CN108753773B

CN108753773B - CD19-CAR-T cell capable of interfering IFN-gama expression and application thereof

Info

Publication number: CN108753773B
Application number: CN201810412693.3A
Authority: CN
Inventors: 谭毅; 郭红; 张慧慧
Original assignee: Qilu Cell Therapy Technology Co ltd
Current assignee: Qilu Cell Therapy Technology Co ltd
Priority date: 2018-05-03
Filing date: 2018-05-03
Publication date: 2021-03-30
Anticipated expiration: 2038-05-03
Also published as: CN108753773A

Abstract

The invention discloses two shRNAs interfering the expression of human IFN-gama genes, which are shown as SEQ ID NO 1 and SEQ ID NO 2. The invention also discloses a lentivirus expression vector which comprises shRNA for interfering the expression of human IFN-gama gene and a nucleotide segment which is shown as SEQ ID NO. 4 and used for coding the chimeric antigen receptor. The invention also discloses a CD19-CAR-T cell for interfering the expression of the human IFN-gama gene, which is a T lymphocyte containing a lentivirus expression vector, or a T lymphocyte integrating shRNA for interfering the expression of the human IFN-gama gene in a chromosome and a nucleotide fragment for coding a chimeric antigen receptor. IFN-gama shRNA is introduced into the CAR-T cell, the IFN-gama shRNA is co-expressed while the CAR is expressed, and the release of the IFN-gama is inhibited from the source through a gene silencing technology, so that the influence of CRS is reduced, and the safety of CAR-T treatment is improved. It can be expected that the compound has important application in preparing medicaments for preventing, treating and assisting in treating malignant tumors.

Description

CD19-CAR-T cell capable of interfering IFN-gama expression and application thereof

Technical Field

The invention relates to preparation and application of a Chimeric Antigen Receptor T cell (CAR-T) of a targeting CD19 for interfering IFN-gama expression, belonging to the fields of genetic engineering and cell biology.

Background

In 1989, the concept of chimeric antigen receptor engineered T cells (CAR-T) was first proposed with the aim of establishing adoptive cell therapy for tumor specific recognition capability. The first generation of CARs was designed by simply replacing the extracellular domain portion of the tcr (cell receptor) with a single-chain antibody-derived scFv (single-chain antibody variable region fragment), retaining the transmembrane structure and CD3zeta region for intracellular signaling, and some groups used FcR for intracellular signaling. The clinical trial results for the first generation of CARs were very disappointing, and the best clinical trial had only long-term remission in 2/11 patients. With increasing immunological awareness, it was found that the CD3zeta signal, although capable of inducing T cell activation and transient proliferation, later induced T cell anergy (anergy). In 1998, two laboratories reported that the CD28 signaling domain could provide an ancillary costimulatory function for CD3zeta signaling. Thus, the design of second generation CARs introduced portions of costimulatory molecules, such as the intracellular signaling domain of CD28 or CD137(4-1BB), on a first generation basis. Third generation CARs incorporate 2 functional signaling domains of costimulatory molecules, including CD27, CD28, 4-1BB, ICOS or OX40 (15-18). The majority of the experiments now performed clinically are the design of second generation CARs.

The efficacy of CAR-T has been recognized by the industry as the first CAR-T drug approved by the FDA in the United states, Kymriah (tisagenlecucel), for treatment of relapsed or refractory (r/r) childhood and young adult B-cell acute lymphoblastic leukemia, on 30/8.2017. This is the first approved immune cell drug. One more than 10 months later, day 18, FDA approved the kit pharmaceutical yescatta (axicabagene ciloleucel, KTE-C10) for the treatment of patients with specific types of adult large B cell lymphoma, including diffuse large B cell lymphoma, transformed follicular lymphoma, primary mediastinal B cell lymphoma, who either failed to respond to other therapies or relapsed after receiving at least 2 treatment regimens. Yescatta is the FDA approved CAR-T therapy of clause 2.

Concomitant with the effectiveness of CD-19-CAR-T therapy is its side effects and cytotoxicity. Because CD19 is selected as a target point during design, the imported T cells can kill normal B cells existing in the body together with tumor cells, and after the tumor disappears, as long as CAR-T cells still exist, no normal B cells in the body survive, and the patient needs to inject immunoglobulin at intervals to maintain basic humoral immunity. On the other hand, in the initial phase of CAR-T cell transfusion, patients develop Cytokine Release Syndrome (CRS) due to the massive expansion of T cells and the secretion of a large number of cytokines during tumor killing by T cells, which is manifested by fever, hypotension, hypoxia and a significant increase in serum levels of certain cytokines including interferon-gamma (IFN-gama), fractal chemokines (Fracktalkine), granulocyte-macrophage growth factor (GM-CSF), interleukin-5 (IL-5), interleukin-6 (IL-6), human FMS-like tyrosine kinase 3 ligand (Flt-3L) and interleukin-10 (IL-10). Juno Therapeutics, headquartered in seattle, published a clinical trial JCAR015 in 2016, three patients in front of each other (four confirmed later) died after CAR-T cell therapy, and the FDA immediately discontinued the clinical trial of JCAR015, Juno later explained because of neurotoxins caused by the use of the chemotherapeutic drug fludarabine, but many also hypothesized that patients died due to the strong cytokine storm (CRS) produced by infused T cells. The CD-19 targeted product CLT019 by Novartis, switzerland, although providing relief in 80% of patients, also causes Cytokine Release (CRS) in nearly half of the patients. In patients with CRS, T cells release excessive inflammatory factors, resulting in an exaggerated inflammatory response and fever, which if severe can be life threatening. Similarly, KTE-C19, a product used by Kitepharma, Calif., for the treatment of non-Hodgkin's lymphoma, in the United states, resulted in the occurrence of neurological side effects in 1/3 patients, CRS in 1/5 patients, and death in two patients tested.

Generally, clinical symptoms of CRS are rapidly reversed by large doses of steroid hormones, such as prednisone. However, steroid drugs significantly inhibit the proliferation of CAR-T cells in vivo, leading to a higher recurrence rate in patients, affecting the efficacy of CAR-T therapy. The currently clinically better therapeutic agent for CRS treatment is a blocking monoclonal antibody drug (Tocilizumab) for interleukin-6 receptor (IL-6R). Clinical tests prove that the inhibitor can rapidly solve the toxic and side effects brought by CRS after IL-6 receptor blocking and has no influence on CAR-T cell in-vivo proliferation. Tocillizumab is currently approved by the FDA for use in treating CRS risk that may occur with CAR-T therapy. However, Tocilizumab is expensive and not a small economic burden to the patient, so if the release of IFN-gama cytokines can be cut off from the source, the risk of CRS can be greatly reduced, enhancing the safety of CAR-T therapy.

