CN110684120B

CN110684120B - Chimeric antigen receptor targeting GPC3 and application thereof

Info

Publication number: CN110684120B
Application number: CN201910967265.1A
Authority: CN
Inventors: 张坤
Original assignee: Sinoneural Shanghai Cell And Gene Engineering Holdings Co ltd
Current assignee: Sinoneural Shanghai Cell And Gene Engineering Holdings Co ltd
Priority date: 2019-10-12
Filing date: 2019-10-12
Publication date: 2023-03-07
Anticipated expiration: 2039-10-12
Also published as: CN110684120A

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

Chimeric antigen receptor targeting GPC3 and application thereof

Technical Field

The invention relates to the field of tumor immunotherapy, in particular to a GPC 3-targeted chimeric antigen receptor and application thereof.

Background

Chimeric Antigen Receptor T-cells (CAR-T) are constructed by introducing a CAR gene fused thereto into the genome of a host T lymphocyte in the form of a nucleic acid. The CAR-T shows favorable treatment effect in the application of hemangioma, and the remission rate of the CAR-T is up to more than 90 percent especially for B lymphocyte leukemia patients who relapse and are difficult to treat. However, in the application of solid tumors, CAR-T has not yet achieved a good clinical efficacy in treating solid tumors due to the lack of tumor-specific antigens, etc.

Disclosure of Invention

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

in one aspect, embodiments of the present invention provide a GPC 3-targeted chimeric antigen receptor 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, and 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 secretion region is shown as SEQ ID NO: shown in fig. 8.

Further, the chimeric antigen receptor targeting GPC3 further comprises a co-stimulatory factor linked at one end to the other end of the CD28 transmembrane region, the co-stimulatory factor being at least one of CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, NKG2D, and B7-H3.

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

In another aspect, the embodiments of the present invention provide a use of the above chimeric antigen receptor targeting GPC3, including: the chimeric antigen receptor targeting GPC3 is used as an antitumor drug.

Further, the tumor comprises: liver cancer, lung cancer, stomach cancer, breast cancer, melanoma, ovarian cancer, yolk sac tumor and neuroblastoma.

Preferably, the tumor is liver cancer.

The technical scheme provided by the embodiment of the invention has the following beneficial effects: the embodiment of the invention provides a GPC 3-targeted chimeric antigen receptor, wherein GPC3 antigen is specifically and highly expressed in primary liver cancer HCC and can be detected at the initial stage of tumor, particularly, the positive rate is very high in small liver cancer and alpha fetoprotein AFP-negative HCC patients, and the GPC3 level in liver cancer tissues is obviously increased, so that the GPC3 antigen of the chimeric antigen receptor provided by the embodiment of the invention is taken as a target point and can be recognized, so that CAR-T cells constructed by adopting the novel chimeric antigen receptor have good killing capacity on GPC3 target protein and can play a stronger anti-tumor effect, and meanwhile, the GPC 3-targeted chimeric antigen receptor can also secrete a cell chemotactic factor CCL19, and the generated chemotactic factor is expected to promote immune cells to infiltrate into a tumor microenvironment, inhibit the growth rate of tumor and reduce the volume of tumor while the anti-tumor activity is not changed, so that the GPC 3-targeted chimeric antigen receptor has stronger anti-solid tumor activity, thereby having good application prospect.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

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

FIG. 2 is an agarose gel electrophoresis image provided by an embodiment of the invention;

FIG. 3 is a graph of CAR19 lentivirus transfection efficiency provided by an embodiment of the invention;

FIG. 4 is a graph comparing the secretion of IL-2 provided by the examples of the present invention;

FIG. 5 is a graph comparing IFN γ secretion amounts provided in examples of the present invention;

FIG. 6 is a comparison graph of cell killing efficiency provided by the present invention, wherein A is the killing ability of CAR-T cells to HepG2 cells, A is the killing ability of control cells to HepG2 cells, the abscissa is the number ratio of effector cells to target cells, and the ordinate is the cell killing efficiency in%.

