CN108342363B

CN108342363B - Transgenic lymphocytes co-expressing anti-MSLN chimeric antigen receptor and immune checkpoint inhibitory molecules and uses thereof

Info

Publication number: CN108342363B
Application number: CN201710056395.0A
Authority: CN
Inventors: 严勇朝; 朱益林; 陈思毅
Original assignee: Beijing Marino Biotechnology Pty Ltd
Current assignee: Beijing Marino Biotechnology Pty Ltd
Priority date: 2017-01-25
Filing date: 2017-01-25
Publication date: 2021-02-12
Anticipated expiration: 2037-01-25
Also published as: WO2018137295A1; CN108342363A

Abstract

The present invention provides a transgenic lymphocyte with a silenced cellular immune checkpoint and expression of a chimeric antigen receptor, a construct, and a therapeutic composition for treating cancer. Wherein the chimeric antigen receptor comprises: an extracellular region comprising a heavy chain variable region and a light chain variable region of a single chain antibody that specifically recognizes the antigen MSLN; a transmembrane region attached to the extracellular region and embedded in the cell membrane of the T lymphocyte; an intracellular domain associated with said transmembrane region, said intracellular domain comprising an intracellular segment of CD28 and a CD3 zeta chain. The transgenic lymphocyte has the characteristic of resisting tumor cell mediated immunosuppression, obviously enhances the killing capability to tumor cells, and particularly has obvious directional killing effect on tumors with high expression of MSLN.

Description

Transgenic lymphocytes co-expressing anti-MSLN chimeric antigen receptor and immune checkpoint inhibitory molecules and uses thereof

Technical Field

The present invention relates to the field of biomedicine, in particular, the invention relates to a T lymphocyte, a lentivirus, a transgenic lymphocyte, a construct, a therapeutic composition for treating cancer and a method for increasing lymphocyte activity.

Background

Mesothelin (MSLN) is a differentiated antigen whose expression in normal human tissues is restricted to only the pleural, pericardial and peritoneal lining of mesothelial cells. However, interstitial element is highly expressed in a variety of human cancer tissues, including almost all mesothelioma and pancreatic cancers, and about 70% ovarian cancer and about 50% lung adenocarcinoma, as well as other cancers, such as cholangiocarcinoma, gastric cancer, intestinal cancer, esophageal cancer, breast cancer. The mesenchyme gene encodes a 71kDa precursor protein which is then processed into an abscission fragment of 31kDa, known as Megakaryocyte Promoting Factor (MPF), and a protein fragment of 40kDa, known as metaxin, which is immobilized on the cell membrane by the anchoring action of glycosyl-phosphatidylinositol (GPI).

Taking mesothelioma as an example, mesothelioma is classified into pleural mesothelioma and peritoneal mesothelioma, pleural mesothelioma is a primary pleural tumor and is classified into a limited type (mostly benign) and a diffuse type (both malignant), wherein the diffuse type malignant mesothelioma is one of tumors with the worst breast prognosis. Peritoneal mesothelioma refers to a tumor that originates in peritoneal mesothelial cells. The clinical manifestations are not characteristic, and the common symptoms and signs are: abdominal pain, ascites, abdominal distension, abdominal mass, etc. There is currently no effective method for the treatment of malignant pleural mesothelioma. The treatment methods include palliative treatment, surgical treatment, chemotherapy, radiotherapy and the like, and the radical pleuropneumectomy is generally advocated for the stage I patients with relatively limited tumors. For patients in stage II, III and IV, radical surgery is not meaningful, and only palliative surgery is performed. In fact, most patients are already in stage II by the time the disease is clearly diagnosed. The rapidly increasing pleural effusion often causes severe dyspnea in patients, and palliative surgery only temporarily improves the quality of life of these late stage patients, but not a radical cure.

Therefore, the development of a treatment method aiming at tumors with high expression of metaplanin is particularly urgent.

Disclosure of Invention

The present application is based on the discovery and recognition by the inventors of the following facts and problems:

however, metaplanin is highly expressed in a variety of human cancer tissues, including almost all mesothelioma and pancreatic cancers, and about 70% ovarian cancer and about 50% lung adenocarcinoma, as well as other cancers, such as cholangiocarcinoma, gastric cancer, intestinal cancer, esophageal cancer, breast cancer. Thus, matrixins represent a highly attractive target in the field of tumor immunotherapy.

Based on the above findings, the inventors propose a construct carrying a nucleic acid molecule that silences a cellular immune checkpoint and a nucleic acid molecule encoding a chimeric antigen receptor that specifically binds the antigen MSLN, and a transgenic lymphocyte formed upon introduction of this construct. Therefore, the constructs and transgenic lymphocytes proposed by the invention can be used for the immunotherapy of adoptive T cells of tumors, particularly interstitial-positive tumors; the transgenic lymphocyte provided by the invention has strong specific killing capacity on tumors with high expression of mesothelin, and has weaker killing on mesothelial cells with normal MSLN expression level.

In a first aspect of the invention, the invention features a T lymphocyte. According to an embodiment of the invention, the cellular immune checkpoint of the T lymphocyte is silenced; and expressing a chimeric antigen receptor, wherein the chimeric antigen receptor comprises: an extracellular region comprising a heavy chain variable region and a light chain variable region of a single chain antibody that specifically recognizes the antigen MSLN; a transmembrane region attached to the extracellular region and embedded in the cell membrane of the T lymphocyte; an intracellular domain associated with said transmembrane region, said intracellular domain comprising an intracellular segment of CD28 and a CD3 zeta chain. Wherein the cellular immune checkpoint comprises at least one of a cell surface or intracellular immune checkpoint. According to the embodiment of the invention, the T lymphocyte has the characteristic of resisting tumor cell mediated immunosuppression, the proliferation capacity in vitro and the proliferation and survival capacity in a tumor patient are obviously improved, the killing capacity on the tumor cell is obviously enhanced, and particularly, the T lymphocyte has an obvious directional killing effect on the tumor cell with high expression of MSLN.

In a second aspect of the invention, a lentivirus is presented. According to an embodiment of the invention, the lentivirus carries the following nucleic acid molecules: a nucleic acid molecule encoding a chimeric antigen receptor having the amino acid sequence of SEQ ID NO: 1, and the nucleic acid molecule encoding the chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO: 2; and a nucleic acid molecule that silences a cellular immune checkpoint, the nucleic acid molecule that silences a cellular immune checkpoint having a nucleotide sequence selected from the group consisting of SEQ ID NO: 3 to 135.

MVLLVTSLLLCELPHPAFLLIPDIQAQVQLVQSGAEVKRPGASVQVSCRASGYSINTYYMQWVRQAPGAGLEWMGVINPSGVTSYAQKFQGRVTLTNDTSTNTVYMQLNSLTSADTAVYYCARWALWGDFGMDVWGKGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSTLSASIGDRVTITCRASEGIYHWLAWYQQKPGKAPKLLIYKASSLASGAPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQYSNYPLTFGGGTKLEIKRASFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO：1)。

ATGGTTCTGCTGGTGACATCTCTCCTGCTCTGTGAACTGCCTCATCCCGCTTTTCTGCTCATTCCCGACATTCAGGCTCAAGTCCAACTGGTCCAAAGTGGTGCTGAAGTCAAACGCCCGGGTGCCTCCGTCCAAGTCTCCTGCCGTGCCTCTGGCTACTCGATTAACACCTATTACATGCAGTGGGTCCGTCAAGCACCGGGTGCAGGTCTGGAATGGATGGGTGTCATCAATCCGTCCGGCGTGACCTCATATGCGCAGAAATTTCAAGGTCGCGTTACCCTGACGAACGATACCAGCACGAATACCGTCTACATGCAGCTGAACTCTCTGACGAGTGCAGACACCGCGGTGTATTACTGCGCACGTTGGGCACTGTGGGGCGATTTCGGCATGGATGTTTGGGGCAAAGGTACGCTGGTGACCGTTAGCTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTAGTGGCGGTGGCGGTTCTGATATTCAGATGACGCAAAGCCCGTCTACCCTGAGTGCCTCCATTGGTGACCGTGTTACGATCACCTGTCGCGCATCCGAAGGCATCTATCATTGGCTGGCTTGGTACCAGCAAAAACCGGGTAAAGCGCCGAAACTGCTGATCTATAAAGCAAGTTCCCTGGCATCGGGTGCTCCGAGCCGCTTTTCAGGTTCGGGTAGCGGCACCGATTTCACGCTGACCATCTCATCGCTGCAGCCGGACGATTTCGCTACCTACTACTGCCAACAATACTCAAACTACCCGCTGACCTTCGGTGGAGGGACCAAGCTGGAGATCAAACGTGCTAGCTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAACAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA(SEQ ID NO：2)。

