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T cell receptor for recognizing EBV antigen short peptide and application thereof

A technology of cell receptors and cells, applied in the direction of receptors/cell surface antigens/cell surface determinants, applications, antibody medical components, etc., can solve problems such as long time, harsh conditions, TIL application restrictions, etc., and achieve low toxicity and side effects , strong tumor-specific effect

Pending Publication Date: 2021-01-29
魏放
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the harsh conditions for the isolation and culture of TIL, it takes a long time to reach the number of cells for clinical treatment, and more importantly, so far, the tumor tissue that can successfully isolate TIL is still very limited, so that TIL has a great role in tumors. Clinical application is limited

Method used

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  • T cell receptor for recognizing EBV antigen short peptide and application thereof
  • T cell receptor for recognizing EBV antigen short peptide and application thereof
  • T cell receptor for recognizing EBV antigen short peptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 - Construction of retroviral vectors to express EBV-specific TCR genes

[0079] Methods for cloning and recombination of genes are well known in the art, and are described in, for example, (Sambrook and Russell et al., "Molecular Cloning-A Laboratory Manual" (Molecular Cloning-A Laboratory Manual) (Third Edition) (2001) CSHL Press) There are detailed descriptions in standard manuals.

[0080] Specifically, total RNA was extracted from EBV-specific cytotoxic T lymphocytes (CTL) with RNApure cultured cell total RNA extraction kit (Beijing, Tiangen Biochemical Technology Co., Ltd.). The cDNA was synthesized using clontech's SMARTerRACE cDNA amplification kit. The primers used for cDNA amplification were: 3'-primers designed at the C-terminal constant region of the human TCR gene, and 5'-primers using the SMARTerRACE cDNA amplification kit In SMARTer Oligo, the sequence of the TCR with a complete 5' end is obtained by rapidly amplifying the cDNA. Amplified produ...

Embodiment 2

[0095] Preparation of embodiment 2-TCR-retrovirus

[0096] Preparation of recombinant plasmids: Transform Dh5α competent cells with the recombinant plasmid pBabe-TCR obtained in Example 1, spread evenly on the LB solid medium plate containing ampicillin, culture at 37°C for 24 hours, pick a single colony to In LB liquid medium containing ampicillin, shake culture at 37°C and 220rpm / min for 14-16h, and extract the plasmid.

[0097] Packaging of recombinant plasmids: Take phenix cells in the logarithmic growth phase as packaging cells, inoculate them into a 10 cm dish containing medium (IMDM containing 10% FBS), and when the cell density reaches 80-85%, precipitate with standard calcium phosphate The recombinant plasmid pBabe-TCR described in Example 1 was transfected by the method. After culturing for 8-9 hours, remove the medium containing the transfection reagent and replace with fresh complete medium. After 24 hours, the culture solution was collected and filtered with a 0...

Embodiment 3-E

[0098] Example 3- Preparation of EBV-specific TCR-T cells and expression analysis of TCR

[0099] Peripheral blood from healthy volunteers was collected, and human peripheral blood mononuclear cells (PBMC) were obtained by using a lymphocyte separation tube (made by Shenzhen Daktronics). Adjust the cell density to 1 x 10 6 cells / ml, and OKT-3 antibody (30ng / ml) and IL-2 (600U / ml) were added to the cell culture medium to activate the T cells therein. After 48 hours, the retrovirus was taken out from the -80°C low-temperature refrigerator, and quickly thawed in a 37°C water bath. In a 24-well plate coated with RetroNectin (Takara) in advance, place 0.5×10 6 Add 1.5ml of virus supernatant to PBMC, and add IL-2 (600U / ml) at the same time, gently blow and mix, and centrifuge at 930g at 32°C for 90 minutes. Then placed at 37°C, 5% CO 2 continue to grow in the incubator. After 24 hours, the culture supernatant containing the virus was replaced with fresh medium, and the culture ...

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Abstract

The present invention provides a T cell receptor (TCR) capable of binding to an oligopeptide derived from an early antigen BMLF1 of the EB virus (EBV), the antigen oligopeptide having the amino acid sequence GLCTLVAML (SEQ ID No: 1), which can form a complex with HLA-A0201 and is presented together to the surface of a cell. The invention also provides nucleic acid molecules encoding the TCRs and vectors comprising the nucleic acid molecules, as well as cells for transducing the TCRs of the invention. The invention also provides application of the TCR, the nucleic acid molecule, the vector andthe cell in preparation of EBV specific T cells and in treatment of EBV related malignant tumors.

Description

technical field [0001] The present invention relates to a T cell receptor (TCR) capable of specifically recognizing a short peptide derived from the early EBV antigen BMLF1. The present invention also relates to transducing the above-mentioned TCR to obtain EBV-specific T cells and its use in treating and / or preventing diseases related to EBV. Background technique [0002] A large number of studies have shown that Epstein-Barr virus (EBV) is closely related to a variety of tumors, including Burkitt's lymphoma, which is highly prevalent in African children, and nasopharyngeal carcinoma, which is only highly prevalent in my country (Griffin BE&Xue SA, Ann Med., 1998, 30(3): 249-259; Kieff E & Rickinson AB, Fields Virology, Vol 2.4thed: Lippincott: Philadelphia, PA; 2001: 2343-2446). EBV exists in almost 100% of highly malignant NK / T cell lymphoma / leukemia (Kwong YL, Leukemia, 2005, 19(12):2186-2194). About 90-95% of the world's population is infected by EBV. Once infected, EB...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/725C07K19/00C12N15/12A61K39/00A61P35/00
CPCC07K14/7051A61K39/001102A61P35/00A61K2039/5158C07K2319/00
Inventor 薛少安聂苏秦
Owner 魏放
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