CN108342363A

CN108342363A - It co-expresses anti-MSLN Chimeric antigen receptors and immunologic test point inhibits the transgenosis lymphocyte and application thereof of molecule

Info

Publication number: CN108342363A
Application number: CN201710056395.0A
Authority: CN
Inventors: 严勇朝; 朱益林; 陈思毅
Original assignee: Beijing Horsepower Nuo Biotechnology Co Ltd
Current assignee: Beijing Horsepower Nuo Biotechnology Co Ltd
Priority date: 2017-01-25
Filing date: 2017-01-25
Publication date: 2018-07-31
Anticipated expiration: 2037-01-25
Also published as: WO2018137295A1; CN108342363B

Abstract

The present invention proposes a kind of transgenosis lymphocyte, a kind of construct and a kind of therapeutic combination for the treatment of cancer, the cellular immunity checkpoint of the transgenosis lymphocyte and is silenced and expresses Chimeric antigen receptor.Wherein, the Chimeric antigen receptor includes：Extracellular region, the extracellular region include the heavy chain variable region and light chain variable region of single-chain antibody, the single-chain antibody specific recognition antigen MSLN；Transmembrane region, the transmembrane region are connected with the extracellular region, and are embedded into the cell membrane of the T lymphocytes；Intracellular region, the intracellular region are connected with the transmembrane region, and the intracellular region includes the intracellular section and CD3 ζ chains of CD28.The transgenosis lymphocyte, which has, resists the immunosuppressive characteristic that tumour cell mediates, and is significantly increased to the killing ability of tumour cell, and the tumour of especially high expression MSLN has significant orientation lethal effect.

Description

It co-expresses anti-MSLN Chimeric antigen receptors and immunologic test point inhibits the transgenosis of molecule Lymphocyte and application thereof

Technical field

The present invention relates to biomedicine fields, in particular it relates to a kind of T lymphocytes, a kind of slow virus, one kind Transgenosis lymphocyte, a kind of construct, a kind of therapeutic combination for treating cancer and a kind of raising lymphocyte activity Method.

Background technology

Between quality (mesothelin, MSLN) be a kind of differentiation antigen, it is only limitted in the expression of human normal tissues The mesothelial cell of pleura, pericardium and peritonaeum lining.However, the quality but high expression in a variety of human cancer tissues, including Almost all of celiothelioma and cancer of pancreas and about 70% oophoroma and about 50% adenocarcinoma of lung and other cancers, such as bile duct Cancer, gastric cancer, intestinal cancer, the cancer of the esophagus, breast cancer.Interstitial plain gene encodes the precursor protein of 71KDa, and precursor protein is then processed to Fall off segment and the protein fragments of 40KDa of 31KDa, the segment that falls off of 31KDa are referred to as megakaryocyte-potentiating factor (MPF), And the protein fragments of 40KDa are referred to as a quality, quality passes through glycosyl-phosphatidyl inositol (glycosyl- Phosphatidylinositol, GPI) anchoring effect be fixed on cell membrane.

By taking celiothelioma as an example, it is that pleura is primary that celiothelioma, which is divided into mesothelioma of pleura and peritoneal mesothelioma, mesothelioma of pleura, Tumour is divided into topical type (mostly benign) and diffuses type (being all pernicious), wherein it is chest prognosis to diffuse type malignant mesothelioma One of the worst tumour.Peritoneal mesothelioma refers to the tumour for being primary in Peritoneal Mesothelial Cells.Clinical manifestation does not have characteristic, often The sings and symptoms seen have：Abdominal pain, ascites, abdominal distension and abdominal mass etc..The treatment of malignant pleural mesothelioma, does not still have at present Effective radical cure method.In therapy, there are palliative therapy, surgical intervention, chemotherapy and radiotherapy etc., it is considered that For the I phase patients of the opposite limitation of tumour, opinion does the pleuropulmonary resection effected a radical cure.For II, III, IV phase patient, radical-ability Operation is nonsensical, only implements palliative operation.In fact, when most patients clarify a diagnosis to disease, it has been in II is more than the phase.The hydrothorax increased rapidly often results in the serious expiratory dyspnea of patient, and palliative operation can only temporarily improve these evenings The quality of life of phase patient, and can not effect a radical cure.

It can be seen that exploitation be directed between quality height expression tumour therapy it is particularly urgent.

Invention content

The application is made to the discovery of following facts and problem and understanding based on inventor：

Between the quality but high expression in a variety of human cancer tissues, including almost all of celiothelioma and cancer of pancreas peace treaty 70% oophoroma and about 50% adenocarcinoma of lung and other cancers, such as cholangiocarcinoma, gastric cancer, intestinal cancer, the cancer of the esophagus, breast cancer. Therefore, quality represents a target spot for having huge attraction in immunotherapy of tumors field.

Based on above-mentioned discovery, inventors herein proposes a kind of nucleic acid molecules carrying silenced cell immunologic test point and encode embedding The transgenosis lymphocyte formed after the construct of the nucleic acid molecules of conjunction antigen receptor and a kind of importing with this construct, coding Chimeric antigen receptor molecule of the antigen binding MSLN.Therefore, construct and transgenosis lymphocyte proposed by the present invention is available In tumour, especially between quality positive tumor adoptive T cell immunization therapy；Transgenosis lymphocyte pair proposed by the invention The specific killing ability of quality tumour is strong between height expression, has weaker killing to the mesothelial cell of normal MSLN expressions.

In the first aspect of the present invention, the present invention proposes a kind of T lymphocytes.According to an embodiment of the invention, the T The cellular immunity checkpoint of lymphocyte is silenced；And expression Chimeric antigen receptor, wherein the Chimeric antigen receptor packet It includes：Extracellular region, the extracellular region include the heavy chain variable region and light chain variable region of single-chain antibody, and the single-chain antibody specificity is known Other antigen MSLN；Transmembrane region, the transmembrane region are connected with the extracellular region, and are embedded into the cell membrane of the T lymphocytes In；Intracellular region, the intracellular region are connected with the transmembrane region, and the intracellular region includes the intracellular section and CD3 ζ of CD28 Chain.Wherein, cellular immunity checkpoint includes cell surface or at least one intracellular immunologic test point.Reality according to the present invention Example is applied, the T lymphocytes of the embodiment of the present invention, which have, resists the immunosuppressive characteristic that tumour cell mediates, proliferation in vitro Ability, the proliferation in tumour patient body and survival ability significantly improve, and are significantly increased to the killing ability of tumour cell, especially There is significant orientation lethal effect to the tumour cell of high expression MSLN.

In the second aspect of the present invention, the present invention proposes a kind of slow virus.According to an embodiment of the invention, the slow disease Poison carries following nucleic acid molecules：The nucleic acid molecules of encoding chimeric antigen receptor, the Chimeric antigen receptor have SEQ ID NO：1 Shown in amino acid sequence, the nucleic acid molecules of the encoding chimeric antigen receptor have SEQ ID NO：Nucleotides sequence shown in 2 Row；And the nucleic acid molecules of silenced cell immunologic test point, the nucleotide of the nucleic acid molecules of the silenced cell immunologic test point Sequence is selected from SEQ ID NO：At least one of 3~135.

MVLLVTSLLLCELPHPAFLLIPDIQAQVQLVQSGAEVKRPGASVQVSCRASGYSINTYYMQWVRQAPGAGLEWMGVI NPSGVTSYAQKFQGRVTLTNDTSTNTVYMQLNSLTSADTAVYYCARWALWGDFGMDVWGKGTLVTVSSGGGGSGGGG SGGGGSDIQMTQSPSTLSASIGDRVTITCRASEGIYHWLAWYQQKPGKAPKLLIYKASSLASGAPSRFSGSGSGTDF TLTISSLQPDDFATYYCQQYSNYPLTFGGGTKLEIKRASFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF PEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO：1).

ATGGTTCTGCTGGTGACATCTCTCCTGCTCTGTGAACTGCCTCATCCCGCTTTTCTGCTCATTCCCGACATTCAGGC TCAAGTCCAACTGGTCCAAAGTGGTGCTGAAGTCAAACGCCCGGGTGCCTCCGTCCAAGTCTCCTGCCGTGCCTCTG GCTACTCGATTAACACCTATTACATGCAGTGGGTCCGTCAAGCACCGGGTGCAGGTCTGGAATGGATGGGTGTCATC AATCCGTCCGGCGTGACCTCATATGCGCAGAAATTTCAAGGTCGCGTTACCCTGACGAACGATACCAGCACGAATAC CGTCTACATGCAGCTGAACTCTCTGACGAGTGCAGACACCGCGGTGTATTACTGCGCACGTTGGGCACTGTGGGGCG ATTTCGGCATGGATGTTTGGGGCAAAGGTACGCTGGTGACCGTTAGCTCTGGTGGTGGTGGTTCTGGTGGTGGTGGT AGTGGCGGTGGCGGTTCTGATATTCAGATGACGCAAAGCCCGTCTACCCTGAGTGCCTCCATTGGTGACCGTGTTAC GATCACCTGTCGCGCATCCGAAGGCATCTATCATTGGCTGGCTTGGTACCAGCAAAAACCGGGTAAAGCGCCGAAAC TGCTGATCTATAAAGCAAGTTCCCTGGCATCGGGTGCTCCGAGCCGCTTTTCAGGTTCGGGTAGCGGCACCGATTTC ACGCTGACCATCTCATCGCTGCAGCCGGACGATTTCGCTACCTACTACTGCCAACAATACTCAAACTACCCGCTGAC CTTCGGTGGAGGGACCAAGCTGGAGATCAAACGTGCTAGCTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGA CGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGG CCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGG GACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAACAAACGGGGCAGAAAGAAAC TCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTT CCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGG CCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGG ACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATG GCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCT CAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA(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 an embodiment of the invention, the slow virus of the present invention is imported into the transgenosis lymphocyte obtained by lymphocyte, It, which has, resists the immunosuppressive characteristic that tumour cell mediates, proliferative capacity in vitro, the proliferation in tumour patient body It is significantly increased with survival ability, the killing ability of tumour cell is significantly increased, especially had to the tumour cell of high expression MSLN There is significant orientation lethal effect.

In the third aspect of the present invention, the present invention proposes a kind of slow virus.According to an embodiment of the invention, the slow disease Poison, which carries, contains SEQ ID NO：136, nucleotide sequence shown in 137,138,139,140 or 141.

ATGGTTCTGCTGGTGACATCTCTCCTGCTCTGTGAACTGCCTCATCCCGCTTTTCTGCTCATTCCCGACATTCAGGC TCAAGTCCAACTGGTCCAAAGTGGTGCTGAAGTCAAACGCCCGGGTGCCTCCGTCCAAGTCTCCTGCCGTGCCTCTG GCTACTCGATTAACACCTATTACATGCAGTGGGTCCGTCAAGCACCGGGTGCAGGTCTGGAATGGATGGGTGTCATC AATCCGTCCGGCGTGACCTCATATGCGCAGAAATTTCAAGGTCGCGTTACCCTGACGAACGATACCAGCACGAATAC CGTCTACATGCAGCTGAACTCTCTGACGAGTGCAGACACCGCGGTGTATTACTGCGCACGTTGGGCACTGTGGGGCG ATTTCGGCATGGATGTTTGGGGCAAAGGTACGCTGGTGACCGTTAGCTCTGGTGGTGGTGGTTCTGGTGGTGGTGGT AGTGGCGGTGGCGGTTCTGATATTCAGATGACGCAAAGCCCGTCTACCCTGAGTGCCTCCATTGGTGACCGTGTTAC GATCACCTGTCGCGCATCCGAAGGCATCTATCATTGGCTGGCTTGGTACCAGCAAAAACCGGGTAAAGCGCCGAAAC TGCTGATCTATAAAGCAAGTTCCCTGGCATCGGGTGCTCCGAGCCGCTTTTCAGGTTCGGGTAGCGGCACCGATTTC ACGCTGACCATCTCATCGCTGCAGCCGGACGATTTCGCTACCTACTACTGCCAACAATACTCAAACTACCCGCTGAC CTTCGGTGGAGGGACCAAGCTGGAGATCAAACGTGCTAGCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTC CTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGG GGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGT GATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGA CTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTC AGCCGCAGCGCAGATGCTCCAGCCTACCAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGA GGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAG AGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGA AGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCA GGCCCTGCCGCCTCGGTAATCCTACTGCgtcgaGCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTC CCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATT GTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGC AGGCATGCTGGGGATGCGGTGGGCTCTATGGGTCGACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCT TCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGT ACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATC ATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCTCCCCA GGCGCAGATCAAAGAGAGTTCAAGAGACTCTCTTTGATCTGCGCCTTTTTT(SEQ ID NO：136).

ATGGTTCTGCTGGTGACATCTCTCCTGCTCTGTGAACTGCCTCATCCCGCTTTTCTGCTCATTCCCGACATTCAGGC TCAAGTCCAACTGGTCCAAAGTGGTGCTGAAGTCAAACGCCCGGGTGCCTCCGTCCAAGTCTCCTGCCGTGCCTCTG GCTACTCGATTAACACCTATTACATGCAGTGGGTCCGTCAAGCACCGGGTGCAGGTCTGGAATGGATGGGTGTCATC AATCCGTCCGGCGTGACCTCATATGCGCAGAAATTTCAAGGTCGCGTTACCCTGACGAACGATACCAGCACGAATAC CGTCTACATGCAGCTGAACTCTCTGACGAGTGCAGACACCGCGGTGTATTACTGCGCACGTTGGGCACTGTGGGGCG ATTTCGGCATGGATGTTTGGGGCAAAGGTACGCTGGTGACCGTTAGCTCTGGTGGTGGTGGTTCTGGTGGTGGTGGT AGTGGCGGTGGCGGTTCTGATATTCAGATGACGCAAAGCCCGTCTACCCTGAGTGCCTCCATTGGTGACCGTGTTAC GATCACCTGTCGCGCATCCGAAGGCATCTATCATTGGCTGGCTTGGTACCAGCAAAAACCGGGTAAAGCGCCGAAAC TGCTGATCTATAAAGCAAGTTCCCTGGCATCGGGTGCTCCGAGCCGCTTTTCAGGTTCGGGTAGCGGCACCGATTTC ACGCTGACCATCTCATCGCTGCAGCCGGACGATTTCGCTACCTACTACTGCCAACAATACTCAAACTACCCGCTGAC CTTCGGTGGAGGGACCAAGCTGGAGATCAAACGTGCTAGCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTC CTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGG GGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGT GATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGA CTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTC AGCCGCAGCGCAGATGCTCCAGCCTACCAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGA GGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAG AGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGA AGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCA GGCCCTGCCGCCTCGGTAATCCTACTGCgtcgaGCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTC CCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATT GTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGC AGGCATGCTGGGGATGCGGTGGGCTCTATGGGTCGACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCT TCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGT ACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATC ATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCTCCCCA ACACAGACGCCATGATTTGCTTCAAGAGAGCAAATCATGGCGTCTGTGTTTTTTT(SEQ ID NO：137).

