US 11,718,827 B2
LRFFT2 cell
Shunchang Jiao, Beijing (CN); Rong Zhang, Beijing (CN); Tianfu Zhang, Beijing (CN); Zishan Zhou, Beijing (CN); Jiasen Xie, Beijing (CN); Ziming Wu, Beijing (CN); and Gang Peng, Beijing (CN)
Assigned to Beijing DCTY Biotech Co., Ltd., Beijing (CN); and Shunchang Jiao, Beijing (CN)
Filed by BEIJING DCTY BIOTECH CO., LTD., Beijing (CN); and Shunchang Jiao, Beijing (CN)
Filed on Jul. 11, 2019, as Appl. No. 16/508,740.
Claims priority of application No. 201811153257.5 (CN), filed on Sep. 30, 2018.
Prior Publication US 2020/0102537 A1, Apr. 2, 2020
Int. Cl. C12N 15/85 (2006.01); C12N 5/0783 (2010.01); C07K 14/725 (2006.01); C12N 7/00 (2006.01); C12N 15/11 (2006.01); C12N 15/86 (2006.01); G01N 33/50 (2006.01)
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).