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T cell receptor identifying SSX2 antigen oligopeptide

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

Active Publication Date: 2019-10-18
XLIFESC LTD
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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 receptor identifying SSX2 antigen oligopeptide
  • T cell receptor identifying SSX2 antigen oligopeptide
  • T cell receptor identifying SSX2 antigen oligopeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0145] Example 1 Cloning SSX2 Antigen Short Peptide-Specific T Cells

[0146] Peripheral blood lymphocytes (PBL) from healthy volunteers with genotype HLA-A0201 were stimulated with the synthetic short peptide KASEKIFYV (SEQ ID NO.:9; Beijing Saibaisheng Gene Technology Co., Ltd.). The KASEKIFYV short peptide was annealed with biotin-labeled HLA-A0201 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.

[0147] The function and specificity of the T cell clone were further detected by ELISPOT experiment. Those skilled in the art are familiar with the method of using ELISPOT assay to ...

Embodiment 2

[0149] Example 2 Obtaining the construction of the TCR gene and vector of the SSX2 antigen short peptide-specific T cell clone

[0150] with Quick-RNA TM MiniPrep (ZYMO research) extracted the total RNA of the antigenic short peptide KASEKIFYV-specific and HLA-A0201-restricted T cell clones screened in Example 1. The cDNA was synthesized using clontech's SMART RACE 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. It should be noted that this sequence is complementary and does not contain introns. 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 , Figure 1d , Figure 1e and Figure 1f They are the amino acid sequence of TCRα chain variable domain, the nucleotide sequence of TCRα chain variable...

Embodiment 3

[0160] Example 3 Expression, refolding and purification of SSX2 antigen short peptide-specific soluble TCR

[0161] In order to obtain a soluble TCR molecule, the α and β chains of the TCR molecule of the present invention may only include their variable domains and part of the constant domains respectively, and a cysteine ​​residue is introduced into the constant domains of the α and β chains respectively To form an artificial interchain disulfide bond, the positions of the introduced cysteine ​​residues are Thr48 of TRAC*01 exon 1 and Ser57 of TRBC2*01 exon 1; the amino acid sequence and nucleotides of the α chain sequence as Figure 4a and Figure 4b As shown, the amino acid sequence and nucleotide sequence of its β chain are as follows Figure 5a and Figure 5b shown. The target gene sequences of the above TCRα and β chains were synthesized and inserted into the expression vector pET28a+ (Novagene) respectively by the standard method described in "Molecular Cloning a L...

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Abstract

The invention provides a T cell receptor (TCR) capable of being specifically bound with oligopeptide KASEKIFYV derived from an SSX2 (synovial sarcoma X breakpoint 2) antigen. The antigen oligopeptideKASEKIFYV and HLA (human leukocyte antigen) A0201 can form a compound and be present to a cell surface. The invention further provides a nucleic acid molecule for encoding the TCR, and a carrier comprising the nucleic acid molecule. In addition, the invention further provides a cell for conducting the TCR.

Description

technical field [0001] The present invention relates to TCRs capable of recognizing short peptides derived from SSX2 antigens, the present invention also relates to SSX2-specific T cells obtained by transducing the above TCRs, and their use in the prevention and treatment of SSX2-related diseases. Background technique [0002] SSX2 is the synovial sarcoma X breakpoint, also known as HOM-MEL-40. SSX2 is one of ten highly homologous nucleic acid proteins of the SSX2 family. The SSX protein is a tumor-testis antigen expressed only in tumor cells and testicular germ cells without MHC expression. SSX2 is expressed in a variety of human cancer cells including, but not limited to, melanoma, head and neck cancer, lymphoma, various myelomas, pancreatic cancer, prostate cancer, sarcoma, hepatocellular carcinoma, and colon cancer. KASEKIFYV (SEQ ID NO:9) is a short peptide derived from the SSX2 antigen and is a target for the treatment of SSX2-related diseases. For the treatment of ...

Claims

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Application Information

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IPC IPC(8): C07K14/725C12N15/12A61K38/17A61P35/00A61P37/02
CPCC07K14/7051A61P35/00A61P37/02A61K38/00
Inventor 李懿胡静孙含丽
Owner XLIFESC LTD
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