ATGGTTCTGCTGGTGACATCTCTCCTGCTCTGTGAACTGCCTCATCCCGCTTTTCTGCTCATTCCCGACATTCAGGC TCAAGTCCAACTGGTCCAAAGTGGTGCTGAAGTCAAACGCCCGGGTGCCTCCGTCCAAGTCTCCTGCCGTGCCTCTG GCTACTCGATTAACACCTATTACATGCAGTGGGTCCGTCAAGCACCGGGTGCAGGTCTGGAATGGATGGGTGTCATC AATCCGTCCGGCGTGACCTCATATGCGCAGAAATTTCAAGGTCGCGTTACCCTGACGAACGATACCAGCACGAATAC CGTCTACATGCAGCTGAACTCTCTGACGAGTGCAGACACCGCGGTGTATTACTGCGCACGTTGGGCACTGTGGGGCG ATTTCGGCATGGATGTTTGGGGCAAAGGTACGCTGGTGACCGTTAGCTCTGGTGGTGGTGGTTCTGGTGGTGGTGGT AGTGGCGGTGGCGGTTCTGATATTCAGATGACGCAAAGCCCGTCTACCCTGAGTGCCTCCATTGGTGACCGTGTTAC GATCACCTGTCGCGCATCCGAAGGCATCTATCATTGGCTGGCTTGGTACCAGCAAAAACCGGGTAAAGCGCCGAAAC TGCTGATCTATAAAGCAAGTTCCCTGGCATCGGGTGCTCCGAGCCGCTTTTCAGGTTCGGGTAGCGGCACCGATTTC ACGCTGACCATCTCATCGCTGCAGCCGGACGATTTCGCTACCTACTACTGCCAACAATACTCAAACTACCCGCTGAC CTTCGGTGGAGGGACCAAGCTGGAGATCAAACGTGCTAGCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTC CTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGG GGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGT GATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGA CTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTC AGCCGCAGCGCAGATGCTCCAGCCTACCAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGA GGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAG AGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGA AGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCA GGCCCTGCCGCCTCGGTAATCCTACTGCgtcgaGCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTC CCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATT GTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGC AGGCATGCTGGGGATGCGGTGGGCTCTATGGGTCGACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCT TCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGT ACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATC ATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCTCCCCG CATCACTTGGGATTAATATTCAAGAGATATTAATCCCAAGTGATGCTTTTT(SEQ ID NO：138).

ATGGTTCTGCTGGTGACATCTCTCCTGCTCTGTGAACTGCCTCATCCCGCTTTTCTGCTCATTCCCGACATTCAGGC TCAAGTCCAACTGGTCCAAAGTGGTGCTGAAGTCAAACGCCCGGGTGCCTCCGTCCAAGTCTCCTGCCGTGCCTCTG GCTACTCGATTAACACCTATTACATGCAGTGGGTCCGTCAAGCACCGGGTGCAGGTCTGGAATGGATGGGTGTCATC AATCCGTCCGGCGTGACCTCATATGCGCAGAAATTTCAAGGTCGCGTTACCCTGACGAACGATACCAGCACGAATAC CGTCTACATGCAGCTGAACTCTCTGACGAGTGCAGACACCGCGGTGTATTACTGCGCACGTTGGGCACTGTGGGGCG ATTTCGGCATGGATGTTTGGGGCAAAGGTACGCTGGTGACCGTTAGCTCTGGTGGTGGTGGTTCTGGTGGTGGTGGT AGTGGCGGTGGCGGTTCTGATATTCAGATGACGCAAAGCCCGTCTACCCTGAGTGCCTCCATTGGTGACCGTGTTAC GATCACCTGTCGCGCATCCGAAGGCATCTATCATTGGCTGGCTTGGTACCAGCAAAAACCGGGTAAAGCGCCGAAAC TGCTGATCTATAAAGCAAGTTCCCTGGCATCGGGTGCTCCGAGCCGCTTTTCAGGTTCGGGTAGCGGCACCGATTTC ACGCTGACCATCTCATCGCTGCAGCCGGACGATTTCGCTACCTACTACTGCCAACAATACTCAAACTACCCGCTGAC CTTCGGTGGAGGGACCAAGCTGGAGATCAAACGTGCTAGCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTC CTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGG GGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGT GATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGA CTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTC AGCCGCAGCGCAGATGCTCCAGCCTACCAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGA GGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAG AGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGA AGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCA GGCCCTGCCGCCTCGGTAATCCTACTGCgtcgaGCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTC CCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATT GTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGC AGGCATGCTGGGGATGCGGTGGGCTCTATGGGTCGACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCT TCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGT ACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATC ATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCTCCCCA GGCGCAGATCAAAGAGAGTTCAAGAGACTCTCTTTGATCTGCGCCTTTTTTAGCTATCGATAGCTAAAAAAACACAG ACGCCATGATTTGCTCTCTTGAAGCAAATCATGGCGTCTGTGTTGGGGAAGATCTGTGGTCTCATACAGAACTTATA AGATTCCCAAATCCAAAGACATTTCACGTTTATGGTGATTTCCCAGAACACATAGCGACATGCAAATATTGCAGGGC GCCACTCCCCTGTCCCTCACAGCCATCTTCCTGCCAGGGCGCACGCGCGCTGGGTGTTCCCGCCTAGTGACACTGGG CCCGCGATTCCTTGGAGCGGGTTGATGACGTCAGCGTTCGAATTGTCGAC(SEQ ID NO：139).

ATGGTTCTGCTGGTGACATCTCTCCTGCTCTGTGAACTGCCTCATCCCGCTTTTCTGCTCATTCCCGACATTCAGGC TCAAGTCCAACTGGTCCAAAGTGGTGCTGAAGTCAAACGCCCGGGTGCCTCCGTCCAAGTCTCCTGCCGTGCCTCTG GCTACTCGATTAACACCTATTACATGCAGTGGGTCCGTCAAGCACCGGGTGCAGGTCTGGAATGGATGGGTGTCATC AATCCGTCCGGCGTGACCTCATATGCGCAGAAATTTCAAGGTCGCGTTACCCTGACGAACGATACCAGCACGAATAC CGTCTACATGCAGCTGAACTCTCTGACGAGTGCAGACACCGCGGTGTATTACTGCGCACGTTGGGCACTGTGGGGCG ATTTCGGCATGGATGTTTGGGGCAAAGGTACGCTGGTGACCGTTAGCTCTGGTGGTGGTGGTTCTGGTGGTGGTGGT AGTGGCGGTGGCGGTTCTGATATTCAGATGACGCAAAGCCCGTCTACCCTGAGTGCCTCCATTGGTGACCGTGTTAC GATCACCTGTCGCGCATCCGAAGGCATCTATCATTGGCTGGCTTGGTACCAGCAAAAACCGGGTAAAGCGCCGAAAC TGCTGATCTATAAAGCAAGTTCCCTGGCATCGGGTGCTCCGAGCCGCTTTTCAGGTTCGGGTAGCGGCACCGATTTC ACGCTGACCATCTCATCGCTGCAGCCGGACGATTTCGCTACCTACTACTGCCAACAATACTCAAACTACCCGCTGAC CTTCGGTGGAGGGACCAAGCTGGAGATCAAACGTGCTAGCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTC CTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGG GGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGT GATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGA CTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTC AGCCGCAGCGCAGATGCTCCAGCCTACCAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGA GGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAG AGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGA AGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCA GGCCCTGCCGCCTCGGTAATCCTACTGCgtcgaGCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTC CCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATT GTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGC AGGCATGCTGGGGATGCGGTGGGCTCTATGGGTCGACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCT TCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGT ACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATC ATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCTCCCCA GGCGCAGATCAAAGAGAGTTCAAGAGACTCTCTTTGATCTGCGCCTTTTTTAGCTATCGATAGCTAAAAAGCATCAC TTGGGATTAATATCTCTTGAATATTAATCCCAAGTGATGCGGGGAAGATCTGTGGTCTCATACAGAACTTATAAGAT TCCCAAATCCAAAGACATTTCACGTTTATGGTGATTTCCCAGAACACATAGCGACATGCAAATATTGCAGGGCGCCA CTCCCCTGTCCCTCACAGCCATCTTCCTGCCAGGGCGCACGCGCGCTGGGTGTTCCCGCCTAGTGACACTGGGCCCG CGATTCCTTGGAGCGGGTTGATGACGTCAGCGTTCGAATTGTCGAC(SEQ ID NO：140).

ATGGTTCTGCTGGTGACATCTCTCCTGCTCTGTGAACTGCCTCATCCCGCTTTTCTGCTCATTCCCGACATTCAGGC TCAAGTCCAACTGGTCCAAAGTGGTGCTGAAGTCAAACGCCCGGGTGCCTCCGTCCAAGTCTCCTGCCGTGCCTCTG GCTACTCGATTAACACCTATTACATGCAGTGGGTCCGTCAAGCACCGGGTGCAGGTCTGGAATGGATGGGTGTCATC AATCCGTCCGGCGTGACCTCATATGCGCAGAAATTTCAAGGTCGCGTTACCCTGACGAACGATACCAGCACGAATAC CGTCTACATGCAGCTGAACTCTCTGACGAGTGCAGACACCGCGGTGTATTACTGCGCACGTTGGGCACTGTGGGGCG ATTTCGGCATGGATGTTTGGGGCAAAGGTACGCTGGTGACCGTTAGCTCTGGTGGTGGTGGTTCTGGTGGTGGTGGT AGTGGCGGTGGCGGTTCTGATATTCAGATGACGCAAAGCCCGTCTACCCTGAGTGCCTCCATTGGTGACCGTGTTAC GATCACCTGTCGCGCATCCGAAGGCATCTATCATTGGCTGGCTTGGTACCAGCAAAAACCGGGTAAAGCGCCGAAAC TGCTGATCTATAAAGCAAGTTCCCTGGCATCGGGTGCTCCGAGCCGCTTTTCAGGTTCGGGTAGCGGCACCGATTTC ACGCTGACCATCTCATCGCTGCAGCCGGACGATTTCGCTACCTACTACTGCCAACAATACTCAAACTACCCGCTGAC CTTCGGTGGAGGGACCAAGCTGGAGATCAAACGTGCTAGCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTC CTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGG GGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGT GATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGA CTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTC AGCCGCAGCGCAGATGCTCCAGCCTACCAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGA GGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAG AGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGA AGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCA GGCCCTGCCGCCTCGGTAATCCTACTGCGTCGAGCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTC CCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATT GTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGC AGGCATGCTGGGGATGCGGTGGGCTCTATGGGTCGACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCT TCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGT ACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATC ATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCTCCCCA GGCGCAGATCAAAGAGAGTTCAAGAGACTCTCTTTGATCTGCGCCTTTTTTAGCTATCGATAGCTAAAAAGCCTAGA GAAGTTTCAGGGAATCTCTTGAATTCCCTGAAACTTCTCTAGGCGGGGAAGATCTGTGGTCTCATACAGAACTTATA AGATTCCCAAATCCAAAGACATTTCACGTTTATGGTGATTTCCCAGAACACATAGCGACATGCAAATATTGCAGGGC GCCACTCCCCTGTCCCTCACAGCCATCTTCCTGCCAGGGCGCACGCGCGCTGGGTGTTCCCGCCTAGTGACACTGGG CCCGCGATTCCTTGGAGCGGGTTGATGACGTCAGCGTTCGAATTGTCGAC(SEQ ID NO：141).

In the fourth aspect of the present invention, the present invention proposes a kind of transgenosis lymphocyte.According to an embodiment of the invention, The lymphocyte cell immunologic test point is silenced；And expression Chimeric antigen receptor, the Chimeric antigen receptor include：Born of the same parents Outskirt, the extracellular region include the heavy chain variable region and light chain variable region of antibody, and the antibody can be with specific for tumour antigen In conjunction with；Transmembrane region；And intracellular region, the intracellular region include co-stimulators intracellular section, wherein the antibody is single-stranded Antibody, the tumour antigen are MSLN.Inventor has surprisingly found that cellular immunity checkpoint is silenced and expresses the embedding of anti-MSLN Close in-vitro multiplication ability, the proliferation in tumour patient body and the survival ability of the lymphocyte of antigen receptor and in neoplastic disease The specific killing ability to tumour cell in human body greatly improves, and especially has to the tumour cell of high expression MSLN notable Orientation lethal effect.

According to an embodiment of the invention, above-mentioned transgenosis lymphocyte can also have following additional technical feature at least it One：

According to an embodiment of the invention, the lymphocyte cell immunologic test point independently selected from CTLA4, PD1, At least one of TIM3, BTLA, LAG-3, IRAK-M, SOCS1, A20, CBL-B.Wherein, CTLA4, PD1, TIM3, BTLA, LAG-3 is cell surface immunologic test point, and IRAK-M, SOCS1, A20, CBL-B are intracellular immunity checkpoint.The present invention is implemented The immunologic test point of example has the function of negative regulation and weakens cellullar immunologic response, is matched by corresponding on tumour cell The specific bond of body, the downward for causing T lymphoproliferatives to react, the secretion of cell factor reduce and the incapability of T cell or Apoptosis.According to an embodiment of the invention, the successful silence of the cell surface of the embodiment of the present invention or intracellular immunity checkpoint, into One step improves the effect of transgenosis lymphocyte resists the immunosupress that tumour mediates, and transgenosis lymphocyte expands in vitro And proliferation in tumour patient body and survival ability, the orientation lethal effect of tumour cell is further strengthened.

According to an embodiment of the invention, it is by shRNA, instead that immunologic test point in the lymphocyte cell surface, which is silenced, What at least one phosphorothioate odn, ribozyme, dominant negative mutations, CRISPR and Zinc finger nuclease were realized.According to an embodiment of the invention, originally The successful silence of the cellular immunity checkpoint of inventive embodiments, the lymphocyte for being remarkably improved the embodiment of the present invention resist tumour The immunosuppressive characteristic mediated, further improves orientation lethal effect of the transgenosis lymphocyte to tumour cell.

