Targeted CD19 humanized scFv chimeric antigen recepter T cell and preparation method and application

A chimeric antigen receptor, humanized technology, applied in the fields of genetic engineering and cell biology, can solve the problems of inability to activate and persist CAR-T cells, improve survival time, enhance therapeutic effect, reduce immunogen sexual effect

Inactive Publication Date: 2020-01-31
山东省齐鲁细胞治疗工程技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, the CD19 antigen receptor used in CAR-T technology uses mouse genes, such as the mouse monoclonal antibody FMC63, but such mouse gene fragments may cause human anti-mouse antibodies (HAMA, Human anti-mouse antibodies)
Studies have shown that the immunogenicity of murine CAR sequences may cause CAR-T cells to fail to activate and persist

Method used

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  • Targeted CD19 humanized scFv chimeric antigen recepter T cell and preparation method and application
  • Targeted CD19 humanized scFv chimeric antigen recepter T cell and preparation method and application
  • Targeted CD19 humanized scFv chimeric antigen recepter T cell and preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: Plasmid vector construction

[0041] After the anti-CD19 scFv fragment is humanized, the anti-hCD19 scFv is obtained, the Mlu I restriction site and the CD8 transmembrane signal peptide are inserted before the fragment, and the BamH I restriction site is inserted after the fragment (such as figure 1 Shown), handed over to the gene company (Jinweizhi) to synthesize. The pUC57-Amp plasmid containing Mlu I+CD8a-hCD19scFv+ BamH I was synthesized by a gene company and was digested with Mlu I and BamH I. The digestion effect was identified by agarose gel electrophoresis, and the modified gene fragment was obtained by gel recovery, such as figure 1 shown. At the same time, the existing lentiviral backbone plasmid pHR (see patent CN 108753774 A) containing the CD8 transmembrane region, 4-1BB costimulatory signal region and CD3 Zeta TCR activation region was double digested with Mlu I and BamH I, and agar Gel recovery of long fragments after gel electrophoresis i...

Embodiment 2

[0042] Embodiment 2: lentivirus preparation and titer detection:

[0043] The lentiviral expression vector carrying the target gene, the pCMV vector and the pMD.2G vector were mixed and transfected into 293FT cells (purchased from ATCC). After 6h-8h after transfection, they were replaced with complete medium for culture, and collected after 48h For the culture medium, keep the supernatant after centrifugation and filter the supernatant with a 0.45 μm filter, keep the filtrate, and the filtrate is the solution of the recombinant lentivirus. Lentivirus concentration was carried out according to the instructions of Lenti-XTM Concentrator (Takara, cat: 631231).

[0044] The titer of the virus was determined by the gradient dilution method, and 1 × 10 5 For each K562 cell, take the concentrated virus and dilute it 10 times, add 1 μL, 3 μL, 10 μL, 30 μL virus respectively, and store at 37°C in 5% CO 2 After culturing for 48 hours, 200 μL of cell liquid was taken from each well for...

Embodiment 3

[0045] Example 3 Preparation of CAR-T cells and detection of CAR positive rate

[0046] 1. Preparation of anti-hCD19CAR-T

[0047] Take 50mL of fresh blood and conduct density gradient centrifugation with lymphocyte separation medium (Tianjin Haoyang) to separate mononuclear cells. Divide mononuclear cells into 1-2 x 10 6 / mL resuspended CTS TM AIM V TM SFM medium (GIBCO, Cat. No. A3021002). At the same time, 5% ICS (GIBCO, A2596101), CD3 monoclonal antibody (Ebioscience) 50ng / mL and CD28 monoclonal antibody (Ebioscience) 50ng / mL were added to activate T lymphocytes, and cultured at 37°C with 5% CO2 for 48 hours.

[0048] After 2 days of culture, collect the cells and resuspend the cells to 1x10 6 / mL, according to MOI=5, add the two kinds of concentrated lentiviruses in Example 2, and at the same time add a final concentration of 500U / mL IL-2 (Quangang) and 4ug / mL polybrene (Sigma), mix well, 37 ° C 5% CO2 Cultivate for 6-8 hours, centrifuge at 300g for 5 minutes to c...

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Abstract

The invention belongs to the technical fields of genetic engineering andcell biology, and particularly relates to a targeted CD19 humanized scFv chimeric antigen recepter T cell and a preparation method and application. The targeted CD19 humanized scFv chimeric antigen recepter T cell specially lies in that a lentivirus expression vector containing a targeted CD19 humanized scFv chimeric antigen recepter (anti-hCD19CAR) gene structure is constructed, and in a lentiviruses mediating manner, the anti-hCD19CAR expression vectors are transduced into the T cell. The anti-hCD19CAR modified T cell can specifically kill CD19 positive tumor cells, can effectively prevent a human-anti-mouse antibody caused by a murine CD19 gene, can reduce immunogenicity of the murine CAR, can improve the survival time of CAR-T cells, can be combined with the humanized CD19 protein in a high specificity manner, can strengthen the treatment effects of the CAR-T, and can improve the CAR-T treatment safety and validity.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and cell biology, and in particular relates to a humanized scFv chimeric antigen receptor (anti-hCD19CAR) T cell targeting CD19, a preparation method and application thereof. Background technique [0002] In view of the poor curative effect of traditional chemotherapy, radiotherapy and hematopoietic stem cell transplantation on hematological malignancies, that is, the complete response rate (Complete response, CR) or high recurrence rate, such as acute lymphoblastic leukemia (ALL), although the CR rate after chemotherapy is good However, the recurrence rate of about 70% and the 3-year disease-free survival rate (Disease free survival, DFS) of only about 30% make researchers still need to further explore better treatment methods. [0003] With the development of tumor immunology theory and technology, cellular immunotherapy has made great progress in recent years, and was listed as the...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/62C12N5/10A61K39/00A61P35/00A61P35/02
CPCA61P35/00A61P35/02A61K39/001112C07K14/7051C07K16/2803C07K2317/622C07K2319/03C07K2319/10C12N15/86C12N2510/00C12N2740/15043
Inventor 谭毅孔群芳张慧慧韩镇
Owner 山东省齐鲁细胞治疗工程技术有限公司
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