CN108753773A

CN108753773A - Interfere CD19-CAR-T cells and its application of IFN-gama expression

Info

Publication number: CN108753773A
Application number: CN201810412693.3A
Authority: CN
Inventors: 谭毅; 郭红; 张慧慧
Original assignee: Shandong Qilu Cell Therapy Engineering Technology Co Ltd
Current assignee: Shandong Qilu Cell Therapy Engineering Technology Co Ltd
Priority date: 2018-05-03
Filing date: 2018-05-03
Publication date: 2018-11-06
Anticipated expiration: 2038-05-03
Also published as: CN108753773B

Abstract

The invention discloses the shRNA of two kinds of interference people's IFN-gama gene expressions, such as SEQ ID NO:1, shown in 2.The invention also discloses a kind of Lentivirals, and it includes the shRNA for having interference people's IFN-gama gene expressions, and such as SEQ ID NO:The nucleotide fragments of encoding chimeric antigen receptor shown in 4.The invention also discloses a kind of CD19-CAR-T cells of interference people's IFN-gama gene expressions, to include the T lymphocytes of Lentiviral, or the T lymphocytes of the shRNA of interference people's IFN-gama gene expressions and the nucleotide fragments of encoding chimeric antigen receptor are integrated in chromosome.The CAR-T cells of the present invention introduce IFN-gama shRNA, and the shRNA of IFN-gama is co-expressed while expressing CAR, inhibit the release of IFN-gama from source by gene silent technology, to reduce the influence of CRS, improve the safety of CAR-T treatments.It is contemplated that preventing, treating preparing, having important application in the drug of adjuvant therapy of malignant tumor.

Description

Interfere CD19-CAR-T cells and its application of IFN-gama expression

Technical field

The present invention relates to the Chimeric antigen receptor T cell (Chimeric of the targeting CD19 of interference IFN-gama expression Antigen Receptor Tcell, CAR-T) preparation and application, belong to genetic engineering and cell biology.

Background technology

1989, Chimeric antigen receptor transformation T cell (CAR-T) this concept was put forward for the first time, it is therefore an objective to establish tumour The adoptive cellular therapy of specific recognition capability.The design of first generation CAR is simply to use monoclonal antibody specific source ScFv (single-chain antibody variable region fragment) section replaces TCR (Tcell receptor) Extracellular part, remain the regions CD3 ζ that transmembrane structure and intracellular transmit signal, some seminar's intracellular signal transfer parts FcR can also be used by dividing.But the clinical test results of first generation CAR are very disappointing, a best clinical test of effect Also only having 2/11 patient has long-term alleviation.As what is recognized immunology deepens continuously, it has been found that though CD3 ζ signals So it is capable of the activation of inducing T cell and of short duration proliferation, but understands the incapability (anergy) of inducing T cell later.1998, there are two Laboratory reports CD28 signal domains can provide the costimulation function of auxiliary for CD3 ζ signals.Therefore, second generation CAR Design introduces the part of costimulatory molecules on the basis of the first generation, such as the intracellular signal structure of CD28 or CD137 (4-1BB) Domain.And third generation CAR be then add the function signal structural domain of 2 costimulatory molecules, including CD27, CD28,4-1BB, ICOS or OX40 (15-18).The mainly design of second generation CAR clinically tested now.

The effect of CAR-T, on August 30th, 2017, U.S. FDA had approved first CAR-T medicines recognized by the industry Object --- Kymriah (tisagenlecleucel), for treating relapsed or stubborn (r/r) children and Young Adults B cell Acute lymphoblastic leukemia.This is first granted immunocyte drug.October after one wheat harvesting period 18, FDA approvals Kite pharmacy Yescarta (axicabtagene ciloleucel, KTE-C10), for treat to other therapies without response or Person received the specific type recurred after at least two kinds of therapeutic schemes adult large B cell lymphoid tumor patient, including the big B of diffusivity is thin Born of the same parents' lymthoma, transformant follicular lymphoma, primary mediastinal B-cell lymphoma.Yescarta is the 2nd section of CAR-T of FDA approvals Therapy.

What is coexisted with the validity of CD-19-CAR-T therapies is its side effect and cytotoxicity.Due to being chosen when design CD19 is target spot, and the T cell of input can kill itself existing normal B cells in vivo together with tumour cell, and swollen After tumor disappears, normal B cells survival is not had as long as CAR-T cells still remain, in body, patient needs one timing of interval Between injecting immune globulin to maintain basic humoral immunity.On the other hand, initial stage is inputted in CAR-T cells, due to T cell Large amount of cell factor can be secreted in large amplification and T cell killing neoplastic process, patient will appear cytokine release synthesis Disease (Cytokine Release Syndrome, CRS) is embodied in certain thin in fever, low blood pressure, anoxic and serum The horizontal significant raising of intracellular cytokine, these factors include interferon-γ (IFN-gama), divide shape chemotactic factor (CF) (Fracktalkine), grain-macrophage growth factor (GM-CSF), interleukin 5 (IL-5), interleukin-6 (IL-6), people's FMS-like tyrosine kinase 3 ligand (Flt-3L) and interleukin 10 (IL-10).At the beginning of 7 months 2016, general headquarters It has issued and has been reported in the clinical test JCAR015 of the said firm's progress positioned at the Juno Therapeutics of Seattle, successively had Death occurs after receiving CAR-T cell therapies for three patients's (being proved to be four afterwards), and FDA stopped JCAR015's immediately Clinical test, it is because leading to neurotoxin using chemotherapeutics fludarabine, but also have many people to guess that Juno, which is then explained, Being strong cellular factor storm (CRS) caused by the T cell due to input causes patient dead.Famous Novartis of Switzerland (Novartis) although the products C LT019 of the targeting CD-19 of company can allow 80% conditions of patients to be eased, simultaneously There is cytohormone release disease (CRS) in the patient for also resulting in nearly half.There is the patient's body of CRS, T cell can discharge excessively Inflammatory factor, lead to too drastic inflammatory reaction and fever, it is serious if entail dangers to life.Likewise, California, USA KitePharma companies are used to treat the product K TE-C19 of non Hodgkin lymphom, and 1/3 patient can be caused nerveous system occur System side effect, 1/5 patient there is CRS, also two patients are dead in testing.

Usually, using large dosage of steroid hormone, such as prednisone, the clinical symptoms of CRS can be reversed rapidly.But It is that steroid medicine can significantly inhibit the proliferation of CAR-T cells in vivo so that patient has higher recurrence rate, influences CAR-T The effect for the treatment of.The blocking list that the preferable medicines of CRS are interleukin-6 receptors (IL-6R) is clinically handled now Clonal antibody drug (Tocilizumab).Existing clinical test proves, can solve what CRS was brought rapidly after blocking IL-6 receptors Toxic side effect and CAR-T cell proliferation in vivo is not influenced.Official approval Tocilizumab is used to treat FDA at present In CAR-T therapies it is possible that CRS risks.But Tocilizumab is expensive, is no small economy for patient Burden can largely mitigate CRS's if it is possible to cut off the release of IFN-gama cell factors from source Risk, the safety of enhancing CAR-T treatments.

RNA interference (RNA interference, RNAi) be one kind is prevalent in organism, sequence-specific is strong, The gene silencing mechanism of post-transcriptional level.Currently, using more universal performance interference effect small RNA molecular in animal body Mainly there are siRNA (small interfering RNA), shRNA (short hairpin RNA) and amiRNA (artificial microRNA) etc..It is earliest by external system using the method for gene function in RNAi technology research organism The single-stranded siRNA molecule got ready is directly injected into nematode body, although being used in gene functional research later, this side The interference timeliness of method is relatively short, and the concentration for improving siRNA to reach effectively interference often has cytotoxicity.Present The method that RNAi technology uses is mostly to build a specific RNAi expression vector, can be by after the vector introduction animal and plant cells Rna plymerase ii type or type III promoter transcription go out corresponding shRNA, amiRNA etc., and then interfere the expression of its target gene.

Currently, be commonly used to structure shRNA interference expression vectors method mainly have it is following two：(1) oligonucleotides moves back Pyrogenic process, common practice are two specific Oligonucleolide primers of chemical synthesis, anneal to form both ends with glutinous in vitro Property end double-strand shRNA segments, then connect conversion again with the carrier of cohesive terminus,cohesive termini with by digestion processing, contained There is the recombinant plasmid of shRNA infection expression units.(2) PCR amplification method：Design pair for amplification control shRNA expression promoters PCR primer, wherein forward primer are matched with promoter specificity, and 5 ' end of reverse primer is additionally plus for the dry of target gene Target sequence is disturbed, PCR product is cloned on corresponding carrier, obtains shRNA interference expression vectors.

