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TCR capable of recognizing preferentially expressed antigen of melanoma (PRAME) short chain polypeptide and coding sequence of TCR

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

Active Publication Date: 2019-04-05
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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  • TCR capable of recognizing preferentially expressed antigen of melanoma (PRAME) short chain polypeptide and coding sequence of TCR
  • TCR capable of recognizing preferentially expressed antigen of melanoma (PRAME) short chain polypeptide and coding sequence of TCR
  • TCR capable of recognizing preferentially expressed antigen of melanoma (PRAME) short chain polypeptide and coding sequence of TCR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0144] Example 1 Cloning of PRAME Antigen Short Peptide-Specific T Cells

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

[0146] 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 ELISPO...

Embodiment 2

[0148] Example 2 Obtaining the construction of TCR gene and carrier of PRAME antigen short peptide-specific T cell clone

[0149] with Quick-RNA TM MiniPrep (ZYMO research) extracted the total RNA of the short antigen peptide VLDGLDVLL-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 TCR α chain variable domain, the nucleotide sequence of TCR α chain variable domain...

Embodiment 3

[0159] Example 3 Expression, refolding and purification of PRAME antigen short peptide-specific soluble TCR

[0160] 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 5bThe 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 vector pET28a+ (Novagene )...

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Abstract

The invention provides a T cell receptor (TCR) capable of specifically combining short chain polypeptide VLDGLDVLL derived from preferentially expressed antigen of melanoma (PRAME), the antigen shortchain polypeptide VLDGLDVLL and HLA A0201 can form a compound and be presented to the cell surface together. The invention further provides a nucleic acid molecule encoding the TCR and a carrier containing the nucleic acid molecule. In addition, the invention further provides cells transducing the TCR.

Description

technical field [0001] The present invention relates to TCRs capable of recognizing short peptides derived from PRAME antigens. The present invention also relates to PRAME-specific T cells obtained by transducing the above TCRs, and their use in the prevention and treatment of PRAME-related diseases. Background technique [0002] PRAME is a preferentially expressed antigen of melanoma (PRAME), which is expressed in 88% of primary and 95% of metastatic melanoma (Ikeda H, etal. Immunity, 1997, 6(2): 199-208) , normal skin tissue and benign melanocytes are not expressed. After PRAME is produced in cells, it is degraded into small molecular polypeptides, and combines with MHC (major histocompatibility complex) molecules to form a complex, which is presented to the cell surface. VLDGLDVLL (SEQ ID NO:9) is a short peptide derived from the PRAME antigen and is a target for the treatment of PRAME-associated diseases. In addition to melanoma, PRAME is also expressed in a variety of...

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
CPCC12N15/86C07K14/7051A61K38/00C12N2740/15043C12N2510/00
Inventor 李懿胡静相瑞瑞孙含丽
Owner XLIFESC LTD
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