CN111574634B

CN111574634B - Double-target chimeric antigen receptor simultaneously targeting mesothelin and FAP and application thereof

Info

Publication number: CN111574634B
Application number: CN202010463122.XA
Authority: CN
Inventors: 郭福春
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2019-12-16
Filing date: 2020-05-27
Publication date: 2022-04-19
Anticipated expiration: 2040-05-27
Also published as: WO2021120526A1; CN111574634A

Abstract

The invention belongs to the field of genetic engineering, and particularly relates to a double-target chimeric antigen receptor for simultaneously targeting mesothelin and FAP and application thereof. Aiming at the phenomenon that expression of tumor cells and fibroblast FAP in solid tumors is up-regulated, the invention provides a double-target chimeric antigen receptor which simultaneously targets mesothelin and FAP and a host cell thereof. The double-target chimeric antigen receptor disclosed by the invention can be competitively combined with FAP, an inhibition signal of FAP is converted into an activation signal, the killing activity of T cells is enhanced, and meanwhile, 4-1BB introduced downstream of the receptor can promote the proliferation and survival of the T cells. The invention also provides host cells expressing the dual-target antigen receptor and application of the host cells in preventing or treating malignant tumors/solid tumors.

Description

Double-target chimeric antigen receptor simultaneously targeting mesothelin and FAP and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to a double-target chimeric antigen receptor for simultaneously targeting mesothelin and FAP, an immunoreaction cell for expressing the chimeric antigen receptor, preparation of the chimeric antigen receptor and the immunoreaction cell, and application of the chimeric antigen receptor and the immunoreaction cell in diagnosis/treatment of malignant tumors.

Background

In 2013, the journal of Science lists tumor immunotherapy as the first ten technological advances in the year. Chimeric Antigen Receptor (CAR) T cell therapy is an important tumor immunotherapy. The antigen antibody scFv fragment is used for linking the intracellular activation and proliferation signals of the T cell to form a Chimeric Antigen Receptor (CAR) -modified T cell (CAR-T), so that the T cell can obtain the antigen specific recognition killing capability independent of HLA (human leukocyte antigen) limitation, the killing activity of the T cell mainly depends on the single-chain receptor recognition antigen on the surface of the CAR-T cell, and the T cell has specific killing activity. Clinical studies have demonstrated that such CAR-ts are able to proliferate, survive and develop immunological memory in vivo, showing potent anti-tumor activity in hematological tumor clinical trials. The CAR-T cell products kymeriah and yescata were approved by the FDA for clinical treatment of hematological tumors in 2017. However, the use of CAR-T cell therapy in solid tumors has progressed slowly compared to the breakthrough progress made in hematological tumor therapy.

Studies have shown that CAR-T cells present several challenges for treating solid tumors: (1) solid tumors lack the desired target antigen. The therapeutic target of most solid tumors is Tumor-associated antigen (TAA). Targeting TAAs is potentially toxic due to their expression in normal tissues. Moreover, there is significant heterogeneity in the expression intensity and distribution of solid tumor antigens. The existing CAR-T cells targeting a single antigen cannot eliminate all tumor cells, and tumor immune escape is easy to cause. (2) The abnormal tissue structure and protein expression of the solid tumor make CAR-T cells difficult to reach tumor sites through circulatory systems such as blood vessels. (3) In solid tumors, the local inhibitory immune microenvironment inhibits anti-tumor T cell activity, such as MDSCs, tregs, and tumor-associated fibroblasts, among others. Several clinical trials have shown that CAR-T cells are able to penetrate inside solid tumors and exert antigen-targeted anti-tumor activity, but with few exceptions, the efficacy in patients is mild and transient. Therefore, how to overcome the inhibitory immune microenvironment is critical for the success of CAR-T cell therapy in solid tumors.

Mesothelin is a glycoprotein anchored to the cell membrane via the phosphatidylinositol domain (GPI) and is highly expressed in a variety of tumor tissues, but small amounts are expressed in normal pleural, pericardial and peritoneal mesothelial cells. The mesothelin gene encodes a 69kDa precursor protein, which is processed to form a 40kDa membrane-associated protein and a 31kDa megakaryocyte stimulating factor (MPF), and is commonly referred to as mesothelin, which is a membrane-anchored fragment and is divided into three regions, I, II, and III, depending on its protein structure. On one hand, the compound can activate intracellular signal pathways of NFKB, MAPK and PI3K, and promote cell proliferation and apoptosis resistance; on the other hand, the interaction with CA125/MUC16 leads to abnormal cell adhesion and promotes cancer cell metastasis. Mesothelin is overexpressed in a variety of malignancies (mesothelioma, ovarian cancer, pancreatic cancer, gastric cancer, cholangiocarcinoma, etc.) due to its limited distribution in normal tissues, and is therefore a very potential tumor-specific therapeutic target.

At present, a plurality of CAR-T cell researches taking mesothelin as a target point are carried out, and a plurality of clinical tests are carried out at home and abroad, mainly aiming at pancreatic cancer, mesothelioma, lung cancer and breast cancer. However, these solid tumors have a characteristic feature that they have a relatively rich Tumor stroma, which contains a large number of Tumor-associated fibroblasts (TAF).

Fibroblast Activation Protein (FAP, also known as seprase, is a 95kDa type II transmembrane serine protease structurally related to dipeptidyl peptidase IV (DPPIV/CD26) FAP is expressed in reactive stromal fibroblasts in tumor tissue and wound healing FAP is also an endopeptidase that can degrade gelatin, type I and IV collagen, fibronectin and laminin, as well as several peptide hormones (e.g., neuropeptide Y, brain natriuretic peptide, substance P, peptide YY and incotins). FAP's stromal-degrading activity can contribute to tumor cell migration and invasion, furthermore FAP can enhance tumor cell growth by limiting the development of anti-tumor immunity. And has no obvious toxic and side effects.

Aiming at the characteristics of rich solid tumor stroma and high FAP expression of tumor-associated fibroblasts, a double-target CAR viral vector which simultaneously targets mesothelin and FAP is designed and constructed in the previous period. The invention takes mesothelin and FAP double-targeting CAR-T (dual CAR-T) as an example, and shows that the FAP target-containing double-target chimeric antigen receptor can eliminate tumor cells, relieve the immunosuppression of interstitial cells inside tumors, prevent and treat malignant/solid tumors and is used for clinically relevant prevention and treatment.

Disclosure of Invention

Aiming at the characteristics of rich solid tumor stroma and high tumor-related fibroblast expression FAP, the invention provides a gene engineering modified double-target chimeric antigen receptor and a host cell thereof.

The first technical problem to be solved by the invention is as follows: the double-target chimeric antigen receptor can be combined with two different targets to transmit two signals.

The invention relates to a gene engineering modified double-target chimeric antigen receptor, which is formed by connecting a chimeric antigen receptor 1 for recognizing mesothelin and a chimeric antigen receptor 2 for recognizing fibroblast activation protein FAP through a connecting peptide.

Wherein, in the above-mentioned genetic engineering modified double-target chimeric antigen receptor, the chimeric antigen receptor 2 includes: single chain antibodies, hinge regions, transmembrane regions and intracellular domains of FAP.

Further, the single-chain antibody of FAP refers to the single-chain antibody of FAP capable of binding to FAP molecules on the surface of tumor cells or immune cells.

Further, the hinge region is a human immunoglobulin Fc fragment (IgG1 Fc fragment).

Further, the transmembrane region is a leukocyte differentiation antigen 8(CD8) transmembrane region.

Further, the intracellular domain is a 4-1BB intracellular domain.

Wherein, the chimeric antigen receptor 2 comprises the following components: scFv of human FAP, IgG1 Fc segment, CD8 transmembrane region, and 4-1BB costimulatory molecule peptide segment.

Specifically, the amino acid sequence of the chimeric antigen receptor 2 is SEQ ID NO: 1 is shown.

