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T cell receptor and application thereof

A cell receptor and expression vector technology, applied to T cell receptors and their application fields, can solve problems such as normal cell damage

Active Publication Date: 2022-03-22
特赛免疫(广州)科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, for the treatment of the above diseases, chemotherapy and radiotherapy can be used, but all of them will cause damage to the normal cells of the body.

Method used

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  • T cell receptor and application thereof
  • T cell receptor and application thereof
  • T cell receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1 Clone-specific T cells

[0086] 1. Experimental method

[0087] Peripheral blood mononuclear cells (PBMC) from healthy volunteers genotyped for HLA-A*11:01 were stimulated by synthetic CT83 polypeptide LASSILCALIVFWKYRRFQR (GenScript Biotechnology Co., Ltd.).

[0088] The INF-γ expression of specific T cells was detected by ELISPOT kit (Dayou, catalog number 2110005), CD3+ T cells were first circled by flow cytometry, and CD8+CD137+ T cells were sorted out by flow cytometry cell( figure 1 ), and then amplified with high-dose IL2 (PeproTech, catalog number 200-02-50UG).

[0089] 2. Experimental results

[0090] The results of flow cytometry sorting were as follows figure 1 As shown, sorting out 5×10 4 CD3+CD8+CD137+ T cells, expanded to 2×10 5 .

Embodiment 2

[0091] Example 2 Acquisition of TCR gene of CT83-specific CD8+ T cell clone and construction of vector

[0092] 1. Acquisition of TCR genes of CT83-specific CD8+ T cell clones

[0093] 1. Experimental method

[0094] Sequence analysis was performed on the obtained TCR by performing single-cell transcriptome and TCR library sequencing on the T cells obtained by sorting and expanding by flow cytometry in Example 1.

[0095] 2. Experimental results

[0096] The results showed that the amino acid sequences of the α chain and β chain with the highest proportion (20.8%) of CDR3 clones of TCR are shown in SEQ ID NO: 3 and SEQ ID NO: 7. The nucleotide sequences of the coding genes are respectively shown in SEQ ID NO: 4 and SEQ ID NO: 8. Wherein the amino acid sequence is as shown in SEQ ID NO: 3, the amino acid sequence of the variable domain of the alpha chain is shown in SEQ ID NO: 1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO: 2 respectively; the amin...

Embodiment 3

[0123] Example 3 TCR retrovirus packaging and primary T cell transfection

[0124] 1. Preparation of retrovirus by GALV cells

[0125] 1. Experimental method

[0126] A retrovirus containing the gene encoding the TCR of Example 1 was packaged using a retrovirus packaging system. GALV cells themselves have been transduced with packaging plasmids, only need to add the target plasmid, using Lipofectamine TM 3000 kit (Invitrogen, catalog number L3000015) was used to transduce the pMP71-LNGFR-T2A-TRB-P2A-TRA and pMP71-eGFP plasmids prepared in Example 2 into GALV cells.

[0127] For transfection, cells were seeded on day 0 in six-well plates at 1.0 × 10 6 GALV cells per well, so that the cells are evenly distributed on the culture dish, and the confluence is slightly higher than 70%. Transduce the plasmid on the first day, and prepare the solution (the amount per well of a six-well plate is): Solution A: 250 μL Opti-MEM (Gibco, catalog number 31985-070) + 7.5 μL Lipofectamine ...

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Abstract

The invention discloses a T cell receptor (TCR) and an application thereof, and the TCR can be specifically combined with a CT83 antigen short peptide CT8314-22 / HLA-A * 11: 01 compound; t cells transduced with the TCR can be specifically activated by dendritic cells expressing a CT8314-22 / HLA-A * 11: 01 compound, and have a powerful killing effect on target cells (such as cancer cells) positive to CT83 and HLA-A * 11: 01; the invention provides a new way for TCR-T cell immunotherapy of CT83 cancer testis antigen related diseases.

Description

technical field [0001] The invention relates to the technical field of immunotherapy, in particular to a T cell receptor (TCR) and its application. Background technique [0002] Testis antigen 83 (CT83), as an endogenous protein, is highly expressed in various malignant tumor cells, and belongs to the class of cancer testis antigens. After CT83 is produced in cells, it is degraded into small molecule short peptides of different lengths. Among them, the short peptides that can bind to HLA molecules form complexes with them and are transported and presented to the cell surface. CT83 was first discovered by Fukuyama et al. in 2006 [FUKUYAMA T, HANAGIRI T, TAKENOYAMA M, et al. Identification of a new cancer / germLine gene, CT83, encoding an antigen recognized by autologous CTInduced on human lung adenocarcinoma. 2006,66(9):4922-8.]; later studies have shown that CT83 in 81% gastric cancer [Shida A, Futawatari N, Fukuyama T, et al.Frequent High Expression of Kita-Kyushu Lung Canc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/725C12N15/12C12N15/867C12N5/10A61K39/00A61P35/00
CPCC07K14/7051C12N15/86C12N5/0636A61K39/0011A61P35/00C12N2740/10043C12N2740/15043C12N2510/00
Inventor 李亮平李清扬
Owner 特赛免疫(广州)科技有限公司
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