RNA interference (RNAi) is a gene silencing mechanism that is ubiquitous in organisms, highly sequence specific, and post-transcriptional. Currently, small RNA molecules that are commonly used to exert interference action mainly include sirna (small interfering RNA), shrna (short hairpin RNA), and amirna (artificial microrna) in animals. The first method of using RNAi technology to research gene function in vivo is to inject single-chain siRNA molecule prepared in vitro directly into nematode body, and although it is used later in gene function research, the interference timeliness of this method is relatively short, and it often has cytotoxicity to increase siRNA concentration for effective interference. Most of the prior RNAi techniques adopt a method of constructing a specific RNAi expression vector, and the vector can be introduced into animal and plant cells to transcribe corresponding shRNA, amiRNA and the like by an RNA polymerase II type or III type promoter so as to interfere the expression of a target gene of the shRNA, the amiRNA and the like.

At present, the following two methods are mainly used for constructing shRNA interference expression vectors: (1) an oligonucleotide annealing method comprises the steps of chemically synthesizing two specific oligonucleotide primers, annealing under an in vitro condition to form a double-stranded shRNA segment with two sticky ends at two ends, and then connecting and transforming the double-stranded shRNA segment with a vector which is subjected to enzyme digestion treatment and also has the sticky ends to obtain a recombinant plasmid containing a shRNA infection expression unit. (2) PCR amplification method: designing a pair of PCR primers for amplifying and controlling the shRNA expression promoter, wherein the forward primer is specifically matched with the promoter, an interference target sequence aiming at a target gene is additionally added at the 5' tail end of the reverse primer, and cloning the PCR product to a corresponding vector to obtain the shRNA interference expression vector.

The interferon-gama (IFN-gama) is used as a Cytokine playing an important role in Cytokine Release Syndrome (CRS), shRNA aiming at the IFN-gama is designed, and the Release of the IFN-gama is inhibited from the source through a gene silencing technology, so that the influence of the CRS is reduced, and the method is a beneficial idea for improving the safety of CAR-T and reducing toxic and side effects.

Disclosure of Invention

Aiming at the prior art, the inventor successfully screens 2 shRNAs capable of interfering IFN-gama gene expression through intensive research and creative work, and co-expresses the shRNAs and CD19 CAR. The inventor finds that the release amount of factors such as IL-6, IFN-gama and the like is reduced in the process of killing tumor cells expressing CD19 by CD19CAR-T cells co-expressing shRNA interfering IFN-gama gene expression, so that the influence of CRS is reduced at the source, and the safety in CAR-T treatment is improved.

The invention is realized by the following technical scheme:

two kinds of shRNAs interfering the expression of human IFN-gama gene are named as IFN-gama shRNA-1 and IFN-gama shRNA-3, the nucleotide sequences of the IFN-gama shRNA-1 are shown as follows (the sequences involved in the invention are 5 '-3' if not specifically stated), and the nucleotide sequence of the IFN-gama shRNA-3 is shown as follows.

IFN-gama shRNA-1 (shown as SEQ ID NO: 1):

GCGGCCGCCGCAGAGCCAAATTGTCTCCTTTTCAAGAGAAAGGAGACAATTTGGCTCTGCTTTTT。

IFN-gama shRNA-3 (shown as SEQ ID NO: 2):

GCGGCCGCCCATTCAGATGTAGCGGATAATTTCAAGAGAATTATCCGCTACATCTGAATGTTTTT。

the sequence of IFN-gama shRNA-1 comprises siRNA aiming at human IFN-gama gene: GCAGAGCCAAATTGTCTCCTT are provided. The sequence of IFN-gama shRNA-3 comprises siRNA aiming at human IFN-gama gene: CATTCAGATGTAGCGGATAAT are provided.

A primer for preparing the IFN-gama shRNA-1 which is the shRNA for interfering the expression of the human IFN-gama gene has a nucleotide sequence shown as follows (shown as SEQ ID NO: 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。

a primer for preparing the IFN-gama shRNA-3 which is the shRNA for interfering the expression of the human IFN-gama gene has a nucleotide sequence shown as follows (shown as SEQ ID NO: 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 for preparing the shRNA interfering the expression of the human IFN-gama gene by using the shRNA primer comprises the following steps: the 4 primers (synthesized by Jinzhi Biotechnology, Inc., Suzhou) were quickly centrifuged in dry powder and diluted to 100uM,1 with water^st，2^nd，3^rd，4^th2.5ul of each primer and 10ul of four primers in total are taken, water is added to 50ul, the mixture is boiled for 5-10 minutes, the mixture is placed in a pot to be naturally cooled overnight to room temperature, and through the process, four primers are annealed to form a section of DNA double-stranded short fragment.

A chimeric antigen receptor comprising the following cis-tandem domains: a signal peptide, a tag gene, a single chain antibody, an extracellular hinge region, a transmembrane region, and an intracellular signal region, wherein the single chain antibody comprises a light chain variable region (VL), a heavy chain variable region (VH), and a hinge region connecting them.

The antigen recognized by the single-chain antibody is selected from any one or more of CD19, CD20, CEA, GD2, CD22, CD23, CD30, CD33, CD44v7/8, CD70, VEGFR1, VEGFR2, IL-11R alpha, EGP-2, EGP-40, FBP and GD3 (the antigens are all known in the prior art), preferably CD19, in this case, the amino acid sequence of the light chain variable region of the single-chain antibody is shown below, the amino acid sequence of the heavy chain variable region is shown below, and the amino acid sequence of the hinge region is shown below.

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

KLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT。

amino acid sequence of heavy chain variable region of single chain antibody:

EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS。

amino acid sequence of hinge region of single chain antibody: GGGGSGGGGSGGGGS.

The amino acid sequence of the signal peptide is as follows: MALPVTALLLPLALLLHAARPEQ are provided.

The amino acid sequence of the tag gene is as follows: KLISEEDLTS are provided.

