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Human-derived CD-19 chimeric antigen receptor T lymphocyte vector and application thereof

A chimeric antigen receptor, CD-19 technology, applied in the application, animal cells, vertebrate cells, etc., can solve the problems of reducing the recurrence rate and the recurrence rate of patients from time to time, so as to increase the transformation efficiency and breakthrough inhibition Restriction of the microenvironment, optimization of the effect of the carrier

Inactive Publication Date: 2018-11-16
山东省医学科学院附属医院 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

2) Break through the tumor microenvironment and reduce the recurrence rate: In the current trials, although the response rate and complete remission rate are maintained at a high level, and the effect of removing residual lesions is better, the recurrence rate of patients still occurs from time to time

Method used

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  • Human-derived CD-19 chimeric antigen receptor T lymphocyte vector and application thereof
  • Human-derived CD-19 chimeric antigen receptor T lymphocyte vector and application thereof
  • Human-derived CD-19 chimeric antigen receptor T lymphocyte vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Construction of anti-Nucleosome-FNEDB-CD19 CAR gene

[0034](1) Design of anti-Nucleosome-FNEDB-CD19 CAR gene: the sequence consists of the ScFv gene of CD19 (SEQ ID NO 1), the ScFv gene of FNEDB (SEQ ID NO 3), and the nucleosome (SEQ ID NO 2) ScFv base, cytoplasmic region 4-1BB (ENST00000374478), CD3zeta gene (ENST00000392122), and CAR structural gene CD8 sequence (ENST00000283635) are connected. The two gene sequences are connected by 40-50NT nucleotide sequences, and HindⅢ and EcoRI restriction enzyme cutting sites were respectively introduced at both ends of the sequence.

[0035] (2) The designed anti-Nucleosome-FNEDB-CD19 CAR gene was synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd., and the designed gene was on the plasmid HF392 provided by the company.

[0036] (3) According to the gene sequence in the genebank library, primers for the anti-Nucleosome-FNEDB-CD19 CAR gene were designed, the upstream primer P1 (5′-CTTAAGCCTATGCAGGTCCAACT...

Embodiment 2

[0038] Construction of anti-Nucleosome-FNEDB-CD19 CAR lentiviral expression vector

[0039] (1) Construction of anti-Nucleosome-FNEDB-CD19 CAR FNEDB lentiviral expression vector:

[0040] Cloning the anti-Nucleosome-FNEDB-CD19 CAR gene fragment into the lentiviral vector pc DNA TM For 3.1(+), the selected insertion site is Nhe I / Bam HI. 293T cells were cultured with RPMI1640+10% FBS. After the cells were 90% confluent, use lipo2000 to mix the transfection plasmids and transfect 293T cells. The transfection method is operated according to the standard of the Invitrogen manufacturer. The plasmids include: expression plasmids, pMDLg / pRRE, pRSV-Rev, and pMD2.0G, and the plasmids are mixed according to the molar ratio. Harvest the lentiviral supernatant after 24-48 hours. Then add Clontech's Lenti-X concentrator, 1500g after mixing, 4°C. Centrifuge for 45 minutes, remove the supernatant, and obtain the lentiviral pellet, that is, the anti-Nucleosome-FNEDB-CD19 CAR lentiviral e...

Embodiment 3

[0044] Cell killing experiment of anti-Nucleosome-FNEDB-CD19 CAR lentiviral expression vector:

[0045] (1) Preparation of CAR-T cells:

[0046] Collect 50-60ml of venous blood from the patient; centrifuge at 2000rpm for 10min, draw plasma, and bathe in water at 56°C for 30min; dilute the blood sediment by one time with PBS, add 20ml of lymphocyte layering solution, centrifuge at 2000rpm for 20min; collect buffy coat cells , washed 3 times with PBS, and then counted cells using trypan blue; resuspended cells in GT-551 culture medium, selected the culture flask coated with anti-CD3 monoclonal antibody, transferred the cells, and added 500U / ml rIL-2, placed in 37°C, 5% CO 2 cultured in an incubator. On the second day of culturing the cells, add 200 μL of the lentiviral vector obtained in Example 2 of the present invention and 2 μL of 1×10 -6 U protamine, after 12 hours of co-cultivation, change the medium, and add 500U / ml rIL-2; on the 4th day of culture, transfer to a cultur...

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Abstract

The invention discloses a human-derived CD-19 chimeric antigen receptor T lymphocyte vector which is characterized by comprising a plasmid vector for coding single-chain variable region genes of CD19monoclonal antibodies, nucleosomal histone monoclonal antibodies and FNEDB monoclonal antibodies. The vector is a lentiviral vector. The invention further discloses application of the human-derived CD-19 chimeric antigen receptor T lymphocyte vector in preparation of CATR cells. The vector is used for transduction of T lymphocytes, natural killer cells and mononuclear cells, and is applied to treating tumors, preferably treating reoccurrence in malignant lymphoma drugs as well as neoplastic hematologic disorders and lymphomas which are refractory and unsuccessful in bone marrow transplantationmatching. The human-derived CD-19 chimeric antigen receptor T lymphocyte vector disclosed by the invention has the beneficial effects that the vector constructed in the invention can achieve the effects of enhancing the targeted CD19 antigen transgenic T lymphocyte specificity, overcoming the tumor microenvironment inhibited immune cellular function and controlling cytokine storm in clinic treatment.

Description

technical field [0001] The present invention relates to the field of chimeric antigen receptors. Specifically, the present invention relates to a humanized CD-19 chimeric antigen receptor T lymphocyte carrier and its application. Background technique [0002] Hematological malignancies and lymphomas are relatively common among cancers. The incidence rates among Chinese people are 2.76 per 100,000 and 6.91 per 100,000, respectively. It is roughly estimated that the annual incidence is about 37,000 and 93,000. Most patients cannot be cured by traditional chemotherapy and radiotherapy. Bone marrow / peripheral blood stem cell transplantation is considered to be an effective way to cure hematological tumors. Less than 10%, and the bone marrow matching is difficult, the cost is high, and immunosuppressants need to be taken all year round after transplantation. [0003] In recent years, CAR-T cells have made breakthroughs in the treatment of blood tumors and lymphomas, bringing ho...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10A61K35/17A61P35/00
CPCA61K35/17A61P35/00C12N5/0636C12N5/0646C12N15/86C12N2510/00C12N2740/15043
Inventor 徐忠法杨美家尹鸿萍张志杰刘永军王岩甄亚男
Owner 山东省医学科学院附属医院
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