CN112210539A - Fourth-generation CAR-T cell and application thereof - Google Patents

Abstract

The invention provides fourth generation CAR-T cells expressing a chimeric antigen receptor that specifically binds antigen, secreted IL-7 and NFAT-regulated CCL 19; the signal peptide of the IL-7 is an IL-10 signal peptide; the promoter of the CCL19 is an NFAT regulatory promoter. According to the invention, the original signal peptide of IL-7 is changed into the signal peptide of T cell secretory protein IL-10, and the NFAT regulated promoter is adopted as the promoter of CCL19, so that the CAR-T cell can secrete IL-7 to the outside of the cell, and the CCL19 is conditionally expressed, thereby promoting the proliferation of the CAR-T cell, recruiting immune cells to the inside of a tumor to the maximum extent, and remarkably improving the anti-tumor efficacy of the CAR-T.

Description

Fourth-generation CAR-T cell and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and relates to a fourth generation CAR-T cell and application thereof.

Background

Chimeric antigen receptor T cell (CAR-T) immunotherapy is a technique that induces T cell activation by expressing Chimeric antigen receptor molecules that specifically recognize and bind to tumor antigens on T cell membranes, thereby achieving specific killing of tumor cells. The technology is one of the most promising tumor immunotherapy at present, and has made a great breakthrough in the field of leukemia treatment.

However, there are still problems with this technique in the treatment of solid tumors. Researchers are trying to improve and optimize CAR-T therapy to improve the therapeutic effect of CAR-T on solid tumors by increasing the affinity of chimeric antigen receptors, knocking out CAR-T cell PD-1, and the like.

The CAR-T cells constructed by CN109153989A can continuously secrete IL-7 and CCL19, so that the in-vitro expansion capacity of the CAR-T cells is improved, more peripheral blood circulation T cells and dendritic cells are recruited into tumors, and the anti-tumor effect of the CAR-T cells is enhanced. However, when CAR-T cells remain in a place with many immune cells such as spleen and lymph node, the secreted CCL19 recruits peripheral blood circulating T cells and dendritic cells to a specific site, and decreases the ratio of T cells and dendritic cells in blood circulation, thereby decreasing the probability of T cells and dendritic cells being recruited into a tumor.

Therefore, the construction of a novel CAR-T cell can not only secrete IL-7 and CCL19, but also recruit peripheral blood immune cells into the interior of a tumor to the maximum extent, and has important significance in the field of solid tumor treatment.

Disclosure of Invention

Aiming at the defects and practical requirements of the prior art, the invention provides a fourth generation CAR-T cell and application thereof, wherein the signal peptide of IL-7 is optimized to be the signal peptide of T cell secretory protein IL-10, and the continuous secretion of CCL19 is optimized to be NFAT regulatory secretion, so that the anti-tumor effect of the CAR-T cell is obviously improved, and the occurrence probability of inflammatory side reactions is reduced.

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

in a first aspect, the invention provides a fourth generation CAR-T cell expressing a chimeric antigen receptor that specifically binds an antigen, secreted IL-7 and NFAT-regulated CCL 19;

the signal peptide of the IL-7 is an IL-10 signal peptide;

the promoter of the CCL19 is an NFAT regulatory promoter.

In the invention, the IL-7 signal peptide is replaced by the signal peptide of the T cell secretory protein IL-10, so that the CAR-T cell secretes exogenous IL-7 to the outside of the cell, and the IL-7 enhances the in vitro proliferation capacity of the T cell; the NFAT regulatory promoter is used for regulating the expression of T cells to CCL19, CCL19 is not expressed when CAR-T cells are in a resting state, when CAR-T cells recognize tumor antigens and are activated, the NFAT regulatory promoter 3 XNFAT-RE-IL-2 starts transcription and translation of CCL19, CCL19 starts expression, and the effect that peripheral blood immune cells are recruited into tumors to the maximum extent is achieved.

Preferably, the IL-10 signal peptide comprises the amino acid sequence shown in SEQ ID NO. 1;

SEQ ID NO:1：MHSSALLCCLVLLTGVRA。

preferably, the IL-7 comprises an amino acid sequence shown in SEQ ID NO 2;

SEQ ID NO:2：

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH。

preferably, CCL19 comprises the amino acid sequence set forth in SEQ ID NO 3;

SEQ ID NO:3：

MALLLALSLLVLWTSPAPTLSGTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQLCAPPDQPWVERIIQRLQRTSAKMKRRSS。

preferably, the chimeric antigen receptor comprises a signal peptide, an antigen binding domain, a transmembrane domain and a signaling domain.