Interferon-gama (IFN-gama) is used as cytokine release syndrome (Cytokine Release Syndrome, CRS) in the cell factor that plays a significant role, design passes through gene silencing skill for the shRNA of IFN-gama Art inhibits the release of IFN-gama from source, to reduce the influence of CRS, is to improve CAR-T safeties, reduces toxic side effect Beneficial to thinking.

Invention content

For the above-mentioned prior art, the present inventor passes through in-depth study and performing creative labour, successfully screens 2 kinds of shRNA that can interfere with IFN-gama gene expressions are gone out, and the shRNA and CD19CAR have been co-expressed.The hair of the present invention A person of good sense has found, has co-expressed the CD19CAR-T cells of the shRNA of interference IFN-gama gene expressions, and killing expression CD19's is swollen The burst size of the factors such as IL-6 during oncocyte, IFN-gama reduces, and to reduce the influence of CRS on source, carries Safety in high CAR-T treatments.

The present invention is achieved by the following technical solutions：

The shRNA of people's IFN-gama gene expressions is interfered, there are two types of, it is respectively designated as IFN-gama shRNA-1, IFN- The nucleotide sequence of gama shRNA-3, IFN-gama the shRNA-1 (sequence involved by the present invention, if without spy as follows Do not mentionlet alone bright, be 5 ' -3 '), the nucleotide sequence of IFN-gama shRNA-3 is as follows.

IFN-gama shRNA-1 (such as SEQ ID NO:Shown in 1)：

GCGGCCGCCGCAGAGCCAAATTGTCTCCTTTTCAAGAGAAAGGAGACAATTTGGCTCTGCTTTTT。

IFN-gama shRNA-3 (such as SEQ ID NO:Shown in 2)：

GCGGCCGCCCATTCAGATGTAGCGGATAATTTCAAGAGAATTATCCGCTACATCTGAATGTTTTT。

In the sequence of IFN-gama shRNA-1, including for the siRNA of people's IFN-gama genes： GCAGAGCCAAATTGTCTCCTT.In the sequence of IFN-gama shRNA-3, including for the siRNA of people's IFN-gama genes： CATTCAGATGTAGCGGATAAT。

It is a kind of be used to prepare above-mentioned interference people IFN-gama gene expressions shRNA --- IFN-gama shRNA-1's draws Object, nucleotide sequence (such as SEQ ID NO as follows:Shown in 5~8)：

hIFN-gama-shRNA1-1^st：GGCCGCCGCAGAGCCAAATTGTCTCCTTTTCAAGAGA；

hIFN-gama-shRNA1-2^nd：AAGGAGACAATTTGGCTCTGCTTTTTGC；

hIFN-gama-shRNA1-3^rd：AAGGAGACAATTTGGCTCTGCGGC；

hIFN-gama-shRNA1-4^th：GGCCGCAAAAAGCAGAGCCAAATTGTCTCCTTTCTCTTGAA.

It is a kind of be used to prepare above-mentioned interference people IFN-gama gene expressions shRNA --- IFN-gama shRNA-3's draws Object, nucleotide sequence (such as SEQ ID NO as follows:Shown in 9~12)：

hIFN-gama-shRNA3-1^st：GGCCGCCCATTCAGATGTAGCGGATAATTTCAAGAGA；

hIFN-gama-shRNA3-2^nd：ATTATCCGCTACATCTGAATGTTTTTGC；

hIFN-gama-shRNA3-3^rd：ATTATCCGCTACATCTGAATGGGC；

hIFN-gama-shRNA3-4^th：GGCCGCAAAAACATTCAGATGTAGCGGATAATTCTCTTGAA.

The method that the shRNA of interference people's IFN-gama gene expressions is prepared using above-mentioned shRNA primers：Above-mentioned 4 are drawn Object (is synthesized) dry powder rapid centrifugation by the Suzhou bio tech ltd Jin Weizhi, is diluted with water 100uM later, and 1^st, 2^nd, 3^rd, 4^th, respectively take 2.5ul, four total 10ul, moisturizing to 50ul to boil 5~10 minutes, be placed on natural cooling in pot and stay overnight extremely Room temperature, by the process, four primers complete annealing, form section of DNA double-strand short-movie section.

A kind of Chimeric antigen receptor, including following cis- concatenated structural domain：Signal peptide, label gene, single-chain antibody, born of the same parents Outer hinge area, transmembrane region and intracellular signal area, wherein single-chain antibody include light chain variable region (VL), heavy chain variable region (VH) with And connect their hinge area.

The antigen that the single-chain antibody is identified be selected from CD19, CD20, CEA, GD2, CD22, CD23, CD30, CD33, Any one or two kinds in CD44v7/8, CD70, VEGFR1, VEGFR2, IL-11R α, EGP-2, EGP-40, FBP, GD3 with Upper (these antigens are existing in the prior art), preferably CD19, at this point, the amino of the light chain variable region of the single-chain antibody Acid sequence is as follows, and the amino acid sequence of heavy chain variable region is as follows, and the amino acid sequence of hinge area is as follows.

The amino acid sequence of the light chain variable region of single-chain antibody：

KLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGT DYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT。

The amino acid sequence of the heavy chain variable region of single-chain antibody：

EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQ VFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS。

The amino acid sequence of the hinge area of single-chain antibody：GGGGSGGGGSGGGGS.

The amino acid sequence of the signal peptide is：MALPVTALLLPLALLLHAARPEQ.

The amino acid sequence of the label gene is：KLISEEDLTS.

In extracellular hinge area, the extracellular hinge area of CD28 or the extracellular hinge area of CD4 of the extracellular hinge area selected from CD8 Any one or two or more, the extracellular hinge area of preferably CD8；The amino acid sequence of the extracellular hinge area of the CD8 is： TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI。

Any one in the transmembrane region of transmembrane region of the transmembrane region selected from CD8, the transmembrane region of CD28 or CD4 or two kinds More than；It is preferred that CD8 transmembrane regions；The amino acid sequence of the CD8 transmembrane regions is：YIWAPLAGTCGVLLLSLVITLYC.

The intracellular signal area is selected from CD28, CD134/OX40, CD137/4-1BB, LCK, ICOS, DAP10, CD3 ζ and Fc Any one in ε RI γ or two or more, preferably 4-1BB intracellular signals area and CD3 ζ intracellular signals area, or：CD28 intracellulars are believed Number area and CD3 ζ intracellular signals area；The amino acid sequence in 4-1BB intracellular signals area and CD3 ζ intracellular signals area difference is as follows It is shown.

The amino acid sequence in 4-1BB intracellular signals area： SLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE。

The amino acid sequence in CD3 ζ intracellular signals area：

LRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRG。

Above-mentioned amino acid sequence is existing in the prior art.

Preferably, the Chimeric antigen receptor, amino acid sequence (such as SEQ ID NO as follows:Shown in 3).

SEQ ID NO:3：

MALPVTALLLPLALLLHAARPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKL LIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSE VKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQV FLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCSLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE LRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRG。

A kind of nucleotide fragments of the above-mentioned Chimeric antigen receptor of coding, nucleotide sequence (such as SEQ ID as follows NO:Shown in 4).

SEQ ID NO:4：

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGCAGAAGCTGAT CAGCGAGGAGGACCTGACTAGTGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAG TCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTT AAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGA TTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGT ACACGTTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGA TCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTC AGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAA TATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGC CAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGG TGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCAGGATCCACCACGACGCCAGCGC CGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCG GGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGG GGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCTCCCTAAAACGGGGCAGAAAGAAACTCCTGTATATATTCA AACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAA GGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTA TAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGG GAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGT GAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAA GGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGCTAA。

A kind of Lentiviral, it includes the shRNA for having above-mentioned interference people IFN-gama gene expressions, shRNA tables Up to frame, and (Lentiviral can express chimeric antigen to the nucleotide fragments of the above-mentioned Chimeric antigen receptor of coding simultaneously Receptor and IFN-gama shRNA)；The shRNA expression cassettes include that shRNA expresses promoter, and shRNA expression promoter is selected from Any one in U6, H1 or 7SK, preferably U6；The nucleotide sequence of the U6 promoters is as follows.Slow virus expression carries The construction method of body is conventional method.

