ROTACs leverage signaling-incompetent R-spondin for targeted protein degradation

Proteolysis-targeting chimeras (PROTACs) are an emerging technology for therapeutic intervention but options to target cell surface proteins and receptors remain limited. Here we introduce ROTACs, bispecific WNT- and BMP signaling-disabled R-spondin (RSPO) chimeras, which leverage the specificity of these stem cell growth factors for ZNRF3/RNF43 E3 transmembrane ligases, to target degradation of transmembrane proteins. As proof of concept, we targeted the immune checkpoint protein programmed death ligand 1 (PD-L1), a prominent cancer therapeutic target, with a bispecific RSPO2 chimera, R2PD1. The R2PD1 chimeric protein bound PD-L1 and at picomolar concentration induced its lysosomal degradation. In three melanoma cell lines, R2PD1 induced between 50-90% PD-L1 protein degradation. PD-L1 degradation was strictly dependent on ZNRF3/RNF43. We conclude that signaling-disabled ROTACs represent a novel strategy to target cell surface proteins for degradation.