Sampathkumar, P.; Jung, H.; Chen, H.; Zhang, Z.; Suen, N.; Yang, Y.; Huang, Z.; Lopez, T.; Benisch, R.; Lee, S.-J.; Ye, J.; Yeh, W.-C.; Li, Y.

Molecules that facilitate targeted protein degradation (TPD) offer great promise as novel therapeutics. Human hepatic lectin, asialoglycoprotein receptor (ASGR) is selectively expressed on hepatocytes. We have previously engineered an anti-ASGR1 antibody-mutant RSPO2 (RSPO2RA) fusion protein (called SWEETS) to drive tissue-specific degradation of ZNRF3/RNF43 E3-ubiquitin ligases, leading to hepatocyte specific enhanced Wnt signaling, proliferation, and restored liver function in mouse models. Such an antibody-RSPO2RA fusion molecule is currently in human clinical trials. In the current study, we identified two new ASGR1 and ASGR1/2 specific antibodies, 8M24 and 8G8. High-resolution crystal structures of ASGR1:8M24 and ASGR2:8G8 complexes revealed that these antibodies bind to distinct epitopes on the opposite sides of ASGR, away from the substrate binding site. Both antibodies enhanced Wnt-activity when assembled as SWEETS molecules with RSPO2RA through specific effects sequestering E3 ligases. In addition, 8M24-RSPO2RA and 8G8-RSPO2RA efficiently downregulated ASGR1 through TPD mechanisms. These results demonstrate the possibility of combining different therapeutic effects and different degradation mechanisms in a single molecule.