METDTLLLWVLLLWVPGSTGAQVQLKQSGAELVKPGASVKLSCKTSGYTFTENIIHWVKQRSGQGLEWIGWFHPGSGSIKYNEKKDKATLTADKSSSTVYMELSRLTSEDSAVYFCARHGGTGRGAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDILMTQSPASSVVSLSGQRATISCRASKSVSTSAYSYMHWYQQKPGQPPKLLIYLASNLESGVPPRFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPYTFGGGTKLEIKDPAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL。

Further, the coding nucleotide sequence of the chimeric antigen receptor 2 is SEQ ID NO: 2, as shown in the figure:

atggagacagatacactgctgctgtgggtgctgctgctgtgggtgcccggcagcaccggcgctcaagtgcagctgaaacagtccggcgccgaactggtgaaacccggcgccagcgtgaagctgagctgcaagaccagcggatacaccttcaccgagaacatcatccactgggtgaagcagaggagcggccaaggcctcgagtggattggatggttccatcccggcagcggcagcatcaagtacaatgagaagaaagacaaggccacactgaccgctgacaagagctcctccaccgtgtatatggagctgtctagactgacaagcgaggacagcgccgtgtacttctgcgctagacatggaggcaccggaagaggagccatggactattggggccaaggcacaagcgtcacagtgtcctccggcggaggaggcagcggaggaggcggaagcggcggcggaggcagcgacattctgatgacccaatcccccgcctccagcgtggtgtccctctccggacagagagctaccatcagctgtagagctagcaagtccgtgagcacatccgcctacagctacatgcactggtaccagcagaaacccggacaacctcccaagctgctgatctatctggccagcaatctcgagagcggcgtgcctcctagattttccggctccggcagcggcacagacttcaccctcaacatccatcccgtggaggaagaggatgccgccacatactactgccagcattctagagagctgccctacacctttggaggcggcaccaaactggagatcaaagatcccgccgagcctaagagccccgacaagacccacacatgtcctccttgtcccgcccctcccgtggctggacctagcgtgtttctgttcccccccaagcccaaggacacactgatgatcgctagaacacccgaggtgacatgcgtcgtggtggacgtctcccacgaagaccccgaggtgaagttcaactggtacgtggacggagtggaagtccacaacgccaaaacaaagcctagagaggagcagtacaactccacatatagagtggtgtccgtgctgacagtgctccaccaagactggctgaacggcaaggagtacaagtgcaaggtcagcaacaaggctctgcccgcccctattgagaagaccatcagcaaggccaagggccagcctagagaaccccaagtctataccctccctccctctagagacgagctcaccaagaatcaagtgtctctgacatgtctggtcaagggcttttaccctagcgacattgctgtggagtgggagtccaacggccagcccgagaacaactacaagaccacaccccccgtgctggatagcgacggcagcttctttctgtattccaagctgaccgtggataagagcagatggcagcaaggcaacgtgtttagctgtagcgtcatgcacgaggctctgcacaaccactatacccagaagtctctgtctctgtcccccggcaagaagatctacatctgggctcctctggctggaacatgcggcgtgctgctgctgtctctggtcatcacactgtactgtaagaggggcagaaagaagctgctctacatcttcaagcagcccttcatgagacccgtccagacaacccaagaggaggacggatgcagctgcagatttcccgaagaggaggagggaggatgcgagctctga。

wherein, the chimeric antigen receptor 1 comprises: a single chain antibody capable of binding a tumor specific antigen or a tumor associated antigen, a hinge region, a transmembrane region, and an intracellular immunoreceptor tyrosine activation motif.

Wherein the tumor specific antigen or tumor associated antigen is (EGFR), epithelial cell adhesion molecule (EpCAM), mesothelin (mesothelin), interleukin 13 receptor alpha 2(IL-13R alpha 2), epidermal growth factor receptor 2(ERBB2), epidermal growth factor receptor 3(ERBB3), epidermal growth factor receptor 4(ERBB4), vascular endothelial growth factor receptor 1(VEGFR1), vascular endothelial growth factor receptor 2(VEGFR2), ganglioside antigen G_D2(GD2), Folate Receptor (FR), Prostate Specific Membrane Antigen (PSMA), melanin precursor protein (gp100), mucin 1(MUC1), mucin 16(MUC16), carbonic anhydrase 9(CA9), L1 cell adhesion molecule (CD171), L1 cell adhesion molecule (CD125), leukocyte differentiation antigen 15-3(CD15-3), leukocyte differentiation antigen 19-9(CD19-9), NY-ESO-1, MART-1, MAGE4, leukocyte differentiation antigen 19(CD19), leukocyte differentiation antigen 20(CD20), leukocyte differentiation antigen 22(CD22), leukocyte differentiation antigen 30(CD30), leukocyte differentiation antigen 33(CD33), carcinoembryonic antigen (CEA), leukocyte differentiation antigen 38(CD38), leukocyte differentiation antigen 138(CD138), leukocyte differentiation antigen 123(CD123), EPH receptor A2(EPHA2), or insulin-like growth factor receptor (IGFR).

Wherein the single-chain antibody capable of binding to a tumor specific antigen or a tumor associated antigen is a single-chain antibody capable of binding to an Epidermal Growth Factor Receptor (EGFR) family protein, including EGFR, human epidermal growth factor receptor-2 (HER2), epidermal growth factor receptor 3(ERBB3), epidermal growth factor receptor 4(ERBB4) or epidermal growth factor receptor variant III (EGFRvIII), epithelial cell adhesion molecule (EpCAM), mesothelin (mesothelin), interleukin 13 receptor alpha 2(IL-13R alpha 2), vascular endothelial growth factor receptor 1(VEGFR1), vascular endothelial growth factor receptor 2(VEGFR2), ganglioside antigen G (G-1)_D2(GD2), Folate Receptor (FR), Prostate Specific Membrane Antigen (PSMA),Melanin precursor protein (gp100), mucin 1(MUC1), mucin 16(MUC16), carbonic anhydrase 9(CA9), L1 cell adhesion molecule (CD171), L1 cell adhesion molecule (CD125), leukocyte differentiation antigen 15-3(CD15-3), leukocyte differentiation antigen 19-9(CD19-9), NY-ESO-1, MART-1, MAGE4, leukocyte differentiation antigen 19(CD19), leukocyte differentiation antigen 20(CD20), leukocyte differentiation antigen 22(CD22), leukocyte differentiation antigen 30(CD30), leukocyte differentiation antigen 33(CD33), carcinoembryonic antigen (CEA), leukocyte differentiation antigen 38(CD38), leukocyte differentiation antigen 138(CD138), leukocyte differentiation antigen 123(CD123), EPH receptor a2(EPHA2), or insulin-like growth factor receptor (IGFR). Preferably, the single chain antibody is mesothelin scFv.

Wherein the transmembrane region is at least one transmembrane region selected from the group consisting of leukocyte differentiation antigen 28(CD28), leukocyte differentiation antigen 8(CD8), leukocyte differentiation antigen 3 ζ (CD3 ζ), leukocyte differentiation antigen 134(CD134), leukocyte differentiation antigen 137(CD137), induced T cell costimulatory molecule (ICOS), DNAX activating protein (DAP10) and leukocyte differentiation antigen 27(CD 27).

Wherein the hinge region is CD8 a.

Preferably, the transmembrane regions of the

chimeric antigen receptors

1 and 2 are selected from different transmembrane regions. More preferably, the transmembrane region of the chimeric antigen receptor 1 is the CD28 transmembrane region, and the transmembrane region of the chimeric antigen receptor 2 is the CD8 transmembrane region.

Wherein the intracellular immunoreceptor tyrosine activation motif comprises an immunoreceptor tyrosine activation motif signal chain selected from the group consisting of CD3 ζ and Fc ε RI.

Wherein, the chimeric antigen receptor 1 comprises the following components: signal peptide, scFv of human mesothelin, CD8a hinge region, CD28 transmembrane region, CD3 zeta binding domain.