GGCCAGGATGGTTCTTAGACT(SEQ ID NO：3)。

GGATTTCCAGTGGCGAGAGAA(SEQ ID NO：4)。

GCCUGUGUUCUCUGUGGACUAUG(SEQ ID NO：5)。

GGUGCUGCUAGUCUGGGUCCUGG(SEQ ID NO：6)。

GACAGAGAGAAGGGCAGAAGUGC(SEQ ID NO：7)。

CAGCUUCUCCAACACAUCGGAGA(SEQ ID NO：8)。

CCGUGUCACACAACUGCCCAACG(SEQ ID NO：9)。

UAUGCCACCAUUGUCUUUCCUAG(SEQ ID NO：10)。

UGCUAAACUGGUACCGCAUGAGC(SEQ ID NO：11)。

GUGACAGAGAGAAGGGCAGAAGU(SEQ ID NO：12)。

CUGAGGAUGGACACUGCUCUUGG(SEQ ID NO：13)。

AUCGGAGAGCUUCGUGCUAAACU(SEQ ID NO：14)。

GGCAACGGAACCCAGATTTAT(SEQ ID NO：15)。

GGAACCCAAATTACGTGTACT(SEQ ID NO：16)。

GAACCCAAATTACGTGTACTA(SEQ ID NO：17)。

GGGAGAAGACTATATTGTACA(SEQ ID NO：18)。

GACGTTTATAGCCGAAATGAT(SEQ ID NO：19)。

GACACTAATACACCAGGTAGA(SEQ ID NO：20)。

ACCUCACUAUCCAAGGACUGAGG(SEQ ID NO：21)。

AUGAGUUGACCUUCCUAGAUGAU(SEQ ID NO：22)。

GGGGAAUGAGUUGACCUUCCUAG(SEQ ID NO：23)。

CUCUGGAUCCUUGCAGCAGUUAG(SEQ ID NO：24)。

CUCCUCUGGAUCCUUGCAGCAGU(SEQ ID NO：25)。

UUUGUGUGUGAGUAUGCAUCUCC(SEQ ID NO：26)。

CACCUCCAGUGGAAAUCAAGUGA(SEQ ID NO：27)。

CACGGGACUCUACAUCUGCAAGG(SEQ ID NO：28)。

UUCUGACUUCCUCCUCUGGAUCC(SEQ ID NO：29)。

AAGUCUGUGCGGCAACCUACAUG(SEQ ID NO：30)。

GGTCGGTCAGAATGCCTATCT(SEQ ID NO：31)。

GCCAATGACTTACGGGACTCT(SEQ ID NO：32)。

GCAGAGGGAATTCGCTCAGAA(SEQ ID NO：33)。

GGAAATTCGGGCACATCATAT(SEQ ID NO：34)。

GATTAAGAGATGACTGGACTA(SEQ ID NO：35)。

GAGATGACTGGACTAGGTCTA(SEQ ID NO：36)。

AGGAAAUUCGGGCACAUCAUAUG(SEQ ID NO：37)。

GACUGAUGAAAGGGAUGUGAAUU(SEQ ID NO：38)。

GCCACUGAUUUUCAAAGAGAUCU(SEQ ID NO：39)。

AGCAGAGUUUUCCCAUUUUCAGA(SEQ ID NO：40)。

AACUUAAACAGGCAUGUCAUUGC(SEQ ID NO：41)。

UUCAGAAGAUAAUGACUCACAUG(SEQ ID NO：42)。

GCCUCUGUAUUUAAGCCAACAGA(SEQ ID NO：43)。

UGCUCAUGUGAUUGUGGAGUAGA(SEQ ID NO：44)。

AUGUUUUCACAUCUUCCCUUUGA(SEQ ID NO：45)。

GAGAGACUUCACUGCAGCCUUUC(SEQ ID NO：46)。

GATTGCCTCTACTCATCACTA(SEQ ID NO：47)。

UCCUAAUGACAAUGGGUCAUACC(SEQ ID NO：48)。

AAGACAUUGCCUGCCAUGCUUGG(SEQ ID NO：49)。

GUCAUACCGCUGUUCUGCAAAUU(SEQ ID NO：50)。

CUCCUGUAUAGUUUACUUCCUUU(SEQ ID NO：51)。

UACCGCUGUUCUGCAAAUUUUCA(SEQ ID NO：52)。

AAAACAAACCAGGCAUUGUUUAU(SEQ ID NO：53)。

AACUAGAAUGCCCUGUGAAAUAC(SEQ ID NO：54)。

GUGACUUGGUGCAAGCUCAAUGG(SEQ ID NO：55)。

AUCCAUGGGAAAGAAUCAUGUGA(SEQ ID NO：56)。

UGGUGCAAGCUCAAUGGAACAAC(SEQ ID NO：57)。

GCTGCTCACCCTTATGAACCT(SEQ ID NO：58)。

AGGACAUGGUGGUGGACGAGUGC(SEQ ID NO：59)。

UGCUCUUCCUGCACGAUAUCAGU(SEQ ID NO：60)。

ACCUCUACUGGUUCCUGUACAUC(SEQ ID NO：61)。

CCCUCCAACUCUGCUCCUCUAGG(SEQ ID NO：62)。

CCCUGAGUGGACAGUCGUCUUCG(SEQ ID NO：63)。

CUGCUCCAGGGAAGCUUCUAUGG(SEQ ID NO：64)。

CGCUCAAGGUCCUGUAUGCCACC(SEQ ID NO：65)。

GAGUUCACCAAGCUCAACAUUUA(SEQ ID NO：66)。

UGCUGCUGCUCACCCUUAUGAAC(SEQ ID NO：67)。

CCCAUCUCCGUGCUCUUCUUUGA(SEQ ID NO：68)。

GGGACATCGTCGAGCTATTCA(SEQ ID NO：69)。

GGACATCGTCGAGCTATTCAT(SEQ ID NO：70)。

GCCAATGTCACCGTGGATAAT(SEQ ID NO：71)。

GTCATCTGTGGCAGTATATCA(SEQ ID NO：72)。

GGATGTAGAGTAGTGTTAGAT(SEQ ID NO：73)。

GGCAAAGTTAAGACCATCAAT(SEQ ID NO：74)。

GACCAAATCCACGCTCAATTA(SEQ ID NO：75)。

UACUGCUUAAAUCUUCCAUCAGC(SEQ ID NO：76)。

GACUGAGAAGUUCUGUCUGAUUU(SEQ ID NO：77)。

CUGUUUCAUCACCCAAACAUACU(SEQ ID NO：78)。

GAAGAUCCUCCCACAUCACUAAA(SEQ ID NO：79)。

UAGACCAAGGUAAAAGUGGAACA(SEQ ID NO：80)。

AAGAGGUUUUUAUCUGAGCUUGA(SEQ ID NO：81)。

UACCUGCACAACGUUCAACCAUG(SEQ ID NO：82)。

GCCUGGAUUCAUGUCUCUCAUUU(SEQ ID NO：83)。

CCCUCGGAAUUUCUCUGCCAAGC(SEQ ID NO：84)。

UGCUGAAGAUCCUCCCACAUCAC(SEQ ID NO：85)。

GCACCTCCTACCTCTTCATGT(SEQ ID NO：86)。

CGCACUUCCGCACAUUCCGUUCG(SEQ ID NO：87)。

GGGGAGGGUCUCUGGCUUUAUUU(SEQ ID NO：88)。

CAGCAUUAACUGGGAUGCCGUGU(SEQ ID NO：89)。

CCAGGACCUGAACUCGCACCUCC(SEQ ID NO：90)。

UACAUAUACCCAGUAUCUUUGCA(SEQ ID NO：91)。

GCCGACAAUGCAGUCUCCACAGC(SEQ ID NO：92)。

CCCCUGGUUGUUGUAGCAGCUUA(SEQ ID NO：93)。

CUGCUGUGCAGAAUCCUAUUUUA(SEQ ID NO：94)。

UGGGAUGCCGUGUUAUUUUGUUA(SEQ ID NO：95)。

UCGCACCUCCUACCUCUUCAUGU(SEQ ID NO：96)。

GCGGAAAGCTGTGAAGATACG(SEQ ID NO：97)。

ACAAAGCCCTCATCGACAGAA(SEQ ID NO：98)。

ATGCCACTTCTCAGTACATGT(SEQ ID NO：99)。

GTGGACTTCAGTACAACTCAC(SEQ ID NO：100)。

GTGGAATTTACTTGCCTCTCC(SEQ ID NO：101)。

GTTGGATGAAGCTAACTTACC(SEQ ID NO：102)。

ACTGGGAAGACGTGTAACTCT(SEQ ID NO：103)。

AAGGAATTGCATCCAAGGTAT(SEQ ID NO：104)。

GGAATTGCATCCAAGGTATAC(SEQ ID NO：105)。

GGATGAGACTGGCAATGGTCA(SEQ ID NO：106)。

UCCUCAGUUUCGGGAGAUCAUCC(SEQ ID NO：107)。

GAGUCUCUCAAAUCUCAGGAAUU(SEQ ID NO：108)。

AGCUCUAGUCCUUUUUGUGUAAU(SEQ ID NO：109)。

CACUGGAAAUGUUCAGAACUUGC(SEQ ID NO：110)。

AUGAUGAAUGGGACAAUCUUAUC(SEQ ID NO：111)。

CACACUGUGUUUCAUCGAGUACA(SEQ ID NO：112)。

GCAGAACCAUCCAUGGACUGUGA(SEQ ID NO：113)。

AAAGAUGUGGCCUUUUGUGAUGG(SEQ ID NO：114)。

UUCAGAACUUGCCAGUUUUGUCC(SEQ ID NO：115)。

AUGAGACUGGCAAUGGUCACAGG(SEQ ID NO：116)。

GTCAATTCCAGGGAGATAACT(SEQ ID NO：117)。

GCCTGGAAGCAATGGCTCTAA(SEQ ID NO：118)。

GCACCAAACCCGGAAGCTATA(SEQ ID NO：119)。

GTTGCACTCGATTGGGACAGT(SEQ ID NO：120)。

GGATTATGTGAACCTACACCT(SEQ ID NO：121)。

GGAATCACAGCGAGTTCAAAT(SEQ ID NO：122)。

GCAAGGCATAGTCTCATTGAA(SEQ ID NO：123)。

GGTGAAGAGAGCCTTAGAGAT(SEQ ID NO：124)。

GTGAAGAGAGCCTTAGAGATA(SEQ ID NO：125)。

AGGAGCUAAGGUCUUUUCCAAUG(SEQ ID NO：126)。

AUGUCGAUGCAAAAAUUGCAAAA(SEQ ID NO：127)。

GUCACAUGCUGGCAGAAAUCAAA(SEQ ID NO：128)。

UCCAGGUUACAUGGCAUUUCUCA(SEQ ID NO：129)。

UUGAACUUUGAACCUGUGAAAUG(SEQ ID NO：130)。

UCCACAUCAACAGCUAAAUCAUU(SEQ ID NO：131)。

AUGCUGGCAGAAAUCAAAGCAAU(SEQ ID NO：132)。

UGCAGAGAAUGACAAAGAUGUCA(SEQ ID NO：133)。

GGCAGAACUCACCAGUCACAUCA(SEQ ID NO：134)。

UCGGUCCUGUGAUAAUGGUCACU(SEQ ID NO：135)。

According to the embodiment of the invention, the transgenic lymphocyte obtained by introducing the lentivirus into the lymphocyte has the characteristic of resisting tumor cell-mediated immunosuppression, the proliferation capability in vitro and the proliferation and survival capability in a tumor patient are obviously enhanced, the killing capability on the tumor cell is obviously enhanced, and particularly, the transgenic lymphocyte has an obvious directional killing effect on the tumor cell with high expression of MSLN.

In a third aspect of the invention, a lentivirus is provided. According to an embodiment of the invention, the lentivirus carries a polypeptide comprising SEQ ID NO: 136. 137, 138, 139, 140 or 141.

ATGGTTCTGCTGGTGACATCTCTCCTGCTCTGTGAACTGCCTCATCCCGCTTTTCTGCTCATTCCCGACATTCAGGCTCAAGTCCAACTGGTCCAAAGTGGTGCTGAAGTCAAACGCCCGGGTGCCTCCGTCCAAGTCTCCTGCCGTGCCTCTGGCTACTCGATTAACACCTATTACATGCAGTGGGTCCGTCAAGCACCGGGTGCAGGTCTGGAATGGATGGGTGTCATCAATCCGTCCGGCGTGACCTCATATGCGCAGAAATTTCAAGGTCGCGTTACCCTGACGAACGATACCAGCACGAATACCGTCTACATGCAGCTGAACTCTCTGACGAGTGCAGACACCGCGGTGTATTACTGCGCACGTTGGGCACTGTGGGGCGATTTCGGCATGGATGTTTGGGGCAAAGGTACGCTGGTGACCGTTAGCTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTAGTGGCGGTGGCGGTTCTGATATTCAGATGACGCAAAGCCCGTCTACCCTGAGTGCCTCCATTGGTGACCGTGTTACGATCACCTGTCGCGCATCCGAAGGCATCTATCATTGGCTGGCTTGGTACCAGCAAAAACCGGGTAAAGCGCCGAAACTGCTGATCTATAAAGCAAGTTCCCTGGCATCGGGTGCTCCGAGCCGCTTTTCAGGTTCGGGTAGCGGCACCGATTTCACGCTGACCATCTCATCGCTGCAGCCGGACGATTTCGCTACCTACTACTGCCAACAATACTCAAACTACCCGCTGACCTTCGGTGGAGGGACCAAGCTGGAGATCAAACGTGCTAGCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACCAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGGTAATCCTACTGCgtcgaGCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGGTCGACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCTCCCCAGGCGCAGATCAAAGAGAGTTCAAGAGACTCTCTTTGATCTGCGCCTTTTTT(SEQ ID NO：136)。

ATGGTTCTGCTGGTGACATCTCTCCTGCTCTGTGAACTGCCTCATCCCGCTTTTCTGCTCATTCCCGACATTCAGGCTCAAGTCCAACTGGTCCAAAGTGGTGCTGAAGTCAAACGCCCGGGTGCCTCCGTCCAAGTCTCCTGCCGTGCCTCTGGCTACTCGATTAACACCTATTACATGCAGTGGGTCCGTCAAGCACCGGGTGCAGGTCTGGAATGGATGGGTGTCATCAATCCGTCCGGCGTGACCTCATATGCGCAGAAATTTCAAGGTCGCGTTACCCTGACGAACGATACCAGCACGAATACCGTCTACATGCAGCTGAACTCTCTGACGAGTGCAGACACCGCGGTGTATTACTGCGCACGTTGGGCACTGTGGGGCGATTTCGGCATGGATGTTTGGGGCAAAGGTACGCTGGTGACCGTTAGCTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTAGTGGCGGTGGCGGTTCTGATATTCAGATGACGCAAAGCCCGTCTACCCTGAGTGCCTCCATTGGTGACCGTGTTACGATCACCTGTCGCGCATCCGAAGGCATCTATCATTGGCTGGCTTGGTACCAGCAAAAACCGGGTAAAGCGCCGAAACTGCTGATCTATAAAGCAAGTTCCCTGGCATCGGGTGCTCCGAGCCGCTTTTCAGGTTCGGGTAGCGGCACCGATTTCACGCTGACCATCTCATCGCTGCAGCCGGACGATTTCGCTACCTACTACTGCCAACAATACTCAAACTACCCGCTGACCTTCGGTGGAGGGACCAAGCTGGAGATCAAACGTGCTAGCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACCAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGGTAATCCTACTGCgtcgaGCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGGTCGACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCTCCCCAACACAGACGCCATGATTTGCTTCAAGAGAGCAAATCATGGCGTCTGTGTTTTTTT(SEQ ID NO：137)。

ATGGTTCTGCTGGTGACATCTCTCCTGCTCTGTGAACTGCCTCATCCCGCTTTTCTGCTCATTCCCGACATTCAGGCTCAAGTCCAACTGGTCCAAAGTGGTGCTGAAGTCAAACGCCCGGGTGCCTCCGTCCAAGTCTCCTGCCGTGCCTCTGGCTACTCGATTAACACCTATTACATGCAGTGGGTCCGTCAAGCACCGGGTGCAGGTCTGGAATGGATGGGTGTCATCAATCCGTCCGGCGTGACCTCATATGCGCAGAAATTTCAAGGTCGCGTTACCCTGACGAACGATACCAGCACGAATACCGTCTACATGCAGCTGAACTCTCTGACGAGTGCAGACACCGCGGTGTATTACTGCGCACGTTGGGCACTGTGGGGCGATTTCGGCATGGATGTTTGGGGCAAAGGTACGCTGGTGACCGTTAGCTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTAGTGGCGGTGGCGGTTCTGATATTCAGATGACGCAAAGCCCGTCTACCCTGAGTGCCTCCATTGGTGACCGTGTTACGATCACCTGTCGCGCATCCGAAGGCATCTATCATTGGCTGGCTTGGTACCAGCAAAAACCGGGTAAAGCGCCGAAACTGCTGATCTATAAAGCAAGTTCCCTGGCATCGGGTGCTCCGAGCCGCTTTTCAGGTTCGGGTAGCGGCACCGATTTCACGCTGACCATCTCATCGCTGCAGCCGGACGATTTCGCTACCTACTACTGCCAACAATACTCAAACTACCCGCTGACCTTCGGTGGAGGGACCAAGCTGGAGATCAAACGTGCTAGCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACCAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGGTAATCCTACTGCgtcgaGCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGGTCGACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCTCCCCGCATCACTTGGGATTAATATTCAAGAGATATTAATCCCAAGTGATGCTTTTT(SEQ ID NO：138)。

ATGGTTCTGCTGGTGACATCTCTCCTGCTCTGTGAACTGCCTCATCCCGCTTTTCTGCTCATTCCCGACATTCAGGCTCAAGTCCAACTGGTCCAAAGTGGTGCTGAAGTCAAACGCCCGGGTGCCTCCGTCCAAGTCTCCTGCCGTGCCTCTGGCTACTCGATTAACACCTATTACATGCAGTGGGTCCGTCAAGCACCGGGTGCAGGTCTGGAATGGATGGGTGTCATCAATCCGTCCGGCGTGACCTCATATGCGCAGAAATTTCAAGGTCGCGTTACCCTGACGAACGATACCAGCACGAATACCGTCTACATGCAGCTGAACTCTCTGACGAGTGCAGACACCGCGGTGTATTACTGCGCACGTTGGGCACTGTGGGGCGATTTCGGCATGGATGTTTGGGGCAAAGGTACGCTGGTGACCGTTAGCTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTAGTGGCGGTGGCGGTTCTGATATTCAGATGACGCAAAGCCCGTCTACCCTGAGTGCCTCCATTGGTGACCGTGTTACGATCACCTGTCGCGCATCCGAAGGCATCTATCATTGGCTGGCTTGGTACCAGCAAAAACCGGGTAAAGCGCCGAAACTGCTGATCTATAAAGCAAGTTCCCTGGCATCGGGTGCTCCGAGCCGCTTTTCAGGTTCGGGTAGCGGCACCGATTTCACGCTGACCATCTCATCGCTGCAGCCGGACGATTTCGCTACCTACTACTGCCAACAATACTCAAACTACCCGCTGACCTTCGGTGGAGGGACCAAGCTGGAGATCAAACGTGCTAGCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACCAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGGTAATCCTACTGCgtcgaGCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGGTCGACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCTCCCCAGGCGCAGATCAAAGAGAGTTCAAGAGACTCTCTTTGATCTGCGCCTTTTTTAGCTATCGATAGCTAAAAAAACACAGACGCCATGATTTGCTCTCTTGAAGCAAATCATGGCGTCTGTGTTGGGGAAGATCTGTGGTCTCATACAGAACTTATAAGATTCCCAAATCCAAAGACATTTCACGTTTATGGTGATTTCCCAGAACACATAGCGACATGCAAATATTGCAGGGCGCCACTCCCCTGTCCCTCACAGCCATCTTCCTGCCAGGGCGCACGCGCGCTGGGTGTTCCCGCCTAGTGACACTGGGCCCGCGATTCCTTGGAGCGGGTTGATGACGTCAGCGTTCGAATTGTCGAC(SEQ ID NO：139)。

ATGGTTCTGCTGGTGACATCTCTCCTGCTCTGTGAACTGCCTCATCCCGCTTTTCTGCTCATTCCCGACATTCAGGCTCAAGTCCAACTGGTCCAAAGTGGTGCTGAAGTCAAACGCCCGGGTGCCTCCGTCCAAGTCTCCTGCCGTGCCTCTGGCTACTCGATTAACACCTATTACATGCAGTGGGTCCGTCAAGCACCGGGTGCAGGTCTGGAATGGATGGGTGTCATCAATCCGTCCGGCGTGACCTCATATGCGCAGAAATTTCAAGGTCGCGTTACCCTGACGAACGATACCAGCACGAATACCGTCTACATGCAGCTGAACTCTCTGACGAGTGCAGACACCGCGGTGTATTACTGCGCACGTTGGGCACTGTGGGGCGATTTCGGCATGGATGTTTGGGGCAAAGGTACGCTGGTGACCGTTAGCTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTAGTGGCGGTGGCGGTTCTGATATTCAGATGACGCAAAGCCCGTCTACCCTGAGTGCCTCCATTGGTGACCGTGTTACGATCACCTGTCGCGCATCCGAAGGCATCTATCATTGGCTGGCTTGGTACCAGCAAAAACCGGGTAAAGCGCCGAAACTGCTGATCTATAAAGCAAGTTCCCTGGCATCGGGTGCTCCGAGCCGCTTTTCAGGTTCGGGTAGCGGCACCGATTTCACGCTGACCATCTCATCGCTGCAGCCGGACGATTTCGCTACCTACTACTGCCAACAATACTCAAACTACCCGCTGACCTTCGGTGGAGGGACCAAGCTGGAGATCAAACGTGCTAGCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACCAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGGTAATCCTACTGCgtcgaGCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGGTCGACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCTCCCCAGGCGCAGATCAAAGAGAGTTCAAGAGACTCTCTTTGATCTGCGCCTTTTTTAGCTATCGATAGCTAAAAAGCATCACTTGGGATTAATATCTCTTGAATATTAATCCCAAGTGATGCGGGGAAGATCTGTGGTCTCATACAGAACTTATAAGATTCCCAAATCCAAAGACATTTCACGTTTATGGTGATTTCCCAGAACACATAGCGACATGCAAATATTGCAGGGCGCCACTCCCCTGTCCCTCACAGCCATCTTCCTGCCAGGGCGCACGCGCGCTGGGTGTTCCCGCCTAGTGACACTGGGCCCGCGATTCCTTGGAGCGGGTTGATGACGTCAGCGTTCGAATTGTCGAC(SEQ ID NO：140)。

ATGGTTCTGCTGGTGACATCTCTCCTGCTCTGTGAACTGCCTCATCCCGCTTTTCTGCTCATTCCCGACATTCAGGCTCAAGTCCAACTGGTCCAAAGTGGTGCTGAAGTCAAACGCCCGGGTGCCTCCGTCCAAGTCTCCTGCCGTGCCTCTGGCTACTCGATTAACACCTATTACATGCAGTGGGTCCGTCAAGCACCGGGTGCAGGTCTGGAATGGATGGGTGTCATCAATCCGTCCGGCGTGACCTCATATGCGCAGAAATTTCAAGGTCGCGTTACCCTGACGAACGATACCAGCACGAATACCGTCTACATGCAGCTGAACTCTCTGACGAGTGCAGACACCGCGGTGTATTACTGCGCACGTTGGGCACTGTGGGGCGATTTCGGCATGGATGTTTGGGGCAAAGGTACGCTGGTGACCGTTAGCTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTAGTGGCGGTGGCGGTTCTGATATTCAGATGACGCAAAGCCCGTCTACCCTGAGTGCCTCCATTGGTGACCGTGTTACGATCACCTGTCGCGCATCCGAAGGCATCTATCATTGGCTGGCTTGGTACCAGCAAAAACCGGGTAAAGCGCCGAAACTGCTGATCTATAAAGCAAGTTCCCTGGCATCGGGTGCTCCGAGCCGCTTTTCAGGTTCGGGTAGCGGCACCGATTTCACGCTGACCATCTCATCGCTGCAGCCGGACGATTTCGCTACCTACTACTGCCAACAATACTCAAACTACCCGCTGACCTTCGGTGGAGGGACCAAGCTGGAGATCAAACGTGCTAGCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACCAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGGTAATCCTACTGCGTCGAGCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGGTCGACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCTCCCCAGGCGCAGATCAAAGAGAGTTCAAGAGACTCTCTTTGATCTGCGCCTTTTTTAGCTATCGATAGCTAAAAAGCCTAGAGAAGTTTCAGGGAATCTCTTGAATTCCCTGAAACTTCTCTAGGCGGGGAAGATCTGTGGTCTCATACAGAACTTATAAGATTCCCAAATCCAAAGACATTTCACGTTTATGGTGATTTCCCAGAACACATAGCGACATGCAAATATTGCAGGGCGCCACTCCCCTGTCCCTCACAGCCATCTTCCTGCCAGGGCGCACGCGCGCTGGGTGTTCCCGCCTAGTGACACTGGGCCCGCGATTCCTTGGAGCGGGTTGATGACGTCAGCGTTCGAATTGTCGAC(SEQ ID NO：141)。

In a fourth aspect of the invention, the invention features a transgenic lymphocyte. According to embodiments of the invention, the lymphocyte immune checkpoint is silenced; and expressing a chimeric antigen receptor comprising: an extracellular region comprising a heavy chain variable region and a light chain variable region of an antibody, the antibody capable of specifically binding to a tumor antigen; a transmembrane region; and an intracellular region comprising an intracellular segment of an immune co-stimulatory molecule, wherein the antibody is a single chain antibody and the tumor antigen is MSLN. The inventor surprisingly found that the cell immune check point is silenced and the in vitro proliferation capacity of lymphocyte of the anti-MSLN chimeric antigen receptor, the proliferation and survival capacity in tumor patients and the specific killing capacity to tumor cells in tumor patients are greatly improved, and particularly the cell immune check point has obvious directional killing effect to the tumor cells with high expression of MSLN.

