CN112592408B

CN112592408B - Single-chain antibody targeting c-Met, chimeric antigen receptor, recombinant vector, CAR-T cell and application

Info

Publication number: CN112592408B
Application number: CN202011613534.3A
Authority: CN
Inventors: 焦顺昌; 陈静远; 梁皓
Original assignee: Beijing Dingcheng Taiyuan Biotechnology Co ltd
Current assignee: Beijing Dingcheng Taiyuan Biotechnology Co ltd
Priority date: 2020-07-20
Filing date: 2020-07-20
Publication date: 2021-08-17
Anticipated expiration: 2040-07-20
Also published as: CN112592408A; CN111848797A; CN111848797B; CN112552404A; CN112552404B

Abstract

The invention provides a single-chain antibody targeting c-Met, a chimeric antigen receptor, a recombinant vector, a CAR-T cell and application, and belongs to the technical field of tumor treatment, wherein the amino acid sequence of the single-chain antibody is shown as SEQ ID No. 3; the chimeric antigen receptor comprises a CD8a signal peptide, a Flag tag, the single-chain antibody, a CD8a hinge region-transmembrane region, a 4-1BB functional region and a CD3 functional region which are connected in sequence; the chimeric antigen receptor targeting c-Met can be combined with the c-Met antigen with high specificity, and the T cell modified by the chimeric antigen receptor targeting c-Met can kill c-Met high-expression tumor cells in a high-efficiency targeting manner, so that a relatively ideal curative effect is achieved in vivo and in vitro.

Description

Single-chain antibody targeting c-Met, chimeric antigen receptor, recombinant vector, CAR-T cell and application

The invention is a divisional application of an invention patent with the application number of CN202010697872.3 and the name of 'a single-chain antibody targeting c-Met, a chimeric antigen receptor, a recombinant vector, CAR-T cells and application', namely the invention patent with the application date of 2020, 07, 20 days.

Technical Field

The invention belongs to the technical field of tumor treatment, and particularly relates to a c-Met-targeted single-chain antibody, a chimeric antigen receptor, a recombinant vector, CAR-T cells and application thereof.

Background

The c-Met gene is located on chromosome 7 (7q21-q31), and is composed of 21 exons and 20 introns, and is approximately 120kDa in length. The c-Met primary transcript produces a 150kDa polypeptide which is then partially glycosylated to form a 170kDa single chain precursor protein. The precursor protein is further glycosylated to form an approximately 190kDa heterodimer consisting of a 50kDa extracellular chain (. alpha. -chain) and a 145kDa transmembrane chain (. beta. -chain) linked together (Maulik G, Shrikhide A, Kijima T, et al. role of the platelet Growth Factor receptor, c-Met, in-oncogenesis and pore for therapeutic inhibition. cell Growth Factor Rev,2002,13(1): 41-59.; Sattler M, Ma PC, Salgia R. therapeutic targeting of the platelet Growth Factor Met. cancer Tree Res,2004,119: 121-138.). The transmembrane strand (β -strand) includes a SEMA domain (SEMA), a PSI domain (PSI), 4 immunoglobulin-like repeat domains (IPTs), a transmembrane domain, a Juxtamebrancein (JM), a tyrosine kinase domain (TK), and a carboxyl-terminal tail region (Carboxylterminal, CT). Among them, the SEMA domain is one of the important elements for ligand binding, and is considered as the binding site of HGF, while the PSI domain functions to allow the extracellular segment of c-Met to bind well to the ligand. The membrane-proximal domain (JM) contains two protein phosphorylation sites: s985 and Y1003. Phosphorylation of the S985 site can negatively regulate kinase activity, and a Y1003 residue is combined with c-Cbl after phosphorylation. c-Cbl is an E3 ubiquitin ligase that can bind to E2 ubiquitin conjugating enzyme via the finger ring domain, thereby dictating intracellular uptake and ultimately leading to substrate ubiquitination and degradation. Phosphorylation of the tyrosine kinase domain activation sites Y1230, Y1234 and Y1235 promote activation of tyrosine kinases. After c-Met binds HGF, the activation residues Y1234 and Y1235 of c-Met are phosphorylated, and further, intracellular SH2 domain (Srchomatology-2, SH2) and other specific signal effector molecules are recruited to activate downstream signal pathways (Cecchi F, Rabe DC, Bottaro DP. targeting the HGF/Met signaling pathway in cancer therapy. Ext Opin Targets,2012,16(6): 553-572.).

In addition to being involved in regulating normal cell adhesion, differentiation and migration, c-Met is also involved in malignant transformation of cells. The HGF activated c-Met receptor directly acts with some target proteins in cells, can activate various cascade pathways of signal transduction including Ras/MAPK and PI3K/PKB and regulate the proliferation and differentiation, morphological change, motor invasion and the like of various malignant tumor cells, thereby promoting the infiltration and metastasis of tumors. Multiple studies have shown that aberrant expression of c-Met is closely associated with the development, progression and metastasis of a variety of tumors.

CAR-T, a chimeric antigen receptor modified T cell, is a human T lymphocyte that stably integrates the CAR gene into the genome by in vitro gene editing techniques. CAR-T combines the targeting effect of antibodies and the effector functions of T lymphocytes to recognize target antigens with high specificity in a non-MHC restricted manner to kill tumor cells. Currently, CAR-T has made a breakthrough progress in the treatment of hematological cancers, with two CAR-T cell products targeting CD19 in succession being marketed. However, for solid tumors, the application effect of CAR-T is influenced due to the limitation of factors such as tumor microenvironment, and scientists in various countries try to solve the problem.

