Chimeric Antigen Receptor Expressed on the Surface of T Lymphocytes and Its Application

A chimeric antigen receptor and lymphocyte technology, applied in the field of genetically modified cells and tumor treatment, can solve the problem of not being able to target CAR-T cells at the same time

Active Publication Date: 2022-03-18
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

[0005] There are currently no CAR-T cells that can simultaneously target MUC1 and NKG2D ligands

Method used

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  • Chimeric Antigen Receptor Expressed on the Surface of T Lymphocytes and Its Application
  • Chimeric Antigen Receptor Expressed on the Surface of T Lymphocytes and Its Application
  • Chimeric Antigen Receptor Expressed on the Surface of T Lymphocytes and Its Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Preparation of T lymphocytes targeting MUC1 and NKG2D ligands and T lymphocytes targeting MUC1

[0028] 1. Synthesis and Amplification

[0029] 1) Synthesize the nucleotide sequence (SEQ ID NO: 2) encoding the chimeric antigen receptor targeting MUC1 and NKG2D ligands of the present invention, and the chimeric antigen receptor 1 (SCFV-IgD–CD28– The nucleotide sequence (SEQID NO:3) of 4-1BB-CD3ζ) was synthesized by Suzhou Synbio Biotechnology Co., Ltd.;

[0030] 2) After amplifying SEQ ID NO: 2 and SEQ ID NO: 3, clone them into the lentiviral expression vector pGreen puro (purchased from Aidi Gene (Add gene) company), respectively. The structure diagram of pGreen puro is as follows Figure 5 As shown, SEQ ID NO: 2 and SEQ ID NO: 3 were connected to the vector through restriction sites BamHI and EcoRI to obtain recombinant lentiviral vector 1 and recombinant lentiviral vector 2 respectively, and the recombinant vectors were obtained after restriction restrictio...

Embodiment 2

[0057] Example 2 The present invention provides verification of the effect of genetically modified CD8+ T lymphocytes in treating tumors in vitro

[0058] 1. The breast cancer cell line MCF-7 stably expressing MUC1 and NKG2D ligands was used as target cells (purchased from ATCC) and divided into 3 groups.

[0059] The CD8+ T lymphocytes targeting MUC1 and NKG2D ligands prepared in Example 1 were used as effector cells 1; the CD8+ T lymphocytes targeting MUC1 prepared in Example 1 were used as effector cells 2; there were also uninfected virus CD8+ T cells as effector cells 3;

[0060] 1) In the experimental group (Anti MUC1 / NKG2D ligand CAR-T cells / MCF-7), the effector cell 1 was added to the first group of target cells at an effect-to-target ratio of 5:1, 10:1, and 20:1;

[0061] 2) For control group 1 (Anti MUC1CAR-T cells / MCF-7), 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;

[0062] 3) Control group 2...

Embodiment 3

[0072] Example 3 The present invention provides verification of the effect of gene-modified T lymphocytes in treating tumors in vivo

[0073] In 20 nude mice (6 weeks old, body weight 18 ~ 20g, purchased from Guangdong Provincial Medical Experimental Animal Center) subcutaneously injected 5x 10 6 MCF-7 breast cancer cells (purchased from ATCC), observe whether tumors appear in mice, and wait for tumors to grow to 60mm 3 size, they were randomly divided into 4 groups.

[0074] 1) For the control group (control / MCF-7), 200ul of normal saline was injected into the tail vein twice a week;

[0075] 2) In the T cell therapy group (T cell / MCF-7), 1×10 CD8+ T cells without virus infection were injected into the tail vein respectively. 7 each time, 2 times a week;

[0076] 3) The T lymphocyte therapy group targeting MUC1 (Anti MUC1CAR-T cells / MCF-7), the T lymphocytes targeting MUC1 prepared in Example 1 were injected into the tail vein 1×10 7 each time, 2 times a week;

[0077]4)...

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Abstract

The present invention provides chimeric antigen receptors expressed on the surface of T lymphocytes and applications thereof. The chimeric antigen receptors include sequentially connected: an extracellular binding region, a hinge region and an intracellular signal region, wherein the extracellular binding The region contains proteins that specifically recognize MUCl and NKG2D ligands. The chimeric antigen receptor is expressed on the surface of T lymphocytes and has the effect of simultaneously specifically recognizing the proteins of MUCl and NKG2D ligands.

Description

technical field [0001] The invention relates to the technical field of genetically modified cells and tumor treatment, in particular to chimeric antigen receptors expressed on the surface of T lymphocytes and applications thereof. Background technique [0002] CAR-T therapy, the full name is Chimeric Antigen Receptor T-Cell Immunotherapy, chimeric antigen receptor T cell immunotherapy. The main principle is to isolate immune T cells from cancer patients, use genetic engineering technology to introduce a chimeric antibody that can recognize tumor cells and activate T cells at the same time, that is, prepare CAR-T cells, and then expand them The CAR-T cells are infused back into the patient to fight cancer cells through 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 activat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K35/17A61P35/00
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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