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T cell receptor (TCR) capable of identifying NY-ESO-1 antigen short-peptides

A variable, β-chain technology, applied in the field of TCR, can solve problems such as normal cell damage

Active Publication Date: 2017-05-10
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 (TCR) capable of identifying NY-ESO-1 antigen short-peptides
  • T cell receptor (TCR) capable of identifying NY-ESO-1 antigen short-peptides
  • T cell receptor (TCR) capable of identifying NY-ESO-1 antigen short-peptides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0144] Example 1 Cloning NY-ESO-1 Antigen Short Peptide-Specific T Cells

[0145] Peripheral blood lymphocytes (PBL) from healthy volunteers with genotype HLA-A0201 were stimulated with a synthetic short peptide SLLMWITQC (SEQ ID NO.:9; Beijing Saibaisheng Gene Technology Co., Ltd.). The SLLMWITQC 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.

Embodiment 2

[0146] Example 2 Obtaining the TCR gene and carrier construction of NY-ESO-1 antigen short peptide-specific T cell clone

[0147] with Quick-RNA TM MiniPrep (ZYMO research) extracted the total RNA of the short antigen peptide SLLMWITQC-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 TCR α chain variable domain amino acid sequence, TCR α chain variable domain nucleotide sequence, TCR α chain ...

Embodiment 3

[0157] Example 3 Expression, refolding and purification of NY-ESO-1 antigen short peptide-specific soluble TCR

[0158] 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 The introduced cysteine ​​residues are shown in bold and underlined letters. The target gene sequences of the above-mentioned TCR α and β chains were synthesized and inserted into the expression vector pET28...

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Abstract

The invention provides a T cell receptor (TCR) capable of being specifically combined with short-peptide SLLMWITQC derived from a NY-ESO-1 antigen. The antigen short-peptide SLLMWITQC and HLA A0201 can form a compound and are presented to the surfaces of cells together. The invention further provides nucleic acid molecules for encoding the TCR and a carrier comprising the nucleic acid molecules. In addition, the invention further provides cells for transducing the TCR.

Description

technical field [0001] The present invention relates to a TCR capable of recognizing short peptides derived from the NY-ESO-1 antigen. The present invention also relates to NY-ESO-1 specific T cells obtained by transducing the above TCR, and their role in the prevention and treatment of NY-ESO- 1 Use in related diseases. Background technique [0002] NY-ESO-1 belongs to the cancer-testis antigen (Cancer-Testis Antigen, CTA) family, which can be expressed in testis, ovarian tissue and many different types of tumor tissues, but not in other normal tissues. strong tumor antigens. NY-ESO-1 is an endogenous antigen, which is degraded into small molecular polypeptides after being produced in cells, and combines with MHC (major histocompatibility complex) molecules to form a complex, which is presented to the cell surface. SLLMWITQC is a short peptide derived from NY-ESO-1 antigen, which is a target for the treatment of NY-ESO-1 related diseases. Studies have shown that NY-ESO-1...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/725C12N15/12C12N15/867C12N5/10A61K38/17A61P35/00A61P37/02
CPCA61K38/00C07K14/7051C07K2319/00A61K35/17A61K38/17A61K48/00C12N5/10C12N15/867
Inventor 李懿相瑞瑞吴万里林燕梅李思韵
Owner XLIFESC LTD
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