The nucleotide sequence of U6 promoters：

AAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGAT AATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGT AGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTG GGTTTATATATCTTGTGGAAAGGACG。

Preferably, the structure of the Lentiviral is pHR-antiCD19CAR-U6-IFN-gama shRNA-1, Or pHR-antiCD19CAR-U6-IFN-gama shRNA-3.

A kind of screening technique of shRNA, using pHR-IFN-gama-GFP and pHR-antiCD19CAR-U6-IFN-gama The mode of shRNA plasmid co-transfections, by calcium phosphate transfection method, by CaCl₂, two kinds of plasmids and water mix according to a certain percentage, It is slowly dropped in the culture supernatant of 293FT cells, harvests 293FT cells afterwards for 24 hours, pass through RT-PCR and flow cytometer detection respectively Method detects the expression of IL-6 in mRNA and protein level.

The slow virus carrier pHR-IFN-gama-GFP, it includes have the IFN-gama genes and GFP genes (can be same When express IFN-gama and GFP).The IFN-gama genes, nucleotide sequence are as follows.The GFP genes, nucleosides Acid sequence is as follows.

The nucleotide sequence of IFN-gama genes：

ATGAAGTACACCAGCTACATCCTGGCCTTTCAGCTGTGCATCGTGCTGGGCAGCCTGGGCTGCTACTGCCAGGACCC CTACGTGAAGGAGGCCGAGAACCTGAAGAAGTACTTCAACGCCGGCCACAGCGACGTGGCCGACAACGGCACCCTGT TCCTCGGCATCCTGAAGAACTGGAAGGAGGAGAGCGACAGGAAGATCATGCAGTCCCAGATCGTGAGCTTCTACTTC AAGCTGTTCAAGAATTTCAAGGACGACCAGAGCATCCAGAAGAGCGTGGAGACCATCAAGGAGGACATGAACGTGAA GTTTTTCAATAGCAACAAGAAGAAGAGGGACGACTTCGAGAAGCTGACCAACTACAGCGTGACCGACCTGAATGTGC AGAGGAAGGCCATCCACGAACTGATCCAGGTGATGGCCGAGCTGAGCCCTGCCGCCAAGACCGGCAAGAGGAAGAGG AGCCAGATGCTGTTCAGGGGCAGGAGGGCCAGCCAG。

The nucleotide sequence of GFP genes：

GTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAA GTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCA AGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCAC ATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGA CGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCG ACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCC GACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGA CCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCG CCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTC GGCATGGACGAGCTGTACAAG。

The method of the expression that IFN-gama is detected by RT-PCR methods is as follows：After harvesting 293FT cells, extraction is total RNA(MiniBEST UniversalRNA,TAKARA,cat:9767), reverse transcription at cDNA (FastKing RT Kit, Tiangeng, cat：KR11601), using the primer of IFN-gama, IFN-gama in mRNA level in-site is measured by the method for quantitative fluorescent PCR It expresses (primer that reference gene β-actin need to be used).

The primer of the IFN-gama, including forward primer and reverse primer, nucleotide sequence are as follows：IFN- gama-F:TCAGCTCTGCATCGTTTTGG；IFN-gama-R:GTTCCATTATCCGCTACATCTGAA.

The primer of the reference gene β-actin, nucleotide sequence are as follows：action-F： CGCCCCAGGCACCAGGGC；action-R：GCTGGGGTGTTGAAGGT.

The method of the expression of the streaming method detection IFN-gama is as follows：After harvesting 293FT cells, the fluorescence of GFP is detected Expression.

A kind of slow virus expression kit, including above-mentioned Lentiviral.

A method of utilizing above-mentioned Lentiviral Prepare restructuring slow virus：By the target gene of carrying It is transfected into 293FT cells after Lentiviral, pCMV carriers and the mixing of pMD.2G carriers, 6~8h is changed to after transfection Complete medium culture collects culture solution after 48h, and supernatant is retained after centrifugation and is filtered with 0.45 μm of filtering head, reservation filtrate, institute State the solution that filtrate is recombinant slow virus.

A kind of CD19-CAR-T cells of interference IFN-gama expression, to include the T leaching of above-mentioned Lentiviral The shRNA of interference people's IFN-gama gene expressions and the core of encoding chimeric antigen receptor are integrated in bar cell or chromosome The T lymphocytes (IFN-gama shRNA and Chimeric antigen receptor can be expressed simultaneously) of acid fragments.The preparation of the recombination T cell Method is conventional method.

The CD19-CAR-T cells of the interference IL-6 expression are pernicious in preparation prevention and/or treatment and/or auxiliary treatment Application in the drug of tumour；Preferably, the malignant tumour is selected from acute lymphoblastic leukemia and/or chronic lymphocytic Leukaemia.

The invention has the advantages that：

1. the present invention provides a kind of method that shRNA is inserted into expression vector, i.e., synthesize one section by 4 primer annealing methods DNA double chain short-movie section.

2. the present invention provides a kind of effective ways of detection screening shRNA, i.e., by expressing IFN-gama shRNA's The carrier pHR-IFN- of carrier pHR-antiCD19CAR-U6-IFN-gama shRNA and expression IFN-gama-GFP fusion proteins The mode of gama-GFP cotransfections detects IFN-gama in mRNA and protein level respectively by the method for RT-PCR and streaming The efficiency of shRNA gene silencings successfully filters out the shRNA of energy effective reticence IFN-gama gene expressions.

3. compared with existing antiCD19CAR-T cells, invention introduces IFN-gama shRNA, in expression CAR While, the shRNA of IFN-gama is co-expressed, inhibits the release of IFN-gama from source by gene silent technology, to drop The influence of low CRS improves the safety of CAR-T treatments.

4. compared with existing antiCD19CAR-T cells, pHR-antiCD19CAR-U6-IFN-gama shRNA-T are thin Born of the same parents co-express the shRNA of IFN-gama while expressing CAR, the experimental results showed that, the shRNA of the IFN-gama of coexpression IL-6 can be reduced, the generation of the release of the factors such as IFN-gama, control CRS side effects does not influence while increasing its safety The expression of CAR does not influence the characteristic of its killing etc., exceeds the expectation of those skilled in the art.

Some essential terms involved in the present invention are described as follows；

Term " shRNA " (shorthairpinRNA), i.e. short hairpin RNA.ShRNA includes two short inverted repeats. The shRNA being cloned into shRNA expression vectors includes two short inverted repeats, and centre is by a stem ring (loop) sequence separates , hairpin structure is formed, is controlled by III promoters of pol.Then tanscription terminations of the 5-6 T as RNA polymerase III is connected again Son.

Term " U6 promoters " is one kind in III promoters of pol, and position and base sequence are highly conserved, can be thin Intracellular efficient transcription downstream structural sequence.It is the common promoters of shRNA.

Term " Chimeric antigen receptor " is artificial reconstructed receptor, can be by the specificity of tumor cell surface antigen point Sub (such as antibody) is anchored on immunocyte (such as T cell), is made immunocyte identification tumour antigen or viral antigen and is killed swollen Oncocyte or the cell of virus infection.

Term " single-chain antibody " (single-chain antibody variable region fragment, scFv) is Finger is formed by antibody light chain variable region (areas VL) amino acid sequence and heavy chain variable region (areas VH) amino acid sequence through hinge connection, With the antibody fragment for combining antigenic capacity.

Term " Linker " or hinge are the polypeptide fragments between the different albumen of connection or polypeptide, and the purpose is to make to be connected Albumen or polypeptide keep respective space conformation, to maintain the function or activity of albumen or polypeptide.

Term " CD19 " refers to human leukocytes differentiation antigen 19, it NCBIGeneBank ID number be 930, have 2 it is different Structure body (cDNA sequence/protein sequence), respectively NM_001178098.1/NP_001171569.1, NM_001770.5/NP_ 001761.3.When referring to the amino acid sequence of CD19 comprising, the overall length of CD19 albumen or the CD19 with CD19 functions Segment；It further include the fusion protein of the overall length or segment.Also, it will be appreciated by those skilled in the art that in the amino acid of CD19 In sequence, can it is naturally-produced or be artificially introduced mutation or variation (including but not limited to replacing, lack and or add), without shadow Ring its biological function.Further include corresponding in its natural or artificial variants also, when describing the protein sequence fragments of CD19 Sequence fragment.