Detailed Description

To make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Examples

The embodiment of the invention provides a chimeric antigen receptor targeting GPC3, which comprises: a signal peptide, a single-chain antibody targeting GPC3 (Glypican-3), a Myc label, a CD8 alpha hinge region, a CD28 transmembrane region and a common domain which are connected in sequenceThe polypeptide comprises a stimulating factor, an intracellular signal domain, a P2A connecting peptide and a CCL19 secretion region, wherein the nucleotide sequence of the signal peptide is shown as SEQ ID NO:1, and 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 secretion region is shown as SEQ ID NO: shown in fig. 8. When implemented, single chain antibodies targeted to GPC3 include light chain V _L Variable region, inter-Linker and GPC3 Single chain antibody heavy chain V _H Variable region three parts.

Further, one end of a costimulatory factor is linked to the other end of the CD28 transmembrane domain, and the costimulatory factor is at least one of CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, NKG2D and B7-H3. In this case, the two or more costimulatory factors are sequentially linked end to end and then linked to the CD8 α transmembrane region and intracellular signaling region, respectively.

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

The embodiment of the invention provides application of a chimeric antigen receptor targeting GPC3, which comprises the following steps: the chimeric antigen receptor targeting GPC3 is used as an antitumor drug. The tumor comprises liver cancer, lung cancer, gastric cancer, breast cancer, melanoma, ovarian cancer, yolk sac tumor and neuroblastoma, preferably, the tumor is liver cancer.

When the method is implemented, the NCBI website database is searched for: human CD8 alpha signal region, human CD28 hinge region, CD28 intracellular region, human CD3 zeta intracellular signal region, human CCL19 gene sequence information, heavy chain of anti-GPC 3 monoclonal antibody and light chain variable region of anti-GPC 3 monoclonal antibody, wherein the sequences are subjected to codon optimization on the website http:// sg. The gene totally synthesizes the gene sequence of the chimeric antigen receptor, and the structure is as follows: the signal peptide-single chain antibody of targeted GPC 3-Myc tag-CD8 alpha hinge region-CD 28 transmembrane region-costimulatory factor CD 28-intracellular signal domain CD3 zeta-connecting peptide P2A-CCL19 secretion region specifically is as follows: the CD8 alpha leader-GPC3 scFv-Myc tag-CD8 alpha hinge-CD28-CD3 zeta-P2A-CCL 19 is marked as CAR19, the structure of the chimeric antigen receptor is shown in figure 1, and the nucleotide sequence of the chimeric antigen receptor is shown as SEQ ID NO:10, and correspondingly, the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO:11, respectively.

The portions of the chimeric antigen receptor may be linked together directly or via a linker sequence. The linker sequence may be a flexible sequence suitable for use in antibody light chain variable regions and heavy chain variable regions as known in the art, e.g., G and S containing (G) ₄ S) ₃ A linker sequence.

The gene plasmid vector of the chimeric antigen receptor is prepared by the following specific method:

the method comprises the steps of amplifying and obtaining a CAR19 gene sequence through PCR (Polymerase Chain Reaction), adding a restriction enzyme site Xba I and a restriction enzyme site BamH I at two ends of the CAR19 gene respectively to obtain a product to be restricted, and performing Xba I and BamH I double restriction enzyme Reaction on the product and a lentiviral vector plasmid pCDH-EF1-MCS-T2A-copGFP respectively to obtain a restriction enzyme fragment containing CAR19 and a restriction enzyme fragment containing pCDH-EF 1-MCS-T2A-copGFP. The enzyme digestion reaction conditions are as follows: the enzyme digestion temperature is 37 ℃, and the enzyme digestion time is 30min. The enzyme digestion system with the total volume of 50 mu L comprises: 5 μ L of 10 × buffer; 5. Mu.g of DNA to be cleaved with an enzyme; 2 μ L of XbaI enzyme; 2 μ L of BamH I enzyme; the volume of the digestion system was made up to 50. Mu.L with deionized water.

And respectively carrying out electrophoresis on the enzyme-digested fragment containing the CAR19 and the enzyme-digested fragment containing the pCDH-EF1-MCS-T2A-copGFP by using agarose gel with the concentration of 1%, respectively cutting nucleic acid bands of the enzyme-digested fragment containing the CAR19 and the enzyme-digested fragment containing the pCDH-EF1-MCS-T2A-copGFP after the electrophoresis is finished, respectively putting the nucleic acid bands into two clean EP tubes, and then purifying and recovering DNA in the agarose gel to obtain the CAR19 enzyme-digested product and the pCDH-EF1-MCS-T2A-copGFP enzyme-digested product.

