Products and preparation method of double chimeric antigen receptor gene modified T lymphocyte targeting breast cancer stem cells

A breast cancer stem cell and chimeric antigen receptor technology, applied in the field of T lymphocytes, can solve the problems of poor specificity of targeted tumor cell killing and limited clinical treatment of malignant tumors.

Inactive Publication Date: 2016-09-21
泰州市数康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims at the poor killing specificity of targeting tumor cells in the existing clinical application of T lymphocyte technology; and the insufficiency of activating the patient's autoi...

Method used

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  • Products and preparation method of double chimeric antigen receptor gene modified T lymphocyte targeting breast cancer stem cells
  • Products and preparation method of double chimeric antigen receptor gene modified T lymphocyte targeting breast cancer stem cells
  • Products and preparation method of double chimeric antigen receptor gene modified T lymphocyte targeting breast cancer stem cells

Examples

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preparation example Construction

[0052] The invention relates to a method for preparing T lymphocytes modified with chimeric antigen receptor genes targeting breast cancer stem cells, which is characterized in that the gene recombination of bispecific chimeric antigen receptors containing CD47 and TAZ is recombined into viruses The carrier is transfected into human T lymphocytes, which highly express the bispecific chimeric antigen receptor gene, which can specifically bind to breast cancer cells and breast cancer stem cells expressing CD47 and TAZ, and activate the first signal and co-stimulatory signal in vivo and in vitro Antitumor cytotoxic activity was elicited in the assay.

[0053] The specific chimeric antigen receptor gene that specifically binds CD47 according to the present invention includes a single-chain antibody that specifically binds CD47 and is connected in series with two intracellular domains of co-stimulatory molecules (CD28 and CD137) and the intracellular signal region of CD3ζ Fragments...

Embodiment 1

[0119] Example 1 Preparation of Bispecific Chimeric Antigen Receptor Gene-Modified T Lymphocytes

[0120] Human IFNγ-signal peptide-scFv(CD47)-CD8a hinge region-CD28-CD137-CD3ζ intracellular region, and scFv(TAZ)-IgD hinge region-TET2 are constructed by gene fragments of Furin cleavage site and connecting sequence.

[0121] Link human IFNγ-signal peptide-scFv(CD47)-CD8a hinge region-CD28-CD137-CD3ζ intracellular region, and scFv(TAZ)-IgD hinge region-TET2 through Furin cleavage site and linker sequence (Linker) , form a recombinant gene sequence, the gene sequence is obtained by chemical synthesis, and form a complete cDNA of the bispecific chimeric antigen receptor gene, that is, Di-CAR; Di-CAR lentiviral vector construction and packaging methods and effects are described in Example 3 The method is the same, the obtained Di-CAR lentivirus is obtained by taking the supernatant to measure the titer by fluorescence method, the virus is divided into 50 μl 2E+8TU / ml, and stored in...

Embodiment 2

[0125] Example 2 Western blotting and flow cytometry detection of bispecific chimeric antigen receptor gene-modified T lymphocytes

[0126] Take the T lymphocytes obtained from the advanced breast cancer patients prepared in Example 1 and normal healthy people on the 14th day of culture and obtain each 1×10 6 , Western blotting Western blot analysis was carried out after sonication, and the method is shown in Example 1 of the effect.

[0127] Figure 8 Western blotting was used to detect the expression level of Di-CAR fusion protein in T lymphocytes from breast cancer patients transfected with Di-CAR lentivirus, and the expression level of Di-CAR fusion protein in Escherichia coli (positive control), that is, the western blotting result graph, in Two bands appeared in both patients, one of which was CD47-CAR with about 479 amino acid residues and a molecular weight of about 53kD; the other was TAZ-CAR with about 417 amino acid residues and a molecular weight of about 46kD. T...

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Abstract

The invention provides a preparation method of a double chimeric antigen receptor gene modified T lymphocyte targeting breast cancer stem cells. The preparation method is characterized in that integrin-associated protein (CD47) and transcriptional coactivator (TAZ) are both highly expressed in breast cancer tissues, especially in the breast cancer stem cells; by established CD47 and TAZ over-expressed breast cancer cells, higher proliferation and metastasis capacity and cancer stem cell features are shown. Extracellular domain of the two breast cancer stem cell antigens CD47 and TAZ are targeted to generate monoclonal strains in specific binding under immunity action, and single-chain antibodies in specific binding with the CD47 and TAZ by genetic recombination are obtained to construct a human CD47 and TAZ containing double chimeric antigen receptor gene recombined to a virus vector to transfect human T lymphocyte. By high expression of the double chimeric antigen receptor gene in specific binding with human CD47 and TAZ expressing breast cancer stem cells, a first signal and a costimulatory signal can be activated to trigger anti-breast-cancer cytotoxicity, and high cytotoxicity in in-vivo and in-vitro anti-cancer experiments is achieved.

Description

technical field [0001] The present invention relates to a method for preparing chimeric antigen receptor gene-modified T lymphocytes targeting breast cancer stem cells, which includes the following features: breast cancer tissue and breast cancer stem cells specifically highly express integrin-related protein (CD47) and Transcriptional coactivator (TAZ), breast cancer cells overexpressing CD47 and TAZ constructed show stronger proliferation, metastasis and tumor stem cell characteristics; the present invention targets breast cancer stem cell antigen CD47 and TAZ ectodomain, and obtains the The specific binding single-chain antibody is constructed to contain the double-specific chimeric antigen receptor (Di-CAR) gene (double chimeric antigen receptor, Di-CAR) against human CD47 and TAZ, recombined into the virus vector and transfected into human T lymphocytes, which can specifically bind Breast cancer stem cells expressing human CD47 and TAZ activate immune signals in vitro and...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/62C12N15/867A61K35/17A61P35/00
CPCA61K35/17C07K14/7051C07K14/70521C07K14/70578C07K16/2896C07K2317/622C07K2319/02C07K2319/33C12N15/86C12N2510/00C12N2740/15043
Inventor 王锦杰宋现让
Owner 泰州市数康生物科技有限公司
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