Further, the amino acid sequence of the chimeric antigen receptor 1 is SEQ ID NO: 3, showing:

MALPVTALLLPLALLLHAARPQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSSGILGSGGGGSGGGGSGGGGSQPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAYRHQALPPRGSGATNFSLLKQAGDVEENPGP。

further, the nucleotide sequence of the chimeric antigen receptor 1 is SEQ ID NO: 4, and (2) is as follows:

atggctttacccgttaccgctctcttattacctctggctctgctgctgcatgccgccagacccgcagcagagcggacccggtttagtgacccctagccagactttaagcctcacatgcgccatttccggcgacagcgtcagcagcaacagcgccacttggaactggattcgtcaatccccctctcgtggtttagaatggctgggaaggacctactatcgtagcaagtggtacaacgactacgctgtgagcgtgaagtctcgtatgtccatcaaccccgacacaagcaagaaccagttctctttacagctcaactccgtcacacccgaagacaccgctgtctactattgcgctcgtggcatgatgacatactattacggcatggacgtgtggggacaaggcactaccgtgacagtgtcctccggcatcctcggttccggcggcggcggcagcggcggcggaggctccggcggtggcggcagccaacccgttctgacacagagcagcagcctcagcgcttcccccggagcttccgcttctttaacttgtacattacgtagcggaattaacgtgggcccctacagaatctactggtatcaacagaagcccggaagccccccccagtatttactcaactacaagagcgactccgacaaacagcaaggtagcggcgtgccctctcgtttttccggctccaaggatgcctccgccaatgccggcgtgctgctgattagcggtttaaggtccgaggatgaggctgattactactgtatgatttggcatagcagcgccgccgtgtttggcggcggaacacagctgaccgtgctgaccacaacacccgctcccagacctcctacccccgctcctaccatcgccagccagcctctgtctttaagacccgaggcttgtagacccgctgctggcggagctgtgcacaccagaggtttagactttgcttgtgatttctgggtgctcgtggtcgtgggaggcgttttagcttgctacagcttattagtgaccgtggcctttatcattttctgggtgaggtccaaaagatctcgtctgctgcactccgactacatgaatatgacacctaggaggcccggtcccactcgtaagcactaccagccttacgccccccccagagacttcgccgcttatcgttccgtgagagtgaagttttctcgttccgccgatgctcccgcttaccagcaaggtcagaaccagctgtacaacgaattaaatctgggtcgtagagaggagtacgacgttttagataagaggaggggcagagatcccgaaatgggcggcaagcccagaaggaaaaaccctcaagagggcctctacaatgagctgcagaaggacaagatggccgaggcttattccgaaatcggcatgaaaggcgagagaagaaggggcaagggacatgatggtttataccaaggtttaagcacagccaccaaagacacctacgacgcctacagacatcaagctctcccccccaga。

wherein the connecting peptide is at least one of Furin-T2A or P2A.

The amino acid sequence of the connecting peptide T2A is shown in SEQ ID NO: 5 is shown in the specification; the nucleotide sequence of the connecting peptide T2A is shown as SEQ ID NO: and 6. The amino acid sequence of the connecting peptide Furin is shown as SEQ ID NO: 7 is shown in the specification; the nucleotide sequence of the connecting peptide Furin is shown as SEQ ID NO: shown in fig. 8.

SEQ ID NO: amino acid sequence of 5-linker peptide T2A:

SGSGEGRGSLLTCGDVEENPGP。

SEQ ID NO: 6 nucleotide sequence of the linker peptide T2A:

agcggcagcggcgagggaagaggaagcctgctgacctgcggcgatgtggaggagaatcccggcccc。

SEQ ID NO: 7 amino acid sequence of the linker peptide Furin:

RRKR。

SEQ ID NO: 8 nucleotide sequence of the connecting peptide Furin:

aggaggaagaga。

the chimeric antigen receptor 1 and the chimeric antigen receptor 2 of the present invention are expressed together by a single vector.

The invention also provides an expression vector which is an expression vector for simultaneously expressing the chimeric antigen receptor 1 and the chimeric antigen receptor 2. Furthermore, the expression vector is a eukaryotic or prokaryotic expression vector, and the eukaryotic expression vector is a plasmid; the prokaryotic expression vector is a viral vector, and the viral vector comprises retrovirus, recombinant lentivirus and recombinant adenovirus; further, the virus vector is pWPXld.

The invention also provides a host cell containing the expression vector. Preferably, the host cell is an immune response cell. Preferably T cells, monocytes, natural killer cells. More preferably T cells and natural killer cells.

The invention also provides the double-target chimeric antigen receptor, a recombinant vector containing the chimeric antigen receptor and application of a host cell containing the recombinant vector in preparation of drugs for preventing or treating malignant tumors.

In the above application, the malignant tumor is a solid tumor, and particularly may be at least one of lung cancer, hepatocellular carcinoma, colon cancer, rectal cancer, breast cancer, ovarian cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, renal cancer, pancreatic cancer, or prostate cancer.

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

the invention can express two antigen receptors in host cells simultaneously by constructing a recombinant vector containing a double-target-point chimeric antigen receptor expression unit, wherein one antigen receptor is a receptor which is combined with tumor specific antigen or tumor-associated antigen and can generate specific targeting effect on tumor cells, the other antigen receptor is an FAP receptor and is mainly expressed in tumor-associated fibroblasts, and when an antigen receptor 2 is contacted with immunosuppressive cells in a tumor microenvironment, an immunosuppressive signal of FAP can be inverted into an activation signal. The double-target specific binding form of the method can target tumor cells and also aims at immunosuppressive cells of a tumor microenvironment, and has better effect on preventing and treating solid tumors rich in interstitial tissues.

Drawings

FIG. 1 is a schematic representation of the framework of the chimeric antigen receptor of the present invention (in which the variable region can be replaced by any single-chain antibody fragment);

FIG. 2 is a diagram showing an embodiment mode of a dual-target CAR that binds mesothelin and FAP;

FIG. 3 is a graph showing detection of T cell surface CAR expression after transfection;

FIG. 4 is a chart showing the detection of tumor cell and fibroblast antigens;

FIG. 5 is a graph showing the in vitro killer cytokine detection of cells;

FIG. 6 shows the results of CAR-T cell killing in vitro;

FIG. 7 shows the results of a mixed vaccination subcutaneous model of CAR-T treated human SKOV3 and fibroblasts.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings. In the following examples, the experimental conditions are not specified and are well known to those skilled in the artKnown conventional conditions, for example, Sambrook J, Russell D.W.,2001, Molecular Cloning: A laboratory manual (3)^rded), the conditions described in Spring Harbor Laboratory Press, or according to the manufacturer's recommendations.

EXAMPLE 1 construction of recombinant Lentiviral vectors for Dual-target chimeric antigen receptors

Constructing a recombinant vector of the double-target chimeric antigen receptor, wherein an expression frame is as follows: the sequence from end 5 to end 3 is: mesothelin scFv-CD8a hinge region-CD 28 transmembrane region-CD 3 ζ -Furin-T2A-FAP scFv-IgG1 Fc-CD8 transmembrane region-4-1 BB.

The amino acid sequence of the mesothelin signal peptide is SEQ ID NO: 9 is as follows:

MALPVTALLLPLALLLHAARP。

the coding nucleotide sequence of the signal peptide of the mesothelin is SEQ ID NO: 10, and:

Atggctttacccgttaccgctctcttattacctctggctctgctgctgcatgccgccagaccc。

the amino acid sequence of the scFv of mesothelin is SEQ ID NO: 11, and:

QVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSSGILGSGGGGSGGGGSGGGGSQPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVL。

the encoding nucleotide sequence of the mesothelin scFv is SEQ ID NO: 12, and: caagttcaactgcagcagagcggacccggtttagtgacccctagccagactttaagcctcacatgcgccatttccggcgacagcgtcagcagcaacagcgccacttggaactggattcgtcaatccccctctcgtggtttagaatggctgggaaggacctactatcgtagcaagtggtacaacgactacgctgtgagcgtgaagtctcgtatgtccatcaaccccgacacaagcaagaaccagttctctttacagctcaactccgtcacacccgaagacaccgctgtctactattgcgctcgtggcatgatgacatactattacggcatggacgtgtggggacaaggcactaccgtgacagtgtcctccggcatcctcggttccggcggcggcggcagcggcggcggaggctccggcggtggcggcagccaacccgttctgacacagagcagcagcctcagcgcttcccccggagcttccgcttctttaacttgtacattacgtagcggaattaacgtgggcccctacagaatctactggtatcaacagaagcccggaagccccccccagtatttactcaactacaagagcgactccgacaaacagcaaggtagcggcgtgccctctcgtttttccggctccaaggatgcctccgccaatgccggcgtgctgctgattagcggtttaaggtccgaggatgaggctgattactactgtatgatttggcatagcagcgccgccgtgtttggcggcggaacacagctgaccgtgctg are provided.

