CN110684120B - A chimeric antigen receptor targeting GPC3 and its application - Google Patents

Abstract

The invention discloses a GPC 3-targeted chimeric antigen receptor and application thereof, and belongs to the field of tumor immunotherapy. The chimeric antigen receptor includes: 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. The chimeric antigen receptor can recognize GPC3 antigen, CAR-T cells constructed by the novel chimeric antigen receptor have good killing capacity on GPC3 target protein, and meanwhile, the chimeric antigen receptor targeting GPC3 can also secrete chemotactic factor CCL19, and the generated chemotactic factor is expected to promote immune cells to infiltrate into a tumor microenvironment while the anti-tumor activity is not changed, so that the growth speed of tumor is inhibited, the volume of tumor is reduced, the chimeric antigen receptor targeting GPC3 has stronger anti-solid tumor activity, and therefore, the application prospect is good.

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-Cell (CAR-T) is constructed by introducing a CAR gene fused into the host T lymphocyte genome in the form of nucleic acid. The application of CAR-T in hematological tumors has shown gratifying therapeutic effects, especially for relapsed and refractory B-lymphoblastic leukemia patients, with a remission rate of over 90%. However, in terms of 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.

Contents of the invention

In order to solve the problems of the prior art, an embodiment of the present invention provides a chimeric antigen receptor targeting GPC3 and its application. Described technical scheme is as follows:

On the one hand, an embodiment of the present invention provides a chimeric antigen receptor targeting GPC3, said chimeric antigen receptor comprising: sequentially connected signal peptide, single-chain antibody targeting GPC3, Myc tag, CD8α hinge region , CD28 transmembrane region, co-stimulatory 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, and the single target 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 co-stimulatory factor is CD28, and the nucleotide sequence of the co-stimulatory 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 GPC3-targeting chimeric antigen receptor, the application comprising: using the GPC3-targeting chimeric antigen receptor as an anti-tumor 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 effect brought by the technical solution provided by the embodiment of the present invention is: the embodiment of the present invention provides a chimeric antigen receptor targeting GPC3, and the GPC3 antigen is specifically highly expressed in primary liver cancer HCC, and in Tumors can be detected in the early stage, especially in small liver cancer and alpha-fetoprotein AFP-negative HCC patients, the positive rate is very high, and the level of GPC3 in liver cancer tissue is significantly increased, so the chimeric antigen receptor provided by the embodiment of the present invention has the following The GPC3 antigen is used as the target and can recognize the GPC3 antigen, which makes the CAR-T cells constructed with this novel chimeric antigen receptor have good killing ability against the GPC3 target protein and exert a stronger anti-tumor effect. The chimeric antigen receptor targeting GPC3 can also secrete the cell chemokine CCL19. While not changing the anti-tumor activity, the chemokine produced is expected to promote the infiltration of immune cells into the tumor microenvironment, inhibit the growth rate of tumors, and shrink tumors The volume makes the chimeric antigen receptor targeting GPC3 have stronger anti-solid tumor tumor activity, thus showing a good application prospect.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings that need to be used in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those skilled in the art, other drawings can also be obtained based on these drawings without creative effort.

Figure 1 is a schematic diagram of the structure of the chimeric antigen receptor provided by the embodiment of the present invention;

Fig. 2 is the agarose gel electrophoresis picture provided by the embodiment of the present invention;

Figure 3 is a graph of CAR19 lentivirus transfection efficiency provided by the embodiment of the present invention;

Figure 4 is a comparison chart of IL-2 secretion provided by the embodiments of the present invention;

Figure 5 is a comparison chart of IFNγ secretion provided by the embodiment of the present invention;

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

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the implementation manner of the present invention will be further described in detail below in conjunction with the accompanying drawings.

Example

An embodiment of the present invention provides a chimeric antigen receptor targeting GPC3, the chimeric antigen receptor comprising: sequentially connected signal peptide, single-chain antibody targeting GPC3 (Glypican-3), Myc tag, CD8α hinge region, CD28 transmembrane region, co-stimulatory 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, 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 nucleotide sequence 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 sequence The list is 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 in the sequence listing: 8. When implemented, the single-chain antibody targeting GPC3 includes three parts: the _VL variable region of the light chain, the Inter-Linker and the _VH variable region of the heavy chain of the GPC3 single-chain antibody.