The extracellular hinge region is selected from any one or more of an extracellular hinge region of CD8, an extracellular hinge region of CD28 or an extracellular hinge region of CD4, preferably an extracellular hinge region of CD 8; the amino acid sequence of the extracellular hinge region of CD8 is: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI are provided.

The transmembrane region is selected from any one or more of a transmembrane region of CD8, a transmembrane region of CD28 or a transmembrane region of CD 4; preferably the CD8 transmembrane region; the amino acid sequence of the CD8 transmembrane region is as follows: YIWAPLAGTCGVLLLSLVITLYC are provided.

The intracellular signal region is selected from any one or more than two of CD28, CD134/OX40, CD137/4-1BB, LCK, ICOS, DAP10, CD3zeta and Fc epsilon RI gamma, preferably 4-1BB intracellular signal region and CD3zeta intracellular signal region, or: a CD28 intracellular signaling region and a CD3 ζ intracellular signaling region; the amino acid sequences of the 4-1BB intracellular signal region and the CD3 ζ intracellular signal region are shown below, respectively.

Amino acid sequence of 4-1BB intracellular signal region: SLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE are provided.

Amino acid sequence of intracellular signaling region of CD3 ζ:

LRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRG。

the above amino acid sequences are known in the prior art.

Preferably, the amino acid sequence of the chimeric antigen receptor is shown as follows (shown as SEQ ID NO: 3).

SEQ ID NO:3：

MALPVTALLLPLALLLHAARPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCSLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRG。

A nucleotide fragment for coding the chimeric antigen receptor has a nucleotide sequence shown as the following (shown as SEQ ID NO: 4).

SEQ ID NO:4：

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGCAGAAGCTGATCAGCGAGGAGGACCTGACTAGTGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCAGGATCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCTCCCTAAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGCTAA。

A slow virus expression vector, which comprises the shRNA for interfering the expression of the human IFN-gama gene, a shRNA expression frame and a nucleotide fragment for coding the chimeric antigen receptor (the slow virus expression vector can simultaneously express the chimeric antigen receptor and the IFN-gama shRNA); the shRNA expression cassette comprises a shRNA expression promoter, and the shRNA expression promoter is selected from any one of U6, H1 or 7SK, preferably U6; the nucleotide sequence of the U6 promoter is shown below. The construction method of the lentivirus expression vector is a conventional method.

Nucleotide sequence of U6 promoter:

AAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGGTTTATATATCTTGTGGAAAGGACG。

preferably, the structure of the lentivirus expression vector is pHR-anti CD19CAR-U6-IFN-gama shRNA-1 or pHR-anti CD19CAR-U6-IFN-gama shRNA-3.

A shRNA screening method, which adopts a mode of co-transfection of pHR-IFN-gama-GFP and pHR-anti CD19CAR-U6-IFN-gama shRNA plasmids and utilizes a calcium phosphate transfection method to screen CaCl₂And mixing the two plasmids and water according to a certain proportion, slowly dripping the mixture into the culture supernatant of 293FT cells, harvesting the 293FT cells after 24h, and detecting the expression of IL-6 at the mRNA and protein level by RT-PCR and flow detection methods respectively.

The lentiviral vector pHR-IFN-gama-GFP comprises an IFN-gama gene and a GFP gene (the IFN-gama and the GFP can be expressed simultaneously). The nucleotide sequence of the IFN-gama gene is shown as follows. The nucleotide sequence of the GFP gene is shown below.

Nucleotide sequence of IFN-gama gene:

ATGAAGTACACCAGCTACATCCTGGCCTTTCAGCTGTGCATCGTGCTGGGCAGCCTGGGCTGCTACTGCCAGGACCCCTACGTGAAGGAGGCCGAGAACCTGAAGAAGTACTTCAACGCCGGCCACAGCGACGTGGCCGACAACGGCACCCTGTTCCTCGGCATCCTGAAGAACTGGAAGGAGGAGAGCGACAGGAAGATCATGCAGTCCCAGATCGTGAGCTTCTACTTCAAGCTGTTCAAGAATTTCAAGGACGACCAGAGCATCCAGAAGAGCGTGGAGACCATCAAGGAGGACATGAACGTGAAGTTTTTCAATAGCAACAAGAAGAAGAGGGACGACTTCGAGAAGCTGACCAACTACAGCGTGACCGACCTGAATGTGCAGAGGAAGGCCATCCACGAACTGATCCAGGTGATGGCCGAGCTGAGCCCTGCCGCCAAGACCGGCAAGAGGAAGAGGAGCCAGATGCTGTTCAGGGGCAGGAGGGCCAGCCAG。

nucleotide sequence of GFP gene:

GTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG。

the method for detecting the expression of IFN-gama by the RT-PCR method comprises the following steps: after harvesting 293FT cells, total RNA (MiniBEST Universal RNA, TAKARA, cat:9767) was extracted, reverse transcribed into cDNA (FastKing RT Kit, Tiangen, cat: KR11601), and IFN-gama expression at the mRNA level (primers for the internal reference gene. beta. -actin were used) was determined by fluorescent quantitative PCR using IFN-gama primers.

The IFN-gama primer comprises a forward primer and a reverse primer, and the nucleotide sequence of the primer is as follows: TCAGCTCTGCATCGTTTTGG parts of IFN-gama-F; GTTCCATTATCCGCTACATCTGAA is IFN-gama-R.

The nucleotide sequence of the primer of the reference gene beta-actin is shown as follows: action-F: CGCCCCAGGCACCAGGGC, respectively; action-R: GCTGGGGTGTTGAAGGT are provided.

The method for detecting the expression of IFN-gama by the flow method comprises the following steps: after harvesting 293FT cells, fluorescence expression of GFP was detected.

A lentivirus expression kit comprises the lentivirus expression vector.

A method for preparing recombinant lentivirus by using the lentivirus expression vector comprises the following steps: and (2) mixing the lentivirus expression vector carrying the target gene, a pCMV vector and a pMD.2G vector, transfecting into 293FT cells, replacing with a complete culture medium for culture after 6-8h after transfection, collecting a culture solution after 48h, centrifuging, reserving a supernatant, filtering by using a 0.45-micrometer filter head, and reserving a filtrate, wherein the filtrate is a solution of the recombinant lentivirus.

A CD19-CAR-T cell for interfering IFN-gama expression is a T lymphocyte containing the lentivirus expression vector or a T lymphocyte integrating shRNA for interfering human IFN-gama gene expression and a nucleotide fragment for coding a chimeric antigen receptor (capable of simultaneously expressing the IFN-gama shRNA and the chimeric antigen receptor). The recombinant T cell is prepared by a conventional method.