According to an embodiment of the invention, the co-stimulators intracellular section independently selected from 4-1BB, OX-40, At least one of CD40L, CD27, CD30, CD28 and their derivative.The co-stimulators born of the same parents of the embodiment of the present invention The expression of inner segment and the silence joint of cellular immunity checkpoint have the function of positive regulation and enhancing cellullar immunologic response, make The transgenosis lymphopoiesis for obtaining the embodiment of the present invention is more notable to the orientation lethal effect effect of tumour；The present invention is implemented The joint of the expression of the co-stimulators intracellular section of example and the silence of cellular immunity checkpoint so that the embodiment of the present invention The proliferative capacity of transgenosis lymphocyte and more notable to the orientation lethal effect of tumour.

According to an embodiment of the invention, the lymphocyte cell immunologic test point is CTLA4, PD1, CBL-B.Wherein, CTLA4, PD1 are cell surface immunologic test points, and CBL-B is intracellular immunity checkpoint.According to an embodiment of the invention, this hair The lymphocyte cell surface immunologic test point CTLA4 or PD1 of bright embodiment be silenced or lymphocyte cell in immunologic test Point is that CBL-B is silenced, and prevents PD1 or CTLA4 developed by molecule and its respective ligand PD-L1 and PD-L2 or CD80 and CD86 Combination, to effectively inhibit incapability or apoptosis to T lymphocytes, or pass through CBL-B silences, enhancing T cell receptor letter Number conduction so that proliferation and survival ability of the transgenosis lymphocyte in tumour patient body are further enhanced, to tumour Orientation lethal effect effect it is more notable.

According to an embodiment of the invention, immunologic test point, which is silenced, in the lymphocyte cell is realized by shRNA 's.According to an embodiment of the invention, the shRNA of the embodiment of the present invention carry specific silenced cell surface or it is intracellular at least it The shRNA of the shRNA of 1 immunologic test point, the embodiment of the present invention have silenced cell surface or intracellular efficiently, specific At least one immunologic test point effect, the successful silence of cellular immunity checkpoint, i.e. cell surface or intracellular immunity inspection The successful silence of point, prevents the specific binding of immunologic test point and respective ligand, to effectively inhibit immunologic test point The negative regulation mechanism of or apoptosis incompetent to T lymphocytes etc., so that the transgenosis lymphocyte of the embodiment of the present invention is swollen Tumor proliferation in patient body and survival ability are further enhanced, and coordinate the antigen targeting of Chimeric antigen receptor so that this The transgenosis lymphocyte of inventive embodiments is more notable to the orientation lethal effect effect of tumour.

According to an embodiment of the invention, the co-stimulators intracellular section is the intracellular section of 4-1BB or CD28.This hair The co-stimulators intracellular section of the Chimeric antigen receptor of transgenosis lymphocyte in bright is the intracellular of CD28 or 4-1BB Section.According to an embodiment of the invention, co-stimulators intracellular section is the intracellular section of CD28 or 4-1BB, is further enhanced The orientation lethal effect of the transgenosis lymphocyte of the embodiment of the present invention.

According to an embodiment of the invention, the lymphocyte is CD3⁺T lymphocytes or natural killer cells are killed naturally Hinder T cell.The cellular immunity checkpoint of the above-mentioned lymphocyte of the embodiment of the present invention is silenced, while expressing antigentic specificity Chimeric antigen receptor, such as the Chimeric antigen receptor of the MSLN antigentic specificities of the embodiment of the present invention, above-mentioned lymphocyte it is thin The targeting of born of the same parents' immunologic cytotoxicity effect is stronger, and proliferation and survival ability in tumour patient body are further enhanced, to swollen The orientation lethal effect effect of tumor is more notable.

In the ninth aspect of the present invention, the present invention proposes a kind of construct.According to an embodiment of the invention, the structure Body includes：First nucleic acid molecules, the first nucleic acid molecule encoding Chimeric antigen receptor；And second nucleic acid molecules, described 2 nucleic acid molecules silenced cell immunologic test points.Wherein, for example preceding institute of the cellular immunity checkpoint, the Chimeric antigen receptor It states.It according to an embodiment of the invention, can after the construct of the embodiment of the present invention successfully imports the lymphocyte of the embodiment of the present invention The Chimeric antigen receptor of effective reticence cell surface or at least one intracellular immunologic test point and expression antigentic specificity, to The lymphocyte of the embodiment of the present invention is to tumour cell, and the orientation lethal effect of the tumour cell of especially high expression MSLN is more Significantly.

According to an embodiment of the invention, above-mentioned construct can further include following additional technical feature at least it One：

According to an embodiment of the invention, it is characterised in that first nucleic acid molecules are set with second nucleic acid molecules Silenced cell immunologic test point and the expression Chimeric antigen receptor in the lymphocyte in front.Reality according to the present invention Example is applied, the lymphocyte of above-mentioned first nucleic acid molecules and the second nucleic acid molecules, the cell table of lymphocyte are successfully provided with Face or at least one intracellular immunologic test point are by success silence, while in lymphocytic cell surface successful expression antigen-specific Sex-mosaicism antigen receptor, it is stronger and special with lethality such as the Chimeric antigen receptor of the MSLN specificity of the embodiment of the present invention Anisotropic stronger tumor-killing effect.

According to an embodiment of the invention, the construct further comprises：First promoter, first promoter and institute The first nucleic acid molecules are stated to be operably connected；And second promoter, second promoter can with second nucleic acid molecules It is operatively connected.According to an embodiment of the invention, the introducing of the first promoter and the second promoter so that the first nucleic acid molecules And the second nucleic acid molecules expression independently, be effectively ensured Chimeric antigen receptor antigen targeting biological action and With effective reticence cellular immunity checkpoint so that the targeting of the lymphocyte of the embodiment of the present invention is stronger, to tumour Lethal effect, it is especially more notable to the orientation killing of the tumour cell of high expression MSLN.

According to an embodiment of the invention, first promoter, second promoter are separately selected from U6, H1, CMV, EF-1, LTR, RSV promoter.According to an embodiment of the invention, the above-mentioned promoter of the embodiment of the present invention has to start and imitate The characteristics of rate height, high specificity, to ensure that the efficient silence of cellular immunity checkpoint and the efficient table of Chimeric antigen receptor It reaches, it is big to the in-vitro multiplication ability, the proliferation in tumour patient body and survival ability of the lymphocyte of the embodiment of the present invention It is big to improve, it is more notable to the orientation fragmentation effect of tumour.

According to an embodiment of the invention, the carrier of the construct is non-pathogenic virus carrier.Non-pathogenic virus carries Body is introduced into the duplication and amplification efficiency for substantially increasing construct in lymphocyte, to substantially increase cellular immunity inspection High efficient expression of the silence and Chimeric antigen receptor made an inventory of in lymphocyte so that Lymphocyte Proliferation in Vitro ability is swelling Tumor proliferation in patient body and survival ability greatly improve, and the targeting of lymphocyte further enhances, to tumour cell Lethal effect is more notable.

According to an embodiment of the invention, the viral vectors includes being selected from retrovirus vector, slow virus carrier or gland At least one of viral related viral vectors.The carrier of the virus of the embodiment of the present invention is in virus packaging and course of infection, disease Malicious infection scope is extensive, can not only infect terminally differentiated cells, but also can infect the cell in division stage, genome can both be integrated It to host chromosome, and can be free in except host chromosome, so that wide spectrum can be realized and efficient efficiency of infection, cellular immunity High efficient expression of the checkpoint by efficient silence and Chimeric antigen receptor in lymphocyte so that the lymph of the embodiment of the present invention is thin In-vitro multiplication ability, the proliferation in tumour patient body and the survival ability of born of the same parents greatly improves, the targeting of lymphocyte into One step enhances, especially more notable to the orientation lethal effect of the tumour cell of high expression MSLN to tumour cell.

In the tenth aspect of the present invention, the present invention, which proposes, a kind of preparing foregoing T lymphocytes or transgenosis The method of lymphocyte.According to an embodiment of the invention, the method includes：By foregoing construct or noted earlier Slow virus be introduced into lymphocyte or T lymphocytes.The construct or slow virus are successfully introduced into above-mentioned lymphocyte Or in T lymphocytes, the cellular immunity inspection for realizing lymphocyte is silenced expression with Chimeric antigen receptor, to this Transgenosis lymphocyte or T lymphocytes prepared by the preparation methods of the inventive embodiments proliferation internal and external in tumour patient And survival ability greatly improves in tumour patient body, transgenosis lymphocyte or T lymphocytes are to tumour cell, especially to height The targeting killing effect for expressing the tumour cell of MSLN is stronger.

In the eleventh aspect of the present invention, the present invention proposes a kind of therapeutic combination for treating cancer.According to this The embodiment of invention, the therapeutic combination include：Above-mentioned construct, slow virus, T lymphocytes or transgenosis lymph are thin Born of the same parents.The composition of any one of the above therapeutic combination can be achieved transgenosis lymphocyte or T lymphocytes cell surface or High efficient expression of the silence and Chimeric antigen receptor of intracellular immunity checkpoint in transgenosis lymphocyte or T lymphocytes, So that gained transgenosis lymphocyte or T lymphocytes have the significant immunosupress resisted tumour cell and mediated, Survival ability greatly improves in the proliferation and tumour patient body of tumour patient in vitro and in vivo, transgenosis lymphocyte or T lymphs The targeting killing effect of cells against tumor cells is stronger, and the therapeutic combination of the treating cancer of the embodiment of the present invention is to tumour cell Targeting killing effect significantly increase, especially the targeting killing effect of the tumour cell of high expression MSLN is significantly increased.

According to an embodiment of the invention, above-mentioned therapeutic combination can further include following additional technical feature at least One of：

According to an embodiment of the invention, the cancer includes being selected from celiothelioma, cancer of pancreas, oophoroma, cholangiocarcinoma, lung cancer, Gastric cancer, intestinal cancer, at least one of the cancer of the esophagus and breast cancer.Above-mentioned tumour cell has the specificity overexpression of MSLN, the present invention The therapeutic combination of embodiment can make the silence and high efficient expression antigen on lymphocyte cell surface or intracellular immunity checkpoint Specific chimeric antigen receptor, such as the antigen-specific chimerics the MSLN antigen receptor of the embodiment of the present invention, gained lymphocyte or T Lymphocyte has the significant immunosuppressive characteristic resisted tumour cell and mediated, the survival ability in the microenvironment of tumour It greatly improves, gained lymphocyte or T lymphocytes are stronger to the targeting killing effect of the tumour cell of high expression MSLN.

In the twelveth aspect of the present invention, the present invention proposes a kind of method improving lymphocyte activity, the lymph Cell carries Chimeric antigen receptor, according to an embodiment of the invention, the method includes：Make the cellular immunity of the lymphocyte Checkpoint is silenced.The cellular immunity checkpoint, the lymphocyte and the Chimeric antigen receptor be as defined above, The lymphocyte activity include the Lymphocyte Proliferation in Vitro ability, the proliferation in tumour patient body and survival ability with And at least one of orientation killing ability of the lymphocyte in tumour patient body.According to an embodiment of the invention, this hair The cell surface of the lymphocyte of bright embodiment or intracellular immunity checkpoint are silenced, and lymphocyte is activated, is Hypertrophic anti- It should raise, cytokine secretion increases, anti-tune dies ability enhancing so that the lymphocyte of the embodiment of the present invention expands in vitro, Survival ability greatly improves in proliferation and tumour patient body in tumour patient body, above-mentioned lymphocyte cell immunologic test point Silence cooperation lymphocyte Chimeric antigen receptor antigentic specificity effect, to realize be effective against tumour cell mediate Immunosupress, the targeting killing effect of the tumour cell of high expression MSLN is significantly increased.

According to an embodiment of the invention, the method for above-mentioned raising lymphocyte activity can further include following additional At least one technical characteristic：

According to an embodiment of the invention, the tumour includes being selected from celiothelioma, cancer of pancreas, oophoroma, cholangiocarcinoma, lung cancer, Gastric cancer, intestinal cancer, at least one of the cancer of the esophagus and breast cancer.Above-mentioned tumor cell specific height expresses MSLN.The embodiment of the present invention Raising lymphocyte activity method, make the Chimeric antigen receptor of Expressions In Lymphocytes MSLN antigentic specificities, while making leaching The immunologic test point of bar cell is silenced, and the method for the raising lymphocyte activity of the embodiment of the present invention is further improved to height Express MSLN tumour cell orientation kill ability, such as above-mentioned celiothelioma, cancer of pancreas, oophoroma, cholangiocarcinoma, lung cancer, gastric cancer, Intestinal cancer, the cancer of the esophagus or breast cancer cell.

It should be noted that the term " cellular immunity checkpoint " used in the present invention includes cell surface immunologic test Point and intracellular immunity checkpoint, " cell surface immunologic test point " is a kind of memebrane protein of lymphocytic cell surface, with tumour The ligand interaction expressed on cell, can inhibit antitumor lymphocyte reaction." intracellular immunity checkpoint " is a kind of A kind of intracellular protein, the cellular signal transduction mechanism albumen of negative regulation of such cellular immunity checkpoint albumen, can inhibit anti- Tumor lympha cell effect.

Description of the drawings

Fig. 1 is that the Chimeric antigen receptor according to the ... of the embodiment of the present invention for co-expressing MSLN antigentic specificities and the silence mankind are thin The structural schematic diagram of the slow virus carrier of born of the same parents' immunologic test point；

Fig. 2 is the Chimeric antigen receptor and silence PD1 of coexpression MSLN antigentic specificities according to the ... of the embodiment of the present invention Lymphocyte, the result figure of proliferative capacity enhancing；

Fig. 3 is the Chimeric antigen receptor and silence PD1 of coexpression MSLN antigentic specificities according to the ... of the embodiment of the present invention Lymphocyte, interferon-γ secrete the result figure increased；And

Fig. 4 is the Chimeric antigen receptor and silence PD1 of coexpression MSLN antigentic specificities according to the ... of the embodiment of the present invention Lymphocyte, the result figure of killing tumor cell ability enhancing.

Specific implementation mode

The embodiment of the present invention is described below in detail, the embodiments described below is exemplary, it is intended to for explaining this Invention, and be not considered as limiting the invention.

