期刊
PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY
卷 50, 期 3, 页码 252-259出版社
TAYLOR & FRANCIS INC
DOI: 10.1080/10826068.2019.1692217
关键词
Cancer; immunotherapy; nanobody; phage display; programmed death receptor 1 (PD-1)
类别
资金
- Science and Technology Planning Projects of Guangdong Province [2014A020210027]
- Major Scientific Research Projects of the Universities of Guangdong Province [2016KZDXM042]
- Natural Science Foundation of Guangdong Province [2015A030310120]
- National Natural Science Foundation of China [81273423]
Targeting the interaction interface is an effective strategy to obtain programmed death receptor 1 (PD-1)/PD-1 ligand 1 (PD-L1) nanobody blockers. To validate this strategy, the interaction interface between PD-1 and the PD-L1 extracellular domain were analyzed using Cn3D 4.1. The peptide PD-1(125-136) located at the interface of PD-1 was selected as the antigen to screen nanobodies from a humanized nanobody phage display library. Six different nanobodies were screened, with molecular weights of 12 similar to 13 kDa, excluding a single basic protein. The nanobody with the longest CDR3 region, termed PD-1-Nb-B20, was selected for further analysis. For mass production, the C-terminal His6-tagged nanobody coding sequence was optimized and cloned into pET-21b for over-expression under the T7 promoter in Escherichia coli BL21 (DE3). PD-1-Nb-B20 was expressed and pancreatic adenocarcinoma cells BxPC-3 over-expressing PD-L1 were selected for nanobody competitive inhibition assays. The purified nanobodies significantly inhibited PD-1 binding to the surface of target cells, indicating their ability to block the PD-1/PD-L1 interaction.
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