The application of the CD19-CAR-T cell interfering IL-6 expression in the preparation of drugs for preventing and/or treating and/or adjunctively treating malignant tumors; preferably, the malignancy is selected from acute lymphocytic leukemia and/or chronic lymphocytic leukemia.

The invention has the following beneficial effects:

1. the invention provides a method for inserting shRNA into an expression vector, namely a section of DNA double-stranded short segment is synthesized by a 4-primer annealing method.

2. The invention provides an effective method for detecting and screening shRNA, namely, by means of cotransfection of a vector pHR-anti CD19CAR-U6-IFN-gama shRNA expressing IFN-gama-GFP and a vector pHR-IFN-gama-GFP expressing IFN-gama-GFP fusion protein, through RT-PCR and flow methods, the efficiency of IFN-gama shRNA gene silencing is detected at the mRNA and protein levels respectively, and the shRNA capable of effectively silencing IFN-gama gene expression is successfully screened.

3. Compared with the existing anti CD19CAR-T cell, the invention introduces IFN-gama shRNA, co-expresses the IFN-gama shRNA while expressing CAR, and inhibits the release of IFN-gama from the source through a gene silencing technology, thereby reducing the influence of CRS and improving the safety of CAR-T treatment.

4. Compared with the existing anti CD19CAR-T cell, the pHR-anti CD19CAR-U6-IFN-gama shRNA-T cell co-expresses the IFN-gama shRNA while expressing CAR, and experimental results show that the co-expressed IFN-gama shRNA can reduce the release of factors such as IL-6 and IFN-gama, control the generation of CRS side effects, increase the safety, and simultaneously, does not influence the expression of CAR, does not influence the characteristics of killing and the like, and is unexpected for the technical personnel in the field.

Some common terms involved in the present invention are described below;

the term "shRNA" (shorthairpinRNA), i.e., short hairpin RNA. The shRNA includes two short inverted repeats. The shRNA cloned into the shRNA expression vector comprises two short inverted repeat sequences, the middle of the two short inverted repeat sequences is separated by a stem-loop (loop) sequence to form a hairpin structure, and the hairpin structure is controlled by a pol III promoter. Then 5-6T are connected as the transcription terminator of RNA polymerase III.

The term "U6 promoter" is one of pol III promoters, which is highly conserved in position and base sequence and is capable of efficiently transcribing a downstream structural gene in a cell. Is a promoter commonly used for shRNA.

The term "chimeric antigen receptor" is an artificially engineered receptor that is capable of anchoring a specific molecule (e.g., an antibody) that recognizes a tumor cell surface antigen onto an immune cell (e.g., a T cell) such that the immune cell recognizes the tumor antigen or a viral antigen and kills the tumor cell or a virally infected cell.

The term "single-chain antibody" (scFv) refers to an antibody fragment having the ability to bind to an antigen, which is formed by the amino acid sequence of the variable region of the light chain (VL region) and the amino acid sequence of the variable region of the heavy chain (VH region) of an antibody, which are linked by a hinge.

The term "Linker" or hinge is a polypeptide segment that links different proteins or polypeptides, with the purpose of maintaining the linked proteins or polypeptides in their respective spatial conformations, to maintain the function or activity of the protein or polypeptide.

The term "CD 19" refers to human leukocyte differentiation antigen 19, which has an ID number of 930 in NCBIGeneBank, 2 isoforms (cDNA sequence/protein sequence), NM-001178098.1/NP-001171569.1, NM-001770.5/NP-001761.3, respectively. When referring to the amino acid sequence of CD19, it includes the full length of the CD19 protein or a fragment of CD19 with CD19 function; fusion proteins of the full-length or fragment are also included. Also, it is understood by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and/or additions) may be naturally occurring or artificially introduced into the amino acid sequence of CD19 without affecting its biological function. Also, when a fragment of the protein sequence of CD19 is described, the corresponding sequence fragment in natural or artificial variants thereof is also included.

Drawings

FIG. 1: IFN-gama shRNA and CD19CAR double expression vector structure diagram.

FIG. 2: pHR-IFN-gama-GFP vector pattern diagram.

FIG. 3: pHR-IFN-gama-GFP transfection efficiency flow chart.

FIG. 4: pHR-anti CD19CAR-U6-IFN-gama shRNA transfection efficiency flow chart.

FIG. 5: detecting an IFN-gama expression pattern by an RT-PCR method after cotransfection.

FIG. 6: detecting IFN-gama expression diagram by a co-transfection post-flow method, wherein the left diagram is a transfection efficiency flow diagram, and the right diagram is a transfection efficiency bar diagram.

FIG. 7: pHR-anti CD19CAR-U6-IFN-gama shRNA-T cell positive rate flow detection map

FIG. 8: K562-CD19 monoclonal flow assay.

FIG. 9: pHR-anti CD19CAR-U6-IFN-gama shRNA-T cell specific killing CD19 positive tumor cell flow chart.

FIG. 10: statistical map of specific killing of CD19 positive tumor cells by pHR-anti CD19CAR-U6-IFN-gama shRNA-T cells.

FIG. 11: and (3) detecting the content of the pHR-anti CD19CAR-U6-IFN-gama shRNA-T cell factor by an RT-PCR method.

FIG. 12: and (3) detecting the content of the pHR-anti CD19CAR-U6-IFN-gama shRNA-T cell factor by ELISA.

Detailed Description

The present invention will be further described with reference to the following examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruker et al, Huang Petang et al) or according to the product instructions.

The instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal manner unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like described in the following examples are conventional experimental methods, detection methods, and the like in the prior art.

Example 1: synthesis of IFN-gama shRNA capable of being cloned into vector

The sequences of IFN-gama shRNA were searched on the Sigma-Aldrich China network, and 4 of them (gama-1, gama-2, gama-3, and gama-4) were selected, and their nucleotide sequences were as follows.

gama-1：CCGGGCAGAGCCAAATTGTCTCCTTCTCGAGAAGGAGACAATTTGGCTCTGCTTTTTG。

gama-2：CCGGTATAAGTGAAGTGATACTATCCTCGAGGATAGTATCACTTCACTTATATTTTTG。

gama-3：CCGGCATTCAGATGTAGCGGATAATCTCGAGATTATCCGCTACATCTGAATGTTTTTG。

gama-4：CCGGGGTTGTCCTGCCTGCAATATTCTCGAGAATATTGCAGGCAGGACAACCTTTTTG。

Wherein the underlined sequences are the sequences of the siRNAs in each.