According to an embodiment of the present invention, the above-mentioned transgenic lymphocyte may further have at least one of the following additional technical features:

according to an embodiment of the invention, the lymphocyte cellular immune checkpoint is independently selected from at least one of CTLA4, PD1, TIM3, BTLA, LAG-3, IRAK-M, SOCS1, a20, CBL-B. Wherein CTLA4, PD1, TIM3, BTLA and LAG-3 are cell surface immune check points, and IRAK-M, SOCS1, A20 and CBL-B are intracellular immune check points. The immune checkpoint of the embodiments of the invention has the effect of negatively regulating and attenuating cellular immune responses, which through specific binding to the corresponding ligand on tumor cells, results in down-regulation of T-lymphocyte proliferative responses, decreased secretion of cytokines, and T-cell anergy or apoptosis. According to embodiments of the present invention, successful silencing of immune checkpoints on the cell surface or within the cell according to embodiments of the present invention further enhances the efficacy of the transgenic lymphocytes against tumor-mediated immunosuppression, and further enhances the in vitro expansion and proliferation and viability of the transgenic lymphocytes in tumor patients, and the targeted killing of tumor cells.

According to embodiments of the invention, the lymphocyte cell surface immune checkpoint is silenced by at least one of shRNA, antisense nucleic acid, ribozyme, dominant negative mutation, CRISPR, and zinc finger nuclease. According to the embodiment of the invention, successful silencing of the cell immune checkpoint of the embodiment of the invention can significantly improve the property of the lymphocyte of the embodiment of the invention against tumor-mediated immunosuppression, and further improve the targeted killing effect of the transgenic lymphocyte on the tumor cell.

According to an embodiment of the invention, the intracellular segment of the immune co-stimulatory molecule is independently selected from at least one of 4-1BB, OX-40, CD40L, CD27, CD30, CD28 and derivatives thereof. The expression of the intracellular segment of the immune co-stimulatory molecule and the silencing combination of the cell immune check point have the functions of forward regulation and enhancing cell immune response, so that the directional killing effect of the proliferation of the transgenic lymphocyte on the tumor is more obvious; the combination of the expression of the intracellular segment of the immune co-stimulatory molecule and the silencing of the cell immune checkpoint in the embodiment of the invention enables the proliferation capacity of the transgenic lymphocyte and the directional killing effect on the tumor to be more obvious.

According to an embodiment of the invention, the lymphocyte immune checkpoint is CTLA4, PD1, CBL-B. Wherein CTLA4 and PD1 are cell surface immune checkpoints, and CBL-B is an intracellular immune checkpoint. According to the embodiment of the invention, the lymphocyte cell surface immune checkpoint CTLA4 or PD1 is silenced, or the lymphocyte intracellular immune checkpoint is CBL-B silenced, so that the combination of the expression of PD1 or CTLA4 molecules and corresponding ligands of PD-L1 and PD-L2 or CD80 and CD86 is prevented, the T lymphocyte can be effectively inhibited from incapacity or apoptosis, or the T cell receptor signaling is enhanced through the silencing of the CBL-B, the proliferation and the survival capacity of the transgenic lymphocyte in a tumor patient can be further improved, and the targeted killing effect on the tumor is more remarkable.

According to embodiments of the invention, silencing of the immune checkpoint in the lymphocyte is achieved by shRNA. According to embodiments of the invention, the shRNAs of the embodiments carry shRNAs that specifically silence at least one immune checkpoint on the cell surface or within the cell, have the functions of efficiently and specifically silencing at least one immune checkpoint on the cell surface or within the cell, successfully silence the cellular immune checkpoint, i.e., successful silencing of immune checkpoints on the cell surface or within the cell, prevents specific binding of the immune checkpoint to the corresponding ligand, thereby effectively inhibiting the negative regulation and control mechanism of T lymphocyte incapability or apoptosis and the like of the immune inspection point, further, the proliferation and the viability of the transgenic lymphocyte in the tumor patient are further improved, and the transgenic lymphocyte is matched with the antigen targeting of the chimeric antigen receptor, the effect of the directional killing effect of the transgenic lymphocyte on the tumor is more obvious.

According to an embodiment of the invention, the intracellular segment of an immune co-stimulatory molecule is an intracellular segment of 4-1BB or CD 28. The intracellular segment of the immune co-stimulatory molecule of the chimeric antigen receptor of the transgenic lymphocytes of the invention is that of CD28 or 4-1 BB. According to embodiments of the invention, the intracellular segment of the immune co-stimulatory molecule is that of CD28 or 4-1BB, further enhancing the targeted killing effect of the transgenic lymphocytes of embodiments of the invention.

According to an embodiment of the invention, said lymphocyte is CD3⁺T lymphocytes or natural killer cells or natural killer T cells. The lymphocytes of the embodiments of the invention have silent cellular immune checkpoints and simultaneously express an antigen-specific chimeric antigen receptor, such as those of the inventionThe MSLN antigen-specific chimeric antigen receptor of the embodiment has stronger targeting of the cell immune killing effect of the lymphocytes, further improves the proliferation and survival capacity in the body of a tumor patient, and has more obvious effect of directionally killing the tumor.

In a ninth aspect of the invention, the invention provides a construct. According to an embodiment of the invention, the construct comprises: a first nucleic acid molecule encoding a chimeric antigen receptor; and a second nucleic acid molecule that silences a cellular immune checkpoint. Wherein the cellular immune checkpoint and the chimeric antigen receptor are as described above. According to the embodiment of the invention, after the construct of the embodiment of the invention is successfully introduced into the lymphocyte of the embodiment of the invention, at least one immune checkpoint on the surface of the cell or in the cell can be effectively silenced, and the chimeric antigen receptor expressing the specificity of the antigen can be effectively expressed, so that the lymphocyte of the embodiment of the invention has more remarkable directional killing effect on tumor cells, especially tumor cells highly expressing MSLN.

According to an embodiment of the invention, the above-mentioned construct may further comprise at least one of the following additional technical features:

an embodiment according to the invention is characterized in that said first nucleic acid molecule and said second nucleic acid molecule are arranged to silence a cellular immune checkpoint in said lymphocytes as described above and to express said chimeric antigen receptor. According to the embodiment of the present invention, the lymphocyte successfully provided with the first nucleic acid molecule and the second nucleic acid molecule can successfully silence at least one of the immune check points on the cell surface or in the cell of the lymphocyte, and successfully express the antigen-specific chimeric antigen receptor on the lymphocyte surface, such as the MSLN-specific chimeric antigen receptor of the embodiment of the present invention, which has stronger lethality and stronger tumor killing effect.

According to an embodiment of the invention, the construct further comprises: a first promoter operably linked to the first nucleic acid molecule; and a second promoter operably linked to the second nucleic acid molecule. According to the embodiment of the invention, the introduction of the first promoter and the second promoter enables the first nucleic acid molecule and the second nucleic acid molecule to be independently expressed respectively, so that the biological effect of the antigen targeting of the chimeric antigen receptor is effectively ensured, and the cell immune check point is effectively silenced, so that the lymphocyte targeting effect of the embodiment of the invention is stronger, the killing effect on tumors is more remarkable, and especially the targeted killing effect on tumor cells with high expression of MSLN is more remarkable.

According to an embodiment of the invention, the first promoter and the second promoter are each independently selected from the group consisting of U6, H1, CMV, EF-1, LTR, RSV promoters. According to the embodiment of the invention, the promoter has the characteristics of high starting efficiency and strong specificity, so that the high-efficiency silencing of a cell immune check point and the high-efficiency expression of a chimeric antigen receptor are ensured, the in-vitro proliferation capacity of the lymphocyte, the proliferation and the survival capacity of a tumor patient are greatly improved, and the targeted killing effect on the tumor is more obvious.

According to an embodiment of the invention, the vector of the construct is a non-pathogenic viral vector. The introduction of the non-pathogenic virus vector greatly improves the replication and amplification efficiency of the construct in lymphocytes, thereby greatly improving the silence of cell immune check points and the high-efficiency expression of chimeric antigen receptors in the lymphocytes, greatly improving the in vitro proliferation capacity of the lymphocytes and the proliferation and survival capacity in tumor patients, further enhancing the targeting effect of the lymphocytes and having more remarkable killing effect on the tumor cells.

According to an embodiment of the invention, the viral vector comprises at least one selected from a retroviral vector, a lentiviral vector or an adeno-associated viral vector. In the virus packaging and infection process, the virus vector of the embodiment of the invention has wide virus infection range, can infect terminal differentiated cells and cells in a division stage, the genome of the virus vector can be integrated into a host chromosome and can be dissociated outside the host chromosome, thus realizing broad-spectrum and high-efficiency infection efficiency, and cell immune check points are efficiently silenced and chimeric antigen receptors are efficiently expressed in lymphocytes, so that the in-vitro proliferation capacity, the proliferation and survival capacity in tumor patients are greatly improved, the targeting effect of the lymphocytes is further enhanced, and the targeted killing effect on tumor cells, particularly tumor cells with high expression of MSLN, is more obvious.

In a tenth aspect of the invention, the invention provides a method of preparing a T lymphocyte or a transgenic lymphocyte as hereinbefore described. According to an embodiment of the invention, the method comprises: introducing the construct or lentivirus into lymphocytes or T lymphocytes. The construct or the lentivirus is successfully introduced into the lymphocyte or the T lymphocyte, so that the cell immunity of the lymphocyte is silenced and the expression of a chimeric antigen receptor is realized, the proliferation of the transgenic lymphocyte or the T lymphocyte prepared by the preparation method of the embodiment of the invention in vivo and in vitro of a tumor patient and the survival capability of the transgenic lymphocyte or the T lymphocyte in vivo of the tumor patient are greatly improved, and the target killing effect of the transgenic lymphocyte or the T lymphocyte on the tumor cell, especially on the tumor cell with high expression of MSLN is stronger.

In an eleventh aspect of the invention, a therapeutic composition for treating cancer is presented. According to an embodiment of the invention, the therapeutic composition comprises: the above construct, lentivirus, T lymphocyte or transgenic lymphocyte. The composition of any one of the therapeutic compositions can realize the silencing of cell surfaces or intracellular immune check points of the transgenic lymphocytes or T lymphocytes and the high-efficiency expression of the chimeric antigen receptor in the transgenic lymphocytes or T lymphocytes, so that the obtained transgenic lymphocytes or T lymphocytes have obvious resistance to tumor cell-mediated immunosuppression, the proliferation in vitro and in vivo of tumor patients and the survival capability in vivo of the tumor patients are greatly improved, and the targeted killing effect of the transgenic lymphocytes or T lymphocytes on the tumor cells is stronger.

According to an embodiment of the present invention, the above-mentioned therapeutic composition may further comprise at least one of the following additional technical features:

according to an embodiment of the present invention, the cancer includes at least one selected from mesothelioma, pancreatic cancer, ovarian cancer, cholangiocarcinoma, lung cancer, stomach cancer, intestinal cancer, esophageal cancer, and breast cancer. The tumor cells have the specificity and high expression of the MSLN, the treatment composition provided by the embodiment of the invention can silence the immune check points on the surfaces of the lymphocytes or in the cells and efficiently express the antigen-specific chimeric antigen receptor, for example, the MSLN antigen-specific chimeric antigen receptor provided by the embodiment of the invention, the obtained lymphocytes or T lymphocytes have the characteristic of obviously resisting tumor cell-mediated immunosuppression, the survival capability in the microenvironment of the tumor is greatly improved, and the obtained lymphocytes or T lymphocytes have stronger target killing effect on the MSLN-highly-expressed tumor cells.

In a twelfth aspect of the invention, the invention provides a method of increasing the activity of lymphocytes carrying a chimeric antigen receptor, according to an embodiment of the invention, the method comprising: silencing a cellular immune checkpoint of the lymphocyte. Said cellular immune checkpoint, said lymphocyte and said chimeric antigen receptor are as previously defined, and said lymphocyte activity comprises at least one of the ability of said lymphocyte to proliferate in vitro, the ability to proliferate and survive in a tumor patient, and the ability of said lymphocyte to kill directionally in a tumor patient. According to the embodiment of the invention, the cell surface or intracellular immune check point of the lymphocyte is silenced, the lymphocyte is activated, the proliferative reaction is up-regulated, the cytokine secretion is increased, and the anti-apoptosis capacity is enhanced, so that the in vitro amplification, the proliferation in a tumor patient and the survival capability in the tumor patient of the lymphocyte are greatly improved.

According to an embodiment of the present invention, the method for increasing lymphocyte activity may further include at least one of the following additional technical features:

according to an embodiment of the present invention, the tumor includes at least one selected from mesothelioma, pancreatic cancer, ovarian cancer, cholangiocarcinoma, lung cancer, stomach cancer, intestinal cancer, esophageal cancer, and breast cancer. The tumor cells specifically and highly express MSLN. The method for improving the activity of the lymphocytes, provided by the embodiment of the invention, enables the lymphocytes to express the MSLN antigen-specific chimeric antigen receptor and simultaneously silences immune check points of the lymphocytes.

It should be noted that the term "cellular immune checkpoint" as used in the present invention includes both cell surface immune checkpoints, which are membrane proteins on the surface of lymphocytes that interact with ligands expressed on tumor cells and inhibit anti-tumor lymphocyte responses, and intracellular immune checkpoints. An "intracellular immune checkpoint" is an intracellular protein that is a negatively regulated cellular signaling machinery protein that inhibits the anti-tumor lymphocyte response.

Drawings

FIG. 1 is a schematic structural diagram of a chimeric antigen receptor co-expressing MSLN antigen-specific and a lentiviral vector silencing a human cell immune checkpoint in accordance with an embodiment of the invention;

FIG. 2 is a graph of the results of enhanced proliferative capacity of lymphocytes co-expressing a chimeric antigen receptor specific for an MSLN antigen and silencing PD1, according to an embodiment of the invention;

FIG. 3 is a graph of the results of increased interferon- γ secretion of lymphocytes co-expressing a chimeric antigen receptor specific for an MSLN antigen and silencing PD1, in accordance with an embodiment of the invention; and

FIG. 4 is a graph of the results of enhanced ability to kill tumor cells of lymphocytes co-expressing a chimeric antigen receptor specific for the MSLN antigen and silencing PD1, in accordance with an embodiment of the invention.

Detailed Description

The following detailed description of embodiments of the invention is intended to be illustrative, and is not to be construed as limiting the invention.

T lymphocytes or transgenic lymphocytes

In one aspect of the invention, the invention features a T lymphocyte or a transgenic lymphocyte. According to embodiments of the invention, the cellular immune checkpoint of the T lymphocytes of the embodiments of the invention is silenced; and expressing a chimeric antigen receptor, wherein the chimeric antigen receptor comprises: the extracellular region comprises a heavy chain variable region and a light chain variable region of a single-chain antibody, and the single-chain antibody specifically recognizes the antigen MSLN; a transmembrane region which is linked to the extracellular region and is embedded in the cell membrane of the T lymphocyte; the intracellular domain, which is associated with the transmembrane region, and which comprises the intracellular segment of CD28 or 4-1BB, as well as the CD3 zeta chain. Wherein the cellular immune checkpoint comprises an immune checkpoint on the surface of or within a cell. The T lymphocyte or the transgenic lymphocyte cell immune check point of the embodiment of the invention is silenced and jointly expresses the MSLN antigen specific chimeric antigen receptor, the proliferation and the survival capacity of the T lymphocyte or the transgenic lymphocyte in vivo and in vitro of a tumor patient and the killing capacity of the T lymphocyte or the transgenic lymphocyte in vivo of the tumor patient on specific tumor cells are obviously enhanced, and particularly, the specific killing effect on the tumor cells efficiently expressing the MSLN is greatly improved.

