LYTACs that engage the asialoglycoprotein receptor for targeted protein degradation

Abstract

Selective protein degradation platforms have afforded new development opportunities for therapeutics and tools for biological inquiry. The first lysosome targeting chimeras (LYTACs) targeted extracellular and membrane proteins for degradation by bridging a target protein to the cation-independent mannose-6-phosphate receptor (CI-M6PR). Here, we developed LYTACs that engage the asialoglycoprotein receptor (ASGPR), a liver-specific lysosomal targeting receptor, to degrade extracellular proteins in a cell type-specific manner. We conjugated binders to a tri-GalNAc motif that engages ASGPR to drive downregulation of proteins. Degradation of EGFR by GalNAc-LYTAC attenuated EGFR signaling compared to inhibition with an antibody. Furthermore, we demonstrated that a LYTAC comprising a 3.4 kDa peptide binder linked to a tri GalNAc ligand degrades integrins and reduces cancer cell proliferation. Degradation with a single tri-GalNAc ligand prompted site-specific conjugation on antibody scaffolds, which improved the pharmacokinetic profile of GalNAc-LYTACs in vivo. GalNAc-LYTACs thus represent an avenue for cell-type restricted protein degradation.Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Degradation from the outside in: Targeting extracellular and membrane proteins for degradation through the endolysosomal pathway

“…Site-specific conjugates altered the pharmacokinetic profile of LYTACs in vivo. Whereas nonspecific Ctx-GalNAc conjugates cleared rapidly from plasma before 6 h, site-specific Ctx-GalNAc showed sustained presence for over 72 h (Ahn et al, 2021). This result demonstrated that the clearance regime of a LYTAC can be tuned by varying the number of ligands per antibody.…”

“…We showed internalization of a fluorophore-labeled IgG in a hepatocellular carcinoma (HCC) cell line, HEPG2, mediated by GalNAc-LYTACs. The internalization by a GalNAc-LYTAC was dramatically more efficient than that of an M6Pn-LYTAC in HEPG2 cells, likely due to the higher surface expression of ASGPR compared with M6PR on these hepatocytes (Ahn et al, 2021).…”

“…For membrane targets, LYTACs need to bind both the target and the LTR in cis, and the degradation of a target may be target and LTR dependent. For example, both Ctx-GalNAc and Ctx-M6Pn degraded greater than 50% of EGFR after 12 h of treatment and reached maximum degradation (70%-80%) after 24 h. However, Ptz-GalNAc removed greater than 50% of surface HER2 within 2 h, whereas Ptz-M6Pn took more than 24 h to achieve similar levels of removal (Ahn et al, 2021). The difference in kinetics between GalNAc-LYTAC and M6Pn-LY-TAC for HER2 indicates that the degradation rate may depend on the LTR.…”

Emerging protein degradation strategies: expanding the scope to extracellular and membrane proteins

“…Studies also revealed that triantenerrary GalNAc (tri-GalNAc) ligands engage ASGPR with higher affinity compared to GalNAc 65 , 66 . Tri-GalNAc ligands are synthesized from peracetylated GalNAc to tri-GalNAc-DBCO in 8 steps, and then are conjugated to azides on non-specifically labeled antibodies 23 . Tang's group directly used the commercially available Tri-GalNAc-COOH.…”

“…Bertozzi's group established the first-generation LYTAC, M6Pn-LYTAC, combining the ligand of the cation-independent mannose-6-phosphate receptor (CI-M6PR) with an extracellular protein binder 21 . Pioneering work from research groups of Bertozzi, Spiegel and Tang developed the second-generation LYTAC, GalNAc-LYTAC, with the advantage of cell-specific degradation by engaging the liver-specific asialoglycoprotein receptor (ASGPR) 23 - 25 . Wells and colleagues used the AbTAC, a bispecific antibody, to expand the scope of targeted degradation via transmembrane E3 ligases 22 .…”