The invention adopts a traditional method of cloning into a vector to insert two DNA oligonucleotides coding IFN-gamasiRNA sequences into the vector. The DNA oligonucleotide contains a sense siRNA sequence 21 nucleotides long, is linked to its reverse complementary antisense siRNA sequence through a 9-nucleotide spacer sequence TTCAAGAGA that Ambion scientists have successfully used, adds 5T's as a termination signal at the 3' end of its oligonucleotide, and adds the required restriction enzyme site NotI for cloning into a vector during design.

The invention adopts a 4-primer annealing synthesis method, and respectively designs 4 primers for each shRNA sequence, wherein the IFN-gama shRNA-1 primer sequence is shown as SEQ ID NO. 5-8. The sequence of the IFN-gama shRNA-2 primer is shown below. The sequence of the IFN-gama shRNA-3 primer is shown as SEQ ID NO 9-12. The sequence of the IFN-gama shRNA-4 primer is shown below.

The sequence of the IFN-gama shRNA-2 primer is 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。

the sequence of the IFN-gama shRNA-4 primer is 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 each shRNA are synthesized by Suzhou Jinweizhi Biotechnology limited, and then dry powder of the synthesized 4 primers is quickly centrifuged and diluted to 100uM,1 by adding water^st，2^nd，3^rd，4^th2.5ul of each of the four water-soluble granules and 10ul of the four water-soluble granules are taken, water is supplemented to 50ul, and the mixture is boiled for 5-10 ulAnd (3) placing the mixture in a pot, naturally cooling the mixture overnight to room temperature, and completing annealing of the four primers through the process to form a DNA double-stranded short fragment.

The sequence of the DNA double-chain short segment formed by the process, IFN-gama shRNA-1, is shown as SEQ ID NO. 1; IFN-gama shRNA-2, the sequence of which is shown as follows; IFN-gama shRNA-3, the sequence of which is shown in SEQ ID NO 2; IFN-gama shRNA-4, the sequence of which is shown below.

IFN-gama shRNA-2：

GCGGCCGCCTATAAGTGAAGTGATACTATCTTCAAGAGAGATAGTATCACTTCACTTATATTTTT。

IFN-gama shRNA-4：

GCGGCCGCCGGTTGTCCTGCCTGCAATATTTTCAAGAGAAATATTGCAGGCAGGACAACCTTTTT。

Example 2: construction of IFN-gama shRNA and CD19CAR double expression vector

As shown in figure 1, CD8 transmembrane signal peptide, myc label, anti-CD19Scfv, CD8 transmembrane region, 4-1BB costimulatory signal region, CD3ZetaTCR activation region, U6 promoter and IFN-gama shRNA are cloned into a lentiviral backbone plasmid pHR in sequence to obtain pHR-anti CD19CAR-U6-IFN-gama shRNA plasmid (four IFN-gama shRNAs are involved in the experiment, so four plasmids are obtained, respectively as follows).

The nucleotide sequence of CD8amyc-anti-CD19Scfv-CD8TM-4-1BB-CD3Zeta-U6-IFN-gama shRNA-1 is shown below (as shown in SEQ ID NO: 13).

SEQ ID NO:13：

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGCAGAAGCTGATCAGCGAGGAGGACCTGACTAGTGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCAGGATCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCTCCCTAAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGCTAAAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGGTTTATATATCTTGTGGAAAGGACGGCGGCCGCCGCAGAGCCAAATTGTCTCCTTTTCAAGAGAAAGGAGACAATTTGGCTCTGCTTTTT。

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

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGCAGAAGCTGATCAGCGAGGAGGACCTGACTAGTGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCAGGATCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCTCCCTAAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGCTAAAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGGTTTATATATCTTGTGGAAAGGACGGCGGCCGCCTATAAGTGAAGTGATACTATCTTCAAGAGAGATAGTATCACTTCACTTATATTTTT。

the nucleotide sequence of CD8amyc-anti-CD19Scfv-CD8TM-4-1BB-CD3Zeta-U6-IFN-gama shRNA-3 is shown below (as shown in SEQ ID NO: 14).

SEQ ID NO:14：

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGCAGAAGCTGATCAGCGAGGAGGACCTGACTAGTGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCAGGATCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCTCCCTAAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGCTAAAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGGTTTATATATCTTGTGGAAAGGACGGCGGCCGCCCATTCAGATGTAGCGGATAATTTCAAGAGAATTATCCGCTACATCTGAATGTTTTT。

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

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGCAGAAGCTGATCAGCGAGGAGGACCTGACTAGTGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCAGGATCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCTCCCTAAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGCTAAAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGGTTTATATATCTTGTGGAAAGGACGGCGGCCGCCGGTTGTCCTGCCTGCAATATTTTCAAGAGAAATATTGCAGGCAGGACAACCTTTTT。

example 3: preparation of pHR-IFN-gama-GFP selection plasmid

As shown in FIG. 2, IFN-gama and GFP were cloned into the lentiviral vector backbone plasmid pHR in sequence to obtain the pHR-IFN-gama-GFP plasmid. GFP in the pHR-IFN-gama-GFP is a reporter gene and is used for detecting the gene silencing efficiency of IFN-gama.

The nucleotide sequence of the IFN-gama-GFP fusion protein is shown below:

ATGAAGTACACCAGCTACATCCTGGCCTTTCAGCTGTGCATCGTGCTGGGCAGCCTGGGCTGCTACTGCCAGGACCCCTACGTGAAGGAGGCCGAGAACCTGAAGAAGTACTTCAACGCCGGCCACAGCGACGTGGCCGACAACGGCACCCTGTTCCTCGGCATCCTGAAGAACTGGAAGGAGGAGAGCGACAGGAAGATCATGCAGTCCCAGATCGTGAGCTTCTACTTCAAGCTGTTCAAGAATTTCAAGGACGACCAGAGCATCCAGAAGAGCGTGGAGACCATCAAGGAGGACATGAACGTGAAGTTTTTCAATAGCAACAAGAAGAAGAGGGACGACTTCGAGAAGCTGACCAACTACAGCGTGACCGACCTGAATGTGCAGAGGAAGGCCATCCACGAACTGATCCAGGTGATGGCCGAGCTGAGCCCTGCCGCCAAGACCGGCAAGAGGAAGAGGAGCCAGATGCTGTTCAGGGGCAGGAGGGCCAGCCAGGGATCCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA。

example 4: screening of pHR-anti CD19CAR-U6-IFN-gama shRNA lentiviral vector for effectively silencing IFN-gama expression

(1) Transfection of pHR-IFN-gama-GFP selection plasmid

Using calcium phosphate transfection method to add CaCl₂Mixing the plasmid and water according to a certain proportion, slowly dripping into the culture supernatant of 293FT cells, detecting 293FT by flow after 24h, and detecting the fluorescence expression of IFN-gama-GFP fusion protein. As shown in FIG. 3, the transfection efficiency of pHR-IFN-gama-GFP was 48.3%, which enabled successful transfection.