Description of the drawings

Fig. 1：IFN-gama shRNA and CD19CAR dual-expression vector structure charts.

Fig. 2：PHR-IFN-gama-GFP carrier mode figures.

Fig. 3：PHR-IFN-gama-GFP transfection efficiency streaming figures.

Fig. 4：PHR-antiCD19CAR-U6-IFN-gama shRNA transfection efficiency streaming figures.

Fig. 5：RT-PCR methods detection IFN-gama expression figures after cotransfection.

Fig. 6：Streaming method detection IFN-gama expression figures after cotransfection, wherein left figure is transfection efficiency streaming figure, and right figure is Transfection efficiency bar chart.

Fig. 7：PHR-antiCD19CAR-U6-IFN-gama shRNA-T cell positive rate flow cytometer detection figures

Fig. 8：K562-CD19 monoclonal flow cytometer detection figures.

Fig. 9：The tumour that pHR-antiCD19CAR-U6-IFN-gama shRNA-T cell-specifics kill the CD19 positives is thin Born of the same parents' streaming figure.

Figure 10：PHR-antiCD19CAR-U6-IFN-gama shRNA-T cell-specifics kill the tumour of the CD19 positives Cell counts figure.

Figure 11：RT-PCR methods detect pHR-antiCD19CAR-U6-IFN-gama shRNA-T cytokine contents.

Figure 12：ELISA detects pHR-antiCD19CAR-U6-IFN-gama shRNA-T cytokine contents.

Specific implementation mode

With reference to embodiment, the present invention is further illustrated.It will be understood to those of skill in the art that following reality It applies example and is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Particular technique or condition are not specified in embodiment Person, according to technology described in document in the art or condition (such as with reference to works such as J. Pehanorm Brookers, what Huang Peitang etc. was translated 《Molecular Cloning:A Laboratory guide》, the third edition, Science Press) or carry out according to product description.

Involved instrument, reagent, material etc., are existing in the prior art unless otherwise noted in following embodiments Conventional instrument, reagent, material etc., can be obtained by regular commercial sources.Involved experimental method in following embodiments, inspection Survey method etc. is unless otherwise noted existing routine experiment method in the prior art, detection method etc..

Embodiment 1：The synthesis of the IFN-gama shRNA of carrier can be cloned into

In the sequence of Sigma-Aldrich China official online enquiries IFN-gama shRNA, 4 (gama- therein are chosen 1, gama-2, gama-3, gama-4), nucleotide sequence difference is as follows.

gama-1：CCGGGCAGAGCCAAATTGTCTCCTTCTCGAGAAGGAGACAATTTGGCTCTGCTTTTTG。

gama-2：CCGGTATAAGTGAAGTGATACTATCCTCGAGGATAGTATCACTTCACTTATATTTTTG。

gama-3：CCGGCATTCAGATGTAGCGGATAATCTCGAGATTATCCGCTACATCTGAATGTTTTTG。

gama-4：CCGGGGTTGTCCTGCCTGCAATATTCTCGAGAATATTGCAGGCAGGACAACCTTTTTG。

Wherein, the sequence that underscore marks is the sequence of every middle siRNA.

The present invention uses traditional method for being cloned into carrier, by DNA widow's core of two coding IFN-gamasiRNA sequences Thuja acid is inserted into carrier.The DNA oligonucleotides contains the just siRNA sequence of 21 nucleotide length, passes through the section of Ambion The intervening sequence TTCAAGAGA for 9 nucleotide that scholar has successfully used and the antisense siRNA sequence phase of its reverse complemental Even, 5 T are added as termination signal in 3 ' ends of its oligonucleotides, and is added in design and is cloned into digestion needed for carrier Site NotI.

The present invention uses 4 primer annealing synthetic methods, 4 primers of respectively every shRNA sequence design, IFN-gama ShRNA-1 primer sequences such as SEQ ID NO:Shown in 5~8.IFN-gama shRNA-2 primer sequences are as follows.IFN-gama ShRNA-3 primer sequences such as SEQ ID NO:Shown in 9~12.IFN-gama shRNA-4 primer sequences are as follows.

IFN-gama shRNA-2 primer sequences are as follows：

hIFN-gama-shRNA2-1^st：GGCCGCCTATAAGTGAAGTGATACTATCTTCAAGAGA；

hIFN-gama-shRNA2-2^nd：GATAGTATCACTTCACTTATATTTTTGC；

hIFN-gama-shRNA2-3^rd：GATAGTATCACTTCACTTATAGGC；

hIFN-gama-shRNA2-4^th：GGCCGCAAAAATATAAGTGAAGTGATACTATCTCTCTTGAA.

IFN-gama shRNA-4 primer sequences are as follows：

hIFN-gama-shRNA4-1^st：GGCCGCCGGTTGTCCTGCCTGCAATATTTTCAAGAGA；

hIFN-gama-shRNA4-2^nd：AATATTGCAGGCAGGACAACCTTTTTGC；

hIFN-gama-shRNA4-3^rd：AATATTGCAGGCAGGACAACCGGC；

hIFN-gama-shRNA4-4^th：GGCCGCAAAAAGGTTGTCCTGCCTGCAATATTTCTCTTGAA.

4 primers of every shRNA are synthesized by the Suzhou bio tech ltd Jin Weizhi, then draw 4 of synthesis Object dry powder rapid centrifugation, is diluted with water 100uM later, and 1^st, 2^nd, 3^rd, 4^th, 2.5ul, four total 10ul, moisturizing is respectively taken to arrive 50ul boils 5~10 minutes, is placed on natural cooling in pot and stays overnight to room temperature, and by the process, four primers complete annealing, shape At section of DNA double-strand short-movie section.

The DNA double chain short-movie section formed after the process, IFN-gama shRNA-1, sequence such as SEQ ID NO:1 It is shown；IFN-gama shRNA-2, sequence are as follows；IFN-gama shRNA-3, sequence such as SEQ ID NO:2 institutes Show；IFN-gama shRNA-4, sequence are as follows.

IFN-gama shRNA-2：

GCGGCCGCCTATAAGTGAAGTGATACTATCTTCAAGAGAGATAGTATCACTTCACTTATATTTTT。

IFN-gama shRNA-4：

GCGGCCGCCGGTTGTCCTGCCTGCAATATTTTCAAGAGAAATATTGCAGGCAGGACAACCTTTTT。

Embodiment 2：The structure of IFN-gama shRNA and CD19CAR dual-expression vectors

As shown in Figure 1, CD8 is worn film signal peptide, myc labels, anti-CD19Scfv, CD8 transmembrane region, 4-1BB costimulations Signaling zone and the active regions CD3ZetaTCR, U6 promoters, IFN-gama shRNA are sequentially cloned into slow virus skeleton plasmid pHR In, obtaining pHR-antiCD19CAR-U6-IFN-gama shRNA plasmids, (the involved IFN-gama shRNA of experiment have four Kind, thus obtained plasmid also there are four types of, respectively it is as follows).

The nucleotide of CD8amyc-anti-CD19Scfv-CD8TM-4-1BB-CD3Zeta-U6-IFN-gama shRNA-1 Sequence (such as SEQ ID NO as follows:Shown in 13).

SEQ ID NO:13：

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGCAGAAGCTGAT CAGCGAGGAGGACCTGACTAGTGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAG TCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTT AAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGA TTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGT ACACGTTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGA TCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTC AGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAA TATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGC CAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGG TGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCAGGATCCACCACGACGCCAGCGC CGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCG GGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGG GGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCTCCCTAAAACGGGGCAGAAAGAAACTCCTGTATATATTCA AACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAA GGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTA TAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGG GAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGT GAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAA GGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGCTAAAAGGTCGGGCAGGAAGAGGGCCTATTTC CCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACA AAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAA AATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGGTTTATATATCTTGTGGAAAGGACGGC GGCCGCCGCAGAGCCAAATTGTCTCCTTTTCAAGAGAAAGGAGACAATTTGGCTCTGCTTTTT。

The nucleotide of CD8amyc-anti-CD19Scfv-CD8TM-4-1BB-CD3Zeta-U6-IFN-gama shRNA-2 Sequence is as follows：

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGCAGAAGCTGAT CAGCGAGGAGGACCTGACTAGTGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAG TCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTT AAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGA TTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGT ACACGTTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGA TCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTC AGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAA TATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGC CAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGG TGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCAGGATCCACCACGACGCCAGCGC CGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCG GGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGG GGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCTCCCTAAAACGGGGCAGAAAGAAACTCCTGTATATATTCA AACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAA GGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTA TAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGG GAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGT GAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAA GGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGCTAAAAGGTCGGGCAGGAAGAGGGCCTATTTC CCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACA AAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAA AATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGGTTTATATATCTTGTGGAAAGGACGGC GGCCGCCTATAAGTGAAGTGATACTATCTTCAAGAGAGATAGTATCACTTCACTTATATTTTT。

The nucleotide of CD8amyc-anti-CD19Scfv-CD8TM-4-1BB-CD3Zeta-U6-IFN-gama shRNA-3 Sequence (such as SEQ ID NO as follows:Shown in 14).

