Covalently Engineered Nanobody Chimeras for Targeted Membrane Protein Degradation

Abstract

The targeted degradation of membrane proteins would afford an attractive and general strategy for treating various diseases that remain difficult with the current proteolysis-targeting chimera (PROTAC) methodology. We herein report a covalent nanobody-based PROTAC strategy, termed GlueTAC, for targeted membrane protein degradation with high specificity and efficiency. We first established a mass-spectrometry-based screening platform for the rapid development of a covalent nanobody (GlueBody) that allowed proximity-enabled cross-linking with surface antigens on cancer cells. By conjugation with a cell-penetrating peptide and a lysosomal-sorting sequence, the resulting GlueTAC chimera triggered the internalization and degradation of programmed death-ligand 1 (PD-L1), which provides a new avenue to target and degrade cell-surface proteins.

Targeted protein degradation: mechanisms, strategies and application

“…To demonstrate the effectiveness of GlueTAC, the authors developed a GlueTAC molecule targeting PD-L1. 88 This GlueTAC molecule is more effective in reducing the level of PD-L1 in cells and inhibiting tumor growth in immunodeficient mice, in comparison with FDA-approved antibody against PD-L1, Atezolizumab. 88

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“… 88 This GlueTAC molecule is more effective in reducing the level of PD-L1 in cells and inhibiting tumor growth in immunodeficient mice, in comparison with FDA-approved antibody against PD-L1, Atezolizumab. 88

Fig. 9 Schematic representation of GlueTAC.

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“…Recently, another lysosome-based strategy, termed GlueTAC, has been developed to degrade cell-surface proteins 88 (Fig. 9 ).…”

Creating a Selective Nanobody Against 3-Nitrotyrosine Containing Proteins

“…As noted in the introduction, generating covalent Nb binders (i.e., “GlueBodies”) through incorporating ncAAs with crosslinking abilities (ncAA-CL), is a means to target proteins for degradation ( Zhang et al, 2021 ). Nbs that target oxPTMs for degradation would be powerful tools for discovering possible physiological changes triggered by specific oxidized proteins.…”

“…Additionally, Nbs are well suited for binding epitopes that are inaccessible to traditional Abs due to their protruding, convex paratopes ( Muyldermans 2013 ; Pardon et al, 2014 ). The ability to generate nitroTyr-protein specific Nbs would allow the full suite of Nb capabilities to be harnessed for both in vitro and in cell work, including for instance, the modulation of protein activity by targeting nitroTyr proteins with a covalent nanobody (or “GlueBody”) for degradation in order to observe the downstream effects on redox signaling ( Bery et al, 2019 ; Cheloha et al, 2020 ; de Beer and Giepmans 2020 ; Zhang et al, 2021 ).…”