(2) Transfection of pHR-anti CD19 CAR-U6-IFN-gamamshRNA plasmid

Using calcium phosphate transfection method to add CaCl₂The plasmid and water were mixed at a certain ratio, and slowly dropped into the culture supernatant of 293FT cells, after 24 hours, 293FT cells were taken, and the CAR-positive cell ratio was detected by flow cytometry (Fluorescein (FITC) Affinipure Goat Anti-Mouse IgG, F (ab')2fragment specific, jackson immunoresearch, cat: 115-095-006). As shown in FIG. 4, the transfection efficiencies of the 4 pHR-anti CD19CAR-U6-IFN-gama shRNA plasmids were 19.8%, 25%, 20.6%, and 15.7%, respectively, as compared with the negative control group. All were successfully transfected.

(3) Screening IFN-gama shRNA by RT-PCR method

CaCl is co-transfected by pHR-IFN-gama-GFP and pHR-anti CD19CAR-U6-IFN-gama shRNA plasmid through calcium phosphate transfection method₂The 2 plasmids and water were mixed in a certain ratio, slowly added dropwise to the culture supernatant of 293FT cells, after 24h 293FT cells were harvested, total RNA was extracted (MiniBEST Universal RNA, TAKARA, cat:9767), reverse-transcribed into cDNA (FastKing RT Kit, Tiangen, cat: KR11601), and IFN-gama primers (as shown above) were used to determine IFN-gama expression at the mRNA level by the fluorescent quantitative PCR method (primers for. beta. -actin, as shown above), which is an internal reference gene.

As shown in FIG. 5, the level of IFN-gama in 293FT after cotransfection with pHR-anti CD19CAR-U6-IFN-gama shRNA-1 plasmid, pHR-anti CD19CAR-U6-IFN-gama shRNA-3 plasmid and pHR-IFN-gama-GFP was significantly reduced compared to 293FT transfected with pHR-IFN-gama-GFP alone. Statistical analysis showed significant differences in IFN-gama levels. The 2 pHR-anti CD19 CAR-U6-IFN-gamamshRNA can effectively reduce the expression of IFN-gama in pHR-IFN-gama-GFP at the mRNA level, and play a role in gene silencing.

(4) Screening of IFN-gama shRNA by flow method

CaCl is co-transfected by pHR-IFN-gama-GFP and pHR-anti CD19CAR-U6-IFN-gama shRNA plasmid through calcium phosphate transfection method₂And mixing the 2 plasmids and water according to a certain proportion, slowly dripping the mixture into the culture supernatant of 293FT cells, detecting 293FT by a flow method after 24 hours, and detecting the fluorescence expression of the IFN-gama-GFP fusion protein. As shown in FIG. 6, compared with the transfection efficiency of pHR-IFN-gama-GFP alone of 17%, the transfection efficiency of GFP after cotransfection of pHR-anti CD19CAR-U6-IFN-gama shRNA-1 plasmid, pHR-anti CD19CAR-U6-IFN-gama shRNA-3 plasmid and pHR-IFN-gama-GFP was significantly reduced to 7.55%, 4.16%. The 2 pHR-anti CD19CAR-U6-IFN-gama shRNAs are shown to be capable of effectively reducing the protein expression of IFN-gama in pHR-IFN-gama-GFP and playing a role in gene silencing.

Example 5: lentivirus packaging and concentration

And (2) mixing the target gene-carrying lentivirus expression vector (pHR-anti CD19CAR-U6-IFN-gama shRNA-1 plasmid and pHR-anti CD19CAR-U6-IFN-gama shRNA-3 plasmid), the pCMV vector and the pMD.2G vector, transfecting into 293FT cells, culturing in a complete culture medium for 6-8h after transfection, collecting a culture solution after 48h, centrifuging, retaining a supernatant, filtering by using a 0.45 mu m filter, and retaining a filtrate, wherein the filtrate is a recombinant lentivirus solution.

Lenti-XTMConcentor (takara, cat:631231) instructions for lentivirus concentration.

Example 6: preparation of T cells of IFN-gama shRNA and CD19CAR double expression vector

50mL of fresh blood was taken, and mononuclear cells were separated by density gradient centrifugation using a lymphocyte separation medium (tertiary amine). Resuspending the mononuclear cells into X-VIVO15 medium (Lonza), adding CD3 monoclonal antibody and CD28 monoclonal antibody to activate T lymphocytes, and 5% CO at 37 ℃₂The culture was carried out for 48 hours.

Take 2X 10⁶The 2 lentiviruses concentrated in example 5 were added at the MOI of 5, together with IL-2 and polybrene, and mixed well at 37 ℃ with 5% CO₂Culturing for 6-8 hr, centrifuging at 300g for 5min, and changing the culture medium to fresh X-VIVO15 (containing IL-2).

Adding fresh X-VIVO15 culture medium (containing IL-2) every 2-3 days, and maintaining cell density at 1 × 10⁶about/mL, 10-12 days of amplification.

Collecting T cells cultured for 48h after virus infection and T cells not infected with virus, each 5 × 10⁵Each sample was incubated with Fluorescein AffiniP Goat Anti-Mouse IgG, F (ab')2fragmentspecific1ul for 15min at room temperature in the absence of light, centrifuged, and the pellet resuspended in 200. mu.L PBS and then tested on the machine (Millipore guava easy CyteHT). As shown in FIG. 7, the ratio of pHR-anti CD19CAR-U6-IFN-gama shRNA-T cells in 2 types of T cells cultured for 48 hours after virus infection was 84.9% and 86.6%, and the infection efficiency was very high. The 2T cells prepared in this example were designated pHR-anti CD19CAR-U6-IFN-gama shRNA-1-T and pHR-anti CD19CAR-U6-IFN-gama shRNA-3-T as cells used in the examples that follow.