The amino acid sequence of the CD8a hinge region is SEQ ID NO: 13, and:

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD。

the encoding nucleotide sequence of the CD8a hinge region is SEQ ID NO: 14, in the following:

accacaacacccgctcccagacctcctacccccgctcctaccatcgccagccagcctctgtctttaagacccgaggcttgtagacccgctgctggcggagctgtgcacaccagaggtttagactttgcttgtgat。

the amino acid sequence of the CD28 transmembrane region is SEQ ID NO: 15, and:

FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS。

the nucleotide sequence of the CD28 transmembrane region is SEQ ID NO: 16 is as follows:

ttctgggtgctcgtggtcgtgggaggcgttttagcttgctacagcttattagtgaccgtggcctttatcattttctgggtgaggtccaaaagatctcgtctgctgcactccgactacatgaatatgacacctaggaggcccggtcccactcgtaagcactaccagccttacgccccccccagagacttcgccgcttatcgttcc。

the amino acid sequence of CD3 ζ is SEQ ID NO: 17 shows:

VRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAYRHQALPPR。

the nucleotide sequence of CD3 ζ is SEQ ID NO: 18, and:

gtgagagtgaagttttctcgttccgccgatgctcccgcttaccagcaaggtcagaaccagctgtacaacgaattaaatctgggtcgtagagaggagtacgacgttttagataagaggaggggcagagatcccgaaatgggcggcaagcccagaaggaaaaaccctcaagagggcctctacaatgagctgcagaaggacaagatggccgaggcttattccgaaatcggcatgaaaggcgagagaagaaggggcaagggacatgatggtttataccaaggtttaagcacagccaccaaagacacctacgacgcctacagacatcaagctctcccccccaga。

the amino acid of Furin-T2A is SEQ ID NO: 19, and:

RRKRSGSGEGRGSLLTCGDVEENPGP。

the coding nucleotide sequence of Furin-T2A is SEQ ID NO: 20, and:

agcggcagcggcgagggaagaggaagcctgctgacctgcggcgatgtggaggagaatcccggccccaggaggaagaga。

the amino acid sequence of the signal peptide of FAP is SEQ ID NO: 21, and:

METDTLLLWVLLLWVPGSTGA。

the nucleotide sequence of the signal peptide of FAP is SEQ ID NO: 22, in which:

atggagacagatacactgctgctgtgggtgctgctgctgtgggtgcccggcagcaccggcgct。

the amino acid sequence of FAP scFv is SEQ ID NO: 23, in the following steps:

QVQLKQSGAELVKPGASVKLSCKTSGYTFTENIIHWVKQRSGQGLEWIGWFHPGSGSIKYNEKKDKATLTADKSSSTVYMELSRLTSEDSAVYFCARHGGTGRGAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDILMTQSPASSVVSLSGQRATISCRASKSVSTSAYSYMHWYQQKPGQPPKLLIYLASNLESGVPPRFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPYTFGGGTKLEIK。

the coding nucleotide sequence of FAP scFv is SEQ ID NO: 24, showing:

caagtgcagctgaaacagtccggcgccgaactggtgaaacccggcgccagcgtgaagctgagctgcaagaccagcggatacaccttcaccgagaacatcatccactgggtgaagcagaggagcggccaaggcctcgagtggattggatggttccatcccggcagcggcagcatcaagtacaatgagaagaaagacaaggccacactgaccgctgacaagagctcctccaccgtgtatatggagctgtctagactgacaagcgaggacagcgccgtgtacttctgcgctagacatggaggcaccggaagaggagccatggactattggggccaaggcacaagcgtcacagtgtcctccggcggaggaggcagcggaggaggcggaagcggcggcggaggcagcgacattctgatgacccaatcccccgcctccagcgtggtgtccctctccggacagagagctaccatcagctgtagagctagcaagtccgtgagcacatccgcctacagctacatgcactggtaccagcagaaacccggacaacctcccaagctgctgatctatctggccagcaatctcgagagcggcgtgcctcctagattttccggctccggcagcggcacagacttcaccctcaacatccatcccgtggaggaagaggatgccgccacatactactgccagcattctagagagctgccctacacctttggaggcggcaccaaactggagatcaaa。

the amino acid sequence of the IgG1 Fc fragment is SEQ ID NO: 25, and:

DPAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKK

the nucleotide sequence of the IgG1 Fc segment is SEQ ID NO: 26, as shown:

gatcccgccgagcctaagagccccgacaagacccacacatgtcctccttgtcccgcccctcccgtggctggacctagcgtgtttctgttcccccccaagcccaaggacacactgatgatcgctagaacacccgaggtgacatgcgtcgtggtggacgtctcccacgaagaccccgaggtgaagttcaactggtacgtggacggagtggaagtccacaacgccaaaacaaagcctagagaggagcagtacaactccacatatagagtggtgtccgtgctgacagtgctccaccaagactggctgaacggcaaggagtacaagtgcaaggtcagcaacaaggctctgcccgcccctattgagaagaccatcagcaaggccaagggccagcctagagaaccccaagtctataccctccctccctctagagacgagctcaccaagaatcaagtgtctctgacatgtctggtcaagggcttttaccctagcgacattgctgtggagtgggagtccaacggccagcccgagaacaactacaagaccacaccccccgtgctggatagcgacggcagcttctttctgtattccaagctgaccgtggataagagcagatggcagcaaggcaacgtgtttagctgtagcgtcatgcacgaggctctgcacaaccactatacccagaagtctctgtctctgtcccccggcaagaag。

the amino acid sequence of the CD8 transmembrane region is SEQ ID NO: 27 shows that:

IYIWAPLAGTCGVLLLSLVITLYC。

the nucleotide sequence of the CD8 transmembrane region is SEQ ID NO: 28, and:

atctacatctgggctcctctggctggaacatgcggcgtgctgctgctgtctctggtcatcacactgtactgt。

the amino acid sequence of 4-1BB is SEQ ID NO: 29, and:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL。

the nucleotide sequence of 4-1BB is SEQ ID NO: 30, and (b):

aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg。

synthesizing the double-target chimeric antigen receptor according to the sequence, and concretely operating as follows:

one, Meso Single CAR, FAP Single CAR and Meso-FAP Dual CAR Gene acquisition

And obtaining the Meso-FAP double CAR gene fragment by adopting a whole gene synthesis method.

At the same time, primers were designed to amplify Meso single CAR fragments as follows:

5' primer: SEQ ID NO: 31

aggtttaaactacggATGGCTTTACCCGTTACCGCTC。

3' primer: SEQ ID NO: 32

atgactagtcccgggTCATCTGGGGGGGAGAGCTTGA。

The reaction conditions were as follows:

and (3) PCR reaction: denaturation at 94 ℃ for 30 seconds; annealing at 60 ℃ for 30 seconds; extension at 68 ℃ for 1 min. The reaction was carried out for 25 cycles. Then further extension at 72 ℃ for 10 minutes.

The following primers were designed to amplify FAP single CAR fragments:

5' primer: SEQ ID NO: 33

aggtttaaactacggATGGAGACAGATACACTGCTGC。

3' primer: SEQ' ID NO: 34

atgactagtcccgggTCACAGTTCACATCCTCCTTCT。

The reaction conditions were as follows:

and (3) PCR reaction: denaturation at 94 ℃ for 30 seconds; annealing at 60 ℃ for 30 seconds; extension at 68 ℃ for 2 minutes. The reaction was carried out for 25 cycles. Then further extension at 72 ℃ for 10 minutes.

The following primers were designed to amplify Meso-FAP dual CAR fragments:

5' primer: SEQ ID NO: 35

aggtttaaactacggATGGCTTTACCCGTTACCGCTC。

3' primer: SEQ ID NO: 36

atgactagtcccgggTCACAGTTCACATCCTCCTTCT。

The reaction conditions were as follows:

and (3) PCR reaction: denaturation at 94 ℃ for 30 seconds; annealing at 60 ℃ for 30 seconds; extension at 68 ℃ for 3 minutes. The reaction was carried out for 25 cycles. Then further extension at 72 ℃ for 10 minutes.