The DNA fragments resulting in the CAR19 cleavage product and pCDH-EF1-MCS-T2A-copGFP cleavage product were ligated overnight at 16 ℃ to form pCDH-EF1-CAR19-T2A-copGFP. Wherein, the connection system with the total volume of 10 mu L comprises: 1 u L pCDH-EF1-MCS-T2A-CopGFP enzyme cutting product, 7 u L CAR19 enzyme cutting product, 1 u L T4 DNA ligase and 1 u L10 x T4 DNA ligase Buffe r.

The ligation products were transferred to Stbl3 competent cells (purchased from TRANSGEN BIOTECH) as follows:

stbl3 competent cells stored in a-80 ℃ refrigerator were taken out and thawed on ice. The ligation product was added to Stbl3 competent cells, ice-cooled for 30min, heat-shocked at 42 ℃ for 45s, and then ice-cooled for 2min to obtain the transformed product.

Adding the transformed product into 900 μ L liquid LB culture medium without antibiotic, fermenting and culturing at 37 deg.C with shaking table rotation speed of 200rpm for 45min to obtain fermentation liquid. The preparation method of the liquid LB culture medium without antibiotics comprises the following steps: 5g of imported yeast extract, 10g of imported peptone, 10g of anhydrous sodium chloride and 1L of sterile water are uniformly mixed, and the mixture is sterilized at 121 ℃ for 20min for use.

And centrifuging the fermentation liquor at 4000rpm for 5min, discarding the supernatant, retaining the precipitate, and resuspending the precipitate by adopting 100 mu L of liquid LB culture medium to obtain a resuspension solution.

The resuspended solution was applied to Amp-resistant solid LB plate medium (purchased from Komaga, shanghai, microbial technologies, ltd.), and the solid LB plate medium was placed in a bacterial incubator at 37 ℃ for overnight culture.

Positive clones were picked on solid LB plate medium.

The obtained positive clone is identified by the following specific method:

the obtained positive clone is subjected to double enzyme digestion reaction of Xba I and BamH I, the specific operation refers to the double enzyme digestion reaction of the substance to be digested, the enzyme digestion product obtained by the positive clone is subjected to agarose gel electrophoresis to identify a target fragment, and the target fragment with the size of about 2000bp is obtained, and the result is shown in figure 2. The target sequence is known to be CAR19 gene through sequencing identification.

And (3) plasmid extraction: and (3) preparing the positive clone with correct sequencing into an original bacterial liquid, inoculating the original bacterial liquid into 100mL of Amp resistant liquid LB culture medium, and carrying out overnight culture at 37 ℃ with the rotation speed of a shaking table of 200rpm to obtain original bacterial fermentation liquid.

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

Plasmid of the thallus is extracted by using an endotoxin-free plasmid macroextraction kit (purchased from Tiangen company), and the specific method is carried out according to the instruction of the kit.

CAR19 lentiviral vector packaging: at about 8.5X 10 per dish 6h before transfection ⁶ 293T cells were seeded into 10cm diameter dishes. Ensure that the confluency of the cells is about 80% during transfection and the cells are uniformly distributed in a culture dish.

Preparing a solution A and a solution B, wherein the solution A comprises: 4mL of 2 XHEPES buffer (8 dishes packaged together), solution B comprising: 72ug of plasmid (target plasmid), 37.04ug of packaged plasmid PLP1, 34.8ug of packaged plasmid PLP2, 24.08ug of packaged plasmid PLP-VSVG, and 400 μ L2.5M calcium ion solution, with a total volume of 4mL of solution B. And fully and uniformly mixing the solution B, adding the solution B into the solution A dropwise while slightly swirling the solution A, and standing for 3-5 min to obtain a mixed solution. The mixture was vortexed gently, added dropwise to 293T cell-containing dishes, 1mL of the mixture was added to each dish, the dishes were gently shaken back and forth to distribute the mixture evenly on the surface of the dishes (care was taken not to rotate the dishes while shaking), and the dishes were incubated in an incubator at 37 ℃. After 12h of culture, the medium was replaced with fresh one and the culture was continued. After 48h of culture, the supernatant containing CAR19 lentivirus was collected. Centrifuging at 1500rpm/min for 5min, collecting supernatant, and filtering with 0.45 μm filter membrane to obtain filtrate containing CAR19 lentivirus.