Disclosure of Invention

In view of the above, the present invention aims to provide a c-Met-targeted single-chain antibody, a chimeric antigen receptor, a recombinant vector, a CAR-T cell and applications thereof; the chimeric antigen receptor targeting c-Met can be combined with the c-Met antigen with high specificity, and the T cell modified by the chimeric antigen receptor targeting c-Met can kill c-Met high-expression tumor cells in a high-efficiency targeting manner, so that a relatively ideal curative effect is achieved in vivo and in vitro.

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

the invention provides a single-chain antibody of targeted c-Met, and the amino acid sequence of the single-chain antibody is shown in SEQ ID No. 3.

The invention provides a gene for coding the single-chain antibody, and the nucleotide sequence of the gene is shown in SEQ ID No. 6.

The invention provides a chimeric antigen receptor targeting c-Met, which comprises a CD8a signal peptide, a Flag tag, a single-chain antibody, a CD8a hinge region-transmembrane region, a 4-1BB functional region and a CD3 functional region which are connected in sequence.

Preferably, the single chain antibody is a combination of a hinge-linked antibody light chain variable region and an antibody heavy chain variable region.

Preferably, the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID No. 9.

The invention provides a gene for coding the chimeric antigen receptor, and the nucleotide sequence of the gene is shown in SEQ ID No. 12.

The invention provides a recombinant vector targeting c-Met, which comprises a gene of a chimeric antigen receptor and an initial vector.

Preferably, the initial vector is a lentiviral vector.

The invention provides a chimeric antigen receptor T cell of targeted c-Met, which is obtained by transfecting the T cell with a recombinant vector of the targeted c-Met.

The invention provides the single-chain antibody, a gene for coding the single-chain antibody, the chimeric antigen receptor, a gene for coding the chimeric antigen receptor, the recombinant vector and application of the chimeric antigen receptor T cell in preparing a medicament for treating tumors.

The invention has the beneficial effects that: the single-chain antibody 3D88 targeting c-Met has extremely strong binding capacity with c-Met antigen, and has no obvious binding with control antigen and cell lysate; has good specific targeting function to c-Met antigen. The chimeric antigen receptor T cells targeting c-Met prepared by the invention have stronger killing effect on c-Met positive H1299 cells, and a control cell Mock-T does not kill, wherein the 2D81CAR-T cells show the strongest killing effect, compared with positive control CAR-T, the ability of the 2D81CAR-T cells to secrete cytokines such as IFN-gamma and TNF for enhancing the activity of T cells is obviously enhanced, the IL-10(IL-10 plays an immunosuppressive role) which is less secreted by the 2D81CAR-T cells than the positive control CAR-T cells, the survival period of tumor-bearing mice is obviously prolonged by 2D81CAR-T, the mice in the normal saline group die at 42 days, the mice in the Mock-T group die at 47 days, and the mice in the 2D81CAR-T group still have 40 percent survival time at 55 days of the experimental end point, it can be seen that 2D81CAR-T cells have good cancer-inhibiting effect on c-Met positive tumors.

Drawings

FIG. 1 shows the results of ELISA assay;

FIG. 2 is a CAR plasmid map;

FIG. 3 shows the results of enzyme digestion electrophoresis;

FIG. 4 is a flow chart of CAR expression rate;

FIG. 5 shows the result of the detection of killing effect of CAR-T cells on c-Met-positive cells H1299;

FIG. 6 shows the results of cytokine detection in 2D81CAR-T cells;

FIG. 7 shows tumor volume changes in mice;

fig. 8 shows the survival results of mice.

Detailed Description

The invention provides a single-chain antibody of targeted c-Met, wherein the amino acid sequence of the single-chain antibody is shown as one of SEQ ID No. 1-SEQ ID No. 3.

In the present invention, the c-Met targeting single chain antibody includes 2a45, 2D81 and 3D 88; the amino acid sequence of the single-chain antibody 2A45 is shown in SEQ ID No.1, and specifically comprises the following steps:

DIQMTQTTSSLSASLGDRVTISCRASQDINNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDVATYFCQLGNTLFTFGSGTKLEIKSSGGGGSGGGGGGSSRSSEVQLQQSGPELVKPGSSVKISCKASGYSFTGYFMNWVMQSHGKSLEWIGRINPNNGDTFYNQKFKGKATLTVDKSSNTAHMEIRSLASEDSAVYYCARMKLIGSYMDYWGQGTSVTVSS。

in the present invention, the single-chain antibody 2a45 comprises a light chain region, a hinge region and a heavy chain region connected in this order; the amino acid sequence of the light chain region is shown as SEQ ID No.13, and specifically comprises the following steps:

DIQMTQTTSSLSASLGDRVTISCRASQDINNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDVATYFCQLGNTLFTFGSGTKLEIK；

the amino acid sequence of the hinge region is shown as SEQ ID No.14, and specifically comprises the following steps:

SSGGGGSGGGGGGSSRSS；

the amino acid sequence of the heavy chain region is shown as SEQ ID No.15, and specifically comprises the following steps:

EVQLQQSGPELVKPGSSVKISCKASGYSFTGYFMNWVMQSHGKSLEWIGRINPNNGDTFYNQKFKGKATLTVDKSSNTAHMEIRSLASEDSAVYYCARMKLIGSYMDYWGQGTSVTVSS。

in the invention, the nucleotide sequence of the gene for coding the single-chain antibody 2A45 is shown as SEQ ID No.4, and the specific steps are as follows:

gatatccagatgactcaaactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattaacaattatttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctactacacatcaagattacactcaggtgtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatgttgccacttacttttgccaactgggtaatacgctattcacgttcggctcggggacaaagttggaaataaaatctagtggtggcggtggttcgggcggtggtggaggtggtagttctagatcttccgaggttcagctgcagcagtctggacctgagctggtgaagcctgggtcttcagtgaagatttcctgcaaggcctctggttactcatttactggctactttatgaactgggtgatgcagagccatggaaagagccttgagtggattggacgtattaatcctaacaatggtgatactttttacaaccagaagttcaagggcaaggccacattgactgtagacaaatcctctaatacagcccacatggagatccggagcctggcatctgaggactctgcagtctattattgtgcaagaatgaagctaattgggtcctatatggactactggggtcaaggaacctcagtcaccgtctcctca。

in the invention, the amino acid sequence of the single-chain antibody 2D81 is shown as SEQ ID No.2, and specifically comprises the following steps:

QIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWDSNPYLFGGGTKLEIKSSGGGGSGGGGGGSSRSSQVQLQQSGAELMKPGASVKISCKATGYTFSTYWIEWVKQRPGHGLEWMGEILPGSDYTNYNEQFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCARPGYADYWGQGTTLTVSS。

in the present invention, the single-chain antibody 2D81 comprises a light chain region, a hinge region and a heavy chain region connected in this order; the amino acid sequence of the light chain region is shown as SEQ ID No.16, and specifically comprises the following steps:

QIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWDSNPYLFGGGTKLEIK；

SSGGGGSGGGGGGSSRSS；

the amino acid sequence of the heavy chain region is shown as SEQ ID No.17, and specifically comprises the following steps:

QVQLQQSGAELMKPGASVKISCKATGYTFSTYWIEWVKQRPGHGLEWMGEILPGSDYTNYNEQFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCARPGYADYWGQGTTLTVSS。

in the invention, the nucleotide sequence of the single-chain antibody 2D81 gene is shown as SEQ ID No.5, and the specific steps are as follows:

caaattgttctctctcagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccacatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtgggatagtaacccatatttgttcggaggggggaccaagctggaaataaaatctagtggtggcggtggttcgggcggtggtggaggtggtagttctagatcttcccaggttcaactgcagcagtctggagctgagctgatgaagcctggggcctcagtgaagatctcctgcaaggctactggctacacattcagtacttactggatagagtgggtaaaacagaggcctggacatggccttgagtggatgggagagattttacctggaagtgattatactaactacaatgagcagttcaagggcaaggccacattcactgcagatacatcctccaacacagcctacatgcaactcagcagcctgacatctgaggactctgccgtctattactgtgcaagacccggctacgcggactactggggccaaggcaccactctcacagtctcctca。

in the invention, the amino acid sequence of the single-chain antibody 3D88 is shown as SEQ ID No.3, and specifically comprises the following steps:

DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPERLISLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPLTFGAGTKLELKSSGGGGSGGGGGGSSRSSEVQLQQSGAELMKPGASVKISCKATGDTISFNWIEWVKQRPGHGLEWIGEILPESIISKKYNEKFKGKATFTADTSSNTVYMQLSSLTSEDSAVYFCASGGYYGSLDFWGQGTPLTVSS。

in the present invention, the single-chain antibody 3D88 includes a light chain region, a hinge region, and a heavy chain region connected in this order; the amino acid sequence of the light chain region is shown as SEQ ID No.18, and specifically comprises the following steps:

DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPERLISLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPLTFGAGTKLELK；

SSGGGGSGGGGGGSSRSS；

the amino acid sequence of the heavy chain region is shown as SEQ ID No.19, and specifically comprises the following steps:

EVQLQQSGAELMKPGASVKISCKATGDTISFNWIEWVKQRPGHGLEWIGEILPESIISKKYNEKFKGKATFTADTSSNTVYMQLSSLTSEDSAVYFCASGGYYGSLDFWGQGTPLTVSS。

in the invention, the nucleotide sequence of the single-chain antibody 3D88 gene is shown as SEQ ID No.6, and the specific steps are as follows:

gacgttgtgatgacccagactccgctcactttgtcggttaccattggacaaccagcctccatctcttgcaagtcaagtcagagcctcttagatagtgatggaaagacatatttgaattggttgttacagaggccaggccagtctccagagcgcctaatctctctggtgtctaaactggactctggagtccctgacaggttcactggcagtggatcggggacagatttcacactgaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggtacacattttcctctcacgttcggtgctgggaccaagctggagctgaaatctagtggtggcggtggttcgggcggtggtggaggtggtagttctagatcttccgaggttcagctgcagcagtctggagctgagctgatgaagcctggggcctcagtgaagatatcctgtaaggcaactggcgacacaatcagtttcaactggatagagtgggtaaagcagaggcctggacatggccttgagtggattggagagattttacctgaaagtattattagtaagaagtacaatgagaagttcaagggcaaggccacattcactgcagatacatcctccaatacagtatacatgcaactcagcagcctgacatctgaggactctgccgtctatttctgtgcaagcgggggttactacggtagccttgacttctggggccaaggcacccctctcacagtctcctca。

the invention also provides a chimeric antigen receptor targeting c-Met, which comprises a CD8a signal peptide, a Flag tag, a single-chain antibody, a CD8a hinge region-transmembrane region, a 4-1BB functional region and a CD3 functional region which are connected in sequence.

In the invention, the amino acid sequences of the CD8a signal peptide, the Flag tag, the CD8a hinge region-transmembrane region, the 4-1BB functional region and the CD3 functional region are respectively shown as SEQ ID No. 20-SEQ ID No.24, and the nucleotide sequences of the genes correspondingly coding the CD8a signal peptide, the Flag tag, the CD8a hinge region-transmembrane region, the 4-1BB functional region and the CD3 functional region are shown as SEQ ID No. 25-SEQ ID No. 29.