SEQ ID NO:14：

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGCAGAAGCTGAT CAGCGAGGAGGACCTGACTAGTGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAG TCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTT AAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGA TTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGT ACACGTTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGA TCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTC AGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAA TATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGC CAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGG TGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCAGGATCCACCACGACGCCAGCGC CGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCG GGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGG GGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCTCCCTAAAACGGGGCAGAAAGAAACTCCTGTATATATTCA AACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAA GGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTA TAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGG GAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGT GAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAA GGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGCTAAAAGGTCGGGCAGGAAGAGGGCCTATTTC CCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACA AAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAA AATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGGTTTATATATCTTGTGGAAAGGACGGC GGCCGCCCATTCAGATGTAGCGGATAATTTCAAGAGAATTATCCGCTACATCTGAATGTTTTT。

The nucleotide of CD8amyc-anti-CD19Scfv-CD8TM-4-1BB-CD3Zeta-U6-IFN-gama shRNA-4 Sequence is as follows：

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGCAGAAGCTGAT CAGCGAGGAGGACCTGACTAGTGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAG TCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTT AAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGA TTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGT ACACGTTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGA TCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTC AGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAA TATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGC CAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGG TGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCAGGATCCACCACGACGCCAGCGC CGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCG GGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGG GGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCTCCCTAAAACGGGGCAGAAAGAAACTCCTGTATATATTCA AACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAA GGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTA TAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGG GAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGT GAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAA GGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGCTAAAAGGTCGGGCAGGAAGAGGGCCTATTTC CCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACA AAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAA AATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGGTTTATATATCTTGTGGAAAGGACGGC GGCCGCCGGTTGTCCTGCCTGCAATATTTTCAAGAGAAATATTGCAGGCAGGACAACCTTTTT。

Embodiment 3：PHR-IFN-gama-GFP screens the preparation of plasmid

As shown in Fig. 2, IFN-gama, GFP are sequentially cloned into slow virus skeleton plasmid pHR, pHR-IFN- is obtained Gama-GFP plasmids.GFP is reporter gene in the pHR-IFN-gama-GFP, for detecting the gene silencing of IFN-gama Efficiency.

The nucleotide sequence of IFN-gama-GFP fusion proteins is as follows：

ATGAAGTACACCAGCTACATCCTGGCCTTTCAGCTGTGCATCGTGCTGGGCAGCCTGGGCTGCTACTGCCAGGACCC CTACGTGAAGGAGGCCGAGAACCTGAAGAAGTACTTCAACGCCGGCCACAGCGACGTGGCCGACAACGGCACCCTGT TCCTCGGCATCCTGAAGAACTGGAAGGAGGAGAGCGACAGGAAGATCATGCAGTCCCAGATCGTGAGCTTCTACTTC AAGCTGTTCAAGAATTTCAAGGACGACCAGAGCATCCAGAAGAGCGTGGAGACCATCAAGGAGGACATGAACGTGAA GTTTTTCAATAGCAACAAGAAGAAGAGGGACGACTTCGAGAAGCTGACCAACTACAGCGTGACCGACCTGAATGTGC AGAGGAAGGCCATCCACGAACTGATCCAGGTGATGGCCGAGCTGAGCCCTGCCGCCAAGACCGGCAAGAGGAAGAGG AGCCAGATGCTGTTCAGGGGCAGGAGGGCCAGCCAGGGATCCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGT GGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATG CCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACC ACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCAT GCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGT TCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCAC AAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTT CAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACG GCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGAT CACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA。

Embodiment 4：The pHR-antiCD19CAR-U6-IFN-gama shRNA slow virus of effective reticence IFN-gama expression The screening of carrier

(1) pHR-IFN-gama-GFP screens the transfection of plasmid

Using calcium phosphate transfection method, by CaCl₂, plasmid and water mix according to a certain percentage, be slowly dropped to 293FT cells Culture supernatant in, for 24 hours after flow cytometer detection 293FT, detect the luciferase expression of IFN-gama-GFP fusion proteins.As shown in Figure 3 The transfection efficiency of pHR-IFN-gama-GFP is 48.3%, being capable of Successful transfection.

(2) transfection of pHR-antiCD19CAR-U6-IFN-gamashRNA plasmids

Using calcium phosphate transfection method, by CaCl₂, plasmid and water mix according to a certain percentage, be slowly dropped to 293FT cells Culture supernatant in, take 293FT cells, the cell proportion (Fluorescein of the flow cytomery CAR positives afterwards for 24 hours (FITC) AffiniPure Goat Anti-Mouse IgG, F (ab') 2fragment specific, jackson Immunoresearch, cat：115-095-006).As shown in figure 4, being compared with negative control group, 4 kinds of pHR- The transfection efficiency of antiCD19CAR-U6-IFN-gama shRNA plasmids is respectively 19.8%, 25%, 20.6%, 15.7%.All It being capable of Successful transfection.

(3) RT-PCR methods screening IFN-gama shRNA

Using the side of pHR-IFN-gama-GFP and pHR-antiCD19CAR-U6-IFN-gama shRNA plasmid co-transfections Formula, by calcium phosphate transfection method, by CaCl₂, 2 kinds of plasmids and water mix according to a certain percentage, be slowly dropped to 293FT cells In culture supernatant, 293FT cells, extraction total serum IgE (MiniBEST Universal RNA, TAKARA, cat are harvested afterwards for 24 hours: 9767), reverse transcription is at cDNA (FastKing RT Kit, Tiangeng, cat：KR11601), using the primer of IFN-gama (as before It is shown), the expression of IFN-gama in mRNA level in-site is measured by the method for quantitative fluorescent PCR (need to use reference gene β-actin Primer, as previously shown).

As shown in figure 5, being compared with the 293FT that pHR-IFN-gama-GFP is individually transfected, pHR-antiCD19CAR-U6- IFN-gama shRNA-1 plasmids, pHR-antiCD19CAR-U6-IFN-gama shRNA-3 plasmids and pHR-IFN-gama- The level of IFN-gama is substantially reduced in 293FT after GFP cotransfections.Statistical analysis shows that the level of IFN-gama has significantly Difference.Show that this 2 kinds of pHR-antiCD19CAR-U6-IFN-gamashRNA can effectively reduce pHR-IFN- in mRNA level in-site The expression of IFN-gama in gama-GFP, plays the effect of gene silencing.

(4) screening of streaming method IFN-gama shRNA

Using the side of pHR-IFN-gama-GFP and pHR-antiCD19CAR-U6-IFN-gama shRNA plasmid co-transfections Formula, by calcium phosphate transfection method, by CaCl₂, 2 kinds of plasmids and water mix according to a certain percentage, be slowly dropped to 293FT cells In culture supernatant, rear flow cytometer detection 293FT, detects the luciferase expression of IFN-gama-GFP fusion proteins for 24 hours.As shown in fig. 6, and The transfection efficiency that pHR-IFN-gama-GFP individually transfects 17% is compared, pHR-antiCD19CAR-U6-IFN-gama shRNA- GFP after 1 plasmid, pHR-antiCD19CAR-U6-IFN-gama shRNA-3 plasmids and pHR-IFN-gama-GFP cotransfections It is 7.55%, 4.16% that transfection efficiency, which is remarkably decreased,.Show this 2 kinds of pHR-antiCD19CAR-U6-IFN-gama shRNA energy The protein expression for effectively reducing IFN-gama in pHR-IFN-gama-GFP, plays the effect of gene silencing.

