Antibody targeting of E3 ubiquitin ligases for receptor degradation

Marei, Hadir; Tsai, Wen‐Ting K.; Kee, Yee-Seir; Ruiz, Karen; He, Jieyan; Cox, C. J.; Sun, Tao; Penikalapati, Sai C.; Dwivedi, Pankaj; Choi, Meena; Kan, David; Sáenz-López, Pablo; Dorighi, Kristel M.; Zhang, Pamela; Kschonsak, Yvonne T.; Kljavin, Noelyn M.; Amin, Dhara N.; Kim, Ingrid; Mancini, Andrew G.; Nguyen, Thao; Wang, Chunling; Janezic, Eric M.; Doan, Alexander; Mai, Elaine; Xi, Hongkang; Gu, Chen; Heinlein, Melanie; Biehs, Brian; Wu, Juan; Lehoux, Isabelle; Harris, S.F.; Comps-Agrar, Laëtitia; Seshasayee, Dhaya; Sauvage, Frédéric J. de; Grimmer, Matthew; Li, Jing; Agard, Nicholas J.; Melo, Felipe de Sousa e

doi:10.1038/s41586-022-05235-6

Cited by 110 publications

(96 citation statements)

References 35 publications

(42 reference statements)

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“…We then used the expressed phages to select for peptides that bound to the extracellular domain of ZNRF3, a membrane-bound E3 ubiquitin ligase that has been used to create proteolysis-targeted chimeras (PROTACs) for degradation of oncoproteins. 33 Each selection was performed in duplicate to ensure reproducibility. After 3 rounds of selection, we observed strong enrichment of phages that bound to the protein target, which was indicated by the percent of phages eluted at the end of the selection (Figure 4B).…”

Section: Resultsmentioning

confidence: 99%

An Amber-Encoding Helper Phage for More Efficient Phage Display of Noncanonical Amino Acids

Hampton¹,

Cho²,

Geng³

et al. 2022

Preprint

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In the past two decades, phage display has emerged as a powerful technique for the identification of antibodies and peptide ligands for therapeutic targets. Using the amber suppression-based noncanonical amino acid (ncAA) mutagenesis approach, we and others have shown that the chemical space in phage display can be significantly expanded for drug discovery. However, the use of amber codon in phages results in poor phage yields and requires tedious processes to enrich amber codon-containing (amber obligate) phage clones. In this work, we demonstrate the development of a novel helper phage, CMa13ile40, for rapid and continuous enrichment of amber obligate phage clones and efficient production of ncAA-containing phages. CMa13ile40 was constructed by the insertion of a Candidatus Methanomethylophilus alvus pyrrolysyl-tRNA synthetase/PylT gene cassette into a helper phage genome. The afforded novel helper phage allowed for a continuous amber codon enrichment strategy for two different phage display libraries and demonstrated a 100-fold increase in selectivity for packaging of library plasmids in comparison with original helper phage plasmids. To demonstrate the applicability of the system, CMa13ile40 was used to create two phage-displayed peptide libraries containing two separate ncAAs, Nϵ-tert-butoxycarbonyl-lysine (BocK) and Nϵ-allyloxycarbonyl-lysine (AllocK), respectively. These were then used to identify peptide ligands that bind to the extracellular domain of ZNRF3, a membrane-bound E3 ligase. Each selection showed differential enrichment of unique sequences dependent upon the ncAA used. Using biolayer interferometry, enriched peptides from both selections were confirmed to have low micromolar affinity for ZNRF3 and this affinity is dependent on the presence of the ncAA used for selection. Our results clearly show that ncAAs in phages provide unique interactions for selection of peptides that are different from each other and from canonical amino acids. As an effective tool for phage display, we believe that CMa13ile40 can be broadly applied to a wide variety of applications.

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Section: Resultsmentioning

confidence: 99%

An Amber-Encoding Helper Phage for More Efficient Phage Display of Noncanonical Amino Acids

Hampton¹,

Cho²,

Geng³

et al. 2022

Preprint

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“…While similar approaches (AbTACs, PROTABs) have been reported during the course of this study to degrade PD-L1 or IGF1R, they are mainly limited by using WNT responsive E3 ligase RNF43 and ZNRF3 (Cotton et al, 2021;Marei et al, 2022) and rely on human IgG antibody scaffolds to generate heterobifunctional targeting molecules. Antibody-derived biologics are generally more constrained by their inherent structural properties including large size (150 kDa), formatting and modularity that might limit the applicability for tumor therapy.…”