In the present invention, the two sides of the single-chain antibody preferably further comprise a first enzyme cutting site and a second enzyme cutting site; the first enzyme cutting site and the second enzyme cutting site are different enzyme cutting sites. In the specific implementation process of the invention, the first enzyme cutting site is an EcoRI enzyme cutting site (GAATTC, SEQ ID No.30), and the second enzyme cutting site is a BamHI enzyme cutting site (GGATCCA, SEQ ID No. 31).

In the present invention, when the single-chain antibody is the single-chain antibody 2a45, the amino acid sequence of the chimeric antigen receptor 2a45 is shown in SEQ ID No.7, which is specifically as follows:

MALPVTALLLPLALLLHAARPDYKDDDDKEFDIQMTQTTSSLSASLGDRVTISCRASQDINNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDVATYFCQLGNTLFTFGSGTKLEIKSSGGGGSGGGGGGSSRSSEVQLQQSGPELVKPGSSVKISCKASGYSFTGYFMNWVMQSHGKSLEWIGRINPNNGDTFYNQKFKGKATLTVDKSSNTAHMEIRSLASEDSAVYYCARMKLIGSYMDYWGQGTSVTVSSGSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*。

in the invention, the nucleotide sequence for coding the chimeric antigen receptor 2A45 is shown as SEQ ID No.10, and the nucleotide sequence is as follows:

atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgattacaaggatgacgacgataaggaattcgatatccagatgactcaaactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattaacaattatttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctactacacatcaagattacactcaggtgtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatgttgccacttacttttgccaactgggtaatacgctattcacgttcggctcggggacaaagttggaaataaaatctagtggtggcggtggttcgggcggtggtggaggtggtagttctagatcttccgaggttcagctgcagcagtctggacctgagctggtgaagcctgggtcttcagtgaagatttcctgcaaggcctctggttactcatttactggctactttatgaactgggtgatgcagagccatggaaagagccttgagtggattggacgtattaatcctaacaatggtgatactttttacaaccagaagttcaagggcaaggccacattgactgtagacaaatcctctaatacagcccacatggagatccggagcctggcatctgaggactctgcagtctattattgtgcaagaatgaagctaattgggtcctatatggactactggggtcaaggaacctcagtcaccgtctcctcaggatccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctag。

in the invention, when the single-chain antibody is the single-chain antibody 2D81, the amino acid sequence of the chimeric antigen receptor 2D81 is shown as SEQ ID No.8, and the specific amino acid sequence is as follows: MALPVTALLLPLALLLHAARPDYKDDDDKEFQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWDSNPYLFGGGTKLEIKSSGGGGSGGGGGGSSRSSQVQLQQSGAELMKPGASVKISCKATGYTFSTYWIEWVKQRPGHGLEWMGEILPGSDYTNYNEQFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCARPGYADYWGQGTTLTVSSGSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT are provided.

In the invention, the nucleotide sequence of the chimeric antigen receptor 2D81 is shown as SEQ ID No.11, and the specific steps are as follows:

atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgattacaaggatgacgacgataaggaattccaaattgttctctctcagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccacatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtgggatagtaacccatatttgttcggaggggggaccaagctggaaataaaatctagtggtggcggtggttcgggcggtggtggaggtggtagttctagatcttcccaggttcaactgcagcagtctggagctgagctgatgaagcctggggcctcagtgaagatctcctgcaaggctactggctacacattcagtacttactggatagagtgggtaaaacagaggcctggacatggccttgagtggatgggagagattttacctggaagtgattatactaactacaatgagcagttcaagggcaaggccacattcactgcagatacatcctccaacacagcctacatgcaactcagcagcctgacatctgaggactctgccgtctattactgtgcaagacccggctacgcggactactggggccaaggcaccactctcacagtctcctcaggatccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctag。

in the present invention, when the single-chain antibody is the single-chain antibody 3D88, the amino acid sequence of the chimeric antigen receptor 3D88 is shown in SEQ ID No.9, which is specifically as follows:

MALPVTALLLPLALLLHAARPDYKDDDDKEFDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPERLISLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPLTFGAGTKLELKSSGGGGSGGGGGGSSRSSEVQLQQSGAELMKPGASVKISCKATGDTISFNWIEWVKQRPGHGLEWIGEILPESIISKKYNEKFKGKATFTADTSSNTVYMQLSSLTSEDSAVYFCASGGYYGSLDFWGQGTPLTVSSGSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*。

in the invention, the nucleotide sequence of the chimeric antigen receptor 3D88 is shown as SEQ ID No.12, which is as follows:

atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgattacaaggatgacgacgataaggaattcgacgttgtgatgacccagactccgctcactttgtcggttaccattggacaaccagcctccatctcttgcaagtcaagtcagagcctcttagatagtgatggaaagacatatttgaattggttgttacagaggccaggccagtctccagagcgcctaatctctctggtgtctaaactggactctggagtccctgacaggttcactggcagtggatcggggacagatttcacactgaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggtacacattttcctctcacgttcggtgctgggaccaagctggagctgaaatctagtggtggcggtggttcgggcggtggtggaggtggtagttctagatcttccgaggttcagctgcagcagtctggagctgagctgatgaagcctggggcctcagtgaagatatcctgtaaggcaactggcgacacaatcagtttcaactggatagagtgggtaaagcagaggcctggacatggccttgagtggattggagagattttacctgaaagtattattagtaagaagtacaatgagaagttcaagggcaaggccacattcactgcagatacatcctccaatacagtatacatgcaactcagcagcctgacatctgaggactctgccgtctatttctgtgcaagcgggggttactacggtagccttgacttctggggccaaggcacccctctcacagtctcctcaggatccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctag。

the invention provides a recombinant vector targeting c-Met, which comprises a chimeric antigen receptor gene and an initial vector. In the present invention, the initial vector is preferably a lentiviral vector; the chimeric antigen receptor gene is preferably recombined between the XbaI and SalI sites of the lentiviral vector. The preparation method of the recombinant vector targeting c-Met is not particularly limited, and the conventional preparation method of the recombinant lentiviral vector in the field can be adopted. In the present invention, it is preferable to ligate the CD8a signal peptide, Flag tag, CD8a hinge region-transmembrane region, 4-1BB functional region and CD3 functional region to the initial vector before ligating the single-chain antibody thereto. The method of the present invention is not particularly limited, and a conventional method of the present invention may be used.

