CN111848818A

CN111848818A - Enhanced immune cell and application thereof

Info

Publication number: CN111848818A
Application number: CN202010761426.4A
Authority: CN
Inventors: 汤朝阳; 秦乐; 吴迪; 邓殷健; 吴海鹏; 王翠花; 冯世忠; 冯嘉昆; 其他发明人请求不公开姓名
Original assignee: Guangdong Zhaotai In Vivo Biomedical Technology Co ltd
Current assignee: Guangdong Zhaotai In Vivo Biomedical Technology Co ltd
Priority date: 2020-07-31
Filing date: 2020-07-31
Publication date: 2020-10-30

Abstract

The invention provides an enhanced immune cell and application thereof, wherein the immune cell expresses a chimeric antigen receptor which comprises an antigen binding domain, a transmembrane domain and a signal transduction domain; the antigen binding domain includes anti-GPC 3 single chain antibody and NKG 2D. The chimeric antigen receptor has a targeting effect on GPC3 specific tumor cells and heterogeneous tumor cells, and the constructed immune cells expressing the chimeric antigen receptor can target the tumor cells expressing specific antigens and simultaneously mobilize the functions of endogenous NKG2D to target the tumor cells with heterogeneity, thereby realizing the effects of removing and killing the specific tumor cells and the heterogeneous tumor cells.

Description

Enhanced immune cell and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and relates to an enhanced immune cell and application thereof.

Background

In recent years, with the development of tumor immunology theory and clinical technology, Chimeric antigen receptor T-cell immunotherapy (CAR-T) has become one of the most promising tumor immunotherapy. At present, CAR-T cell therapy has been widely applied to the treatment of B cell malignant tumors, and CAR-T cells targeting CD19 are precursors for the treatment of B cell malignant tumors by CAR-T therapy, and provide an effective scheme for the treatment of B cell malignant tumors.

Although CAR-T cells can target tumor cells that express specific antigens, many tumor cells do not express specific, specific tumor antigens, and tumor heterogeneity is a major obstacle facing cell therapy for solid tumors. Increasing the ability of chimeric antigen receptor molecules to recognize different targets is one of the solutions to the above problems, e.g., CAR-T cells can be constructed with reference to receptors in innate immunity that recognize a broad spectrum of antigenic proteins, thereby enhancing the ability of traditional CAR-T cells to recognize tumors that express no specific antigen.

Disclosure of Invention

Aiming at the defects and actual needs of the prior art, the invention provides an enhanced immune cell and application thereof, wherein the enhanced immune cell has a targeted clearing and killing effect on tumor cells expressing specific antigens and heterogeneous tumor cells.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a chimeric antigen receptor comprising an antigen binding domain, a transmembrane domain, and a signaling domain;

the antigen binding domain includes anti-GPC 3 single chain antibody and NKG 2D.

In the invention, the chimeric antigen receptor of which the antigen binding domain is anti-GPC 3 single-chain antibody and NKG2D has targeting effect on GPC3 specific tumor cells and heterogeneous tumor cells, and the recognition capability of the traditional CAR molecule on non-specific antigen expressing tumors is obviously enhanced.

Preferably, the anti-GPC 3 single-chain antibody comprises the amino acid sequence shown in SEQ ID NO. 1;

SEQ ID NO:1：

DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHVPPTFGSGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGASVKLSCKASGYTFTDYEMHWVKQTPVHGLKWIGALDPKTGDTAYSQKFKGKATLTADKSSSTAYMELRSLTSEDSAVYYCTRFYSYTYWGQGTLVTVSA。

preferably, the NKG2D comprises the amino acid sequence shown in SEQ ID NO. 2;

SEQ ID NO:2：

FNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQ NASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTII EMQKGDCALYASSFKGYIENCSTPNTYICMQRTV。

preferably, the anti-GPC 3 single-chain antibody and NKG2D are linked by a linking peptide.

Preferably, the transmembrane domain comprises CD28 and/or CD8 a.

Preferably, the antigen binding domain and transmembrane domain are connected by a hinge region.

Preferably, the hinge region comprises an IgG1 hinge region and/or a CD8 a hinge region.

