Design, synthesis and biological evaluation of PD-1 derived peptides as inhibitors of PD-1/PD-L1 complex formation for cancer therapy.

Over the past few years, many molecules such as monoclonal antibodies, affibodies, nanobodies, and small compounds have been designed and tested as inhibitors of PD-1/PD-L1 complex formation. Some of them have been successfully implemented into clinical oncology practice. However, the majority of these compounds have disadvantages and limitations, such as high production price, potential for immunogenicity and/or prolonged clearance. Thus, new inhibitors of the PD-1/PD-L1 immune checkpoints are needed. Recently, peptides emerged as potential novel approach for blocking receptor/ligand interaction. In the presented studies we have designed, synthesised and tested peptides, which are potential inhibitors of the PD-1/PD-L1 axis. The amino acid sequences of the designed peptides were based on the binding sites of PD-1 to PD-L1, as determined by the crystal structure of the protein complex and also based on MM/GBSA analysis. Interactions of the peptides with PD-L1 protein were confirmed using SPR, while their inhibitory properties were studied using cell-based PD-1/PD-L1 immune checkpoint blockade assays. The characterization of the peptides has shown that the peptides PD-1(119-142), PD-1(119-142) and PD-1(122-138) strongly bind to PD-L1 protein and disrupt the interaction of the proteins. PD-1(122-138) peptide was shown to have the best inhibitory potential from the panel of peptides. Its 3D NMR structure was determined and the binding site to PD-L1 was established using molecular modelling methods. Our results indicate that the PD-1 derived peptides are able to mimic the PD-1 protein and inhibit PD-1/PD-L1 complex formation.