Date of Award

Fall 2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Engineering (ENAS)

First Advisor

Carson, Richard

Abstract

Camelid single-domain antibody fragments (“nanobodies”), first described in the late 1980s, have proven particularly useful as both research tools and therapeutic agents. Tremendous progress has been made over the past three decades to understand these 15-kDa immunoglobulin domains derived from camelid heavy chain antibodies, from their structure, evolution, and biochemical properties to their various applications as a versatile binding moiety. Commercialization of nanobodies exploded in 2001, as a growing body of literature revealed salient features of nanobodies, including favorable solubility, stability, high-affinity, and deep penetration of its structure to active sites of enzymes. The approval of the first nanobody-based therapeutic, Caplacizumab, in 2019 cemented the clinical viability of this molecular format. Despite their advantageous utilities, nanobodies are available for only a handful of human antigens, limiting their widespread application. This is in part due to technological limitations in nanobody discovery, which entails a long, labor-intensive, and expensive process in a highly-specialized environment. The discovery and availability of nanobodies to a broader range of biological targets will spark accelerated development of nanobody-based diagnostics and therapeutics. In this thesis, I will describe a new technological approach to screen specific nanobodies for thousands of antigenic targets and how the molecular tools discovered by this technique may expand the toolbox and future applications of synthetic biology. In Chapter 1, I present a brief introduction to nanobodies as a class of molecules, with a particular focus on the technological approaches for nanobody discovery, and through that lens address the current challenges with scaling the nanobody discovery efforts. In Chapter 2, I introduce the development of HAPPY, a novel technique for high-throughput nanobody discovery using both yeast and phage surface display methodologies, and validate the utility of this technique by screening nanobodies for close to 100 antigens at once. In Chapter 3, I present the application of HAPPY to screen nanobody candidates for 1,000+ human cell surface and secreted proteins that are of important physiological and pathological functions. In Chapter 4, I illustrate how nanobodies may be applied to build diagnostic assays for SARS-CoV-2, and describe how these assays are used to evaluate population seroprevalence during the COVID-19 pandemic. Finally, in Chapter 5, I provide a summary of my thesis work and discuss potential future directions.

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