Construction of lentiviral recombinant plasmids of two, Meso-CAR, FAP-CAR and Meso-FAP Dual CAR genes

As mentioned above, in the total gene synthetic fragment, there is no enzyme cutting site, we use homologous recombination method to insert the fragment directly into the multiple cloning site EcoR I/BamH I of eukaryotic expression vector pWPXld (purchased from Addge company, USA), and the enzyme cutting site will disappear after recombination. The construction process is as follows:

1. construction of recombinant Lentiviral plasmid pWPXld-Single CAR

The lentiviral vector pWPXld is linearized by restriction enzymes EcoR1 and BamH1, a single CAR gene amplification fragment is inserted into EcoR I/BamH I sites of pWPXld by a Tiangen easy Geno rapid recombinant cloning kit (Tiangen Biochemical technology Co., Ltd., VI201), a recombinant product pWPXld-single CAR is transformed into escherichia coli Stbl3, and 30 clones are randomly selected for sequencing identification. The sequencing results were consistent with the designed full-length sequence of the single CAR.

2. Construction of recombinant lentiviral plasmid pWPXld-Dual CAR

The lentiviral vector pWPXld is linearized by restriction enzymes EcoR1 and BamH1, the full-length double CAR gene amplification fragment is inserted into EcoR I/BamH I sites of pWPXld by using Tiangen easy recombinant cloning kit (Tiangen Biochemical technology Co., Ltd., VI201), the recombinant product pWPXld-double CAR is transformed into Escherichia coli Stbl3, and 30 clones are randomly selected for sequencing identification. The sequencing results were consistent with the designed full-length sequence of the dual CAR.

Plasmids were extracted and purified using a plasmid purification kit from Qiagen, the purification procedure was referred to kit instructions, and high quality recombinant expression vectors were obtained, and the results of the inserted target fragments are shown in FIG. 1.

Example 2 Lentiviral packaging and expression of T cell CAR

1. Culture and passage of 293T cells:

DMEM containing 10% fetal calf serum and 100U/ml penicillin streptomycin and pancreatin were preheated in a 37 ℃ water bath. After the 293T cells grew to 80% -90% density, the medium was aspirated off the dish with a sterile pipette, and 2ml of pancreatin was added to briefly rinse out the remaining medium from the dish, which was then aspirated and removed. 1ml of pancreatin was dropped into the culture dish, and the cells were observed under a mirror until the cells were separated by rounding, and pancreatin was aspirated. 5ml of fresh complete medium was added to the dish and the cells were gently blown down. The cell suspension was divided into other dishes and medium was added to reach 10ml per dish. The culture dish was shaken 3 times in a cross-like manner, the cells were shaken up, observed under a mirror, and then placed in an incubator at 37 ℃. Observing the cell state, and carrying out next subculture when the cells grow to 80% -90%.

2. Obtaining a lentivirus stock solution:

day 1: planking. Digestion of 293T cells at a good density of 80%, passage 1:3, approximately 1.0X 107cells/20ml/15cm dish, 5% CO₂Incubated overnight at 37 ℃. The cell density is about 50-70% (not more than 70%) in 16 h. Day 2: and (4) transfection. 2 hours before transfection, the medium was changed with pre-warmed antibiotic-free 10% DMEM high-sugar medium, 20 ml/dish. All transfection reagents and plasmids were allowed to equilibrate at room temperature. Transfection: a. the following DNA mixture (per 15cm plate) was prepared in 50ml BD tubes, with psPAX2 (packing plasmid)22.5 ug; pMD.2G (envelope plasmid)11.25 ug; pWPXld (lentivirus vector)22.5 ug. b. Adding water to constant volume of 1125 ul. c.2.5M CaCl 2125 ul was added dropwise to the DNA solution and vortexed for 5 s. The BD tube was placed on a vortex apparatus (4 th), and 2 XBBS (1250ul) solution was added drop by drop to the DNA-CaCl2 mixture, shaking for 5 s. e. The mixture was allowed to stand at room temperature for 15 minutes. 2.25ml of the transfection mixture was dropped into the dish, mixed by gentle shaking in a crisscross (10 times each) and 3% CO₂And cultured at 37 ℃. After 12h, the medium was aspirated, and 15ml of pre-warmed 2% DMEM medium, 5% CO, was added gently₂Culturing at 37 deg.C for 48 h. Day 4: 48hr after transfection, cell supernatant was collected and 15ml of preheated DMEM medium containing 2% FBS and 5% CO was added₂Culturing at 37 ℃; the virus supernatant was filtered through a 0.45 μm filter and stored at 4 ℃. Day 5: after transfection for 72hr, the virus supernatant was collected, filtered through a 0.45 μm filter, and stored at 4 ℃.

3. Concentration of lentivirus:

the instrument comprises the following steps: the ultra-high speed centrifuge instrument comprises a rotor and a sleeve matched with the ultra-high speed centrifuge tube and a balance for balancing. The sleeve and balance were UV sterilized for 30 min. Viral suspension filtered through a 0.45um filter was added to the centrifuge tube and rigorously trimmed. And (3) mounting each sleeve after being balanced into a rotor of a centrifuge, and centrifuging for 2 hours at 20 ℃ and 70000 g. After centrifugation, the medium was decanted and the centrifuge tube inverted and the remaining medium was aspirated off on sterile filter paper. The viral pellet was resuspended using sterile PBS (200 ul/tube), and the resuspended virus was dispensed into EP tubes (100 ul/tube), stored at-80 ℃ in an ultra-low temperature freezer.

4. Isolation of human peripheral blood T lymphocytes:

extracting peripheral blood of healthy people by 15-20ml, and collecting with a green cover anticoagulation tube. The peripheral blood with the same volume is slowly added into the FICOLL lymphocyte separation liquid, and the mixture is centrifuged for 30min at the temperature of 32 ℃ and 1000g, and the adding/reducing speed is 3. After centrifugation, the blood was seen to be divided into 3 layers, and the middle buffy coat layer was taken. The aspirated lymphocytes were added to 20mL serum-free and antibiotic-free X-VIVO medium and centrifuged at 500g for 10 min. Discard the supernatant, resuspend the lymphocyte pellet with 10mL sterile schizophrenic solution, incubate for 2-3 min. Centrifuge 500g, 10 min. The supernatant was discarded, washed twice with 1640 medium, counted, and added with CD3/CD28 Dynabeads (life technologies 11131D) (cells: beads ═ 1: 3), and cultured in suspension with a 5% human fetal bovine serum-containing X-vivo medium supplemented with 50U/ml IL-2, 10ng/ml IL-7, and 10ng/ml IL 15.

5. T cell transfection and CAR expression assay:

one day prior to viral infection, six well plates were coated with fibronectin (RetroNectin) dilutions (50ug/ml) and left overnight at 4 ℃. On the day of infection, fibronectin dilutions were aspirated, the six-well plates were blocked with PBS containing 2% BSA (bovine serum albumin) for 30min, the supernatant was discarded, and 3 washes were performed with PBS. The cells were centrifuged and the T cells resuspended in medium containing viral supernatant (MOI 1: 10), mixed well, added to a six-well plate and centrifuged at 32 ℃, 1000g, 2 h. Taking out six-hole plate, placing at 37 deg.C and 5% CO₂And (5) culturing in a cell culture box.

Meso single CAR, FAP single CAR and Meso-FAP double CAR concentration virus infected T cells 48h before flow detection, anti-human Fab antibody detected Meso-CAR expression, and FITC-labeled FAP protein detected FAP-CAR expression (see figure 3). The results show that: in T cells transfected with FAP single CAR, 73.83% CAR expression was detected; in T cells transfected with Meso single CAR, 62.28% of CAR expression was detected; in T cells transfected with Meso-FAP dual CAR, 52.14% FAP CAR and 54.68% Meso CAR expression were detected, respectively; the detection result shows that: we successfully constructed single CAR and dual CAR T cells targeting FAP and MSLN.

Example 3 Performance assay of mesothelin and FAP Dual-target CAR-T cells

1. Target cell identification

And determining high expression FAP of the primary tumor-associated fibroblasts by flow detection. SKOV3 cells are negative in MSLN expression, and are transformed into MSNL full-length genes through lentivirus infection to construct a SKOV3 cell line for stably expressing MSLN (see figure 4).