Preferably, the signalling domain comprises any one of CD3 ζ, 4-1BB, CD28, TLR1, TLR2, CD27, OX40 or DAP10, or a combination of at least two.

Preferably, the chimeric antigen receptor further comprises a signal peptide.

Preferably, the signal peptide comprises a CD8 a signal peptide.

Preferably, the chimeric antigen receptor consists of a CD8 a signal peptide, an anti-GPC 3 single chain antibody, a linker peptide, NKG2D, CD8 a, 4-1BB and CD3 ζ in tandem.

Preferably, the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 3;

SEQ ID NO:3：

MALPVTALLLPLALLLHAARPDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHVPPTFGSGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGASVKLSCKASGYTFTDYEMHWVKQTPVHGLKWIGALDPKTGDTAYSQKFKGKATLTADKSSSTAYMELRSLTSEDSAVYYCTRFYSYTYWGQGTLVTVSAGGGGSGGGGSGGGGSGGGGSFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

in a second aspect, the present invention provides a coding gene encoding the chimeric antigen receptor of the first aspect.

Preferably, the coding gene comprises a nucleic acid sequence shown as SEQ ID NO. 4;

SEQ ID NO:4：

atggcactgcctgtgactgccctgctgctccctctcgcactcctgctgcacgcagcccgcccagatgttgtgatgacccaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcttgcagatctagtcagagccttgtacacagtaatggaaacacctatttacattggtacctgcagaagccaggccagtctccaaagctcctgatctacaaagtttccaaccgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttatttctgctctcaaaatacacatgttcctcctacgttcggatcggggaccaagctggaaataaaaggtggaggcggttcaggcggaggtggcagcggcggtggcgggtcgcaggttcaactgcagcagtctggggctgagctggtgaggcctggggcttcagtgaagctgtcctgcaaggcttcgggctacacatttactgactatgaaatgcactgggtgaagcagacacctgtgcatggcctaaaatggattggagctcttgatcctaaaactggtgatactgcctacagtcagaagttcaagggcaaggccacactgactgcagacaaatcctccagcacagcctacatggagctccgcagcctgacatctgaggactctgccgtctattactgtacaagattctactcctatacttactggggccaagggactctggtcactgtctctgcaggtggaggcggcagtggcggaggtgggagcggagggggcggttccggtggcgggggatctttcaaccaagaagttcaaattcccttgaccgaaagttactgtggcccatgtcctaaaaactggatatgttacaaaaataactgctaccaattttttgatgagagtaaaaactggtatgagagccaggcttcttgtatgtctcaaaatgccagccttctgaaagtatacagcaaagaggaccaggatttacttaaactggtgaagtcatatcattggatgggactagtacacattccaacaaatggatcttggcagtgggaagatggctccattctctcacccaacctactaacaataattgaaatgcagaagggagactgtgcactctatgcctcgagctttaaaggctatatagaaaactgttcaactccaaatacgtacatctgtatgcaaaggactgtgaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaagagaggcaggaagaagctgctgtacatcttcaagcagcccttcatgcgccccgtgcagacaacccaggaggaggacggctgcagctgtcggttcccagaggaggaggagggaggatgtgagctgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc。

in a third aspect, the present invention provides an expression vector, wherein the expression vector is a lentiviral vector comprising the coding gene of the second aspect.

In a fourth aspect, the present invention provides a recombinant lentivirus which is a mammalian cell transfected with an expression vector and a helper plasmid as described in the third aspect.

In a fifth aspect, the present invention provides a chimeric antigen receptor immune cell expressing the chimeric antigen receptor of the first aspect.

Preferably, the chimeric antigen receptor immune cell has the coding gene of the second aspect integrated into its genome.

Preferably, the chimeric antigen receptor immune cell comprises the expression vector of the third aspect and/or the recombinant lentivirus of the fourth aspect.

Preferably, the chimeric antigen receptor immune cell is a T cell expressing the chimeric antigen receptor of the first aspect.

Preferably, the chimeric antigen receptor immune cell is an NK cell expressing the chimeric antigen receptor of the first aspect.