The filtrate containing the CAR19 lentivirus was transferred to an ultracentrifuge tube and a 20% concentration of sucrose was carefully layered on the bottom of the ultracentrifuge tube (1 mL of sucrose per 8mL of filtrate containing the lentivirus). Equilibrating the ultracentrifuge tube with PBS (phosphate buffer saline), centrifuging at 27600rpm/min for 2h at 4 deg.C, carefully removing the ultracentrifuge tube, decanting the supernatant, inverting the ultracentrifuge tube to remove the residual supernatant and retaining the precipitate. Adding 150 mu LPBS into an ultracentrifuge tube, lightly blowing and beating the bottom of the ultracentrifuge tube for several times by using a micropipette gun to dissolve precipitates in PBS to obtain concentrated CAR19 lentivirus (a gene plasmid vector of a chimeric antigen receptor), subpackaging the concentrated CAR19 lentivirus into the centrifuge tube when the implementation is carried out, and storing at-80 ℃.

CAR19 lentiviral titer assay: concentrated CAR19 lentivirus 0.5. Mu.L, 5. Mu.L and 50. Mu.L were used to infect 293T cells (1X 10) ⁵ One/hole) 24h, changing the solution after 24h, extracting cell genome DNA after 72h, and diluting the genome DNA concentration to 5-100 ng/. Mu.L. Using TransLv ^TM Lentivirus qPCR transcription Kit (available from TransGen) was performed according to the instructions. The CAR19 lentivirus titer is 5 multiplied by 10 according to detection ⁸ TU/mL。

The T cell of the chimeric antigen receptor is prepared by the following specific method:

preparation of PBMC (Peripheral Blood Mononulear Cell): 20mL of peripheral blood of the volunteer was collected, the peripheral blood was added into a 50mL centrifuge tube containing heparin, centrifuged at 2000rpm for 10min, and the upper plasma was transferred to a new centrifuge tube for cryopreservation. Adding physiological saline preheated at 37 ℃ and equal in volume to the sediment into a centrifuge tube, fully and uniformly mixing, and carrying out heavy suspension on the blood cell sediment to obtain heavy suspension cell sap. Another 50mL centrifuge tube was added with 20mL of the pre-warmed lymphocyte separation medium. 20mL of resuspended cell fluid was slowly added to the upper layer of lymphocyte separation fluid. Centrifuge at 800rpm for 20min. Sucking the upper plasma layer at a constant speed, stopping sucking the plasma when the plasma is 2-3 cm away from the tunica albuginea layer, quickly sucking the tunica albuginea layer cells, transferring to another new 50mL centrifuge tube, supplementing the volume to 45mL by using normal saline, centrifuging at 1200rpm for 5min, and repeating for 2 times for cleaning the cells. The cell pellet was resuspended using RPMI1640+ FBS medium at 10% concentration and the T cell number was calculated. In this example, T cell numberIs 1.5X 10 ⁷ And (4) respectively.

CAR19 lentivirus transfection of human T cells: in this example, the T cell density was adjusted to 1X 10 ⁶ and/mL, inoculating the T cells into anti-human 50ng/mL CD3 antibody and 50ng/mL CD28 antibody according to 1 mL/hole, adding 200IU/mL interleukin 2, and stimulating and culturing for 48h. Two days after T cell activation, CAR19 lentivirus was transfected with an infection factor of MOI =5 and 8 μ g/mL polybrene was added and cultured in an incubator at 37 ℃. After 24h of transfection, the culture medium is changed, and the growth condition of the cells is continuously observed, wherein the culture time is 8-13 days. Resulting in transfected CAR-T cells.

CAR19 lentivirus transfection efficiency assay: after transfection was complete, the transfected cells were observed periodically using an inverted fluorescence microscope. Transfected CAR-T cells were aspirated, the pellet was collected by centrifugation at 1000rpm for 5min, and the pellet was washed with PBS solution. The proportion of cells expressing GFP fluorescence of transfected CAR-T cells was detected using the flow cytometer FITC channel. The transfection efficiency is shown in FIG. 3.