Tumors can avoid immune surveillance, shutting down the immune killing response of lymphocytes to them by stimulating the expression of their immunosuppressive receptors; as an immune negative regulatory mechanism, activated Cytotoxic T Lymphocytes (CTLs) also express a regulatory mechanism of negative regulation, i.e. immune checkpoint molecules on the cell surface or within the cell. Programmed cell death 1 receptor (PD1) as embodied herein is expressed on activated CTLs, which interact with programmed death ligand 1(PD-L1) expressed on tumor cells and inhibit anti-tumor T cell responses. Many tumors express PD-L1, and binding of PD-L1 to its ligand PD-1 results in down-regulation of the proliferative response of CTLs, decreased secretion of cytokines, and anergy or apoptosis of T cells. Cytotoxic T lymphocyte antigen 4(CTLA4) of the present embodiments is another key negative regulator of T cells, which can inhibit T cell activation by interacting with ligands B7.1, B7.2(CD80 and CD86) expressed on antigen presenting cells. CBL-B in cytotoxic T lymphocytes of the embodiments of the invention (E3 ubiquitin protein ligase CBL-B) is another key negative regulator in cells that inhibits T cell activity by inhibiting T Cell Receptor (TCR) signaling. Therefore, the immune checkpoints of the T lymphocytes or transgenic lymphocytes of the embodiments of the invention are silenced, and the proliferation and viability of the T lymphocytes or transgenic lymphocytes in the tumor patient are significantly improved.

In addition, according to an embodiment of the present invention, the antibody of the extracellular region of the chimeric antigen receptor is a single chain antibody. The inventors have discovered that single chain antibodies can remove non-specifically reactive competitive surface proteins, while single chain antibodies are more permeable to tumor tissue to increase the therapeutic concentration of the drug. The transgenic lymphocyte of the embodiment of the invention expresses the chimeric antigen receptor of the single-chain antibody, thereby greatly improving the directional killing effect of the transgenic lymphocyte on the targeted tumor cell.

According to some further embodiments of the invention, the binding antigen of the antibody is MSLN. Therefore, the transgenic lymphocyte of the embodiment of the invention has directional killing effect on cells expressing the antigen MSLN, the specific binding effect of the antigen antibody is stronger, and the directional killing effect of the transgenic lymphocyte of the embodiment of the invention on MSLN antigen expressing tumor cells is greatly improved.

According to some further embodiments of the invention, the lymphocyte cellular immune checkpoint comprises a cell surface and an intracellular immune checkpoint, the lymphocyte cellular surface immune checkpoint of embodiments of the invention is independently selected from at least one of CTLA4, PD1, TIM3, BTLA, LAG-3, and the lymphocyte intracellular immune checkpoint is independently selected from at least one of IRAK-M, SOCS1, a20, CBL-B. The molecules can be specifically combined with antigens expressed by tumor cells, inhibit the activation of lymphocytes and promote the incapacity or apoptosis of the lymphocytes, thereby negatively regulating and weakening the cellular immune response. According to the embodiment of the invention, the successful silencing of the cell surface or intracellular immune check points further improves the proliferation and survival capacity of the transgenic lymphocytes in the tumor patients, and further enhances the directional killing effect on the tumor cells.

According to some further embodiments of the invention, the lymphocyte cell surface immune checkpoint is silenced by at least one of shRNA, antisense nucleic acid, ribozyme, dominant negative mutation, zinc finger nuclease, and CRISPR.

The small hairpin RNA or short hairpin RNA (shRNA) is an introduction form of siRNA (small interfering RNA), wherein the siRNA is a small RNA molecule (consisting of 21-25 nucleotides) and is processed by Dicer (enzyme with specific shearing action on double-stranded RNA in RNAase III family); siRNA plays a central role in the RNA silencing pathway, degrading specific messenger RNA (mrna), and post-transcriptional regulation.

The antisense nucleic acid comprises antisense RNA and antisense DNA, wherein the antisense RNA refers to a small molecule RNA or an oligonucleotide fragment which can be completely complementary with mRNA, the antisense DNA refers to a short DNA molecule which can be combined with a sense strand in a gene DNA double strand in a complementary way, and the antisense RNA and the antisense DNA mainly play a role through translation of the mRNA and transcription of the gene DNA; the antisense nucleic acid can prevent ribosome from being combined with mRNA by forming steric hindrance effect through being combined with target mRNA, and can activate endogenous RNA enzyme or ribozyme after being combined with mRNA, so as to degrade mRNA; the antisense DNA binds specifically to the regulatory region of the gene DNA duplex to form a DNA trimer, or binds to the coding region of DNA, terminating the elongation of the mRNA chain being transcribed; antisense nucleic acids also inhibit post-transcriptional mRNA processing modifications such as 5 'capping, 3' tailing, intermediate splicing, and internal base methylation, and prevent transport of mature mRNA from the nucleus into the cytoplasm, and thus antisense RNA is an effective technique for silencing a gene of interest.

The ribozyme is an RNA molecule with catalytic function, is a biocatalyst, can degrade specific mRNA sequence, participates in RNA self-shearing and processing process by catalyzing transphosphorylation and phosphodiester bond hydrolysis reaction, has more stable spatial structure compared with general antisense RNA, is not easy to be attacked by RNA enzyme, and more importantly, can be separated from a hybrid chain after cutting off mRNA, and can be recombined and cut off other mRNA molecules.

The dominant negative mutation means that some signal transduction proteins have no functions after mutation, and can inhibit or block the action of wild type signal transduction proteins in the same cell, and the mutation is mainly realized by forming dimers with the wild type proteins, so that the mutation has high toxicity and can obviously inhibit or block the action of target signal transduction proteins in the cell.

The zinc finger nuclease consists of a DNA recognition domain and a non-specific endonuclease, wherein the DNA recognition domain is formed by connecting a series of Cys2-His2 zinc finger proteins in series (generally 3-4), each zinc finger protein recognizes and is combined with a specific triplet base, the zinc finger proteins form an alpha-beta secondary structure, 16 amino acid residues of an alpha helix determine the DNA binding specificity of the zinc finger, the skeleton structure is conservative, and new DNA binding specificity can be obtained by changing an amino acid introduction sequence determining the DNA binding specificity, so that different amino acid introduction sequences can be designed aiming at different target genes, and the specific silencing of different target genes is realized.

CRISPR (Clustered regularly interspersed short palindromic repeats) is a gene editor, a system used by bacteria to protect themselves against viruses. It can be used to delete, add, activate or repress target genes of other organisms, including target genes in human cells.

CRISPR clusters are a family of specific DNA repeats widely present in the bacterial and archaeal genomes, the sequences of which consist of a Leader (Leader), multiple short highly conserved repeats (Repeat) and multiple spacers (Spacer). The leader region is generally positioned AT the upstream of the CRISPR cluster, is a region rich in AT with the length of 300-500 bp, and is considered to be a promoter sequence of the CRISPR cluster. The length of the repeated sequence region is 21-48 bp, and the repeated sequence region contains a palindromic sequence and can form a hairpin structure. The repeated sequences are separated by a spacer with the length of 26-72 bp. The Spacer region is composed of captured exogenous DNA, and when the exogenous DNA containing the same sequence invades, the exogenous DNA can be recognized by a bacterial organism and is cut to silence the expression of the exogenous DNA, so that the aim of protecting the safety of the bacterial organism is fulfilled. By analyzing the flanking sequences of the CRISPR cluster, a polymorphic family gene exists nearby. The proteins encoded by this family all contain functional domains (with nuclease, helicase, integrase and polymerase activities) that can interact with nucleic acids and work in concert with CRISPR regions, and are therefore named CRISPR associations (CRISPR assocteds), abbreviated as Cas. Cass currently discovered include various types, such as Cas 1-Cas 10. Cas genes and CRISPR (clustered regularly interspaced short palindromic repeats) are evolved together to form a highly conserved system. When bacteria resist the invasion of exogenous DNA such as bacteriophage, CRISPR is transcribed into long RNA precursor (Pre RISPR RNA, Pre-crRNA) under the control of leader region, then processed into a series of short mature crRNA containing conserved repetitive sequence and spacer region, finally recognized and combined to the complementary exogenous DNA sequence to play shearing action. processing of pre-crRNA is involved by Cas9 in the Cas family. Cas9 contains RuvC at the amino terminus and HNH2 unique active sites in the middle of the protein that play a role in crRNA maturation and double-stranded DNA cleavage. At the same time as the transcription of the pre-crRNA, a Trans-activating crRNA (tracrRNA) complementary to its repeat sequence is also transcribed and stimulates Cas9 and double-stranded RNA-specific RNase III nuclease to process the pre-crRNA. After maturation, the crRNA, tracrRNA and Cas9 form a complex that recognizes and binds to the crRNA complementary sequence, then unzips the DNA double strand to form an R-loop, allowing the crRNA to hybridize to the complementary strand, leaving the other strand in a free single-stranded state, then cleaves the crRNA complementary DNA strand by the HNH active site in Cas9, the RuvC active site cleaves the non-complementary strand, and finally introduces a DNA Double Strand Break (DSB). By artificially designing RNA, sgRNA (short guide RNA) with a guiding function can be transformed to guide the Cas9 to cut a target gene of the DNA in a fixed point manner.

In summary, the shRNA, the antisense nucleic acid, the ribozyme, the dominant negative mutation, the CRISPR, and the zinc finger nuclease are effective means for specifically silencing the target gene, and the means for silencing the gene is not particularly limited, and those skilled in the art can select the target gene according to specific experimental objectives and conditions, for example, at least one of the shRNA, the antisense nucleic acid, the ribozyme, the dominant negative mutation, the CRISPR, and the zinc finger nuclease used in the embodiments of the present invention, to achieve specific silencing of the target gene.

According to embodiments of the invention, silencing of lymphocyte cell surface or intracellular immune checkpoints is preferably achieved using shRNA. The siRNA molecules carried by ShRNA are typically a double region of between 10 and 30 base pairs in length. The PD1 or CTLA4 or CBL-B siRNA of the embodiment of the invention is designed to be homologous to the coding region of PD1 or CTLA4 or CBL-B mRNA, and inhibit gene expression through mRNA degradation. The siRNA is associated with a multiple protein complex called the induced RNA silencing complex (RISC), during which the sense strand is cleaved by the enzyme. The activated RISC directs RISC to the corresponding mRNA based on sequence homology; the same nuclease cleavage targets PD1 or CTLA4 or CBL-B mRNA, resulting in silencing of the specific gene PD1 or CTLA4 or CBL-B, inhibiting expression of the specific gene PD1 or CTLA4 or CBL-B. The siRNA is introduced into cells in the form of shRNA (the shRNA comprises a nucleotide siRNA sequence of about 18-23, and then a nucleotide ring with the length of 9-15 and a reverse complement of the siRNA sequence), and the shRNA is designed to better avoid a matching point in a 3' UTR cell gene; proper chain selection is ensured. One single siRNA molecule can be repeatedly applied to the division of multi-targeted mRNA molecules. RNAi (RNA interference) can be induced by introducing synthetic siRNA. According to the embodiment of the invention, the shRNA adopted in the embodiment of the invention has the function of efficiently and specifically silencing the cell surface or intracellular immune check point, and the successful silencing of the cell surface or intracellular immune check point, so that the transgenic lymphocyte has the obvious characteristic of resisting tumor-mediated immunosuppression, the proliferation and the survival capability in a tumor patient are further improved, and the effect of directionally killing the tumor is more obvious.

Further, according to embodiments of the invention, the immune co-stimulatory molecule intracellular segment is independently selected from at least one of 4-1BB, OX-40, CD40L, CD27, CD30, CD28, and derivatives thereof. The combination of the expression of the intracellular segment of the immune co-stimulatory molecule and the silencing of at least one immune check point on the cell surface or in the cell has the functions of forward regulation and enhancing cellular immune response, so that the transgenic lymphocyte has the obvious characteristic of resisting tumor-mediated immunosuppression, the proliferation and the survival capability in a tumor patient are further improved, and the directional killing effect on the tumor with high expression of MSLN is more obvious.

According to some further embodiments of the invention, the lymphocyte cell surface immune checkpoint is preferably CTLA4 or PD1 and the lymphocyte intracellular immune checkpoint is preferably CBL-B. According to the embodiment of the invention, the cell surface immune checkpoint CTLA4 or PD1 of the lymphocyte is silenced or the cell immune checkpoint CBL-B is silenced, so that the transgenic lymphocyte has more remarkable property of resisting tumor-mediated immunosuppression, the proliferation and the survival capacity of the transgenic lymphocyte in a tumor patient are further improved, and the effect of the targeted killing on the tumor is more remarkable.

According to an embodiment of the invention, the lymphocyte of the embodiment of the invention is CD3⁺Lymphocytes or natural killer cells or natural killer T cells. CD3⁺Lymphocytes are total T cells, natural killer cells are one of the immune cells, non-specifically recognize the target cells, and natural killer T cells are a subset of T cells with T cells and natural killer cell receptors. The immune check points in the lymphocytes are silenced and express chimeric antigen receptors, so that the targeted killing performance of the cell immunity of the lymphocytes is stronger, and the killing effect on tumor cells is more obvious.

Lentiviruses or constructs

In another aspect of the invention, the invention features a lentivirus or construct. According to an embodiment of the invention, the lentivirus or construct carries the following nucleic acid molecules: a nucleic acid molecule encoding a chimeric antigen receptor having the amino acid sequence of SEQ ID NO: 1, and the nucleic acid molecule encoding the chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO: 2; and a nucleic acid molecule that silences a cell surface or intracellular immune checkpoint, the nucleic acid molecule that silences a cell surface immune checkpoint having a nucleotide sequence selected from the group consisting of SEQ ID NO: 3-68, and the nucleotide sequence of the nucleic acid molecule that silences an intracellular immune checkpoint is a nucleotide sequence selected from SEQ ID NO: 69 to 135. Wherein, SEQ ID NO: 3-14 are human programmed death receptor 1(PD1) siRNA nucleotide sequences, SEQ ID NO: 15-30 are human cytotoxic T lymphocyte-associated antigen 4(CTLA4) siRNA sequences, SEQ ID NOs: 31-46 are human T cell immunoglobulin mucin molecule 3(TIM3) siRNA sequences, SEQ ID NO: 47-57 are human T lymphocyte attenuation factor (BTLA) siRNA sequences, SEQ ID NO: 58-68 are human lymphocyte activation gene 3 protein (LAG-3) siRNA sequences, SEQ ID NO: 69-85 is a human IRAK-M (interleukin-1 receptor associated kinase 3) siRNA nucleotide sequence, SEQ ID NO: 86-96 are human SOCS1 (cytokine signal transduction inhibitor 1) siRNA sequences, SEQ ID NO: 97-116 are human A20 (tumor necrosis factor-alpha inducing protein A20) siRNA sequences, SEQ ID NO: 117-135 is human CBL-B (E3 ubiquitin protein ligase CBL-B) siRNA sequence, according to the embodiment of the present invention, the lentivirus or the construct of the embodiment of the present invention is introduced into the transgenic lymphocyte obtained from the lymphocyte, the immune checkpoint PD1, CTLA4, TIM3, BTLA, LAG-3 or the intracellular immune checkpoint IRAK-M, SOCS1, a20, CBL-B on the cell surface is specifically silenced and inhibited to express, and the anti-MSLN chimeric antigen receptor is expressed on the cell surface, so that the transgenic lymphocyte of the embodiment of the present invention has significant efficacy of resisting tumor-mediated immune inhibition, enhanced anti-apoptosis ability and proliferation ability, and significantly improved directional killing ability, and the proliferation and survival ability and killing ability of the transgenic lymphocyte of the embodiment of the present invention in vivo and in vitro of tumor patients are greatly improved, especially has obvious specific killing effect on tumor cells with high expression of MSLN.