Further, one end of the co-stimulatory factor is connected to the other end of the CD28 transmembrane region, and the co-stimulatory factor is at least one of CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, NKG2D and B7-H3. When implemented, the co-stimulatory factors may be a combination of two or more of the above-mentioned co-stimulatory factors. At this time, the two or more co-stimulatory factors are sequentially connected end to end, and then their two ends are respectively connected to the CD8α transmembrane region and the intracellular signaling domain.

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

The application of the chimeric antigen receptor targeting GPC3 provided by the embodiment of the present invention includes: using the chimeric antigen receptor targeting GPC3 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, search in the NCBI website database: 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 The sequence information of the heavy chain and the light chain variable region of the anti-GPC3 monoclonal antibody. These sequences are codon optimized on the website http://sg.idtdna.com/site to ensure that the encoded amino acid sequence remains unchanged It is more suitable for expression in human cells. Gene fully synthesized chimeric antigen receptor gene sequence, its structure is: signal peptide-single-chain antibody targeting GPC3-Myc label-CD8α hinge region-CD28 transmembrane region-co-stimulatory factor CD28-intracellular signal domain CD3ζ-connection Peptide P2A-CCL19 secretion 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 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 connected through a linker sequence. The linker sequence may be a flexible sequence known in the art suitable for the light chain variable region and the heavy chain variable region of an antibody, such as a (G ₄ S) ₃ linker sequence containing G and S.

The gene plasmid carrier of this chimeric antigen receptor is prepared, and the specific method is as follows:

PCR (Polymerase Chain Reaction, Polymerase Chain Reaction) amplifies and obtains the CAR19 gene sequence, respectively adds restriction site Xba I and restriction site BamH I to the two ends of the CAR19 gene to obtain the product to be digested, and 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 enzyme digestion reaction conditions are: enzyme digestion temperature 37°C, enzyme digestion time 30min. The enzyme 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; make up the volume of the enzyme digestion system to 50 μL with deionized water.

The enzyme-cut fragments containing CAR19 and the enzyme-cut fragments containing pCDH-EF1-MCS-T2A-copGFP were electrophoresed on 1% agarose gel respectively. After electrophoresis, the enzyme-cut fragments containing CAR19 and pCDH- The nucleic acid bands of the restriction fragment of EF1-MCS-T2A-copGFP were excised, and placed in two clean EP tubes, 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 obtained from the digested product of CAR19 and the digested product of pCDH-EF1-MCS-T2A-copGFP were ligated overnight at 16°C to form pCDH-EF1-CAR19-T2A-copGFP. Among them, 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 r .

Transfer the ligation product 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 refrigerator and thaw on ice. The ligation product was added to Stbl3 competent cells, and 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 liquid LB medium without adding antibiotics, and the rotation speed of the shaker was 200 rpm at 37° C., and the fermentation was carried out for 45 minutes to obtain a fermentation broth. The preparation method of the liquid LB medium without adding antibiotics includes: taking 5g of imported yeast extract, 10g of imported peptone, 10g of anhydrous sodium chloride and 1L of sterile water, mixing them, and sterilizing at 121°C for 20 minutes before use.

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

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

Pick positive clones on solid LB plate medium.

Identify the positive clones obtained, the specific method is as follows:

The obtained positive clones were subjected to Xba I and BamH I double enzyme digestion reactions. For specific operations, refer to the above-mentioned double enzyme digestion reactions of the objects to be digested. The digested products obtained from the positive clones were identified by agarose gel electrophoresis. The target fragment was obtained. The target fragment with a size of about 2000bp is shown in Figure 2. After sequencing and identification, it can be known that the target sequence is the CAR19 gene.

Plasmid extraction: The positive clones with correct sequencing were prepared as 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 liquid.

The original bacteria fermentation liquid was centrifuged at 4000rpm for 10min, the supernatant was discarded, and the precipitate (bacteria) was retained.

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

CAR19 lentiviral vector packaging: 6 hours before transfection, inoculate 293T cells into a culture dish with a diameter of 10 cm at about 8.5×10 ⁶ cells per dish. Make sure that the confluence of the cells is around 80% and that they are evenly distributed in the culture dish during transfection.

