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T-cell receptors that recognize short peptides of the mage-a1 antigen

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

Active Publication Date: 2020-09-25
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 receptors that recognize short peptides of the mage-a1 antigen
  • T-cell receptors that recognize short peptides of the mage-a1 antigen
  • T-cell receptors that recognize short peptides of the mage-a1 antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0145] Example 1 Cloning of MAGE-A1 Antigen Short Peptide-Specific T Cells

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

[0147] Example 2 Construction of TCR gene and vector for obtaining MAGE-A1 antigen short peptide-specific T cell clone

[0148] with Quick-RNA TM MiniPrep (ZYMO research) extracted the total RNA of the short antigenic peptide KVLEYVIKV-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,...

Embodiment 3

[0158] Example 3 Expression, refolding and purification of MAGE-A1 antigen short peptide-specific soluble TCR

[0159] 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. By the standard method described in > (Molecular Cloning a Laboratory Manual) (third edition, Sambrook and Russell), the target gen...

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Abstract

The invention provides a TCR (T Cell Receptor) which is capable of specifically binding oligopeptides KVLEYVIKV derived from an MAGE-A1 (Melanoma antigen A1). A compound can be formed by the oligopeptides KVLEYVIKV derived from the MAGE-A1 and an HLA (Human Leukocyte Antigen) A0201, and the oligopeptides KVLEYVIKV and the HLA A0201 can be transferred to cell surfaces together. The invention also provides a nucleic acid molecule for coding the TCR and a carrier comprising the nucleic acid molecule. 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 MAGE-A1 antigen, and the present invention also relates to MAGE-A1-specific T cells obtained by transducing the above TCR, and their use in the prevention and treatment of MAGE-A1-related diseases . Background technique [0002] As an endogenous tumor antigen, MAGE-A1 is degraded into small molecular polypeptides after being generated in cells, and combines with MHC (major histocompatibility complex) molecules to form a complex, which is presented to the cell surface. Studies have shown that KVLEYVIKV is a short peptide derived from MAGE-A1. MAGE-A1 protein is expressed in a variety of tumor types, including melanoma, and other solid tumors such as gastric cancer, lung cancer, esophageal cancer, bladder cancer, squamous cell carcinoma of the head and neck, etc. For the treatment of the above diseases, methods such as chemotherapy and radiotherapy can be used...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/725C12N15/13A61K38/17A61P35/00A61P37/02
CPCA61K38/00C07K14/7051
Inventor 李懿林燕梅相瑞瑞
Owner XLIFESC LTD
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