According to an embodiment of the invention, the lentivirus or construct of an embodiment of the invention carries a polypeptide comprising SEQ ID NO: 136. 137, 138, 139, 140 or 141. Wherein, SEQ ID NO: 136 is a nucleic acid molecule that co-expresses an anti-MSLN chimeric antigen receptor and silenced cell immune checkpoint PD-1 (MSLN-CAR/iPD1), SEQ ID NO: 137 is a nucleic acid molecule that co-expresses an anti-MSLN chimeric antigen receptor and silences the cellular immune checkpoint CBL-B (MSLN-CAR/iCBL-B), SEQ ID NO: 138 is a nucleic acid molecule that co-expresses an anti-MSLN chimeric antigen receptor and silences the cellular immune checkpoint CTLA4 (MSLN-CAR/itla 4), SEQ ID NO: 139 is a nucleic acid molecule that co-expresses an anti-MSLN chimeric antigen receptor, silences the cellular immune checkpoint PD1, and silences another cellular immune checkpoint CBL-B (MSLN-CAR/iPD1-CBL-B), SEQ ID NO: 140 is a nucleic acid molecule that co-expresses an anti-MSLN chimeric antigen receptor, silences a cellular immune checkpoint PD1, and silences another cellular immune checkpoint CTLA4 (MSLN-CAR/iPD1-CTLA4), SEQ ID NO: 141 is a nucleic acid molecule that co-expresses an anti-MSLN chimeric antigen receptor, a first silent cellular immune checkpoint of PD1, and a second silent cellular immune checkpoint of PD1 (MSLN-CAR/iPD1-PD 1). According to the embodiment of the invention, the immune check point PD1 or CTLA4 or CBL-B of the cell of the transgenic lymphocyte obtained by introducing the lentivirus into the lymphocyte is specifically silenced, and the anti-MSLN chimeric antigen receptor is expressed, so that the transgenic lymphocyte has obvious effect of resisting tumor-mediated immunosuppression, the anti-apoptosis capacity and the proliferation capacity of the transgenic lymphocyte are enhanced, and the directional killing capacity of the transgenic lymphocyte is obviously improved, thereby greatly improving the proliferation and survival capacity of the transgenic lymphocyte in vitro and in vivo of a tumor patient and the killing capacity of the transgenic lymphocyte in vivo of the tumor patient, and particularly having obvious effect of specifically killing the tumor cell with high expression of the MSLN.

According to the embodiment of the present invention, the expression of the cell chimeric antigen receptor and the surface or intracellular immune checkpoint shRNA independently is realized by the following method, wherein the expression refers to the expression of protein and the transcription of RNA.

A promoter: a first promoter operably linked to a nucleic acid molecule encoding a chimeric antigen receptor; and a second promoter operably linked to a nucleic acid molecule that silences a cellular immune checkpoint. According to the embodiment of the invention, the adopted first promoter and the adopted second promoter are respectively and independently selected from U6, CMV, H1, EF-1, LTR, RSV promoter, and the introduction of the first promoter and the second promoter, so that the nucleic acid molecule for coding the chimeric antigen receptor and the nucleic acid molecule for silencing the cell immune checkpoint are respectively and independently expressed, the cell surface or the cell immune checkpoint is effectively silenced, the high-efficiency expression of the chimeric antigen receptor is ensured, the survival rate of the lymphocyte in the tumor environment is greatly improved, the targeting effect of the lymphocyte is stronger, and the specific killing effect on the tumor is more obvious.

According to embodiments of the invention, a third promoter independently selected from at least one of U6, CMV, H1, EF-1, LTR, RSV promoters may be further introduced, the third promoter is operably linked to a nucleic acid molecule that silences an immune checkpoint of a cell, the third promoter and the second promoter are different from the nucleic acid molecule that silences the cell to which they are linked, and the third promoter and the second promoter each initiate an shRNA that silences a different immune checkpoint.

By introducing the first promoter, the second promoter or the third promoter, the cell surface or the intracellular immune check point is efficiently silenced, and the chimeric antigen receptor is efficiently expressed on the transgenic lymphocyte membrane of the embodiment of the invention, so that the immune negative regulation of the immune check point is efficiently inhibited, the biological effect of the chimeric antigen receptor is ensured, the survival rate of the lymphocyte in a tumor environment is greatly improved, and the targeted killing effect of the lymphocyte is more remarkable.

Additionally, according to embodiments of the invention, the vector of the construct of the embodiments of the invention is a non-pathogenic viral vector. The nonpathogenic viral vector greatly improves the replication and amplification efficiency of the construct in the lymphocyte, and further greatly improves the proliferation and survival capacity of the lymphocyte in a tumor patient, further enhances the targeting effect of the lymphocyte and has more obvious killing effect on the tumor cell.

According to an embodiment of the invention, the viral vector of the construct of an embodiment of the invention is selected from at least one of a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector. According to the embodiment of the invention, in the virus packaging and infection process, the virus vector of the embodiment of the invention has wide virus infection range, can infect terminal differentiated cells, can infect cells in a division stage, can be integrated into host chromosomes, can be dissociated outside the host chromosomes, and realizes broad-spectrum and high-efficiency infection efficiency, so that immune check points on the surfaces of the cells or in the cells are efficiently silenced, and chimeric antigen receptors are efficiently expressed in lymphocytes.

According to an embodiment of the present invention, taking the construction of a lentiviral vector as an example, the inventors have inserted a nucleic acid of interest into the viral genome at the location of certain viral sequences in order to construct a lentiviral vector, thereby generating a replication-deficient virus. To generate virions, the inventors further constructed a packaging cell line (containing the gag, pol and env genes, but not the LTR and packaging components). The inventors introduced a recombinant plasmid containing the gene of interest, along with the lentiviral LTR and the packaging sequence, into a packaging cell line. The packaging sequence allows the recombinant plasmid RNA transcript to be packaged into viral particles and then secreted into the culture medium. Further, the inventors collected a matrix containing the recombinant lentivirus, selectively concentrated, and used for gene transfer. Slow vectors can infect a variety of cell types, including both dividing and non-dividing cells.

In addition, according to the embodiment of the present invention, the lentivirus of the embodiment of the present invention is a complex lentivirus, and contains other genes with regulatory and structural functions in addition to the common lentivirus genes gag, pol and env. Lentiviral vectors are well known to those skilled in the art and include: human immunodeficiency viruses HIV-1, HIV-2 and simian immunodeficiency virus SIV. The lentivirus vector is generated by multiple attenuation of HIV pathogenic genes, such as complete deletion of genes env, vif, vpr, vpu and nef, so that the lentivirus vector forms a biosafety vector. The recombinant lentiviral vector can infect non-dividing cells, and can be used for gene transfer and nucleic acid sequence expression in vivo and in vitro. For example: in a suitable host cell, two or more vectors with packaging functions (gag, pol, env, rev and tat) are capable of infecting non-dividing cells. Targeting of recombinant viruses is achieved by binding of antibodies or specific ligands (targeting specific cell type receptors) to membrane proteins. At the same time, targeting of the recombinant virus allows the vector to be specifically targeted by inserting an effective sequence (including regulatory regions) into the viral vector, along with another gene encoding a ligand for the receptor on the specific target cell. Various useful lentiviral vectors, as well as vectors produced by various methods and procedures, etc., for altering expression in a cell. According to the embodiment of the invention, the lentiviral vector of the embodiment of the invention can effectively transport and co-express shRNA (a transport form of siRNA), and the small shRNA can effectively inhibit the expression of PD1 or CTLA4 or CBL-B.

In accordance with embodiments of the present invention, adeno-associated viral vectors (AAV) of embodiments of the invention can be constructed using the DNA of one or more of the well-known serological adeno-associated viral vectors. One skilled in the art constructs a suitable adeno-associated viral vector to carry and co-express a small hairpin RNA that inhibits expression of the PD1 or CTLA4 or CBL-B genes.

In addition, embodiments of the present invention also include a minigene. A minigene is meant to refer to a combination of selected nucleotide sequences and necessary associated linking sequences operable to direct the transformation, transcription and/or expression of a gene product in a host cell in vivo or in vitro. The "operably linked" sequences are used to include expression control sequences for the contiguous gene of interest and expression control sequences that function to control the gene of interest in trans or remotely.

In addition, the vectors of the embodiments of the present invention also include conventional control elements that allow transcription, transformation, and/or expression of small hairpin RNAs during cell transfection with plasmid vectors or/and infection of cells with viral vectors. A wide variety of expression control sequences (including native, inducible and/or tissue-specific promoters) may be used. According to an embodiment of the invention, the promoter expressing the shRNA is an RNA polymerase promoter. Also, according to an embodiment of the present invention, the promoter is a RAN polymerase promoter selected from U6, H1, pol I, pol II and pol III. According to an embodiment of the invention, the promoter is a tissue specific promoter. According to an embodiment of the invention, the promoter is an inducible promoter. According to an embodiment of the invention, the promoter is selected from promoters based on the chosen vector. According to an embodiment of the invention, when a lentiviral vector is selected, the promoter is a U6, H1, CMV IE gene, EF-1 α, ubiquitin C, or phosphoglycerate kinase (PGK) promoter. Other conventional expression control sequences include selectable markers or reporter genes, including nucleotide sequences encoding geneticin, hygromycin, ampicillin or puromycin resistance, and the like. Other components of the vector include an origin of replication.

Techniques for constructing vectors are well known to those skilled in the art and include conventional cloning techniques such as shRNA, polymerase chain reaction and any suitable method of providing the desired nucleotide sequence as used in the examples herein.

According to embodiments of the invention, the inventors have constructed viral vectors that co-express small hairpin rna (shrna) (to suppress immune checkpoints) and Chimeric Antigen Receptors (CARs). The small hairpin RNA that delivers siRNA that silence PD1 or CTLA4 or CBL-B and the viral vector or plasmid expressing the Chimeric Antigen Receptor (CAR) of the present embodiments are complexed, and the viral vector or plasmid can be conjugated to polymers or other materials to increase its stability, or to assist in its targeted movement.

Method for preparing transgenic lymphocyte

In another aspect of the invention, the invention provides a method of preparing a T lymphocyte or a transgenic lymphocyte as described above. According to an embodiment of the invention, the method comprises: introducing the construct or lentivirus into lymphocytes or T lymphocytes. The mode of introduction may be selected from the group consisting of induction or viral infection of the host cell. The construct or the lentivirus of the embodiment of the invention is successfully introduced into the lymphocyte or the T lymphocyte, so that the expression of a chimeric antigen receptor aiming at the antigen MSLN and the silencing of a cell surface or an intracellular immune check point of the lymphocyte are realized, the obtained lymphocyte or the T lymphocyte has the obvious effect of resisting tumor-mediated immunosuppression, the in-vivo and in-vitro proliferation of a tumor patient and the in-vivo survival capability of the tumor patient are greatly improved, and the targeted killing effect of the lymphocyte or the T lymphocyte on the tumor cell, particularly on the tumor cell highly expressing the MSLN is stronger.

Therapeutic compositions for the treatment of cancer

In another aspect of the invention, the invention features a therapeutic composition for treating cancer. According to an embodiment of the invention, the therapeutic composition comprises: the above construct, the above lentivirus, the above T lymphocyte or the above transgenic lymphocyte. The composition of any one of the above therapeutic compositions can realize the high-efficiency expression of the antigen MSLN chimeric antigen receptor in the transgenic lymphocyte or T lymphocyte and the silencing of the cell surface or intracellular immune check point of the transgenic lymphocyte or T lymphocyte, so that the obtained transgenic lymphocyte or T lymphocyte is amplified in vitro, the proliferation in a tumor patient and the survival capability in the tumor patient are greatly improved, and the targeted killing effect of the transgenic lymphocyte or T lymphocyte on the MSLN high-expression tumor cell is stronger.

According to embodiments of the invention, therapeutic compositions of embodiments of the invention are provided to a patient for application to a biocompatible solution or to an acceptable pharmaceutical carrier. Various therapeutic compositions are prepared to be suspended or dissolved in a pharmaceutically or physiologically acceptable carrier, such as physiological saline; isotonic saline solution or other more obvious formulations specific to the person in the field. The appropriate carrier will depend to a large extent on the route of administration. Other isotonic sterile injection solutions, both aqueous and anhydrous, and sterile suspensions, both aqueous and anhydrous, are pharmaceutically acceptable carriers.

According to embodiments of the invention, a sufficient number of viral vectors are transduced into the targeted T cells and provide sufficient strength of the transgene, silencing PD1 or CTLA4 or CBL-B and expressing the characteristic anti-MSLN chimeric antigen receptor. The dosage of the therapeutic agent depends primarily on the condition being treated, age, weight, and health of the patient, and may cause variability among patients.

These methods of silencing PD1 or CTLA4 or CBL-B and expressing the unique chimeric antigen receptor for the antigen MSLN are part of a combination therapy. These viral vectors and anti-tumor T cells for adoptive immunotherapy can be performed alone or in combination with other methods of treating cancer. Under appropriate conditions, one method of treatment involves the use of one or more drug therapies.

According to an embodiment of the present invention, the cancer includes at least one selected from mesothelioma, pancreatic cancer, ovarian cancer, cholangiocarcinoma, lung cancer, stomach cancer, intestinal cancer, esophageal cancer, and breast cancer. The following biological effects: the silencing of the cell immune check point and the high-efficiency expression of the combined chimeric antigen receptor in the transgenic lymphocyte or the T lymphocyte greatly improve the survival capability of the obtained lymphocyte or the T lymphocyte in the microenvironment of the tumor, have stronger target killing effect on the tumor cell by the lymphocyte or the T lymphocyte, and particularly have more obvious killing effect on the tumor cell with high expression of MSLN.

Method for increasing lymphocyte activity

In a further aspect of the invention there is provided a method of increasing the activity of a lymphocyte carrying a chimeric antigen receptor, the method comprising, according to an embodiment of the invention, silencing a cellular immune checkpoint in said lymphocyte, the cellular immune checkpoint, the lymphocyte, the chimeric antigen receptor being as hereinbefore defined. According to an embodiment of the present invention, the lymphocyte activity of the embodiment of the present invention includes at least one of the ability of lymphocytes to proliferate in vitro, the ability to proliferate and survive in a tumor patient, and the ability of lymphocytes to kill in a tumor patient. According to embodiments of the invention, the lymphocytes of the embodiments of the invention have silent cellular immune checkpoints, are activated, have upregulated proliferative responses, have increased cytokine secretion, and have enhanced anti-apoptotic capacity. The lymphocyte provided by the embodiment of the invention can be amplified and proliferated in vitro, and the target killing effect on tumor cells is obviously enhanced.

The scheme of the invention will be explained with reference to the examples.

It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are carried out according to techniques or conditions described in literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruke et al, Huang Petang et al) or according to product instructions. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1

The cell lines and basic experimental techniques used in the examples of the invention are as follows:

lentiviral production and transduction of human T lymphocytes

Replication-deficient lentiviral vectors were generated and harvested by centrifugation for transduction of human T lymphocytes. The experimental procedures for the generation and collection of lentiviral vectors are briefly described below: 293T cells were plated on cell culture dishes with a bottom area of 150-cm square and were virally transduced using Express-In (purchased from Open Biosystems/Thermo Scientific, Waltham, Mass.) according to the instructions. Each plate of cells were loaded with 15. mu.g of lentiviral transgenic plasmid, 5. mu.g of pVSV-G (VSV glycoprotein expression plasmid), 10. mu.g of pCMVR8.74 plasmid (Gag/Pol/Tat/Rev expression plasmid) and 174. mu.l of Express-In (concentration 1. mu.g/microliter). Supernatants were collected at 24 hours and 48 hours, respectively, and centrifuged using an ultracentrifuge at 28,000 rpm (centrifuge rotor Beckman SW 32Ti, available from Beckman Coulter, break, CA) for 2 hours. Finally, the viral plasmid pellet was resuspended in 0.75ml of RPMI-1640 medium.

Human primary T lymphocytes were isolated from healthy volunteer donors. Human T lymphocytes were cultured in RPMI-1640 medium and stimulated to activate using monoclonal antibody-coated beads (purchased from Invitrogen, Carlsbad, CA) against CD3 and CD 28. 18-24 hours after the activation of the human T lymphocyte,t lymphocytes were transduced by spin-seeding, as follows: in a 24-well plate, each well was plated with 0.5X 10⁶T lymphocytes, 0.75ml of the above-mentioned resuspended viral supernatant and Polybrene (concentration 8. mu.g/ml) were added to each well of cells. The mixture of cells and viral plasmids was centrifuged in a bench top centrifuge (from Sorvall ST 40; Thermo Scientific) at room temperature and 2500rpm for 90 minutes. Human recombinant interleukin-2 (IL-2; from Novartis, Basel, Switzerland) was added to the T lymphocyte culture medium every 2-3 days, the final concentration of IL-2 was 100-IU/ml, and the cell density was maintained at 0.5X 10 during the T lymphocyte culture⁶～1×10⁶And/ml. Once transduced T lymphocytes become dormant, e.g., cells grow slower and become smaller, wherein the cell growth rate and size are assessed by a Coulter Counter (available from Beckman Counter), or transduced T lymphocytes at a planned time point, can be used for functional analysis.