T lymphocytes or transgenosis lymphocyte

In one aspect of the invention, the present invention proposes a kind of T lymphocytes or transgenosis lymphocyte.According to the present invention Embodiment, the cellular immunity checkpoint of the T lymphocytes of the embodiment of the present invention is silenced；And expression Chimeric antigen receptor, Wherein, Chimeric antigen receptor includes：Extracellular region, extracellular region includes the heavy chain variable region and light chain variable region of single-chain antibody, single-stranded Antibody specificity identifies antigen MSLN；Transmembrane region, transmembrane region are connected with extracellular region, and are embedded into the cell of institute's T lymphocytes In film；Intracellular region, intracellular region are connected with transmembrane region, and intracellular region includes the intracellular section and CD3 ζ chains of CD28 or 4-1BB.Its In, cellular immunity checkpoint includes cell surface or intracellular immunologic test point.The T lymphocytes of the embodiment of the present invention turn Gene lymphocyte cell immunologic test point is silenced the Chimeric antigen receptor of Combined expression MSLN antigentic specificities, and the present invention is real Apply example T lymphocytes or transgenosis lymphocyte in the internal and external proliferation of tumour patient and survival ability and in tumour The killing ability to specific tumor cell in patient body significantly increases, especially the spy to the tumour cell of high efficient expression MSLN Anisotropic fragmentation effect greatly improves.

Tumour can close lymphocyte to it to avoid immunosurveillance by stimulating the expression of its inhibitive ability of immunity receptor Immunologic cytotoxicity reaction；As immune negative regulator mechanism, the cytotoxic T lymphocyte (CTLs) of activation also expresses negative regulation Regulatory agency, i.e. cell surface or intracellular immunologic test point molecule.As the embodiment of the present invention apoptosis 1 by Body (PD1) expression interacts on activation CTLs with the programmed death ligand 1 (PD-L1) expressed on tumour cell, can To inhibit antitumor t cell responses.Many tumours express PD-L1, and the combination of PD-L1 and its ligand PD-1 lead to CTLs hyperplasia Property reaction downward, cell factor secretion reduce and T cell incapability or apoptosis.The cytotoxic T of the embodiment of the present invention drenches Bar cellular antigens 4 (CTLA4) are the key that the negative regulatory factors of another T cell, can inhibit T cell activation, by with It expresses the interaction of ligand B7.1, B7.2 (CD80 and CD86) on antigen presenting cell and inhibits the activation of T cell.This CBL-B (E3 ubiquitin protein ligase CBL-B) in the cytotoxic T lymphocyte of inventive embodiments is intracellular another Crucial negative regulatory factor, by inhibiting T cell receptor (TCR) signal transduction, to inhibit the activity of T cell.Therefore, this hair The T lymphocytes of bright embodiment or the immunologic test point of transgenosis lymphocyte are silenced, and T lymphocytes or transgenosis lymph are thin The proliferation in tumour patient body and survival ability of born of the same parents significantly improves.

In addition, according to an embodiment of the invention, the antibody of above-mentioned Chimeric antigen receptor extracellular region is single-chain antibody.Inventor It was found that single-chain antibody can remove the competitive surface protein of nonspecific reaction, while single-chain antibody is more easy to infiltration tumor tissues Increase drug concentration levels.The Chimeric antigen receptor of the transgenosis Expressions In Lymphocytes single-chain antibody of the embodiment of the present invention, significantly Improve orientation lethal effect of the transgenosis lymphocyte to targets neoplastic cells.

The combination antigen of other embodiment according to the present invention, above-mentioned antibody is MSLN.Therefore the embodiment of the present invention Transgenosis lymphocyte there is directionality lethal effect, the specific binding of antigen-antibody for the cell of expression antigen MSLN Act on stronger, the transgenosis lymphocyte for substantially increasing the embodiment of the present invention kills the orientation of MSLN antigen presentation tumour cells Wound acts on.

The cellular immunity checkpoint of other embodiment according to the present invention, lymphocyte includes cell surface and cell Interior immunologic test point, the lymphocyte cell surface immunologic test point of the embodiment of the present invention independently selected from CTLA4, PD1, At least one TIM3, BTLA, LAG-3, in lymphocyte cell immunologic test point independently selected from IRAK-M, SOCS1, A20, At least one of CBL-B.Above-mentioned molecule can be combined with the antigentic specificity of tumor cells expression, inhibit the work of lymphocyte Change, promote the incapability or apoptosis of lymphocyte, to negative regulation and weakens cellullar immunologic response.Implementation according to the present invention The successful silence of example, above-mentioned cell surface or intracellular immunity checkpoint, further improves transgenosis lymphocyte in tumour Proliferation in patient body and survival ability further strengthen the orientation lethal effect of tumour cell.

Other embodiment according to the present invention, the lymphocyte cell surface immunologic test point quilt of the embodiment of the present invention Silence is realized by least one shRNA, antisense nucleic acid, ribozyme, dominant negative mutations, Zinc finger nuclease and CRISPR.

Children purpura nephritis or short hairpin RNA (shRNA) are the importing forms of siRNA (siRNA), and siRNA is a kind of small RNA molecule (is made of) 21~25 nucleotide, (is made with specific cleavage to double-stranded RNA in III families of RNAase by Dicer Enzyme) it is process；SiRNA plays central role in RNA silence accesses, degrades to specific mRNA (mRNA), is Regulate and control after transcriptional level.

Antisense nucleic acid includes antisense RNA and antisense DNA, and antisense RNA refers to can be with one section of small molecule of mRNA complete complementaries RNA or oligonucleotide fragment, antisense DNA refer to can be combined with the sense strand complementation in gene DNA double-strand short and small DNA points Son, antisense RNA and antisense DNA are mainly played a role by the translation of mRNA and the transcription of gene DNA；Antisense nucleic acid one Aspect to form space steric effect by being combined with said target mrna, and ribosomes is prevented to be combined with mRNA, and on the other hand it is tied with mRNA Endogenous RNA enzyme or ribozyme are activated after conjunction, and then the mRNA that degrades；The control region specific bond of antisense DNA and gene DNA double helix DNA tripolymers are formed, or are combined with DNA encoding area, the extension for the mRNA chains transcribed is terminated；Antisense nucleic acid, which may also suppress, to be turned The processing modification of mRNA after record, such as the ends 5' cap, the ends 3' tailing, intermediate montage and internal base methylate, and prevent maturation MRNA is transported from nucleus into cytoplasm, and therefore, antisense RNA is a kind of technology of effective silence target gene.

Ribozyme is the RNA molecule for having catalysis, is biocatalyst, degradable special mRNA sequence, and ribozyme is logical It crosses catalysis and turns phosphate and phosphodiester bond hydrolysis participation RNA itself shearings, process, with general antisense RNA phase There is relatively stable space structure than, ribozyme, be not easily susceptible to the attack of RNA enzyme, it is often more important that, ribozyme after cutting off mRNA, It can be escaped from hybridization chain again, recombine and cut others mRNA molecules.

Dominant negative mutation refers to not only own reactive energy after the mutation of certain signal transducers, moreover it is possible to inhibit or block same The effect of one intracellular wild type signal transducer, it is mainly real by way of forming dimer with wild-type protein Existing, this mutation toxic effect is big, can significantly inhibit or block the effect of intracellular echo signal transducin.

Zinc finger nuclease identifies that domain and a non-specific nucleic acid restriction endonuclease are constituted by a DNA, and DNA identifies that domain is by one Serial Cys2-His2 zinc finger proteins are composed in series (general 3~4), and each zinc finger protein identifies and combines one special three Conjuncted base, zinc finger protein form alpha-beta-β secondary structures, and it is special to determine that the DNA of zinc finger is combined for wherein 16 amino acid residues of α spirals The opposite sex, skeleton structure is conservative, is tied to determining that the change of the amino acid calling sequence of DNA binding specificities can obtain new DNA Specificity is closed, so as to design different amino acid calling sequences for different target gene, realizes different target gene Specific silence.

CRISPR (Clustered regularly interspaced short palindromic repeats, rule The short palindrome in cluster interval repeats), it is a kind of gene editing device, is bacterium to protect themselves against a system of virus.It The target gene of other organisms can be used for deleting, adding, activating or inhibiting, these target genes include the mesh in people's cell Mark gene.

CRISPR clusters are a special repetitive dna sequence families being widely present in bacterium and Archimycetes genome, Sequence is by a leader (Leader), multiple short and highly conserved repetitive sequences (Repeat) and multiple spacer regions (Spacer) it forms.Leader is normally at CRISPR clusters upstream, is considered rich in the region that AT length is 300~500bp It may be the promoter sequence of CRISPR clusters.Repetitive sequence section length is 21~48bp can form hair fastener knot containing palindromic sequence Structure.It is separated by the spacer region that length is 26~72bp between repetitive sequence.The regions Spacer are made of the exogenous DNA captured, when It when exogenous DNA containing same sequence is invaded, can be identified by bacterium body, and carry out shearing and be allowed to expression silencing, reach protection The purpose of inherently safe.By being found to the flanking sequence analysis of CRISPR clusters, there is a polymorphism family base in its vicinity Cause.The protein of family coding, which contains the functional domain that can be had an effect with nucleic acid, (has nuclease, unwindase, integrase With polymerase isoreactivity), and play a role jointly with the regions CRISPR, therefore it is named as CRISPR association bases (CRISPR Associated), it is abbreviated as Cas.Presently found Cas includes the multiple types such as Cas1~Cas10.Cas genes and CRISPR Common evolutionary collectively forms a highly conserved system.When bacterium resists the invasion of the exogenous DNAs such as bacteriophage, in leader Regulation and control under, CRISPR is transcribed into long RNA precursors (Pre RISPR RNA, pre-crRNA), is subsequently processed into a series of The short ripe crRNA containing conservative repetitive sequence and spacer region, finally identifies and is attached to the exogenous DNA array being complementary to Upper performance shear action.The processing of pre-crRNA is participated in by the Cas9 in Cas families.Cas9 contains the RuvC in amino terminal With HNH2 unique active sites in the middle part of protein, play a role in crRNA maturations and double-stranded DNA shearing.pre- While crRNA is transcribed, with the trans-activation crRNA of its repetitive sequence complementation (Trans-activating crRNA, TracrRNA) also transcription comes out, and Cas9 and double-stranded RNA specificity RNase III nucleases is excited to carry out pre-crRNA Processing.After processing is ripe, crRNA, tracrRNA and Cas9 form complex, identify and be incorporated into the sequence of crRNA complementations, so After unlock DNA double chain, form R-loop, so that crRNA and complementary strand thereof, another chain is kept free single-chain state, then The complementary dna chain of crRNA is sheared by the HNH active sites in Cas9, RuvC active sites are sheared incomplementarity chain, are eventually introduced DNA double chain is broken (DSB).By engineer RNA, sgRNA (the short guide to be formed with guiding function can be transformed RNA), to guide Cas9 to cut the pinpoint target gene of DNA.

In conclusion shRNA, antisense nucleic acid, ribozyme, dominant negative mutations, CRISPR, Zinc finger nuclease are specific silence The means of the effective means of target gene, cryptiogene are not particularly limited, and those skilled in the art can be according to specific experiment Purpose and condition selection, shRNA, antisense nucleic acid, ribozyme, dominant negative mutations as used by the embodiment of the present invention, CRISPR or At least one of Zinc finger nuclease realizes the specific silence of target gene.

According to an embodiment of the invention, lymphocyte cell surface or intracellular immunity checkpoint are silenced and preferably use ShRNA is realized.SiRNA molecule entrained by ShRNA is typically the dual zone of base-pair of the length between 10 and 30. PD1 or CTLA4 or the CBL-B siRNA of the embodiment of the present invention are designed to the volume for being derived from PD1 or CTLA4 or CBL-B mRNA Code region, by the degradation of mRNA come inhibition of gene expression.SiRNA, which is associated with, to be referred to as inducing RNA silencing complex (RISC) Multiplexed protein compound, positive-sense strand is by enzymatic lysis during this period.It is based on sequence homology in the RISC being activated, guides RISC To corresponding mRNA；Identical nucleic acid cleavage targets PD1 or CTLA4 or CBL-B mRNA, generate specific gene PD1 or CTLA4 or CBL-B silences inhibit the expression of specific gene PD1 or CTLA4 or CBL-B.SiRNA is imported carefully in the form of shRNA (shRNA includes the nucleotide siRNA sequence of about 18-23, the nucleotide ring and a siRNA of one 9-15 length of heel to born of the same parents The reversed supplement of sequence), the design of shRNA preferably avoids the match point in 3 ' UTR cytogenes；It ensures appropriate Chain selects.One single siRNA molecule can be repeated the division applied to more targeting mRNA molecules.RNAi (RNA interference) can lead to The mode for crossing introducing synthesis siRNA is induced.According to an embodiment of the invention, the shRNA of the embodiment of the present invention is constantly originated from carefully Intracellular, therefore its effect is more lasting, to extend the shRNA periods, shRNA used in the embodiment of the present invention has efficient, special The success of the effect on anisotropic silenced cell surface or intracellular immunity checkpoint, cell surface or intracellular immunity checkpoint is heavy It is silent so that transgenosis lymphocyte has the significant immunosuppressive characteristic resisted tumour and mediated, in tumour patient body Proliferation and survival ability are further enhanced, more notable to the orientation lethal effect effect of tumour.

In addition, according to an embodiment of the invention, the co-stimulators intracellular section is independently selected from 4-1BB, OX- 40, at least one of CD40L, CD27, CD30, CD28 and their derivative.The expression of co-stimulators intracellular section There is positive regulation and enhancing cellullar immunologic response with the silence joint of cell surface or at least one intracellular immunologic test point Effect so that transgenosis lymphocyte has the significant immunosuppressive characteristic resisted tumour and mediated, in neoplastic disease human body Interior proliferation and survival ability is further enhanced, more aobvious to the orientation lethal effect effect of the tumour of high expression MSLN It writes.

Other embodiment according to the present invention, the lymphocyte cell surface preferred CTLA4 or PD1 of immunologic test point, The preferred CBL-B of immunologic test point in lymphocyte.According to an embodiment of the invention, lymphocyte cell surface immunologic test point CTLA4 or PD1 is silenced or intracellular immunity checkpoint CBL-B is silenced so that transgenosis lymphocyte has more significantly Resistance tumour mediate immunosuppressive characteristic, proliferation and survival ability in tumour patient body are further carried Height, it is more notable to the orientation lethal effect effect of tumour.

According to an embodiment of the invention, the lymphocyte of the embodiment of the present invention is CD3⁺Lymphocyte or natural killer cells Or natural killer T cells.CD3⁺Lymphocyte is total T cell, and natural killer cells is one kind of immunocyte, and non-specificity is known Other target cell, natural killer T cells are that have T cell and the T cell subgroup of Natural Killer Cell Receptors.In above-mentioned lymphocyte Immunologic test point is silenced and expresses Chimeric antigen receptor so that the target killing of the cellular immunity of above-mentioned lymphocyte is more By force, more notable to the lethal effect effect of tumour cell.

