CN109750066A

CN109750066A - Secreting type anti-immunity checkpoint antibody, immunologic test intracellular point inhibit the coexpression and its application of molecule and tEGFR molecule

Info

Publication number: CN109750066A
Application number: CN201711058873.8A
Authority: CN
Inventors: 黄雪芬; 陈思毅
Original assignee: Ai Shengxu Co
Current assignee: Ai Shengxu Co
Priority date: 2017-11-01
Filing date: 2017-11-01
Publication date: 2019-05-14

Abstract

The invention proposes a kind of expression vector, slow virus, transgenic cell, circulating antibodies and for the therapeutic combination for the treatment of cancer.The expression vector carries following nucleic acid molecules: (a) the first nucleic acid molecules, and the first nucleic acid molecule encoding secreting type immunologic test point inhibits molecule fragment；(b) the second nucleic acid molecules, second nucleic acid molecules are for immunologic test point in silenced cell, optionally, further carry third nucleic acid molecules, the third nucleic acid molecule encoding nonfunctional EGFR.The expression vector of the embodiment of the present invention is imported in recipient cell, secreting type anti-immunity checkpoint antibody high efficient expression and is secreted into extracellular in recipient cell, the immunologic test point molecule of recipient cell is thwarted by specific silencing by the Immune escaping mechanism that immunologic test point mediates simultaneously.The expression vector of the embodiment of the present invention is imported into recipient cell, in lymphocyte, the proliferative capacity and effector function of lymphocyte enhance, more powerful to the specific killing of tumour cell more effective.

Description

Secreting type anti-immunity checkpoint antibody, immunologic test intracellular point inhibit molecule and tEGFR The coexpression and its application of molecule

Technical field

The present invention relates to biomedicine fields, in particular it relates to co-express secreting type anti-immunity checkpoint antibody, Immunologic test point intracellular inhibits cell and its application of molecule and tEGFR surface markers, more particularly it relates to express Carrier, slow virus, transgenic cell, circulating antibody and the therapeutic combination for treating cancer.

Background technique

Cancer, since genes within cells mutation leads to a kind of disease of uncontrolled cellular proliferation.Human health is had become at present Significant threat, be one of the main reason for leading to human death.The World Health Organization (WHO) is in " the global cancer report delivered Accuse 2014 " in point out, global cancer patients in 2012 and death are all increasing sharply.Therefore, efficiently special cancer is found Disease treatment method has great clinical value.

Traditional tumor therapeuticing method mainly includes operation, radiation and chemotherapy, but these types of method all has biggish office Sex-limited, such as due to the proximal end invasion of cancer cell or far-end transfer, the tumour metastasis and recurrence rate after operation excision is higher, and puts It treats and chemotherapy will cause serious damage for the normal cell especially hemopoietic system and immune system of body itself, therefore Patient for metastases have occurred also is extremely difficult to preferable late result.With the further investigation of tumor cells mechanism With the further development of biotechnology, targeted drug treatment and immunization therapy play more and more in the comprehensive treatment of tumors Big effect.Targeted therapies mainly include monoclonal antibody and small molecule targeted drug, and immunotherapy mainly include cell because Sub- therapy, immunologic test point monoclonal antibody, adoptive immunotherapy, tumor vaccine etc., by transferring the anti tumor immune response of body, To control and killing tumor cell, therefore efficient height, high specificity, the good advantage of tolerance have in oncotherapy Wide prospect.

However, the immunotherapy of tumour is only effective to some patients, still need further to further investigate and develop, Lai Zengqiang Clinical efficacy.

Summary of the invention

The present invention is directed to solve at least some of the technical problems in related technologies.

In the first aspect of the present invention, the invention proposes a kind of expression vectors.According to an embodiment of the invention, the table Following nucleic acid molecules: (a) the first nucleic acid molecules, the first nucleic acid molecule encoding secreting type immunologic test point are carried up to carrier Inhibit molecule fragment；(b) the second nucleic acid molecules, second nucleic acid molecules are for immunologic test point in silenced cell, optionally Ground further carries third nucleic acid molecules, the third nucleic acid molecule encoding nonfunctional EGFR surface markers.It will be of the invention real Apply example expression vector import recipient cell in, secreting type anti-immunity checkpoint antibody in recipient cell high efficient expression and point Secrete extracellular, while the immunologic test point molecule of recipient cell is by specific silencing, immune is escaped by what immunologic test point mediated Ease mechanism is thwarted.The expression vector of the embodiment of the present invention, which is imported recipient cell, leads to lymphocyte in lymphocyte Proliferative capacity enhancing, it is more powerful to the specific killing of tumour cell more effective.

According to an embodiment of the invention, above-mentioned expression vector can further include following one or more supplementary technologies Feature:

According to an embodiment of the invention, it includes: anti-immunity checkpoint that the secreting type immunologic test point, which inhibits molecule fragment, Single-chain antibody molecules and IgG Fc, optionally, the IgG Fc are IgG1Fc or IgG4Fc.The immune inspection of secreting type as a result, It makes an inventory of and molecule fragment is inhibited to be secreted into extracellularly, and specifically bound with the immunologic test of cell surface point, and then effectively hinder Hold back the Immune escaping mechanism of immunologic test point mediation.

According to an embodiment of the invention, the immunologic test point molecule includes being selected from CTLA4, PD-1, PD-L1, PD-L2, At least one of TIM3, BTLA, LAG-3, IRAK-M, SOCS1, A20, CBL-B.The expression vector of the embodiment of the present invention is led Enter recipient lymphocytes, on the one hand, secreting type immunologic test point inhibits molecule fragment to secret out of rear and above-mentioned immunologic test point minute Son combines, and then the immunologic escape of Reverse transcriptase tumour cell, on the other hand, the immunologic test point quilt of recipient lymphocytes Silencing, the Immune escaping mechanism of cell mediated are suppressed again, so lymphocyte to the specific killing of tumour cell into One step significantly increases.

According to an embodiment of the invention, it is anti-PD-L1 single-chain antibody that the secreting type immunologic test point, which inhibits molecule fragment, With IgG1Fc fusion protein molecule.

According to an embodiment of the invention, the secreting type immunologic test point inhibit molecule fragment be anti-PD-1 single-chain antibody and IgG4 Fc fusion protein molecule.

According to an embodiment of the invention, the secreting type immunologic test point inhibit molecule fragment be anti-LAG3 single-chain antibody and IgG4Fc fusion protein molecule,

According to an embodiment of the invention, it is anti-CTLA 4 single-chain antibody that the secreting type immunologic test point, which inhibits molecule fragment, With IgG4Fc fusion protein molecule.

According to an embodiment of the invention, the silencing be by shRNA, antisense nucleic acid, ribozyme, dominant negative mutations, What at least one CRISPR-Cas9, CRISPR-Cpf1 and Zinc finger nuclease were realized, it is preferable that the silencing is to pass through shRNA It realizes.At least one of through the above way, it can effectively realize effective inhibition to intracellular immunity checkpoint molecule.

According to an embodiment of the invention, first nucleic acid molecules have nucleotides sequence shown in NO:1~5 SEQ ID Column.

ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCT CCTGATCCCAGACATTGTGCTCACCCAATCTCCAGCTTCTTTGGCTCTGTCTCCCGGGG AGAGAGCCACCCTCTCCTGCAGAGCCACTGAAAGTGTTGAATACTATGGCACAAGTTT AGTGCAGTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGC ATCCAGCGTAGATTCTGGGGTCCCTTCCAGGTTTAGTGGCAGTGGGTCTGGGACAGAC TTCACCCTCACCATCAATTCTCTGGAGGAGGAGGATGCTGCAATGTATTTCTGTCAGC AAAGTAGGAGGGTTCCGTACACGTTCGGACAGGGGACCAAGCTGGAGATAAAAGGC TCCACCTCTGGATCCGGCAAGCCCGGATCTGGCGAGGGATCCACCAAGGGCGAGGTC CAGCTGGTGCAGTCTGGAGCTGAGGTGAAAAAGCCTGGGGCTTCAGTGAAGATGTCC TGCAAGGCTTCTGGATACACATTCACTAGCTATGTTATGCACTGGGTGAAGCAGGCCC CTGGGCAGCGCCTTGAGTGGATTGGATATGTTAATCCTTTCAATGATGGTACTAAGTAC AATGAGATGTTCAAAGGCAGGGCCACACTGACTTCAGACAAATCCACCAGCACAGCC TACATGGAGCTCAGCAGCCTGAGGTCTGAGGACACTGCGGTCTATTACTGTGCAAGAC AGGCTTGGGGTTACCCCTGGGGCCAAGGGACTCTGGTCACTGTCTCTTCTGCGGCCG CAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGT CAGTCTTCCTCTTCCCCCCAAAACCCAAGGACCAACTGATGATCTCCCGGACCCCTGA GGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTG GTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGT ACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGA ATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGA AAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCC CATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCT TCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGCTGCA TGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAG G CAGCGGC (SEQ ID NO: 1)。

ATGGTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCC TCCTGATCCCAGAGATTGTGCTGACACAGTCTCCTGCTACCTTATCTCTGTCTCCAGGG CAGAGGCTCACCATCTCATGCAGGGCCAGCCAAAGTGTCAGTACATCTGGCTATAGTT ATATGCACTGGTACCAACAGAAACCAGACCAGTCCCCCAAACTCCTCATCAAGTTTGG CTCCAACCTAGAATCTGGCATCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGA CTTCACCCTCACCATCTCTTCTCTGGAGCCTGAGGATTTTGCAACATATTACTGTCAGC ACAGTTGGGAGATTCCGTACACGTTCGGACAGGGGACCAAGCTGGAAATAAAAGGCT CCACCTCTGGATCCGGCAAGCCCGGATCTGGCGAGGGATCCACCAAGGGCCAGGTCC AGCTTGTGCAGTCTGGGCATGAAGTGAAACAGCCTGGGGCCTCAGTGAAGATGTCCT GCAAGGCTTCTGGCTACAGTTTTACTAGCTCCTGGATACACTGGGTGAGACAGGCTCC TGGACAGGGTCTGGAATGGATTGGATACATTTATCCTAGCACTGGTTTTACTGAGTACA ATCAGAAGTTCAAGGACAGGGCCACATTGACTGCAGACAAATCCACCAGCACAGCCT ACATGGAACTGAGCAGCCTGAGATCTGAGGACACTGCAGTCTATTACTGTGCAAGATG GAGGGACAGCTCGGGCTACCATGCTATGGACTACTGGGGTCAAGGAACCCTGGTCAC CGTCTCCTCAGCTAGCCCCCCATGCCCATCATGCCCAGCACCTGAGTTCCTGGGGGGA CCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCC CTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTC AACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGA GCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTG GCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCAT CGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCC TGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCA AAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACA GCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCG TGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCG G GTAAA (SEQ ID NO:2).

ATGGCTCTGCCCGTGACCGCTCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGCTG CTAGACCCGACATTGTGCTGACCCAGTCCCCAGACTCCCTGGCCGTGTCTCTGGGAGA GAGGGCCACCATCAACTGCAGAGCCTCTGAGAGCGTGGAGTACTATGGCACATCCCT GATGCAGTGGTATCAGCAGAAGCCTGGCCAGCCCCCTAAGCTGCTGATCTATGCCGCC AGCAATGTGGAGTCCGGCGTGCCAGACAGGTTCTCCGGCTCTGGCAGCGGCACCGAC TTCACCCTGACAATCAGCTCCCTGCAGGCAGAGGACGTGGCCGTGTACTATTGCCAGC AGTCCCGCAAGGACCCCAGCACCTTCGGCGGAGGCACAAAGGTGGAGATCAAGGGC TCCACCTCTGGCAGCGGCAAGCCCGGCTCTGGAGAGGGCAGCACAAAGGGACAGGT GCAGCTGGTGCAGTCTGGAGCAGAGGTGAAGAAGCCTGGCGCATCCGTGAAGGTGT CTTGTAAGGCCAGCGGCTACACCTTCACAAGCTATAACATGCACTGGGTGCGGCAGGC CCCTGGCCAGGGCCTGGAGTGGATCGGCGACATCTACCCAGGCCAGGGCGATACCTC CTATAATCAGAAGTTTAAGGGCAGAGCCACCATGACAGCCGACAAGTCCACCTCTACA GTGTACATGGAGCTGTCTAGCCTGAGGAGCGAGGACACAGCCGTGTACTATTGCGCCC GCGTGGGAGGAGCATTCCCTATGGACTATTGGGGCCAGGGCACCCTGGTGACAGTGT CCTCTGCTAGCCCACCATGCCCTTCCTGTCCTGCACCAGAGTTTCTGGGCGGCCCAAG CGTGTTCCTGTTTCCTCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCAGAG GTGACATGCGTGGTGGTGGACGTGTCTCAGGAGGACCCCGAGGTGCAGTTCAACTGG TACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAGCCTCGGGAGGAGCAGTTT AACTCTACCTACAGAGTGGTGAGCGTGCTGACAGTGCTGCACCAGGATTGGCTGAAC GGCAAGGAGTATAAGTGCAAGGTGAGCAATAAGGGCCTGCCCAGCTCCATCGAGAAG ACCATCTCCAAGGCCAAGGGCCAGCCCAGAGAGCCTCAGGTGTACACACTGCCCCCT TCTCAGGAGGAGATGACCAAGAACCAGGTGAGCCTGACATGTCTGGTGAAGGGCTTC TATCCCAGCGACATCGCCGTGGAGTGGGAGTCCAATGGCCAGCCTGAGAACAATTAC AAGACCACACCACCCGTGCTGGACTCTGATGGCAGCTTCTTTCTGTATTCCAGGCTGA CCGTGGATAAGTCTCGCTGGCAGGAGGGCAACGTGTTCAGCTGTTCCGTGATGCACG AAGCACTGCACAACCACTACACTCAGAAGTCACTGTCCCTGTCACTGGGCAAG (SEQ ID NO:3).

ATGGCTCTGCCCGTGACCGCTCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGCTG CCAGACCTGAAATTGTCCTGACCCAGAGCCCCGCAACACTGTCCCTGAGCCCCGGCG AAAGAGCCACCCTGTCCTGTAGAGCCTCCCAGAGCATCAGCTCCTACCTGGCCTGGTA TCAGCAGAAGCCAGGACAGGCTCCACGACTGCTGATCTACGACGCATCCAACCGGGC CACAGGGATTCCAGCTAGATTCTCAGGAAGCGGCTCCGGGACTGACTTTACCCTGAC AATCTCTAGTCTGGAGCCTGAAGATTTCGCCGTGTACTATTGCCAGCAGCGGTCTAAC TGGCCACTGACCTTTGGACAGGGCACAAATCTGGAGATTAAGGGCTCTACCAGTGGG TCAGGAAAACCCGGCTCCGGGGAAGGATCTACAAAGGGACAGGTGCAGCTGCAGCA GTGGGGAGCAGGGCTGCTGAAACCTAGTGAGACTCTGTCACTGACCTGTGCCGTCTA CGGCGGGAGCTTCTCCGATTACTATTGGAACTGGATTCGACAGCCCCCTGGAAAGGGC CTGGAGTGGATCGGGGAAATTAATCACAGGGGATCTACCAACAGTAATCCCTCACTGA AAAGCCGCGTCACTCTGAGCCTGGACACCTCCAAGAATCAGTTCTCTCTGAAACTGA GAAGTGTGACAGCCGCTGATACTGCTGTCTACTATTGCGCATTTGGCTATAGCGATTAT GAATACAACTGGTTTGATCCTTGGGGGCAGGGGACTCTGGTCACTGTCTCCTCCGCTA GCCCACCATGCCCTTCCTGTCCTGCACCAGAGTTTCTGGGCGGCCCAAGCGTGTTCCT GTTTCCTCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCAGAGGTGACATG CGTGGTGGTGGACGTGTCTCAGGAGGACCCCGAGGTGCAGTTCAACTGGTACGTGGA TGGCGTGGAGGTGCACAATGCCAAGACCAAGCCTCGGGAGGAGCAGTTTAACTCTAC CTACAGAGTGGTGAGCGTGCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAGGA GTATAAGTGCAAGGTGAGCAATAAGGGCCTGCCCAGCTCCATCGAGAAGACCATCTCC AAGGCCAAGGGCCAGCCCAGAGAGCCTCAGGTGTACACACTGCCCCCTTCTCAGGAG GAGATGACCAAGAACCAGGTGAGCCTGACATGTCTGGTGAAGGGCTTCTATCCCAGC GACATCGCCGTGGAGTGGGAGTCCAATGGCCAGCCTGAGAACAATTACAAGACCACA CCACCCGTGCTGGACTCTGATGGCAGCTTCTTTCTGTATTCCAGGCTGACCGTGGATA AGTCTCGCTGGCAGGAGGGCAACGTGTTCAGCTGTTCCGTGATGCACGAAGCACTGC ACAACCACTACACTCAGAAGTCACTGTCCCTGTCACTGGGCAAG (SEQ ID NO:4).

ATGGTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCT CCTGATCCCAGAGATCGTGCTGACCCAGTCCCCCGGCACCCTGTCCCTGTCCCCCGGC GAGCGGGCCACCCTGTCCTGCCGGGCCTCCCAGTCCGTGGGCTCCTCCTACCTGGCCT GGTACCAGCAGAAGCCCGGCCAGGCCCCCCGGCTGCTGATCTACGGCGCCTTCTCCC GGGCCACCGGCATCCCCGACCGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCC TGACCATCTCCCGGCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTACGG CTCCTCCCCCTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGGGCTCCACCTC TGGATCCGGCAAGCCCGGATCTGGCGAGGGATCCACCAAGGGCCAGGTGCAGCTGGT GGAGTCCGGCGGCGGCGTGGTGCAGCCCGGCCGGTCCCTGCGGCTGTCCTGCGCCGC CTCCGGCTTCACCTTCTCCTCCTACACCATGCACTGGGTGCGGCAGGCCCCCGGCAAG GGCCTGGAGTGGGTGACTTTCATCTCCTACGACGGCAACAACAAGTACTACGCCGAC TCCGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTG CAGATGAACTCCCTGCGGGCCGAGGACACCGCCATCTACTACTGCGCCCGGACCGGC TGGCTGGGCCCCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCCGCT AGCCCCCCATGCCCATCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCC TGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTG CGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGA TGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCA CGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGG AGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCT CCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGG AGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCA GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACC ACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGG ACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTC TGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID NO:5).

Wherein, SEQ ID NO:1 is that coding secreting type immunologic test point inhibits molecule fragment-anti human PD-L 1 single-chain antibody With the nucleotide sequence of human IgG1 Fc fusion protein molecule (Anti-PD-L1scFv-IgG1Fc)；SEQ ID NO:2 is coding Secreting type immunologic test point inhibits the anti-human PD-1 single-chain antibody of molecule fragment-and human IgG 4Fc fusion protein molecule (Anti-PD- Nucleotide sequence 1scFv-IgG4Fc)；SEQ ID NO:3 is that coding secreting type immunologic test point inhibits molecule fragment-anti-human The nucleotide sequence of TIM3 single-chain antibody and human IgG 4Fc fusion protein molecule (Anti-PD-1scFv-IgG4Fc)；SEQ ID NO:4 is that coding secreting type immunologic test point inhibits the anti-human LAG3 single-chain antibody of molecule fragment-and human IgG 4Fc fusion protein minute The nucleotide sequence of sub (Anti-LAG3scFv-IgG4Fc)；SEQ ID NO:5 is coding secreting type immunologic test point inhibition minute The nucleosides of sub-piece-anti-human CTLA4 single-chain antibody and human IgG 4Fc fusion protein molecule (Anti-CTLA4scFv-IgG4Fc) Acid sequence.

According to an embodiment of the invention, second nucleic acid molecules have nucleotides sequence shown in NO:6~138 SEQ ID Column.

GGCCAGGATGGTTCTTAGACT (SEQ ID NO:6).

GGATTTCCAGTGGCGAGAGAA (SEQ ID NO:7).

GCCUGUGUUCUCUGUGGACUAUG (SEQ ID NO:8).

GGUGCUGCUAGUCUGGGUCCUGG (SEQ ID NO:9).

GACAGAGAGAAGGGCAGAAGUGC (SEQ ID NO:10).

CAGCUUCUCCAACACAUCGGAGA (SEQ ID NO:11).

CCGUGUCACACAACUGCCCAACG (SEQ ID NO:12).

UAUGCCACCAUUGUCUUUCCUAG (SEQ ID NO:13).

UGCUAAACUGGUACCGCAUGAGC (SEQ ID NO:14).

GUGACAGAGAGAAGGGCAGAAGU (SEQ ID NO:15).

CUGAGGAUGGACACUGCUCUUGG (SEQ ID NO:16).

AUCGGAGAGCUUCGUGCUAAACU (SEQ ID NO:17).

GGCAACGGAACCCAGATTTAT (SEQ ID NO:18).

GGAACCCAAATTACGTGTACT (SEQ ID NO:19).

GAACCCAAATTACGTGTACTA (SEQ ID NO:20).

GGGAGAAGACTATATTGTACA (SEQ ID NO:21).

GACGTTTATAGCCGAAATGAT (SEQ ID NO:22).

GACACTAATACACCAGGTAGA (SEQ ID NO:23).

ACCUCACUAUCCAAGGACUGAGG (SEQ ID NO:24).

AUGAGUUGACCUUCCUAGAUGAU (SEQ ID NO:25).

GGGGAAUGAGUUGACCUUCCUAG (SEQ ID NO:26).

CUCUGGAUCCUUGCAGCAGUUAG (SEQ ID NO:27).

CUCCUCUGGAUCCUUGCAGCAGU (SEQ ID NO:28).

UUUGUGUGUGAGUAUGCAUCUCC (SEQ ID NO:29).

CACCUCCAGUGGAAAUCAAGUGA (SEQ ID NO:30).

CACGGGACUCUACAUCUGCAAGG (SEQ ID NO:31).

UUCUGACUUCCUCCUCUGGAUCC (SEQ ID NO:32).

AAGUCUGUGCGGCAACCUACAUG (SEQ ID NO:33).

GGTCGGTCAGAATGCCTATCT (SEQ ID NO:34).

GCCAATGACTTACGGGACTCT (SEQ ID NO:35).

GCAGAGGGAATTCGCTCAGAA (SEQ ID NO:36).

GGAAATTCGGGCACATCATAT (SEQ ID NO:37).

GATTAAGAGATGACTGGACTA (SEQ ID NO:38).

GAGATGACTGGACTAGGTCTA (SEQ ID NO:39).

AGGAAAUUCGGGCACAUCAUAUG (SEQ ID NO:40).

GACUGAUGAAAGGGAUGUGAAUU (SEQ ID NO:41).

GCCACUGAUUUUCAAAGAGAUCU (SEQ ID NO:42).

AGCAGAGUUUUCCCAUUUUCAGA (SEQ ID NO:43).

AACUUAAACAGGCAUGUCAUUGC (SEQ ID NO:44).

UUCAGAAGAUAAUGACUCACAUG (SEQ ID NO:45).

GCCUCUGUAUUUAAGCCAACAGA (SEQ ID NO:46).

UGCUCAUGUGAUUGUGGAGUAGA (SEQ ID NO:47).

AUGUUUUCACAUCUUCCCUUUGA (SEQ ID NO:48).

GAGAGACUUCACUGCAGCCUUUC (SEQ ID NO:49).

GATTGCCTCTACTCATCACTA (SEQ ID NO:50).

UCCUAAUGACAAUGGGUCAUACC (SEQ ID NO:51).

AAGACAUUGCCUGCCAUGCUUGG (SEQ ID NO:52).

GUCAUACCGCUGUUCUGCAAAUU (SEQ ID NO:53).

CUCCUGUAUAGUUUACUUCCUUU (SEQ ID NO:54).

UACCGCUGUUCUGCAAAUUUUCA (SEQ ID NO:55).

AAAACAAACCAGGCAUUGUUUAU (SEQ ID NO:56).

AACUAGAAUGCCCUGUGAAAUAC (SEQ ID NO:57).

GUGACUUGGUGCAAGCUCAAUGG (SEQ ID NO:58).

AUCCAUGGGAAAGAAUCAUGUGA (SEQ ID NO:59).

UGGUGCAAGCUCAAUGGAACAAC (SEQ ID NO:60).

GCTGCTCACCCTTATGAACCT (SEQ ID NO:61).

AGGACAUGGUGGUGGACGAGUGC (SEQ ID NO:62).

UGCUCUUCCUGCACGAUAUCAGU (SEQ ID NO:63).

ACCUCUACUGGUUCCUGUACAUC (SEQ ID NO:64).

CCCUCCAACUCUGCUCCUCUAGG (SEQ ID NO:65).

CCCUGAGUGGACAGUCGUCUUCG (SEQ ID NO:66).

CUGCUCCAGGGAAGCUUCUAUGG (SEQ ID NO:67).

CGCUCAAGGUCCUGUAUGCCACC (SEQ ID NO:68).

GAGUUCACCAAGCUCAACAUUUA (SEQ ID NO:69).

UGCUGCUGCUCACCCUUAUGAAC (SEQ ID NO:70).

CCCAUCUCCGUGCUCUUCUUUGA (SEQ ID NO:71).

GGGACATCGTCGAGCTATTCA (SEQ ID NO:72).

GGACATCGTCGAGCTATTCAT (SEQ ID NO:73).

GCCAATGTCACCGTGGATAAT (SEQ ID NO:74).

GTCATCTGTGGCAGTATATCA (SEQ ID NO:75).

GGATGTAGAGTAGTGTTAGAT (SEQ ID NO:76).

GGCAAAGTTAAGACCATCAAT (SEQ ID NO:77).

GACCAAATCCACGCTCAATTA (SEQ ID NO:78).

UACUGCUUAAAUCUUCCAUCAGC (SEQ ID NO:79).

GACUGAGAAGUUCUGUCUGAUUU (SEQ ID NO:80).

CUGUUUCAUCACCCAAACAUACU (SEQ ID NO:81).

GAAGAUCCUCCCACAUCACUAAA (SEQ ID NO:82).

UAGACCAAGGUAAAAGUGGAACA (SEQ ID NO:83).

AAGAGGUUUUUAUCUGAGCUUGA (SEQ ID NO:84).

UACCUGCACAACGUUCAACCAUG (SEQ ID NO:85).

GCCUGGAUUCAUGUCUCUCAUUU (SEQ ID NO:86).

CCCUCGGAAUUUCUCUGCCAAGC (SEQ ID NO:87).

UGCUGAAGAUCCUCCCACAUCAC (SEQ ID NO:88).

GCACCTCCTACCTCTTCATGT (SEQ ID NO:89).

CGCACUUCCGCACAUUCCGUUCG (SEQ ID NO:90).

GGGGAGGGUCUCUGGCUUUAUUU (SEQ ID NO:91).

CAGCAUUAACUGGGAUGCCGUGU (SEQ ID NO:92).

CCAGGACCUGAACUCGCACCUCC (SEQ ID NO:93).

UACAUAUACCCAGUAUCUUUGCA (SEQ ID NO:94).

GCCGACAAUGCAGUCUCCACAGC (SEQ ID NO:95).

CCCCUGGUUGUUGUAGCAGCUUA (SEQ ID NO:96).

CUGCUGUGCAGAAUCCUAUUUUA (SEQ ID NO:97).

UGGGAUGCCGUGUUAUUUUGUUA (SEQ ID NO:98).

UCGCACCUCCUACCUCUUCAUGU (SEQ ID NO:99).

GCGGAAAGCTGTGAAGATACG (SEQ ID NO:100).

ACAAAGCCCTCATCGACAGAA (SEQ ID NO:101).

ATGCCACTTCTCAGTACATGT (SEQ ID NO:102).

GTGGACTTCAGTACAACTCAC (SEQ ID NO:103).

GTGGAATTTACTTGCCTCTCC (SEQ ID NO:104).

GTTGGATGAAGCTAACTTACC (SEQ ID NO:105).

ACTGGGAAGACGTGTAACTCT (SEQ ID NO:106).

AAGGAATTGCATCCAAGGTAT (SEQ ID NO:107).

GGAATTGCATCCAAGGTATAC (SEQ ID NO:108).

GGATGAGACTGGCAATGGTCA (SEQ ID NO:109).

UCCUCAGUUUCGGGAGAUCAUCC (SEQ ID NO:110).

GAGUCUCUCAAAUCUCAGGAAUU (SEQ ID NO:111).

AGCUCUAGUCCUUUUUGUGUAAU (SEQ ID NO:112).

CACUGGAAAUGUUCAGAACUUGC (SEQ ID NO:113).

AUGAUGAAUGGGACAAUCUUAUC (SEQ ID NO:114).

CACACUGUGUUUCAUCGAGUACA (SEQ ID NO:115).

GCAGAACCAUCCAUGGACUGUGA (SEQ ID NO:116).

AAAGAUGUGGCCUUUUGUGAUGG (SEQ ID NO:117).

UUCAGAACUUGCCAGUUUUGUCC (SEQ ID NO:118).

AUGAGACUGGCAAUGGUCACAGG (SEQ ID NO:119).