The flow cytometer used in the examples of the present application was BD facscan II (available from BD Biosciences), and flow cytometry data was analyzed using flowjoversion 7.2.5 software (available from Tree Star, Ashland, OR).

Measuring cytokine secretion

T cells non-transduced or transduced with the chimeric antigen receptor plasmid (cell number 1X 10) 2 to 7 days after plasmid transduction⁶Per well) were co-cultured with human pleural mesothelioma cells, human pleural mesothelioma cells (from ATCC), varying the ratio of different effective target cells during the experiment. Using a specific enzyme-linked immunosorbent assay (cytokine enzyme-linked immunosorbent assay kit, available from R)&D Systems, inc., Minneapolis, MN, USA) examined the production of cytokines in cell supernatants. The cell supernatants were taken from the supernatants of the cells after 24 hours, 48 hours and 72 hours of culture, and the results were measured for the production of a representative cytokine (interferon-gamma) (IFN γ).

The brief assay procedure is as follows: 100 microliter/hole cytokine dilution (such as IFN gamma) or cell supernatant to be tested as a series of standard controls are added to the microplate, and the microplate is left at room temperature for 2 hours. After 2 hours, the solution in the microplate was aspirated off and the microplate was rinsed four times with 400. mu.l of washing solution. After washing, 200. mu.l of enzyme-linked anti-cytokine antibody was added to each well of the microplate. The standing at room temperature was continued for 2 hours, after which 200. mu.l of the substrate solution was added to each well. After the addition of the substrate solution, the microplate was left at room temperature for 30 minutes, and then 50. mu.l of the termination reaction solution was added to each well. The optical density per well of the microplate was determined within 30 minutes. The microplate reader was set at 450 nm.

Chromium Release test

In the examples, the cytotoxic activity of anti-MSLN chimeric antigen receptor T cells (anti-MSLN CAR T lymphocytes) was assessed using a 4-hour 51-chromium release assay. The method comprises the following specific steps: target test cells were labeled with 51Cr at 37 degrees celsius for 1 hour. After labeling, cells were rinsed in RPMI medium containing 10% Fetal Calf Serum (FCS). After the washing, the cells were resuspended in the same medium at a concentration of 1X 10⁵And/ml. After transduction, T cells were added to the target test cell suspension at various target-effective cell ratios (T: E) and the cells were seeded in 96-wells at 200. mu.l per well volume. Cells were cultured in a 37 degree incubator for 4 hours. After 4 hours, 30 microliters of supernatant from each well was removed and placed in a counter 96-well plate for counting analysis. The analytical instrument was a top-count NXT micro scintillation counter (from Packard Bioscience). The number of effector cells in all count wells was calculated based on the total number of T cells. The labeled target test cell is MSLN⁺MSTO-211H (human pleural mesothelioma cells, human pleural mesothelioma cells (from ATCC)).

Example 2 construction of vectors Co-expressing silenced cellular immune checkpoint shRNA and anti-MSLN chimeric antigen receptor

In this example, the inventors cloned a zeta-chain sequence encoding a combination of a single-chain antibody against human MSLN, a 4-1BB intracellular fragment and a T cell receptor into a lentiviral vector (lentiviral vector) containing an EF-1 promoter, selected restriction enzymes XbaI and NotI and XhoI during cloning, and generated a lentiviral plasmid (LV-MSCAR) expressing an anti-MSLN chimeric antigen receptor by digestion, ligation, selection and amplification of the desired plasmid. Sequences comprising U6 promoter and human PD1 shRNA (iPD1) or CBL-B shRNA (iCBL-B) or CTLA4 shRNA (iCTLA4) were cloned into LV-MSLNCAR vector plasmids to construct LV-MSLNCAR/iPD 1 or LV-MSLNCAR/iCBL or LV-MSLNCAR/i CTLA 4. FIG. 1 is a schematic representation of a lentiviral vector comprising a sequence encoding an anti-MSLN chimeric antigen receptor, a U6 or H1 promoter sequence, a PD1 shRNA or CBL-B shRNA or CTLA4 shRNA sequence. The sequence of the anti-MSLN chimeric antigen receptor is under the start control of a promoter EF-1, and the sequence of CTLA4, PD1 or CBL-B shRNA is under the start control of a promoter U6 or H1.

Example 3 Co-expression of PD1 shRNA and anti-MSLN chimeric antigen receptor in T lymphocytes with higher cell proliferation potency

In this example, peripheral blood lymphocytes are taken from an unknown donor. Peripheral blood lymphocytes are separated by gradient centrifugation, Ficoll-Hypaque. Activated T lymphocytes were transduced with lentiviral vector, expanded in vitro, in the presence of T lymphocyte activating factor magnetic beads CD3/CD28 (purchased from Invitrogen, Carlsbad, Calif.) as described in example 1. 2-7 days after transduction with lentiviral vector, T cells were transduced (cell number 1X 10)⁶Hole) and MSLN⁺MSTO-211H was co-cultured and after 4 days, cell number was currently detected by flow meter. The results of the experiment are shown in FIG. 2. The results in FIG. 2 show that the number of T lymphocytes transduced with LV-MSLN CAR/iPD1 was significantly increased compared to the number of T lymphocytes transduced with LV-MSLN CAR or empty LV-GFP. The notation represents the mean ± SD of each 3 wells. (P)<0.05；LV-MSLN CAR/iPD1vs.LV-MSLN CAR).

Example 4 Co-expression of PD1 shRNA and anti-MSLN chimeric antigen receptor in T lymphocytes with increased cytokine secretion

Activated T lymphocytes are transduced by lentiviral vectors and cultured in vitro in the presence of T lymphocyte activating factor magnetic beads CD3/CD28, as described in example 1. 2-7 days after transduction with lentiviral vector, T cells were transduced (cell number 1X 10)⁶Hole) and MSLN⁺MSTO-211H cells were co-cultured and after 4 days, cytokine secretion was detected by ELISA. The results of the experiment are shown in FIG. 3. FIG. 3 results show that T lymphocytes transduced with LV-MSLN CAR/iPD1 secreted more IFN γ (P) than T lymphocytes transduced with LV-MSLN CAR or empty LV-GFP (P)<0.05; LV-MSLN CAR/iPD1 vs. LV-MSLN CAR). This demonstrates that T lymphocytes transduced with LV-MSLN CAR/iPD1 have a significantly improved cytokine-producing ability compared to T lymphocytes transduced with LV-MSLN CAR.

Example 5 enhanced cytolytic capacity of T lymphocytes co-expressing PD1 shRNA and anti-MSLN chimeric antigen receptor.

In this example, peripheral blood lymphocytes are taken from an unknown donor. Peripheral blood lymphocytes are separated by gradient centrifugation, Ficoll-Hypaque. T lymphocytes and T cell activator magnetic beads CD3/CD28 (purchased from Invitrogen, Carlsbad, Calif.) in 5% CO₂Incubation was carried out at 37 ℃ for 72 hours in RPMI medium 1640 (ex Invitrogen Gibco Cat. No.12633-012) supplemented with 2mmol/L glutamine, 10% high temperature inactivated Fetal Calf Serum (FCS) (ex Sigma-Aldrich Co.), and 100U/ml penicillin/streptomycin double antibody. After 72 hours of activation, the cells were rinsed with a washing solution and the magnetic beads were washed away. T cells were seeded on recombinant fibronectin fragment (FNch-296; Retronectin) cell culture dishes and transduced with lentiviruses, LV-MSLN CAR/iPD1, LV-MSLN CAR or empty-load (LV-GFP) transduction procedures as described in example 1. Transduced T cells were cultured in RPMI-1640 medium and treated with recombinant human IL-2 factor (100 ng/ml; purchased from R)&D Systems) for 7-10 days, and then performing a functional test experiment. The inventors measured the transduction of different lentivirus T cells (effector cells) against MSLN⁺Killing effect of MSTO-211H target cells, wherein the ratio of target-effect cells is 1: 25 or 1:5, the measurement method adopts standard 4-hour⁵¹Chromium release method, 4-hour⁵¹The chromium release method is as described in example 1. The results are shown in FIG. 4. As shown in FIG. 4, T lymphocytes co-expressing anti-MSLN chimeric antigen receptor and PD1 shRNA (iPD1) can more effectively kill the anti-MSLN chimeric antigen receptor than T lymphocytes expressing the anti-MSLN chimeric antigen receptor aloneMSLN⁺MSTO-211H target cells. Idle lentivirus transduced T lymphocytes (control LV-GFP T lymphocytes) vs MSLN⁺MSTO-211H cells did not have significant killing effect. The statistical data represent the mean ± SEM of three wells.

Example 6T cells co-expressing CBL-B shRNA and anti-MSLN chimeric antigen receptor, T cells co-expressing CTLA4 shRNA and anti-MSLN chimeric antigen receptor, T cells co-expressing PD1 shRNA, CBL-B shRNA and anti-MSLN chimeric antigen receptor, and T cells co-expressing PD1 shRNA, CTLA4 shRNA and anti-MSLN chimeric antigen receptor, have enhanced lytic capacity and have characteristics of more cytokine secretion and stronger cell proliferation.

In this example, the inventors also examined the tumor lysis capacity, cytokine secretion capacity and cell proliferation capacity of T cells co-expressing CBL-B shRNA and anti-MSLN chimeric antigen receptor, T cells co-expressing CTLA4 shRNA and anti-MSLN chimeric antigen receptor, T cells co-expressing 2 shRNA (PD1 shRNA and CBL-B shRNA or PD1 and CTLA4 shRNA or 2 PD1 shRNAs against different PD1 regions) and anti-MSLN chimeric antigen receptor. The experimental procedure was the same as in examples 3, 4 and 5. The above T cells have enhanced cytolytic capacity, more cytokine secretion and stronger cell proliferation than T cells expressing anti-MSLN chimeric antigen receptor alone. T cells co-expressing 2 shRNAs (PD1 shRNA and CTLA4 shRNA or PD1 shRNA and CBL-B shRNA or 2 PD1 shRNAs aiming at different PD1 regions) and anti-MSLN chimeric antigen receptor have stronger cytolytic capacity, more cytokine secretion and stronger cell proliferation than T cells co-expressing 1 shRNA (PD1 shRNA or CBL-B shRNA or CTLA4 shRNA) and anti-MSLN chimeric antigen receptor.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

SEQUENCE LISTING

<110> Beijing horse Biotechnology Ltd

<120> Co-expression of anti-MSLN chimeric antigen receptor and immune checkpoint inhibitory molecule

<130> PIDC1153486

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<170> PatentIn version 3.3

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Ser Gly Ala Glu Val Lys Arg Pro Gly Ala Ser Val Gln Val Ser Cys

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Gln Ala Pro Gly Ala Gly Leu Glu Trp Met Gly Val Ile Asn Pro Ser

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Ser Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Trp Ala Leu Trp Gly

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Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

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Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Gly Ile Tyr His Trp

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Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

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Tyr Lys Ala Ser Ser Leu Ala Ser Gly Ala Pro Ser Arg Phe Ser Gly

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

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Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asn Tyr Pro Leu

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

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Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro

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

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

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

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Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg

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

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

370 375 380

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

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Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr

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<213> Artificial

<220>

<223> human cytotoxic T lymphocyte-associated antigen 4(CTLA4) siRNA sequence

<400> 29

uucugacuuc cuccucugga ucc 23

<210> 30

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human cytotoxic T lymphocyte-associated antigen 4(CTLA4) siRNA sequence

<400> 30

aagucugugc ggcaaccuac aug 23

<210> 31

<211> 21

<212> DNA

<213> Artificial

<220>

<223> human T cell immunoglobulin mucin molecule 3(TIM3) siRNA sequence

<400> 31

ggtcggtcag aatgcctatc t 21

<210> 32

<211> 21

<212> DNA

<213> Artificial

<220>

<223> human T cell immunoglobulin mucin molecule 3(TIM3) siRNA sequence

<400> 32

gccaatgact tacgggactc t 21

<210> 33

<211> 21

<212> DNA

<213> Artificial

<220>

<223> human T cell immunoglobulin mucin molecule 3(TIM3) siRNA sequence

<400> 33

gcagagggaa ttcgctcaga a 21

<210> 34

<211> 21

<212> DNA

<213> Artificial

<220>

<223> human T cell immunoglobulin mucin molecule 3(TIM3) siRNA sequence

<400> 34

ggaaattcgg gcacatcata t 21

<210> 35

<211> 21

<212> DNA

<213> Artificial

<220>

<223> human T cell immunoglobulin mucin molecule 3(TIM3) siRNA sequence

<400> 35

gattaagaga tgactggact a 21

<210> 36

<211> 21

<212> DNA

<213> Artificial

<220>

<223> human T cell immunoglobulin mucin molecule 3(TIM3) siRNA sequence

<400> 36

gagatgactg gactaggtct a 21

<210> 37

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human T cell immunoglobulin mucin molecule 3(TIM3) siRNA sequence

<400> 37

aggaaauucg ggcacaucau aug 23

<210> 38

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human T cell immunoglobulin mucin molecule 3(TIM3) siRNA sequence

<400> 38

gacugaugaa agggauguga auu 23

<210> 39

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human T cell immunoglobulin mucin molecule 3(TIM3) siRNA sequence

<400> 39

gccacugauu uucaaagaga ucu 23

<210> 40

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human T cell immunoglobulin mucin molecule 3(TIM3) siRNA sequence

<400> 40

agcagaguuu ucccauuuuc aga 23

<210> 41

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human T cell immunoglobulin mucin molecule 3(TIM3) siRNA sequence

<400> 41

aacuuaaaca ggcaugucau ugc 23

<210> 42

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human T cell immunoglobulin mucin molecule 3(TIM3) siRNA sequence

<400> 42

uucagaagau aaugacucac aug 23

<210> 43

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human T cell immunoglobulin mucin molecule 3(TIM3) siRNA sequence

<400> 43

gccucuguau uuaagccaac aga 23

<210> 44

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human T cell immunoglobulin mucin molecule 3(TIM3) siRNA sequence

<400> 44

ugcucaugug auuguggagu aga 23

<210> 45

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human T cell immunoglobulin mucin molecule 3(TIM3) siRNA sequence

<400> 45

auguuuucac aucuucccuu uga 23

<210> 46

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human T cell immunoglobulin mucin molecule 3(TIM3) siRNA sequence

<400> 46

gagagacuuc acugcagccu uuc 23

<210> 47

<211> 21

<212> DNA

<213> Artificial

<220>

<223> human T lymphocyte attenuation factor (BTLA) siRNA sequence

<400> 47

gattgcctct actcatcact a 21

<210> 48

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human T lymphocyte attenuation factor (BTLA) siRNA sequence

<400> 48

uccuaaugac aaugggucau acc 23

<210> 49

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human T lymphocyte attenuation factor (BTLA) siRNA sequence

<400> 49

aagacauugc cugccaugcu ugg 23

<210> 50

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human T lymphocyte attenuation factor (BTLA) siRNA sequence

<400> 50

gucauaccgc uguucugcaa auu 23

<210> 51

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human T lymphocyte attenuation factor (BTLA) siRNA sequence

<400> 51

cuccuguaua guuuacuucc uuu 23

<210> 52

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human T lymphocyte attenuation factor (BTLA) siRNA sequence

<400> 52

uaccgcuguu cugcaaauuu uca 23

<210> 53

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human T lymphocyte attenuation factor (BTLA) siRNA sequence

<400> 53

aaaacaaacc aggcauuguu uau 23

<210> 54

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human T lymphocyte attenuation factor (BTLA) siRNA sequence

<400> 54

aacuagaaug cccugugaaa uac 23

<210> 55

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human T lymphocyte attenuation factor (BTLA) siRNA sequence

<400> 55

gugacuuggu gcaagcucaa ugg 23

<210> 56

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human T lymphocyte attenuation factor (BTLA) siRNA sequence

<400> 56

auccauggga aagaaucaug uga 23

<210> 57

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human T lymphocyte attenuation factor (BTLA) siRNA sequence

<400> 57

uggugcaagc ucaauggaac aac 23

<210> 58

<211> 21

<212> DNA

<213> Artificial

<220>

<223> human lymphocyte activation gene 3 protein (LAG 1) siRNA sequence

<400> 58

gctgctcacc cttatgaacc t 21

<210> 59

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human lymphocyte activation gene 3 protein (LAG 1) siRNA sequence

<400> 59

aggacauggu gguggacgag ugc 23

<210> 60

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human lymphocyte activation gene 3 protein (LAG 1) siRNA sequence

<400> 60

ugcucuuccu gcacgauauc agu 23

<210> 61

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human lymphocyte activation gene 3 protein (LAG 1) siRNA sequence