Example 7: in vitro killing test of pHR-anti CD19CAR-U6-IFN-gama shRNA-T cells on tumor cell lines

1. Construction of K562 cells (target cells) stably expressing CD19 Gene

(1) Synthesizing DNA coding sequences of the CD19 extracellular region and the transmembrane region, inserting the DNA coding sequences into a pHR vector, and constructing the obtained vector which is named as pHR-CD 19.

The nucleotide coding sequence of the extracellular region and transmembrane region of CD19 is shown as follows:

ATGCCACCTCCTCGCCTCCTCTTCTTCCTCCTCTTCCTCACCCCCATGGAAGTCAGGCCCGAGGAACCTCTAGTGGTGAAGGTGGAAGAGGGAGATAACGCTGTGCTGCAGTGCCTCAAGGGGACCTCAGATGGCCCCACTCAGCAGCTGACCTGGTCTCGGGAGTCCCCGCTTAAACCCTTCTTAAAACTCAGCCTGGGGCTGCCAGGCCTGGGAATCCACATGAGGCCCCTGGCCATCTGGCTTTTCATCTTCAACGTCTCTCAACAGATGGGGGGCTTCTACCTGTGCCAGCCGGGGCCCCCCTCTGAGAAGGCCTGGCAGCCTGGCTGGACAGTCAATGTGGAGGGCAGCGGGGAGCTGTTCCGGTGGAATGTTTCGGACCTAGGTGGCCTGGGCTGTGGCCTGAAGAACAGGTCCTCAGAGGGCCCCAGCTCCCCTTCCGGGAAGCTCATGAGCCCCAAGCTGTATGTGTGGGCCAAAGACCGCCCTGAGATCTGGGAGGGAGAGCCTCCGTGTCTCCCACCGAGGGACAGCCTGAACCAGAGCCTCAGCCAGGACCTCACCATGGCCCCTGGCTCCACACTCTGGCTGTCCTGTGGGGTACCCCCTGACTCTGTGTCCAGGGGCCCCCTCTCCTGGACCCATGTGCACCCCAAGGGGCCTAAGTCATTGCTGAGCCTAGAGCTGAAGGACGATCGCCCGGCCAGAGATATGTGGGTAATGGAGACGGGTCTGTTGTTGCCCCGGGCCACAGCTCAAGACGCTGGAAAGTATTATTGTCACCGTGGCAACCTGACCATGTCATTCCACCTGGAGATCACTGCTCGGCCAGTACTATGGCACTGGCTGCTGAGGACTGGTGGCTGGAAGGTCTCAGCTGTGACTTTGGCTTATCTGATCTTCTGCCTGTGTTCCCTTGTGGGCATTCTTCATCTTCAAAGAGCCCTGGTCCTGAGG。

(2) lentiviral packaging and concentration

And mixing the pHR-CD19 vector, the pCMV vector and the pMD.2G vector, transfecting into 293FT cells, replacing with a complete culture medium for culture after 6-8h after transfection, collecting a culture solution after 48h, centrifuging, reserving a supernatant, filtering by using a 0.45 mu m filter, and reserving a filtrate, wherein the filtrate is a solution of the recombinant lentivirus.

(3) Preparation of K562 cells expressing CD19

1640(Gibco) Medium resuspended K562 cells to 1X10⁶Perml, lentivirus (MOI5-10), 5% CO at 37 deg.C₂Culturing in an incubator for 6-8h, and centrifuging to change the culture solution into fresh K562 cell proliferation culture solution. Adding fresh K562 fine powder every 2-3 daysCell proliferation culture medium for maintaining cell density at 0.5 × 10⁶about/mL. After 5 generations, monoclonal screening was performed using limiting dilution. After the monoclonal cells grow to a certain amount, the cells are screened by a flow cytometer (anti-CD19PE, biolegend cat: 302254), and the cell clone with high expression level and high purity, namely the K562 cell stably expressing CD19, is named as K562-CD19 (shown in FIG. 8) and is used as the target cell in the embodiment.

In vitro validation of the killing function of pHR-anti CD19CAR-U6-IFN-gama shRNA-T

Inoculating K562 cells and constructed K562-CD19 cells into a 96-well plate according to the ratio of 1:1, and inoculating pHR-anti CD19CAR-U6-IFN-gama shRNA-1-T, pHR-anti CD19CAR-U6-IFN-gama shRNA-3-T, antiCD19CAR-T cells and T cells which are cultured for 9 days according to the ratio of E: T ═ 1:1 and 3:1 respectively. 5% CO at 37 ℃₂After 24 hours of incubation, 100. mu.L of supernatant was aspirated from each well and stored at-80 ℃ for further use. The remaining cells were added to each well of PE anti-humanCD19(Biolegend) and FITCCanti-humanCD 3(Biolegend), incubated for 15min at room temperature in the absence of light, centrifuged, and the pellet resuspended in 200. mu.L PBS and then tested on the machine (Millipore guava easy CyteHT). As shown in FIG. 9 and FIG. 10, the pHR-anti CD19CAR-U6-IFN-gama shRNA-T cells in group 2 specifically kill K562-CD19 cells, the average values of specific killing efficiencies were 56.7%, 100%, 63.3% and 100% when the effective target ratio was 1:1 and 3:1, respectively, and the killing efficiency increased with the increase of the effective target ratio. Statistical analysis showed that there was a significant difference in specific killing efficiency between the pHR-anti cd19CAR-U6-IFN-gama shRNA-T cell and T cell groups in group 2, whereas there was no significant difference in specific killing efficiency between the pHR-anti cd19CAR-U6-IFN-gama shRNA-T and anti cd19CAR-T cell groups.

Example 8: detection of content of pHR-anti CD19 CAR-U6-IFN-gamamshRNA-T cell cytokine

1) RT-PCR method for detecting pHR-anti CD19CAR-U6-IFN-gama shRNA-T cytokine release

pHR-anti CD19 CAR-U6-IFN-gamamshRNA-1-T, pHR-anti CD19CAR-U6-IFN-gama shRNA-3-T and anti CD19CAR-T cells cultured for 9 days are taken, total RNA (MiniBEST Universal RNA, TAKARA, cat:9767) is extracted and is reversely transcribed into cDNA (FastKing RTKit, Tiangen, cat: KR11601), and the expression of IL-6, IFN-gama and IL-2 at the mRNA level is determined by a fluorescent quantitative PCR method by using a primer (shown below) for IL-6, a primer for IFN-gama (shown above), a primer for IL-2 (shown below) and a primer for housekeeping gene beta-action (shown above).

