Human papillomavirus-specific t-cell receptor and its anti-tumor use

A human papilloma virus and cell receptor technology, applied in the field of medicine, can solve the problems of inactivation of p53, hinder the inhibitory effect of p53 protein, etc., and achieve the effects of high binding characteristics and high infection efficiency

Active Publication Date: 2022-06-07
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The early gene product E6 protein can combine with p53 protein to form a complex, resulting in the inactivation of p53, thereby hindering the inhibitory effect of p53 protein in the process of cell carcinogenesis

Method used

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  • Human papillomavirus-specific t-cell receptor and its anti-tumor use
  • Human papillomavirus-specific t-cell receptor and its anti-tumor use
  • Human papillomavirus-specific t-cell receptor and its anti-tumor use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1. HPV virus E6 antigen TV-10 polypeptide-specific T cell sorting and TCR gene cloning

[0066] First, the inventor synthesized the HPV-16 virus E6 antigen HLA-A2 restriction epitope polypeptides, and used these polypeptides to immunize mice, and selected the mutant polypeptides with immunogenicity through ELISPOT experiments. The amino acid sequence of the polypeptide is shown in SEQ ID NO: 21, i.e., HLA-A2 restriction epitope polypeptide OF the present invention TV-10, hereinafter also referred to as polypeptide TV-10 or TV-10 polypeptide.

[0067] Subsequently, a tetramer of TV-10 polypeptide and mouse HLA-A2 was prepared, and CD3 was selected from postimmune mouse splenocytes by staining together with CD3 and CD8 antibodies + CD8 + T cells, sorted to obtain TV-10 epitope-specific T cells. The details are as follows.

[0068] Preparation of 1.TV-10 / HLA-A2 tetramer

[0069] The binding characteristics of polypeptide TV-10 and HLA-A2, as well as specific T cell resp...

Embodiment 2

[0107] Example 2. TV-10 peptide / HLA-A2 tetramer binding experiment with cells expressing m2P8D TCR

[0108] In the present embodiment, the inventor further confirmed that the screened m2P8D TCR has a specific binding for TV-10 polypeptide / HLA-A2.

[0109] Validation of 1.m2P8D TCR binding specificity

[0110] First, the α chain and β chain variable region (V region) of m2P8D TCR (amino acid sequences are shown in SEQ ID NO:1, SEQID NO:5, respectively, and the α chain and β chain constant region (C region) genes of human TCR (synthesized by Hongxun Biotechnology) are connected to obtain m2P8D chimeric TCRα and β chain sequences, the amino acid sequence of the chimeric sequence is shown in SEQ ID NO:9, and its nucleic acid sequence is shown in SEQ IDNO:10.

[0111]The α chain and β chain of m2P8D TCR are connected in the middle of the P2A sequence (the amino acid sequence of the P2A sequence is shown in SEQ ID NO:26), and the lentiviral expression plasmid pCDH (purchased from Invi...

Embodiment 3

[0117] Example 3. Effector cells expressing m2P8D TCR and target cells expressing E6-TV-10 / HLA-A2

[0118] In the present embodiment, the m2P8D TCR gene is introduced into the Jurkat T cell line (purchased from the Shanghai Cell Bank) as a effector cell; E6-TV-10 / HLA-A2 was introduced into K562 cells (purchased from the Shanghai Cell Bank) as target cells. The effect of m2P8D TCR and target cells expressing E6-TV-10 / HLA-A2 was evaluated by detecting the level of IL-2 after effector cell and target cell action. The specific operation is as follows.

[0119] 1. E6-TV-10 / HLA-A2 target cell preparation

[0120] According to eukaryotic cell expression codons to optimize the DNA sequence encoding the genes of polypeptide TV-10 and HLA-A2 heavy chain and β2m, the resulting nucleic acid sequence is called TV-10 / HLA-A2-E6 SCT in turn, the sequence is shown in SEQ ID NO:12, and the amino acid sequence encoded is shown in SEQ ID NO:11.

[0121] The above DNA was synthesized by Hongxun Biotech ...

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Abstract

The present invention provides human papillomavirus E6 gene T cell epitope-specific T cell receptor murine m2P8D TCR, its humanized form h2P8D TCR, and their antigen-binding fragments. The two T cell receptors are composed of α , β two peptide chains, and also provide nucleic acid encoding them, the vector containing the nucleic acid, the host cell containing the vector and their anti-tumor application; also provide the T cell epitope specific T cell of human papillomavirus E6 gene Cell receptor murine m2P8D TCR, its humanized form h2P8D TCR and methods for their preparation. The two specific T cell receptors and antigen-binding fragments thereof can serve as immune effector activators to stimulate the immune response of the body, thereby producing anti-tumor and other diseases.

Description

Technical field [0001] The present invention belongs to the field of medicine, specifically relates to a T cell receptor capable of specifically identifying the T cell epitope of a human papillomavirus gene (TCR) or an antigen-binding fragment thereof. Background [0002] In 2011, cancer surpassed heart disease as the world's leading cause of death. The WHO announced in December 2013 that the number of new cancer patients worldwide has exceeded 14 million per year, a significant increase from the 2008 statistic of 12.7 million. As of 2020, cancer kills 9.6 million people a year, and about $1 trillion a year worldwide is invested in cancer treatment. [0003] In the early 1980s, Allison and other researchers identified the genetic structure of the alphaβT cell receptor (TCR), which is responsible for recognizing antigens on the surface of T cells. In the late 1980s, Boone, Rosenberg, Old and others found that there are some tumor-specific antigens in different tumor patients, whic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/725C12N15/12C12N15/867A61K38/17A61P35/00A61P31/20G01N33/574G01N33/569
CPCC07K14/7051C12N15/86A61P35/00A61P31/20G01N33/57411G01N33/57484G01N33/56983C12N2740/15043C12N2800/107A61K38/00G01N2333/025
Inventor 高福孙萌谭曙光张贺琳王杰
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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