<400> 61

accucuacug guuccuguac auc 23

<210> 62

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human lymphocyte activation gene 3 protein (LAG 1) siRNA sequence

<400> 62

cccuccaacu cugcuccucu agg 23

<210> 63

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human lymphocyte activation gene 3 protein (LAG 1) siRNA sequence

<400> 63

cccugagugg acagucgucu ucg 23

<210> 64

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human lymphocyte activation gene 3 protein (LAG 1) siRNA sequence

<400> 64

cugcuccagg gaagcuucua ugg 23

<210> 65

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human lymphocyte activation gene 3 protein (LAG 1) siRNA sequence

<400> 65

cgcucaaggu ccuguaugcc acc 23

<210> 66

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human lymphocyte activation gene 3 protein (LAG 1) siRNA sequence

<400> 66

gaguucacca agcucaacau uua 23

<210> 67

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human lymphocyte activation gene 3 protein (LAG 1) siRNA sequence

<400> 67

ugcugcugcu cacccuuaug aac 23

<210> 68

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human lymphocyte activation gene 3 protein (LAG 1) siRNA sequence

<400> 68

cccaucuccg ugcucuucuu uga 23

<210> 69

<211> 21

<212> DNA

<213> Artificial

<220>

<223> human IRAK-M siRNA (human interleukin-1 receptor associated kinase 3) nucleotide sequence

<400> 69

gggacatcgt cgagctattc a 21

<210> 70

<211> 21

<212> DNA

<213> Artificial

<220>

<400> 70

ggacatcgtc gagctattca t 21

<210> 71

<211> 21

<212> DNA

<213> Artificial

<220>

<400> 71

gccaatgtca ccgtggataa t 21

<210> 72

<211> 21

<212> DNA

<213> Artificial

<220>

<400> 72

gtcatctgtg gcagtatatc a 21

<210> 73

<211> 21

<212> DNA

<213> Artificial

<220>

<400> 73

ggatgtagag tagtgttaga t 21

<210> 74

<211> 21

<212> DNA

<213> Artificial

<220>

<400> 74

ggcaaagtta agaccatcaa t 21

<210> 75

<211> 21

<212> DNA

<213> Artificial

<220>

<400> 75

gaccaaatcc acgctcaatt a 21

<210> 76

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 76

uacugcuuaa aucuuccauc agc 23

<210> 77

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 77

gacugagaag uucugucuga uuu 23

<210> 78

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 78

cuguuucauc acccaaacau acu 23

<210> 79

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 79

gaagauccuc ccacaucacu aaa 23

<210> 80

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 80

uagaccaagg uaaaagugga aca 23

<210> 81

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 81

aagagguuuu uaucugagcu uga 23

<210> 82

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 82

uaccugcaca acguucaacc aug 23

<210> 83

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 83

gccuggauuc augucucuca uuu 23

<210> 84

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 84

cccucggaau uucucugcca agc 23

<210> 85

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 85

ugcugaagau ccucccacau cac 23

<210> 86

<211> 21

<212> DNA

<213> Artificial

<220>

<223> human SOCS1 siRNA (human cytokine Signal transduction inhibitor 1) sequence

<400> 86

gcacctccta cctcttcatg t 21

<210> 87

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 87

cgcacuuccg cacauuccgu ucg 23

<210> 88

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 88

ggggaggguc ucuggcuuua uuu 23

<210> 89

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 89

cagcauuaac ugggaugccg ugu 23

<210> 90

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 90

ccaggaccug aacucgcacc ucc 23

<210> 91

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 91

uacauauacc caguaucuuu gca 23

<210> 92

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 92

gccgacaaug cagucuccac agc 23

<210> 93

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 93

ccccugguug uuguagcagc uua 23

<210> 94

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 94

cugcugugca gaauccuauu uua 23

<210> 95

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 95

ugggaugccg uguuauuuug uua 23

<210> 96

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 96

ucgcaccucc uaccucuuca ugu 23

<210> 97

<211> 21

<212> DNA

<213> Artificial

<220>

<223> human A20 siRNA (human tumor necrosis factor-alpha inducing protein A20) sequence

<400> 97

gcggaaagct gtgaagatac g 21

<210> 98

<211> 21

<212> DNA

<213> Artificial

<220>

<400> 98

acaaagccct catcgacaga a 21

<210> 99

<211> 21

<212> DNA

<213> Artificial

<220>

<400> 99

atgccacttc tcagtacatg t 21

<210> 100

<211> 21

<212> DNA

<213> Artificial

<220>

<400> 100

gtggacttca gtacaactca c 21

<210> 101

<211> 21

<212> DNA

<213> Artificial

<220>

<400> 101

gtggaattta cttgcctctc c 21

<210> 102

<211> 21

<212> DNA

<213> Artificial

<220>

<400> 102

gttggatgaa gctaacttac c 21

<210> 103

<211> 21

<212> DNA

<213> Artificial

<220>

<400> 103

actgggaaga cgtgtaactc t 21

<210> 104

<211> 21

<212> DNA

<213> Artificial

<220>

<400> 104

aaggaattgc atccaaggta t 21

<210> 105

<211> 21

<212> DNA

<213> Artificial

<220>

<400> 105

ggaattgcat ccaaggtata c 21

<210> 106

<211> 21

<212> DNA

<213> Artificial

<220>

<400> 106

ggatgagact ggcaatggtc a 21

<210> 107

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 107

uccucaguuu cgggagauca ucc 23

<210> 108

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 108

gagucucuca aaucucagga auu 23

<210> 109

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 109

agcucuaguc cuuuuugugu aau 23

<210> 110

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 110

cacuggaaau guucagaacu ugc 23

<210> 111

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 111

augaugaaug ggacaaucuu auc 23

<210> 112

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 112

cacacugugu uucaucgagu aca 23

<210> 113

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 113

gcagaaccau ccauggacug uga 23

<210> 114

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 114

aaagaugugg ccuuuuguga ugg 23

<210> 115

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 115

uucagaacuu gccaguuuug ucc 23

<210> 116

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 116

augagacugg caauggucac agg 23

<210> 117

<211> 21

<212> DNA

<213> Artificial

<220>

<223> human CBL-B siRNA (E3 ubiquitin protein ligase CBL-B) sequence

<400> 117

gtcaattcca gggagataac t 21

<210> 118

<211> 21

<212> DNA

<213> Artificial

<220>

<223> human CBL-B siRNA (E3 ubiquitin protein ligase CBL-B) sequence

<400> 118

gcctggaagc aatggctcta a 21

<210> 119

<211> 21

<212> DNA

<213> Artificial

<220>

<223> human CBL-B siRNA (E3 ubiquitin protein ligase CBL-B) sequence

<400> 119

gcaccaaacc cggaagctat a 21

<210> 120

<211> 21

<212> DNA

<213> Artificial

<220>

<223> human CBL-B siRNA (E3 ubiquitin protein ligase CBL-B) sequence

<400> 120

gttgcactcg attgggacag t 21

<210> 121

<211> 21

<212> DNA

<213> Artificial

<220>

<223> human CBL-B siRNA (E3 ubiquitin protein ligase CBL-B) sequence

<400> 121

ggattatgtg aacctacacc t 21

<210> 122

<211> 21

<212> DNA

<213> Artificial

<220>

<223> human CBL-B siRNA (E3 ubiquitin protein ligase CBL-B) sequence

<400> 122

ggaatcacag cgagttcaaa t 21

<210> 123

<211> 21

<212> DNA

<213> Artificial

<220>

<223> human CBL-B siRNA (E3 ubiquitin protein ligase CBL-B) sequence

<400> 123

gcaaggcata gtctcattga a 21

<210> 124

<211> 21

<212> DNA

<213> Artificial

<220>

<223> human CBL-B siRNA (E3 ubiquitin protein ligase CBL-B) sequence

<400> 124

ggtgaagaga gccttagaga t 21

<210> 125

<211> 21

<212> DNA

<213> Artificial

<220>

<223> human CBL-B siRNA (E3 ubiquitin protein ligase CBL-B) sequence

<400> 125

gtgaagagag ccttagagat a 21

<210> 126

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human CBL-B siRNA (E3 ubiquitin protein ligase CBL-B) sequence

<400> 126

aggagcuaag gucuuuucca aug 23

<210> 127

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human CBL-B siRNA (E3 ubiquitin protein ligase CBL-B) sequence

<400> 127

augucgaugc aaaaauugca aaa 23

<210> 128

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human CBL-B siRNA (E3 ubiquitin protein ligase CBL-B) sequence

<400> 128

gucacaugcu ggcagaaauc aaa 23

<210> 129

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human CBL-B siRNA (E3 ubiquitin protein ligase CBL-B) sequence

<400> 129

uccagguuac auggcauuuc uca 23

<210> 130

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human CBL-B siRNA (E3 ubiquitin protein ligase CBL-B) sequence

<400> 130

uugaacuuug aaccugugaa aug 23

<210> 131

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human CBL-B siRNA (E3 ubiquitin protein ligase CBL-B) sequence

<400> 131

uccacaucaa cagcuaaauc auu 23

<210> 132

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human CBL-B siRNA (E3 ubiquitin protein ligase CBL-B) sequence

<400> 132

augcuggcag aaaucaaagc aau 23

<210> 133

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human CBL-B siRNA (E3 ubiquitin protein ligase CBL-B) sequence

<400> 133

ugcagagaau gacaaagaug uca 23

<210> 134

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human CBL-B siRNA (E3 ubiquitin protein ligase CBL-B) sequence