And (3) detecting the secretion of the chemotactic factor of the CAR-T cell, wherein the detection comprises the following steps:

to determine whether CAR-T cells transfected with CAR19 lentivirus were activated efficiently, this example tested CAR-T cells co-cultured with target cells and the secreted amounts of IFN γ and IL-2 were determined by ELISA kits, respectively. Specifically, each well will be 1 × 10, respectively ⁶ Individual CAR-T cells and 1X 10 cells per well ⁵ Each target cell was seeded in 6-well plates at 37 ℃ with 5% CO ₂ And culturing for 24h. The culture supernatant was aspirated, centrifuged at 1000rpm for 5min to remove cell pellet, and the culture supernatant was harvested. Culture supernatants were assayed for IFN γ and IL-2 according to the ELISA kit instructions. As shown in FIG. 4 and FIG. 5, FIG. 4 is a graph comparing the secretion amounts of IL-2, and FIG. 5 is a graph comparing the secretion amounts of IFN γ. Since IFN γ and IL-2 secretion were detected, it could be demonstrated that CAR-T cells after CAR19 lentivirus transfection had been effectively activated.

In vitro anti-tumor effect: (1) group (2): out of the 96-well plates, 40 wells were taken and divided into eight groups of five wells, with 200. Mu.L of medium (denoted Ab) added to each group, and 200. Mu.L of medium added to each groupThe groups were all supplemented with 100. Mu.L of medium and 100. Mu.L of 1X 10 ⁴ Target cells (designated Ack) which were HepG2 cells containing the GPC3 target protein, and the third group to which 100. Mu.L of the medium and 100. Mu.L of 1X 10 cells were added ⁴ An effector cell (designated Acn), wherein the effector cell is CAR-T cell transfected with CAR structure, and 100. Mu.L of 1X 10 cells were added to the fourth group ⁴ One target cell and 100. Mu.L of 1X 10 ⁴ Effector cells (As, effector cell count: target cell count = 1: 1), and 100. Mu.L of medium and 100. Mu.L of 5X 10 cells were added to the fifth group ⁴ Each effector cell (designated Acn), 100. Mu.L of 1X 10 cells were added to the sixth group ⁴ Target cells and 100. Mu.L of 5X 10 ⁴ Effector cells (As, effector cell count: target cell count = 5: 1), 100. Mu.L of medium and 100. Mu.L of 1X 10 cells were added to the seventh group ⁵ Each effector cell (designated Acn), in the eighth group was added 100. Mu.L of 1X 10 ⁴ One target cell and 100. Mu.L of 1X 10 ⁵ Effector cells (As, effector cell count: target cell count = 10: 1).

(2) Group (control group): an additional 40 wells of the 96-well plate were divided into eight groups of five multiple wells each, in the same grouping method as in group (1), but different from group (1) in that the effector cells were CAR-T cells transfected with CAR structure or uninfected T cells.

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

The embodiment of the invention provides a chimeric antigen receptor targeting GPC3, the GPC3 antigen is specifically and highly expressed in primary liver cancer HCC, can be detected in the initial stage of tumor, and has high positive rate particularly in small liver cancer and alpha fetoprotein AFP negative HCC patients. The GPC3 level in liver cancer tissues is obviously increased, so that the chimeric antigen receptor provided by the embodiment of the invention takes a GPC3 antigen as a target spot and can recognize the GPC3 antigen, so that CAR-T cells constructed by adopting the novel chimeric antigen receptor have good killing capacity on GPC3 target protein and can play a stronger anti-tumor effect, meanwhile, the chimeric antigen receptor targeting GPC3 can also secrete a cell chemotactic factor CCL19, and the generated chemotactic factor is expected to promote immune cells to infiltrate into a tumor microenvironment, inhibit the growth rate of tumors and reduce the volume of the tumors while the anti-tumor activity is not changed, so that the chimeric antigen receptor targeting GPC3 has stronger anti-solid tumor activity, and thus, the application prospect is good.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.

Sequence listing

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cttactgccg acgagtcaac aagcacggct tatatggaac tttccagctt gcggagcgaa 720

gacaccgccg tgtattattg taccgaattc ggtggcgaac aaaagttgat ttctgaagaa 780

gatttgagat ctgccctgag caactccatc atgtacttca gccacttcgt gccggtcttc 840

ctgccagcga agcccaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 900

gcgtcgcagc ccctgtccct gcgcccagag gcgagccggc cagcggcggg gggcgcagtg 960

cacacgaggg ggctggacgt caagcccttt tgggtgctgg tggtggttgg tggagtcctg 1020

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 (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

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, and 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, and 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 GPC 3-targeted chimeric antigen receptor of claim 1, wherein one end of the costimulatory factor is linked 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.

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: shown at 9.

4. Use of a GPC 3-targeted chimeric antigen receptor according to any of claims 1 to 3 for the preparation of a medicament, 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.