GTCAATTCCAGGGAGATAACT (SEQ ID NO:120).

GCCTGGAAGCAATGGCTCTAA (SEQ ID NO:121).

GCACCAAACCCGGAAGCTATA (SEQ ID NO:122).

GTTGCACTCGATTGGGACAGT (SEQ ID NO:123).

GGATTATGTGAACCTACACCT (SEQ ID NO:124).

GGAATCACAGCGAGTTCAAAT (SEQ ID NO:125).

GCAAGGCATAGTCTCATTGAA (SEQ ID NO:126).

GGTGAAGAGAGCCTTAGAGAT (SEQ ID NO:127).

GTGAAGAGAGCCTTAGAGATA (SEQ ID NO:128).

AGGAGCUAAGGUCUUUUCCAAUG (SEQ ID NO:129).

AUGUCGAUGCAAAAAUUGCAAAA (SEQ ID NO:130).

GUCACAUGCUGGCAGAAAUCAAA (SEQ ID NO:131).

UCCAGGUUACAUGGCAUUUCUCA (SEQ ID NO:132).

UUGAACUUUGAACCUGUGAAAUG (SEQ ID NO:133).

UCCACAUCAACAGCUAAAUCAUU (SEQ ID NO:134).

AUGCUGGCAGAAAUCAAAGCAAU (SEQ ID NO:135).

UGCAGAGAAUGACAAAGAUGUCA (SEQ ID NO:136).

GGCAGAACUCACCAGUCACAUCA (SEQ ID NO:137).

UCGGUCCUGUGAUAAUGGUCACU (SEQ ID NO:138).

Wherein, NO:6~17 SEQ ID are mankind's programmed death receptor 1 (PD1) siRNA nucleotide sequence, SEQ ID NO:18~33 are human cell poison T lymphocyte antigen 4 (CTLA4) siRNA sequences, and NO:34~49 SEQ ID are people 3 (TIM3) siRNA sequence of class T cell immunoglobulin mucoprotein molecule, NO:50~60 SEQ ID are mankind's T lymphocytes Decay factor (BTLA) siRNA sequence, NO:61~71 SEQ ID are 3 albumen of Human Lymphocytes activating gene (LAG1) SiRNA sequence, NO:72~88 SEQ ID are mankind IRAK-M (interleukin 1 receptor associated kinase 3) siRNA nucleotide Sequence, NO:89~99 SEQ ID are mankind SOCS1 (Suppressor of Cytokine Signaling 1) siRNA sequence, SEQ ID NO:100~119 are mankind A20 (tumor necrosis factor-alpha inducible protein A20) siRNA sequence, NO:120~138 SEQ ID It is mankind CBL-B (E3 ubiquitin protein ligase CBL-B) siRNA sequence.

According to an embodiment of the invention, the third nucleic acid molecules have nucleotide sequence shown in SEQ ID NO:139.

ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCCG CACGCCCTGGGAGTCGCAAAGTCTGTAATGGGATCGGCATCGGCGAGTTCAAGGACA GCCTGTCCATCAACGCCACCAATATCAAGCACTTTAAGAATTGCACATCTATCAGCGGC GACCTGCACATCCTGCCAGTGGCCTTCCGGGGCGATTCTTTTACCCACACACCCCCTC TGGACCCTCAGGAGCTGGATATCCTGAAGACCGTGAAGGAGATCACAGGCTTCCTGC TGATCCAGGCCTGGCCTGAGAACAGAACCGATCTGCACGCCTTTGAGAATCTGGAGA TCATCCGGGGCAGAACAAAGCAGCACGGCCAGTTCTCCCTGGCCGTGGTGTCTCTGA ACATCACCAGCCTGGGCCTGAGGTCCCTGAAGGAGATCTCTGACGGCGATGTGATCAT CTCCGGCAACAAGAACCTGTGCTACGCCAACACAATCAATTGGAAGAAGCTGTTTGG CACCTCTGGCCAGAAGACAAAGATCATCTCTAACCGGGGCGAGAATAGCTGCAAGGC AACCGGACAGGTGTGCCACGCACTGTGCAGCCCAGAGGGATGTTGGGGCCCAGAGC CACGGGACTGCGTGAGCTGTAGAAACGTGTCCAGGGGCCGCGAGTGCGTGGATAAGT GTAATCTGCTGGAGGGCGAGCCAAGGGAGTTCGTGGAGAACTCCGAGTGCATCCAGT GTCACCCCGAGTGCCTGCCTCAGGCCATGAACATCACCTGTACAGGCCGCGGCCCCG ACAATTGCATCCAGTGTGCCCACTATATCGATGGCCCTCACTGCGTGAAGACCTGTCC AGCCGGCGTGATGGGCGAGAACAATACACTGGTGTGGAAGTACGCAGACGCAGGAC ACGTGTGCCACCTGTGCCACCCCAATTGCACCTATGGCTGTACAGGACCAGGCCTGGA GGGATGCCCAACCAACGGCCCTAAGATCCCAAGCATCGCCACAGGCATGGTGGGGGC ACTGCTGCTGCTGCTGGTGGTGGCTCTGGGGATTGGGCTGTTTATGAGAAGGTAA (SEQ ID NO:139)。

According to an embodiment of the invention, nonfunctional EGFR (tEGFR) receptor lack the end N- ligand binding domain and into the cell by Body tyrosine kinase activity, but the transmembrane region including Wild type EGFR receptor and the complete sequence in conjunction with anti-egfr antibodies, So nonfunctional EGFR receptor can be used as the suicide label of lymphocyte.The slow virus of the embodiment of the present invention imports receptor lymph In cell, the expression of nonfunctional EGFR receptor can be in the targeting killing effect to tumour cell that lymphocyte is effectively ensured Under the premise of, if serious adverse reaction occurs in patient, lymphocyte can be removed by anti-egfr antibodies, and then improve the present invention The safety of the treatment tumour patient such as slow virus, lymphocyte of embodiment.

According to an embodiment of the invention, the expression vector further comprises: the first promoter, first promoter with First nucleic acid molecules are operably connected；Second promoter, second promoter can be grasped with second nucleic acid molecules Make ground connection；And optional third promoter, the optional third promoter and the third nucleic acid molecules are operationally Connection.Above-mentioned first, second and optional third promoter can separately start expression first, second and optionally Third nucleic acid molecules, and then be more conducive to the regulation of corresponding nucleic developed by molecule.

According to the embodiment that we are bright, first promoter, second promoter, third promoter difference are only On the spot it is selected from U6, H1, CMV, EF-1, LTR or RSV promoter.Inventors have found that U6, H1, CMV, EF-1, LTR or RSV are opened Mover can efficiently start expression first, second and optional third nucleic acid molecules, first, second and optional third The expression efficiency of nucleic acid molecules significantly improves.

According to an embodiment of the invention, the expression vector includes the first nucleic acid molecules, the second nucleic acid molecules and third core Acid molecule, and the expression vector further comprises: internal ribosome entry site sequence, and the internal ribosome enters Site sequence is arranged between first nucleic acid molecules and the third nucleic acid molecules, the internal ribosome entry site With nucleotide sequence shown in SEQ ID NO:140.

CCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGC CGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGG GCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCG CCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTT CTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTG GCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGG CACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCT CCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGG ATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAA CGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAAT ATGGCCACAACC(SEQ ID NO:140).

The introducing of internal ribosome entry site sequence, so that the starting expression of third nucleic acid molecules does not depend on 5 ' cap knots Structure, and first and the proportional expression of third nucleic acid molecules, and then it is more conducive to expression regulation, transgenosis leaching obtained The Therapeutic safety of bar cell is higher.

According to an embodiment of the invention, the expression vector includes the first nucleic acid molecules, the second nucleic acid molecules and third core Acid molecule, and the expression vector further comprises: the 4th nucleic acid molecules, the 4th nucleic acid molecules setting is described the Between one nucleic acid molecules and the third nucleic acid molecules or between multiple first nucleic acid molecules, and the 4th nucleic acid Molecule encoding link peptide.The introducing of link peptide is so that expressed secreting type immunologic test point inhibits molecule fragment and idle Energy EGFR is in non-fused state, and expressed multiple recombinant proteins are in non-fused state.

According to a particular embodiment of the invention, the link peptide has amino acid sequence shown in NO:141~144 SEQ ID Column.

E G R G S L L T C G D V E E N P G P (SEQ ID NO:141).

A T N F S L L K Q A G D V E E N P G P (SEQ ID NO:142).

Q C T N Y A L L K L A G D V E S N P G P (SEQ ID NO:143).

V K Q T L N F D L L K L AG D V E S N P G P (SEQ ID NO:144).

Link peptide with amino acid sequence shown in NO:141~144 SEQ ID, the link peptide can it is described by It is cut in body cell.The introducing of link peptide is so that expressed secreting type immunologic test point inhibits molecule fragment and idle Energy EGFR is in non-fused state, and it is in non-fused state that expressed multiple secreting type immunologic test points, which inhibit molecule fragment,.

According to an embodiment of the invention, the expression vector is non-pathogenic virus carrier.

According to the specific embodiment of invention, the viral vectors include selected from retrovirus vector, slow virus carrier or At least one of adeno-associated virus (AAV) carrier.The pathogenic sites of construct carrier in inventive embodiments are by modification or dash forward Become, has lost the pathogenic of virus, and then treatment in the case where non-pathogenic virus according to an embodiment of the present invention is carrier mediated Safety is higher.For the carrier of the virus of the embodiment of the present invention in virus packaging and course of infection, virus-infected area is extensive, Not only terminally differentiated cells can be infected, but also the cell in division stage can be infected, can not only be integrated into host chromosome, but also can dissociate Except host chromosome, and then it can realize wide spectrum and efficient efficiency of infection.

In the second aspect of the present invention, the invention proposes a kind of slow virus.According to an embodiment of the invention, the slow disease Poison carries the nucleic acid with nucleotide sequence shown in NO:145~149 SEQ ID.

ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCT CCTGATCCCAGACATTGTGCTCACCCAATCTCCAGCTTCTTTGGCTCTGTCTCCCGGGG AGAGAGCCACCCTCTCCTGCAGAGCCACTGAAAGTGTTGAATACTATGGCACAAGTTT AGTGCAGTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGC ATCCAGCGTAGATTCTGGGGTCCCTTCCAGGTTTAGTGGCAGTGGGTCTGGGACAGAC TTCACCCTCACCATCAATTCTCTGGAGGAGGAGGATGCTGCAATGTATTTCTGTCAGC AAAGTAGGAGGGTTCCGTACACGTTCGGACAGGGGACCAAGCTGGAGATAAAAGGC TCCACCTCTGGATCCGGCAAGCCCGGATCTGGCGAGGGATCCACCAAGGGCGAGGTC CAGCTGGTGCAGTCTGGAGCTGAGGTGAAAAAGCCTGGGGCTTCAGTGAAGATGTCC TGCAAGGCTTCTGGATACACATTCACTAGCTATGTTATGCACTGGGTGAAGCAGGCCC CTGGGCAGCGCCTTGAGTGGATTGGATATGTTAATCCTTTCAATGATGGTACTAAGTAC AATGAGATGTTCAAAGGCAGGGCCACACTGACTTCAGACAAATCCACCAGCACAGCC TACATGGAGCTCAGCAGCCTGAGGTCTGAGGACACTGCGGTCTATTACTGTGCAAGAC AGGCTTGGGGTTACCCCTGGGGCCAAGGGACTCTGGTCACTGTCTCTTCTGCGGCCG CAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGT CAGTCTTCCTCTTCCCCCCAAAACCCAAGGACCAACTGATGATCTCCCGGACCCCTGA GGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTG GTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGT ACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGA ATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGA AAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCC CATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCT TCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGCTGCA TGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGG CAGCGGCGAGGGCAGGGGCAGCCTGCTGACCTGCGGCGACGTGGAGGAGAACCCCG GCCCCATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGC CGCACGCCCTGGGAGTCGCAAAGTCTGTAATGGGATCGGCATCGGCGAGTTCAAGGA CAGCCTGTCCATCAACGCCACCAATATCAAGCACTTTAAGAATTGCACATCTATCAGCG GCGACCTGCACATCCTGCCAGTGGCCTTCCGGGGCGATTCTTTTACCCACACACCCCC TCTGGACCCTCAGGAGCTGGATATCCTGAAGACCGTGAAGGAGATCACAGGCTTCCT GCTGATCCAGGCCTGGCCTGAGAACAGAACCGATCTGCACGCCTTTGAGAATCTGGA GATCATCCGGGGCAGAACAAAGCAGCACGGCCAGTTCTCCCTGGCCGTGGTGTCTCT GAACATCACCAGCCTGGGCCTGAGGTCCCTGAAGGAGATCTCTGACGGCGATGTGAT CATCTCCGGCAACAAGAACCTGTGCTACGCCAACACAATCAATTGGAAGAAGCTGTT TGGCACCTCTGGCCAGAAGACAAAGATCATCTCTAACCGGGGCGAGAATAGCTGCAA GGCAACCGGACAGGTGTGCCACGCACTGTGCAGCCCAGAGGGATGTTGGGGCCCAG AGCCACGGGACTGCGTGAGCTGTAGAAACGTGTCCAGGGGCCGCGAGTGCGTGGATA AGTGTAATCTGCTGGAGGGCGAGCCAAGGGAGTTCGTGGAGAACTCCGAGTGCATCC AGTGTCACCCCGAGTGCCTGCCTCAGGCCATGAACATCACCTGTACAGGCCGCGGCC CCGACAATTGCATCCAGTGTGCCCACTATATCGATGGCCCTCACTGCGTGAAGACCTG TCCAGCCGGCGTGATGGGCGAGAACAATACACTGGTGTGGAAGTACGCAGACGCAGG ACACGTGTGCCACCTGTGCCACCCCAATTGCACCTATGGCTGTACAGGACCAGGCCTG GAGGGATGCCCAACCAACGGCCCTAAGATCCCAAGCATCGCCACAGGCATGGTGGGG GCACTGCTGCTGCTGCTGGTGGTGGCTCTGGGGATTGGGCTGTTTATGAGAAGGTAAT CCTACTGCGAATTCTCGAGCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCC CTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAA AATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGG TGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGAT GCGGTGGGCTCTATGGGTCGACAGATCTCAAGGTCGGGCAGGAAGAGGGCCTATTTC CCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTA ATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTT CTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTA ACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCTC CCCAACACAGACGCCATGATTTGCTTCAAGAGAGCAAATCATGGCGTCTGTGTTTT TT T (SEQ ID NO:145).

ATGgTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCT CCTGATCCCAGAGATTGTGCTGACACAGTCTCCTGCTACCTTATCTCTGTCTCCAGGGC AGAGGCTCACCATCTCATGCAGGGCCAGCCAAAGTGTCAGTACATCTGGCTATAGTTA TATGCACTGGTACCAACAGAAACCAGACCAGTCCCCCAAACTCCTCATCAAGTTTGGC TCCAACCTAGAATCTGGCATCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACCCTCACCATCTCTTCTCTGGAGCCTGAGGATTTTGCAACATATTACTGTCAGCA CAGTTGGGAGATTCCGTACACGTTCGGACAGGGGACCAAGCTGGAAATAAAAGGCTC CACCTCTGGATCCGGCAAGCCCGGATCTGGCGAGGGATCCACCAAGGGCCAGGTCCA GCTTGTGCAGTCTGGGCATGAAGTGAAACAGCCTGGGGCCTCAGTGAAGATGTCCTG CAAGGCTTCTGGCTACAGTTTTACTAGCTCCTGGATACACTGGGTGAGACAGGCTCCT GGACAGGGTCTGGAATGGATTGGATACATTTATCCTAGCACTGGTTTTACTGAGTACAA TCAGAAGTTCAAGGACAGGGCCACATTGACTGCAGACAAATCCACCAGCACAGCCTA CATGGAACTGAGCAGCCTGAGATCTGAGGACACTGCAGTCTATTACTGTGCAAGATGG AGGGACAGCTCGGGCTACCATGCTATGGACTACTGGGGTCAAGGAACCCTGGTCACC GTCTCCTCAGCTAGCCCCCCATGCCCATCATGCCCAGCACCTGAGTTCCTGGGGGGAC CATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCC TGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCA ACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGC AGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGC TGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCG AGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTG CCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA GGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCA GGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGA TGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGTA AAGGCAGCGGCGAGGGCAGGGGCAGCCTGCTGACCTGCGGCGACGTGGAGGAGAA CCCCGGCCCCATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCTCTGGCTCTGCTGCTG CACGCCGCACGCCCTGGGAGTCGCAAAGTCTGTAATGGGATCGGCATCGGCGAGTTC AAGGACAGCCTGTCCATCAACGCCACCAATATCAAGCACTTTAAGAATTGCACATCTA TCAGCGGCGACCTGCACATCCTGCCAGTGGCCTTCCGGGGCGATTCTTTTACCCACAC ACCCCCTCTGGACCCTCAGGAGCTGGATATCCTGAAGACCGTGAAGGAGATCACAGG CTTCCTGCTGATCCAGGCCTGGCCTGAGAACAGAACCGATCTGCACGCCTTTGAGAAT CTGGAGATCATCCGGGGCAGAACAAAGCAGCACGGCCAGTTCTCCCTGGCCGTGGTG TCTCTGAACATCACCAGCCTGGGCCTGAGGTCCCTGAAGGAGATCTCTGACGGCGAT GTGATCATCTCCGGCAACAAGAACCTGTGCTACGCCAACACAATCAATTGGAAGAAG CTGTTTGGCACCTCTGGCCAGAAGACAAAGATCATCTCTAACCGGGGCGAGAATAGC TGCAAGGCAACCGGACAGGTGTGCCACGCACTGTGCAGCCCAGAGGGATGTTGGGG CCCAGAGCCACGGGACTGCGTGAGCTGTAGAAACGTGTCCAGGGGCCGCGAGTGCG TGGATAAGTGTAATCTGCTGGAGGGCGAGCCAAGGGAGTTCGTGGAGAACTCCGAGT GCATCCAGTGTCACCCCGAGTGCCTGCCTCAGGCCATGAACATCACCTGTACAGGCCG CGGCCCCGACAATTGCATCCAGTGTGCCCACTATATCGATGGCCCTCACTGCGTGAAG ACCTGTCCAGCCGGCGTGATGGGCGAGAACAATACACTGGTGTGGAAGTACGCAGAC GCAGGACACGTGTGCCACCTGTGCCACCCCAATTGCACCTATGGCTGTACAGGACCA GGCCTGGAGGGATGCCCAACCAACGGCCCTAAGATCCCAAGCATCGCCACAGGCATG GTGGGGGCACTGCTGCTGCTGCTGGTGGTGGCTCTGGGGATTGGGCTGTTTATGAGAA GGTAATCCTACTGCGAATTCTCGAGCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGT TTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCT AATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGT GGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGG GGATGCGGTGGGCTCTATGGGTCGACAGATCTCAAGGTCGGGCAGGAAGAGGGCCTA TTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGA ATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATA ATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTAC CGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACA CCTCCCCAACACAGACGCCATGATTTGCTTCAAGAGAGCAAATCATGGCGTCTGTG TT TTTTT (SEQ ID NO: 146)。

ATGGCTCTGCCCGTGACCGCTCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGCTG CTAGACCCGACATTGTGCTGACCCAGTCCCCAGACTCCCTGGCCGTGTCTCTGGGAGA GAGGGCCACCATCAACTGCAGAGCCTCTGAGAGCGTGGAGTACTATGGCACATCCCT GATGCAGTGGTATCAGCAGAAGCCTGGCCAGCCCCCTAAGCTGCTGATCTATGCCGCC AGCAATGTGGAGTCCGGCGTGCCAGACAGGTTCTCCGGCTCTGGCAGCGGCACCGAC TTCACCCTGACAATCAGCTCCCTGCAGGCAGAGGACGTGGCCGTGTACTATTGCCAGC AGTCCCGCAAGGACCCCAGCACCTTCGGCGGAGGCACAAAGGTGGAGATCAAGGGC TCCACCTCTGGCAGCGGCAAGCCCGGCTCTGGAGAGGGCAGCACAAAGGGACAGGT GCAGCTGGTGCAGTCTGGAGCAGAGGTGAAGAAGCCTGGCGCATCCGTGAAGGTGT CTTGTAAGGCCAGCGGCTACACCTTCACAAGCTATAACATGCACTGGGTGCGGCAGGC CCCTGGCCAGGGCCTGGAGTGGATCGGCGACATCTACCCAGGCCAGGGCGATACCTC CTATAATCAGAAGTTTAAGGGCAGAGCCACCATGACAGCCGACAAGTCCACCTCTACA GTGTACATGGAGCTGTCTAGCCTGAGGAGCGAGGACACAGCCGTGTACTATTGCGCCC GCGTGGGAGGAGCATTCCCTATGGACTATTGGGGCCAGGGCACCCTGGTGACAGTGT CCTCTGCTAGCCCACCATGCCCTTCCTGTCCTGCACCAGAGTTTCTGGGCGGCCCAAG CGTGTTCCTGTTTCCTCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCAGAG GTGACATGCGTGGTGGTGGACGTGTCTCAGGAGGACCCCGAGGTGCAGTTCAACTGG TACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAGCCTCGGGAGGAGCAGTTT AACTCTACCTACAGAGTGGTGAGCGTGCTGACAGTGCTGCACCAGGATTGGCTGAAC GGCAAGGAGTATAAGTGCAAGGTGAGCAATAAGGGCCTGCCCAGCTCCATCGAGAAG ACCATCTCCAAGGCCAAGGGCCAGCCCAGAGAGCCTCAGGTGTACACACTGCCCCCT TCTCAGGAGGAGATGACCAAGAACCAGGTGAGCCTGACATGTCTGGTGAAGGGCTTC TATCCCAGCGACATCGCCGTGGAGTGGGAGTCCAATGGCCAGCCTGAGAACAATTAC AAGACCACACCACCCGTGCTGGACTCTGATGGCAGCTTCTTTCTGTATTCCAGGCTGA CCGTGGATAAGTCTCGCTGGCAGGAGGGCAACGTGTTCAGCTGTTCCGTGATGCACG AAGCACTGCACAACCACTACACTCAGAAGTCACTGTCCCTGTCACTGGGCAAGGGCA GCGGCGAGGGCAGGGGCAGCCTGCTGACCTGCGGCGACGTGGAGGAGAACCCCGGC CCCATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCCG CACGCCCTGGGAGTCGCAAAGTCTGTAATGGGATCGGCATCGGCGAGTTCAAGGACA GCCTGTCCATCAACGCCACCAATATCAAGCACTTTAAGAATTGCACATCTATCAGCGGC GACCTGCACATCCTGCCAGTGGCCTTCCGGGGCGATTCTTTTACCCACACACCCCCTC TGGACCCTCAGGAGCTGGATATCCTGAAGACCGTGAAGGAGATCACAGGCTTCCTGC TGATCCAGGCCTGGCCTGAGAACAGAACCGATCTGCACGCCTTTGAGAATCTGGAGA TCATCCGGGGCAGAACAAAGCAGCACGGCCAGTTCTCCCTGGCCGTGGTGTCTCTGA ACATCACCAGCCTGGGCCTGAGGTCCCTGAAGGAGATCTCTGACGGCGATGTGATCAT CTCCGGCAACAAGAACCTGTGCTACGCCAACACAATCAATTGGAAGAAGCTGTTTGG CACCTCTGGCCAGAAGACAAAGATCATCTCTAACCGGGGCGAGAATAGCTGCAAGGC AACCGGACAGGTGTGCCACGCACTGTGCAGCCCAGAGGGATGTTGGGGCCCAGAGC CACGGGACTGCGTGAGCTGTAGAAACGTGTCCAGGGGCCGCGAGTGCGTGGATAAGT GTAATCTGCTGGAGGGCGAGCCAAGGGAGTTCGTGGAGAACTCCGAGTGCATCCAGT GTCACCCCGAGTGCCTGCCTCAGGCCATGAACATCACCTGTACAGGCCGCGGCCCCG ACAATTGCATCCAGTGTGCCCACTATATCGATGGCCCTCACTGCGTGAAGACCTGTCC AGCCGGCGTGATGGGCGAGAACAATACACTGGTGTGGAAGTACGCAGACGCAGGAC ACGTGTGCCACCTGTGCCACCCCAATTGCACCTATGGCTGTACAGGACCAGGCCTGGA GGGATGCCCAACCAACGGCCCTAAGATCCCAAGCATCGCCACAGGCATGGTGGGGGC ACTGCTGCTGCTGCTGGTGGTGGCTCTGGGGATTGGGCTGTTTATGAGAAGGTAATCC TACTGCGAATTCTCGAGCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCT CCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAAT GAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGG GGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCG GTGGGCTCTATGGGTCGACAGATCTCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCAT GATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTT GACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTT GGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACT TGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCTCCCC AACACAGACGCCATGATTTGCTTCAAGAGAGCAAATCATGGCGTCTGTGTTTTTTT (SEQ ID NO:147).

ATGGCTCTGCCCGTGACCGCTCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGCTG CCAGACCTGAAATTGTCCTGACCCAGAGCCCCGCAACACTGTCCCTGAGCCCCGGCG AAAGAGCCACCCTGTCCTGTAGAGCCTCCCAGAGCATCAGCTCCTACCTGGCCTGGTA TCAGCAGAAGCCAGGACAGGCTCCACGACTGCTGATCTACGACGCATCCAACCGGGC CACAGGGATTCCAGCTAGATTCTCAGGAAGCGGCTCCGGGACTGACTTTACCCTGAC AATCTCTAGTCTGGAGCCTGAAGATTTCGCCGTGTACTATTGCCAGCAGCGGTCTAAC TGGCCACTGACCTTTGGACAGGGCACAAATCTGGAGATTAAGGGCTCTACCAGTGGG TCAGGAAAACCCGGCTCCGGGGAAGGATCTACAAAGGGACAGGTGCAGCTGCAGCA GTGGGGAGCAGGGCTGCTGAAACCTAGTGAGACTCTGTCACTGACCTGTGCCGTCTA CGGCGGGAGCTTCTCCGATTACTATTGGAACTGGATTCGACAGCCCCCTGGAAAGGGC CTGGAGTGGATCGGGGAAATTAATCACAGGGGATCTACCAACAGTAATCCCTCACTGA AAAGCCGCGTCACTCTGAGCCTGGACACCTCCAAGAATCAGTTCTCTCTGAAACTGA GAAGTGTGACAGCCGCTGATACTGCTGTCTACTATTGCGCATTTGGCTATAGCGATTAT GAATACAACTGGTTTGATCCTTGGGGGCAGGGGACTCTGGTCACTGTCTCCTCCGCTA GCCCACCATGCCCTTCCTGTCCTGCACCAGAGTTTCTGGGCGGCCCAAGCGTGTTCCT GTTTCCTCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCAGAGGTGACATG CGTGGTGGTGGACGTGTCTCAGGAGGACCCCGAGGTGCAGTTCAACTGGTACGTGGA TGGCGTGGAGGTGCACAATGCCAAGACCAAGCCTCGGGAGGAGCAGTTTAACTCTAC CTACAGAGTGGTGAGCGTGCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAGGA GTATAAGTGCAAGGTGAGCAATAAGGGCCTGCCCAGCTCCATCGAGAAGACCATCTCC AAGGCCAAGGGCCAGCCCAGAGAGCCTCAGGTGTACACACTGCCCCCTTCTCAGGAG GAGATGACCAAGAACCAGGTGAGCCTGACATGTCTGGTGAAGGGCTTCTATCCCAGC GACATCGCCGTGGAGTGGGAGTCCAATGGCCAGCCTGAGAACAATTACAAGACCACA CCACCCGTGCTGGACTCTGATGGCAGCTTCTTTCTGTATTCCAGGCTGACCGTGGATA AGTCTCGCTGGCAGGAGGGCAACGTGTTCAGCTGTTCCGTGATGCACGAAGCACTGC ACAACCACTACACTCAGAAGTCACTGTCCCTGTCACTGGGCAAGGGCAGCGGCGAGG GCAGGGGCAGCCTGCTGACCTGCGGCGACGTGGAGGAGAACCCCGGCCCCATGGCT CTGCCCGTCACCGCTCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCCGCACGCCCTG GGAGTCGCAAAGTCTGTAATGGGATCGGCATCGGCGAGTTCAAGGACAGCCTGTCCA TCAACGCCACCAATATCAAGCACTTTAAGAATTGCACATCTATCAGCGGCGACCTGCA CATCCTGCCAGTGGCCTTCCGGGGCGATTCTTTTACCCACACACCCCCTCTGGACCCT CAGGAGCTGGATATCCTGAAGACCGTGAAGGAGATCACAGGCTTCCTGCTGATCCAG GCCTGGCCTGAGAACAGAACCGATCTGCACGCCTTTGAGAATCTGGAGATCATCCGG GGCAGAACAAAGCAGCACGGCCAGTTCTCCCTGGCCGTGGTGTCTCTGAACATCACC AGCCTGGGCCTGAGGTCCCTGAAGGAGATCTCTGACGGCGATGTGATCATCTCCGGC AACAAGAACCTGTGCTACGCCAACACAATCAATTGGAAGAAGCTGTTTGGCACCTCT GGCCAGAAGACAAAGATCATCTCTAACCGGGGCGAGAATAGCTGCAAGGCAACCGG ACAGGTGTGCCACGCACTGTGCAGCCCAGAGGGATGTTGGGGCCCAGAGCCACGGG ACTGCGTGAGCTGTAGAAACGTGTCCAGGGGCCGCGAGTGCGTGGATAAGTGTAATC TGCTGGAGGGCGAGCCAAGGGAGTTCGTGGAGAACTCCGAGTGCATCCAGTGTCACC CCGAGTGCCTGCCTCAGGCCATGAACATCACCTGTACAGGCCGCGGCCCCGACAATT GCATCCAGTGTGCCCACTATATCGATGGCCCTCACTGCGTGAAGACCTGTCCAGCCGG CGTGATGGGCGAGAACAATACACTGGTGTGGAAGTACGCAGACGCAGGACACGTGTG CCACCTGTGCCACCCCAATTGCACCTATGGCTGTACAGGACCAGGCCTGGAGGGATGC CCAACCAACGGCCCTAAGATCCCAAGCATCGCCACAGGCATGGTGGGGGCACTGCTG CTGCTGCTGGTGGTGGCTCTGGGGATTGGGCTGTTTATGAGAAGGTAATCCTACTGCG AATTCTCGAGCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGT GCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAA ATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGG ACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGC TCTATGGGTCGACAGATCTCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCC TTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGT AAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAG TTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAG TATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCTCCCCAACACA GACGCCATGATTTGCTTCAAGAGAGCAAATCATGGCGTCTGTGTTTTTTT (SEQ ID NO:148).

ATGGTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCC TCCTGATCCCAGAGATCGTGCTGACCCAGTCCCCCGGCACCCTGTCCCTGTCCCCCGG CGAGCGGGCCACCCTGTCCTGCCGGGCCTCCCAGTCCGTGGGCTCCTCCTACCTGGCC TGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGGCTGCTGATCTACGGCGCCTTCTCC CGGGCCACCGGCATCCCCGACCGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACC CTGACCATCTCCCGGCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTACG GCTCCTCCCCCTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGGGCTCCACCT CTGGATCCGGCAAGCCCGGATCTGGCGAGGGATCCACCAAGGGCCAGGTGCAGCTGG TGGAGTCCGGCGGCGGCGTGGTGCAGCCCGGCCGGTCCCTGCGGCTGTCCTGCGCCG CCTCCGGCTTCACCTTCTCCTCCTACACCATGCACTGGGTGCGGCAGGCCCCCGGCAA GGGCCTGGAGTGGGTGACTTTCATCTCCTACGACGGCAACAACAAGTACTACGCCGA CTCCGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCT GCAGATGAACTCCCTGCGGGCCGAGGACACCGCCATCTACTACTGCGCCCGGACCGG CTGGCTGGGCCCCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCCGCT AGCCCCCCATGCCCATCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCC TGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTG CGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGA TGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCA CGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGG AGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCT CCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGG AGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCA GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACC ACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGG ACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTC TGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGGCAGCGGCG AGGGCAGGGGCAGCCTGCTGACCTGCGGCGACGTGGAGGAGAACCCCGGCCCCATG GCTCTGCCCGTCACCGCTCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCCGCACGCC CTGGGAGTCGCAAAGTCTGTAATGGGATCGGCATCGGCGAGTTCAAGGACAGCCTGT CCATCAACGCCACCAATATCAAGCACTTTAAGAATTGCACATCTATCAGCGGCGACCT GCACATCCTGCCAGTGGCCTTCCGGGGCGATTCTTTTACCCACACACCCCCTCTGGAC CCTCAGGAGCTGGATATCCTGAAGACCGTGAAGGAGATCACAGGCTTCCTGCTGATCC AGGCCTGGCCTGAGAACAGAACCGATCTGCACGCCTTTGAGAATCTGGAGATCATCC GGGGCAGAACAAAGCAGCACGGCCAGTTCTCCCTGGCCGTGGTGTCTCTGAACATCA CCAGCCTGGGCCTGAGGTCCCTGAAGGAGATCTCTGACGGCGATGTGATCATCTCCGG CAACAAGAACCTGTGCTACGCCAACACAATCAATTGGAAGAAGCTGTTTGGCACCTC TGGCCAGAAGACAAAGATCATCTCTAACCGGGGCGAGAATAGCTGCAAGGCAACCGG ACAGGTGTGCCACGCACTGTGCAGCCCAGAGGGATGTTGGGGCCCAGAGCCACGGG ACTGCGTGAGCTGTAGAAACGTGTCCAGGGGCCGCGAGTGCGTGGATAAGTGTAATC TGCTGGAGGGCGAGCCAAGGGAGTTCGTGGAGAACTCCGAGTGCATCCAGTGTCACC CCGAGTGCCTGCCTCAGGCCATGAACATCACCTGTACAGGCCGCGGCCCCGACAATT GCATCCAGTGTGCCCACTATATCGATGGCCCTCACTGCGTGAAGACCTGTCCAGCCGG CGTGATGGGCGAGAACAATACACTGGTGTGGAAGTACGCAGACGCAGGACACGTGTG CCACCTGTGCCACCCCAATTGCACCTATGGCTGTACAGGACCAGGCCTGGAGGGATGC CCAACCAACGGCCCTAAGATCCCAAGCATCGCCACAGGCATGGTGGGGGCACTGCTG CTGCTGCTGGTGGTGGCTCTGGGGATTGGGCTGTTTATGAGAAGGTAATCCTACTGCG AATTCTCGAGCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGT GCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAA ATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGG ACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGC TCTATGGGTCGACAGATCTCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCC TTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGT AAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAG TTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAG TATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCTCCCCAACACA GACGCCATGATTTGCTTCAAGAGAGCAAATCATGGCGTCTGTGTTTTTTT (SEQ ID NO:149).

Wherein, SEQ ID NO:145 is α PD-L1Ab/iCbl/M (tEGFR) (α PD-L1scFv-IgG1Fc- (2A)-M (tEGFR)-PolyA- (U6)-Cbl-b-shRNA) nucleotide sequence；SEQ ID NO:146 is α PD-1Ab/iCbl/M (α PD1scFv-IgG4Fc- (2A)-M (tEGFR)-PolyA- (U6)-iCbl-b) nucleotide sequence；SEQ ID NO:147 is α The nucleotide sequence of TIM3Ab/iCbl/M (α Tim3scFv-IgG4Fc- (2A)-M (tEGFR)-PolyA- (U6)-iCbl-b)； LSEQ ID NO:148 is α LAG3Ab/iCbl/M (α LAG3scFv-IgG4Fc- (2A)-M (tEGFR)-PolyA- (U6)- ICbl-b nucleotide sequence)；LSEQ ID NO:149 is α CTLA4Ab/iCbl/M (α CTLA4scFv-IgG4Fc- (2A)-M (tEGFR)-PolyA- (U6)-iCbl-b) nucleotide sequence.Above-mentioned slow virus according to an embodiment of the present invention imports lymph Cell or dip dyeing lymphocyte are, it can be achieved that secreting type immunologic test point noted earlier inhibits molecule fragment in lymphocyte Expression, immunologic test point intracellular noted earlier inhibit the expression of molecule, height of the optional nonfunctional EGFR on recipient cell after birth Effect expression, realizes checking for immunosuppression mechanism, lymphocyte obtained to tumor cell specific kill it is powerful, effective, Safety.

In the third aspect of the present invention, the invention proposes a kind of transgenic cells.According to an embodiment of the invention, described Transgenic cell is obtained by the way that mentioned-above expression vector or mentioned-above slow virus are imported recipient cell.Root Recipient cell is imported according to the above-mentioned slow virus of the embodiment of the present invention or expression vector, before transgenic cell obtained can be realized Secreting type immunologic test point described in face inhibits the expression of molecule fragment, height of the optional nonfunctional EGFR on recipient cell after birth Effect expression, and realize checking for immunosuppression mechanism.

According to an embodiment of the invention, above-mentioned recipient cell be stem cell, immunocyte, tumor-infiltrated T lymphocyte,

Periphery blood T lymphocyte, Natural killer T cells, natural killer cells, bone-marrow-derived lymphocyte, thick liquid cell, tumour The special T lymph of the special killing cell of killing cell, the Tumor mutations neoantigen of correlation antigen specific, tumor associated antigen The special T lymphocyte of cell, Tumor mutations neoantigen.It should be noted that tumor associated antigen described herein is special Killer T cell refer to specificity for expression tumor associated antigen tumour killer T cell；Tumour described herein The special killer T cell of mutation neoantigen refers to specificity for the killer T cell of the tumour of expression Tumor mutations neoantigen； The special T lymphocyte of tumor associated antigen described herein refers to specificity for the tumour for expressing tumor associated antigen T lymphocyte；The special T lymphocyte of Tumor mutations neoantigen refers to specificity for the swollen of expression Tumor mutations neoantigen The T lymphocyte of tumor.

According to an embodiment of the invention, the recipient cell is PD-1⁺Immunocyte, tumor-infiltrated PD-1⁺CD3⁺T lymph Cell, tumor-infiltrated PD-1⁺CD8⁺T lymphocyte, peripheral blood PD-1⁺CD3⁺T lymphocyte or peripheral blood PD-1⁺CD8⁺T lymph Cell.

In the fourth aspect of the present invention, the invention proposes a kind of secreting type immunologic test points to inhibit molecule fusion protein. According to an embodiment of the invention, the fusion protein is to be secreted by mentioned-above transgenic cell, and can be described as secreting type Anti-immunity checkpoint antibody.Secreting type fusion protein according to an embodiment of the present invention can effective antagonism tumour cell immune escaping Ease, and then immune system can be enhanced using secreting type fusion protein according to an embodiment of the present invention, the immune of tumour cell is killed Wound.

In the fifth aspect of the invention, the invention proposes a kind of secreting type immunologic test points to inhibit molecule fusion protein. According to an embodiment of the invention, the fusion protein includes immunologic test point molecule extracellular fragment and IgG Fc.

According to an embodiment of the invention, above-mentioned secreting type immunologic test point inhibits molecule fusion protein that can also further wrap Include at least one following additional technical feature:

According to an embodiment of the invention, the IgG Fc is IgG1Fc or IgG4Fc.

According to an embodiment of the invention, the fusion protein includes: PD-1 extracellular fragment and IgG1Fc.

According to an embodiment of the invention, the fusion protein includes: PD-L1 extracellular fragment and IgG4Fc.

According to an embodiment of the invention, the fusion protein includes: PD-L2 extracellular fragment and IgG4Fc.

According to an embodiment of the invention, the fusion protein includes: TIM3 extracellular fragment and IgG1Fc.

According to an embodiment of the invention, the fusion protein includes: LAG3 extracellular fragment and IgG1Fc.

In the sixth aspect of the present invention, the invention proposes a kind of therapeutic combinations for treating cancer.According to this hair Bright embodiment, the composition include: mentioned-above expression vector, mentioned-above slow virus, mentioned-above turn base Because cell or mentioned-above secreting type immunologic test point inhibit molecule fusion protein.It is controlled using according to an embodiment of the present invention It is high to treat composition, the specific killing that can effectively realize and promote immune system to cancer cell, and degree of safety.

Detailed description of the invention

Fig. 1 is recombined lentivirus vector structural schematic diagram according to an embodiment of the present invention；

Fig. 2 is PD-1 according to an embodiment of the present invention⁺CD8⁺T cell has killing activity to same patient's tumour cell Result figure；

Fig. 3 is slow virus carrier LV- α PDL1/iCbl/M transduction enhancing PD-1 according to an embodiment of the present invention⁺CD8⁺T cell Killing activity, but PD-1 is not remarkably reinforced^-CD8⁺T cell and CD8 is not separated⁺The result figure of T cell killing activity；And

Fig. 4 is slow virus carrier LV- α PDL1/iCbl/M transduction according to an embodiment of the present invention, than slow virus carrier LV- α PDL1/M or LV-iCbl/M transduction, more effectively enhancing PD-1⁺CD8⁺T cell is to the same active knot of patient's tumor cytotoxicity Fruit figure.

Specific embodiment

The embodiment of the present invention is described below in detail.The embodiments described below is exemplary, and is only used for explaining this hair It is bright, and be not considered as limiting the invention.Particular technique or condition are not specified in embodiment, according in the art Technology or conditions described in document (such as with reference to J. Pehanorm Brooker etc. write, Huang Peitang etc. translate " Molecular Cloning: A Laboratory refers to South ", the third edition, Science Press) or carry out according to product description.Reagents or instruments used without specified manufacturer, Being can be with conventional products that are commercially available.

Expression vector

In the first aspect of the present invention, the invention proposes a kind of expression vectors.According to an embodiment of the invention, the table Following nucleic acid molecules: (a) the first nucleic acid molecules, the first nucleic acid molecule encoding secreting type immunologic test point are carried up to carrier Inhibit molecule fragment；(b) the second nucleic acid molecules, second nucleic acid molecules are for immunologic test point in silenced cell, optionally Ground further carries third nucleic acid molecules, the third nucleic acid molecule encoding nonfunctional EGFR.By the table of the embodiment of the present invention Up in vector introduction recipient cell, secreting type anti-immunity checkpoint antibody high efficient expression and is secreted into cell in recipient cell Outside, while the immunologic test of recipient cell point molecule is by specific silencing, the Immune escaping mechanism quilt mediated by immunologic test point It checks.The expression vector of the embodiment of the present invention is imported into recipient cell, in lymphocyte, the proliferative capacity of lymphocyte increases By force, more powerful to the specific killing of tumour cell more effective.

Human antibodies have the foundation structure of a 150kDa, this foundation structure includes two light chain immunoglobulins and two A heavy chain immunoglobulin, is connected between heavy chain and light chain with covalent bond or non-covalent bond, and then results in three independent eggs The white area area-Liang Ge Fab and an area Fc.The area Fab is connected with the area Fc by the flexible interconnecting piece as hinge area.It is anti- The area body Zhong Fab is the same structure, and there are a specific antigen binding site, the interaction in the area Fc and ligand in the area Fab Site, the site can inductive effect device function, including cellular Fc Receptor and C1q Complement.The physiology of therapeutic antibodies Activity is by two independent native immunoglobulin mechanisms mediates: the effect of therapeutic antibodies is by its specificity and and target The divalent of antigen, which combines, to be caused and (e.g., blocks or neutralize target antigen or induce cell apoptosis), can also be by Fc and effector ligand The effector functions for the immunocomplex activation that (Fc receptor and C1q component) is formed cause.