Prepare solution A and solution B, wherein, solution A includes: 4mL of 2×HEPES buffer buffer (the amount packed 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 is 4 mL. Mix solution B thoroughly, and while vortexing solution A gently, add solution B dropwise to solution A, and let it stand for 3-5 minutes to obtain a mixed solution. Gently vortex the mixed solution, add the mixed solution drop by drop to the culture dish containing 293T cells, add 1mL of the mixed solution to each culture dish, gently shake the culture dish back and forth to make the mixed solution evenly distributed on the surface of the culture dish (note Do not rotate the Petri dish when shaking), and place the Petri dish in a 37°C incubator for cultivation. After culturing for 12 h, replace with fresh medium and continue culturing. After 48 h of culture, the supernatant containing CAR19 lentivirus was collected. After centrifugation at 1500rpm/min for 5min, the supernatant was retained, and the supernatant was filtered through a filter membrane with a specification of 0.45 μm to obtain a filtrate containing CAR19 lentivirus.

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

Detection of CAR19 lentivirus titer: 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, extract the genomic DNA of the cells after 72 hours, and extract the genomic DNA Concentration diluted to 5 ~ 100ng/μL. TransLv ^TM Lentivirus qPCR Titration Kit (purchased from TransGen) was used, and the specific method was carried out according to its instructions. The titer of the CAR19 lentivirus was 5×10 ⁸ TU/mL after detection.

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, centrifuge at 2000 rpm for 10 min, and transfer the upper layer of plasma to a new centrifuge tube Freeze. Add the same volume as the sediment into the centrifuge tube, and add physiological saline preheated at 37°C, mix thoroughly, 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 medium. Slowly add 20mL of the resuspended cell solution to the upper layer of the lymphocyte separation medium. Centrifuge at 800 rpm for 20 min. Aspirate the upper layer of plasma at a constant speed, stop the plasma when the plasma is 2-3 cm away from the buffy coat, then quickly suck off the cells in the buffy coat, transfer them to another new 50mL centrifuge tube, and replenish the volume to 45mL, centrifuge at 1200rpm for 5min, repeat twice to wash the cells. Use RPMI1640+10% FBS medium to resuspend the cell pellet, and count the number of T cells. In this example, the number of T cells is 1.5×10 ⁷ .

Human T cells transfected with CAR19 lentivirus: In this example, the T cell density was adjusted to 1×10 ⁶ /mL, and T cells were inoculated with anti-human 50ng/mL CD3 antibody and 50ng/mL CD28 at 1mL/well Add 200 IU/mL interleukin 2 to the antibody, and stimulate the culture for 48 hours. After T cell activation and culture for two days, the CAR19 lentivirus was transfected at an infection coefficient of MOI=5, and 8 μg/mL polybrene was added, and cultured in a 37°C incubator. After 24 hours of transfection, the medium was replaced, and the growth of the cells was continuously observed, and the culture time was 8-13 days. The transfected CAR-T cells were obtained.

Detection of CAR19 lentiviral transfection efficiency: After the transfection was completed, the transfected cells were observed under an inverted fluorescence microscope at regular intervals. Aspirate the transfected CAR-T cells, centrifuge at 1000rpm for 5min to collect the precipitate, and wash the precipitate with PBS solution. The proportion of cells expressing GFP fluorescence in the transfected CAR-T cells was detected using the FITC channel of the flow cytometer. The transfection efficiency is shown in Figure 3.

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

In order to detect whether 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 inoculated into 6-well plates, and cultured at 37 °C in 5% CO ₂ for 24 h. Aspirate the culture supernatant, centrifuge at 1000 rpm for 5 min to remove the cell pellet, and harvest the culture supernatant. IFNγ and IL-2 in the culture supernatant were detected according to the instructions of the ELISA kit. As shown in Figure 4 and Figure 5, Figure 4 is a comparison chart of IL-2 secretion, and Figure 5 is a comparison chart of IFNγ secretion. Since the secretion of IFNγ and IL-2 was detected, it can be proved that the CAR-T cells transfected with CAR19 lentivirus have been effectively activated.

Anti-tumor effect in vitro: ①Group: Take 40 wells of the 96-well plate and divide them into eight groups. Each group has five duplicate holes. Add 100 μL culture medium and 100 μL 1×10 ⁴ target cells (denoted as Ack) to the second group, the target cells are HepG2 cells containing GPC3 target protein in the target cells, add 100 μL culture medium and 100 μL 1×10 ⁴ cells to the third group Effector cells (denoted as Acn), wherein the effector cells are CAR-T cells transfected with CAR structure, 100 μL 1×10 ⁴ target cells and 100 μL 1×10 ⁴ effector cells (denoted as As , number of effector cells: number of target cells=1:1), 100 μL medium and 100 μL 5× ¹⁰⁴ effector cells (referred to as Acn) were added to the fifth group, and 100 μL 1×104 effector cells were added to the sixth group. ⁴ target cells and 100 μL 5×10 ⁴ effector cells (denoted as As, number of effector cells: number of target cells=5:1), 100 μL medium and 100 μL 1×10 ⁵ effector cells were added to the seventh group For cells (denoted as Acn), 100 μL of 1×10 ⁴ target cells and 100 μL of 1×10 ⁵ effector cells (denoted as As, number of effector cells:number of target cells=10:1) were added to the eighth group.