Embodiment 5：The packaging of slow virus and concentration

By the Lentiviral (pHR-antiCD19CAR-U6-IFN-gama for carrying target gene ShRNA-1 plasmids and pHR-antiCD19CAR-U6-IFN-gama shRNA-3 plasmids), pCMV carriers and pMD.2G carriers it is mixed It is transfected into after conjunction in 293FT cells, 6~8h is changed to complete medium culture after transfection, culture solution is collected after 48h, after centrifugation Retain supernatant and filtered with 0.45 μm of filter, retains filtrate, the filtrate is the solution of recombinant slow virus.

Slow virus concentrates according to Lenti-XTMConcentrator (takara, cat:631231) specification carries out.

Embodiment 6：The preparation of the T cell of IFN-gama shRNA and CD19CAR dual-expression vectors

50mL new bloods are taken, it is single to detach to carry out density gradient centrifugation by lymphocyte separation medium (oceans Tianjin Hao) Nucleus.Mononuclearcell is resuspended into X-VIVO15 culture mediums (Lonza), while adding CD3 monoclonal antibodies and CD28 monoclonal antibodies Activated T lymphocytes, 37 DEG C of 5%CO₂Culture 48 hours.

Take 2 × 10⁶Cell, 2 kinds of slow virus concentrating in embodiment 5 are added according to MOI=5, at the same be added IL-2 and Polybrene, mixing, 37 DEG C of 5%CO₂Culture 6-8 hours, it is that fresh X-VIVO15 culture mediums (contain that liquid is changed in 300g5min centrifugations IL-2)。

Fresh X-VIVO15 culture mediums (containing IL-2) were added per 2-3 days, maintain cell density 1 × 10⁶/ mL or so expands Increase 10-12 days.

The T cell each 5 × 10 of the T cell and uninfecting virus of culture 48h after taking virus to infect⁵, each sample adds FluoresceinAffiniPure Goat Anti-Mouse IgG, F (ab') 2fragmentspecific1ul, room temperature are protected from light It is incubated 15min, centrifugation, precipitation upper machine testing (Millipore guava easyCyteHT) after being resuspended with 200 μ LPBS.As a result As shown in fig. 7,2 kinds of pHR-antiCD19CAR-U6-IFN-gama shRNA-T are thin in the T cell of culture 48h after virus infection The ratio of born of the same parents is 84.9%, 86.6%, and efficiency of infection is very high.Prepared in the embodiment 2 kinds of T cells are named as pHR- AntiCD19CAR-U6-IFN-gama shRNA-1-T and pHR-antiCD19CAR-U6-IFN-gama shRNA-3-T, as Used cell in later embodiment.

Embodiment 7：The Cytotoxicity in vitro of pHR-antiCD19CAR-U6-IFN-gama shRNA-T cells against tumor cells strains Experiment

1. stablizing the structure of the K562 cells (target cell) of expression CD19 genes

(1) the DNA encoding sequence for synthesizing CD19 extracellular regions and transmembrane region, is inserted into pHR carriers, builds the carrier name of acquisition For pHR-CD19.

CD19 extracellular regions and the nucleotide coding sequence of transmembrane region are as follows：

ATGCCACCTCCTCGCCTCCTCTTCTTCCTCCTCTTCCTCACCCCCATGGAAGTCAGGCCCGAGGAACCTCTAGTGGT GAAGGTGGAAGAGGGAGATAACGCTGTGCTGCAGTGCCTCAAGGGGACCTCAGATGGCCCCACTCAGCAGCTGACCT GGTCTCGGGAGTCCCCGCTTAAACCCTTCTTAAAACTCAGCCTGGGGCTGCCAGGCCTGGGAATCCACATGAGGCCC CTGGCCATCTGGCTTTTCATCTTCAACGTCTCTCAACAGATGGGGGGCTTCTACCTGTGCCAGCCGGGGCCCCCCTC TGAGAAGGCCTGGCAGCCTGGCTGGACAGTCAATGTGGAGGGCAGCGGGGAGCTGTTCCGGTGGAATGTTTCGGACC TAGGTGGCCTGGGCTGTGGCCTGAAGAACAGGTCCTCAGAGGGCCCCAGCTCCCCTTCCGGGAAGCTCATGAGCCCC AAGCTGTATGTGTGGGCCAAAGACCGCCCTGAGATCTGGGAGGGAGAGCCTCCGTGTCTCCCACCGAGGGACAGCCT GAACCAGAGCCTCAGCCAGGACCTCACCATGGCCCCTGGCTCCACACTCTGGCTGTCCTGTGGGGTACCCCCTGACT CTGTGTCCAGGGGCCCCCTCTCCTGGACCCATGTGCACCCCAAGGGGCCTAAGTCATTGCTGAGCCTAGAGCTGAAG GACGATCGCCCGGCCAGAGATATGTGGGTAATGGAGACGGGTCTGTTGTTGCCCCGGGCCACAGCTCAAGACGCTGG AAAGTATTATTGTCACCGTGGCAACCTGACCATGTCATTCCACCTGGAGATCACTGCTCGGCCAGTACTATGGCACT GGCTGCTGAGGACTGGTGGCTGGAAGGTCTCAGCTGTGACTTTGGCTTATCTGATCTTCTGCCTGTGTTCCCTTGTG GGCATTCTTCATCTTCAAAGAGCCCTGGTCCTGAGG。

(2) slow virus packaging and concentration

It is transfected into 293FT cells after the pHR-CD19 carriers, pCMV carriers and pMD.2G carriers are mixed, after transfection 6~8h is changed to complete medium culture, and culture solution is collected after 48h, and supernatant is retained after centrifugation and is filtered with 0.45 μm of filter, is protected Reserved filtrate, the filtrate are the solution of recombinant slow virus.

(3) preparation of the K562 cells of expression CD19

K562 cells are resuspended to 1x10 in 1640 (Gibco) culture mediums⁶/ mL, is added slow virus (MOI5-10), 37 DEG C, 5% CO₂Incubator culture 6-8h, it is fresh K562 cell Proliferations culture solution that liquid is changed in centrifugation.Fresh K562 cells were added per 2-3 days to increase Culture solution is grown, maintains cell density in 0.5x10⁶/ mL or so.After 5 generations, monoclonal screening is carried out using limiting dilution assay.Wait for list Clone cell is grown to certain amount, and (anti-CD19PE, biolegend cat is screened by flow cytometer： 302254) cell clone that, expression quantity is high and purity is high, as stablizes the K562 cells of expression CD19, is named as K562-CD19 (as shown in Figure 8), as the target cell in the present embodiment.

The killing ability Validation in vitro of 2.pHR-antiCD19CAR-U6-IFN-gama shRNA-T

The K562-CD19 cells of K562 cells and structure are pressed 1:1 is inoculated in 96 orifice plates, by E:T=1:1,3:1 difference 9 days pHR-antiCD19CAR-U6-IFN-gama shRNA-1-T of inoculated and cultured, pHR-antiCD19CAR-U6-IFN- Gama shRNA-3-T, antiCD19CAR-T cells and T cell.37 DEG C of 5%CO₂Culture 24 hours is drawn per hole on 100 μ L - 80 DEG C of clear liquid saves backup.PE anti-humanCD19 (Biolegend) and FITCanti- are added per hole for remaining cell Human CD3 (Biolegend), room temperature, which is protected from light, is incubated 15min, centrifugation, precipitation upper machine testing after being resuspended with 200 μ L PBS (Millipore guava easyCyteHT).As a result as shown in FIG. 9 and 10,2 groups of pHR-antiCD19CAR-U6-IFN- It is 1 that gama shRNA-T cells have specific killing, effect target ratio to K562-CD19 cells:1,3:Specific killing efficiency is equal when 1 Value be respectively 56.7%, 100% and 63.3%, 100%, killing rate with effect target than increase and increase.Statistical analysis shows, Specific killing efficiency between 2 groups of pHR-antiCD19CAR-U6-IFN-gama shRNA-T cells and T cell group has significantly Difference, and the specificity between pHR-antiCD19CAR-U6-IFN-gama shRNA-T and antiCD19CAR-T groups of cells is killed Hinder efficiency without significant difference.