In a sixth aspect, the present invention provides a method for producing the chimeric antigen receptor immune cell of the fifth aspect, the method comprising the step of introducing the gene encoding the chimeric antigen receptor of the first aspect into an immune cell.

In a seventh aspect, the present invention provides a use of the chimeric antigen receptor of the first aspect, the coding gene of the second aspect, the expression vector of the third aspect, the recombinant lentivirus of the fourth aspect, or the chimeric antigen receptor immune cell of the fifth aspect in the preparation of a medicament for treating a disease.

Preferably, the disease comprises a tumor.

Preferably, the tumor includes any one or a combination of at least two of liver cancer, lung cancer, breast cancer, stomach cancer, nephroblastoma, glioma, neuroblastoma, melanoma, nasopharyngeal carcinoma, mesothelioma, islet cell tumor, retinoblastoma, pancreatic cancer, uterine fibroids, cervical cancer, or thyroid cancer.

Compared with the prior art, the invention has the following beneficial effects:

(1) according to the invention, the NKG2D is embedded with the anti-GPC 3scFv and the signal conduction structural domain to construct a chimeric antigen receptor, and the chimeric antigen receptor has a targeting effect on GPC3 specific tumor cells and heterogeneous tumor cells;

(2) the immune cell expressing the chimeric antigen receptor has the function of targeted elimination of tumor cells expressing specific antigens, can also play a role in recognizing cell stress proteins such as MICA/B antigens, ULBP family antigens and the like by an endogenous NKG2D receptor, and has remarkable elimination and killing functions on specific tumor cells and heterogeneous tumor cells.

Drawings

FIG. 1 is a schematic structural diagram of a dual-targeted CAR molecule combining tumor antigen GPC3scFv and endogenous NKG 2D;

FIG. 2 is a graph of the killing efficiency of CAR-T cells on hepatoma cells at different E: T ratios;

FIG. 3 shows the secretion of IFN-. gamma.after coculture of CAR-T with test cells.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Example 1 construction of CAR molecular vectors

This example first synthesized a fragment with NKG2D and 4-1BB and CD3 ζ intracellular signaling domains, linked to pWPXLD-EGFP vector, followed by PCR-specific amplification of GPC 3-targeted scFv cloned into the above expression vector to obtain CAR molecules with antigen binding domains against GPC3scFv and NKG2D (amino acid sequence shown in SEQ ID NO:3 and nucleic acid sequence shown in SEQ ID NO: 4), and the schematic structure is shown in FIG. 1.

And transferring the constructed plasmid into escherichia coli, selecting a monoclonal with a target plasmid for overnight culture, and extracting the plasmid by using a plasmid extraction kit to obtain the recombinant lentiviral vector.

In this example, CAR (anti GPC3scFv-CD8 alpha-4-1 BB-CD3 zeta) and NKG2D CAR (NKG2D-CD8 alpha-4-1 BB-CD3 zeta) with antigen binding domains against GPC3scFv were constructed simultaneously, and corresponding lentiviral vectors were constructed.

Example 2 Lentiviral packaging

This example performed lentiviral packaging of the lentiviral vector constructed in example 1, as follows:

(1) culturing 293T cells in a 10cm culture dish in a DMEM high-sugar medium + 10% FBS (fetal bovine serum) + 1% double antibody (100 Xpenicillin-streptomycin mixed solution);

(2) when the density of 293T cells in the culture dish reaches 80%, replacing a DMEM high-sugar medium, 1% FBS and 1% double antibody with a culture medium;

(3) after the culture medium is replaced and cultured for 2 hours, preparing a transfection reagent, adding 500 mu L of opti-DMEM into a 15mL centrifuge tube, adding 7.2 mu L of PEI (linear polyethyleneimine) with the concentration of 10 mu g/mu L, slightly mixing uniformly, and standing for 5 min;

(4) putting 500 mu L of opti-DMEM into a 1.5mL centrifuge tube, adding 9 mu g of recombinant lentiviral vector, 3 mu g of pMD2.G helper plasmid and 12 mu g of psPAX into the centrifuge tube, mixing uniformly, adding into a transfection reagent, reversing and mixing uniformly, and standing for 20 min;