The invention provides a chimeric antigen receptor T cell of targeted c-Met, which is obtained by transfecting the T cell with a recombinant vector of the targeted c-Met. In the present invention, the method for preparing the c-Met targeted chimeric antigen receptor T cell preferably comprises the following steps: 1) separating T cells from peripheral blood of a healthy human body and culturing; 2) and (2) performing mixed transfection on the lentiviral vector and the cultured T cells in the step 1) to obtain the chimeric antigen receptor T cells targeting c-Met. The T cell separation method is not particularly limited, and a T cell separation method which is conventional in the field can be adopted; after the separation, the T cells are placed in a complete culture solution of X-VIVO +100U IL2 for suspension, and CD3/CD28 magnetic beads are added for culture; the cultivation is preferably carried out in an incubator, the temperature of the cultivation is preferably 37 ℃, the volume concentration of carbon dioxide of the cultivation is preferably 5%, and the time of the cultivation is preferably 45-50 h, and more preferably 48 h. In the present invention, the ratio of the lentiviral vector to the cultured T cells is calculated based on the MOI of the virus, which is preferably 15. In the invention, the transfection time is preferably 2-4 d, and more preferably 3 d.

The invention provides the single-chain antibody, a gene for coding the single-chain antibody, the chimeric antigen receptor, a gene for coding the chimeric antigen receptor, the recombinant vector and application of the chimeric antigen receptor T cell in preparing a medicament for treating tumors. In the invention, the single-chain antibody has good specific targeting function to the c-Met antigen; the chimeric antigen receptor T cell targeting c-Met has stronger killing effect on c-Met positive cells and has good cancer inhibition effect on c-Met positive tumors; can be applied to the preparation of the medicine for treating the tumor.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Phage display screening of variable region sequences

Total RNA was extracted from spleen and bone marrow of mice immunized with c-Met extracellular antigen by trizol method, and murine antibody variable was amplified using murine antibody scFv gene amplification kit (cat # P001Z)The region gene is formed by connecting an antibody heavy chain and a light chain variable region by a peptide Linker (a hinge region sequence: SSGGGGSGGGGSSRSS) consisting of a plurality of glycin (Gly) and serine (Ser) to form a single-chain antibody gene fragment scFv, cloning the scFv single-chain antibody gene fragment into a phagemid vector pCANTAB5E, constructing an scFv phage display library, and displaying and expressing the single-chain antibody scFv in the phage display library. Then extracting plasmid, electrically converting phage plasmid to construct library with the storage capacity not less than 10⁸. Then, screening the phage library by using an ELISA plate coated c-Met Protein (source: c-MET Protein, Human, recombinant (His tag) Cat:10692-H08H), obtaining positive clones by adopting a panning-amplification-enrichment circulating mode, further detecting the selected positive clones by an ELISA experiment, carrying out PCR (polymerase chain reaction) on the positive clones detected by the ELISA, carrying out enzyme digestion typing, then sequencing, selecting a proper variable region sequence according to a sequencing result, and finally determining phage clones with the best effects of the three strains of 2A45, 2D81 and 3D88, wherein the ELISA result is shown in figure 1. The detection results show that three phage clone strains of 2A45, 2D81 and 3D88 have extremely strong binding capacity with c-Met antigen, but have no obvious binding with control antigen and cell lysate, which indicates that the three phage clone strains have very good specific targeting function for the c-Met antigen.

Example 2

CAR plasmid construction

The CAR sequence construction concept is shown in figure 2. Designing the following amplification primers according to the sequenced 3 scFv sequences targeting c-Met,

2A45-F：taaggaattcgatgttgtgatgacc(SEQ ID No.32)

2A45-R：ggtggatcctgaggagacggtgac(SEQ ID No.33)

2D81-F：taaggaattccagattgttctctctc(SEQ ID No.34)

2D81-R：ggtggatcctgaggagactgtgag(SEQ ID No.35)

3D88-F：aaggaattcgacgttgtgatgacc(SEQ ID No.36)

3D88-R：gtggatcctgaggagactgtgag(SEQ ID No.37)

obtaining 2A45, 2D81 and 3D88 scFv gene fragments carrying enzyme cutting sites through PCR amplification respectively. The gene fragments and PZX-CAR vector (PZX-CAR vector map is shown in figure 2) are respectively subjected to EcoRI and BamHI double enzyme digestion treatment, agarose gel electrophoresis is carried out, and gel cutting is carried out to recover target genes and skeleton plasmids. Thereafter, ligation was performed with high-performance T4 ligase at room temperature for 15min, transformed into Stbl3 competence, allowed to stand on ice for 30min, heat-shocked at 42 ℃ for 90s, and rapidly put into ice for 2 min. Add 500. mu.L LB liquid medium and incubate at 37 ℃ for 60 min. The thalli is collected by centrifugation at 4000rpm for 3min and is evenly coated on a plate containing antibiotics. Selecting a monoclonal strain at night, shaking the strain, extracting plasmids for enzyme digestion identification, wherein the identification result is shown in figure 3,

lanes

1, 2, 3 and 4 respectively correspond to 2A45 CAR, 2D81CAR, 3D88 CAR and positive control CAR, and the size of the cut target fragment is about 1500bp and is consistent with the expectation. And (4) sending the strains successfully identified by enzyme digestion to a sequencing company for sequencing.