Preferably, the signal peptide comprises a GM-CSF signal peptide.

Preferably, the antigen binding domain binds to a tumor surface antigen comprising any one of CD19, CD20, CD22, CD30, CEA, EGFR, BRAF, HER-2, Mesothelin, MUC1, PSCA, GPC3, TERT, PTEN, PD-1, PD-L1 or VEGF, preferably any one of CD19, MUC1, GPC3, Mesothelin, PSCA or HER2, more preferably PSCA.

Preferably, the antigen binding domain is a single chain antibody targeting PSCA.

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

Preferably, the signaling domain comprises any one of, or a combination of at least two of, the CD28 endodomain, CD3 zeta, TLR2, 4-1BB, TLR1, CD27, OX40, or DAP10, preferably the combination of CD28 endodomain and CD3 zeta.

Preferably, the chimeric antigen receptor consists of a GM-CSF signal peptide, an anti-PSCA single chain antibody, a CD28 transmembrane domain, a CD28 intracellular domain, and a CD3 zeta tandem.

Preferably, the chimeric antigen receptor comprises an amino acid sequence shown as SEQ ID NO. 4;

SEQ ID NO:4：

MLLLVTSLLLCELPHPAFLLDIQLTQSPSSLSASVGDRVTITCSASSSVRFIHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSSPFTFGQGTKVEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWVAWIDPENGDTEFVPKFQGRATISADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQGTLVTVSSEPKSCDKTHTCPPCIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

in a second aspect, the present invention provides a lentiviral vector comprising a chimeric antigen receptor encoding gene, an IL-10 signal peptide encoding gene, an IL-7 encoding gene, an NFAT regulated promoter and a CCL19 encoding gene.

Preferably, the chimeric antigen receptor encoding gene comprises a nucleic acid sequence shown as SEQ ID NO. 5;

SEQ ID NO:5：

atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctggacattcagctgacccaatctccaagctctttgtccgcctctgtgggggatagggtcaccatcacctgcagtgccagttcaagtgtaagattcattcactggtaccagcagaaaccaggaaaagctcccaaaagactcatctatgacacatccaaactggcttctggcgtcccttctaggttcagtggctccgggtctgggacagacttcaccctcaccattagcagtctgcagccggaagatttcgccacctattactgtcagcagtggagtagtagcccattcacgttcggacaggggaccaaggtggagataaaaggcagtactagcggcggtggctccggaggcggctccggaggtggcggcagctcagaggttcagctggtggagtctgggggtggccttgtgcagccagggggctcactccgtttgtcctgcgcagcttctggcttcaacattaaagactactatatacactgggtgcgtcaggcccctggtaagggcctggaatgggttgcatggattgatcctgagaatggtgacactgaatttgtcccgaagttccagggccgtgccactataagcgcagacacatccaaaaacacagcctacctgcagatgaacagcctgcgtgctgaggacactgccgtctattattgtaaaacgggggggttctggggtcaaggaaccctggtcaccgtctcgagcgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc。

preferably, the IL-10 signal peptide coding gene comprises a nucleic acid sequence shown in SEQ ID NO. 6;

SEQ ID NO:6：

atgcacagctcagcactgctctgttgcctggtcctcctgactggggtgagggcc。

preferably, the IL-7 encoding gene comprises a nucleic acid sequence shown in SEQ ID NO. 7;

SEQ ID NO:7：

gattgtgatattgaaggtaaagatggcaaacaatatgagagtgttctaatggtcagcatcgatcaattattggacagcatgaaagaaattggtagcaattgcctgaataatgaatttaacttttttaaaagacatatctgtgatgctaataaggaaggtatgtttttattccgtgctgctcgcaagttgaggcaatttcttaaaatgaatagcactggtgattttgatctccacttattaaaagtttcagaaggcacaacaatactgttgaactgcactggccaggttaaaggaagaaaaccagctgccctgggtgaagcccaaccaacaaagagtttggaagaaaataaatctttaaaggaacagaaaaaactgaatgacttgtgtttcctaaagagactattacaagagataaaaacttgttggaataaaattttgatgggcactaaagaacac。

preferably, the NFAT regulated promoter comprises the nucleic acid sequence set forth in SEQ ID NO 8;

