab) was added to the first group. In the second group, 100 μL of culture medium and 100 μL of 1×10 ⁴ target cells (marked as Ack) were added. The target cells were HepG2 cells containing GPC3 target protein. In the third group, 100 μL of culture medium and 100 μL of 1×10 ⁴ were added. 100 μL of 1×10 ⁴ target cells and 100 μL of 1×10 ⁴ effector cells (referred to as As) were added to the fourth group. , the number of effector cells: the number of target cells = 1:1), 100 μL medium and 100 μL 5×10 ⁴ effector cells (marked as Acn) were added to the fifth group, and 100 μL 1×10 were added to the sixth group. ⁴ target cells and 100 μL of 5×10 ⁴ effector cells (marked as As, the number of effector cells: the number of target cells = 5:1), 100 μL of medium and 100 μL of 1×10 ⁵ effectors were added to the seventh group Cells (marked as Acn), 100 μL of 1×10 ⁴ target cells and 100 μL of 1×10 ⁵ effector cells were added to the eighth group (marked as As, number of effector cells:number of target cells=10:1).

② Group (control group): The other 40 wells of the 96-well plate were divided into eight groups, and each group was set with five duplicate wells. The grouping method was the same as that of group ①, but the difference from group ① was that the effector cells were transfected CAR-T cells in CAR structure or uninfected T cells.

After incubating the 96-well plate for 4 hours, 20uL of CCK-8 solution was added to each well, and the 96-well plate was incubated in an incubator for 4 hours. Absorbance was measured at 450 nm with a microplate reader. Calculate the cell killing efficiency of ① group and ② group respectively according to the absorbance = [1-(As-Acn)/(Ack-Ab)]×100%, where As is the test well (medium containing target cells, effector cells and CCK-8 solution), Ack is the target cell control well (medium containing target cells and CCK-8 solution), Can is the effector cell control well (medium containing effector cells, CCK-8 solution), and Ab is blank Control (medium without cells and CCK-8 solution). FIG. 6 is a cell killing efficiency diagram provided by the embodiment of the present invention. As shown in FIG. 6 , the killing efficiency of HepG2 cells by CAR-T cells obtained by using the chimeric antigen receptor targeting GPC3 provided by the embodiment of the present invention is significantly higher than that of HepG2 cells. In the control group, it can be seen that the GPC3-targeting chimeric antigen receptor provided in the embodiment of the present invention has a good anti-tumor effect on the GPC3 target protein.

The embodiment of the present invention provides a chimeric antigen receptor targeting GPC3. GPC3 antigen is specifically highly expressed in primary liver cancer HCC, and can be detected at the early stage of tumor, especially in small liver cancer and alpha-fetoprotein The positive rate is high in AFP-negative HCC patients. The level of GPC3 is significantly increased in liver cancer tissue. Therefore, the chimeric antigen receptor provided in the embodiment of the present invention uses the GPC3 antigen as a target and can recognize the GPC3 antigen, which makes the CAR constructed by using the new chimeric antigen receptor. -T cells have good killing ability to GPC3 target proteins and can exert stronger anti-tumor effect. At the same time, the chimeric antigen receptor targeting GPC3 can also secrete the chemokine CCL19, which does not change the anti-tumor activity at the same time. , the chemokine produced is expected to promote the infiltration of immune cells into the tumor microenvironment, inhibit the growth rate of the tumor, and reduce the size of the tumor, so that the GPC3-targeting chimeric antigen receptor has stronger anti-solid tumor tumor activity, thereby showing good application prospects.

The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

sequence listing

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gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1080

agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgcccagg gcctacccgc 1140

aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cggaagagtg 1200

aagttcagca ggagcgcaga cgcccccgcg taccagcagg gccagaacca gctctataac 1260

gagctcaatc taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac 1320

cctgagatgg ggggaaagcc gagaaggaag aaccctcagg aaggcctgta caatgaactg 1380

cagaaagata agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg 1440

ggcaaggggc acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac 1500

gcccttcaca tgcaggccct gccccctcgc ggaagcggag ctactaactt cagcctgctg 1560

aagcaggctg gagacgtgga ggagaaccct ggacctatgg ccctgctact ggccctcagc 1620

ctgctggttc tctggacttc cccagcccca actctgagtg gcaccaatga tgctgaagac 1680

tgctgcctgt ctgtgaccca gaaacccatc cctgggtaca tcgtgaggaa cttccactac 1740

cttctcatca aggatggctg cagggtgcct gctgtagtgt tcaccacact gaggggccgc 1800

cagctctgtg cacccccaga ccagccctgg gtagaacgca tcatccagag actgcagagg 1860

acctcagcca agatgaagcg ccgcagcagt taa 1893

<210> 11

<211> 650

<212> PRT

<213> Artificial Sequence

<400> 11

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

1 5 10 15

His Ala Ala Ala Pro Ala Val Val Met Thr Gly Ser Pro Leu Ser Leu

20 25 30

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

35 40 45

Ser Leu Val His Ser Ala Ala Ala Thr Thr Leu His Thr Thr Leu Gly

50 55 60

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

65 70 75 80

Pro Ser Gly Val Pro Ala Ala Pro Ser Gly Ser Gly Ser Gly Thr Ala

85 90 95

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

100 105 110

Thr Cys Ser Gly Ala Thr His Val Pro Pro Thr Pro Gly Gly Gly Thr

115 120 125

Leu Leu Gly Ile Leu Ala Gly Gly Cys Gly Gly Cys Gly Gly Gly Gly

130 135 140

Gly Thr Thr Cys Thr Gly Gly Thr Gly Gly Cys Gly Gly Cys Gly Gly

145 150 155 160

Cys Ala Gly Cys Gly Gly Cys Gly Gly Thr Gly Gly Ala Gly Gly Ala

165 170 175

Thr Cys Ala Gly Val Gly Leu Val Gly Ser Gly Ala Gly Val Leu Leu

180 185 190

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

195 200 205

Thr Ala Thr Gly Met His Thr Val Ala Gly Ala Pro Gly Gly Gly Leu

210 215 220

Gly Thr Met Gly Ala Leu Ala Pro Leu Thr Gly Ala Thr Ala Thr Ser

225 230 235 240

Gly Leu Pro Leu Gly Ala Val Thr Leu Thr Ala Ala Gly Ser Thr Ser

245 250 255

Thr Ala Thr Met Gly Leu Ser Ser Leu Ala Ser Gly Ala Thr Ala Val

260 265 270

Thr Thr Cys Thr Gly Gly Leu Leu Ile Ser Gly Gly Ala Leu Ala Leu

275 280 285

Ser Ala Ser Ile Met Thr Pro Ser His Pro Val Pro Val Pro Leu Pro

290 295 300

Ala Leu Pro Thr Thr Thr Pro Ala Pro Ala Pro Pro Thr Pro Ala Pro

305 310 315 320

Thr Ile Ala Ser Gly Pro Leu Ser Leu Ala Pro Gly Ala Ser Ala Pro

325 330 335

Ala Ala Gly Gly Ala Val His Thr Ala Gly Leu Ala Leu Pro Pro Thr

340 345 350

Val Leu Val Val Val Gly Gly Val Leu Ala Cys Thr Ser Leu Leu Val

355 360 365

Thr Val Ala Pro Ile Ile Pro Thr Val Ala Ser Leu Ala Ser Ala Leu

370 375 380

Leu His Ser Ala Thr Met Ala Met Thr Pro Ala Ala Pro Gly Pro Thr

385 390 395 400

Ala Leu His Thr Gly Pro Thr Ala Pro Pro Ala Ala Pro Ala Ala Thr

405 410 415

Ala Ser Ala Val Leu Pro Ser Ala Ser Ala Ala Ala Pro Ala Thr Gly

420 425 430

Gly Gly Gly Ala Gly Leu Thr Ala Gly Leu Ala Leu Gly Ala Ala Gly

435 440 445

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

450 455 460

Gly Leu Pro Ala Ala Leu Ala Pro Gly Gly Gly Leu Thr Ala Gly Leu

465 470 475 480

Gly Leu Ala Leu Met Ala Gly Ala Thr Ser Gly Ile Gly Met Leu Gly

485 490 495

Gly Ala Ala Ala Gly Leu Gly His Ala Gly Leu Thr Gly Gly Leu Ser

500 505 510

Thr Ala Thr Leu Ala Thr Thr Ala Ala Leu His Met Gly Ala Leu Pro

515 520 525

Pro Ala Gly Ser Gly Ala Thr Ala Pro Ser Leu Leu Leu Gly Ala Gly

530 535 540

Ala Val Gly Gly Ala Pro Gly Pro Met Ala Leu Leu Leu Ala Leu Ser

545 550 555 560

Leu Leu Val Leu Thr Thr Ser Pro Ala Pro Thr Leu Ser Gly Thr Ala

565 570 575

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

580 585 590

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

595 600 605

Val Pro Ala Val Val Pro Thr Thr Leu Ala Gly Ala Gly Leu Cys Ala

610 615 620

Pro Pro Ala Gly Pro Thr Val Gly Ala Ile Ile Gly Ala Leu Gly Ala

625 630 635 640

Thr Ser Ala Leu Met Leu Ala Ala Ser Ser

645 650

Claims (6)

1. A chimeric antigen receptor targeted to GPC3, comprising: the polypeptide comprises a signal peptide, a single-chain antibody targeting GPC3, a Myc tag, a CD8 alpha hinge region, a CD28 transmembrane region, a costimulatory factor, an intracellular signal domain, a P2A connecting peptide and a CCL19 secretion region which are connected in sequence, wherein the nucleotide sequence of the signal peptide is shown as SEQ ID NO: 1, the nucleotide sequence of the single-chain antibody targeting GPC3 is shown as SEQ ID NO: 2, the nucleotide sequence of the Myc label is shown as SEQ ID NO: 3, the nucleotide sequence of the CD8 alpha hinge region is shown as SEQ ID NO: 4, the nucleotide sequence of the CD28 transmembrane region is shown as SEQ ID NO: 5, the nucleotide sequence of the intracellular signal domain is shown as SEQ ID NO: 6, the nucleotide sequence of the P2A connecting peptide is shown as SEQ ID NO: 7, the nucleotide sequence of the CCL19 secretory domain is shown as SEQ ID NO: shown in fig. 8.

2. The chimeric antigen receptor targeting GPC3 according to claim 1, wherein one end of the costimulatory factor is linked to the other end of the CD28 transmembrane region, the costimulatory factor being at least one of CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, NKG2D, and B7-H3.

3. The chimeric antigen receptor targeting GPC3 of claim 2, wherein the costimulatory factor is CD28, and the nucleotide sequence of the costimulatory factor is as shown in SEQ ID NO: shown at 9.

4. Use of a chimeric antigen receptor targeting GPC3 according to any one of claims 1 to 3, comprising: the chimeric antigen receptor targeting GPC3 is used as an antitumor drug.

5. The use of claim 4, wherein the tumor comprises: liver cancer, lung cancer, stomach cancer, breast cancer, melanoma, ovarian cancer, yolk sac tumor and neuroblastoma.

6. The use of claim 5, wherein the tumor is liver cancer.

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