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

After incubating the 96-well plate for 4 hours, 20 uL of CCK-8 solution was added to each well, and the 96-well plate was incubated in the incubator for 4 hours. Absorbance was measured at 450 nm with a microplate reader. According to the absorbance, calculate the cell killing efficiency of group ① and group ② respectively=[1-(As-Acn)/(Ack-Ab)]×100%, where As is the test well (medium containing target cells, effector cells and CCK-8 solution), Ack is target cell control well (medium containing target cells and CCK-8 solution), Can is effector cell control well (medium containing effector cells, CCK-8 solution), Ab is blank Control (medium without cells and CCK-8 solution). Figure 6 is a diagram of the cell killing efficiency provided by the embodiment of the present invention. As shown in Figure 6, the CAR-T cells obtained by using the chimeric antigen receptor targeting GPC3 provided by the embodiment of the present invention have significantly higher killing efficiency on 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. The GPC3 antigen is highly specifically expressed in primary liver cancer HCC, and can be detected in the early stage of the tumor, especially in small liver cancer and alpha-fetoprotein The positive rate is high in AFP-negative HCC patients. The level of GPC3 in liver cancer tissue is significantly increased, so the chimeric antigen receptor provided by the embodiment of the present invention targets the GPC3 antigen and can recognize the GPC3 antigen, which makes the CAR constructed by the novel chimeric antigen receptor -T cells have a good ability to kill the GPC3 target protein, and can exert a stronger anti-tumor effect. At the same time, the chimeric antigen receptor targeting GPC3 can also secrete the cell chemokine CCL19, without changing the anti-tumor activity , the chemokines produced are expected to promote the infiltration of immune cells into the tumor microenvironment, inhibit the growth rate of tumors, and reduce the volume of tumors, so that the chimeric antigen receptor targeting GPC3 has stronger anti-solid tumor tumor activity, thus showing A good application prospect.

The above descriptions 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 range.

sequence listing

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gcgtcgcagc ccctgtccct gcgcccagag gcgagccggc cagcggcggg gggcgcagtg 960

cacacgaggg ggctggacgt caagcccttt tgggtgctgg tggtggttgg tggagtcctg 1020

gcttgctata gcttgctagt aacagtggcc tttaattattt 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 (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 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 targeting GPC3, characterized in that, the chimeric antigen receptor comprises: a signal peptide connected in sequence, a single-chain antibody targeting GPC3, a Myc tag, a CD8α hinge region, a CD28 span Membrane region, co-stimulatory factor, intracellular signal domain, P2A linking peptide and CCL19 secretory region, the nucleotide sequence of the signal peptide is shown in SEQ ID NO: 1 in the sequence listing, and the single-chain antibody targeting GPC3 The nucleotide sequence of the Myc tag 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 is shown in the sequence The list is shown in SEQ ID NO: 4, 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 SEQ ID in the sequence listing As shown in 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. 2. The chimeric antigen receptor targeting GPC3 according to claim 1, wherein one end of the co-stimulatory factor is connected to the other end of the CD28 transmembrane region, and the co-stimulatory factor is CD27, At least one of CD28, 4-1BB, OX40, CD30, CD40, ICOS, NKG2D and B7-H3. 3. The chimeric antigen receptor targeting GPC3 according to claim 2, wherein the costimulatory factor is CD28, and the nucleotide sequence of the costimulatory factor is as shown in SEQ ID NO: 9 in the sequence listing shown. 4. An application of the chimeric antigen receptor targeting GPC3 according to any one of claims 1 to 3 in the preparation of medicine, characterized in that, the application comprises: using the chimeric antigen receptor targeting GPC3 Antigen receptors as antineoplastic agents. 5. The application according to claim 4, wherein the tumors include: liver cancer, lung cancer, gastric cancer, breast cancer, melanoma, ovarian cancer, yolk sac tumor and neuroblastoma. 6. The use according to claim 5, characterized in that the tumor is liver cancer.

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