Specific T cell receptor targeting NY-ESO-1(157-165) epitope and anti-tumor application

[0051] Example 1. Preparation of NY-ESO-1(157-165) polypeptide / HLA-A2 tetramer and antigen-specific T cell sorting and TCR gene cloning

[0052] In this example, by preparing NY-ESO-1(157-165) / HLA-A2 tetramer, staining mouse splenocytes after immunization with CD3 and CD8 antibodies, and sorting NY- ESO-1(157-165) specific T cells for subsequent TCR screening.

[0053] 1. Preparation of NY-ESO-1(157-165) / HLA-A2 tetramer

[0054] (1) Refolding and purification of NY-ESO-1(157-165) / HLA-A2 complex

[0055] Optimize β2m (β2-microglobulin, HLA-A2 light chain gene) (Uniprot: P61769) and HLA-A2 heavy chain gene (Uniprot: P01892) according to prokaryotic codons, and add Express the sequence of biotin-specific binding polypeptide (Biotin-tag), synthesize the DNA sequence respectively, and introduce the enzyme cutting sites Nde I and Xho I respectively, wherein the Nde I enzyme cutting site is located at the 5' end of the sequence, and the enzyme cutting site Point Xho I is loca

[0097] Example 2. NY-ESO-1(157-165) / HLA-A2 tetramer binding experiment with cells expressing YW-TCR-1 TCR

[0098] In order to further confirm that the screened YW-TCR-1 TCR can specifically recognize NY-ESO-1(157-165) / HLA-A2, in this example, the α chain and β chain variable regions of YW-TCR-1 TCR (V region) (ie, SEQ ID NO: 1 and SEQ ID NO: 2) gene and human TCR α chain and β chain constant region (C region) gene (synthesized by Hongxun Biological Co., Ltd.) to form YW-TCR-1 TCR ( That is, the gene of SEQ ID NO:9+SEQ ID NO:10) was connected to the pCDH-GFP lentiviral expression plasmid (System Biosciences: Cat. No.: CD527A-1), and the chimeric YW-TCR-1 TCR expression vector (YW-TCR-1 -pCDH). HEK-293T cells (purchased from ATCC) were co-transfected with YW-TCR-1-pCDH plasmid and CD3-CD8-pCDH plasmid expressing CD3 and CD8. After 24 hours of transfection, the cells were centrifuged; washed three times with PBS containing 0.5% BSA, and centrifuged; - CD3 was co-incubated wi

[0100] Example 3. Preparation of YW-TCR-1 TCR protein and determination of its affinity with NY-ESO-1(157-165) / HLA-A2

[0101] The researchers further determined the binding affinity between YW-TCR-1 TCR and NY-ESO-1(157-165) / HLA-A2 at the protein level.

[0102] 1. Preparation of YW-TCR-1 TCR protein

[0103] The extracellular region genes of YW-TCR-1 TCR α chain and β chain were optimized according to prokaryotic codons, and the DNA sequences of the extracellular regions of YW-TCR-1 TCR α chain and β chain were synthesized, respectively, and introduced into restriction enzymes Sites Nde I and Xho I, wherein the Nde I restriction site is located at the 5' end of the sequence, and the restriction site Xho I is located at the 3' end of the sequence. The DNA sequences of the extracellular regions of the synthesized YW-TCR-1 TCR α chain and β chain were cloned into the expression vector pET21 a (Invitrogen) by using restriction sites Nde I and Xho I to establish the YW-TCR-1 TC