The primer sequence of IL-6 in RT-PCR is shown below: IL-6-F AGACAGCCACTCACCTCTTCAG; IL-6-R: TTCTGCCAGTGCCTCTTTGCTG.

The primer sequence for IL-2 in RT-PCR is shown below: AACTCACCAGGATGCTCACATTTA for IL-2-F; IL-2-R: TCCCTGGGTCTTAAGTGAAAGTTT.

As shown in FIG. 11, pHR-anti CD19CAR-U6-IFN-gama shRNA-T cells can significantly reduce the release of IFN-gama, IL-6 and IL-2 at mRNA level and play a role in gene silencing IL-6, compared with CD19CAR-T cells.

2) ELISA method for detecting content of secreted factors in culture supernatant of pHR-anti CD19CAR-U6-IFN-gama shRNA-T cells

The supernatants from the E.T. ═ 3:1 in vitro killing assay of example 7, which were the T cell group, anti CD19CAR-T cell group, pHR-anti CD19CAR-U6-IFN-gama shRNA-1-T cell group, pHR-anti CD19CAR-U6-IFN-gama shRNA-3-T cell group, and the negative control group of tumor cells were collected as Humani L-2ELISAMAX^TMDeluxe(Biolegend，431804)、Human IFN-gama ELISAMAX^TMDeluxe(Biolegend，430104)、HumanIL-6ELISAMAX^TMDeluxe (Biolegend, 430504) kit instructions for the assay of IFN-gama, IL-6 and IL-2 release.

The results are shown in FIG. 12, and pHR-anti CD19CAR-U6-IFN-gama shRNA-T cells can significantly reduce the release of IFN-gama compared with CD19CAR-T cells. Reducing IL-6 release. Without significant effect on IL-2 release.

The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims. All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each such publication, patent, or patent application were specifically and individually indicated to be incorporated by reference.

Sequence listing

<110> Qilu cell therapy engineering Co., Ltd, Shandong province

<120> IFN-gamma expression interfering CD19-CAR-T cell and application thereof

<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. shRNA interfering human IFN-gama gene expression is characterized in that: is IFN-gama shRNA-1 or IFN-gama shRNA-3,

the nucleotide sequence of the IFN-gama shRNA-1 is shown as SEQ ID NO: 1:

GCGGCCGCCGCAGAGCCAAATTGTCTCCTTTTCAAGAGAAAGGAGACAATTTGGCTCTGCTTTTT；

the nucleotide sequence of the IFN-gama shRNA-3 is shown as SEQ ID NO: 2:

GCGGCCGCCCATTCAGATGTAGCGGATAATTTCAAGAGAATTATCCGCTACATCTGAATGTTTTT。

2. the primer for preparing shRNA interfering with human IFN-gama gene expression according to claim 1, which is characterized in that:

the nucleotide sequence of the primer for preparing the IFN-gama shRNA-1 is shown as follows:

hIFN-gama-shRNA1-1st：GGCCGCCGCAGAGCCAAATTGTCTCCTTTTCAAGAGA；

hIFN-gama-shRNA1-2nd：AAGGAGACAATTTGGCTCTGCTTTTTGC；

hIFN-gama-shRNA1-3rd：AAGGAGACAATTTGGCTCTGCGGC；

hIFN-gama-shRNA1-4th：GGCCGCAAAAAGCAGAGCCAAATTGTCTCCTTTCTCTTGAA；

the nucleotide sequence of the primer for preparing the IFN-gama shRNA-3 is shown as follows:

hIFN-gama-shRNA3-1st：GGCCGCCCATTCAGATGTAGCGGATAATTTCAAGAGA；

hIFN-gama-shRNA3-2nd：ATTATCCGCTACATCTGAATGTTTTTGC；

hIFN-gama-shRNA3-3rd：ATTATCCGCTACATCTGAATGGGC；

hIFN-gama-shRNA3-4th：GGCCGCAAAAACATTCAGATGTAGCGGATAATTCTCTTGAA。

3. a lentiviral expression vector, comprising: comprising shRNA interfering with the expression of human IFN-gama gene according to claim 1 and a nucleotide fragment encoding a chimeric antigen receptor; the nucleotide sequence of the nucleotide fragment for coding the chimeric antigen receptor is shown as SEQ ID NO. 4.

4. The lentiviral expression vector of claim 3, wherein: the structure of the lentivirus expression vector is pHR-anti CD19CAR-U6-IFN-gama shRNA-1, and the nucleotide sequence of the lentivirus expression vector is shown as SEQ ID NO. 13;

or: pHR-anti CD19CAR-U6-IFN-gama shRNA-3, and the nucleotide sequence is shown as SEQ ID NO: 14.

5. A lentiviral expression kit, comprising: comprising the lentiviral expression vector of claim 3 or 4.

6. A recombinant lentivirus, characterized in that: the preparation method comprises the following steps: the lentivirus expression vector, the pCMV vector and the pMD.2G vector of claim 3 or 4 are mixed and transfected into 293FT cells, complete culture medium culture is replaced after 6-8h after transfection, culture solution is collected after 48h, supernatant is reserved after centrifugation and filtered by a 0.45-micron filter head, and filtrate is reserved, namely the solution of recombinant lentivirus.

7. A CD19-CAR-T cell that interferes with human IFN-gama gene expression, comprising: a T lymphocyte comprising the lentiviral expression vector of claim 3 or 4, or a T lymphocyte having integrated into its chromosome the shRNA interfering with the expression of the human IFN-gama gene of claim 1 and a nucleotide fragment encoding a chimeric antigen receptor; the nucleotide sequence of the nucleotide fragment for coding the chimeric antigen receptor is shown as SEQ ID NO. 4.

8. Use of the lentiviral expression vector of claim 3 or 4, in the preparation of a medicament for the prevention and/or treatment of a malignant tumor, wherein: the malignant tumor is selected from acute lymphocytic leukemia and/or chronic lymphocytic leukemia.