SEQ ID NO:8：

gctttaaggcaagctttaaggcaagctttaaggcaaagatctagactctagagggtatataatggaagctcgaattccagcttggcattccggtactgttggtaaaaagcttggcaatccggtactgttggtaaagccacc。

preferably, the CCL19 encoding gene comprises a nucleic acid sequence shown as SEQ ID NO. 9;

SEQ ID NO:9：

atggccctgctactggccctcagcctgctggttctctggacttccccagccccaactctgagtggcaccaatgatgctgaagactgctgcctgtctgtgacccagaaacccatccctgggtacatcgtgaggaacttccactaccttctcatcaaggatggctgcagggtgcctgctgtagtgttcaccacactgaggggccgccagctctgtgcacccccagaccagccctgggtagaacgcatcatccagagactgcagaggacctcagccaagatgaagcgccgcagcagt。

preferably, the lentiviral vector further comprises a linker peptide encoding gene.

Preferably, the connecting peptide encoding gene comprises a nucleic acid sequence shown as SEQ ID NO. 10;

SEQ ID NO:10：

aaaattgtcgctcctgtcaaacaaactcttaactttgatttactcaaactggctggggatgtagaaagcaatccaggtcca。

in a third aspect, the invention provides a recombinant lentivirus prepared by co-transfecting a mammalian cell with the lentiviral vector of the second aspect and a packaging helper plasmid.

In a fourth aspect, the invention provides a method of producing a fourth generation CAR-T cell according to the first aspect, the method comprising the step of introducing into the T cell the lentiviral vector according to the second aspect or the recombinant lentivirus according to the third aspect.

In a fifth aspect, the invention provides a pharmaceutical composition comprising a fourth generation CAR-T cell according to the first aspect.

Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

In a sixth aspect, the invention provides a fourth generation CAR-T cell of the first aspect, a lentiviral vector of the second aspect, a recombinant lentivirus of the third aspect or a pharmaceutical composition of the fifth aspect for use in the preparation of a medicament for the treatment of a solid tumor.

Preferably, the solid tumor includes any one of or a combination of at least two of liver cancer, lung cancer, breast cancer, wilms' tumor, 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) the invention constructs the CAR-T cell secreting IL-7 and CCL19, and the CAR-T cell can secrete IL-7 to the outside of the cell by changing the original signal peptide of IL-7 into the signal peptide of the T cell secretory protein IL-10, so that the proliferation of the CAR-T cell is promoted; the NFAT regulatory promoter is used as a promoter of CCL19, so that the CAR-T cell conditionally expresses CCL19, immune cells are recruited into the tumor to the maximum extent, and the anti-tumor efficiency of the CAR-T cell is improved;

(2) compared with the second generation and third generation CAR-T cells, the fourth generation CAR-T cell which secretes IL-7 continuously and expresses CCL19 conditionally has a remarkably enhanced tumor killing effect, and has important significance in the field of solid tumor treatment.

Drawings

FIG. 1 is the results of different CAR-T cells secreting IL-7 and CCL 19;

figure 2 is the T cell recruitment capacity of different CAR-T 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 preparation of CAR-T cells expressing secretory IL-7 and conditional CCL19

(1) Preparation of Lentiviral vectors

The following nucleic acid molecules were synthesized by whole gene:

(ii) a CAR molecule formed by the tandem connection of the nucleic acid sequences of the GM-CSF signal peptide, PSCA scFv, CD28 transmembrane domain, CD28 intracellular domain, CD3 ζ and 2A peptide;

nucleic acid molecules formed by the tandem connection of the nucleic acid sequences of IL-7 signal peptide (SEQ ID NO:11), IL-7 (without IL-7 signal peptide) (SEQ ID NO:7), 2A peptide (SEQ ID NO:10) and CCL19(SEQ ID NO: 9);

③ a secretory IL-7 molecule formed by the tandem connection of the nucleic acid sequences of IL-10 signal peptide (SEQ ID NO:6) and IL-7 (without IL-7 signal peptide) (SEQ ID NO: 7);

a conditional CCL19 molecule formed by connecting a 3 XNFAT-RE-IL 2 promoter (SEQ ID NO:8) and CCL19(SEQ ID NO:9) in series;

SEQ ID NO:11：

atgttccatgtttcttttaggtatatctttggacttcctcccctgatccttgttctgttgccagtagcatcatct。

cloning the first step into a lentivirus expression vector pwpxld-eGFP; sequentially cloning the first and the second into a lentivirus expression vector pwpxld-tCD 19; (iii) cloning of (i) and (iii) sequentially into the lentiviral expression vector pwpxld-tCD19, followed by reverse cloning of (iv) into the end of pwpxld-tCD 19.

