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Chimeric antigen receptor-T cells secreting and expressing IL15RA-IL15 fusion proteins and CCL21 chemotactic factors, and applications thereof

A technology of IL15RA-IL15 and chimeric antigen receptor, which is applied in the direction of chemokines, genetically modified cells, cells modified by introducing foreign genetic materials, etc., can solve the problems of short duration, poor curative effect of solid tumors, and immune escape And other issues

Active Publication Date: 2020-02-21
浙江启新生物技术有限公司
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AI Technical Summary

Problems solved by technology

However, 10-20% of CAR-T therapy is ineffective at initial treatment, 30-50% relapses within one year after complete remission, and the effect on solid tumors is not good, mainly due to the short duration of CAR-T cells in the body, immune escape, and tumor lesions immunosuppressive microenvironment, etc.

Method used

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  • Chimeric antigen receptor-T cells secreting and expressing IL15RA-IL15 fusion proteins and CCL21 chemotactic factors, and applications thereof
  • Chimeric antigen receptor-T cells secreting and expressing IL15RA-IL15 fusion proteins and CCL21 chemotactic factors, and applications thereof
  • Chimeric antigen receptor-T cells secreting and expressing IL15RA-IL15 fusion proteins and CCL21 chemotactic factors, and applications thereof

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Experimental program
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Effect test

Embodiment

[0021] Main experimental materials:

[0022] EcoRI-HF, MluI-HF, NdeI restriction endonuclease (NEB Company), Seamless Cloning Enzyme (He Yuan Biotechnology), High Fidelity Prime GXL STAR Enzyme (TAKARA Company), TransStbl3 Competent Cells (Quanshijin Biotechnology Ltd.), Plasmid Mini Kit I (OMEGA), Plasmid Maxi Kit (QIAGEN), DMEM, RPMI-1640, Opti-MEM medium, Gibco FBS (Thermo Fisher Scientific), Sanger sequencing (Shanghai Sunny Biological Co., Ltd.), Nacl, yeast powder, peptone, EDTA, NaOH (Shanghai Sangon Bioengineering Co., Ltd.), primers (Jiangsu Jinweizhi Biotechnology Co., Ltd.).

[0023] 1. Construction of recombinant plasmids

[0024] ① Construction of Plenti-anti-CD19 CAR-IL15Rα(sushi domain)-IL15-CCL21 recombinant plasmid: IL15Rα(sushi domain)-IL15-CCL21 (SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.3, SEQ ID NO. ID NO.4, SEQ ID NO.5 nucleotide sequence end-to-end connection), using Ndel and MluI-HF double enzyme digestion plenti-EF1a-anti-CD19CAR vector and...

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Abstract

The invention discloses chimeric antigen receptor-T cells secreting and expressing IL15RA-IL15 fusion proteins and CCL21 chemotactic factors as well as application thereof. Super IL15 can promote activation and proliferation of T (especially memory T) cells, NK cells and NKT cells; and CCL21 can recruit peripheral CCR7-positive initial T cells, memory T cells and dendritic cells (DC) into lymphoidtissue or tumor lesions so as to activate active anti-tumor immune response (such as cancer memory T cells) in the body, thereby significantly improving efficiency and remission rate, as well as reducing recurrence rate after remission.

Description

technical field [0001] The invention belongs to the field of biotechnology engineering, and specifically relates to the construction and application of the fourth generation chimeric antigen receptor (CAR)-T cells (secreting and expressing IL15Rα (sushi domain)-IL15 (N72D) fusion protein and CCL21 chemokine) (mainly targeting B cell malignancies as an example). Background technique [0002] CAR-T therapy is a kind of cellular immunotherapy based on chimeric antigen receptor (CAR). Through in vitro gene transfer technology, the gene sequence encoding chimeric antigen receptor (CAR) is transfected into T cells to generate tumor-specific T cells that can bind to target antigens. The fourth-generation CAR-T cells can significantly improve the immunosuppressive microenvironment of tumor lesions through the complete first (tumor-associated antigen), second (co-stimulatory factor) and third signals (such as IL-7 and CCL19), and then Activate the body's intrinsic active anti-tumor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N5/10C12N15/62
CPCC07K16/2803C07K14/7051C07K14/7155C07K14/5443C07K14/521C12N5/0636C07K2319/33C07K2319/03C12N2510/00
Inventor 高基民魏成赵爱
Owner 浙江启新生物技术有限公司
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