Compact clustering of highly oligomerized anti-DR5 nanobodies effectively drives apoptotic cancer cell death, significantly suppressing tumor growth

Death receptors (DRs) are attractive targets for cancer therapy due to their tight regulation by apoptosis-inducing ligands and their high expression on many cancer cells. Here, we employ a high affinity anti-DR5 nanobody (aDR5Nb) as a DR5 agonist and generate various oligomeric aDR5Nb clusters with different oligomerization states and arrangements, using a variety of scaffold proteins. Hexameric and higher-oligomeric aDR5Nb clusters efficiently initiate DR5-mediated apoptotic signals in lung and breast cancer cells, leading to significant apoptotic cancer cell death. Compact aDR5Nb clusters containing the same number of aDR5Nb in a cluster exhibit superior cytotoxic effects compared to more spatially dispersed clusters, demonstrating that both the number and compactness of aDR5Nb clusters are critical for driving robust DR5-mediated cancer cell death. Notably, highly compact and oligomerized dodecameric aDR5Nb clusters are required to overcome resistance in TRAIL-resistant lung and breast cancer cells. Compact dodecameric aDR5Nb clusters substantially suppress tumor growth in a TRAIL-resistant lung cancer xenograft model without notable side effects. Our study clearly demonstrates the importance of both oligomerization and the compactness of aDR5Nb clusters in enhancing apoptotic signaling and antitumor activity, offering a promising strategy for improving DR5-mediated cancer therapies by optimizing the nanoscale environment of cancer cells.