2. In vitro killing ability of CAR-T cells, and measurement of IL-2 and IFN-gamma secretion.

Cytokine release was detected using human IL-2 and IFN γ ELISA Kit (Thermo). Cell supernatants were collected and stored at-20 ℃. During detection, 50ul of sample analysis buffer solution and 50ul of sample are mixed, added into a detection plate and incubated overnight at 4 ℃; washing the plate for 5 times, removing the supernatant by suction, and adding a biotinylated antibody working solution (100 ul/well); sealing the reaction hole with sealing plate gummed paper, and incubating for 60 minutes at room temperature; washing the plate for 5 times, removing the supernatant, adding the working solution (100 ul/well) of the enzyme conjugate, and incubating for 20 minutes at room temperature in the dark; washing the plate for 5 times, adding a color developing agent TMB100 ul/hole, and incubating for 20 minutes at room temperature in a dark place; add stop solution 50 ul/well and mix well and measure OD450 value immediately. And judging the result that the result is valid when the value of the multiple wells is within the difference range of 20%, calculating the supernatant concentration according to a standard curve, and multiplying the final concentration by the dilution factor. The results in fig. 5 show that when Meso single CAR and Meso-FAP dual-targeted CAR-T cells were incubated with MSLN positive SKOV3 cells, T cell proliferation and killing could be activated efficiently, releasing a large amount of IL-2 and IFN- γ; FAP single CAR and Meso-FAP dual-targeted CAR-T cells can also activate T cells when co-incubated with fibroblasts, but because the intracellular segment of FAP lacks the CD3 zeta chain, its intensity is significantly weaker than the activation of Meso CAR structure.

3. Determination of the killing capacity of CAR-T cells in vitro.

Using Cell Trace^TMCFSE Cell promotion Kit (Thermo) and Cell Trace^TMFar Red Cell Proliferation Kit (Thermo) stained effector and target cells, respectively. Effector cells (e.g., control T cells, FAP CAR T, Meso CAR T cells, or Meso-FAP dual CAR T cells) and target cells (e.g., tumor fibroblasts, SKOV3-MSLN, and SKOV3 cells) were added to 12-well plates at an effective target ratio of 1:1, 2:1, 4:1, 8:1, with the number of target cells per well being 1 × 10⁶Control wells are added which are effective only in cells or target cells. Wherein SKOV3-MSLN and tumor fibroblast are target cells, and SKOV3 is negative cellCell, observing flow results, wherein the death or proliferation condition of target cells reflects the in-vitro killing capacity of CAR-T, and the results are shown in figure 6, wherein the Meso CAR T cells and the Meso-FAP double CAR T cells have obvious killing effect on MSLN positive SKOV 3; all T cells have no specific killing effect on MSLN and FAP expression negative SKOV3 cells; the FAP CAR T and Meso-FAP double CAR T cells have a certain killing effect on fibroplast, but compared with specific killing, the effect is obviously weaker, and the indirect killing effect induced by the fact that T cells are activated and further secrete cytokines due to antigen combination is supposed to be achieved.

4. CAR-T cells treated the human SKOV3 ovarian cancer subcutaneous model.

A MSLN positive human ovarian cancer model enriched in tumor-associated fibroblasts. In the existing research, a subcutaneous tumor model is established by adopting a mode of directly inoculating SKOV3 cells, the influence of tumor interstitial cells on treatment is ignored, female NOD.Cg-PrkdcscilIL 2rgtmWjl/SzJ (NSG) mice with the age of 8-12 weeks are selected, and 1 × 10 mice are inoculated⁶MSLN positive SKOV3 cells and 1X 10⁶Fibroblast 1:1, mixed inoculation under the skin to build a human ovarian cancer model. When the tumor grows to 50-100mm ³5 × 106 different kinds of T cells were injected intravenously; the same dose was injected 2 days later. Tumor size was measured every 2 days from the 1 st CAR-T injection. As is evident from the results of fig. 7, Meso-FAP dual-targeted CAR-T cells have significantly better therapeutic efficacy than FAP CAR T and Meso CAR T cells.

From the above test results, it can be seen that: the double-targeting CAR-T cells of mesothelin and FAP can effectively recognize and kill MSLN positive tumor cells, can be activated (release cytokines and do not directly kill) by FAP positive cells (figure 2), and has in-vivo curative effect obviously superior to single-targeting CAR-T cells of MSLN and better than control T cells. The invention constructs a double-target chimeric antigen receptor containing MSLN and FAP, and experiments prove that the double-target chimeric antigen receptor has in vitro anti-tumor effect, higher anti-tumor activity is verified in vivo experiments, and a more effective treatment method is provided for solid tumors rich in stroma.

Sequence listing

<110> Sichuan university Hospital in western China

<120> Dual-target chimeric antigen receptor targeting mesothelin and FAP simultaneously and use thereof

<130> A200356K (preface)

<141> 2020-05-27

<150> 201911291969.8

<151> 2019-12-16

<160> 36

<170> SIPOSequenceListing 1.0

<210> 1

<211> 568

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Ala Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu

20 25 30

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

35 40 45

Thr Phe Thr Glu Asn Ile Ile His Trp Val Lys Gln Arg Ser Gly Gln

50 55 60

Gly Leu Glu Trp Ile Gly Trp Phe His Pro Gly Ser Gly Ser Ile Lys

65 70 75 80

Tyr Asn Glu Lys Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser

85 90 95

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

100 105 110

Val Tyr Phe Cys Ala Arg His Gly Gly Thr Gly Arg Gly Ala Met Asp

115 120 125

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Leu Met Thr

145 150 155 160

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

165 170 175

Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser Ala Tyr Ser Tyr

180 185 190

Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

195 200 205

Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Pro Arg Phe Ser Gly

210 215 220

Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu

225 230 235 240

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg Glu Leu Pro Tyr

245 250 255

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Asp Pro Ala Glu Pro

260 265 270

Lys Ser Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro

275 280 285

Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

290 295 300

Leu Met Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

305 310 315 320

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

325 330 335

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

340 345 350

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

355 360 365

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

370 375 380

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

385 390 395 400

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

405 410 415

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

420 425 430

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

435 440 445

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

450 455 460

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

465 470 475 480

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

485 490 495

Leu Ser Pro Gly Lys Lys Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

Glu Glu Glu Gly Gly Cys Glu Leu

565

<210> 2

<211> 1707

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggagacag atacactgct gctgtgggtg ctgctgctgt gggtgcccgg cagcaccggc 60

gctcaagtgc agctgaaaca gtccggcgcc gaactggtga aacccggcgc cagcgtgaag 120

ctgagctgca agaccagcgg atacaccttc accgagaaca tcatccactg ggtgaagcag 180

aggagcggcc aaggcctcga gtggattgga tggttccatc ccggcagcgg cagcatcaag 240

tacaatgaga agaaagacaa ggccacactg accgctgaca agagctcctc caccgtgtat 300

atggagctgt ctagactgac aagcgaggac agcgccgtgt acttctgcgc tagacatgga 360

ggcaccggaa gaggagccat ggactattgg ggccaaggca caagcgtcac agtgtcctcc 420

ggcggaggag gcagcggagg aggcggaagc ggcggcggag gcagcgacat tctgatgacc 480

caatcccccg cctccagcgt ggtgtccctc tccggacaga gagctaccat cagctgtaga 540

gctagcaagt ccgtgagcac atccgcctac agctacatgc actggtacca gcagaaaccc 600

ggacaacctc ccaagctgct gatctatctg gccagcaatc tcgagagcgg cgtgcctcct 660

agattttccg gctccggcag cggcacagac ttcaccctca acatccatcc cgtggaggaa 720

gaggatgccg ccacatacta ctgccagcat tctagagagc tgccctacac ctttggaggc 780

ggcaccaaac tggagatcaa agatcccgcc gagcctaaga gccccgacaa gacccacaca 840

tgtcctcctt gtcccgcccc tcccgtggct ggacctagcg tgtttctgtt cccccccaag 900

cccaaggaca cactgatgat cgctagaaca cccgaggtga catgcgtcgt ggtggacgtc 960

tcccacgaag accccgaggt gaagttcaac tggtacgtgg acggagtgga agtccacaac 1020

gccaaaacaa agcctagaga ggagcagtac aactccacat atagagtggt gtccgtgctg 1080

acagtgctcc accaagactg gctgaacggc aaggagtaca agtgcaaggt cagcaacaag 1140

gctctgcccg cccctattga gaagaccatc agcaaggcca agggccagcc tagagaaccc 1200

caagtctata ccctccctcc ctctagagac gagctcacca agaatcaagt gtctctgaca 1260

tgtctggtca agggctttta ccctagcgac attgctgtgg agtgggagtc caacggccag 1320

cccgagaaca actacaagac cacacccccc gtgctggata gcgacggcag cttctttctg 1380

tattccaagc tgaccgtgga taagagcaga tggcagcaag gcaacgtgtt tagctgtagc 1440

gtcatgcacg aggctctgca caaccactat acccagaagt ctctgtctct gtcccccggc 1500

aagaagatct acatctgggc tcctctggct ggaacatgcg gcgtgctgct gctgtctctg 1560

gtcatcacac tgtactgtaa gaggggcaga aagaagctgc tctacatctt caagcagccc 1620

ttcatgagac ccgtccagac aacccaagag gaggacggat gcagctgcag atttcccgaa 1680

gaggaggagg gaggatgcga gctctga 1707

<210> 3

<211> 527

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

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 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu

20 25 30

Val Thr Pro Ser Gln Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp

35 40 45

Ser Val Ser Ser Asn Ser Ala Thr Trp Asn Trp Ile Arg Gln Ser Pro

50 55 60

Ser Arg Gly Leu Glu Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp

65 70 75 80

Tyr Asn Asp Tyr Ala Val Ser Val Lys Ser Arg Met Ser Ile Asn Pro

85 90 95

Asp Thr Ser Lys Asn Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro

100 105 110

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Met Met Thr Tyr Tyr

115 120 125

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

130 135 140

Gly Ile Leu Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

145 150 155 160

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

165 170 175

Ser Pro Gly Ala Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Ile

180 185 190

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

195 200 205

Pro Pro Gln Tyr Leu Leu Asn Tyr Lys Ser Asp Ser Asp Lys Gln Gln

210 215 220

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

225 230 235 240

Asn Ala Gly Val Leu Leu Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala

245 250 255

Asp Tyr Tyr Cys Met Ile Trp His Ser Ser Ala Ala Val Phe Gly Gly

260 265 270

Gly Thr Gln Leu Thr Val Leu Thr Thr Thr Pro Ala Pro Arg Pro Pro

275 280 285

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

290 295 300

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

305 310 315 320

Phe Ala Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Ala Asp Ala Pro Ala Tyr Gln 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

Tyr Arg His Gln Ala Leu Pro Pro Arg Gly Ser Gly Ala Thr Asn Phe

500 505 510

Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro

515 520 525

<210> 4

<211> 1504

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atggctttac ccgttaccgc tctcttatta cctctggctc tgctgctgca tgccgccaga 60

cccgcagcag agcggacccg gtttagtgac ccctagccag actttaagcc tcacatgcgc 120

catttccggc gacagcgtca gcagcaacag cgccacttgg aactggattc gtcaatcccc 180

ctctcgtggt ttagaatggc tgggaaggac ctactatcgt agcaagtggt acaacgacta 240

cgctgtgagc gtgaagtctc gtatgtccat caaccccgac acaagcaaga accagttctc 300

tttacagctc aactccgtca cacccgaaga caccgctgtc tactattgcg ctcgtggcat 360

gatgacatac tattacggca tggacgtgtg gggacaaggc actaccgtga cagtgtcctc 420

cggcatcctc ggttccggcg gcggcggcag cggcggcgga ggctccggcg gtggcggcag 480

ccaacccgtt ctgacacaga gcagcagcct cagcgcttcc cccggagctt ccgcttcttt 540

aacttgtaca ttacgtagcg gaattaacgt gggcccctac agaatctact ggtatcaaca 600

gaagcccgga agcccccccc agtatttact caactacaag agcgactccg acaaacagca 660

aggtagcggc gtgccctctc gtttttccgg ctccaaggat gcctccgcca atgccggcgt 720

gctgctgatt agcggtttaa ggtccgagga tgaggctgat tactactgta tgatttggca 780

tagcagcgcc gccgtgtttg gcggcggaac acagctgacc gtgctgacca caacacccgc 840

tcccagacct cctacccccg ctcctaccat cgccagccag cctctgtctt taagacccga 900

ggcttgtaga cccgctgctg gcggagctgt gcacaccaga ggtttagact ttgcttgtga 960

tttctgggtg ctcgtggtcg tgggaggcgt tttagcttgc tacagcttat tagtgaccgt 1020

ggcctttatc attttctggg tgaggtccaa aagatctcgt ctgctgcact ccgactacat 1080

gaatatgaca cctaggaggc ccggtcccac tcgtaagcac taccagcctt acgccccccc 1140

cagagacttc gccgcttatc gttccgtgag agtgaagttt tctcgttccg ccgatgctcc 1200

cgcttaccag caaggtcaga accagctgta caacgaatta aatctgggtc gtagagagga 1260

gtacgacgtt ttagataaga ggaggggcag agatcccgaa atgggcggca agcccagaag 1320

gaaaaaccct caagagggcc tctacaatga gctgcagaag gacaagatgg ccgaggctta 1380

ttccgaaatc ggcatgaaag gcgagagaag aaggggcaag ggacatgatg gtttatacca 1440

aggtttaagc acagccacca aagacaccta cgacgcctac agacatcaag ctctcccccc 1500

caga 1504

<210> 5

<211> 22

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Ser Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro

20

<210> 6

<211> 66

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

agcggcagcg gcgagggaag aggaagcctg ctgacctgcg gcgatgtgga ggagaatccc 60

ggcccc 66

<210> 7

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Arg Arg Lys Arg

1

<210> 8

<211> 12

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

aggaggaaga ga 12

<210> 9

<211> 21

<212> PRT

<213> Artificial Sequence (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

20

<210> 10

<211> 63

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atggctttac ccgttaccgc tctcttatta cctctggctc tgctgctgca tgccgccaga 60

ccc 63

<210> 11

<211> 258

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Thr Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Ser Ala Thr Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Val Ser Val Lys Ser Arg Met Ser Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Gly Met Met Thr Tyr Tyr Tyr Gly Met Asp Val

100 105 110

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Ile Leu Gly Ser

115 120 125

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

130 135 140

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

145 150 155 160

Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Ile Asn Val Gly Pro Tyr

165 170 175

Arg Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Gln Tyr Leu

180 185 190

Leu Asn Tyr Lys Ser Asp Ser Asp Lys Gln Gln Gly Ser Gly Val Pro

195 200 205

Ser Arg Phe Ser Gly Ser Lys Asp Ala Ser Ala Asn Ala Gly Val Leu

210 215 220

Leu Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Met

225 230 235 240

Ile Trp His Ser Ser Ala Ala Val Phe Gly Gly Gly Thr Gln Leu Thr

245 250 255

Val Leu

<210> 12

<211> 774

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

caagttcaac tgcagcagag cggacccggt ttagtgaccc ctagccagac tttaagcctc 60

acatgcgcca tttccggcga cagcgtcagc agcaacagcg ccacttggaa ctggattcgt 120

caatccccct ctcgtggttt agaatggctg ggaaggacct actatcgtag caagtggtac 180

aacgactacg ctgtgagcgt gaagtctcgt atgtccatca accccgacac aagcaagaac 240

cagttctctt tacagctcaa ctccgtcaca cccgaagaca ccgctgtcta ctattgcgct 300

cgtggcatga tgacatacta ttacggcatg gacgtgtggg gacaaggcac taccgtgaca 360

gtgtcctccg gcatcctcgg ttccggcggc ggcggcagcg gcggcggagg ctccggcggt 420

ggcggcagcc aacccgttct gacacagagc agcagcctca gcgcttcccc cggagcttcc 480

gcttctttaa cttgtacatt acgtagcgga attaacgtgg gcccctacag aatctactgg 540

tatcaacaga agcccggaag ccccccccag tatttactca actacaagag cgactccgac 600

aaacagcaag gtagcggcgt gccctctcgt ttttccggct ccaaggatgc ctccgccaat 660

gccggcgtgc tgctgattag cggtttaagg tccgaggatg aggctgatta ctactgtatg 720

atttggcata gcagcgccgc cgtgtttggc ggcggaacac agctgaccgt gctg 774

<210> 13

<211> 45

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

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

35 40 45

<210> 14

<211> 135

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

accacaacac ccgctcccag acctcctacc cccgctccta ccatcgccag ccagcctctg 60

tctttaagac ccgaggcttg tagacccgct gctggcggag ctgtgcacac cagaggttta 120

gactttgctt gtgat 135

<210> 15

<211> 68

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

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

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser

20 25 30

Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly

35 40 45

Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala

50 55 60

Ala Tyr Arg Ser

65

<210> 16

<211> 204

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ttctgggtgc tcgtggtcgt gggaggcgtt ttagcttgct acagcttatt agtgaccgtg 60