Example 3

CAR-T cell construction

1. Lentiviral packaging

mu.L of each of the bacterial suspension carrying 2A45 CAR, 2D81CAR, 3D88 CAR and positive control CAR and the different bacterial suspension of lentivirus helper plasmid (SL3, SL4, SL5) were added to different flasks containing 100mL of LB medium (Amp added thereto at a final concentration of 100. mu.g/mL) and cultured on a shaker for 16h at 37 ℃ and 180 rpm. Then, the plasmid was subjected to large-scale extraction using an endotoxin-free plasmid large-scale extraction kit (Tiangen organism). Different CAR plasmids were co-transfected with the packaging plasmid into 293T cells, respectively, and cell supernatants were collected 48h after transfection and centrifuged at 4000rpm for 10 min. The supernatant was collected, filtered through a 0.45 μm filter and the lentiviral pellet was collected by an ultracentrifuge. The centrifugation condition is 4 ℃, 100000 Xg, no brake, and centrifugation for 90 min. The concentrated lentivirus is subpackaged into 0.5mL freezing tubes according to 200 mul per tube, and is subpackaged and frozen at-80 ℃.

T cell isolation

20mL of blood was taken from a healthy human peripheral blood and added to a 50mL centrifuge tube, and 1mL of RosetteSep was added to the blood^TMCocktail was mixed well to give an antibody concentration of 50. mu.L/mL. 20mL of GE Ficoll solution was added to a new 50mL centrifuge tube, 20mL of the diluted blood sample was carefully aspirated with a pipette and slowly added to the top of the Ficoll solution at a ratio of 1:1And (3) a layer. And (3) putting the centrifuge tube added with the blood sample into a centrifuge, rotating at the speed of 400g, and centrifuging for 30 min. The upper plasma and platelets were aspirated with a sterile pipette without touching the mononuclear cell layer, and a second layer of circular opalescent lymphocytes was carefully aspirated into a new centrifuge tube. Three volumes of PBS were added to each tube of lymphocyte suspension to wash the cells, and centrifuged at 400g for 10 min. The supernatant was discarded, and the resulting cells were resuspended in 5mL PBS and centrifuged at 1200rpm for 10 min. Finally, the cells were suspended in X-VIVO +100U IL2 complete medium and counted. 1mL of the fraction was separated to a density of 1X 10⁶T lymphocytes were seeded into 1 well of a 24-well plate and 25. mu.l of CD3/CD28 magnetic beads were added. Cells were cultured in 5% CO₂The mixture was used for lentivirus infection after 48 hours in an incubator at 37 ℃.

3. Viral infection of T cells

The corresponding lentivirus was added at MOI 15, the next day the cells were replenished, each well to 30ml and transferred to a T75 flask and incubated horizontally. After 3 days, the CAR-T cells were transferred into sterile flow tubes and the magnetic beads were removed by placing each tube of cells on a biolegend magnet for 2 min. After that, the partial cell staining flow antibody was used to detect the CAR expression rate, and the detection results are shown in fig. 4, wherein the 2a45 CAR expression rate was 86.4%, the 2D81CAR expression rate was 78.8%, the 3D88 CAR expression rate was 82.7%, and the positive control CAR expression rate was 79.0%.

Example 4

In vitro cell killing

In the white plate, the number of H1299 target cells inoculated in the sample well is 1X 10⁴One well, 5 replicates, while 5 control wells, 100 μ l per well, were set. After overnight, after aspiration of the supernatant, CAR-T cells were added at an effective target ratio of E/T ratio of 5: 1; each well was 100. mu.l. After 6h, centrifuge at 1500rpm for 5min and remove supernatant from all wells as much as possible. Adding 100 mul of lysis solution into each hole, cracking for 10min at room temperature, starting to test on a machine, and adding 100 mul of firefly luciferase detection reagent into each hole. The killing efficiency calculation formula is as follows: killing efficiency (%) × (control well fluorescence intensity-killing fluorescence intensity)/(control well fluorescence intensity-blank well fluorescence intensity) × 100%. The killing result is shown in FIG. 5, the prepared four CAR-T cells all have stronger killing effect on c-Met positive H1299 cells, while the control cell Mock-T has stronger killing effect on c-Met positive H1299 cellsNo killing. Among several CAR-T cells, 2D81CAR-T cells showed the strongest killing effect, better than the CAR-T cells involved in patent 201810687119.9 (positive control CAR-T).

Example 5

Cytokine assay

Mocl-T, positive control CAR-T and 2D81CAR-T were incubated with H1299 for 6H, the supernatant was lysed, and the cytokine content was determined by CBA method, as follows: transferring the standard pellets to a 15ml centrifuge tube, adding 2ml of assay Dilute, standing at room temperature for at least 15min, blowing gently with a gun, not swirling or mixing strongly, and marking as the highest concentration standard. Prepare 8 EP tubes, add 100 μ Ι assay Dilute per tube, aspirate 100 μ Ι from the highest concentration standard into the next tube, 1: 2 gradient dilution up to 1: 256, blow gently with a gun, not vortex or mix strongly, prepare a tube with only Assay plus of 0pg/ml, which is the negative control. Vortex the capturebeds and add 50 μ l per tube for all tubes. Add 50. mu.l of diluted standards or samples to each tube, followed by 50. mu.l of Detection Reagent to all tubes. After mixing, incubation for 3h at room temperature in the dark, at which time flow setup can be performed. Add 1ml wash buffer to each tube, centrifuge for 5min at 200g, carefully aspirate the supernatant and finally add 300. mu.l wash buffer to resuspend the bead pellet. As shown in FIG. 6, the ability of 2D81CAR-T cells and positive control CAR-T cells to secrete cytokines such as IFN-gamma and TNF that enhance the activity of T cells is significantly enhanced, and 2D81CAR-T cells secrete less IL-10 than positive control CAR-T cells (IL-10 plays an immunosuppressive role).