Section: Discussionmentioning

confidence: 97%

“…More recently, targeted protein degradation approaches utilizing lysosomal degradation strategies (LYTAC, KineTac) (Banik et al, 2020; Pance et al, 2022) and proteolysis-targeting antibodies (AbTac, ProTab) using WNT related transmembrane E3 ligases (RNF43, ZNRF3) have emerged (Cotton et al, 2021; Marei et al, 2022). These approaches tether target proteins on the cell surface to either lysosome shuttling receptors or cell-surface E3 ubiquitin ligases to induce membrane clearance.…”

Section: Mainmentioning

confidence: 99%

Receptor Elimination by E3 Ubiquitin Ligase Recruitment (REULR): A Targeted Protein Degradation Toolbox

Siepe

Picton

Garcia

2022

Preprint

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In recent years, targeted protein degradation (TPD) of plasma membrane proteins by hijacking the ubiquitin proteasome system (UPS) or the lysosomal pathway have emerged as novel therapeutic avenues in drug development to address and inhibit canonically difficult targets. While TPD strategies have been successful to target cell surface receptors, these approaches are limited by the availability of suitable binders to generate heterobifunctional molecules. Here, we present the development of a nanobody (VHH) based degradation toolbox termed REULR (Receptor Elimination by E3 Ubiquitin Ligase Recruitment). We generated human and mouse cross-reactive nanobodies against 5 transmembrane PA-TM-RING type E3 Ubiquitin Ligases (RNF218, RNF130, RNF167, RNF43, ZNRF3) covering a broad range and selectivity of tissue expression with which we characterized expression in human and mouse cell lines and immune cells (PBMCs). We demonstrate that heterobifunctional REULR molecules can enforce transmembrane E3 ligase interaction with a variety of disease relevant target receptors (EGFR, EPOR, PD-1) by induced proximity resulting in effective membrane clearance of the target receptor at varying levels. In addition, we designed E3 Ligase self-degrading molecules, "fratricide" REULRs (RNF128, RNF130, RENF167, RNF43, ZNRF3), that allow downregulation of one or several E3 Ligases from the cell surface and consequently modulate receptor signaling strength. REULR molecules represent a VHH-based, modular and versatile "mix and match" targeting strategy for facile modulation of cell surface proteins by induced proximity to transmembrane PA-TM-RING E3 ligases.

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“…Recently, the novel generation of PROTAC compounds targeting plasma membrane integral proteins, based on the use of the plasma membrane-bound E3 ligase-coupled antibodies was reported. 27 This promising strategy needs to be further elaborated and optimized to target cytosolic and nuclear HDAC degraders. [29][30][31][32][33][34][35][36][37][38][39][40] Figure 1 Alterations of HATs and HDACs in cancers (Borrowed from: "Introduction to Epigenetics".…”

Section: Other Questions For Discussion and Future Developmentmentioning

confidence: 99%

Cell biologist’s perspective: frontiers in the development of PROTAC-HDAC degraders

Sobko¹

2022

JSRT

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This “Minireview and Perspective” article describes histone deacetylases (HDACs), as promising specific molecular targets to treat a variety of disease states by downregulating the expression of associated proteins with the use of a new generation of bioengineered compounds called protein targeting chimeras (PROTACs). We present the classification of HDACs, discuss their functions as key epigenetic regulators of gene expression, describe their roles in the biology of aging, describe histone- and nonhistone substrates of HDACs and their functions, and briefly introduce the concept of histone-modifying multiprotein complexes. Insight into the biological functions of specific HDACs comes from genetic knockout studies of individual genes encoding deacetylases. Initially discovered and newly developed HDAC inhibitors are powerful tools to investigate the functions of HDACs in cells and organs, that have also been successfully used in numerous preclinical and clinical studies, as promising drug candidates. We focus on the molecular and cellular mechanisms of their action, and introduce PROTACs, which are bivalent degrader molecules that have been recently developed to target HDACs. We then discuss recent studies focused on designing and testing several classes of selective and nonselective HDAC degraders in terms of their molecular and cellular mechanisms of action. Finally, we present open questions and new perspectives in developing the next generation of HDAC-degraders.

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Antibody targeting of E3 ubiquitin ligases for receptor degradation

Cited by 110 publications

References 35 publications

An Amber-Encoding Helper Phage for More Efficient Phage Display of Noncanonical Amino Acids

An Amber-Encoding Helper Phage for More Efficient Phage Display of Noncanonical Amino Acids

Receptor Elimination by E3 Ubiquitin Ligase Recruitment (REULR): A Targeted Protein Degradation Toolbox

Cell biologist’s perspective: frontiers in the development of PROTAC-HDAC degraders

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