ATNC: Versatile Nanobody Chimeras for Autophagic Degradation of Intracellular Unligandable and Undruggable Proteins

Abstract

Targeted protein degradation (TPD) through the autophagy pathway displays broad substrate scope and is gaining increasing interest in biology and medicine. However, current approaches using small-molecule degraders have limitations due to the lack of versatility, modularity, and ease of implementation and are restricted to addressing only ligandable proteins. Herein, we report a nonsmall molecule-based autophagy-targeting nanobody chimera (ATNC), or phagobody, for selective degradation of intracellular targets, which overcomes these limitations. The core of an ATNC features a nanobody for recruiting proteins as well as an autophagic pathway-directing module. ATNC turns out to be a general, modular, and versatile degradation platform. We show that ATNC can be versatilely implemented in different ways including expressed ATNC intrabodies for ease of use, chemically induced proximity (CIP)-operated logic-gated conditional and tunable degradation, and cyclic cell-penetrating peptide-tethered cell-permeable phagobodies that selectively degrade the undruggable therapeutically relevant HE4 protein, resulting in effective suppression of ovarian cancer cell proliferation and migration. Overall, ATNC represents a general, modular, and versatile targeted degradation platform that degrades unligandable proteins and offers therapeutic potential.

Expanding the horizons of targeted protein degradation: A non-small molecule perspective

“…Furthermore, autophagy-targeting nanobody chimera (ATNC), a versatile and multifunctional autophagy-based targeted degradation platform technology was presented via the adoption of modular construction strategies 185 . The fundamental construction of ATNC was conceived by directly fusing the POI-targeting nanobody to the LC3B protein.…”

Lysosome Targeting Chimaeras for Glut1-Facilitated Targeted Protein Degradation

“…With the deepening exploration of the endocytic-lysosomal and autophagic-lysosomal degradation pathways, targeting protein degradation (TPD) strategies through the lysosomal pathway have arisen in recent years. These encompass protein degradation through the endocytic-lysosomal pathway such as lysosome-targeting chimaeras (LYTAC), − antibody-based PROTAC (AbTAC), bispecific adapter chimeras, , and covalent nanobody-based PROTAC strategy (GlueTAC), as well as technologies for degrading target molecules through the autophagic-lysosomal pathway such as autophagy-tethering compounds (ATTEC), autophagy-targeting chimera (AUTAC , and AUTOTAC), ER-targeting DNA nanodevice for autophagy-dependent degradation, and autophagy-targeting nanobody chimera (ATNC) …”

“…The composition of commonly targeted proteins or lysosomes includes antibodies, − ,, nucleic acid aptamers, , peptides, and small molecules. ,, However, the synthesis process for nucleic acid aptamers and peptides is complex and their production efficiency is low, resulting in high production costs and limited clinical application. Additionally, small molecules have disadvantages such as off-target effects, high toxicity, and rapid metabolism.…”