CPC C12N 5/0636 (2013.01) [C07K 14/7051 (2013.01); C12N 7/00 (2013.01); C12N 15/11 (2013.01); C12N 15/86 (2013.01); G01N 33/505 (2013.01); C12N 2310/20 (2017.05); C12N 2501/998 (2013.01); C12N 2502/11 (2013.01); C12N 2510/00 (2013.01); C12N 2740/15043 (2013.01)] | 5 Claims |
1. An LRFFT2 cell, prepared by a method comprising the steps of:
1) using human peripheral blood for ctDNA sequencing or tumor tissues for whole exome sequencing, and screening out mutation sites;
2) performing antigen epitope prediction according to the mutation sites, and synthesizing a gene sequence of mutant peptides;
3) constructing a lentiviral vector expressing the mutant peptides, and packaging a lentivirus;
4) transfecting an antigen-presenting cell and co-culturing with a Peripheral Blood Mononuclear Cell (PBMC) to obtain an LFF cell;
5) stimulating the LFF cell with the mutant peptides as an antigen to screen out mutant polypeptides recognized by the LFF cell;
6) stimulating the LFF cell with the mutant polypeptides recognized by the LFF cell as an antigen, screening out a specific cell capable of recognizing the mutant polypeptides recognized by the LFF cell, and sequencing and obtaining a high-frequency TCR gene of the specific cell, wherein the high-frequency TCR gene of the specific cell is TCR6 comprising the DNA sequence of SEQ ID NO: 23 and the amino acid sequence of SED ID NO: 24;
7) knocking out an original TCR gene in peripheral blood T cells and transferring the TCR6 to obtain a TCR-T cell; and
8) blocking a suppressive signaling molecule by an antibody in vitro, to obtain an LRFFT2 cell;
wherein the suppressive signaling molecule comprises one or more of PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, and 2B4 (CD244).
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