Harnessing the Lysosomal Sorting Signals of the Cation-Independent Mannose-6-Phosphate Receptor for Targeted Degradation of Membrane Proteins

Abstract

Membrane proteins are a crucial class of therapeutic targets that remain challenging to modulate using traditional occupancy-driven inhibition strategies or current proteolysis-targeting degradation approaches. Here, we report that the inherent endolysosomal sorting machinery can be harnessed for the targeted degradation of membrane proteins. A new degradation technique, termed signal-mediated lysosome-targeting chimeras (SignalTACs), was developed by genetically fusing the signaling motif from the cation-independent mannose-6-phosphate receptor (CI-M6PR) to a membrane protein binder. Antibody-based SignalTACs were constructed with the CI-M6PR signal peptides fused to the C-terminus of both heavy and light chains of IgG. We demonstrated the scope of this platform technology by degrading five pathogenesis-related membrane proteins, including HER2, EGFR, PD-L1, CD20, and CD71. Furthermore, two simplified constructs of SignalTACs, nanobody-based and peptide-based SignalTACs, were created and shown to promote the lysosomal degradation of target membrane proteins. Compared to the parent antibodies, SignalTACs exhibited significantly higher efficiency in inhibiting tumor cell growth both in vitro and in vivo. This work provides a simple, general, and robust strategy for degrading membrane proteins with molecular precision and may represent a powerful platform with broad research and therapeutic applications.

IGF2 Peptide-Based LYTACs for Targeted Degradation of Extracellular and Transmembrane Proteins

“…Bispecific molecules that direct the internalization and degradation of these proteins have shown great promise as a therapeutic approach. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] While such molecules have been reported for some time, [35][36][37] it is only recently that specific endosomal targeting molecules such as LYTAC, KineTAC, etc. have been described in the literature.…”

“…have been described in the literature. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] LYTACs are sugar-conjugated antibodies that bind the target protein and a lysosome-targeting receptor, i.e., cation-independent mannose-6-phosphate receptor (CI-M6PR) 1,5 or asialoglycoprotein receptor (ASGPR), [2][3][4][5] thereby inducing internalization, lysosomal trafficking, and degradation of membrane proteins. The LYTAC compounds described to date are based on conjugates, which would be challenging for large-scale production and therefore not feasible as potential therapeutic agents.…”

“…LYTACs, also called MoDEs for molecular degraders of extracellular proteins 3 , are bifunctional conjugates that simultaneously bind the extracellular domain of a target protein of interest (POI) and a cell-surface lysosome-targeting receptor (LTR) to form a ternary complex, leading to protein internalization via clathrin-mediated endocytosis 7 and subsequent degradation in a lysosome. Currently known LYTACs at the POI-targeting side contain antibodies, small molecules, peptides, aptamers [1][2][3][4][5][6][8][9][10][11][12][13][14][15][16][17] These are linked to the LRK-binding glycopeptides, peptides, aptamers, dendritic DNA, and cytokines that bind to the receptors facilitating endocytosis and lysosomal degradation: the cation-independent mannose-6-phosphate receptor (CI-M6PR), 1,[11][12][13][14] , the liver-specific asialoglycoprotein receptor 3,4,9,15 , integrin 8 , the transmembrane E3 ligase ring finger 43 (RNF43) 10 , surface scavenger receptors, 16 the cytokine decoy recycling receptor CXCR7 17 .…”

“…LYTACs are bifunctional conjugates that bind both the extracellular dom ain of a target protein of interest (POI) and a cell-surface lysosom e-targetińg receptor (LTR) to form a ternary com plex, leading to the internalization of the POI protein via endocytosis [5 ] an d its su b seq u en t d eg rad atio n in ly so so m es. C u rren tly k n o w n LYTA Cs on th e P O I targeting side include antibodies, sm all m olecules, peptides and aptam ers [1,2,[6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22]. These are lin ked to the L T R -b in d in g g ly co p ep tid es, p ep tid es, ap tam ers, d en d ritic D N A , and cy to k in es th at bin d to the recep to rs fa cilita tin g en d o cy to sis an d ly so so m al d eg ra d a tio n : th e catio n -in d ep en d en t m a n n o se-6-p h osp h ate recep to r (C I-M 6P R ) [1,[11][12][13][14]19], the liversp ecific asialo g ly co p ro teiń recep to r [6,7,9,15], iń teg riń [8 ], the tran sm em b ran e E 3 ligase rin g fin g er 43 (R N F43) [10] an d zin c-an d rin g fin g er 3 (Z N R F3) [18], su rface scaven ger receptors [16], the cytokine decoy recycling receptor C X C R 7 [17], angiopep-2 receptor [20], LRP-1 [21] and transferrin receptor 1 (TfR1) [22].…”

Reducing PD-L1 Expression by Degraders and Downregulators as a Novel Strategy to Target the PD-1/PD-L1 Pathway

“…Second, more types of cell-surface LTR should be explored in addition to CI-M6PR, such as liver-specific asialoglycoprotein receptor (ASGPR), , sortillin, and transferrin receptors . Additionally, Jiang and colleagues designed signal-mediated lysosome-targeting chimeras (SignalTACs) that degrade PD-L1 based on the lysosomal sorting signals of CI-M6PR, which may also provide inspiring insights into the development of LYTACs. Third, the PK, pharmacodynamics (PD), and safety of LYTACs need to be further investigated.…”