PD-S15 fusion protein specifically target PD-1 and rapidly expands NK/T cells
10.3872/j.issn.1007-385x.2019.04.004
- VernacularTitle:PD-S15融合蛋白体外特异性靶向PD-1分子并快速扩增NK/T细胞
- Author:
WANG Axiang
1
;
LI Tiepeng
1
;
ZHANG Fang
1
;
WANG Yao
1
;
DU Xuexiang
1
;
GAO Quanli
1
Author Information
1. (Cancer Biotherapy Center, Cancer Hospital of Henan Province, Cancer HospitalAffiliated to Zhengzhou University
- Publication Type:Journal Article
- Keywords:
programmed death receptor-1 (PD-1);
IL-15/IL-15Rα;
PD-S15 fusion protein;
adoptive cell therapy;
antigen-specific T cell
- From:
Chinese Journal of Cancer Biotherapy
2019;26(4):389-395
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To investigate the function of anti-PD-1 (scFv)/IL-15/IL-15Rα-sushi fusion protein (PD-S15) to specifically bind to PD-1 in vitro and to explore its effect on NK/T cell proliferation. Methods: The human anti-PD-1 (scFv) gene sequence and human IL-15/IL-15Rα-sushi fusion gene sequence were synthesized chemically. The recombinant expression plasmid pUC57-PD-S15 was constructed by enzyme digestion and ligation of the two target genes, and then transiently transfected into HEK293T cells by lipofectamineTM 2000. The supernatants of cell culture medium were acquired, and the expression of PD-S15 fusion protein in cell culture supernatants was detected by Wb assay. PBMCs and TILs were cultured in mediums with different proportion of PD-S15/X-VIVOTM15, respectively. Then, the capacity of PD-S15 fusion protein to bind to PD-1 in vitro and its effect on the proliferation of PBMCs and the proportion of CD3+CD8+, CD3+CD4+ and CD3-CD56+ subsets were detected by flow cytometry. The effect of PD-S15 fusion protein on the proliferation of TILs was detected by cytometry. Results: The successful construction of pUC57-PD-S15 eukaryotic expression plasmid was confirmed by double enzyme digestion and sequencing, and then successfully transfected into HEK293T cells. The relative molecular weight of the target protein was approximately 55 000, and was in line with expectations. PD-S15 fusion protein could specifically combine with PD-1 in vitro (P<0.05) and stimulate NK/T cell proliferation (P<0.05). Compared with classical TILs culture method, the efficiency of activation and amplification of T cells in vitro by PD-S15 culturemethodwasbetter (P<0.01). Conclusion: PD-S15 fusion protein can specifically target PD-1 and rapidly expand NK/T cells in vitro, which lays a foundation for the selective expansion of CD8+PD-1+ antigen-specific T lymphocytes from tumor tissues and even peripheral blood.
- Full text:20190404.pdf