Slow virus or construct

In another aspect of this invention, the present invention proposes a kind of slow virus or construct.According to an embodiment of the invention, Slow virus or construct carry following nucleic acid molecules：The nucleic acid molecules of encoding chimeric antigen receptor, Chimeric antigen receptor have SEQ ID NO：The nucleic acid molecules of amino acid sequence shown in 1, encoding chimeric antigen receptor have SEQ ID NO：Nucleotide shown in 2 Sequence；And the nucleic acid molecules on silenced cell surface or intracellular immunity checkpoint, the core of silenced cell surface immunologic test point The nucleotides sequence of acid molecule is classified as selected from SEQ ID NO：At least one of 3~68, the nucleic acid of immunologic test point in silenced cell The nucleotides sequence of molecule is classified as selected from SEQ ID NO：At least one of 69~135.Wherein, SEQ ID NO：3~14 be the mankind Programmed death receptor 1 (PD1) siRNA nucleotide sequences, SEQ ID NO：15~30 be that human cell poison T lymphocytes are related Antigen 4 (CTLA4) siRNA sequence, SEQ ID NO：31~46 be human T cells immunoglobulin mucoprotein molecule 3 (TIM3) SiRNA sequence, SEQ ID NO：47~57 be human T-lymphocyte decay factor (BTLA) siRNA sequence, SEQ ID NO：58 ~68 be 3 albumen of Human Lymphocytes activating gene (LAG-3) siRNA sequence, SEQ ID NO：69~85 be mankind IRAK-M (interleukin 1 receptor associated kinase 3) siRNA nucleotide sequences, SEQ ID NO：86~96 be mankind SOCS1 (cell because Subsignal transduction inhibiting factor 1) siRNA sequence, SEQ ID NO：97~116 be that (tumor necrosis factor-alpha induces egg to mankind A20 White A20) siRNA sequence, SEQ ID NO：117~135 be mankind CBL-B (E3 ubiquitin protein ligase CBL-B) siRNA sequences The slow virus of the embodiment of the present invention or construct are imported the transgenosis obtained by lymphocyte by row according to an embodiment of the invention In lymphocyte, immunologic test point PD1, CTLA4, TIM3, BTLA, LAG-3 of cell surface or intracellular immunity checkpoint IRAK-M, SOCS1, A20, CBL-B are by specific silence and inhibit to express, while expressing the chimeric of anti-MSLN in its cell surface Antigen receptor, to which the transgenosis lymphocyte of the embodiment of the present invention is provided with the immunosuppressive of significant resistance tumour mediation Effect, anti-tune dies ability and proliferative capacity enhancing, orientation killing ability significantly improve, and the transgenosis lymph of the embodiment of the present invention is thin Born of the same parents greatly improve in the internal and external proliferation of tumour patient and survival ability and the killing ability in tumour patient body, especially It is especially pronounced to the tumor cell specific fragmentation effect of high expression MSLN.

According to ground of the invention embodiment, the slow virus of the embodiment of the present invention or construct carry and contain SEQ ID NO：136、 137, nucleotide sequence shown in 138,139,140 or 141.Wherein, SEQ ID NO：136 be the anti-MSLN chimeric antigens of coexpression The nucleic acid molecules (MSLN-CAR/iPD1) of receptor and silenced cell immunologic test point PD-1, SEQ ID NO：137 be that coexpression is anti- The nucleic acid molecules (MSLN-CAR/iCBL-B) of MSLN Chimeric antigen receptors and silenced cell immunologic test point CBL-B, SEQ ID NO：138 be the nucleic acid molecules (MSLN-CAR/ for co-expressing anti-MSLN Chimeric antigen receptors and silenced cell immunologic test point CTLA4 ICTLA4), SEQ ID NO：139 be that the anti-MSLN Chimeric antigen receptors of coexpression, silenced cell immunologic test point PD1 and silence are another The nucleic acid molecules (MSLN-CAR/iPD1-CBL-B) of one cellular immunity checkpoint CBL-B, SEQ ID NO：140 be coexpression The nucleic acid of anti-MSLN Chimeric antigen receptors, silenced cell immunologic test point PD1 and another cellular immunity checkpoint of silence CTLA4 Molecule (MSLN-CAR/iPD1-CTLA4), SEQ ID NO：141 be that the anti-MSLN Chimeric antigen receptors of coexpression, the first silence are thin Nucleic acid molecules (the MSLN-CAR/iPD1- of the nucleic acid molecules of born of the same parents' immunologic test point PD1 and the second silenced cell immunologic test point PD1 PD1).According to an embodiment of the invention, the transgenosis lymph obtained by the slow virus of implementation column of the present invention importing lymphocyte is thin Born of the same parents, the immunologic test point PD1 or CTLA4 or CBL-B of cell is by specific silence and the Chimeric antigen receptor table of anti-MSLN It reaches so that transgenosis lymphocyte has effects that ability and increasing are died in the immunosupress that significantly resistance tumour mediates, anti-tune Grow ability enhancing, orientation killing ability significantly improve so that transgenosis lymphocyte is in tumour patient in vitro and in vivo Proliferation and survival ability and the killing ability in tumour patient body greatly improve, especially the tumour cell to high expression MSLN Specific killing effect it is especially pronounced.

According to an embodiment of the invention, inventor is to realize above-mentioned cell chimerism antigen receptor and table in the following way What face or intracellular immunity checkpoint shRNA were separately expressed, wherein it should be noted that expression herein both refers to egg White expression refers to rna transcription again.

Promoter：First promoter, the first promoter are operably connected with the nucleic acid molecules of encoding chimeric antigen receptor； And second promoter, the second promoter are operably connected with the nucleic acid molecules of silenced cell immunologic test point.According to this hair Bright embodiment, used first promoter, the second promoter are separately selected from U6, CMV, H1, EF-1, LTR, RSV Promoter, the introducing of the first and second promoter so that the nucleic acid molecules and silenced cell of encoding chimeric antigen receptor are immune The expression of the nucleic acid molecules of checkpoint independently, to effective reticence cell surface or intracellular immunity checkpoint, and It ensure that the high efficient expression of Chimeric antigen receptor so that survival rate of the lymphocyte in tumor environment greatly improves, and lymph is thin The targeting of born of the same parents is stronger, more notable to the specific killing action of tumour.

According to an embodiment of the invention, can also be further introduced into third promoter, third promoter independently selected from U6, The nucleic acid minute of the immunologic test point of at least one of CMV, H1, EF-1, LTR, RSV promoter, third promoter and silenced cell The operable connection of son, the nucleic acid molecules of the immunologic test for the silenced cell that third promoter and the second promoter are connected are not Together, the shRNA of silence difference immunologic test point is respectively started in third promoter and the second promoter.

Pass through the introducing of above-mentioned first, second promoter or further third promoter so that cell surface is intracellular Immunologic test point is efficiently expressed the transgenosis Lymphocyte Membrane in the embodiment of the present invention by efficient silence and Chimeric antigen receptor On, to efficiently inhibit the immune negative regulation of immunologic test point and ensure that the biological action of Chimeric antigen receptor, to So that survival rate of the lymphocyte in tumor environment greatly improves, the targeting killing effect of lymphocyte is more notable.

In addition, according to an embodiment of the invention, the carrier of the construct of the embodiment of the present invention is non-pathogenic virus carrier. Non-pathogenic virus carrier substantially increases duplication and amplification efficiency of the construct in lymphocyte, and then the embodiment of the present invention Proliferation and survival ability of the lymphocyte in tumour patient body greatly improve, the targeting of lymphocyte further increases By force, more notable to the lethal effect of tumour cell.

According to an embodiment of the invention, the viral vectors of the construct of the embodiment of the present invention be selected from retrovirus vector, At least one of slow virus carrier, adenovirus vector or adenoassociated virus carrier.According to an embodiment of the invention, of the invention The carrier of the virus of embodiment is in virus packaging and course of infection, and virus-infected area is extensive, and it is thin both to have infected terminal differentiation Born of the same parents, and the cell in division stage can be infected, it can be not only integrated into host chromosome, but also can be free in except host chromosome, it is real Existing wide spectrum and efficient efficiency of infection, to which cell surface or intracellular immunity checkpoint are by efficient silence and Chimeric antigen receptor The high efficient expression in lymphocyte, the proliferation in tumour patient body and survival ability of the lymphocyte of the embodiment of the present invention are big Big to improve, the targeting of lymphocyte further enhances, more notable to the lethal effect of tumour cell.

According to a particular embodiment of the invention, for building a slow virus carrier, inventor is slow in order to build one Purpose nucleic acid is inserted into viral genome by viral vectors in the position of certain virus sequences, to generate replication defective Virus.In order to generate virion, it (comprising gag, pol and env genes, but does not include LTR that inventor builds package cell line in turn With packaging ingredient).Recombinant plasmid containing target gene is concomitantly introduced into packet by inventor together with slow virus LTR and packaging sequence It fills in cell line.Packaging sequence allows recombinant plasmid rna transcription product to be wrapped into virion, is then secreted into culture In base.And then inventor collects the matrix for including recombinant slow virus, selectively concentrates, and it is used for gene transfer.Slow carrier Various kinds of cell type can be infected, including can dividing cell and can not dividing cell.

In addition, according to an embodiment of the invention, the slow virus of the embodiment of the present invention is compound slow virus, in addition to common slow Viral gene gag, pol and env also include other genes of regulation and control and structure function.Slow virus carrier is art technology Known to personnel, slow virus includes：Human immunodeficiency virus HIV -1, HIV -2 and simian immunodeficiency virus SIV.Slow disease Poisonous carrier is generated by Multiple decrements AIDS virus Disease-causing gene, such as all deletes gene env, vif, vpr, vpu and nef, Slow virus carrier is set to form biological safe type carrier.Recombined lentivirus vector can infect non-dividing cell, while can be used for body The transfer of interior and outer-gene and nucleic acid sequence expression.Such as：In suitable host cell, and with packaging function (gag, Pol, env, rev and tat) two or more carriers together, non-dividing cell can be infected.The targeting of recombinant virus, It is to be realized by antibody or particular ligand (targeting particular cell types receptor) and the combination of memebrane protein.Meanwhile recombinating disease The targeting of poison is encoded specific by being inserted into an ordered sequence (including regulatory region) to viral vectors together with another The gene of the ligand of receptor on target cell makes carrier be provided with specific targeting.Various useful slow virus carriers, and it is each The carrier of the generations such as kind method and operation, for changing the expression of cell.According to an embodiment of the invention, the embodiment of the present invention Slow virus carrier effectively can transport and co-express shRNA (transport form of siRNA), the small shRNA can effectively inhibit PD1 or The expression of CTLA4 or CBL-B.

According to an embodiment of the invention, the gland association viral vectors (AAV) of the embodiment of the present invention can be used one or more The DNA structures of known serum type gland association viral vectors.Those skilled in the art build a suitable gland association Viral vectors carries with this and co-expresses children purpura nephritis, which can inhibit PD1 or CTLA4 or CBL-B genes Expression.

In addition, according to an embodiment of the invention, the embodiment of the present invention also includes micro- gene.Micro- gene means with combination (selected nucleotide sequence and operable necessary relevant connection sequence) come instruct conversion, transcription and/or gene outcome exist Expression in vivo or in vitro host cell.It is controlled using the expression that " operable connection " sequence includes continuous target gene Sequence, and act on trans- or far distance controlled target gene expression control sequence.

In addition, the carrier of the embodiment of the present invention further includes conventional control element, in the cell transfecting with plasmid vector together Or/and in the cell infection of viral vectors together, these elements allow transcription, conversion and/or the expression of children purpura nephritis.Largely Expression control sequence (including natural, can induce and/or promoter of specific organization) be likely to be used.It is according to the present invention Embodiment, the promoter for expressing shRNA are RNA polymerase promoter.Meanwhile according to an embodiment of the invention, promoter is choosing From U6, H1, the RAN polymerase promoters of pol I, pol II and pol III.According to an embodiment of the invention, promoter is Tissue-specific promoter.According to an embodiment of the invention, promoter is inducible promoter.According to an embodiment of the invention, Promoter is selected from the promoter based on selected carrier.According to an embodiment of the invention, when selecting slow virus carrier, promoter For U6, H1, CMV IE genes, EF-1 α, ubiquitin C or phosphoglycerokinase (PGK) promoter.Other conventional expression control sequences Including optional label or reporter gene, including encoding geneticin, hygromycin, ampicillin or Puromycine resistance etc. Nucleotide sequence.The other assemblies of carrier include replication orgin.

What the technology of carrier construction was well known to those skilled in the art, these technologies include conventional cloning techniques, such as Nucleotide sequence needed for used shRNA, polymerase chain reaction and any offer appropriate in embodiments of the present invention Method.

According to an embodiment of the invention, inventor constructs coexpression children purpura nephritis (shRNA) (for inhibiting immune inspection Make an inventory of) and Chimeric antigen receptor (CAR) viral vectors.The transport silence PD1 or CTLA4 or CBL-B of the embodiment of the present invention SiRNA children purpura nephritis and expression Chimeric antigen receptor (CAR) viral vectors or plasmid be it is compound, this virus carry Body or plasmid increase its stability in combination with polymer or other materials, or assist its targeting movement.

The method of prepare transgenosis lymphocyte

In another aspect of this invention, the present invention, which proposes, a kind of preparing foregoing T lymphocytes or transgenosis The method of lymphocyte.According to an embodiment of the invention, this method includes：By foregoing construct or foregoing Slow virus is introduced into lymphocyte or T lymphocytes.Incorporation way can be selected from electricity turn or virus infects host cell Mode introduces.The construct of the embodiment of the present invention or slow virus are successfully introduced into above-mentioned lymphocyte or T lymphocytes, realize The expression of Chimeric antigen receptor and the cell surface of lymphocyte or intracellular immunity checkpoint for antigen MSLN are sunk It is silent, so that gained lymphocyte or T lymphocytes have effects that significantly to resist the immunosupress that tumour mediates, swollen Tumor is greatly improved with survival ability in external proliferation and tumour patient body in patient body, and lymphocyte or T lymphocytes are to swollen Oncocyte, it is especially stronger to the targeting killing effect of the tumour cell of high expression MSLN.

