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Isolated T-cell receptor, cells modified therefor, encoding nucleic acid, expression vector, preparation method, pharmaceutical composition and application

A technology of cell receptors and nucleic acids, applied in the biological field, to achieve the effect of avoiding off-target toxic and side effects

Active Publication Date: 2022-05-27
HANGZHOU CONVERD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this high-affinity TCR may cross-react with normal self-proteins and cause killing of normal tissue cells, that is, severe or even fatal off-target toxicity (see the literature "Curr OpinImmunol 2015; 33: 16–22", see the literature "Sci Transl Med.2013; 5(197):197ra103")

Method used

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  • Isolated T-cell receptor, cells modified therefor, encoding nucleic acid, expression vector, preparation method, pharmaceutical composition and application
  • Isolated T-cell receptor, cells modified therefor, encoding nucleic acid, expression vector, preparation method, pharmaceutical composition and application
  • Isolated T-cell receptor, cells modified therefor, encoding nucleic acid, expression vector, preparation method, pharmaceutical composition and application

Examples

Experimental program
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Effect test

preparation example Construction

[0139] Preparation of recombinant TCR lentiviral expression vector: The viral vector used to express TCR is a replication-defective lentiviral vector, including: GFP-expressing lentiviral vector pCDH-EF1α-MCS-(PGK-GFP), which can be purchased from System Biosciences (Cat#CD811A-1); and the vector pCDH-EF1α-MCS that does not express GFP, obtained by removing the PGK promoter and GFP gene on the pCDH-EF1α-MCS-(PGK-GFP) vector using conventional techniques in the art. According to the obtained TCR gene sequence, synthesize the entire gene sequence of TCR β chain and α chain and the cleavable F2A sequence and Furin restriction fragment in between, and link to the multiple cloning site downstream of the EF-1α promoter of the vector , the transcription sequence inserted into TCR is TCRβ chain (without stop codon), Furin digestion fragment, F2A fragment, TCRα chain (for the method, see the document "Gene Ther. 2008 Nov; 15(21): 1411-1423"). The GFP expressing vector is driven by the ...

Embodiment 1

[0142] Example 1: Induction of Her2 / neu 369-377 polypeptide-specific killer T cells from HLA-A2 positive normal donor peripheral blood

[0143] In this example, a low concentration of 1 μg / ml Her2 / neu 369-377 polypeptide was used to induce polypeptide-specific killer T cells from normal PBMC (#2PBMC) after two rounds of in vitro stimulation, and flow cytometry analysis and single cell isolation were performed. The specific method is as described above. The result is as follows:

[0144] figure 1 A Right panel shows that 0.013% of lymphocytes are CD8 positive killer T cells that can bind Her2 / neu 369-377 / HLA-A2 pentamer (ie Her2-E75 pentamer), left panel is not stimulated by Her2 polypeptide of control cells did not appear CD8-positive pentamer-positive cells. The results indicated that the number of specific T cells recognizing the Her2 / neu 369-377 antigen polypeptide was very small in the natural T cell pool. Despite their small numbers, this population of T cells recogni...

Embodiment 2

[0145] Example 2: Acquisition and identification of the full sequence of Her2 / neu 369-377 polypeptide-specific TCR

[0146] This example obtained directly from the Her2 / neu 369-377 polypeptide-specific CTL clone obtained in Example 1, a CTL comprising matching alpha and beta chains (ie, the two chains could together constitute a functional TCR that recognizes the antigenic polypeptide) The complete TCR gene sequence, the encoded TCR is called "Her2 TCR-1B5". The amino acid sequence of the α chain of the TCR is shown in SEQ ID NO: 2, the coding sequence is shown in SEQ ID NO: 1, and the amino acid sequence of the β chain of the TCR is shown in SEQ ID NO: 4, and the coding sequence is shown in SEQ ID NO: 4 ID NO:3. This TCR exists in the peripheral T cell pool of HLA-A2-positive normal people, and will not cross-react with normal cells expressing Her2 / neu protein at a small amount, resulting in autoimmune reactions. The specific detection method is as described above. The res...

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Abstract

The invention provides isolated T cell receptors, modified cells, encoding nucleic acids, expression vectors, preparation methods, pharmaceutical compositions and applications. The isolated T cell receptor (TCR) comprises at least one of an alpha chain and a beta chain, both of which comprise a variable region and a constant region, characterized in that the T cell receptor is capable of Specifically recognize the antigen Her2 / neu expressed by tumor cells, and the amino acid sequence of the variable region of the α chain has at least 98% identity with the amino acid sequence shown in SEQ ID NO: 19, the β chain The amino acid sequence of the variable region has at least 98% identity with the amino acid sequence shown in SEQ ID NO:20. The TCR can specifically recognize tumor antigens while avoiding possible off-target toxic and side effects. Immune cells modified with this TCR have significant anti-tumor effects.

Description

technical field [0001] The present invention belongs to the field of biotechnology, and in particular, relates to isolated T cell receptors, modified cells thereof, encoding nucleic acids, expression vectors, preparation methods, pharmaceutical compositions and applications. Background technique [0002] Her2 / neu (ERBB2) is a transmembrane protein belonging to the EGFR family, which forms dimers with other proteins in the family and regulates cell proliferation, differentiation and carcinogenesis through a series of intracellular signaling pathways (see "Growth Factors, 2008;26:263", "Oncol Biol. Phys, 2004;58:903"). Her2 / neu protein is overexpressed in cancer cells of various epithelial origins, such as breast cancer, gastric cancer, colorectal cancer, ovarian cancer, pancreatic cancer, lung cancer, esophageal cancer, bladder cancer, kidney cancer, etc. (see "Trends in Molecular Med , 2013; 19: 677"), and its expression is relatively uniform in primary and metastatic cance...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/725C12N15/12C12N5/10A61P35/00
CPCA61P35/00C12N5/10
Inventor 侯亚非侯大炜
Owner HANGZHOU CONVERD CO LTD
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