Screening And Activity Study Of Peptide Inhibitors Targeting Immune Checkpoint Protein HVEM

Application of immune checkpoint inhibitors is one of the hot spots in cancer immunotherapy.The interaction of immune checkpoint receptors and ligands can affect the activation and suppression of the immune system.What’s more,the clinical success of antibody drugs proves that blocking the inhibitory immune checkpoint pathway is a promising strategy for cancer immunotherapy.B and T lymphocyte attenuator(BTLA)is an inhibitory receptor similar to cytotoxic T Lymphocyte-Associated Antigen-4(CTLA-4)and programmed cell death protein 1(PD-1),which regulates T cell activation by binding to its unique ligand—herpes virus entry mediator(HVEM)which is often overexpressed by cancer cells to achieve immune escape.This characteristic makes it a potential target for novel immunotherapies.Here,four peptides were screened by phage display technology,the binding affinity between peptides and HVEM was determined by surface plasmon resonance(SPR);the inhibition ability of peptides to the formation of BTLA/HVEM complex was tested by competitive enzyme linked immunosorbent assay(ELISA).Polypeptide P1,whose sequence is GCTCPRGWVCLAREPC,showed the best affinity to HVEM.Further research shows that,P1-l,P1-c(1-3,2-4)and P1-c(1-4,2-3)both have high binding affinity to HVEM,and can effectively interfere with the interaction between BTLA/HVEM.Among that,the affinity between P1-c(1-4,2-3)and HVEM is the highest,the KD value is 1.46×10-7 M.However,in terms of competitive inhibition,P1-l was more dominant,and the semi-inhibitory concentration of P1-l was 1/5 of that of P1-c(1-4,2-3)in the same experimental conditions.We found that the blocking ability was due to the cysteine encompassed in this peptide.In addition,P1 showed no cytotoxicity to human peripheral blood mononuclear cell(PBMC)and no hemolytic activity.furthermore,all three configurations of P1 can relieve the immunosuppression caused by HVEM protein and stimulate the proliferation and activation of PBMC.In conclusion,these data highlight that P1 can relieve immune suppression and release immune system function by blocking BTLA/HVEM interaction,which should be taken in consideration as a potential drug precursor for cancer immunotherapy.