The therapeutic combination for the treatment of cancer

In another aspect of this invention, of the invention to propose a kind of therapeutic combination for treating cancer.According to this The embodiment of invention, the therapeutic combination include：Above-mentioned construct, above-mentioned slow virus, above-mentioned T lymphocytes above-mentioned turn base Because of lymphocyte.The composition of any one of the above therapeutic combination can be achieved turning base for antigen MSLN Chimeric antigen receptors Because in lymphocyte or T lymphocytes high efficient expression and transgenosis lymphocyte or T lymphocyte cells surface or intracellular The silence of immunologic test point, so that gained transgenosis lymphocyte or T lymphocytes expand, in vitro in neoplastic disease human body Survival ability greatly improves in interior proliferation and tumour patient body, and transgenosis lymphocyte or T lymphocytes express MSLN to height Tumour cell targeting killing effect it is stronger.

According to an embodiment of the invention, it is supplied to the therapeutic combination of the embodiment of the present invention of patient, is preferably applied to Bio-compatible solution or acceptable pharmacy delivery vehicle.Various therapeutic combinations as preparation are suspended or are dissolved in medicine Upper or physiologically acceptable carrier, such as physiological saline；Isotonic salting liquid or other people's being proficient in the knowledge of is obvious In formula.Carrier appropriate depends greatly on administration route.Other have water and anhydrous isotonic sterile injection liquid and There are water and anhydrous sterile suspensions, is pharmaceutically acceptable carrier.

According to an embodiment of the invention, sufficient amount of viral vectors is transduceed in targeting T-cells, and is provided sufficiently strong The transgenosis of degree, silence PD1 or CTLA4 or CBL-B and the distinctive anti-MSLN Chimeric antigen receptors of expression.The dosage of therapeutic reagent Depend primarily on treatment situation, age, weight, the health degree of patient, so as to cause the variability of patient.

Silence PD1 or CTLA4 or CBL-B and distinctive these methods for antigen MSLN Chimeric antigen receptors of expression It is a part for combination therapy.These viral vectors and antitumor T cell for adoptive immunotherapy, can be by individually or knot The method for closing other treatment cancer executes together.Under suitable conditions, therapy includes using one or more Medicinal treatment.

According to an embodiment of the invention, the cancer includes being selected from celiothelioma, cancer of pancreas, oophoroma, cholangiocarcinoma, lung cancer, Gastric cancer, intestinal cancer, at least one of the cancer of the esophagus and breast cancer.Following biological effect：The silence of cellular immunity checkpoint, joint are chimeric High efficient expression of the antigen receptor in transgenosis lymphocyte or T lymphocytes so that gained lymphocyte or T lymphocytes exist Survival ability in the microenvironment of above-mentioned tumour greatly improves, the target killing of lymphocyte or T lymphocytes to tumour cell Act on it is stronger, it is especially more notable to the lethal effect of the above-mentioned tumour cell of high expression MSLN.

The method for improving lymphocyte activity

In another aspect of this invention, the present invention proposes a kind of method improving lymphocyte activity, and the present invention is implemented The lymphocyte of example carries Chimeric antigen receptor, and according to an embodiment of the invention, this method includes:Make the thin of the lymphocyte Born of the same parents' immunologic test point is silenced, and cellular immunity checkpoint, lymphocyte, Chimeric antigen receptor are as defined above.According to this The lymphocyte activity of the embodiment of invention, the embodiment of the present invention includes Lymphocyte Proliferation in Vitro ability, in neoplastic disease human body At least one of killing ability of the interior proliferation and survival ability and lymphocyte in tumour patient body.It is according to the present invention The cellular immunity checkpoint of embodiment, the lymphocyte of the embodiment of the present invention is silenced, and lymphocyte is activated, productive reaction Up-regulation, cytokine secretion increase, anti-tune dies ability enhancing.The lymphocyte of the embodiment of the present invention expands and be proliferated in vitro, The targeting killing effect of tumour cell is significantly increased.

The solution of the present invention is explained below in conjunction with embodiment.

It will be understood to those of skill in the art that the following examples are merely to illustrate the present invention, and it should not be regarded as limiting this The range of invention.Particular technique or condition are not specified in embodiment, according to technology or item described in document in the art Part (such as with reference to works such as J. Pehanorm Brookers, what Huang Peitang etc. was translated《Molecular Cloning:A Laboratory guide》, the third edition, Science Press) Or it is carried out according to product description.Reagents or instruments used without specified manufacturer, being can be by acquisition purchased in market Conventional products.

Embodiment 1

Used cell line and basic experiment technology are as described below in the embodiment of the present invention：

The generation of slow virus and the transduction of human T lymphocyte

The slow virus carrier of replication defective is generated, and the transduction for human T lymphocyte is collected by centrifugation in slow virus carrier. The experimentation of generation, the collection of slow virus carrier is briefly described below：It is 150- squares li that 293T cells, which are layered on floor space, In the Tissue Culture Dish of rice, and according to specification, (Open Biosystems/Thermo are purchased from using Express-In Scientific, Waltham, MA) viral transduction is carried out to 293T cells.Often the slow virus transgenosis of 15 micrograms is added in disk cell PCMVR8.74 plasmids (the Gag/Pol/Tat/Rev tables of plasmid, the pVSV-G (VSV P-glycoprotein expressions plasmid) of 5 micrograms, 10 micrograms Up to plasmid) and 174 microlitres of Express-In (a concentration of 1 microgram/microlitre).Supernatant was collected respectively at 24 hours and 48 hours, And using ultracentrifuge 28,000rpm (centrifuge rotor be Beckman SW 32Ti, be purchased from Beckman Coulter, Brea, CA) under conditions of centrifuge 2 hours.Weight finally is carried out to virus particle precipitation with the RPMI-1640 culture mediums of 0.75ml It is outstanding.

The primary T lymphocytes of people are detached from healthy volunteer's donor.Human T lymphocyte's culture is cultivated in RPMI-1640 It is stimulated in base and using the coated pearl of monoclonal antibody (being purchased from Invitrogen, Carlsbad, CA) of AntiCD3 McAb and CD28 Activation.It 18~24 hours after human T lymphocyte's activation, is transduceed, is turned to T lymphocytes using the method for spin-inoculation It is as described below to lead process：In 24- orifice plates, 0.5 × 10 is covered with per hole⁶T lymphocytes are added 0.75ml's into every hole cell The viral supernatants and Polybrene (a concentration of 8 micrograms/ml) of above-mentioned resuspension.The mixed liquor of cell and virus particle it is desk-top from Scheming (is purchased from Sorvall ST 40；Thermo Scientific) in centrifugation, centrifugal condition is room temperature, 2500rpm, and the time is 90 minutes.People's recombination leukocyte mesonium-2 (IL-2；Purchased from Novartis, Basel, Switzerland) every 2~3 days addition T In lymphocyte culture medium, the final concentration of 100-IU/ml of IL-2 keeps the density of cell in T lymphocyte incubations It is 0.5 × 10⁶~1 × 10⁶/ml.Once suspend mode occur in the T lymphocytes transduceed, such as vitro growth rates are slack-off and cell Become smaller, wherein vitro growth rates and size be by Coulter Counter (being purchased from Beckman Coulter) assessment, Or the T lymphocytes transduceed, on the time point that some is planned, T lymphocytes can be used to do work and can analyze.

Flow cytometer used in embodiments herein is that BD FACSCanto II (are purchased from BD Biosciences), and flow cytometric analysis data using FlowJo version 7.2.5 softwares (be purchased from Tree Star, Ashland, OR) it is analyzed.

Measure the secretion of cell factor

2-7 days after plasmid transduction, (number of cells was 1 for non-transduction or the T cell of Chimeric antigen receptor plasmid of having transduceed ×10⁶/ hole) and people's pleural mesothelium oncocyte, human pleural mesothelioma cells (coming from ATCC) co-cultivation, Change different effect target rations in experimentation.Using specific enzyme-linked immunosorbent assay, (cell factor is enzyme-linked to be exempted from Epidemic disease adsorb test agent box, be purchased from R＆D Systems, Inc., Minneapolis, MN, USA) detection cell supernatant in cell because The yield of son.Above-mentioned cell supernatant is derived from the supernatant for having cultivated the cell after 24 hours, 48 hours and 72 hours, measures knot Fruit is used for weighing the yield of representational cell factor (interferon-γ) (IFN γ).

Brief continuous mode is as follows：The cell factor as series of standards control in 100 microlitres/hole is added in ELISA Plate Dilute solution (such as IFN γ) or cell conditioned medium solution to be measured, and ELISA Plate is placed at room temperature 2 hours.It is inhaled after 2 hours and abandons enzyme Solution and the washing lotion rinse ELISA Plate with 400 microlitres, rinse four times in target.After rinse, 200 are added into each hole of ELISA Plate Microlitre enzyme-linked anti-cytokine antibodies.Continue to place 2 hours at room temperature, it is backward per hole in 200 microlitres of bottom is added Object solution.After substrate solution is added, ELISA Plate is placed at room temperature 30 minutes, later, 50 microlitres of end is added into every hole Only reaction solution.Optical density of the ELISA Plate per hole is measured in 30 minutes.Microplate reader is arranged in 450nm.

Chromium release experiment

In embodiment anti-MSLN Chimeric antigen receptors T cell (anti-MSLN is assessed using the analysis of 4-hours, 51 chromium method for releasing CAR T lymphocytes) cytotoxic activity.It is as follows：The label 1 under 37 degrees Celsius is small with 51Cr for target detection cell When.After label, with the RPMI culture medium rinse cells containing 10% fetal calf serum (FCS).After rinse, cell is resuspended in identical Culture medium in, the concentration that cell is resuspended is 1 × 10⁵/ml.T cell is with different target effect cell ratio (T after transduction：E it) is added It it is 200 microlitres per pore volume in target detection cell suspending liquid, and by cell kind in the holes 96-.By cell in 37 degree of incubators Middle culture 4 hours.After 4 hours, 30 microlitres of supernatant is taken out from every hole be put in the 96- microwell plates of counter and count point Analysis.Analytical instrument is the micro- scintillation counters of top counting NXT (being purchased from Packard Bioscience).Effect in all counting holes The number of cell is calculated based on T cell sum.Labeled target detection cell is MSLN⁺MSTO-211H (people's pleuras Mesothelioma cell, human pleural mesothelioma cells (coming from ATCC)).

The carrier of the structure coexpression of embodiment 2 silenced cell immunologic test point shRNA and anti-MSLN Chimeric antigen receptors

In the present embodiment, coding is had sequence, 4-1BB intracellulars section and the T cell of the single-chain antibody of anti-human MSLN by inventor ζ-chain-ordering of receptor combination is cloned on the slow virus carrier containing EF-1 promoters (lentiviral vector), clone In the process, the restricted digestion of selection is XbaI and NotI double digestions and NotI and XhoI double digestions, by digestion, connection, The amplification of screening and purpose plasmid generates the slow virus plasmid (LV-MSLN CAR) for expressing anti-MSLN Chimeric antigen receptors.Including The sequence quilt of U6 promoters and people PD1 shRNA (iPD1) or CBL-B shRNA (iCBL-B) or CTLA4 shRNA (iCTLA4) It clones into LV-MSLN CAR vector plasmids, is built into LV-MSLN CAR/iPD1 or LV-MSLN CAR/iCBL or LV-MSLN CAR/i CTLA4.Fig. 1 is the schematic diagram of slow virus carrier, includes the sequence of the anti-MSLN Chimeric antigen receptors of coding, U6 or H1 are opened Promoter sequences, PD1 shRNA or CBL-B shRNA or CTLA4 shRNA sequences.The sequence of anti-MSLN Chimeric antigen receptors is opening Under the startup regulation and control of mover EF-1, CTLA4, PD1 CBL-B shRNA sequences are under the startup regulation and control of promoter U6 or H1.

Embodiment 3, which co-expresses PD1 shRNA and the T lymphocytes of anti-MSLN Chimeric antigen receptors, has ability of cell proliferation The characteristics of higher

In the present embodiment, peripheral blood lymphocytes is derived from blank blood donor.Peripheral blood lymphocytes by gradient from The heart is detached, Gradient Centrifuge Ficoll-Hypaque.It (is purchased from T lymphocyte activating factor magnetic beads CD3/CD28 Invitrogen, Carlsbad, CA) in the presence of, the T lymphocytes being activated are by slow virus carrier transduction, amplification in vitro training It supports, method is as described in Example 1.2-7 days after slow virus carrier transduction, (number of cells was 1 × 10 to the T cell of transduction⁶/ Hole) and MSLN⁺MSTO-211H is co-cultured, and after 4 days, is detected by streaming instrument before cell number.Experimental result is as shown in Figure 2.Fig. 2 The result shows that T of the T lymphocytes for the LV-MSLN CAR/iPD1 that transduceed than transduceed LV-MSLN CAR or zero load LV-GFP The cell number of lymphocyte dramatically increases.Mark represents the average value ± SD in every 3 holes.(P<0.05；LV-MSLN CAR/ iPD1vs.LV-MSLN CAR).

Embodiment 4, which co-expresses PD1 shRNA and the T lymphocytes of anti-MSLN Chimeric antigen receptors, has cytokine secretion More features

In the presence of T lymphocyte activating factor magnetic bead CD3/CD28, the T lymphocytes being activated pass through slow virus carrier Transduction, amplification in vitro culture, method are as described in Example 1.2-7 days after slow virus carrier transduction, the T cell of transduction was (thin Born of the same parents' number is 1 × 10⁶/ hole) and MSLN⁺MSTO-211H cells co-culture, and after 4 days, point of cell factor is detected by ELISA It secretes.Experimental result is as shown in Figure 3.Fig. 3 the result shows that, the T lymphocyte ratios of the LV-MSLN CAR/iPD1 that transduceed have been transduceed LV- More IFN γ (the P of T lymphocytic emiocytosis of MSLN CAR or zero load LV-GFP<0.05；LV-MSLN CAR/iPD1 vs.LV- MSLN CAR).T lymphocyte of the T lymphocytes of this LV-MSLN CAR/iPD1 that illustrate to have transduceed than the LV-MSLN CAR that transduce Generation Cytokines significantly improve.

Embodiment 5 co-expresses the T lymphocytic tumours cytolytic abilities of PD1 shRNA and anti-MSLN Chimeric antigen receptors Enhancing.