gcctttatca ttttctgggt gaggtccaaa agatctcgtc tgctgcactc cgactacatg 120

aatatgacac ctaggaggcc cggtcccact cgtaagcact accagcctta cgcccccccc 180

agagacttcg ccgcttatcg ttcc 204

<210> 17

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Arg

<210> 18

<211> 339

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gtgagagtga agttttctcg ttccgccgat gctcccgctt accagcaagg tcagaaccag 60

ctgtacaacg aattaaatct gggtcgtaga gaggagtacg acgttttaga taagaggagg 120

ggcagagatc ccgaaatggg cggcaagccc agaaggaaaa accctcaaga gggcctctac 180

aatgagctgc agaaggacaa gatggccgag gcttattccg aaatcggcat gaaaggcgag 240

agaagaaggg gcaagggaca tgatggttta taccaaggtt taagcacagc caccaaagac 300

acctacgacg cctacagaca tcaagctctc ccccccaga 339

<210> 19

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

Arg Arg Lys Arg Ser Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr

1 5 10 15

Cys Gly Asp Val Glu Glu Asn Pro Gly Pro

20 25

<210> 20

<211> 78

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

agcggcagcg gcgagggaag aggaagcctg ctgacctgcg gcgatgtgga ggagaatccc 60

ggccccagga ggaagaga 78

<210> 21

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Ala

20

<210> 22

<211> 63

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

atggagacag atacactgct gctgtgggtg ctgctgctgt gggtgcccgg cagcaccggc 60

gct 63

<210> 23

<211> 246

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

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

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Asn

20 25 30

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

35 40 45

Gly Trp Phe His Pro Gly Ser Gly Ser Ile Lys Tyr Asn Glu Lys Lys

50 55 60

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

65 70 75 80

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

85 90 95

Arg His Gly Gly Thr Gly Arg Gly Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

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

130 135 140

Ser Val Val Ser Leu Ser Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala

145 150 155 160

Ser Lys Ser Val Ser Thr Ser Ala Tyr Ser Tyr Met His Trp Tyr Gln

165 170 175

Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Asn

180 185 190

Leu Glu Ser Gly Val Pro Pro Arg Phe Ser Gly Ser Gly Ser Gly Thr

195 200 205

Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr

210 215 220

Tyr Tyr Cys Gln His Ser Arg Glu Leu Pro Tyr Thr Phe Gly Gly Gly

225 230 235 240

Thr Lys Leu Glu Ile Lys

245

<210> 24

<211> 738

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

caagtgcagc tgaaacagtc cggcgccgaa ctggtgaaac ccggcgccag cgtgaagctg 60

agctgcaaga ccagcggata caccttcacc gagaacatca tccactgggt gaagcagagg 120

agcggccaag gcctcgagtg gattggatgg ttccatcccg gcagcggcag catcaagtac 180

aatgagaaga aagacaaggc cacactgacc gctgacaaga gctcctccac cgtgtatatg 240

gagctgtcta gactgacaag cgaggacagc gccgtgtact tctgcgctag acatggaggc 300

accggaagag gagccatgga ctattggggc caaggcacaa gcgtcacagt gtcctccggc 360

ggaggaggca gcggaggagg cggaagcggc ggcggaggca gcgacattct gatgacccaa 420

tcccccgcct ccagcgtggt gtccctctcc ggacagagag ctaccatcag ctgtagagct 480

agcaagtccg tgagcacatc cgcctacagc tacatgcact ggtaccagca gaaacccgga 540

caacctccca agctgctgat ctatctggcc agcaatctcg agagcggcgt gcctcctaga 600

ttttccggct ccggcagcgg cacagacttc accctcaaca tccatcccgt ggaggaagag 660

gatgccgcca catactactg ccagcattct agagagctgc cctacacctt tggaggcggc 720

accaaactgg agatcaaa 738

<210> 25

<211> 235

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 25

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

1 5 10 15

Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro

20 25 30

Lys Pro Lys Asp Thr Leu Met Ile Ala Arg Thr Pro Glu Val Thr Cys

35 40 45

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

50 55 60

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

65 70 75 80

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

85 90 95

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

100 105 110

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

115 120 125

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

130 135 140

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

145 150 155 160

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

165 170 175

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

180 185 190

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

195 200 205

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

210 215 220

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Lys

225 230 235

<210> 26

<211> 705

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

gatcccgccg agcctaagag ccccgacaag acccacacat gtcctccttg tcccgcccct 60

cccgtggctg gacctagcgt gtttctgttc ccccccaagc ccaaggacac actgatgatc 120

gctagaacac ccgaggtgac atgcgtcgtg gtggacgtct cccacgaaga ccccgaggtg 180

aagttcaact ggtacgtgga cggagtggaa gtccacaacg ccaaaacaaa gcctagagag 240

gagcagtaca actccacata tagagtggtg tccgtgctga cagtgctcca ccaagactgg 300

ctgaacggca aggagtacaa gtgcaaggtc agcaacaagg ctctgcccgc ccctattgag 360

aagaccatca gcaaggccaa gggccagcct agagaacccc aagtctatac cctccctccc 420

tctagagacg agctcaccaa gaatcaagtg tctctgacat gtctggtcaa gggcttttac 480

cctagcgaca ttgctgtgga gtgggagtcc aacggccagc ccgagaacaa ctacaagacc 540

acaccccccg tgctggatag cgacggcagc ttctttctgt attccaagct gaccgtggat 600

aagagcagat ggcagcaagg caacgtgttt agctgtagcg tcatgcacga ggctctgcac 660

aaccactata cccagaagtc tctgtctctg tcccccggca agaag 705

<210> 27

<211> 24

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

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

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 28

<211> 72

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

atctacatct gggctcctct ggctggaaca tgcggcgtgc tgctgctgtc tctggtcatc 60

acactgtact gt 72

<210> 29

<211> 42

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 29

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> 30

<211> 126

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 31

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

aggtttaaac tacggatggc tttacccgtt accgctc 37

<210> 32

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

atgactagtc ccgggtcatc tgggggggag agcttga 37

<210> 33

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

aggtttaaac tacggatgga gacagataca ctgctgc 37

<210> 34

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

atgactagtc ccgggtcaca gttcacatcc tccttct 37

<210> 35

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

aggtttaaac tacggatggc tttacccgtt accgctc 37

<210> 36

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

atgactagtc ccgggtcaca gttcacatcc tccttct 37

Claims

1. A dual-target chimeric antigen receptor targeting mesothelin and FAP simultaneously, characterized in that: the double-target chimeric antigen receptor is formed by connecting a chimeric antigen receptor 1 for recognizing mesothelin and a chimeric antigen receptor 2 for recognizing FAP through a connecting peptide; the amino acid sequence of the chimeric antigen receptor 1 is SEQ ID NO: 3 is shown in the specification; the amino acid sequence of the chimeric antigen receptor 2 is SEQ ID NO: 1 is shown in the specification; the connecting peptide is Furin-T2A.

2. The dual-target chimeric antigen receptor of claim 1, wherein: the composition of the chimeric antigen receptor 2 is as follows: a signal peptide, scFv of human FAP, a human IgG1 Fc segment, a CD8 transmembrane region, and a 4-1BB costimulatory molecule peptide segment.

3. The dual-target chimeric antigen receptor of claim 1, wherein: the composition of the chimeric antigen receptor 1 is as follows: signal peptide, scFv of human mesothelin, CD8a hinge region, CD28 transmembrane region, CD3 zeta binding domain.

4. The dual-target chimeric antigen receptor of claim 1, wherein: the chimeric antigen receptor 1 and the chimeric antigen receptor 2 are expressed together by a vector.

5. An expression vector which simultaneously expresses the dual-target chimeric antigen receptor of any one of claims 1 to 4.

6. A host cell comprising the expression vector of claim 5.

7. Use of the dual-target chimeric antigen receptor of any one of claims 1 to 4, the expression vector of claim 5, the host cell of claim 6 for the preparation of a medicament for the prevention or treatment of a malignant tumor; the malignant tumor is solid tumor.

8. Use according to claim 7, characterized in that: the malignant tumor is at least one of lung cancer, hepatocellular carcinoma, colon cancer, rectal cancer, breast cancer, ovarian cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, renal cancer, pancreatic cancer or prostate cancer.