Single-chain antibody (scFv) is a kind of form of genetic engineering antibody, wherein the domain VH and VL and flexible polypeptide connector phase Even.Compared with whole antibody and Fab, single-chain antibody shows better tissue infiltration pharmacokinetics, and due to antigen binding table Face is not changed and has complete binding specificity.However, the half-life short of single-chain antibody in blood, does not have Fc segment Effector function, because not having Fc molecule fragment in single-chain antibody.

The area Fc of antibody mediates its serum half-life and effector function, such as the cell toxicant (CDC) of Complement Dependent, antibody-dependant Property cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP).Fc segment with single-chain antibody for connecting, to increase The half-life period of single-chain antibody in blood, and there is Fc effector function.Five immunoglobulin like protein (IgM, IgD, IgE, IgG, and IgA) and four IgG subclass (IgG1, IgG2, IgG3, IgG4) are present in the mankind.IgG content highest in human serum.Four subclass IgG1, IgG2, IgG3 and IgG4 are highly conserved, their constant region difference, the domain CH2 especially on hinge.These areas ginseng With combination IgG Fc receptor (Fc γ R) and C1Q.As a result, different subclasses has different effector function triggering FC γ r tables Up to cell, so as to cause the effect of the cytotoxicity and activating complement of phagocytosis or antibody dependent cellular mediation.IgG1 and IgG3 can effectively trigger classical complement activation pathway, but IgG2 and IgG4 and less effectively.IgG is in conjunction with Fc γ Rs or C1q Depending on being located at the residue of hinge area and the domain CH2.Many mutation are imitated in the domain human IgG 1CH2 to change antibody CDC and ADCC It answers.It is worth noting that, the alanine substitution report in position 333 improves ADCC and CDC.

The often high expression in tumour cell of PD-L1 immunologic test point molecule.According to an embodiment of the invention, being directed to tumour Cell express single chain antibody fusion IgG1Fc, the Increased Plasma Half-life of this single-chain antibody, and this single-chain antibody of PD-L1 with The enhancing of PD-L1 binding ability, so that PD-1 and PD-L1 is blocked to interact, enhanced CT L killing tumor cell.IgG1 Fc can Effective trigger effect function, such as the cell toxicant (CDC) of Complement Dependent, antibody-dependent cytotoxicity acts on (ADCC) and antibody Dependent cell swallows (ADCP), carrys out further killing tumor cell.

Immunoregulatory molecules are often expressed in T cell height such as PD-1, Tim-3, LAG3 and CTLA4.IgG4 antibody has Antibody combination block function, and cell-free Fc mediates killing to remove function.For anti-immunity cell surface immunologic test point molecule Antibody, especially those immunocytes for having effector function need to eliminate Fc and killing are mediated to remove function.According to the present invention The antibody of anti-immunity cell surface immunologic test point of embodiment merged with IgG4Fc subclass, Increased Plasma Half-life, scFv antibody Can and PD-1 combination, but not cause effector function damage immunocyte.

According to an embodiment of the invention, the immunologic test point molecule be selected from PD-L1, PD-L2, CTLA4, PD-1, TIM3, At least one of BTLA, LAG-3, IRAK-M, SOCS1, A20, CBL-B.The expression vector of the embodiment of the present invention is imported into receptor Lymphocyte, on the one hand, secreting type immunologic test point inhibits molecule fragment to secret out of rear and above-mentioned immunologic test point molecule knot It closes, and then the immunologic escape of Reverse transcriptase tumour cell, on the other hand, the immunologic test point of recipient lymphocytes is silenced, The Immune escaping mechanism of cell mediated is suppressed again, and then lymphocyte is further to the specific killing of tumour cell It significantly increases.

According to an embodiment of the invention, the secreting type immunologic test point inhibit molecule fragment be anti-PD-1 single-chain antibody and IgG4Fc fusion protein molecule.

According to an embodiment of the invention, the silencing be by shRNA, antisense nucleic acid, ribozyme, dominant negative mutations, What at least one CRISPR-Cas9, CRISPR-Cpf1 and Zinc finger nuclease were realized, it is preferable that the silencing is to pass through shRNA It realizes.By above-mentioned at least one mode, it can be achieved that effective inhibition to intracellular immunity checkpoint molecule.

Children purpura nephritis or short hairpin RNA (shRNA) are the importing forms of siRNA (siRNA), and siRNA is a kind of Small RNA molecular (is made of) 21~25 nucleotide, (is cut to double-stranded RNA with specificity in III family of RNAase by Dicer Cut the enzyme of effect) it is process；SiRNA plays central role in RNA silencing access, drops to specific mRNA (mRNA) Solution, to 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 complementary RNA or oligonucleotide fragment, antisense DNA refer to can combination complementary with the sense strand in gene DNA double-strand short and small DNA point Son, antisense RNA and antisense DNA are mainly to be played a role by the translation of mRNA and the transcription of gene DNA；Antisense nucleic acid On the one hand by forming space steric effect with said target mrna ining conjunction with, prevent ribosomes in conjunction with mRNA, another aspect itself and mRNA In conjunction with rear activation endogenous RNA enzyme or ribozyme, and then the mRNA that degrades；The control region of antisense DNA and gene DNA double helix is special In conjunction with formation DNA tripolymer, or in conjunction with DNA encoding area, the extension for the mRNA chain transcribed is terminated；Antisense nucleic acid may be used also Inhibit the processing modification of mRNA after transcription, such as the end 5' is capped, the end 3' tailing, intermediate montage and internal base methylation, and hinders Only maturation mRNA is transported from nucleus into cytoplasm, and therefore, antisense RNA is a kind of technology of effective silencing target gene.

Ribozyme is the RNA molecule with catalysis, is biocatalyst, degradable special mRNA sequence, and ribozyme is logical It crosses catalysis and turns phosphate and phosphodiester bond hydrolysis participation RNA itself shearing, process, with general antisense RNA phase There is more 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 molecule.

Dominant negative mutation refers to after the mutation of certain signal transducers not only own reactive energy, 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 is made of an identification domain DNA and a non-specific nucleic acid restriction endonuclease, and DNA identifies that domain is by one Serial Cys2-His2 zinc finger protein is 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 structure, and wherein 16 amino acid residues of α spiral determine that the DNA of zinc finger is combined Specificity, skeleton structure is conservative, can obtain new DNA to the change for the amino acid calling sequence for determining DNA binding specificity Binding specificity realizes different purpose bases so as to design different amino acid calling sequences for different target gene The specific silencing of cause.

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

CRISPR cluster is the special repetitive dna sequence family 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 cluster upstream, is the region for being 300~500bp rich in AT length, is recognized For the promoter sequence that may be CRISPR cluster.Repetitive sequence section length can form hair containing palindromic sequence for 21~48bp Card structure.It is separated between repetitive sequence by the spacer region that length is 26~72bp.The region Spacer is by the exogenous DNA group captured At, when containing same sequence exogenous DNA invasion when, can be identified by bacterium body, and carry out shearing be allowed to expression silencing, reach To the purpose of protection inherently safe.Find there is a polymorphism in its vicinity by the flanking sequence analysis to CRISPR cluster Family gene.The family coding protein contain can have an effect with nucleic acid functional domain (have nuclease, unwindase, Integrase and polymerase isoreactivity), and play a role jointly with the region CRISPR, therefore be named as CRISPR association base Because of (CRISPR associated), it is abbreviated as Cas.Presently found Cas includes the multiple types such as Cas1~Cas10.Cas base Cause and CRISPR common evolutionary, collectively form a highly conserved system.When bacterium resists the invasion of the exogenous DNAs such as bacteriophage When, under the regulation of leader, CRISPR is transcribed into long RNA precursor (Pre RISPR RNA, pre-crRNA), then A series of short mature crRNA containing conservative repetitive sequence and spacer region are processed into, finally identifies and is integrated to and be complementary Exogenous DNA array on play shear action.The processing of pre-crRNA is participated in by the Cas9 in Cas family.Cas9 contains The RuvC of amino terminal and HNH2 unique active sites in the middle part of protein, in crRNA maturation and double-stranded DNA shearing It plays a role.While pre-crRNA is transcribed, the trans-activation crRNA (Trans- complementary with its repetitive sequence Activating crRNA, tracrRNA) also transcription comes out, and excites Cas9 and double-stranded RNA specificity RNase III core Sour enzyme processes pre-crRNA.After processing is mature, crRNA, tracrRNA and Cas9 form complex, identify and combine In the sequence of crRNA complementation, DNA double chain is then unlocked, forms R-loop, makes crRNA and complementary strand thereof, another chain is protected Free single-chain state is held, then by the complementary dna chain of the HNH active site shearing crRNA in Cas9, RuvC active site Incomplementarity chain is sheared, DNA double chain fracture (DSB) is eventually introduced.By engineer RNA, being transformed to be formed, there is guidance to make SgRNA (short guide RNA), it is sufficient to Cas9 be guided to cut the pinpoint target gene of DNA.

In conclusion shRNA, antisense nucleic acid, ribozyme, dominant negative mutations, CRISPR Zinc finger nuclease are specific silencing 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, such as shRNA used by the embodiment of the present invention, antisense nucleic acid, ribozyme, dominant negative mutations, CRISPR or At least one of Zinc finger nuclease realizes the specific silencing of target gene.According to an embodiment of the invention, silencing lymph Cellular immunity checkpoint preferably uses shRNA.SiRNA molecule entrained by ShRNA is usually a length between 10 and 30 Base-pair dual zone.The PD1siRNA of the embodiment of the present invention passes through the degradation of mRNA with the coding region for being derived from PD1 Carry out inhibition of gene expression.SiRNA is associated with the multiplexed protein compound for referred to as inducing RNA silencing complex (RISC), herein Period, normal chain was by enzymatic lysis.It is based on sequence homology in the RISC being activated, guides RISC to corresponding mRNA；Identical core Targeting PD1 is cut in sour digestion, generates specific gene PD1 silencing, inhibits the expression of specific gene PD1.SiRNA is in the form of shRNA Import cell (shRNA include about 18-23 nucleotide siRNA sequence, the nucleotide ring of one 9-15 length of heel and this The reverse sequence of a siRNA), the design of shRNA preferably avoids the match point in 3 ' UTR cytogenes；It ensures suitable When chain selection.One single siRNA molecule can be repeated the division applied to multiple targeting mRNA molecules.(RNA is dry by RNAi Disturb) it can be induced by way of introducing and synthesizing siRNA.According to an embodiment of the invention, the shRNA of the embodiment of the present invention is not Stopping pregnancy is from intracellular, therefore its effect is more lasting, thus extend the shRNA period, shRNA tool used in the embodiment of the present invention There are efficient, specific silenced cell immunologic test point, the successful silencing of cellular immunity checkpoint, so that transgenosis Lymphocyte has the significant immunosuppressive characteristic resisting tumour and mediating, in the intracorporal proliferation of tumour patient and existence energy Power is further enhanced, more significant to the orientation lethal effect effect of tumour.

According to an embodiment of the invention, nonfunctional EGFR receptor lacks the end N- ligand binding domain and intracellular receptor tyrosine Kinase activity, but the transmembrane region including Wild type EGFR receptor and the complete sequence in conjunction with anti-egfr antibodies, so nothing Function EGFR receptor can be used as the suicide label of lymphocyte.The slow virus of the embodiment of the present invention imports in recipient lymphocytes, The expression of nonfunctional EGFR receptor can be effectively ensured lymphocyte to the targeting killing effect of tumour cell under the premise of, If serious adverse reaction occurs in patient, lymphocyte can be removed by anti-egfr antibodies, and then improve the embodiment of the present invention The safety of the treatment tumour patient such as slow virus, lymphocyte.

According to a particular embodiment of the invention, the link peptide has amino acid sequence shown in NO:141~144 SEQ ID Column.Link peptide with amino acid sequence shown in NO:141~144 SEQ ID, the link peptide can be in the recipient cells It is cut in born of the same parents.The introducing of link peptide is so that expressed secreting type immunologic test point inhibits molecule fragment and nonfunctional EGFR is in non-fused state, and it is in non-fused state that expressed multiple secreting type immunologic test points, which inhibit molecule fragment,.According to this The embodiment of invention, above-mentioned link peptide are 2A Self cleavage link peptide.2A link peptide is found in stomatopod disease viral (FMDV), Usually with the oligopeptides of 19~22 amino acid, it is located between the memebrane protein of picornavirus family.FMDV virus 2A self cleavage peptide can Self cleavage, and then generate mature virus protein, here it is known translation effects " halting " Or " stopping carrying ".Cleavage site be located at the end C- the last one glycine and the downstream 2B albumen first proline it Between (- LLNFDLLKLAGDVESNPG ↓ P-).2A similar sequence is had found in other virus mRNA molecules currently, having succeeded, Including -1 2A of porcine teschovirus (P2A, sequence is as shown in SEQ ID NO:142), thosea asigna virus 2A (T2A, sequence Column are as shown in SEQ ID NO:141), horse rhinitis A virus 2A (E2A, sequence such as SEQ ID NO:143), cytoplasmic polyhedrosis virus (BmCPV 2A) and flacherie virus (BmIFV 2A) (F2A, sequence such as SEQ ID NO:144).Inventor passes through screening experiment It was found that the link peptide of amino acid sequence shown in NO:141~144 SEQ ID with itself cutting power is arranged in the first core Between acid molecule and third nucleic acid molecules, the function of its own cutting can be played well in recipient cell, be obtained Secreting type fusion protein and nonfunctional EGFR expresses efficiency in lymph in non-fused state and success rate is further shown It writes and improves.

According to the specific embodiment of invention, the viral vectors include selected from retrovirus vector, slow virus carrier or At least one of adeno-associated virus (AAV) carrier.The pathogenic sites of carrier construction in inventive embodiments are by modification or are mutated, The pathogenic of virus is lost, and then the carrier mediated treatment of non-pathogenic virus of the embodiment of the present invention has higher safety Property.The viral vectors of the embodiment of the present invention has virus-infected area extensive, can not only infect terminally differentiated cells, but also can infect Cell in division stage can not only be integrated into host chromosome, but also can be free in except host chromosome, and then can realize wide It composes and efficient efficiency of infection.

