Recombinant expression vector, chimeric antigen receptor T cell with reduced depletion and applications of T cell

A chimeric antigen receptor and expression vector technology, applied in genetically modified cells, cells modified by introducing foreign genetic material, vectors, etc., can solve T cell exhaustion, T cell exhaustion that has not been disclosed, CAR-T cells Loss of effect function, etc.

Inactive Publication Date: 2020-12-15
NANJING FIRST HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, under the continuous stimulation of antigens in the tumor microenvironment, the CAR-T cells infused back into the patient's body will become unresponsive, with more and more inhibitory receptors, and the CAR-T cells will lose their effector functions, resulting in T cell exhaustion.
The key mechanism leading to T cell exhaustion is not disclosed in the prior art

Method used

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  • Recombinant expression vector, chimeric antigen receptor T cell with reduced depletion and applications of T cell
  • Recombinant expression vector, chimeric antigen receptor T cell with reduced depletion and applications of T cell
  • Recombinant expression vector, chimeric antigen receptor T cell with reduced depletion and applications of T cell

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Determine the effective target sequence of the PTPN2 gene, add restriction endonuclease Eco I and BspEI sites at its 5' end and 3' end, synthesize the required DNA double-stranded fragment, and obtain 3 candidate sequences, named sh -PTPN2-1, sh-PTPN2-2, sh-PTPN2-3, the complementary DNA sequences of each group are as follows:

[0054] sh-PTPN2-1:

[0055] 5'-CCGGC GCTATTACTACCTTTAATTCATTTTTTG-3';

[0056] 5'-AATTCAAAAAA AATTAAAGGTAGTAATAGCCC-3';

[0057] sh-PTPN2-2:

[0058] 5'-CCGGC CGTATCAACAATGTTCGATGTTTTTTTTG-3';

[0059] 5'-AATTCAAAAAAATCGAACATTGTTGATACGAA-3';

[0060] sh-PTPN2-3:

[0061] 5'-CCGGC CCAGGATCAATCATGTCATTCTTTTTTG-3';

[0062] 5'-AATTCAAAAAA ATGACATGATTGATCCTGGAT-3'.

[0063] Culture 293T cells in logarithmic growth phase, inoculate 1.3-1.5×10 cells 2 days before transfection 6 Put each cell into a 10cm culture dish, add 10mL DMEM medium containing 10% FBS (fetal bovine serum is heat-inactivated in advance), and wait until the cells grow to 70...

Embodiment 2

[0075] 1. Western-blot detection of the expression of CD3ζ and PTPN2 in CAR-T cells

[0076] Extract bispecific chimeric antigen receptor retroviral plasmid, transfect into human embryonic kidney cell 293T cells with PI transfection reagent, wash once with PBS after 48 hours, lyse cells with cell protein extraction reagent RIPA, extract transfection The proteins of the 293T cells were subjected to gel electrophoresis by 10% SDS-PAGE, transferred to the membrane, incubated with mouse anti-human CD3ζ antibody and PTPN2 antibody at 4°C overnight, and then used horseradish peroxidase-labeled anti-mouse II Anti-incubation for 1h, ECL color development.

[0077] The result is as Figure 5 As shown, the anti-human CD3ζ antibody can detect the expression of CAR molecules, the protein molecular size is consistent with the theory, which is 75kDa, and the position of PTPN2 is about 46kDa.

[0078] 2. Flow cytometric detection of the expression of PTPN2 expression molecules in CAR-T cel...

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Abstract

The invention belongs to the technical field of biology, and specifically relates to a recombinant expression vector, a chimeric antigen receptor T cell with reduced depletion and applications of theT cell. T cell PTPN2 genes derived from healthy people can be knocked out by using a gene editing technology, so that the depletion of CD8 and positive T cells can be reduced, co-expression with CAR on the T cell can be realized, blocking response to tumor checkpoints can be improved, and antitumor immunity can be effectively promoted.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant expression carrier, a chimeric antigen-receiving T cell body with reduced depletion and applications thereof. Background technique [0002] Tumors can directly or indirectly inhibit cytotoxic CD8 by setting immune checkpoints + Activation and function of T cells to evade the immune system. The tumor microenvironment can upregulate ligands of T cell inhibitory receptors, such as programmed cell death protein-1 (PD-1) on tumor cells, which can inhibit T cell signaling and promote T cell tolerance or exhaustion. Immune checkpoint receptors, including PD-1 and cytotoxic T lymphocyte antigen-4 (CTLA-4), can counteract T-cell receptor (TCR) and costimulatory receptor-induced kinase signaling by recruiting phosphatases, thereby Inhibits the duration of T cell responses. [0003] CAR-T therapy is to modify the patient's own immune T cells through genetic engineer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/62C12N15/55C12N5/10A61K39/00A61P35/00
CPCA61K2039/5156A61P35/00A61K39/001102A61K39/001111C07K14/7051C07K14/70517C07K14/70521C07K14/70578C07K16/2827C07K16/30C07K2317/24C07K2317/31C07K2317/622C07K2319/03C07K2319/33C07K2319/74C12N5/0636C12N9/16C12N15/86C12N2510/00C12N2740/15043C12N2800/107C12Y301/03048
Inventor 赵薇
Owner NANJING FIRST HOSPITAL
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