In the present embodiment, peripheral blood lymphocytes is derived from blank blood donor.Peripheral blood lymphocytes by gradient from The heart is detached, Gradient Centrifuge Ficoll-Hypaque.T lymphocytes and t cell activation factor magnetic bead CD3/CD28 (purchases From Invitrogen, Carlsbad, CA) in 5%CO₂, be incubated culture 72 hours under 37 degrees Celsius, culture medium is added with 2mmol/ L glutamine, the mould of fetal calf serum (FCS) (being purchased from Sigma-Aldrich Co.) and 100U/ml of 10% high-temperature inactivation The dual anti-RPMI culture mediums 1640 of element/streptomysin (are purchased from Invitrogen Gibco Cat.no.12633-012).Activation culture After 72 hours, with washing lotion rinse cell, magnetic bead is washed away.T cell kind is being covered with recombination fibronectin fragment (FNch-296； Retronectin) on Tissue Culture Dish, lentiviruses transduction is used in combination, transduction slow virus is respectively LV-MSLN CAR/iPD1, LV- MSLN CAR or zero load (LV-GFP) transductive process are as described in Example 1.T cell culture after transduction is cultivated in RPMI-1640 In base and with the recombinant human IL-2 factors (100ng/ml；Purchased from R＆D Systems) carry out induced amplification 7-10 days, then carry out Functional test is tested.Inventor's measurement transduceed different slow virus T cell (effector cell) to MSLN⁺MSTO-211H targets are thin The lethal effect of born of the same parents, it is 1 that target, which imitates cell proportion,：25 or 1:5, measurement method uses 4-hour of standard⁵¹Chromium method for releasing, 4-hours⁵¹ Chromium method for releasing is as described in Example 1.The results are shown in Figure 4.As shown in figure 4, co-expressing anti-MSLN Chimeric antigen receptors and PD1 The T lymphocytes of the T lymphocytes MSLN Chimeric antigen receptors more anti-than single expression of shRNA (iPD1), more efficiently can be killed Dead MSLN⁺MSTO-211H target cells.The T lymphocytes (control LV-GFP T lymphocytes) of unloaded lentiviruses transduction are to MSLN⁺ MSTO-211H cells are without apparent lethal effect.Statistical data represents the average value ± SEM in three holes.

Embodiment 6 co-expresses the T cell of CBL-B shRNA and anti-MSLN Chimeric antigen receptors, coexpression CTLA4 shRNA With the T cell of anti-MSLN Chimeric antigen receptors, PD1 shRNA, CBL-B shRNA and anti-MSLN Chimeric antigen receptors are co-expressed T cell, co-expresses the cell of the T cell of PD1 shRNA, CTLA4 shRNA and anti-MSLN Chimeric antigen receptors, and solvability increases By force and have the characteristics that cytokine secretion is more and cell Proliferation is stronger.

In the present embodiment, inventor has also investigated coexpression CBL-B shRNA and the T of anti-MSLN Chimeric antigen receptors is thin Born of the same parents, the T cell for co-expressing CTLA4 shRNA and anti-MSLN Chimeric antigen receptors, co-express 2 shRNA (PD1 shRNA and CBL-B shRNA or PD1 shRNA and CTLA4 shRNA or 2 PD1 shRNAs for the different regions PD1) and anti-MSLN Tumor lysis ability, cytokine secretion ability and the ability of cell proliferation of the T cell of Chimeric antigen receptor.Experimentation and reality It is identical to apply example 3,4 and 5.The cytolytic ability of the T cell of above-mentioned T cell MSLN Chimeric antigen receptors more anti-than single expression increases By force, cytokine secretion is more and cell Proliferation is stronger.Co-express 2 shRNA (PD1 shRNA and CTLA4 shRNA or PD1 ShRNA and CBL-B shRNA or 2 PD1 shRNAs for the different regions PD1) and the T of anti-MSLN Chimeric antigen receptors it is thin Born of the same parents are than 1 shRNA (PD1 shRNA or CBL-B shRNA or CTLA4 shRNA) of coexpression and anti-MSLN Chimeric antigen receptors The cytolytic ability of T cell is stronger, and cytokine secretion is more and cell Proliferation is stronger.

In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office It can be combined in any suitable manner in one or more embodiments or example.In addition, without conflicting with each other, the skill of this field Art personnel can tie the feature of different embodiments or examples described in this specification and different embodiments or examples It closes and combines.

Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned Embodiment is changed, changes, replacing and modification.

SEQUENCE LISTING

<110>Beijing horsepower bio tech ltd

<120>It co-expresses anti-MSLN Chimeric antigen receptors and immunologic test point inhibits molecule

<130> PIDC1153486

<160> 141

<170> PatentIn version 3.3

<210> 1

<211> 507

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of Chimeric antigen receptor

<400> 1

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

1 5 10 15

Ala Phe Leu Leu Ile Pro Asp Ile Gln Ala Gln Val Gln Leu Val Gln

20 25 30

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

35 40 45

Arg Ala Ser Gly Tyr Ser Ile Asn Thr Tyr Tyr Met Gln Trp Val Arg

50 55 60

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

65 70 75 80

Gly Val Thr Ser Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Leu Thr

85 90 95

Asn Asp Thr Ser Thr Asn Thr Val Tyr Met Gln Leu Asn Ser Leu Thr

100 105 110

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

115 120 125

Asp Phe Gly Met Asp Val Trp Gly Lys Gly Thr Leu Val Thr Val Ser

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505

<210> 2

<211> 1524

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the nucleic acid molecules of encoding chimeric antigen receptor

<400> 2

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 ttcgtgccgg tcttcctgcc agcgaagccc 840

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 900

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 960

gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 1020

ctgtcactgg ttatcaccct ttactgcaac cacaggaaca aacggggcag aaagaaactc 1080

ctgtatatat tcaaacaacc atttatgaga ccagtacaaa ctactcaaga ggaagatggc 1140

tgtagctgcc gatttccaga agaagaagaa ggaggatgtg aactgagagt gaagttcagc 1200

aggagcgcag acgcccccgc gtaccagcag ggccagaacc agctctataa cgagctcaat 1260

ctaggacgaa gagaggagta cgatgttttg gacaagagac gtggccggga ccctgagatg 1320

gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt acaatgaact gcagaaagat 1380

aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1440

cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1500

atgcaggccc tgccccctcg ctaa 1524

<210> 3

<211> 21

<212> DNA

<213> Artificial

<220>

<223>Mankind's programmed death receptor 1（PD1）SiRNA nucleotide sequences

<400> 3

ggccaggatg gttcttagac t 21

<210> 4

<211> 21

<212> DNA

<213> Artificial

<220>

<223>Mankind's programmed death receptor 1（PD1）SiRNA nucleotide sequences

<400> 4

ggatttccag tggcgagaga a 21

<210> 5

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind's programmed death receptor 1（PD1）SiRNA nucleotide sequences

<400> 5

gccuguguuc ucuguggacu aug 23

<210> 6

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind's programmed death receptor 1（PD1）SiRNA nucleotide sequences

<400> 6

ggugcugcua gucugggucc ugg 23

<210> 7

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind's programmed death receptor 1（PD1）SiRNA nucleotide sequences

<400> 7

gacagagaga agggcagaag ugc 23

<210> 8

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind's programmed death receptor 1（PD1）SiRNA nucleotide sequences

<400> 8

cagcuucucc aacacaucgg aga 23

<210> 9

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind's programmed death receptor 1（PD1）SiRNA nucleotide sequences

<400> 9

ccgugucaca caacugccca acg 23

<210> 10

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind's programmed death receptor 1（PD1）SiRNA nucleotide sequences

<400> 10

uaugccacca uugucuuucc uag 23

<210> 11

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind's programmed death receptor 1（PD1）SiRNA nucleotide sequences

<400> 11

ugcuaaacug guaccgcaug agc 23

<210> 12

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind's programmed death receptor 1（PD1）SiRNA nucleotide sequences

<400> 12

gugacagaga gaagggcaga agu 23

<210> 13

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind's programmed death receptor 1（PD1）SiRNA nucleotide sequences

<400> 13

cugaggaugg acacugcucu ugg 23

<210> 14

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind's programmed death receptor 1（PD1）SiRNA nucleotide sequences

<400> 14

aucggagagc uucgugcuaa acu 23

<210> 15

<211> 21

<212> DNA

<213> Artificial

<220>

<223>Human cell's poison T lymphocyte-associated antigen 4s（CTLA4）SiRNA sequence

<400> 15

ggcaacggaa cccagattta t 21

<210> 16

<211> 21

<212> DNA

<213> Artificial

<220>

<400> 16

ggaacccaaa ttacgtgtac t 21

<210> 17

<211> 21

<212> DNA

<213> Artificial

<220>

<400> 17

gaacccaaat tacgtgtact a 21

<210> 18

<211> 21

<212> DNA

<213> Artificial

<220>

<400> 18

gggagaagac tatattgtac a 21

<210> 19

<211> 21

<212> DNA

<213> Artificial

<220>

<400> 19

gacgtttata gccgaaatga t 21

<210> 20

<211> 21

<212> DNA

<213> Artificial

<220>

<400> 20

gacactaata caccaggtag a 21

<210> 21

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 21

accucacuau ccaaggacug agg 23

<210> 22

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 22

augaguugac cuuccuagau gau 23

<210> 23

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 23

ggggaaugag uugaccuucc uag 23

<210> 24

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 24

cucuggaucc uugcagcagu uag 23

<210> 25

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 25

cuccucugga uccuugcagc agu 23

<210> 26

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 26

uuugugugug aguaugcauc ucc 23

<210> 27

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 27

caccuccagu ggaaaucaag uga 23

<210> 28

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 28

cacgggacuc uacaucugca agg 23

<210> 29

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 29

uucugacuuc cuccucugga ucc 23

<210> 30

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 30

aagucugugc ggcaaccuac aug 23

<210> 31

<211> 21

<212> DNA

<213> Artificial

<220>

<223>Human T cells immunoglobulin mucoprotein molecule 3（TIM3 ）SiRNA sequence

<400> 31

ggtcggtcag aatgcctatc t 21

<210> 32

<211> 21

<212> DNA

<213> Artificial

<220>

<400> 32

gccaatgact tacgggactc t 21

<210> 33

<211> 21

<212> DNA

<213> Artificial

<220>

<400> 33

gcagagggaa ttcgctcaga a 21

<210> 34

<211> 21

<212> DNA

<213> Artificial

<220>

<400> 34

ggaaattcgg gcacatcata t 21

<210> 35

<211> 21

<212> DNA

<213> Artificial

<220>

<400> 35

gattaagaga tgactggact a 21

<210> 36

<211> 21

<212> DNA

<213> Artificial

<220>

<400> 36

gagatgactg gactaggtct a 21

<210> 37

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 37

aggaaauucg ggcacaucau aug 23

<210> 38

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 38

gacugaugaa agggauguga auu 23

<210> 39

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 39

gccacugauu uucaaagaga ucu 23

<210> 40

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 40

agcagaguuu ucccauuuuc aga 23

<210> 41

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 41

aacuuaaaca ggcaugucau ugc 23

<210> 42

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 42

uucagaagau aaugacucac aug 23

<210> 43

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 43

gccucuguau uuaagccaac aga 23

<210> 44

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 44

ugcucaugug auuguggagu aga 23

<210> 45

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 45

auguuuucac aucuucccuu uga 23

<210> 46

<211> 23

<212> DNA

<213> Artificial

<220>

<400> 46

gagagacuuc acugcagccu uuc 23

<210> 47

<211> 21

<212> DNA

<213> Artificial

<220>

<223>Human T-lymphocyte decay factor（BTLA）SiRNA sequence

<400> 47

gattgcctct actcatcact a 21

<210> 48

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Human T-lymphocyte decay factor（BTLA）SiRNA sequence

<400> 48

uccuaaugac aaugggucau acc 23

<210> 49

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Human T-lymphocyte decay factor（BTLA）SiRNA sequence

<400> 49

aagacauugc cugccaugcu ugg 23

<210> 50

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Human T-lymphocyte decay factor（BTLA）SiRNA sequence

<400> 50

gucauaccgc uguucugcaa auu 23

<210> 51

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Human T-lymphocyte decay factor（BTLA）SiRNA sequence

<400> 51

cuccuguaua guuuacuucc uuu 23

<210> 52

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Human T-lymphocyte decay factor（BTLA）SiRNA sequence

<400> 52

uaccgcuguu cugcaaauuu uca 23

<210> 53

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Human T-lymphocyte decay factor（BTLA）SiRNA sequence

<400> 53

aaaacaaacc aggcauuguu uau 23

<210> 54

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Human T-lymphocyte decay factor（BTLA）SiRNA sequence

<400> 54

aacuagaaug cccugugaaa uac 23

<210> 55

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Human T-lymphocyte decay factor（BTLA）SiRNA sequence

<400> 55

gugacuuggu gcaagcucaa ugg 23

<210> 56

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Human T-lymphocyte decay factor（BTLA）SiRNA sequence

<400> 56

auccauggga aagaaucaug uga 23

<210> 57

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Human T-lymphocyte decay factor（BTLA）SiRNA sequence

<400> 57

uggugcaagc ucaauggaac aac 23

<210> 58

<211> 21

<212> DNA

<213> Artificial

<220>

<223>3 albumen of Human Lymphocytes activating gene（LAG1）SiRNA sequence

<400> 58

gctgctcacc cttatgaacc t 21

<210> 59

<211> 23

<212> DNA

<213> Artificial

<220>

<223>3 albumen of Human Lymphocytes activating gene（LAG1）SiRNA sequence

<400> 59

aggacauggu gguggacgag ugc 23

<210> 60

<211> 23

<212> DNA

<213> Artificial

<220>

<223>3 albumen of Human Lymphocytes activating gene（LAG1）SiRNA sequence

<400> 60

ugcucuuccu gcacgauauc agu 23

<210> 61

<211> 23

<212> DNA

<213> Artificial

<220>

<223>3 albumen of Human Lymphocytes activating gene（LAG1）SiRNA sequence

<400> 61

accucuacug guuccuguac auc 23

<210> 62

<211> 23

<212> DNA

<213> Artificial

<220>

<223>3 albumen of Human Lymphocytes activating gene（LAG1）SiRNA sequence

<400> 62

cccuccaacu cugcuccucu agg 23

<210> 63

<211> 23

<212> DNA

<213> Artificial

<220>

<223>3 albumen of Human Lymphocytes activating gene（LAG1）SiRNA sequence

<400> 63

cccugagugg acagucgucu ucg 23

<210> 64

<211> 23

<212> DNA

<213> Artificial

<220>

<223>3 albumen of Human Lymphocytes activating gene（LAG1）SiRNA sequence

<400> 64

cugcuccagg gaagcuucua ugg 23

<210> 65

<211> 23

<212> DNA

<213> Artificial

<220>

<223>3 albumen of Human Lymphocytes activating gene（LAG1）SiRNA sequence

<400> 65

cgcucaaggu ccuguaugcc acc 23

<210> 66

<211> 23

<212> DNA

<213> Artificial

<220>

<223>3 albumen of Human Lymphocytes activating gene（LAG1）SiRNA sequence

<400> 66

gaguucacca agcucaacau uua 23

<210> 67

<211> 23

<212> DNA

<213> Artificial

<220>

<223>3 albumen of Human Lymphocytes activating gene（LAG1）SiRNA sequence

<400> 67

ugcugcugcu cacccuuaug aac 23

<210> 68

<211> 23

<212> DNA

<213> Artificial

<220>

<223>3 albumen of Human Lymphocytes activating gene（LAG1）SiRNA sequence

<400> 68

cccaucuccg ugcucuucuu uga 23

<210> 69

<211> 21

<212> DNA

<213> Artificial

<220>

<223>Mankind IRAK-M siRNA (- 1 receptor-associated kinase 3 of human interleukin) 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>Mankind SOCS1 siRNA（Human cytokine's signal transduction inhibiting factor 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>Mankind A20 siRNA (human tnf-α inducible protein A20) sequence