(2) Recombinant lentivirus packaging

Auxiliary plasmids gag/pol, Rev and VSV-G are mixed with a lentiviral vector according to a proportion, added into serum-free DMEM with a certain volume, uniformly mixed and placed for 15 min; adding the above mixture into 293T cell culture bottle, mixing, and adding 5% CO at 37 deg.C₂Culturing for 6h in a cell culture box; after 6h, replacing a fresh culture medium, continuing to culture, and adding 10mM sodium butyrate solution; after 72h, collecting lentivirus culture supernatant for purification detection.

Lentiviral vectors include those expressing CAR, intact IL-7 and CCL, or those expressing CAR, secreted IL-7, conditional CCL 10.

(3) T cell activation and lentivirus transfection

Separating mononuclear cells from blood by Ficoll density gradient method, removing erythrocytes by lysis with erythrocyte lysate, separating T cells by MACS Pan-T magnetic beads, and diluting with culture medium AIM-V, 5% FBS, 100U/mL penicillin and 0.1mg/mL streptomycin to concentration of 2.5 × 10⁶Per mL for standby; t cells were stimulated using magnetic beads (both gentle and gentle) coated with CD2, CD3, and CD28 antibodies at a 1:2 ratio to T cells and a T cell density of 5X 10⁶Per mL/cm²T cells at 37 ℃ 5% CO₂Culturing and stimulating for 48h in an incubator;

removing the magnetic beads from the T cells to activateThe subsequent T cells were centrifuged at 300g for 5min, the supernatant was removed, resuspended in fresh medium, and different CAR lentiviral vectors, polybrene at 8. mu.g/mL and IL-2 at 300IU/mL were added at a virus loading MOI of 10, 37 ℃ and 5% CO₂After 24h incubation in an incubator, the cells were centrifuged at 300g for 5min, the supernatant removed, the cells resuspended in fresh medium containing 300IU/mL IL-2, 5% CO at 37 ℃₂Culturing in an incubator;

maintenance of CAR-T cell density at 1X 10⁶About one/mL, and carrying out half-amount liquid change every 2-3 days; the subsequent experiments were performed after 10 days of culture.

The CAR-T cells prepared in this example were PSCA CAR-T, PSCA-7X 19CAR-T and PSCA-secIL 7X IL2pro-CCL19 CAR-T, respectively.

Example 2 secretion of IL-7 and CCL19 by CAR-T cells

Three CAR-T cells prepared in example 1 were taken 2X 10 each⁶Culturing the cells in a fresh T cell culture medium for 24 hours, and collecting cell supernatant; take 2X 10 at the same time⁶Culturing individual PSCA-secIL7 × IL2pro-CCL19 CAR-T with equal amount of PSCA positive cell K562-PSCA for 24h, and collecting cell supernatant; using an ELISA kit (R)&D system) the secretion of IL-7 and CCL19 by each cell was examined.

As shown in FIG. 1, the culture supernatant of PSCA CAR-T cells was free of IL-7 and CCL19, the culture supernatant of PSCA-7X 19CAR-T cells was free of CCL19 and IL-7, and the culture supernatant of PSCA-secIL 7X IL2pro-CCL19 CAR-T cells was free of CCL-7 and CCL19, and after coculture with K562-PSCA, PSCA-secIL 7X IL2pro-CCL19 CAR-T secreted IL-7 and CCL 19.

The result shows that PSCA-secIL7 xIL 2pro-CCL19 CAR-T cells can successfully secrete IL-7 to the outside of cells by using IL-10 signal peptide, and meanwhile, the expression of CCL19 is regulated by tumor antigen.

Example 3 in vitro proliferation assay of CAR-T cells

Three CAR-T cells prepared in example 1 were taken 2X 10 each⁶And culturing with fresh T cell culture medium, counting every two days, continuously culturing for 7 days, and analyzing the proliferation condition of the CAR-T cells.