(5) adding all the mixed solution into 293T cells, incubating for 6h, and replacing 7mL of fresh culture medium DMEM high-sugar medium + 1% FBS + 1% double antibody;

(6) collecting supernatant after replacing the culture medium for 24 hours, and replacing 7mL of fresh culture medium DMEM high-sugar medium + 1% FBS + 1% double antibody;

(7) after 24h, collecting the supernatant again, and replacing 7mL of fresh culture medium DMEM high-glucose medium + 1% FBS + 1% double antibody;

(8) supernatants were collected after 24h and cells were discarded;

(9) after the collection of the culture medium supernatant was completed, the culture medium was centrifuged at 2500g for 0.5 hour, and the supernatant was filtered through a 0.45 μm filter to obtain a recombinant lentivirus.

The recombinant lentiviral vector comprises a lentiviral vector containing a coding gene of anti GPC3-NKG2D CAR, a lentiviral vector containing a coding gene of anti GPC3 CAR and a lentiviral vector containing a coding gene of NKG2D CAR, and the pWPXld-eGFP plasmid is an empty vector containing no CAR coding gene.

Example 3T cell activation and lentivirus transfection

(1) After the Pan T cells were separated from the cord blood, the cells were counted and the concentration was adjusted to 1X 10⁶one/mL, followed by addition of 10 μ L of american and whirlwind TransAct T cell reagent to each mL of cell suspension, and 48 hours after activation replaced with fresh medium (IMDM medium + 5% FBS (fetal bovine serum) + 1% diabody (100 x mixed penicillin-streptomycin solution) + IL-2);

(2) after 48h of T cell activation, the beads were removed, 300g was centrifuged for 5min, the supernatant was removed, the T cells were resuspended in fresh medium, CAR-expressing recombinant lentivirus or control lentivirus (MOI 10) was added, polybrene and 300IU/mL IL-2 were added, and the mixture was incubated at 37 ℃ with 5% CO₂Culturing in an incubator;

(3) after 24h, centrifuging for 5min at 300g, removing supernatant, and resuspending T cells in fresh culture medium containing 300IU/mL IL-2 to obtain CAR-T cells.

The CAR-T cells constructed in this example were anti gpc3-NKG2D-CAR-T, antiGPC3-CAR-T, NKG2D-CAR-T, respectively, while a WT control group (transfection blank lentivirus) was set.

Example 4 in vitro assay of killing function of CAR-T cells on hepatoma cells

The anti GPC3-NKG2D-CAR-T, antiGPC3-CAR-T, NKG2D-CAR-T, WT prepared in example 3 was mixed with 1X 10 of⁴Mixing tumor cells HepG2 at E: T ratio of 4:1, 2:1, 1:2, 1:4, and 1:8, adding into 96-well plate, setting 3 multiple wells in each group, centrifuging at 250g for 5min, placing at 37 deg.C and 5% CO₂Co-culturing for 18h in an incubator;

after 18h, 100. mu.L/well Luciferase substrate (1X) was added to the 96-well plate, the cells were resuspended and mixed well, RLU (relative light unit) was immediately measured by a multifunctional microplate reader for 1 second, and the killing effect of anti GPC3-NKG2D-CAR-T, antiGPC3-CAR-T, NKG2D-CAR-T, WT on HepG2 was compared in vitro using the Luciferase (Luciferase) quantitative killing efficiency assessment method, and the killing ratio calculation formula was as follows:

100% × (control well reading-experimental well reading)/control well reading (blank reading without cells negligible)

The results are shown in figure 2, and it can be seen that anti gpc3-NKG2D-CAR-T of endogenous NKG2D synergizes with anti gpc3-NKG2D-CAR-T of endogenous NKG2D, relative to negative control and anti gpc3-CAR-T and NKG2D-CAR-T that can recognize only a single target.

Example 5

HepG2 cells were treated at 5X 10⁵Cell/well density inoculation of 24-well plates, followed by addition of anti GPC3-NKG2D-CAR-T, antiGPC3-CAR-T, NKG2D-CAR-T and WT, in incubator co-culture for 12 h; and detecting the co-culture supernatant by adopting an IFN-gamma ELISA detection kit.

