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Chimeric antigen receptor combination expressed on T lymphocyte surface and application thereof

A technology of chimeric antigen receptors and lymphocytes, which is applied in the field of genetically modified cells and tumor treatment, and can solve the problems of lack of anti-tumor activity of cells and insufficient persistence of CAR-T cells

Active Publication Date: 2019-05-14
深圳市芥至和生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Clinical studies of first-generation CAR-T cells derived from the treatment of B-cell malignancies (by targeting CD20 or CD19 antigens) showed the feasibility of this approach; however, these engineered cells lacked significant antitumor activity, possibly because Insufficient persistence of CAR-T cells

Method used

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  • Chimeric antigen receptor combination expressed on T lymphocyte surface and application thereof
  • Chimeric antigen receptor combination expressed on T lymphocyte surface and application thereof
  • Chimeric antigen receptor combination expressed on T lymphocyte surface and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Preparation of T lymphocytes targeting MUC1 and CD19

[0034] 1. Construction of lentiviral plasmid vector

[0035] 1) Synthesize and amplify the nucleotide sequences SEQ ID NO:3 and SEQ ID NO:4 encoding the chimeric antigen receptor combination targeting MUC1 and CD19 of the present invention, completed by Suzhou Synbio Biotechnology Co., Ltd.;

[0036]2) Ligate the enzyme cutting sites XbaI and BamHI, the binding sequence of the transcription factor Gal4-VP64, and the EF1 promoter, and the enzyme cutting sites NheI and EcoRI, and insert into the lentiviral expression vector pGreen puro (the structure of which is shown in Figure 1A Shown) behind the CMV promoter, the transformed lentiviral expression vector pGreen puro was obtained; the binding sequence of the CMV promoter and restriction site XbaI and BamHI, transcription factor Gal4-VP64, and EF1 promoter, and restriction enzyme digestion The schematic diagram of the linker of site NheI and EcoRI is shown ...

Embodiment 2

[0059] Example 2 The present invention provides verification of the effect of genetically modified CD8+ T lymphocytes in treating tumors in vitro

[0060] a. Raji, a lymphoma cell line stably expressing MUC1 and CD19 (double-positive expression of MUC1 and CD19, purchased from ATCC), was used as target cells and divided into two groups.

[0061] The CD8+ T lymphocytes targeting MUC1 and CD19 prepared in Example 1 (ie switch-CAR-T cells) were used as effector cells 1; CD8+ T cells not infected with the virus were used as effector cells 2;

[0062] 1) In the experimental group (i.e. switch-CAR-T cells / Raji), effector cells 1 were added to the first group of target cells at an effect-to-target ratio of 5:1, 10:1, and 20:1;

[0063] 2) In the control group (i.e. T cells / Raji), effector cells 2 were added to the second group of target cells at an effect-to-target ratio of 5:1, 10:1, and 20:1;

[0064] Specifically, the target cells were mixed at a density of 1x 10 5 cells / ml inoc...

Embodiment 3

[0071] Example 3 The present invention provides verification of the effect of gene-modified T lymphocytes in treating tumors in vivo

[0072] In 30 nude mice (6 weeks old, weighing 18-20g, purchased from Guangdong Provincial Medical Experimental Animal Center), 15 of them were subcutaneously injected with 5x 10 6 Lymphoma cell line Raji (both positive expression of MUC1 and CD19, purchased from ATCC), another 15 Raji cells (positive expression of CD19 only) with siRNA silenced MUC1 expression were subcutaneously injected to observe whether tumors appeared in the mice, and the tumors grew to 60mm 3 size, they were randomly divided into 3 groups.

[0073] a. MUC1 and CD19 double-positive tumor cells transplanted into mice

[0074] 1) For the control group (control / Raji), 200ul of normal saline was injected into the tail vein twice a week;

[0075] 2) In the T cell therapy group (T cell / Raji), 1×10 CD8+ T cells without virus infection were injected into the tail vein respective...

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Abstract

The invention provides a chimeric antigen receptor combination expressed on T lymphocyte surface and an application thereof. The chimeric antigen receptor combination comprises a chimeric antigen receptor 1 and a chimeric antigen receptor 2, wherein the chimeric antigen receptor 1, namely Anti MUC1 synNotch, comprises three parts linked sequentially as follows: SCFV, Notch core and transcription factor Gal4-VP64, which specifically recognize an MUC1 protein; the chimeric antigen receptor 2, namely Anti CD19 CAR, has an extracellular binding region, a hinge region and an intracellular signal region linked sequentially as follows: SCFV, IgD, CD28-4-1BB-CD3 zeta, which specifically recognize a CD19 protein. The chimeric antigen receptor combination is expressed on the surface of T lymphocytesand has the effect of specifically recognizing MUC1 and CD19 proteins.

Description

technical field [0001] The invention relates to the technical field of genetically modified cells and tumor treatment, in particular to a combination of chimeric antigen receptors expressed on the surface of T lymphocytes and applications thereof. Background technique [0002] The targeting, killing activity and persistence of T cells expressed in tumor-specific chimeric antigen receptor (CAR) by gene modification technology developed in recent years have injected new solutions into adoptive cellular immunotherapy. . Chimeric Antigen Receptor (CAR) is mainly composed of two parts, one end is located outside the cell, which can specifically recognize an antigen on the surface of cancer cells; the other end is located inside the cell, containing signal activation elements (such as T cell receptor The Zeta chain of the body) plays a role in transmitting signals to activate T cells. In this way, CAR-expressing T lymphocytes (CAR-T cells) can avoid the restriction of T cell rec...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N7/01C12N5/10A61K35/17A61P35/00
Inventor 刘未斌刘曲波李琼书胡源
Owner 深圳市芥至和生物科技有限公司
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