Stapled peptide PROTAC induced significantly greater anti-PD-L1 effects than inhibitor in human cervical cancer cells

Abstract

IntroductionImmune checkpoint inhibitors (ICIs) are monoclonal antibodies that target immune checkpoints that suppress immune cell activity. Low efficiency and high resistance are currently the main barriers to their clinical application. As a representative technology of targeted protein degradation, proteolysis-targeting chimeras (PROTACs) are considered to have potential for addressing these limitations.MethodsWe synthesized a stapled peptide-based PROTAC (SP-PROTAC) that specifically targeted palmitoyltransferase ZDHHC3 and resulted in the decrease of PD-L1 in human cervical cancer cell lines. Flow cytometry, confocal microscopy, protein immunoblotting, Cellular Thermal Shift Assay (CETSA), and MTT assay analyses were conducted to evaluate the effects of the designed peptide and verify its safety in human cells.ResultsIn cervical cancer celllines C33A and HeLa, the stapled peptide strongly downregulated PD-L1 to < 50% of baseline level at 0.1 μM. DHHC3 expression decreased in both dosedependentand time-dependent manners. MG132, the proteasome inhibitor, can alleviate the SP-PROTAC mediated degradation of PD-L1 in human cancer cells. In a co-culture model of C33A and T cells, treatment with the peptide induced IFN-γ and TNF-α release in a dose-dependent manner by degrading PD-L1. These effects were more significant than that of the PD-L1 inhibitor, BMS-8.ConclusionsCells treated with 0.1 μM of SP-PROTAC or BMS-8 for 4 h revealed that the stapled peptide decreased PD-L1 more effectively than BMS-8. DHHC3-targeting SP-PROTAC decreased PD-L1 in human cervical cancer more effectively than the inhibitor BMS-8.

Targeted Protein Degradation (TPD) for Immunotherapy: Understanding Proteolysis Targeting Chimera-Driven Ubiquitin-Proteasome Interactions

“…A recent study reported on a PROTAC stapled peptide specifically targeting ZDHHC3, a palmitoyltransferase that regulates PD-L1 stability . This stapled peptide PROTAC (SP-PROTAC) was more effective than BMS-8, a PD-L1 inhibitor, in reducing PD-L1 levels and enhancing cytokine production and T-cell activation in human cervical cancer cells . Further, this study showed how SP-PROTACs can act as novel dual agents that both inhibit and degrade PD-L1.…”

“…This peptide-PROTAC system can effectively lower the PD-L1 and PD-1 expression in cervical cancer cells and boost the immune system’s ability to fight tumors. This study shows that peptides can be used as an alternative strategy to design potent and selective PD-L1 and PD-1 degraders …”

PTMs of PD-1/PD-L1 and PROTACs application for improving cancer immunotherapy

“…Compared with BMS-8, an ICI, SP-PROTAC displayed more effective on IFN-γ and TNF-α release. Taken together, SP-PROTAC targeted DHHC3 and alleviated PD-L1 protein levels in human cervical cancer ( 150 ).…”