Results Table 1 shows that PSCA-7X 19CAR-T does not promote proliferation of CAR-T cells due to its lack of IL-7 secretion; the modified PSCA-secIL7 xIL 2pro-CCL19 CAR-T expressed IL-7 can be successfully secreted to the extracellular space, and the proliferation of CAR-T cells is promoted.

TABLE 1

Example 4 in vitro migration assay of CAR-T cells

Three CAR-T cells prepared in example 1 were taken 2X 10 each⁶One of them was cultured in the lower layer of a Transwell device for 24 hours, and simultaneously, equal amounts of PSCA-secIL7 XIL 2pro-CCL19 CAR-T and K562-PSCA were co-cultured in the lower layer for 24 hours, CFSE-labeled wild T cells were cultured in the upper layer for 5 hours, and the number of CFSE-labeled T cells in the lower layer was measured by flow cytometry.

The statistical results are shown in fig. 2, the PSCA-7 × 19CAR-T can recruit more T cells than PSCA CAR-T, and PSCA-secIL7 × IL2pro-CCL19 CAR-T recruits more T cells after being activated by tumor antigen, which indicates that PSCA-secIL7 × IL2pro-CCL19 CAR-T conditionally expresses CCL19, and only after tumor cells are recognized, CCL19 is secreted to play a role in recruitment and avoid inflammatory side reactions.

In conclusion, the fourth generation CAR-T cell can secrete IL-7 outside cells continuously, and the CCL19 is expressed and secreted after a tumor antigen is recognized, so that the anti-tumor effect of the CAR-T cell is improved remarkably, and inflammatory side reactions possibly caused by recruitment of immune cells due to excessive non-tumor tissue environment caused by continuous secretion of CCL19 are avoided.

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> fourth generation CAR-T cell and application thereof

<130> 202009

<160> 11

<170> PatentIn version 3.3

<210> 1

<211> 18

<212> PRT

<213> Artificial sequence

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

1 5 10 15

Arg Ala

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<211> 152

<212> PRT

<213> Artificial sequence

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Asp Cys Asp Ile Glu Gly Lys Asp Gly Lys Gln Tyr Glu Ser Val Leu

1 5 10 15

Met Val Ser Ile Asp Gln Leu Leu Asp Ser Met Lys Glu Ile Gly Ser

20 25 30

Asn Cys Leu Asn Asn Glu Phe Asn Phe Phe Lys Arg His Ile Cys Asp

35 40 45

Ala Asn Lys Glu Gly Met Phe Leu Phe Arg Ala Ala Arg Lys Leu Arg

50 55 60

Gln Phe Leu Lys Met Asn Ser Thr Gly Asp Phe Asp Leu His Leu Leu

65 70 75 80

Lys Val Ser Glu Gly Thr Thr Ile Leu Leu Asn Cys Thr Gly Gln Val

85 90 95

Lys Gly Arg Lys Pro Ala Ala Leu Gly Glu Ala Gln Pro Thr Lys Ser

100 105 110

Leu Glu Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu Asn Asp Leu

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Cys Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys Trp Asn Lys

130 135 140

Ile Leu Met Gly Thr Lys Glu His

145 150

<210> 3

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Met Ala Leu Leu Leu Ala Leu Ser Leu Leu Val Leu Trp Thr Ser Pro

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Ala Pro Thr Leu Ser Gly Thr Asn Asp Ala Glu Asp Cys Cys Leu Ser

20 25 30

Val Thr Gln Lys Pro Ile Pro Gly Tyr Ile Val Arg Asn Phe His Tyr

35 40 45

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

50 55 60

Leu Arg Gly Arg Gln Leu Cys Ala Pro Pro Asp Gln Pro Trp Val Glu

65 70 75 80

Arg Ile Ile Gln Arg Leu Gln Arg Thr Ser Ala Lys Met Lys Arg Arg

85 90 95

Ser Ser

<210> 4

<211> 489

<212> PRT

<213> Artificial sequence

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Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

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

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Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser

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Val Arg Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys

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Arg Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

85 90 95

Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser

100 105 110

Ser Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Ser

115 120 125

Thr Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser

130 135 140

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

145 150 155 160

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

165 170 175

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

180 185 190

Ala Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Phe Val Pro Lys Phe

195 200 205

Gln Gly Arg Ala Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

210 215 220

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

225 230 235 240

Lys Thr Gly Gly Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250 255

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Ile Glu

260 265 270

Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr

275 280 285

Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro

290 295 300

Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu

305 310 315 320

Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

325 330 335

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

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Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