According to a particular embodiment of the invention, for constructing a slow virus carrier, inventor is slow in order to construct 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, inventor constructs package cell line in turn and (comprising gag, pol and env gene, but does not include LTR and packaging ingredient).Inventor draws the recombinant plasmid containing target gene together with slow virus LTR and packaging sequence together Enter in package cell line.Packaging sequence allows recombinant plasmid rna transcription product to be wrapped into virion, is then secreted Into culture medium.And then inventor collects the culture medium comprising recombinant slow virus, is selectively concentrated, and turns for gene It moves.Slow carrier can infect various kinds of cell type, 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 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.Slowly Viral vectors by Multiple decrements AIDS virus Disease-causing gene generate, such as all delete gene env, vif, vpr, vpu and Nef makes slow virus carrier form biological safe type carrier.Recombined lentivirus vector can infect non-dividing cell, can be used simultaneously It is expressed in internal and external gene transfer and nucleic acid sequence.Such as: in a suitable host cell, and have packaging function Two or more carriers of (gag, pol, env, rev and tat) together, can infect non-dividing cell.The target of recombinant virus Tropism is realized by antibody or particular ligand (targeting particular cell types receptor) and the combination of memebrane protein.Meanwhile The targeting of recombinant virus, into viral vectors, is compiled by one ordered sequence (including regulatory region) of insertion together with another The gene of the ligand of receptor on the specific target cell of code, makes the carrier be provided with specific targeting.Various useful slow virus carry The carrier of the generations such as body and various methods and operation, for changing the expression of cell.According to an embodiment of the invention, this hair The slow virus carrier of bright embodiment effectively can transport and co-express shRNA (transport form of siRNA), which can have Effect inhibits the expression of PD1 or CTLA4 or CBL-B.

According to an embodiment of the invention, the embodiment of the present invention gland association viral vectors (AAV) can be used it is one or more The DNA building of known serum type gland association viral vectors.Those skilled in the art construct a suitable gland association Viral vectors carries with this and co-expresses children purpura nephritis, which can inhibit the isogenic expression of PD1.

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 product exist Expression in internal or external host cell.It include the expression control of continuous target gene using " operable connection " sequence Sequence processed, and act on trans- or far distance control 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 the promoter of specific organization) be likely to be used.According to the present invention Embodiment, express shRNA promoter be RNA polymerase promoter.Meanwhile according to an embodiment of the invention, promoter For selected from U6, H1, the RAN polymerase promoter of pol I, pol II and pol III.According to an embodiment of the invention, opening Mover is tissue-specific promoter.According to an embodiment of the invention, promoter is inducible promoter.It is according to the present invention Embodiment, promoter are selected from the promoter based on selected carrier.According to an embodiment of the invention, when selection slow virus carrier When, promoter U6, H1, CMV IE gene, EF-1 α, ubiquitin C or phosphoglycerokinase (PGK) promoter.Other conventional tables It include optional label or reporter gene, including encoding geneticin up to control sequence, hygromycin, ampicillin or purine are mould The nucleotide sequence of plain drug resistance etc..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 immunologic test Point) and optional nonfunctional EGFR receptor and secreting type fusion protein viral vectors.The transport silencing of the embodiment of the present invention The children purpura nephritis of PD1 siRNA and the nucleic acid molecules and expression secreting type fusion egg for expressing optional nonfunctional EGFR receptor White viral vectors or plasmid be it is compound, it is steady that this viral vectors or plasmid increase it in combination with polymer or other materials It is qualitative, or assist its targeting movement.

Slow virus

In the second aspect of the present invention, the invention proposes a kind of slow virus.According to an embodiment of the invention, the slow disease Poison carries the nucleic acid with nucleotide sequence shown in NO:145~149 SEQ ID.It is according to an embodiment of the present invention above-mentioned slow Virus imports lymphocyte or dip dyeing lymphocyte, it can be achieved that secreting type immunologic test point noted earlier inhibits molecule fragment to exist Expression in lymphocyte, expression of the optional nonfunctional EGFR on recipient cell after birth, realizes the resistance of immunosuppression mechanism Hold back, killing to tumor cell specific for lymphocyte obtained is powerful, effective, safe.

Transgenic cell

Periphery blood T lymphocyte, Natural killer T cells, natural killer cells, bone-marrow-derived lymphocyte, thick liquid cell, tumour The special T lymph of the special killing cell of killing cell, the Tumor mutations neoantigen of correlation antigen specific, tumor associated antigen Cell or the special T lymphocyte of Tumor mutations neoantigen.

According to an embodiment of the invention, the recipient cell is PD-1⁺Immunocyte, tumor-infiltrated PD-1⁺CD3⁺T lymph Cell, tumor-infiltrated PD-1⁺CD8⁺T lymphocyte, peripheral blood PD-1⁺CD3⁺T lymphocyte or peripheral blood PD-1⁺CD8+ T lymph Cell.

Autologous tumor lymphocyte infiltration (TIL) adoptive cell therapy has half in treatment metastasis melanin tumor The tumor recurrence of patient is effectively controlled, and about the patient of a quarter can obtain long-term fully control.TIL identifies target 2 major class of molecule: the neoantigen of autoantigen and mutation.However, TIL is a kind of foreign cell group.It has recently been demonstrated that Tumor-infiltrated PD1⁺CD8⁺Cell enrichment is directed to Tumor mutations neoantigen CD8⁺T lymphocyte.In addition, blood of cancer patients In PD1⁺CD8⁺T cell is also enriched for Tumor mutations neoantigen CD8⁺T lymphocyte.Table according to an embodiment of the present invention Up to the intracorporal PD1 of vector introduction cancer patient⁺CD8⁺T lymphocyte is conducive to for Tumor mutations neoantigen, tomour specific Kill the anti-tumor activity of cell (CTLs).

Secreting type immunologic test point inhibits molecule fusion protein

In the fourth aspect of the present invention, the invention proposes a kind of secreting type immunologic test points to inhibit molecule fusion protein. According to an embodiment of the invention, the fusion protein is secreted by mentioned-above transgenic cell.Implement according to the present invention The secreting type fusion protein of example can effective antagonism tumour cell immunologic escape, and then utilize according to an embodiment of the present invention point Immune system can be enhanced to the immunologic cytotoxicity of tumour cell by secreting type fusion protein.

According to an embodiment of the invention, it includes immunologic test point that the secreting type immunologic test point, which inhibits molecule fusion protein, Molecule extracellular fragment and IgG Fc.

According to an embodiment of the invention, the IgG Fc is IgG1Fc or IgG4Fc.

Therapeutic combination

On the other hand, the invention proposes a kind of therapeutic combinations for treating cancer.According to an embodiment of the invention, The composition includes: mentioned-above expression vector, mentioned-above slow virus, mentioned-above transgenic cell or preceding Secreting type immunologic test point described in face inhibits molecule fusion protein.It, can be effective using the therapeutic combination of the embodiment of the present invention It realizes and promotes immune system to the specific killing of cancer cell, and degree of safety is high.

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

According to an embodiment of the invention, sufficient amount of viral vectors is transduceed in targeting T-cells, and provide sufficiently strong The transgenosis of degree, the immunologic tests such as the silencing PD1 point nonfunctional EGFR receptor optional with expression and the distinctive secreting type of expression Immunologic test point inhibits molecule fusion protein.The dosage of therapeutic reagent depends primarily on treatment situation, the age, weight, patient's Health degree, so as to cause the variability of patient.

The immunologic tests such as the silencing PD1 point nonfunctional EGFR receptor optional with expression and the distinctive above-mentioned secreting type of expression It is a part of combination therapy that immunologic test point, which inhibits these methods of molecule fusion protein,.These viral vectors and for adopting The antitumor T cell of immunization therapy can be executed together by method that is independent or combining other treatment cancer.In suitable item Under part, a treatment method may be used in combination one or more medicinal treatments.

According to an embodiment of the invention, the type of institute's treating cancer is not particularly limited.Using according to embodiments of the present invention Therapeutic combination, to the specific killing significant effect of PD-L1 or PD-L2 positive tumor cell.

The method for improving lymphocyte immunity killing ability

Finally, the invention proposes a kind of methods of raising lymphocyte immunity killing ability.Implementation according to the present invention Example, this method comprises: being silenced the cellular immunity checkpoint of the lymphocyte, and makes before the lymphocyte table The secreting type immunologic test point inhibits molecule fusion protein.Utilize the above method according to an embodiment of the present invention, Neng Gouyou Effect improves lymphocyte and kills to the specific immunity of tumour cell.

Used cell line and basic experiment technology are as described below in the examples below:

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

Purpose is the slow virus carrier for generating replication defective, and slow virus carrier is collected by centrifugation and is used for human T lymphocyte Transduction.

The experimentation of generation, the collection of slow virus carrier is briefly described below: 293T cell, which is layered on floor space, is In 150- square centimeters of Tissue Culture Dish, and according to specification, (Open Biosystems/ is purchased from using Express-In Thermo Scientific, Waltham, MA) viral transduction is carried out to 293T cell.The slow virus of 15 μ g is added in every disk cell PCMVR8.74 plasmid (the Gag/Pol/Tat/ of transgenosis plasmid, the pVSV-G (VSV P-glycoprotein expression plasmid) of 5 μ g, 10 μ g Rev expression plasmid) and 174 μ l Express-In (concentration be 1 μ g/ μ l).Respectively at 24 hours and 48 hours collection supernatants, And using ultracentrifuge 28,000rpm (centrifuge rotor be Beckman SW 32Ti, be purchased from Beckman Coulter, Brea, CA) under conditions of be centrifuged 2 hours.Weight finally is carried out to virus particle precipitating with the RPMI-1640 culture medium of 0.75ml It is outstanding.

The primary T lymphocyte of people is separated from HLA-A2+ health and tumour patient Volunteer donor.Human T lymphocyte's training Support in RPMI-1640 culture medium and using the coated pearl of monoclonal antibody of AntiCD3 McAb and CD28 (purchased from Invitrogen, Carlsbad, CA) carry out stimulation activation.18~24 hours after human T lymphocyte's activation, using spin-inoculation method pair T lymphocyte is transduceed, and transductive process is as described below: in 24- orifice plate, every hole is covered with 0.5 × 10⁶T lymphocyte, to The viral supernatants and Polybrene of the above-mentioned resuspension of 0.75ml are added in every hole cell (concentration is 8 μ g/ml).Cell and virus The mixed liquor of plasmid (is purchased from Sorvall ST 40 in desk centrifuge；Thermo Scientific) in centrifugation, centrifugal condition It is room temperature, 2500rpm, the time is 90 minutes.People's recombination leukocyte mesonium-2 (IL-2；Purchased from Novartis, Basel, Switzerland) every in 2~3 days addition T lymphocyte culture solutions, the final concentration of 100-IU/ml of IL-2, in T lymph In cell cultivation process, keeping the density of cell is 0.5 × 10⁶~1 × 10⁶/ml.Once the T lymphocyte transduceed occurs Suspend mode, such as vitro growth rates are slack-off and cell becomes smaller, wherein vitro growth rates and size are to pass through Coulter Counter (be purchased from Beckman Coulter) assessment, or the T lymphocyte transduceed is on the time point that some is planned, T Lymphocyte can be used to do work and can analyze.

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

The cytotoxicity (ADCC) of antibody dependent cellular mediation

In the examples below, using 4 hours-⁵¹Cr- method for releasing assesses anti-EGFR-antibodies inducing expression nonfunctional EGFR The ability of the cell dependent antibody cracking of the lymphocyte of receptor.The human T-lymphocyte of slow virus carrier of having been transduceed is used as Target cell.100μCi Na₂ ⁵¹CrO₄(being purchased from GE Healthcare Life Sciences, Marlborough, MA) calibration 2 ~5 x 10⁶Target cell, calibration condition are that concussion is incubated for 1 hour at 37 DEG C.Cell using PBS rinse three times, and with cultivate Base weight is outstanding, and (cell density is 1x 10⁵/ml).Then, the cell being calibrated is layered in 96- orifice plate that (every hole is covered with 5 × 10³It is a thin Born of the same parents, added with 50 μ l culture mediums), and the anti-EGFR-antibodies (being purchased from Erbitux, Genentech) (final concentration of 20 of 50 μ l are added μ g/ml), 30 minutes of preculture then change culture medium antibody-containing into ordinary culture medium under normal temperature conditions, thus come Detection⁵¹The spontaneous release of Cr.Final concentration of 1% Triton X-100 is added to guarantee⁵¹The maximum burst size of Cr.Following In specific implementation, (every hole 5 × 10 in orifice plate is added in human PBMC (effector cell)⁵A cell) and cultivated cell at 37 DEG C Night.Second day, cell conditioned medium is collected, and calculate cpm using gamma counter and determine with this⁵¹The release of Cr.Cytotoxicity ratio It is calculated with following formula: % Specific lytic=(experiment release cpm data-spontaneous release cpm data)/(maximum release cpm The spontaneous release cpm data of data -) * 100, wherein maximum release cpm data are real by the way that Triton X-100 is added in target cell Existing, spontaneous release cpm data are measured under conditions of no anti-egfr antibodies and effector cell.

Chromium release experiment

Applied for 4-hours in embodiment⁵¹The cytotoxic activity of chromium method for releasing analysis assessment recombinant receptor T cell.Specific steps As follows: target detection cell is used⁵¹Label 1 hour under 37 degrees Celsius Cr.After label, with contain 10% fetal calf serum (FCS) RPMI culture medium rinse cell.After rinse, cell is resuspended in identical culture medium, the concentration that cell is resuspended is 1 × 10⁵/ml.T cell is added in target detection cell suspending liquid after transduction with different effect target cell ratio (E:T), and by cell For kind in the hole 96-, every pore volume is 200 microlitres.Cell is cultivated 4 hours in 37 degree of incubators.After 4 hours, from every hole The 96- microwell plate that the supernatant of 30 microlitres of taking-up is put in counter carries out analysis of accounts.Analysis instrument is the micro- sudden strain of a muscle of top counting NXT Bright counter (being purchased from Packard Bioscience).In all counting holes the number of effector cell be based on T cell sum come It calculates.Labeled target detection cell is PD-L1 positive tumor cell.

