Boosting Robust DR5-Mediated Cell Death in Cancer Cells through Oligomerization of anti-DR5 nanobodies

Abstract

Clustering of death receptors has been important, as it can enhance the effects of death receptor- mediated apoptosis in cancer cells. Several strategies have been performed to recruit death receptors (DRs) on the surface of cancer cells. However, there is a lack of research on how the number and spatial arrangement of ligands in these strategies relates to their ability to trigger apoptosis and whether these conditions overcome TRAIL-resistance. In this work, ST-fused monomeric and dimeric aDR5Nbs were designed, and then ligated monomeric or dimeric aDR5Nb(s) to a SC-fused helix bundle oligomer protein and were able to make from monomeric state up to dodecameric state of aDR5Nb. Using these constructs, i aimed to determine how the oligomeric state of aDR5Nb is related to the killing efficacy. Several experiments revealed that increasing oligomeric state of aDR5Nb is associated with enhanced killing efficacy in TRAIL-sensitive cancer cell lines. In addition, i displayed aDR5Nbs on oligomers with different shapes and widths and investigated how the same number of aDR5Nb in different configurations and orientations affected the killing efficacy. The results showed that narrower inter-ligand distance with compactness of aDR5Nbs is enabled to trigger higher apoptosis-mediated cell death. Finally, i tried to investigate whether the optimal oligomeric state and configuration of aDR5Nb could overcome TRAIL-resistance. The results confirmed that the highest oligomeric state with compact configuration and orientation, oHex/Di-aDR5Nb (x12), could induce apoptosis-mediated cell death at a rapid rate with high efficacy even in TRAIL-resistant cancer cells. My study demonstrates how the positioning and number of aDR5Nb should be adjusted to enhance the existing weak DR5-mediated apoptosis.