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

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Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu

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

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

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

465 470 475 480

Leu His Met Gln Ala Leu Pro Pro Arg

485

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<212> DNA

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<400> 5

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

gacattcagc tgacccaatc tccaagctct ttgtccgcct ctgtggggga tagggtcacc 120

atcacctgca gtgccagttc aagtgtaaga ttcattcact ggtaccagca gaaaccagga 180

aaagctccca aaagactcat ctatgacaca tccaaactgg cttctggcgt cccttctagg 240

ttcagtggct ccgggtctgg gacagacttc accctcacca ttagcagtct gcagccggaa 300

gatttcgcca cctattactg tcagcagtgg agtagtagcc cattcacgtt cggacagggg 360

accaaggtgg agataaaagg cagtactagc ggcggtggct ccggaggcgg ctccggaggt 420

ggcggcagct cagaggttca gctggtggag tctgggggtg gccttgtgca gccagggggc 480

tcactccgtt tgtcctgcgc agcttctggc ttcaacatta aagactacta tatacactgg 540

gtgcgtcagg cccctggtaa gggcctggaa tgggttgcat ggattgatcc tgagaatggt 600

gacactgaat ttgtcccgaa gttccagggc cgtgccacta taagcgcaga cacatccaaa 660

aacacagcct acctgcagat gaacagcctg cgtgctgagg acactgccgt ctattattgt 720

aaaacggggg ggttctgggg tcaaggaacc ctggtcaccg tctcgagcga gcccaaatct 780

tgtgacaaaa ctcacacatg cccaccgtgc attgaagtta tgtatcctcc tccttaccta 840

gacaatgaga agagcaatgg aaccattatc catgtgaaag ggaaacacct ttgtccaagt 900

cccctatttc ccggaccttc taagcccttt tgggtgctgg tggtggttgg gggagtcctg 960

gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1020

agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1080

aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cagagtgaag 1140

ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc agaaccagct ctataacgag 1200

ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 1260

gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 1320

aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1380

aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 1440

cttcacatgc aggccctgcc ccctcgc 1467

<210> 6

<211> 54

<212> DNA

<213> Artificial sequence

<400> 6

atgcacagct cagcactgct ctgttgcctg gtcctcctga ctggggtgag ggcc 54

<210> 7

<211> 456

<212> DNA

<213> Artificial sequence

<400> 7

gattgtgata ttgaaggtaa agatggcaaa caatatgaga gtgttctaat ggtcagcatc 60

gatcaattat tggacagcat gaaagaaatt ggtagcaatt gcctgaataa tgaatttaac 120

ttttttaaaa gacatatctg tgatgctaat aaggaaggta tgtttttatt ccgtgctgct 180

cgcaagttga ggcaatttct taaaatgaat agcactggtg attttgatct ccacttatta 240

aaagtttcag aaggcacaac aatactgttg aactgcactg gccaggttaa aggaagaaaa 300

ccagctgccc tgggtgaagc ccaaccaaca aagagtttgg aagaaaataa atctttaaag 360

gaacagaaaa aactgaatga cttgtgtttc ctaaagagac tattacaaga gataaaaact 420

tgttggaata aaattttgat gggcactaaa gaacac 456

<210> 8

<211> 141

<212> DNA

<213> Artificial sequence

<400> 8

gctttaaggc aagctttaag gcaagcttta aggcaaagat ctagactcta gagggtatat 60

aatggaagct cgaattccag cttggcattc cggtactgtt ggtaaaaagc ttggcaatcc 120

ggtactgttg gtaaagccac c 141

<210> 9

<211> 294

<212> DNA

<213> Artificial sequence

<400> 9

atggccctgc tactggccct cagcctgctg gttctctgga cttccccagc cccaactctg 60

agtggcacca atgatgctga agactgctgc ctgtctgtga cccagaaacc catccctggg 120

tacatcgtga ggaacttcca ctaccttctc atcaaggatg gctgcagggt gcctgctgta 180

gtgttcacca cactgagggg ccgccagctc tgtgcacccc cagaccagcc ctgggtagaa 240

cgcatcatcc agagactgca gaggacctca gccaagatga agcgccgcag cagt 294

<210> 10

<211> 81

<212> DNA

<213> Artificial sequence

<400> 10

aaaattgtcg ctcctgtcaa acaaactctt aactttgatt tactcaaact ggctggggat 60

gtagaaagca atccaggtcc a 81

<210> 11

<211> 75

<212> DNA

<213> Artificial sequence

<400> 11

atgttccatg tttcttttag gtatatcttt ggacttcctc ccctgatcct tgttctgttg 60

ccagtagcat catct 75

Claims (10)

