Targeting the HVEM protein using a fragment of glycoprotein D to inhibit formation of the BTLA/HVEM complex

Cancer immunotherapy using blockade of immune checkpoints is mainly based on monoclonal antibodies. Despite the tremendous success achieved by using those molecules to block immune checkpoint proteins, antibodies possess some weaknesses, which means that there is still a need to search for new compounds as alternatives to antibodies. Many current approaches are focused on use of peptides/peptidomimetics to destroy receptor/ligand interactions. Our studies concern blockade of the BTLA/HVEM complex, which generates an inhibitory effect on the immune response resulting in tolerance to cancer cells. To design inhibitors of such proteins binding we based our work on the amino acid sequence and structure of a ligand of HVEM protein, namely glycoprotein D, which possesses the same binding site on HVEM as BTLA protein.To disrupt the BTLA and HVEM interaction we designed several peptides, all fragments of glycoprotein D, and tested their binding to HVEM using SPR and their ability to inhibit the BTLA/HVEM complex formation using ELISA tests and cellular reporter platforms. That led to identification of two peptides, namely gD(1-36)(K10CD30C) and gD(1-36)(A12C-L25C), which interact with HVEM and possess blocking capacities. Both peptides are not cytotoxic to human PBMCs, and show stability in human plasma. We also studied the 3D structure of the gD (1-36)(K10C-D30C) peptide using NMR and molecular modeling methods. The obtained data reveal that it possesses an unstructured conformation and binds to HVEM in the same location as gD and BTLA. All these results suggest that peptides based on the binding fragment of gD protein represent promising immunomodulation agents for future cancer immunotherapy.