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T cell receptor (TCR) capable of recognizing short peptide of PRAME antigen

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

Active Publication Date: 2019-09-24
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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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 recognizing short peptide of PRAME antigen
  • T cell receptor (TCR) capable of recognizing short peptide of PRAME antigen
  • T cell receptor (TCR) capable of recognizing short peptide of PRAME antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0139] Example 1 Cloning of PRAME Antigen Short Peptide-Specific T Cells

[0140] Peripheral blood lymphocytes (PBL) from healthy volunteers with genotype HLA-A11 were stimulated with synthetic short peptide PRAME 176-184EVLVDLFLK (Beijing Saibaisheng Gene Technology Co., Ltd.). The PRAME176-184EVLVDLFLK short peptide was refolded with biotin-labeled HLA-A*1101 to prepare pHLA monomers. These monomers 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 monoclonal culture by limiting dilution. Monoclonal cells were stained with tetramers and anti-CD8 antibodies, and the double-positive clones screened were as follows: image 3 shown.

Embodiment 2

[0141] Example 2 Obtaining the construction of the TCR gene and carrier of the PRAME antigen short peptide-specific T cell clone of the present invention

[0142] with Quick-RNA TM MiniPrep (ZYMO research) extracted the total RNA of the PRAME 176-184EVLVDLFLK-specific, HLA-A11-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. 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 with Figure 1d They are the amino acid sequence of TCRα chain variable domain, the nucleotide sequence of TCRα chain variable domain, the amino acid sequence of TCRα chain and the nucleotide sequence of TCRα chain; Figure 2a ...

Embodiment 3

[0152] Example 3 Expression, refolding and purification of antigenic short peptide-specific soluble TCR of the present invention

[0153] 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 with Figure 4b As shown, the amino acid sequence and nucleotide sequence of its β chain are as follows Figure 5a with Figure 5b The introduced cysteine ​​residues are shown in bold and underlined letters. The target gene sequences of the above TCRα and β chains were synthesized and inserted into the expression vec...

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Abstract

The invention provides a T cell receptor (TCR) capable of specifically recognizing short peptide EVLVDLFLK derived from a PRAME antigen, particularly provides the TCR which can bond with an EVLVDLFLK-HLA-A*1101 complex presented to cell surfaces. The invention also provides a nucleic acid molecular sequence coding the TCR and a vector containing the nucleic acid molecular sequence.

Description

technical field [0001] The present invention relates to a TCR capable of specifically recognizing short peptides of PRAME antigens, and also relates to PRAME-specific T cells obtained by transducing the above TCR, and their use in preventing and treating PRAME-related diseases. Background technique [0002] As an endogenous tumor antigen, RPAME 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 EVLVDLFLK is a short peptide derived from PRAME. In addition to being expressed in solid tumors such as melanoma, renal cell carcinoma, lung cancer, breast cancer, and medulloblastoma, PRAME antigen is also expressed in some hematological malignancies, such as acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic Cell carcinoma, myeloma, etc. (Kessler JH, et al. J Exp Med, 2001, 193(1): 73-88. ...

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
CPCC07K14/7051C12N15/86C12N5/0636A61K35/17A61P35/00A61P37/02C12N2800/107C12N2740/15043C12N2510/00A61K38/00
Inventor 李懿陈安安
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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