<400> 97

gcggaaagct gtgaagatac g 21

<210> 98

<211> 21

<212> DNA

<213> Artificial

<220>

<223>Mankind A20 siRNA (human tnf-α inducible protein A20) sequence

<400> 98

acaaagccct catcgacaga a 21

<210> 99

<211> 21

<212> DNA

<213> Artificial

<220>

<223>Mankind A20 siRNA (human tnf-α inducible protein A20) sequence

<400> 99

atgccacttc tcagtacatg t 21

<210> 100

<211> 21

<212> DNA

<213> Artificial

<220>

<223>Mankind A20 siRNA (human tnf-α inducible protein A20) sequence

<400> 100

gtggacttca gtacaactca c 21

<210> 101

<211> 21

<212> DNA

<213> Artificial

<220>

<223>Mankind A20 siRNA (human tnf-α inducible protein A20) sequence

<400> 101

gtggaattta cttgcctctc c 21

<210> 102

<211> 21

<212> DNA

<213> Artificial

<220>

<223>Mankind A20 siRNA (human tnf-α inducible protein A20) sequence

<400> 102

gttggatgaa gctaacttac c 21

<210> 103

<211> 21

<212> DNA

<213> Artificial

<220>

<223>Mankind A20 siRNA (human tnf-α inducible protein A20) sequence

<400> 103

actgggaaga cgtgtaactc t 21

<210> 104

<211> 21

<212> DNA

<213> Artificial

<220>

<223>Mankind A20 siRNA (human tnf-α inducible protein A20) sequence

<400> 104

aaggaattgc atccaaggta t 21

<210> 105

<211> 21

<212> DNA

<213> Artificial

<220>

<223>Mankind A20 siRNA (human tnf-α inducible protein A20) sequence

<400> 105

ggaattgcat ccaaggtata c 21

<210> 106

<211> 21

<212> DNA

<213> Artificial

<220>

<223>Mankind A20 siRNA (human tnf-α inducible protein A20) sequence

<400> 106

ggatgagact ggcaatggtc a 21

<210> 107

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind A20 siRNA (human tnf-α inducible protein A20) sequence

<400> 107

uccucaguuu cgggagauca ucc 23

<210> 108

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind A20 siRNA (human tnf-α inducible protein A20) sequence

<400> 108

gagucucuca aaucucagga auu 23

<210> 109

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind A20 siRNA (human tnf-α inducible protein A20) sequence

<400> 109

agcucuaguc cuuuuugugu aau 23

<210> 110

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind A20 siRNA (human tnf-α inducible protein A20) sequence

<400> 110

cacuggaaau guucagaacu ugc 23

<210> 111

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind A20 siRNA (human tnf-α inducible protein A20) sequence

<400> 111

augaugaaug ggacaaucuu auc 23

<210> 112

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind A20 siRNA (human tnf-α inducible protein A20) sequence

<400> 112

cacacugugu uucaucgagu aca 23

<210> 113

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind A20 siRNA (human tnf-α inducible protein A20) sequence

<400> 113

gcagaaccau ccauggacug uga 23

<210> 114

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind A20 siRNA (human tnf-α inducible protein A20) sequence

<400> 114

aaagaugugg ccuuuuguga ugg 23

<210> 115

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind A20 siRNA (human tnf-α inducible protein A20) sequence

<400> 115

uucagaacuu gccaguuuug ucc 23

<210> 116

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind A20 siRNA (human tnf-α inducible protein A20) sequence

<400> 116

augagacugg caauggucac agg 23

<210> 117

<211> 21

<212> DNA

<213> Artificial

<220>

<223>Mankind CBL-B siRNA（E3 ubiquitin protein ligases CBL-B）Sequence

<400> 117

gtcaattcca gggagataac t 21

<210> 118

<211> 21

<212> DNA

<213> Artificial

<220>

<223>Mankind CBL-B siRNA（E3 ubiquitin protein ligases CBL-B）Sequence

<400> 118

gcctggaagc aatggctcta a 21

<210> 119

<211> 21

<212> DNA

<213> Artificial

<220>

<223>Mankind CBL-B siRNA（E3 ubiquitin protein ligases CBL-B）Sequence

<400> 119

gcaccaaacc cggaagctat a 21

<210> 120

<211> 21

<212> DNA

<213> Artificial

<220>

<223>Mankind CBL-B siRNA（E3 ubiquitin protein ligases CBL-B）Sequence

<400> 120

gttgcactcg attgggacag t 21

<210> 121

<211> 21

<212> DNA

<213> Artificial

<220>

<223>Mankind CBL-B siRNA（E3 ubiquitin protein ligases CBL-B）Sequence

<400> 121

ggattatgtg aacctacacc t 21

<210> 122

<211> 21

<212> DNA

<213> Artificial

<220>

<223>Mankind CBL-B siRNA（E3 ubiquitin protein ligases CBL-B）Sequence

<400> 122

ggaatcacag cgagttcaaa t 21

<210> 123

<211> 21

<212> DNA

<213> Artificial

<220>

<223>Mankind CBL-B siRNA（E3 ubiquitin protein ligases CBL-B）Sequence

<400> 123

gcaaggcata gtctcattga a 21

<210> 124

<211> 21

<212> DNA

<213> Artificial

<220>

<223>Mankind CBL-B siRNA（E3 ubiquitin protein ligases CBL-B）Sequence

<400> 124

ggtgaagaga gccttagaga t 21

<210> 125

<211> 21

<212> DNA

<213> Artificial

<220>

<223>Mankind CBL-B siRNA（E3 ubiquitin protein ligases CBL-B）Sequence

<400> 125

gtgaagagag ccttagagat a 21

<210> 126

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind CBL-B siRNA（E3 ubiquitin protein ligases CBL-B）Sequence

<400> 126

aggagcuaag gucuuuucca aug 23

<210> 127

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind CBL-B siRNA（E3 ubiquitin protein ligases CBL-B）Sequence

<400> 127

augucgaugc aaaaauugca aaa 23

<210> 128

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind CBL-B siRNA（E3 ubiquitin protein ligases CBL-B）Sequence

<400> 128

gucacaugcu ggcagaaauc aaa 23

<210> 129

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind CBL-B siRNA（E3 ubiquitin protein ligases CBL-B）Sequence

<400> 129

uccagguuac auggcauuuc uca 23

<210> 130

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind CBL-B siRNA（E3 ubiquitin protein ligases CBL-B）Sequence

<400> 130

uugaacuuug aaccugugaa aug 23

<210> 131

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind CBL-B siRNA（E3 ubiquitin protein ligases CBL-B）Sequence

<400> 131

uccacaucaa cagcuaaauc auu 23

<210> 132

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind CBL-B siRNA（E3 ubiquitin protein ligases CBL-B）Sequence

<400> 132

augcuggcag aaaucaaagc aau 23

<210> 133

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind CBL-B siRNA（E3 ubiquitin protein ligases CBL-B）Sequence

<400> 133

ugcagagaau gacaaagaug uca 23

<210> 134

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind CBL-B siRNA（E3 ubiquitin protein ligases CBL-B）Sequence

<400> 134

ggcagaacuc accagucaca uca 23

<210> 135

<211> 23

<212> DNA

<213> Artificial

<220>

<223>Mankind CBL-B siRNA（E3 ubiquitin protein ligases CBL-B）Sequence

<400> 135

ucgguccugu gauaaugguc acu 23

<210> 136

<211> 2053

<212> DNA

<213> Artificial

<220>

<223>The 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>The 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>The 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>The 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>The 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>The 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 kind of T lymphocytes, which is characterized in that the cellular immunity checkpoint of the T lymphocytes is silenced；

And expression Chimeric antigen receptor, wherein

The Chimeric antigen receptor includes：

Extracellular region, the extracellular region include the heavy chain variable region and light chain variable region of single-chain antibody, the single-chain antibody specificity Identify antigen MSLN；

Transmembrane region, the transmembrane region are connected with the extracellular region, and are embedded into the cell membrane of the T lymphocytes；

Intracellular region, the intracellular region are connected with the transmembrane region, and the intracellular region includes the intracellular section and CD3 ζ of CD28 Chain.

2. a kind of slow virus, which is characterized in that the slow virus carries following nucleic acid molecules：

The nucleic acid molecules of encoding chimeric antigen receptor, the Chimeric antigen receptor have SEQ ID NO：Amino acid sequence shown in 1 The nucleic acid molecules of row, the encoding chimeric antigen receptor have SEQ ID NO：Nucleotide sequence shown in 2；And

The nucleic acid molecules of silenced cell immunologic test point, the nucleotide sequence of the nucleic acid molecules of the silenced cell immunologic test point To be selected from SEQ ID NO：At least one of 3~135.

3. a kind of slow virus, which is characterized in that the slow virus, which carries, contains SEQ ID NO：Nucleotide shown in 136~141 Sequence.

4. a kind of transgenosis lymphocyte, which is characterized in that the lymphocyte cell immunologic test point is silenced；And expression Chimeric antigen receptor, the Chimeric antigen receptor include：

Extracellular region, the extracellular region include the heavy chain variable region and light chain variable region of antibody, and the antibody can be with tumour antigen Specific binding；

Transmembrane region；And

Intracellular region, the intracellular region include co-stimulators intracellular section,

Wherein, the antibody is single-chain antibody, and the tumour antigen is MSLN.

5. transgenosis lymphocyte according to claim 4, which is characterized in that the lymphocyte cell immunologic test point Independently selected from least one of CTLA4, PD1, TIM3, BTLA, LAG-3, IRAK-M, SOCS1, A20, CBL-B.

6. transgenosis lymphocyte according to claim 4, which is characterized in that the lymphocyte cell immunologic test point Be silenced is realized by least one shRNA, antisense nucleic acid, ribozyme, dominant negative mutations, CRISPR and Zinc finger nuclease.

7. transgenosis lymphocyte according to claim 4, which is characterized in that the co-stimulators intracellular section is only On the spot it is selected from 4-1BB, OX-40, CD40L, CD27, CD30, CD28 and at least one of their derivative.

8. transgenosis lymphocyte according to claim 5, which is characterized in that the lymphocyte cell immunologic test point Independently selected from least one of CTLA4, PD1, CBL-B.

9. transgenosis lymphocyte according to claim 6, which is characterized in that the lymphocyte cell immunologic test point Be silenced is realized by shRNA.

10. transgenosis lymphocyte according to claim 7, which is characterized in that the co-stimulators intracellular section It is the intracellular section of 4-1BB or CD28.

11. transgenosis lymphocyte according to claim 4, which is characterized in that the lymphocyte is CD3⁺T lymphs are thin Born of the same parents.

12. transgenosis lymphocyte according to claim 4, which is characterized in that the lymphocyte is that natural kill is thin Born of the same parents.

13. transgenosis lymphocyte according to claim 4, which is characterized in that the lymphocyte is that natural killer T is thin Born of the same parents.

14. a kind of construct, which is characterized in that the construct includes：

First nucleic acid molecules, the first nucleic acid molecule encoding Chimeric antigen receptor；And

Second nucleic acid molecules, the second nucleic acid molecules silenced cell immunologic test point, wherein the cellular immunity checkpoint, The Chimeric antigen receptor is as defined in any one of claim 4~13.

15. construct according to claim 14, it is characterised in that first nucleic acid molecules and second nucleic acid point Son is arranged on expresses the Chimeric antigen receptor and silenced cell is exempted from claim 4~13 any one of them lymphocyte Epidemic disease checkpoint.

16. construct according to claim 14, which is characterized in that further comprise：

First promoter, first promoter are operably connected with first nucleic acid molecules；And

Second promoter, second promoter are operably connected with second nucleic acid molecules.

17. construct according to claim 16, which is characterized in that first promoter and second promoter point Not independently selected from U6, H1, CMV, EF-1, LTR or RSV promoters.

18. construct according to claim 14, which is characterized in that the carrier of the construct is that non-pathogenic virus carries Body.

19. construct according to claim 18, which is characterized in that the viral vectors includes being carried selected from retrovirus At least one of body, slow virus carrier or adeno-associated virus (AAV) carrier.

20. a kind of preparing T lymphocytes described in claim 1 or the leaching of claim 4~13 any one of them transgenosis The method of bar cell, which is characterized in that including：

Claim 14~19 any one of them construct or slow virus according to claim 2 or 3 are introduced into lymph In cell or T lymphocytes.

21. a kind of therapeutic combination for treating cancer, which is characterized in that including：

It is claim 14~19 any one of them construct, slow virus according to claim 2 or 3, described in claim 1 T lymphocytes or claim 4~13 any one of them transgenosis lymphocyte.

22. therapeutic combination according to claim 21, which is characterized in that the cancer includes being selected from celiothelioma, pancreas Cancer, oophoroma, cholangiocarcinoma, lung cancer, gastric cancer, intestinal cancer, at least one of the cancer of the esophagus and breast cancer.

23. a kind of method improving lymphocyte activity, the lymphocyte carry Chimeric antigen receptor, which is characterized in that make The cellular immunity checkpoint of the lymphocyte is silenced,

Institute in the cellular immunity checkpoint, the lymphocyte, any one of the Chimeric antigen receptor such as claim 4~13 Definition,

The lymphocyte activity includes the proliferative capacity of the lymphocyte in vitro, the proliferation in tumour patient body and life Deposit at least one of the orientation killing ability of ability and the lymphocyte in tumour patient body.

24. according to the method for claim 23, which is characterized in that the tumour includes being selected from celiothelioma, cancer of pancreas, ovary Cancer, cholangiocarcinoma, lung cancer, gastric cancer, intestinal cancer, at least one of the cancer of the esophagus and breast cancer.