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 ∼ 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.

靶向相互作用界面是获得程序性死亡受体1（PD-1）/ PD-1配体1（PD-L1）纳米抗体阻断剂的有效策略。为了验证该策略，使用Cn3D 4.1分析了PD-1和PD-L1细胞外域之间的相互作用界面。从人源化的纳米抗体噬菌体展示文库中，选择位于PD-1界面的PD-1 ^125-136肽作为抗原来筛选纳米抗体。筛选了六个不同的纳米抗体，分子量为12〜13 kDa，不包括单个碱性蛋白。选择了具有最长CDR3区的纳米抗体，称为PD-1-Nb-B20，用于进一步分析。为了进行大规模生产，对C端His6标记的纳米抗体编码序列进行了优化，并将其克隆到pET-21b中，以在T7启动子下过量表达。大肠杆菌BL21（DE3）。表达PD-1-Nb-B20，并选择过量表达PD-L1的胰腺腺癌细胞BxPC-3进行纳米抗体竞争抑制试验。纯化的纳米抗体可显着抑制PD-1与靶细胞表面的结合，表明其阻断PD-1 / PD-L1相互作用的能力。