Nano Medical Engineering Laboratory, RIKEN Cluster of Pioneering Research, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering, Waseda University, TWIns, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan.
ACS Chem Biol. 2021 Feb 19;16(2):316-323. doi: 10.1021/acschembio.0c00817. Epub 2021 Jan 15.
Inhibiting the programmed death-1 (PD-1)/programmed death ligand 1 (PD-L1) axis by monoclonal antibodies (mAbs) is a successful cancer immunotherapy. However, mAb-based drugs have various disadvantages including high production costs and large molecular sizes, which motivated us to develop a smaller alternative drug. Since PD-L1 binds PD-1 with moderate affinity, a higher affinity PD-1 variant should serve as a competitive inhibitor of the wild-type PD-1/PD-L1 interaction. In this report, we conducted point mutagenesis of PD-1 to identify potent PD-1 variants with a higher affinity toward PD-L1 and refined the results using a luciferase-based in-cell protein-protein interaction (PPI) assay. As a result, a PD-1 variant was developed that had two mutated amino acids (T76Y, A132V), termed 2-PD-1. 2-PD-1 could bind with PD-L1 at a dissociation constant of 12.74 nM. Moreover, 2-PD-1 successfully inhibited the PD-1/PD-L1 interaction with a half maximal inhibitory concentration of 19.15 nM and reactivated the T cell with a half maximal effective concentration of 136.1 nM. These results show that mutagenesis combined with an in-cell PPI assay verification strategy successfully prepared a non-IgG inhibitor of the PD-1/PD-L1 interaction.
抑制程序性死亡受体 1（PD-1）/程序性死亡配体 1（PD-L1）轴的单克隆抗体（mAbs）是一种成功的癌症免疫疗法。然而，mAb 类药物存在生产成本高和分子尺寸大等各种缺点，这促使我们开发一种更小的替代药物。由于 PD-L1 与 PD-1 结合具有中等亲和力，因此具有更高亲和力的 PD-1 变体应作为野生型 PD-1/PD-L1 相互作用的竞争性抑制剂。在本报告中，我们对 PD-1 进行了点突变，以鉴定对 PD-L1 具有更高亲和力的有效 PD-1 变体，并使用基于荧光素酶的细胞内蛋白质-蛋白质相互作用（PPI）测定法对结果进行了优化。结果，开发了一种具有两个突变氨基酸（T76Y、A132V）的 PD-1 变体，称为 2-PD-1。2-PD-1 与 PD-L1 的解离常数为 12.74 nM。此外，2-PD-1 成功抑制了 PD-1/PD-L1 相互作用，其半最大抑制浓度为 19.15 nM，半最大有效浓度为 136.1 nM，可重新激活 T 细胞。这些结果表明，通过突变体与细胞内 PPI 测定验证策略的结合，成功制备了 PD-1/PD-L1 相互作用的非 IgG 抑制剂。