As shown in FIG. 3, the IFN- γ cytokine level in the co-culture supernatant of anti GPC3-NKG2D-CAR-T and HepG2 was significantly higher than that in the other groups, and some IFN- γ cytokines could be detected in the co-culture supernatant of anti GPC3-CAR-T or NKG2D-CAR-T and HepG 2.

In conclusion, the chimeric antigen receptor disclosed by the invention has a targeting effect on GPC3 specific tumor cells and heterogeneous tumor cells, and the constructed immune cells expressing the chimeric antigen receptor can target the tumor cells expressing specific antigens and simultaneously mobilize the functions of endogenous NKG2D to target the tumor cells with heterogeneity, so that the effects of removing and killing the specific tumor cells and the heterogeneous tumor cells are realized.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

SEQUENCE LISTING

<110> Guangdong Shoutai biomedical science and technology Co., Ltd

<120> an enhanced immune cell and uses thereof

<130>2020

<160>4

<170>PatentIn version 3.3

<210>1

<211>242

<212>PRT

<213> Artificial sequence

<400>1

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

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

115 120 125

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

130 135 140

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

145 150 155 160

Met His Trp Val Lys Gln Thr Pro Val His Gly Leu Lys Trp Ile Gly

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Ser Ala

<210>2

<211>134

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<213> Artificial sequence

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Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro

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Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe

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Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln

3540 45

Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu

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Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile Pro Thr

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Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu

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Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser

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Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile

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Cys Met Gln Arg Thr Val

130

<210>3

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

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

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

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

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

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

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

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Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr

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Tyr His Trp Met Gly Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln

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Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr

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

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

435 440 445

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

450 455 460

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

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Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile

485 490 495

Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp

500 505 510

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

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

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

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

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

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

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

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

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<210>4

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<212>DNA

<213> Artificial sequence

<400>4

atggcactgc ctgtgactgc cctgctgctc cctctcgcac tcctgctgca cgcagcccgc 60

ccagatgttg tgatgaccca aactccactc tccctgcctg tcagtcttgg agatcaagcc 120

tccatctctt gcagatctag tcagagcctt gtacacagta atggaaacac ctatttacat 180

tggtacctgc agaagccagg ccagtctcca aagctcctga tctacaaagt ttccaaccga 240

ttttctgggg tcccagacag gttcagtggc agtggatcag ggacagattt cacactcaag 300

atcagcagag tggaggctga ggatctggga gtttatttct gctctcaaaa tacacatgtt 360

cctcctacgt tcggatcggg gaccaagctg gaaataaaag gtggaggcgg ttcaggcgga 420

ggtggcagcg gcggtggcgg gtcgcaggtt caactgcagc agtctggggc tgagctggtg 480

aggcctgggg cttcagtgaa gctgtcctgc aaggcttcgg gctacacatt tactgactat 540

gaaatgcact gggtgaagca gacacctgtg catggcctaa aatggattgg agctcttgat 600

cctaaaactg gtgatactgc ctacagtcag aagttcaagg gcaaggccac actgactgca 660

gacaaatcct ccagcacagc ctacatggag ctccgcagcc tgacatctga ggactctgcc 720

gtctattact gtacaagatt ctactcctat acttactggg gccaagggac tctggtcact 780

gtctctgcag gtggaggcgg cagtggcgga ggtgggagcg gagggggcgg ttccggtggc 840

gggggatctt tcaaccaaga agttcaaatt cccttgaccg aaagttactg tggcccatgt 900

cctaaaaact ggatatgtta caaaaataac tgctaccaat tttttgatga gagtaaaaac 960

tggtatgaga gccaggcttc ttgtatgtct caaaatgcca gccttctgaa agtatacagc 1020

aaagaggacc aggatttact taaactggtg aagtcatatc attggatggg actagtacac 1080

attccaacaa atggatcttg gcagtgggaa gatggctcca ttctctcacc caacctacta 1140

acaataattg aaatgcagaa gggagactgt gcactctatg cctcgagctt taaaggctat 1200

atagaaaact gttcaactcc aaatacgtac atctgtatgc aaaggactgt gaccacgacg 1260

ccagcgccgc gaccaccaac accggcgccc accatcgcgt cgcagcccct gtccctgcgc 1320

ccagaggcgt gccggccagc ggcggggggc gcagtgcaca cgagggggct ggacttcgcc 1380

tgtgatatct acatctgggc gcccttggcc gggacttgtg gggtccttct cctgtcactg 1440

gttatcaccc tttactgcaa gagaggcagg aagaagctgc tgtacatctt caagcagccc 1500

ttcatgcgcc ccgtgcagac aacccaggag gaggacggct gcagctgtcg gttcccagag 1560

gaggaggagg gaggatgtga gctgagagtg aagttcagca ggagcgcaga cgcccccgcg 1620

taccagcagg gccagaacca gctctataac gagctcaatc taggacgaag agaggagtac 1680

gatgttttgg acaagagacg tggccgggac cctgagatgg ggggaaagcc gagaaggaag 1740

aaccctcagg aaggcctgta caatgaactg cagaaagata agatggcgga ggcctacagt 1800

gagattggga tgaaaggcga gcgccggagg ggcaaggggc acgatggcct ttaccagggt 1860

ctcagtacag ccaccaagga cacctacgac gcccttcaca tgcaggccct gccccctcgc 1920

Claims

1. A chimeric antigen receptor, comprising an antigen binding domain, a transmembrane domain, and a signaling domain;

2. The chimeric antigen receptor according to claim 1, wherein the anti-GPC 3 single-chain antibody comprises the amino acid sequence shown in SEQ id No. 1;

preferably, the NKG2D comprises the amino acid sequence shown in SEQ ID NO. 2;

3. The chimeric antigen receptor according to claim 1 or 2, wherein the transmembrane domain comprises CD28 and/or CD8 a;

preferably, the antigen binding domain and transmembrane domain are connected by a hinge region;

preferably, the hinge region comprises an IgG1 hinge region and/or a CD8 a hinge region;

preferably, the signalling domain comprises any one of CD3 ζ, 4-1BB, CD28, TLR1, TLR2, CD27, OX40 or DAP10, or a combination of at least two;

preferably, the chimeric antigen receptor further comprises a signal peptide;

preferably, the signal peptide comprises a CD8 a signal peptide.

4. The chimeric antigen receptor according to any one of claims 1 to 3, consisting of a CD8 a signal peptide, an anti-GPC 3 single-chain antibody, a linker peptide, NKG2D, CD8 a, 4-1BB, and CD3 ζ in tandem;

preferably, the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 3.

5. A coding gene encoding the chimeric antigen receptor of any one of claims 1 to 4;

preferably, the coding gene comprises a nucleic acid sequence shown as SEQ ID NO. 4.

6. An expression vector, wherein the expression vector is a lentiviral vector comprising the coding gene of claim 5.

7. A recombinant lentivirus, wherein the recombinant lentivirus is a mammalian cell transfected with the expression vector of claim 6 and a helper plasmid.

8. A chimeric antigen receptor immune cell, wherein said chimeric antigen receptor immune cell expresses the chimeric antigen receptor of any one of claims 1-4;

preferably, the chimeric antigen receptor immune cell has the coding gene of claim 5 integrated into its genome;

preferably, the chimeric antigen receptor immune cell comprises the expression vector of claim 6 and/or the recombinant lentivirus of claim 7;

preferably, the chimeric antigen receptor immune cell is a T cell expressing the chimeric antigen receptor of any one of claims 1-4;

preferably, the chimeric antigen receptor immune cell is an NK cell expressing the chimeric antigen receptor of any one of claims 1-4.

9. A method for producing the chimeric antigen receptor immune cell according to claim 8, which comprises the step of introducing the gene encoding the chimeric antigen receptor according to any one of claims 1 to 4 into an immune cell.

10. Use of the chimeric antigen receptor of any one of claims 1 to 4, the coding gene of claim 5, the expression vector of claim 6, the recombinant lentivirus of claim 7 or the chimeric antigen receptor immune cell of claim 8 for the preparation of a medicament for the treatment of a disease;

preferably, the disease comprises a tumor;