Example 6

In vivo experiments in mice

6-8 week male NOG mice were selected as experimental animals. After H1299 cell digestion, the cells were resuspended and washed 2 times with sterile 1 XPBS for viable cell counting, with cell density adjusted to 5X 10⁶At 50. mu.l/cell, the ratio of 1:1 volume of each was mixed with 50. mu.l matrigel, i.e.finally 100. mu.l total volume of cell suspension per mouse was injected. The transplantation site was subcutaneous on the lower right side of the back of the mouse. Tumor volume was measured three times a week with a caliper after tumor implantationThe formula for calculating the tumor volume is 0.5a × b². When the tumor size reaches 100mm³At the same time, press 10⁷CAR-T cells or saline were reinfused alone and tumor size and mouse survival status were continuously observed. As can be seen in FIG. 7, the tumor volume of the mice was effectively controlled after 2D81CAR-T inoculation, while the tumors of the normal saline group and the Mock-T group rapidly increased. As can be seen from FIG. 8, 2D81CAR-T significantly prolonged the survival of tumor-bearing mice, all of the saline mice died at 42 days, all of the Mock-T mice died at 47 days, and 40% of the 2D81CAR-T mice still survived at 55 days at the end of the experiment, demonstrating that 2D81CAR-T cells have a good cancer suppression effect on c-Met positive tumors.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Beijing ancient cooking peptide source Biotechnology Ltd

Jiao Shunchang

<120> c-Met-targeted single-chain antibody, chimeric antigen receptor, recombinant vector, CAR-T cell and application

<160> 25

<170> SIPOSequenceListing 1.0

<210> 3

<211> 249

<212> PRT

<213> Artificial Sequence

<400> 3

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

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Glu Arg Leu Ile Ser Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Thr 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 Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Ser Ser Arg

115 120 125

Ser Ser Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro

130 135 140

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

145 150 155 160

Phe Asn Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu

165 170 175

Trp Ile Gly Glu Ile Leu Pro Glu Ser Ile Ile Ser Lys Lys Tyr Asn

180 185 190

Glu Lys Phe Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn

195 200 205

Thr Val Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val

210 215 220

Tyr Phe Cys Ala Ser Gly Gly Tyr Tyr Gly Ser Leu Asp Phe Trp Gly

225 230 235 240

Gln Gly Thr Pro Leu Thr Val Ser Ser

245

<210> 6

<211> 747

<212> DNA

<213> Artificial Sequence

<400> 6

gacgttgtga tgacccagac tccgctcact ttgtcggtta ccattggaca accagcctcc 60

atctcttgca agtcaagtca gagcctctta gatagtgatg gaaagacata tttgaattgg 120

ttgttacaga ggccaggcca gtctccagag cgcctaatct ctctggtgtc taaactggac 180

tctggagtcc ctgacaggtt cactggcagt ggatcgggga cagatttcac actgaaaatc 240

agcagagtgg aggctgagga tttgggagtt tattattgct ggcaaggtac acattttcct 300

ctcacgttcg gtgctgggac caagctggag ctgaaatcta gtggtggcgg tggttcgggc 360

ggtggtggag gtggtagttc tagatcttcc gaggttcagc tgcagcagtc tggagctgag 420

ctgatgaagc ctggggcctc agtgaagata tcctgtaagg caactggcga cacaatcagt 480

ttcaactgga tagagtgggt aaagcagagg cctggacatg gccttgagtg gattggagag 540

attttacctg aaagtattat tagtaagaag tacaatgaga agttcaaggg caaggccaca 600

ttcactgcag atacatcctc caatacagta tacatgcaac tcagcagcct gacatctgag 660

gactctgccg tctatttctg tgcaagcggg ggttactacg gtagccttga cttctggggc 720

caaggcaccc ctctcacagt ctcctca 747

<210> 9

<211> 505

<212> PRT

<213> Artificial Sequence

<400> 9

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

1 5 10 15

His Ala Ala Arg Pro Asp Tyr Lys Asp Asp Asp Asp Lys Glu Phe Asp

20 25 30

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

35 40 45

Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp

50 55 60

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

65 70 75 80

Glu Arg Leu Ile Ser Leu Val Ser Lys Leu Asp Ser Gly Val Pro Asp

85 90 95

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

100 105 110

Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly Thr

115 120 125

His Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Ser

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Ser Ser Arg Ser

145 150 155 160

Ser Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly

165 170 175

Ala Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Asp Thr Ile Ser Phe

180 185 190

Asn Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp

195 200 205

Ile Gly Glu Ile Leu Pro Glu Ser Ile Ile Ser Lys Lys Tyr Asn Glu

210 215 220

Lys Phe Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr

225 230 235 240

Val Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr

245 250 255

Phe Cys Ala Ser Gly Gly Tyr Tyr Gly Ser Leu Asp Phe Trp Gly Gln

260 265 270

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

275 280 285

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

290 295 300

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

305 310 315 320

Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly

325 330 335

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

340 345 350

Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg

355 360 365

Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro

370 375 380

Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser

385 390 395 400

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

405 410 415

Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg

420 425 430

Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Leu His Met Gln Ala Leu Pro Pro Arg