Embodiment 8：PHR-antiCD19CAR-U6-IFN-gamashRNA-T cell cytokine content detections

1) RT-PCR methods detect pHR-antiCD19CAR-U6-IFN-gama shRNA-T cytokine releases

Take pHR-antiCD19CAR-U6-IFN-gamashRNA-1-T, pHR-antiCD19CAR-U6- of culture 9 days IFN-gama shRNA-3-T and antiCD19CAR-T cells, extraction total serum IgE (MiniBEST Universal RNA, TAKARA,cat:9767), reverse transcription is at cDNA (FastKing RTKit, Tiangeng, cat：KR11601), the primer of IL-6 is utilized The primer (as previously shown) of (as follows), IFN-gama, the primer (as follows) of IL-2, house-keeping gene β-action draw Object (as previously shown) measures the expression of IL-6, IFN-gama, IL-2 in mRNA level in-site by the method for quantitative fluorescent PCR.

The primer sequence of IL-6 is as follows in RT-PCR：IL-6-F:AGACAGCCACTCACCTCTTCAG；IL-6-R: TTCTGCCAGTGCCTCTTTGCTG。

The primer sequence of IL-2 is as follows in RT-PCR：IL-2-F:AACTCACCAGGATGCTCACATTTA；IL-2- R:TCCCTGGGTCTTAAGTGAAAGTTT。

It is compared as shown in figure 11 with CD19CAR-T cells, pHR-antiCD19CAR-U6-IFN-gama shRNA-T are thin Born of the same parents can significantly reduce the release of IFN-gama, IL-6 and IL-2 in mRNA level in-site, play the effect of gene silencing IL-6.

2) secreted in ELISA method detection pHR-antiCD19CAR-U6-IFN-gama shRNA-T cells and supernatants because Sub- content

E in Example 7:T=3:The stayed supernatant of 1 killing experiment in vitro, respectively T cell group, antiCD19CAR-T Groups of cells, pHR-antiCD19CAR-U6-IFN-gama shRNA-1-T groups of cells, pHR-antiCD19CAR-U6-IFN- The supernatant of gama shRNA-3-T groups of cells and the negative control group of tumour cell, by HumanI L- 2ELISAMAX^TMDeluxe (Biolegend, 431804), Human IFN-gama ELISAMAX^TMDeluxe (Biolegend, 430104)、HumanIL-6ELISAMAX^TMDeluxe (Biolegend, 430504) kit specification operates, and detects IFN- The release of gama, IL-6 and IL-2.

As a result as shown in figure 12, pHR-antiCD19CAR-U6-IFN-gama shRNA-T cells and CD19CAR-T cells Compared to the release that can significantly reduce IFN-gama.Reduce the release of IL-6.And the release of IL-2 is had not significant impact.

Above-described embodiment is provided to those skilled in the art, how to implement and use to be advocated with full disclosure and description Embodiment, rather than for limiting range disclosed herein.Obvious modification will to those skilled in the art Within the scope of the appended claims.The all publications, patents and patent applications of this specification citation are incorporated by reference into this Text, as these publications, patents and patent applications respectively particularly and individually show to be incorporated herein by reference.

Sequence table

<110>Shandong Qilu Cell Therapy Engineering Technology Co., Ltd.

<120>Interfere CD19-CAR-T cells and its application of IFN-gama expression

<141> 2018-04-27

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 65

<212> DNA

<213> Artificial Sequence

<400> 1

gcggccgccg cagagccaaa ttgtctcctt ttcaagagaa aggagacaat ttggctctgc 60

ttttt 65

<210> 2

<211> 65

<212> DNA

<213> Artificial Sequence

<400> 2

gcggccgccc attcagatgt agcggataat ttcaagagaa ttatccgcta catctgaatg 60

ttttt 65

<210> 3

<211> 499

<212> PRT

<213> Artificial Sequence

<400> 3

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

1 5 10 15

His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asp

20 25 30

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

35 40 45

Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu

50 55 60

Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr

65 70 75 80

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

85 90 95

Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu

100 105 110

Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr

115 120 125

Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu Ser

145 150 155 160

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

165 170 175

Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln

180 185 190

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

195 200 205

Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys

210 215 220

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

225 230 235 240

Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu

355 360 365

Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys

370 375 380

Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Pro Arg Gly

<210> 4

<211> 1512

<212> DNA

<213> Artificial Sequence

<400> 4

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggagcaga agctgatcag cgaggaggac ctgactagtg acatccagat gacacagact 120

acatcctccc tgtctgcctc tctgggagac agagtcacca tcagttgcag ggcaagtcag 180

gacattagta aatatttaaa ttggtatcag cagaaaccag atggaactgt taaactcctg 240

atctaccata catcaagatt acactcagga gtcccatcaa ggttcagtgg cagtgggtct 300

ggaacagatt attctctcac cattagcaac ctggagcaag aagatattgc cacttacttt 360

tgccaacagg gtaatacgct tccgtacacg ttcggagggg ggaccaagct ggagatcaca 420

ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatctgaggt gaaactgcag 480

gagtcaggac ctggcctggt ggcgccctca cagagcctgt ccgtcacatg cactgtctca 540

ggggtctcat tacccgacta tggtgtaagc tggattcgcc agcctccacg aaagggtctg 600

gagtggctgg gagtaatatg gggtagtgaa accacatact ataattcagc tctcaaatcc 660

agactgacca tcatcaagga caactccaag agccaagttt tcttaaaaat gaacagtctg 720

caaactgatg acacagccat ttactactgt gccaaacatt attactacgg tggtagctat 780

gctatggact actggggcca aggaacctca gtcaccgtct cctcaggatc caccacgacg 840

ccagcgccgc gaccaccaac accggcgccc accatcgcgt cgcagcccct gtccctgcgc 900

ccagaggcgt gccggccagc ggcggggggc gcagtgcaca cgagggggct ggacttcgcc 960

tgtgatatct acatctgggc gcccttggcc gggacttgtg gggtccttct cctgtcactg 1020

gttatcaccc tttactgctc cctaaaacgg ggcagaaaga aactcctgta tatattcaaa 1080

caaccattta tgagaccagt acaaactact caagaggaag atggctgtag ctgccgattt 1140

ccagaagaag aagaaggagg atgtgaactg agagtgaagt tcagcaggag cgcagacgcc 1200

cccgcgtaca agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag 1260

gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga 1320

aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc 1380

tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac 1440

cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc 1500

cctcgcggct aa 1512

<210> 5

<211> 37

<212> DNA

<213> Artificial Sequence

<400> 5

ggccgccgca gagccaaatt gtctcctttt caagaga 37

<210> 6

<211> 28

<212> DNA

<213> Artificial Sequence

<400> 6

aaggagacaa tttggctctg ctttttgc 28

<210> 7

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 7

aaggagacaa tttggctctg cggc 24

<210> 8

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 8

ggccgcaaaa agcagagcca aattgtctcc tttctcttga a 41

<210> 9

<211> 37

<212> DNA

<213> Artificial Sequence

<400> 9

ggccgcccat tcagatgtag cggataattt caagaga 37

<210> 10

<211> 28

<212> DNA

<213> Artificial Sequence

<400> 10

attatccgct acatctgaat gtttttgc 28

<210> 11

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 11

attatccgct acatctgaat gggc 24

<210> 12

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 12

ggccgcaaaa acattcagat gtagcggata attctcttga a 41

<210> 13

<211> 1834

<212> DNA

<213> Artificial Sequence

<400> 13

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggagcaga agctgatcag cgaggaggac ctgactagtg acatccagat gacacagact 120

