Development of a Non-IgG PD-1/PD-L1 Inhibitor by in Silico Mutagenesis and an In-Cell Protein-Protein Interaction Assay.

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.

Conjugation of biphenyl groups with poly(ethylene glycol) to enhance inhibitory effects on the PD-1/PD-L1 immune checkpoint interaction.

Monoclonal antibodies have been developed as anticancer agents to block immune checkpoint pathways associated with programmed cell death 1 (PD-1) and its ligand PD-L1. However, the high cost of antibodies has encouraged researchers to develop other inhibitor types. Here, biphenyl compounds were conjugated with poly(ethylene glycol) (PEG) to enhance the activity of small molecular inhibitors. Immunoassay results revealed the decrease in the inhibition activity following conjugation with linear PEG, suggesting that the PEG moiety reduced the interaction between the biphenyl structure and PD-L1. However, the inhibitory effect on PD-1/PD-L1 interaction was further enhanced by using branched PEG conjugates. The increase in the number of conjugated biphenyl compounds resulted in increased inhibitory activity. The highest IC50 value was 0.33 µM, which was about 5 times higher than that observed for a non-conjugated monovalent compound. The inhibitory activity was more than 20 times the activity reported for the starting compound. Considering the increase in the inhibition activity, this multivalent strategy can be useful in the design of new immune checkpoint inhibitors.