500 505

<210> 12

<211> 1518

<212> DNA

<213> Artificial Sequence

<400> 12

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60

cccgattaca aggatgacga cgataaggaa ttcgacgttg tgatgaccca gactccgctc 120

actttgtcgg ttaccattgg acaaccagcc tccatctctt gcaagtcaag tcagagcctc 180

ttagatagtg atggaaagac atatttgaat tggttgttac agaggccagg ccagtctcca 240

gagcgcctaa tctctctggt gtctaaactg gactctggag tccctgacag gttcactggc 300

agtggatcgg ggacagattt cacactgaaa atcagcagag tggaggctga ggatttggga 360

gtttattatt gctggcaagg tacacatttt cctctcacgt tcggtgctgg gaccaagctg 420

gagctgaaat ctagtggtgg cggtggttcg ggcggtggtg gaggtggtag ttctagatct 480

tccgaggttc agctgcagca gtctggagct gagctgatga agcctggggc ctcagtgaag 540

atatcctgta aggcaactgg cgacacaatc agtttcaact ggatagagtg ggtaaagcag 600

aggcctggac atggccttga gtggattgga gagattttac ctgaaagtat tattagtaag 660

aagtacaatg agaagttcaa gggcaaggcc acattcactg cagatacatc ctccaataca 720

gtatacatgc aactcagcag cctgacatct gaggactctg ccgtctattt ctgtgcaagc 780

gggggttact acggtagcct tgacttctgg ggccaaggca cccctctcac agtctcctca 840

ggatccacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 900

cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 960

gggctggact tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 1020

cttctcctgt cactggttat caccctttac tgcaaacggg gcagaaagaa actcctgtat 1080

atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1140

tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 1200

gcagacgccc ccgcgtacaa gcagggccag aaccagctct ataacgagct caatctagga 1260

cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1320

aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1380

gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1440

ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1500

gccctgcccc ctcgctag 1518

<210> 14

<211> 18

<212> PRT

<213> Artificial Sequence

<400> 14

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Ser Ser Arg

1 5 10 15

Ser Ser

<210> 18

<211> 112

<212> PRT

<213> Artificial Sequence

<400> 18

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

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Glu Arg Leu Ile Ser Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Thr 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 Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 19

<211> 119

<212> PRT

<213> Artificial Sequence

<400> 19

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Asp Thr Ile Ser Phe Asn

20 25 30

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

35 40 45

Gly Glu Ile Leu Pro Glu Ser Ile Ile Ser Lys Lys Tyr Asn Glu Lys

50 55 60

Phe Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Val

65 70 75 80

Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe

85 90 95

Cys Ala Ser Gly Gly Tyr Tyr Gly Ser Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Pro Leu Thr Val Ser Ser

115

<210> 20

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 20

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

1 5 10 15

His Ala Ala Arg Pro

20

<210> 21

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 21

Asp Tyr Lys Asp Asp Asp Asp Lys

1 5

<210> 22

<211> 69

<212> PRT

<213> Artificial Sequence

<400> 22

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Ile Thr Leu Tyr Cys

65

<210> 23

<211> 42

<212> PRT

<213> Artificial Sequence

<400> 23

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 24

<211> 112

<212> PRT

<213> Artificial Sequence

<400> 24

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 25

<211> 63

<212> DNA

<213> Artificial Sequence

<400> 25

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60

ccc 63

<210> 26

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 26

gattacaagg atgacgacga taag 24

<210> 27

<211> 207

<212> DNA

<213> Artificial Sequence

<400> 27

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 180

ctgtcactgg ttatcaccct ttactgc 207

<210> 28

<211> 126

<212> DNA

<213> Artificial Sequence

<400> 28

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 29

<211> 339

<212> DNA

<213> Artificial Sequence

<400> 29

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgctag 339

<210> 30

<211> 6

<212> DNA

<213> Artificial Sequence

<400> 30

gaattc 6

<210> 31

<211> 6

<212> DNA

<213> Artificial Sequence

<400> 31

ggatcc 6

<210> 32

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 32

taaggaattc gatgttgtga tgacc 25

<210> 33

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 33

ggtggatcct gaggagacgg tgac 24

<210> 34

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 34

taaggaattc cagattgttc tctctc 26

<210> 35

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 35

ggtggatcct gaggagactg tgag 24

<210> 36

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 36

aaggaattcg acgttgtgat gacc 24

<210> 37

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 37

gtggatcctg aggagactgt gag 23

Claims

1. A single-chain antibody targeting c-Met is characterized in that the amino acid sequence of the single-chain antibody is shown in SEQ ID No. 3.

2. The gene encoding the single-chain antibody of claim 1, wherein the nucleotide sequence of the gene is represented by SEQ ID No. 6.

3. A chimeric antigen receptor targeting c-Met comprising, in series, a CD8a signal peptide, a Flag tag, the single chain antibody of claim 1, a CD8a hinge region-transmembrane region, a 4-1BB functional region, and a CD3 functional region.

4. The chimeric antigen receptor according to claim 3, wherein the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID No. 9.

5. The gene encoding the chimeric antigen receptor of claim 4, wherein the nucleotide sequence of said gene is represented by SEQ ID No. 12.

6. A recombinant vector targeting c-Met, comprising the gene for the chimeric antigen receptor of claim 5 and an initial vector.

7. The recombinant vector according to claim 6, wherein the initial vector is a lentiviral vector.

8. A c-Met-targeted chimeric antigen receptor T cell obtained by transfecting the T cell with the c-Met-targeted recombinant vector of claim 6 or 7.