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Separated T cell receptor, cells modified by separated T cell receptor, encoding nucleic acid, expression vector, preparation method, pharmaceutical composition and application

A cell receptor and nucleic acid technology, applied in the biological field, to avoid immune rejection, good industrial application prospects, and avoid cytokine storms

Active Publication Date: 2019-05-21
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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  • Separated T cell receptor, cells modified by separated T cell receptor, encoding nucleic acid, expression vector, preparation method, pharmaceutical composition and application
  • Separated T cell receptor, cells modified by separated T cell receptor, encoding nucleic acid, expression vector, preparation method, pharmaceutical composition and application
  • Separated T cell receptor, cells modified by separated T cell receptor, encoding nucleic acid, expression vector, preparation method, pharmaceutical composition and application

Examples

Experimental program
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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: the lentiviral vector pCDH-EF1α-MCS-(PGK-GFP) expressing 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 by using conventional techniques in the art. According to the obtained TCR gene sequence, synthesize the whole gene sequence of TCR β chain and α chain and the cleavable F2A sequence and Furin restriction fragment between them, and link to the multiple cloning site downstream of the EF-1α promoter of the vector , the sequence of transcription inserted into TCR is TCRβ chain (without stop codon), Furin fragment, F2A fragment, and TCRα chain (see the literature "Gene Ther. 2008 Nov; 15(21): 1411-1423"). The vector expressing GFP is driven by the inverted ...

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 Her2 / neu 369-377 polypeptide of 1 μg / ml 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 The right figure shows that 0.013% of lymphocytes are CD8-positive killer T cells that can bind to Her2 / neu 369-377 / HLA-A2 pentamer (ie Her2-E75 pentamer), and the left figure is not stimulated by Her2 polypeptide There were no CD8-positive pentamer-positive cells in the control cells. The results showed that in the natural T cell pool, the number of specific T cells recognizing Her2 / neu 369-377 antigen polypeptide was very small. Although small in number, this group of T cells t...

Embodiment 2

[0145] Example 2: Obtaining and identifying the complete sequence of Her2 / neu 369-377 polypeptide-specific TCR

[0146] In this example, the Her2 / neu 369-377 polypeptide-specific CTL clone obtained in Example 1 is directly obtained from the Her2 / neu 369-377 polypeptide-specific CTL clone comprising matching α and β chains (that is, the two chains can together form a functional TCR that recognizes the antigen 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 shown. This TCR exists in the peripheral T cell pool of HLA-A2 positive normal people, and will not cross-react with normal cells that express Her2 / neu protein in a small amount and cause autoimmune reactions. The specific detect...

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Abstract

The invention provides a separated T cell receptor, cells modified by the separated T cell receptor, encoding nucleic acid, an expression vector, a preparation method, a pharmaceutical composition andapplication. The separated T cell receptor (TCR) includes at least one of an alpha chain and a beta chain, the alpha chain and the beta chain both include variable regions and constant regions, and the separated T cell receptor is characterized in that an antigen Her2 / neu expressed by tumor cells can be specifically identified by the T cell receptor, the consistency of the amino acid sequence ofthe variable regions of the alpha chain and the amino acid sequence shown in SEQ ID NO:19 is 98% or above, and the consistency of the amino acid sequence of the variable regions of the beta chain andthe amino acid sequence shown in SEQ ID NO:20 is 98% or above. The T cell receptor (TCR) can specifically identify the tumor antigen and avoid possible miss toxic and side effects at the same time. The immune cells modified by the T cell receptor (TCR) has a significant anticancer effect.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, relates to isolated T cell receptors, modified cells, 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 a dimer with other proteins in the family and regulates cell proliferation, differentiation and carcinogenesis through a series of intracellular signal transduction pathways (see the literature "Growth Factors, 2008;26:263", "Oncol Biol. Phys, 2004;58:903"). Her2 / neu protein is overexpressed in a variety of epithelial cancer cells, such as breast cancer, gastric cancer, colorectal cancer, ovarian cancer, pancreatic cancer, lung cancer, esophageal cancer, bladder cancer, kidney cancer, etc. (see the literature "Trends in Molecular Med , 2013; 19:677"), and the expression in primary and metastatic cancer cells ...

Claims

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Application Information

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Patent Type & Authority Applications(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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