Construction method and clinical application of novel non-viral vector TSCM gene therapy

A construction method, PB-CAR-TSCM technology, applied in antiviral agents, antibody medical components, biochemical equipment and methods, etc., can solve problems such as short survival time, weak self-renewal ability, poor therapeutic effect, etc., to reduce Cost of production, avoidance of magnetic bead sorting, effects of improved viability

Pending Publication Date: 2021-05-14
路春光
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the traditional CAR-T cell expansion program, after the monocytes activated by CD3 / CD28 monoclonal antibody are further expanded by interleukin 2, most of the T cells are highly differentiated effector / memory T cells with weak self-renewal ability , short survival time in vivo after transplantation, resulting in poor therapeutic effect; in this study, TSCM was used to replace T cells, and the proportion of TSCM after expansion was as high as 44.41%, which greatly improved the survival time of immune cells in vivo after transplantation and improved the therapeutic effect

Method used

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  • Construction method and clinical application of novel non-viral vector TSCM gene therapy
  • Construction method and clinical application of novel non-viral vector TSCM gene therapy
  • Construction method and clinical application of novel non-viral vector TSCM gene therapy

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Experimental program
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Effect test

Embodiment 1

[0041] Construction of the plasmid system:

[0042] Construction of PB-EF1-SP-HER2ScFv-CD8-CD137-CD3-IRES-GFP-T2A-Puro-SV40-PA and PB-EF1-SP-CD19ScFv-CD8-CD137-CD3-IRES-GFP-T2A-Puro-SV40 -PA, enzyme digestion identification and sequencing to verify the correctness of the sequence, large plasmid extraction, plasmid digestion electrophoresis detection, all plasmids are correct, see figure 1 and figure 2 .

Embodiment 2

[0044] Tumor cell line establishment and labeling

[0045] CD19 positivity of Raji cell line and HER2 positivity of SGC-7901 verified by flow cytometry, HER2 expression was up to 99.2% in SGC-7901 cell line, see Figure 4 .

Embodiment 3

[0047] Preparation of TSCM cells

[0048] 100ml of sodium citrate anticoagulated blood was collected, PBMC and UBMC were separated by lymphocyte separation medium, T cells were purified, and cryopreserved. On day D0, TSCM-II was coated into a culture flask, and placed flat in a 37° C. incubator for more than 2 hours. PBMC and UBMC or T cells were resuspended with TSCM-III according to the cell density of 1×106 / mL, and the TSCM cells were expanded and cultured with TSCM-I medium the next day, after 12-15 days of expansion culture, harvested TSCM cells that meet clinical application standards, and the cell preparations are tested for endotoxin, activity and phenotype. The results of cell identification show that the cell activity is over 95%, and it contains a relatively high proportion of CD3+-BV421 / CD4+-APC-Cy / CD8+APC / CD62L+PE-cy7 / CD45RO-Percp-Cy5.5CD / 45RA+FITC / CD95+PE cells;

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Abstract

The invention relates to the technical field of immune cell targeted therapy, and aims to solve the problems that in the traditional CAR-T cell amplification process, after monocytes activated by CD3 / CD28 monoclonal antibodies are further amplified by interleukin 2, most T cells are highly differentiated effect / memory T cells, the self-renewal capability is weak, the in-vivo survival time after transplantation is short, the treatment effect is poor, according to the research, T cells are replaced by the TSCM, the proportion of the amplified TSCM is up to 44.41%, the survival time of in-vivo immune cells after transplantation is greatly improved, and the treatment effect is improved. The capacity of the PiggyBac vector and the capacity of the Sleep Buffer vector are far higher than those of viruses and even can reach 100 kb or above, almost all elements trying to be added can be contained, the packaging link and the sequence specificity requirement do not exist, and the CAR-TSCM vector is the best candidate from the requirement for future research and development of CAR-TSCM or from the possibility of cost reduction and market development.

Description

technical field [0001] The present invention relates to the technical field of immune cell targeted therapy, in particular to an optimized isolation and purification of T cells from fresh or cryopreserved human peripheral blood mononuclear cells or umbilical cord blood mononuclear cells, and T cell induction and expansion of stem cell-like memory T cells (TSCM), a new CAR-TSCM platform built through the transposon vector system, will seize the opportunity in the future competition in the field of cellular immunotherapy. Background technique [0002] The main carrier system currently used by CAR is the application of lentivirus, which has high-efficiency packaging and high-efficiency infection characteristics, making it a viral vector suitable for genetic modification of T cells. [0003] As the types of cancers that need to be treated increase and the requirements for therapeutic effects increase, more challenges are posed to the CART carrier system, such as multi-target sys...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10C12N5/0783A61K39/00A61P35/00A61P31/12A61P31/14A61P31/20A61P1/16A61P31/06A61P31/18
CPCC12N15/85C12N5/0636A61K39/001112A61K39/001106A61P35/00A61P31/12A61P31/14A61P31/20A61P1/16A61P31/06A61P31/18C12N2510/00C12N2501/2307C12N2501/2315C12N2501/2321C12N2501/515C12N2501/51A61K2039/5156
Inventor 路春光
Owner 路春光
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