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T cell receptor for recognizing NY-ESO-1 antigen oligopeptide and coding sequence of T cell receptor

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

Active Publication Date: 2019-12-31
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 for recognizing NY-ESO-1 antigen oligopeptide and coding sequence of T cell receptor
  • T cell receptor for recognizing NY-ESO-1 antigen oligopeptide and coding sequence of T cell receptor
  • T cell receptor for recognizing NY-ESO-1 antigen oligopeptide and coding sequence of T cell receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0145] Example 1 Cloning NY-ESO-1 antigen short peptide-specific T cells

[0146] 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. The double-positive clones obtained through layers of screening still need to meet further functional tests.

[0147] The function and specificity of the T cell clone were further d...

Embodiment 2

[0149] Example 2 Construction of TCR gene and vector for obtaining NY-ESO-1 antigen short peptide-specific T cell clone

[0150] 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 the amino acid sequence of TCRα chain variable domain, the nucleotide sequence of TCRα chain variable domain, t...

Embodiment 3

[0160] Example 3 Expression, refolding and purification of NY-ESO-1 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 has been introduced in 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 nucleotide 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 5bshown. The target gene sequences of the above-mentioned TCRα and β chains were synthesized and inserted into the expression vector pET28a+ (Novagene) respectively by the standard method described in "Molecular Cloning a ...

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Abstract

The present invention provides a T cell receptor (TCR) capable of specifically binding to an oligopeptide SLLMWITQC derived from an NY-ESO-1 antigen, the antigen oligopeptide SLLMWITQC is capable of forming a complex with HLA A0201 and is presented together to the surface of a cell. The invention also provides nucleic acid molecules encoding the TCR and a vector comprising the nucleic acid molecules. In addition, the invention also provides a cell 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 (SEQ ID NO:9) 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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/725C07K19/00A61K35/17A61P35/00A61P37/00
CPCC07K14/7051A61K35/17A61P35/00A61P37/00C07K2319/00
Inventor 李懿胡静
Owner XLIFESC LTD
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