<400> 134

ggcagaacuc accagucaca uca 23

<210> 135

<211> 23

<212> DNA

<213> Artificial

<220>

<223> human CBL-B siRNA (E3 ubiquitin protein ligase CBL-B) sequence

<400> 135

ucgguccugu gauaaugguc acu 23

<210> 136

<211> 2053

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of MSLN-CAR/iPD1

<400> 136

atggttctgc tggtgacatc tctcctgctc tgtgaactgc ctcatcccgc ttttctgctc 60

attcccgaca ttcaggctca agtccaactg gtccaaagtg gtgctgaagt caaacgcccg 120

ggtgcctccg tccaagtctc ctgccgtgcc tctggctact cgattaacac ctattacatg 180

cagtgggtcc gtcaagcacc gggtgcaggt ctggaatgga tgggtgtcat caatccgtcc 240

ggcgtgacct catatgcgca gaaatttcaa ggtcgcgtta ccctgacgaa cgataccagc 300

acgaataccg tctacatgca gctgaactct ctgacgagtg cagacaccgc ggtgtattac 360

tgcgcacgtt gggcactgtg gggcgatttc ggcatggatg tttggggcaa aggtacgctg 420

gtgaccgtta gctctggtgg tggtggttct ggtggtggtg gtagtggcgg tggcggttct 480

gatattcaga tgacgcaaag cccgtctacc ctgagtgcct ccattggtga ccgtgttacg 540

atcacctgtc gcgcatccga aggcatctat cattggctgg cttggtacca gcaaaaaccg 600

ggtaaagcgc cgaaactgct gatctataaa gcaagttccc tggcatcggg tgctccgagc 660

cgcttttcag gttcgggtag cggcaccgat ttcacgctga ccatctcatc gctgcagccg 720

gacgatttcg ctacctacta ctgccaacaa tactcaaact acccgctgac cttcggtgga 780

gggaccaagc tggagatcaa acgtgctagc accactaccc cagcaccgag gccacccacc 840

ccggctccta ccatcgcctc ccagcctctg tccctgcgtc cggaggcatg tagacccgca 900

gctggtgggg ccgtgcatac ccggggtctt gacttcgcct gcgatatcta catttgggcc 960

cctctggctg gtacttgcgg ggtcctgctg ctttcactcg tgatcactct ttactgtaag 1020

cgcggtcgga agaagctgct gtacatcttt aagcaaccct tcatgaggcc tgtgcagact 1080

actcaagagg aggacggctg ttcatgccgg ttcccagagg aggaggaagg cggctgcgaa 1140

ctgcgcgtga aattcagccg cagcgcagat gctccagcct accagcaggg gcagaaccag 1200

ctctacaacg aactcaatct tggtcggaga gaggagtacg acgtgctgga caagcggaga 1260

ggacgggacc cagaaatggg cgggaagccg cgcagaaaga atccccaaga gggcctgtac 1320

aacgagctcc aaaaggataa gatggcagaa gcctatagcg agattggtat gaaaggggaa 1380

cgcagaagag gcaaaggcca cgacggactg taccagggac tcagcaccgc caccaaggac 1440

acctatgacg ctcttcacat gcaggccctg ccgcctcggt aatcctactg cgtcgagcga 1500

ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 1560

tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 1620

tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 1680

gggaagacaa tagcaggcat gctggggatg cggtgggctc tatgggtcga ccaaggtcgg 1740

gcaggaagag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt 1800

tagagagata attagaatta atttgactgt aaacacaaag atattagtac aaaatacgtg 1860

acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga 1920

ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg 1980

gaaaggacga aacacctccc caggcgcaga tcaaagagag ttcaagagac tctctttgat 2040

ctgcgccttt ttt 2053

<210> 137

<211> 2057

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of MSLN-CAR/iCBL-B

<400> 137

atggttctgc tggtgacatc tctcctgctc tgtgaactgc ctcatcccgc ttttctgctc 60

attcccgaca ttcaggctca agtccaactg gtccaaagtg gtgctgaagt caaacgcccg 120

ggtgcctccg tccaagtctc ctgccgtgcc tctggctact cgattaacac ctattacatg 180

cagtgggtcc gtcaagcacc gggtgcaggt ctggaatgga tgggtgtcat caatccgtcc 240

ggcgtgacct catatgcgca gaaatttcaa ggtcgcgtta ccctgacgaa cgataccagc 300

acgaataccg tctacatgca gctgaactct ctgacgagtg cagacaccgc ggtgtattac 360

tgcgcacgtt gggcactgtg gggcgatttc ggcatggatg tttggggcaa aggtacgctg 420

gtgaccgtta gctctggtgg tggtggttct ggtggtggtg gtagtggcgg tggcggttct 480

gatattcaga tgacgcaaag cccgtctacc ctgagtgcct ccattggtga ccgtgttacg 540

atcacctgtc gcgcatccga aggcatctat cattggctgg cttggtacca gcaaaaaccg 600

ggtaaagcgc cgaaactgct gatctataaa gcaagttccc tggcatcggg tgctccgagc 660

cgcttttcag gttcgggtag cggcaccgat ttcacgctga ccatctcatc gctgcagccg 720

gacgatttcg ctacctacta ctgccaacaa tactcaaact acccgctgac cttcggtgga 780

gggaccaagc tggagatcaa acgtgctagc accactaccc cagcaccgag gccacccacc 840

ccggctccta ccatcgcctc ccagcctctg tccctgcgtc cggaggcatg tagacccgca 900

gctggtgggg ccgtgcatac ccggggtctt gacttcgcct gcgatatcta catttgggcc 960

cctctggctg gtacttgcgg ggtcctgctg ctttcactcg tgatcactct ttactgtaag 1020

cgcggtcgga agaagctgct gtacatcttt aagcaaccct tcatgaggcc tgtgcagact 1080

actcaagagg aggacggctg ttcatgccgg ttcccagagg aggaggaagg cggctgcgaa 1140

ctgcgcgtga aattcagccg cagcgcagat gctccagcct accagcaggg gcagaaccag 1200

ctctacaacg aactcaatct tggtcggaga gaggagtacg acgtgctgga caagcggaga 1260

ggacgggacc cagaaatggg cgggaagccg cgcagaaaga atccccaaga gggcctgtac 1320

aacgagctcc aaaaggataa gatggcagaa gcctatagcg agattggtat gaaaggggaa 1380

cgcagaagag gcaaaggcca cgacggactg taccagggac tcagcaccgc caccaaggac 1440

acctatgacg ctcttcacat gcaggccctg ccgcctcggt aatcctactg cgtcgagcga 1500

ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 1560

tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 1620

tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 1680

gggaagacaa tagcaggcat gctggggatg cggtgggctc tatgggtcga ccaaggtcgg 1740

gcaggaagag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt 1800

tagagagata attagaatta atttgactgt aaacacaaag atattagtac aaaatacgtg 1860

acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga 1920

ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg 1980

gaaaggacga aacacctccc caacacagac gccatgattt gcttcaagag agcaaatcat 2040

ggcgtctgtg ttttttt 2057

<210> 138

<211> 2053

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of MSLN-CAR/iCTLA4

<400> 138

atggttctgc tggtgacatc tctcctgctc tgtgaactgc ctcatcccgc ttttctgctc 60

attcccgaca ttcaggctca agtccaactg gtccaaagtg gtgctgaagt caaacgcccg 120

ggtgcctccg tccaagtctc ctgccgtgcc tctggctact cgattaacac ctattacatg 180

cagtgggtcc gtcaagcacc gggtgcaggt ctggaatgga tgggtgtcat caatccgtcc 240

ggcgtgacct catatgcgca gaaatttcaa ggtcgcgtta ccctgacgaa cgataccagc 300

acgaataccg tctacatgca gctgaactct ctgacgagtg cagacaccgc ggtgtattac 360

tgcgcacgtt gggcactgtg gggcgatttc ggcatggatg tttggggcaa aggtacgctg 420

gtgaccgtta gctctggtgg tggtggttct ggtggtggtg gtagtggcgg tggcggttct 480

gatattcaga tgacgcaaag cccgtctacc ctgagtgcct ccattggtga ccgtgttacg 540

atcacctgtc gcgcatccga aggcatctat cattggctgg cttggtacca gcaaaaaccg 600

ggtaaagcgc cgaaactgct gatctataaa gcaagttccc tggcatcggg tgctccgagc 660

cgcttttcag gttcgggtag cggcaccgat ttcacgctga ccatctcatc gctgcagccg 720

gacgatttcg ctacctacta ctgccaacaa tactcaaact acccgctgac cttcggtgga 780

gggaccaagc tggagatcaa acgtgctagc accactaccc cagcaccgag gccacccacc 840

ccggctccta ccatcgcctc ccagcctctg tccctgcgtc cggaggcatg tagacccgca 900

gctggtgggg ccgtgcatac ccggggtctt gacttcgcct gcgatatcta catttgggcc 960

cctctggctg gtacttgcgg ggtcctgctg ctttcactcg tgatcactct ttactgtaag 1020

cgcggtcgga agaagctgct gtacatcttt aagcaaccct tcatgaggcc tgtgcagact 1080

actcaagagg aggacggctg ttcatgccgg ttcccagagg aggaggaagg cggctgcgaa 1140

ctgcgcgtga aattcagccg cagcgcagat gctccagcct accagcaggg gcagaaccag 1200

ctctacaacg aactcaatct tggtcggaga gaggagtacg acgtgctgga caagcggaga 1260

ggacgggacc cagaaatggg cgggaagccg cgcagaaaga atccccaaga gggcctgtac 1320

aacgagctcc aaaaggataa gatggcagaa gcctatagcg agattggtat gaaaggggaa 1380

cgcagaagag gcaaaggcca cgacggactg taccagggac tcagcaccgc caccaaggac 1440

acctatgacg ctcttcacat gcaggccctg ccgcctcggt aatcctactg cgtcgagcga 1500

ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 1560

tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 1620

tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 1680

gggaagacaa tagcaggcat gctggggatg cggtgggctc tatgggtcga ccaaggtcgg 1740

gcaggaagag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt 1800

tagagagata attagaatta atttgactgt aaacacaaag atattagtac aaaatacgtg 1860

acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga 1920

ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg 1980

gaaaggacga aacacctccc cgcatcactt gggattaata ttcaagagat attaatccca 2040

agtgatgctt ttt 2053

<210> 139

<211> 2360

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of MSLN-CAR/iPD1-CBL-B

<400> 139

atggttctgc tggtgacatc tctcctgctc tgtgaactgc ctcatcccgc ttttctgctc 60

attcccgaca ttcaggctca agtccaactg gtccaaagtg gtgctgaagt caaacgcccg 120

ggtgcctccg tccaagtctc ctgccgtgcc tctggctact cgattaacac ctattacatg 180

cagtgggtcc gtcaagcacc gggtgcaggt ctggaatgga tgggtgtcat caatccgtcc 240

ggcgtgacct catatgcgca gaaatttcaa ggtcgcgtta ccctgacgaa cgataccagc 300

acgaataccg tctacatgca gctgaactct ctgacgagtg cagacaccgc ggtgtattac 360

tgcgcacgtt gggcactgtg gggcgatttc ggcatggatg tttggggcaa aggtacgctg 420

gtgaccgtta gctctggtgg tggtggttct ggtggtggtg gtagtggcgg tggcggttct 480

gatattcaga tgacgcaaag cccgtctacc ctgagtgcct ccattggtga ccgtgttacg 540

atcacctgtc gcgcatccga aggcatctat cattggctgg cttggtacca gcaaaaaccg 600

ggtaaagcgc cgaaactgct gatctataaa gcaagttccc tggcatcggg tgctccgagc 660

cgcttttcag gttcgggtag cggcaccgat ttcacgctga ccatctcatc gctgcagccg 720

gacgatttcg ctacctacta ctgccaacaa tactcaaact acccgctgac cttcggtgga 780

gggaccaagc tggagatcaa acgtgctagc accactaccc cagcaccgag gccacccacc 840

ccggctccta ccatcgcctc ccagcctctg tccctgcgtc cggaggcatg tagacccgca 900

gctggtgggg ccgtgcatac ccggggtctt gacttcgcct gcgatatcta catttgggcc 960

cctctggctg gtacttgcgg ggtcctgctg ctttcactcg tgatcactct ttactgtaag 1020

cgcggtcgga agaagctgct gtacatcttt aagcaaccct tcatgaggcc tgtgcagact 1080

actcaagagg aggacggctg ttcatgccgg ttcccagagg aggaggaagg cggctgcgaa 1140

ctgcgcgtga aattcagccg cagcgcagat gctccagcct accagcaggg gcagaaccag 1200

ctctacaacg aactcaatct tggtcggaga gaggagtacg acgtgctgga caagcggaga 1260

ggacgggacc cagaaatggg cgggaagccg cgcagaaaga atccccaaga gggcctgtac 1320

aacgagctcc aaaaggataa gatggcagaa gcctatagcg agattggtat gaaaggggaa 1380

cgcagaagag gcaaaggcca cgacggactg taccagggac tcagcaccgc caccaaggac 1440

acctatgacg ctcttcacat gcaggccctg ccgcctcggt aatcctactg cgtcgagcga 1500

ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 1560

tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 1620

tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 1680

gggaagacaa tagcaggcat gctggggatg cggtgggctc tatgggtcga ccaaggtcgg 1740

gcaggaagag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt 1800

tagagagata attagaatta atttgactgt aaacacaaag atattagtac aaaatacgtg 1860

acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga 1920

ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg 1980

gaaaggacga aacacctccc caggcgcaga tcaaagagag ttcaagagac tctctttgat 2040

ctgcgccttt tttagctatc gatagctaaa aaaacacaga cgccatgatt tgctctcttg 2100

aagcaaatca tggcgtctgt gttggggaag atctgtggtc tcatacagaa cttataagat 2160

tcccaaatcc aaagacattt cacgtttatg gtgatttccc agaacacata gcgacatgca 2220

aatattgcag ggcgccactc ccctgtccct cacagccatc ttcctgccag ggcgcacgcg 2280

cgctgggtgt tcccgcctag tgacactggg cccgcgattc cttggagcgg gttgatgacg 2340

tcagcgttcg aattgtcgac 2360

<210> 140

<211> 2356

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of MSLN-CAR/iPD1-CTLA4

<400> 140

atggttctgc tggtgacatc tctcctgctc tgtgaactgc ctcatcccgc ttttctgctc 60

attcccgaca ttcaggctca agtccaactg gtccaaagtg gtgctgaagt caaacgcccg 120

ggtgcctccg tccaagtctc ctgccgtgcc tctggctact cgattaacac ctattacatg 180

cagtgggtcc gtcaagcacc gggtgcaggt ctggaatgga tgggtgtcat caatccgtcc 240

ggcgtgacct catatgcgca gaaatttcaa ggtcgcgtta ccctgacgaa cgataccagc 300

acgaataccg tctacatgca gctgaactct ctgacgagtg cagacaccgc ggtgtattac 360

tgcgcacgtt gggcactgtg gggcgatttc ggcatggatg tttggggcaa aggtacgctg 420

gtgaccgtta gctctggtgg tggtggttct ggtggtggtg gtagtggcgg tggcggttct 480

gatattcaga tgacgcaaag cccgtctacc ctgagtgcct ccattggtga ccgtgttacg 540

atcacctgtc gcgcatccga aggcatctat cattggctgg cttggtacca gcaaaaaccg 600

ggtaaagcgc cgaaactgct gatctataaa gcaagttccc tggcatcggg tgctccgagc 660

cgcttttcag gttcgggtag cggcaccgat ttcacgctga ccatctcatc gctgcagccg 720

gacgatttcg ctacctacta ctgccaacaa tactcaaact acccgctgac cttcggtgga 780

gggaccaagc tggagatcaa acgtgctagc accactaccc cagcaccgag gccacccacc 840

ccggctccta ccatcgcctc ccagcctctg tccctgcgtc cggaggcatg tagacccgca 900

gctggtgggg ccgtgcatac ccggggtctt gacttcgcct gcgatatcta catttgggcc 960

cctctggctg gtacttgcgg ggtcctgctg ctttcactcg tgatcactct ttactgtaag 1020

cgcggtcgga agaagctgct gtacatcttt aagcaaccct tcatgaggcc tgtgcagact 1080

actcaagagg aggacggctg ttcatgccgg ttcccagagg aggaggaagg cggctgcgaa 1140

ctgcgcgtga aattcagccg cagcgcagat gctccagcct accagcaggg gcagaaccag 1200

ctctacaacg aactcaatct tggtcggaga gaggagtacg acgtgctgga caagcggaga 1260

ggacgggacc cagaaatggg cgggaagccg cgcagaaaga atccccaaga gggcctgtac 1320

aacgagctcc aaaaggataa gatggcagaa gcctatagcg agattggtat gaaaggggaa 1380

cgcagaagag gcaaaggcca cgacggactg taccagggac tcagcaccgc caccaaggac 1440

acctatgacg ctcttcacat gcaggccctg ccgcctcggt aatcctactg cgtcgagcga 1500

ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 1560

tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 1620

tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 1680

gggaagacaa tagcaggcat gctggggatg cggtgggctc tatgggtcga ccaaggtcgg 1740

gcaggaagag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt 1800

tagagagata attagaatta atttgactgt aaacacaaag atattagtac aaaatacgtg 1860

acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga 1920

ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg 1980

gaaaggacga aacacctccc caggcgcaga tcaaagagag ttcaagagac tctctttgat 2040

ctgcgccttt tttagctatc gatagctaaa aagcatcact tgggattaat atctcttgaa 2100

tattaatccc aagtgatgcg gggaagatct gtggtctcat acagaactta taagattccc 2160

aaatccaaag acatttcacg tttatggtga tttcccagaa cacatagcga catgcaaata 2220

ttgcagggcg ccactcccct gtccctcaca gccatcttcc tgccagggcg cacgcgcgct 2280

gggtgttccc gcctagtgac actgggcccg cgattccttg gagcgggttg atgacgtcag 2340

cgttcgaatt gtcgac 2356

<210> 141

<211> 2360

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of MSLN-CAR/iPD1-PD1

<400> 141

atggttctgc tggtgacatc tctcctgctc tgtgaactgc ctcatcccgc ttttctgctc 60

attcccgaca ttcaggctca agtccaactg gtccaaagtg gtgctgaagt caaacgcccg 120

ggtgcctccg tccaagtctc ctgccgtgcc tctggctact cgattaacac ctattacatg 180

cagtgggtcc gtcaagcacc gggtgcaggt ctggaatgga tgggtgtcat caatccgtcc 240

ggcgtgacct catatgcgca gaaatttcaa ggtcgcgtta ccctgacgaa cgataccagc 300

acgaataccg tctacatgca gctgaactct ctgacgagtg cagacaccgc ggtgtattac 360

tgcgcacgtt gggcactgtg gggcgatttc ggcatggatg tttggggcaa aggtacgctg 420

gtgaccgtta gctctggtgg tggtggttct ggtggtggtg gtagtggcgg tggcggttct 480

gatattcaga tgacgcaaag cccgtctacc ctgagtgcct ccattggtga ccgtgttacg 540

atcacctgtc gcgcatccga aggcatctat cattggctgg cttggtacca gcaaaaaccg 600

ggtaaagcgc cgaaactgct gatctataaa gcaagttccc tggcatcggg tgctccgagc 660

cgcttttcag gttcgggtag cggcaccgat ttcacgctga ccatctcatc gctgcagccg 720

gacgatttcg ctacctacta ctgccaacaa tactcaaact acccgctgac cttcggtgga 780

gggaccaagc tggagatcaa acgtgctagc accactaccc cagcaccgag gccacccacc 840

ccggctccta ccatcgcctc ccagcctctg tccctgcgtc cggaggcatg tagacccgca 900

gctggtgggg ccgtgcatac ccggggtctt gacttcgcct gcgatatcta catttgggcc 960

cctctggctg gtacttgcgg ggtcctgctg ctttcactcg tgatcactct ttactgtaag 1020

cgcggtcgga agaagctgct gtacatcttt aagcaaccct tcatgaggcc tgtgcagact 1080

actcaagagg aggacggctg ttcatgccgg ttcccagagg aggaggaagg cggctgcgaa 1140

ctgcgcgtga aattcagccg cagcgcagat gctccagcct accagcaggg gcagaaccag 1200

ctctacaacg aactcaatct tggtcggaga gaggagtacg acgtgctgga caagcggaga 1260

ggacgggacc cagaaatggg cgggaagccg cgcagaaaga atccccaaga gggcctgtac 1320

aacgagctcc aaaaggataa gatggcagaa gcctatagcg agattggtat gaaaggggaa 1380

cgcagaagag gcaaaggcca cgacggactg taccagggac tcagcaccgc caccaaggac 1440

acctatgacg ctcttcacat gcaggccctg ccgcctcggt aatcctactg cgtcgagcga 1500

ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 1560

tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 1620

tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 1680

gggaagacaa tagcaggcat gctggggatg cggtgggctc tatgggtcga ccaaggtcgg 1740

gcaggaagag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt 1800

tagagagata attagaatta atttgactgt aaacacaaag atattagtac aaaatacgtg 1860

acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga 1920

ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg 1980

gaaaggacga aacacctccc caggcgcaga tcaaagagag ttcaagagac tctctttgat 2040

ctgcgccttt tttagctatc gatagctaaa aagcctagag aagtttcagg gaatctcttg 2100

aattccctga aacttctcta ggcggggaag atctgtggtc tcatacagaa cttataagat 2160

tcccaaatcc aaagacattt cacgtttatg gtgatttccc agaacacata gcgacatgca 2220

aatattgcag ggcgccactc ccctgtccct cacagccatc ttcctgccag ggcgcacgcg 2280

cgctgggtgt tcccgcctag tgacactggg cccgcgattc cttggagcgg gttgatgacg 2340

tcagcgttcg aattgtcgac 2360

Claims

1. A T lymphocyte characterized in that its cellular immune checkpoint PD1 is silenced;

and expressing the chimeric antigen receptor, wherein,

the chimeric antigen receptor includes:

an extracellular region comprising a heavy chain variable region and a light chain variable region of a single chain antibody that specifically recognizes the antigen MSLN;

a transmembrane region attached to the extracellular region and embedded in the cell membrane of the T lymphocyte;

an intracellular domain associated with said transmembrane region and comprising an intracellular segment of CD28 and a CD3 zeta chain;

the cell immune checkpoint PD1 of the T lymphocyte is silenced and expresses a chimeric antigen receptor by hybridizing SEQ ID NO: 136 into a T lymphocyte.

2. A lentivirus, wherein said lentivirus carries a nucleic acid sequence comprising SEQ ID NO: 136, or a nucleotide sequence set forth in seq id no.

3. A transgenic lymphocyte characterized in that the lymphocyte immune checkpoint PD1 is silenced; and expressing a chimeric antigen receptor comprising:

an extracellular region comprising a heavy chain variable region and a light chain variable region of an antibody, the antibody capable of specifically binding to a tumor antigen;

a transmembrane region; and

an intracellular region comprising an intracellular segment of an immune co-stimulatory molecule,

wherein the antibody is a single chain antibody, the tumor antigen is MSLN,

the transgenic lymphocyte has its cellular immune checkpoint PD1 silenced and expresses a chimeric antigen receptor by combining SEQ ID NO: 136 into lymphocytes.

4. The transgenic lymphocyte of claim 3, wherein the lymphocyte is CD3⁺T lymphocytes.

5. The transgenic lymphocyte of claim 3, wherein the lymphocyte is a natural killer cell.

6. The transgenic lymphocyte of claim 3, wherein the lymphocyte is a natural killer T cell.

7. A method of producing the T lymphocyte of claim 1 or the transgenic lymphocyte of any one of claims 3 to 6, comprising:

introducing the lentivirus of claim 2 into lymphocytes or T lymphocytes.

8. A therapeutic composition for treating cancer, comprising:

the lentivirus of claim 2, the T lymphocyte of claim 1 or the transgenic lymphocyte of any one of claims 3 to 6.