Engineering Nanobody-targeted Proteolytic Chimeras for Targeted Protein Degradation

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2026-06-06

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2024

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Abstract

Targeted protein degradation (TPD) is an emerging therapeutic strategy with the potential to degrade aberrant proteins. However, the most commonly used TPD technologies leverage proximity-induced ubiquitination and rely on the recruitment of cellular degradation machinery. This fundamentally restricts TPD from being applied in vitro and underscores the need for alternative TPD technologies that can catalyze protein degradation without cellular factors. Here we meet this demand by introducing nanobody-targeted proteolytic chimeras (NanoPROCs), a novel class of proteases with highly specific substrate targeting. NanoPROCs harness nanobodies as the basis of their substrate targeting and include a minimal catalytic structure derived from proteases, enabling them to degrade proteins autonomously. In this work, we developed a computational pipeline for the design of novel NanoPROCs, designed a substrate that can be used to assay for novel NanoPROC activity, and explored the interchangeability of NanoPROC components. Our results support the assembly of stable NanoPROCs from multiple nanobodies and proteolytic domains, further suggesting that NanoPROCs can be tailored for desired substrate and cleavage specificities. As such, we predict that NanoPROCs can be more broadly applied to industrial manufacturing, diagnostic development, and therapeutic contexts than current TPD technologies. Specifically, the ability to autonomously degrade proteins enables NanoPROCs to be applied to peptide purification workflows. In a clinical context, the potential for NanoPROCs to autonomously degrade proteins in the extracellular space enables infectious disease oriented applications, such as viral receptor cleavage and bacterial toxin degradation. Overall, the introduction of NanoPROCs represents a significant advancement in the field of targeted protein degradation, offering a versatile and autonomous approach to protein degradation that can be applied across numerous industrial and disease contexts.

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Yeo, Cadmus (2024). Engineering Nanobody-targeted Proteolytic Chimeras for Targeted Protein Degradation. Master's thesis, Duke University. Retrieved from https://hdl.handle.net/10161/31074.

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