acatcctccc tgtctgcctc tctgggagac agagtcacca tcagttgcag ggcaagtcag 180

gacattagta aatatttaaa ttggtatcag cagaaaccag atggaactgt taaactcctg 240

atctaccata catcaagatt acactcagga gtcccatcaa ggttcagtgg cagtgggtct 300

ggaacagatt attctctcac cattagcaac ctggagcaag aagatattgc cacttacttt 360

tgccaacagg gtaatacgct tccgtacacg ttcggagggg ggaccaagct ggagatcaca 420

ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatctgaggt gaaactgcag 480

gagtcaggac ctggcctggt ggcgccctca cagagcctgt ccgtcacatg cactgtctca 540

ggggtctcat tacccgacta tggtgtaagc tggattcgcc agcctccacg aaagggtctg 600

gagtggctgg gagtaatatg gggtagtgaa accacatact ataattcagc tctcaaatcc 660

agactgacca tcatcaagga caactccaag agccaagttt tcttaaaaat gaacagtctg 720

caaactgatg acacagccat ttactactgt gccaaacatt attactacgg tggtagctat 780

gctatggact actggggcca aggaacctca gtcaccgtct cctcaggatc caccacgacg 840

ccagcgccgc gaccaccaac accggcgccc accatcgcgt cgcagcccct gtccctgcgc 900

ccagaggcgt gccggccagc ggcggggggc gcagtgcaca cgagggggct ggacttcgcc 960

tgtgatatct acatctgggc gcccttggcc gggacttgtg gggtccttct cctgtcactg 1020

gttatcaccc tttactgctc cctaaaacgg ggcagaaaga aactcctgta tatattcaaa 1080

caaccattta tgagaccagt acaaactact caagaggaag atggctgtag ctgccgattt 1140

ccagaagaag aagaaggagg atgtgaactg agagtgaagt tcagcaggag cgcagacgcc 1200

cccgcgtaca agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag 1260

gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga 1320

aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc 1380

tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac 1440

cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc 1500

cctcgcggct aaaaggtcgg gcaggaagag ggcctatttc ccatgattcc ttcatatttg 1560

catatacgat acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag 1620

atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta 1680

aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc 1740

ttgggtttat atatcttgtg gaaaggacgg cggccgccgc agagccaaat tgtctccttt 1800

tcaagagaaa ggagacaatt tggctctgct tttt 1834

<210> 14

<211> 1834

<212> DNA

<213> Artificial Sequence

<400> 14

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggagcaga agctgatcag cgaggaggac ctgactagtg acatccagat gacacagact 120

acatcctccc tgtctgcctc tctgggagac agagtcacca tcagttgcag ggcaagtcag 180

gacattagta aatatttaaa ttggtatcag cagaaaccag atggaactgt taaactcctg 240

atctaccata catcaagatt acactcagga gtcccatcaa ggttcagtgg cagtgggtct 300

ggaacagatt attctctcac cattagcaac ctggagcaag aagatattgc cacttacttt 360

tgccaacagg gtaatacgct tccgtacacg ttcggagggg ggaccaagct ggagatcaca 420

ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatctgaggt gaaactgcag 480

gagtcaggac ctggcctggt ggcgccctca cagagcctgt ccgtcacatg cactgtctca 540

ggggtctcat tacccgacta tggtgtaagc tggattcgcc agcctccacg aaagggtctg 600

gagtggctgg gagtaatatg gggtagtgaa accacatact ataattcagc tctcaaatcc 660

agactgacca tcatcaagga caactccaag agccaagttt tcttaaaaat gaacagtctg 720

caaactgatg acacagccat ttactactgt gccaaacatt attactacgg tggtagctat 780

gctatggact actggggcca aggaacctca gtcaccgtct cctcaggatc caccacgacg 840

ccagcgccgc gaccaccaac accggcgccc accatcgcgt cgcagcccct gtccctgcgc 900

ccagaggcgt gccggccagc ggcggggggc gcagtgcaca cgagggggct ggacttcgcc 960

tgtgatatct acatctgggc gcccttggcc gggacttgtg gggtccttct cctgtcactg 1020

gttatcaccc tttactgctc cctaaaacgg ggcagaaaga aactcctgta tatattcaaa 1080

caaccattta tgagaccagt acaaactact caagaggaag atggctgtag ctgccgattt 1140

ccagaagaag aagaaggagg atgtgaactg agagtgaagt tcagcaggag cgcagacgcc 1200

cccgcgtaca agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag 1260

gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga 1320

aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc 1380

tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac 1440

cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc 1500

cctcgcggct aaaaggtcgg gcaggaagag ggcctatttc ccatgattcc ttcatatttg 1560

catatacgat acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag 1620

atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta 1680

aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc 1740

ttgggtttat atatcttgtg gaaaggacgg cggccgccca ttcagatgta gcggataatt 1800

tcaagagaat tatccgctac atctgaatgt tttt 1834

Claims

1. interfering the shRNA of people's IFN-gama gene expressions, it is characterised in that：For IFN-gama shRNA-1 or IFN-gama The nucleotide sequence of shRNA-3, IFN-gama shRNA-1 are as follows, and the nucleotide sequence of IFN-gama shRNA-3 is as follows It is shown：

IFN-gama shRNA-1, such as SEQ ID NO:Shown in 1：

GCGGCCGCCGCAGAGCCAAATTGTCTCCTTTTCAAGAGAAAGGAGACAATTTGGCTCTGCTTTTT。

IFN-gama shRNA-3, such as SEQ ID NO:Shown in 2：

GCGGCCGCCCATTCAGATGTAGCGGATAATTTCAAGAGAATTATCCGCTACATCTGAATGTTTTT。

2. being used to prepare the primer of the shRNA of interference people's IFN-gama gene expressions described in claim 1, it is characterised in that： It is used to prepare the primer of IFN-gama shRNA-1, nucleotide sequence is as follows：

hIFN-gama-shRNA1-1^st：GGCCGCCGCAGAGCCAAATTGTCTCCTTTTCAAGAGA；

hIFN-gama-shRNA1-2^nd：AAGGAGACAATTTGGCTCTGCTTTTTGC；

hIFN-gama-shRNA1-3^rd：AAGGAGACAATTTGGCTCTGCGGC；

hIFN-gama-shRNA1-4^th：GGCCGCAAAAAGCAGAGCCAAATTGTCTCCTTTCTCTTGAA.

It is used to prepare the primer of IFN-gama shRNA-3, nucleotide sequence is as follows：

hIFN-gama-shRNA3-1^st：GGCCGCCCATTCAGATGTAGCGGATAATTTCAAGAGA；

hIFN-gama-shRNA3-2^nd：ATTATCCGCTACATCTGAATGTTTTTGC；

hIFN-gama-shRNA3-3^rd：ATTATCCGCTACATCTGAATGGGC；

hIFN-gama-shRNA3-4^th：GGCCGCAAAAACATTCAGATGTAGCGGATAATTCTCTTGAA.

3. a kind of Lentiviral, it is characterised in that：It includes the interference people's IFN-gama genes having the right described in requirement 1 The shRNA of expression and the nucleotide fragments of encoding chimeric antigen receptor；The nucleotide piece of the encoding chimeric antigen receptor Section, nucleotide sequence such as SEQ ID NO:Shown in 4.

4. Lentiviral according to claim 3, it is characterised in that：The structure of the Lentiviral is PHR-antiCD19CAR-U6-IFN-gama shRNA-1, the nucleotide sequence such as SEQ ID NO of expressing gene:Shown in 13； Or：PHR-antiCD19CAR-U6-IFN-gama shRNA-3, the nucleotide sequence such as SEQ ID NO of expressing gene:14 institutes Show.

5. a kind of slow virus expresses kit, include the Lentiviral of claim 3 or 4.

6. a kind of recombinant slow virus, which is characterized in that be prepared by the following method to obtain：By the slow virus table of claim 3 or 4 It is transfected into 293FT cells after being mixed up to carrier, pCMV carriers and pMD.2G carriers, 6~8h is changed to complete culture after transfection Base culture collects culture solution after 48h, and supernatant is retained after centrifugation and is filtered with 0.45 μm of filtering head, reservation filtrate, as recombinant lentiviral The solution of virus.

7. a kind of CD19-CAR-T cells of interference people's IFN-gama gene expressions, it is characterised in that：To include claim 3 Or 4 Lentiviral T lymphocytes or chromosome in be integrated with interference people IFN-gama described in claim 1 The T lymphocytes of the shRNA of gene expression and the nucleotide fragments of encoding chimeric antigen receptor；The encoding chimeric antigen The nucleotide fragments of receptor, nucleotide sequence such as SEQ ID NO:Shown in 4.

8. the Lentiviral described in claim 3 or 4 is pernicious swollen in preparation prevention and/or treatment and/or auxiliary treatment Application in the drug of tumor.

9. the CD19-CAR-T cells of interference people's IFN-gama gene expressions described in claim 7 prevent and/or control preparing Application in the drug for the treatment of and/or adjuvant therapy of malignant tumor.

10. application according to claim 8 or claim 9, it is characterised in that：The malignant tumour is selected from the white blood of acute lymphoblastic Disease and/or chronic lymphocytic leukemia.