1. A fourth generation CAR-T cell expressing a chimeric antigen receptor that specifically binds an antigen, secreted IL-7, and NFAT-regulated CCL 19;

the signal peptide of the IL-7 is an IL-10 signal peptide;

the promoter of the CCL19 is an NFAT regulatory promoter.

2. A fourth generation CAR-T cell according to claim 1, wherein the IL-10 signal peptide comprises the amino acid sequence shown in SEQ ID No. 1;

preferably, the IL-7 comprises an amino acid sequence shown in SEQ ID NO 2;

preferably, CCL19 comprises the amino acid sequence set forth in SEQ ID NO 3.

3. A fourth generation CAR-T cell according to claim 1 or 2, wherein the chimeric antigen receptor comprises a signal peptide, an antigen binding domain, a transmembrane domain and a signalling domain;

preferably, the signal peptide comprises a GM-CSF signal peptide;

preferably, the antigen binding domain binds to a tumor surface antigen comprising any one of CD19, CD20, CD22, CD30, CEA, EGFR, BRAF, HER-2, Mesothelin, MUC1, PSCA, GPC3, TERT, PTEN, PD-1, PD-L1, or VEGF, preferably any one of CD19, MUC1, GPC3, Mesothelin, PSCA, or HER2, more preferably PSCA;

preferably, the antigen binding domain is a single chain antibody targeting PSCA;

preferably, the transmembrane domain comprises a CD28 transmembrane domain and/or a CD8 a transmembrane domain, preferably a CD28 transmembrane domain;

preferably, the signaling domain comprises any one of, or a combination of at least two of, the CD28 endodomain, CD3 zeta, TLR2, 4-1BB, TLR1, CD27, OX40, or DAP10, preferably the combination of CD28 endodomain and CD3 zeta.

4. A fourth generation CAR-T cell according to any of claims 1 to 3, wherein the chimeric antigen receptor consists of a GM-CSF signal peptide, an anti-PSCA single chain antibody, a CD28 transmembrane domain, a CD28 endodomain and a CD3 zeta concatemer;

preferably, the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO. 4.

5. A lentiviral vector comprising a chimeric antigen receptor encoding gene, an IL-10 signal peptide encoding gene, an IL-7 encoding gene, an NFAT regulated promoter and a CCL19 encoding gene;

preferably, the chimeric antigen receptor encoding gene comprises a nucleic acid sequence shown as SEQ ID NO. 5;

preferably, the IL-10 signal peptide coding gene comprises a nucleic acid sequence shown in SEQ ID NO. 6;

preferably, the IL-7 encoding gene comprises a nucleic acid sequence shown in SEQ ID NO. 7;

preferably, the NFAT regulated promoter comprises the nucleic acid sequence set forth in SEQ ID NO 8;

preferably, the CCL19 encoding gene comprises a nucleic acid sequence shown as SEQ ID NO. 9.

6. The lentiviral vector of claim 5, wherein the lentiviral vector further comprises a linker peptide encoding gene;

preferably, the connecting peptide encoding gene comprises a nucleic acid sequence shown in SEQ ID NO. 10.

7. A recombinant lentivirus prepared by co-transfecting a mammalian cell with the lentiviral vector of claim 5 or 6 and a packaging helper plasmid.

8. A method of producing a fourth generation CAR-T cell according to any one of claims 1 to 4 comprising the step of introducing into a T cell the lentiviral vector of claim 5 or 6 or the recombinant lentivirus of claim 7.

9. A pharmaceutical composition comprising a fourth generation CAR-T cell according to any of claims 1-4;

preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

10. Use of a fourth generation CAR-T cell according to any of claims 1 to 4, a lentiviral vector according to claim 5 or 6, a recombinant lentivirus according to claim 7 or a pharmaceutical composition according to claim 9 for the manufacture of a medicament for the treatment of a solid tumor;

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

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