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T-cell receptors that recognize short peptides of Epstein-Barr virus

A cell receptor and carrier technology, applied in the field of TCR, can solve problems such as normal cell damage

Active Publication Date: 2020-11-27
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For the treatment of the above diseases, methods such as chemotherapy and radiotherapy can be used, but all of them will cause damage to their own normal cells

Method used

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  • T-cell receptors that recognize short peptides of Epstein-Barr virus
  • T-cell receptors that recognize short peptides of Epstein-Barr virus
  • T-cell receptors that recognize short peptides of Epstein-Barr virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] Example 1 Cloning of EBV-specific T cells

[0127] Peripheral blood lymphocytes (PBL) from healthy volunteers with genotype HLA-A1101 were stimulated with synthetic short peptide EBV LMp2A 340-349 SSSSSCPLSK (Beijing Saibaisheng Gene Technology Co., Ltd.). The EBV LMp2A 340-349 SSSSSCPLSK short peptide was annealed with biotin-labeled HLA-A*1101 to prepare pHLA haploids. These haploids were combined with PE-labeled streptavidin (BD Company) to form PE-labeled tetramers, and the tetramers and anti-CD8-APC double-positive cells were sorted. Sorted cells were expanded and subjected to secondary sorting as described above, followed by monoclonalization by limiting dilution. Monoclonal cells were stained with tetramers, and the double-positive clones screened were as follows: image 3 shown.

Embodiment 2

[0128] Example 2 Obtaining the construction of the TCR gene and vector of EBV-specific T cell clones

[0129] with Quick-RNA TM MiniPrep (ZYMO research) extracted the total RNA of the EBV LMp2A340-349-specific and HLA-A1101-restricted T cell clones screened in Example 1. The cDNA was synthesized using clontech's SMARTRACE cDNA amplification kit, and the primers used were designed at the C-terminal conserved region of the human TCR gene. The sequence was cloned into T vector (TAKARA) for sequencing. After sequencing, the sequence structures of the TCR α chain and β chain expressed by the double-positive clone are shown in Figure 1 and Figure 2, respectively. Figure 1a , Figure 1b , Figure 1c and Figure 1d They are the amino acid sequence of TCRα chain variable domain, the nucleotide sequence of TCRα chain variable domain, the amino acid sequence of TCRα chain and the nucleotide sequence of TCRα chain; Figure 2a , Figure 2b , Figure 2c and Figure 2d They are the ...

Embodiment 3

[0139] Example 3 EBV-specific T cell receptor lentivirus packaging and transfection of primary T cells with EBV TCR

[0140] (a) Preparation of lentivirus by Express-In-mediated transient transfection of 293T / 17 cells

[0141] Lentivirus containing the gene encoding the desired TCR was packaged using a third-generation lentiviral packaging system. Using Express-In-mediated transient transfection (OpenBiosystems), four kinds of plasmids (one containing pLenti-EBVTRA-2A-TRB-IRES-NGFR described in Example 2) 293T / 17 cells were transfected with three lentiviral vectors and three plasmids containing other components necessary for the construction of infectious but non-replicating lentiviral particles.

[0142] For transfection, cells were seeded on day 0, in a 15 cm dish, seeded with 1.7 × 10 7 293T / 17 cells, so that the cells are evenly distributed on the culture dish, and the confluence is slightly higher than 50%. On day 1, transfect plasmids, package pLenti-EBVTRA-2A-TRB-IRE...

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Abstract

Provided is a T cell receptor (TCR) capable of combining peptide derived from Epstein barr virus (EB virus) latent membrane protein (LMP-2). The peptide is presented in the form of a SSCSSCPLSK-HLA A1101 compound. Also provided are a nucleic acid molecule for encoding the TCR, carrier comprising the nucleic acid molecule, and cell for transferring the TCR.

Description

technical field [0001] The present invention relates to a TCR capable of recognizing antigens derived from Epstein-Barr virus (Epstein Barr virus, EBV), and the present invention also relates to EBV-specific T cells obtained by transducing the above-mentioned TCR, and their use in the prevention and treatment of EBV-related diseases . Background technique [0002] EBV is a human herpes virus that is ubiquitous worldwide. Studies have shown that more than 95 percent of adults have antibodies against the virus, meaning they have been infected at some point. Most people who are infected will have EBV in their body for their entire lives, and there are usually few problems. However, in some cases, EBV has been associated with the development of some cancers, including Burkitt's lymphoma, Hodgkin lymphoma, EBV-positive posttransplant lymphoproliferative disease (PTLD), or nasopharyngeal carcinoma Wait. For example, LMP1 and LMP2 are latent membrane proteins belonging to EBV, ...

Claims

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Application Information

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IPC IPC(8): C07K14/725C12N15/12C12N15/86C12N5/10A61K38/17A61P35/00
CPCA61K38/17C12N5/10C12N15/86
Inventor 李懿李友佳
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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