The building coexpression secreting type anti-immunity checkpoint antibody of embodiment 1, the shRNA of silencing immunologic test point intracellular, and The carrier of nonfunctional EGFR receptor

In the present embodiment, inventor will encode anti human PD-L 1 single-chain antibody and human IgG1 Fc fusion gene cloning to containing On the slow virus carrier (lentiviral vector) of EF-1 promoter.In cloning procedure, the restricted digestion of selection is XbaI and NotI double digestion and NotI and XhoI double digestion, by digestion, connection, screening and purpose plasmid amplification, it is raw At slow virus plasmid (the LV- α PDL1scFv-Fc (LV- α PDL1) of expression secreting type anti-immunity checkpoint antibody.Inventor will Anti-human PD-1 single-chain antibody and human IgG 4Fc fusion gene cloning are encoded to the slow virus carrier containing EF-1 promoter On (lentiviral vector).In cloning procedure, the restricted digestion of selection is XbaI and NotI double digestion and NotI With XhoI double digestion, by digestion, connection, screening and purpose plasmid amplification, it is anti-to generate expression secreting type anti-immunity checkpoint Slow virus plasmid (the LV- α PD1scFv-Fc (LV- α PD-1) of body.Sequence comprising IRES and expression nonfunctional EGFR receptor (M) Column are cloned into LV- α PD-L1 or LV- α PD-1 vector plasmid, are built into LV- α PD-L1/M or LV- α PD-1/M.Comprising U6 and The sequence of the shRNA of silencing immunologic test point, as the sequence of Cbl--shRNA is cloned into LV- α PD-L1/M or LV- α PD-1/ M constitutes LV- α PD-L1/iCbl/M or LV- α PD-1/iCbl/M.Fig. 1 is the schematic diagram of slow virus carrier, includes coding secretion The sequence, IRES, volume of type anti-immunity checkpoint (PD-L1, PD-1, TIM3, LAG3, CTLA4) scFv and IgG Fc fusion protein The shRNA of code nonfunctional EGFR receptor sequence (M), U6 and silencing Cbl-B, PD1, CTLA4 or SOCS1.It is anti-to encode secreting type The sequence of immunologic test point antibody is under the starting regulation of promoter EF-1, the sequence of the shRNA of silencing immunologic test point, such as The shRNA sequence of silencing Cbl-B expresses the sequence of nonfunctional EGFR receptor (M) as one under the starting regulation of promoter U6 A individual mRNA transcriptional units after IRES sequence translate.

The PD-1 that embodiment 2 is separated from tumour patient⁺CD8⁺T cell has killing activity to autologous tumor cell.

From HLA-A2⁺Metastasis melanin tumor patient takes PBLs (peripheral blood) and fresh tumor sample with it.By quiet Arteries and veins, which punctures, takes out PBLs, and carries out gradient centrifugation (LSM；ICN Biomedicals Inc.), freezen protective until analysis to With.Fresh tumor sample aseptically shreds, and carries out the digestion process (composition of digestive juice are as follows: RPMI-1640 using enzyme Culture medium contains L-glutamine [Lonza] in culture medium, 1mg/ml clostridiopetidase A IV [Sigma-Aldrich], 30U/ml DNA enzymatic and antibiotic), digestion condition is digestion at room temperature overnight or digests a few hours under the conditions of 37 DEG C, and Having a rest, it is mechanically decoupled to be carried out using MACS (Miltenyi Biotech).It is thin that tumour single cell suspension is used to acquisition Primary Tumor Born of the same parents are and for separating and expanding PD-1⁺CD8⁺T cell.From the PD-1 of tumour single cell suspension⁺CD8⁺⁺T cell is to pass through FCAS screening and the amplification acquisition in T25 flask, the amplification are carried out in T cell culture medium, are contained in culture medium 30ng/ml solubility anti-cd 3 antibodies (OKT3, Miltenyi), 3,000IU/ml recombinant human IL-2 (Chiron), and come from 3 The 3 × 10 of a heteroplastic transplantation contributor⁷PBMCs.After 5 days, fresh culture is every other day replaced, contains IL-2 in culture medium. When cell density is more than 3 × 10⁶When cells/ml, cell is carried out to expand bottle culture.At the 14th -15 day, the cell of massive amplification Freezen protective after counting.Mankind's original is obtained from the single cell suspension in tumor sample by method mechanical or that enzymatic hydrolysis is isolated It can be carried out according to the operating guidance of standard for the method for K-1735.Human primary's K-1735 culture exists In RPMI 1640 containing 10%FBS (Sigma-Aldrich), 100U/ml penicillin and 100 μ g/ml streptomysins, item is cultivated Part is 37 DEG C, 5%CO₂。

In Cytotoxicity tests test, target cell system loads 100 μ Ci (1Ci=37GBq)⁵¹Cr (PerkinElmer), and Elution is twice.The target melanoma cells of label with come from the matched PD-1 of the same patient⁺CD8⁺Effector T cell is in culture dish In be layered in the culture dish of the hole the 96- bottom U- with specified effect/target ratio, 37 DEG C cultivate 4 hours.⁵¹Cr burst size by γ count into Row calculates, and three samples calculate a dissolution rate.Fig. 2 shows PD-1⁺CD8⁺T cell has strong killing autologous tumor cell Activity.On the contrary, PD-1^-CD8⁺T cell and unsegregated CD8⁺T cell does not kill the ability of autologous tumor cell.

3 slow virus carrier LV- α PDL1/iCbl/M of embodiment transduction enhancing PD-1⁺CD8⁺The anti-tumor activity of T cell

The isolated PD1 of metastasis melanin tumor patient⁺CD8⁺T cell, PD1^-CD8⁺T cell and CD8 is not separated⁺T cell Kind is being covered with recombination fibronectin fragment (FN ch-296；Retronectin) on Tissue Culture Dish, and with lentiviruses transduction, Transduction slow virus is respectively LV- α PDL1/M/iCbl or zero load LV-M transduction.It is tried after T cell of transduceing amplification for cell killing Test target melanoma cells with from the same patient as the target cell target cell with 100 μ Ci (1Ci=37 GBq)⁵¹Cr (PerkinElmer) it marks, it is matched after elution twice, from same patient's effector T cell with specified in culture dish Effect/target ratio be layered in the culture dish of the hole the 96- bottom U-, 37 DEG C cultivate 4 hours.⁵¹Cr burst size is calculated by γ, Three samples calculate a dissolution rate.Fig. 3 shows slow virus carrier LV- α PDL1/iCbl/M transduction enhancing PD-1⁺CD8⁺T cell Anti-tumor activity, but PD-1 is not remarkably reinforced^-CD8⁺T cell and CD8 is not separated⁺T cell anti-tumor activity.This result Show that slow virus carrier LV- α PDL1/iCbl/M can effectively enhance PD-1⁺CD8⁺The special lethal effect of T cell tumour.

4 slow virus carrier LV- α PDL1/iCbl/M of embodiment transduction, than slow virus carrier LV- α PDL1/M or LV-iCbl/ M transduction, more effectively enhancing PD-1⁺CD8⁺T cell is to autologous tumor cell killing activity.

The isolated PD1 of metastasis melanin tumor patient⁺CD8⁺T cell and CD8 is not separated⁺T cell kind is being covered with recombination fibre Even protein fragments (FN ch-296；Retronectin) on Tissue Culture Dish, and with lentiviruses transduction, slow virus of transduceing is respectively LV- α PDL1/iCbl/M, LV- α PDL1/M, LV-iCbl/M, or zero load LV-M transduction.Cell is used for after T cell of transduceing amplification Fragmentation test target melanoma cells with from the same patient as target cell.100 μ Ci (1 Ci=of the target cell 37GBq)⁵¹Cr (PerkinElmer) label, it is and matched after elution twice, exist from same patient effect T cell of transduceing It is layered in the culture dish of the hole the 96- bottom U- in culture dish with specified effect/target ratio, is cultivated 4 hours at 37 DEG C.⁵¹Cr burst size passes through γ Counting is calculated, and three samples calculate a dissolution rate.Fig. 4 shows slow virus carrier LV- α PDL1/iCbl/M transduction, than Slow virus carrier LV- α PDL1/M or LV-iCbl/M transduction, more effectively enhancing PD-1⁺CD8⁺T cell is thin to patient's autologous tumor Born of the same parents' killing activity.Slow virus carrier LV- α PDL1/iCbl/M can make the PD-1 of transduction as the result is shown for this⁺CD8⁺T cell expression point Immunologic test point molecule Cbl-B in type PD-L1 antibody and silenced cell is secreted, to effectively promote the lethal effect of tomour specific.

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 embodiment or examples.In addition, without conflicting with each other, this field Technical staff can carry out the feature of different embodiments or examples described in this specification and different embodiments or examples Combination and combination.

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, modifies, replacement and variant.

Claims

1. a kind of expression vector, which is characterized in that the expression vector carries following nucleic acid molecules:

(a) the first nucleic acid molecules, the first nucleic acid molecule encoding secreting type immunologic test point inhibit molecule fragment；

(b) the second nucleic acid molecules, second nucleic acid molecules are used for immunologic test point in silenced cell,

Optionally, third nucleic acid molecules, the third nucleic acid molecule encoding nonfunctional EGFR are further carried.

2. expression vector according to claim 1, which is characterized in that the secreting type immunologic test point inhibits molecule fragment It include: anti-immunity checkpoint single-chain antibody molecules and IgG Fc,

Optionally, the IgG Fc is IgG1Fc or IgG4Fc.

3. expression vector according to claim 1, which is characterized in that the immunologic test point includes being selected from PD-L1, PD- L2, at least one of CTLA4, PD-1, TIM3, BTLA, LAG-3, IRAK-M, SOCS1, A20, CBL-B.

4. expression vector according to claim 1, which is characterized in that the secreting type immunologic test point inhibits molecule fragment For anti-PD-L1 single-chain antibody and IgG1Fc fusion protein molecule,

Optionally, it is anti-PD-1 single-chain antibody and IgG4Fc fusion protein minute that the secreting type immunologic test point, which inhibits molecule fragment, Son,

Optionally, it is anti-TIM3 single-chain antibody and IgG4Fc fusion protein minute that the secreting type immunologic test point, which inhibits molecule fragment, Son,

Optionally, it is anti-LAG3 single-chain antibody and IgG4Fc fusion protein minute that the secreting type immunologic test point, which inhibits molecule fragment, Son,

Optionally, it is anti-CTLA 4 single-chain antibody and IgG4Fc fusion protein that the secreting type immunologic test point, which inhibits molecule fragment, Molecule.

5. expression vector according to claim 1, which is characterized in that the silencing is by shRNA, antisense nucleic acid, core What at least one enzyme, dominant negative mutations, CRISPR-Cas9, CRISPR-Cpf1 and Zinc finger nuclease were realized,

Preferably, the silencing is realized by shRNA.

6. expression vector according to claim 1, which is characterized in that first nucleic acid molecules have SEQ ID NO:1 Nucleotide sequence shown in~5.

7. expression vector according to claim 1, which is characterized in that second nucleic acid molecules have SEQ ID NO:6 Nucleotide sequence shown in~138,

Optionally, the third nucleic acid molecules have nucleotide sequence shown in SEQ ID NO:139.

8. expression vector according to claim 1, which is characterized in that further comprise:

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

Second promoter, second promoter are operably connected with second nucleic acid molecules；And

Optional third promoter, the optional third promoter are operably connected with the third nucleic acid molecules.

9. expression vector according to claim 8, which is characterized in that first promoter, second promoter, institute It states third promoter and is separately selected from U6, H1, CMV, EF-1, LTR or RSV promoter.

10. expression vector according to claim 1, which is characterized in that the expression vector includes the first nucleic acid molecules, the Two nucleic acid molecules and third nucleic acid molecules, the expression vector further comprises:

Internal ribosome entry site sequence, internal ribosome entry site sequence setting first nucleic acid molecules with Between the third nucleic acid molecules, the internal ribosome entry site has nucleotide sequence shown in SEQ ID NO:140.

11. expression vector according to claim 1, which is characterized in that the expression vector includes the first nucleic acid molecules, the Two nucleic acid molecules and third nucleic acid molecules, and the expression vector further comprises:

4th nucleic acid molecules, the 4th nucleic acid molecules are arranged between first nucleic acid molecules and the third nucleic acid molecules Or between multiple first nucleic acid molecules, and the 4th nucleic acid molecule encoding link peptide.

12. expression vector according to claim 11, which is characterized in that the link peptide have SEQ ID NO:141~ Amino acid sequence shown in 144.

13. expression vector according to claim 1, which is characterized in that the expression vector is non-pathogenic virus carrier.

14. expression vector according to claim 13, which is characterized in that the viral vectors includes being selected from retrovirus At least one of carrier, slow virus carrier or adeno-associated virus (AAV) carrier.

15. a kind of slow virus, which is characterized in that the slow virus, which carries, has nucleotide shown in NO:145~149 SEQ ID The nucleic acid of sequence.

16. a kind of transgenic cell, which is characterized in that the transgenic cell is by by any one of claim 1~15 institute Slow virus described in the expression vector or claim 15 stated imports what recipient cell obtained.

17. transgenic cell according to claim 16, which is characterized in that the recipient cell is stem cell,

Optionally, the recipient cell is immunocyte,

Optionally, the recipient cell is tumor-infiltrated T lymphocyte,

Optionally, the recipient cell is periphery blood T lymphocyte,

Optionally, the recipient cell is Natural killer T cells,

Optionally, the recipient cell is natural killer cells,

Optionally, the recipient cell is bone-marrow-derived lymphocyte,

Optionally, the recipient cell is thick liquid cell,

Optionally, the recipient cell is the special killer T cell of tumor associated antigen,

Optionally, the recipient cell is the special killer T cell of Tumor mutations neoantigen,

Optionally, the recipient cell is the special T lymphocyte of tumor associated antigen,

Optionally, the recipient cell is the special T lymphocyte of Tumor mutations neoantigen.

18. transgenic cell according to claim 16, which is characterized in that the recipient cell is PD-1⁺Immunocyte,

Optionally, the recipient cell is tumor-infiltrated PD-1⁺CD3⁺T lymphocyte,

Optionally, the recipient cell is tumor-infiltrated PD-1⁺CD8⁺T lymphocyte,

Optionally, the recipient cell is peripheral blood PD-1⁺CD3⁺T lymphocyte,

Optionally, the recipient cell is peripheral blood PD-1⁺CD8⁺T lymphocyte.

19. a kind of secreting type immunologic test point inhibits molecule fusion protein, which is characterized in that be any by claim 16~18 What the transgenic cell described in was secreted.

20. a kind of secreting type immunologic test point inhibits molecule fusion protein characterized by comprising immunologic test point molecule born of the same parents Outer segment and IgG Fc.

21. fusion protein according to claim 20, which is characterized in that the IgG Fc is IgG1Fc or IgG4Fc,

Optionally, the fusion protein includes: PD-1 extracellular fragment and IgG1Fc,

Optionally, the fusion protein includes: PD-L1 extracellular fragment and IgG4Fc,

Optionally, the fusion protein includes: PD-L2 extracellular fragment and IgG4Fc,

Optionally, the fusion protein includes: TIM3 extracellular fragment and IgG1Fc,

Optionally, the fusion protein includes: LAG3 extracellular fragment and IgG1Fc.

22. a kind of therapeutic combination for treating cancer characterized by comprising

Slow virus, claim 16~18 times described in the described in any item expression vectors of claim 1~14, claim 15 Transgenic cell described in one or the described in any item secreting type immunologic test points of claim 19~21 inhibit molecule fusion Albumen.