Development of a Non-IgG PD-1/PD-L1 Inhibitor by in Silico Mutagenesis and an In-Cell Protein–Protein Interaction Assay,ACS Chemical Biology

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Development of a Non-IgG PD-1/PD-L1 Inhibitor by in Silico Mutagenesis and an In-Cell Protein–Protein Interaction Assay
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2021-01-15 , DOI: 10.1021/acschembio.0c00817
Boyang Ning _{1,

2} , Xueli Ren ₁ , Kyoji Hagiwara ₁ , Shinji Takeoka _{1,

2,

3} , Yoshihiro Ito _{1,

2,

4} , Hideyuki Miyatake _{1,

5}

Affiliation

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 in silico point mutagenesis of PD-1 to identify potent PD-1 variants with a higher affinity toward PD-L1 and refined the in silico 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 in silico mutagenesis combined with an in-cell PPI assay verification strategy successfully prepared a non-IgG inhibitor of the PD-1/PD-L1 interaction.

中文翻译：

通过计算机诱变和细胞内蛋白质-蛋白质相互作用测定开发非IgG PD-1 / PD-L1抑制剂

通过单克隆抗体（mAb）抑制程序性死亡1（PD-1）/程序性死亡配体1（PD-L1）轴是一种成功的癌症免疫疗法。但是，基于mAb的药物具有多种缺点，包括生产成本高和分子大小大，这促使我们开发出较小的替代药物。由于PD-L1以中等亲和力与PD-1结合，因此亲和力更高的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，我们进行了PD-1的计算机硅点诱变，以鉴定对PD-L1具有更高亲和力的有效PD-1变体，并改进了计算机使用基于萤光素酶的细胞内蛋白质-蛋白质相互作用（PPI）分析的结果。结果，开发了PD-1变体，它具有两个突变的氨基酸（T76Y，A132V），称为2-PD-1。2-PD-1可以以12.74 nM的解离常数与PD-L1结合。此外，2-PD-1以最大抑制浓度的一半为19.15 nM成功抑制了PD-1 / PD-L1的相互作用，并以最大有效浓度的一半为136.1 nM重新激活了T细胞。这些结果表明，计算机诱变结合细胞内PPI分析验证策略成功制备了PD-1 / PD-L1相互作用的非IgG抑制剂。

更新日期：2021-02-19

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