Developing Ligation Strategies for Preparing Antibody-Gold Nanocluster Conjugates

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Authors

Golds, Alexandra

Date

2024-10-04

Type

thesis

Language

eng

Keyword

antibody , gold nanocluster , antibody remodeling , cysteine-maleimide ligations , glyco-engineering , bioconjugation , click chemistry

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Abstract

Radiation therapy is one of the most effective and widely used treatments for solid cancers, however, typical therapeutic doses can severely damage surrounding healthy tissues. Recent work has shown that coupling radio-sensitizing agents to targeting molecules for targeted photodynamic therapies (PDTs) can greatly improve the precision and selectivity of radiation treatment. As such, targeted PDTs have emerged as a highly effective alternative treatment for hard-to-treat solid cancers. Gold nanoclusters (AuNCs) show promise as radio- and photo-sensitizers; gold is highly biocompatible and non-toxic, yet has photo-physical properties that aid in tumour killing. While AuNCs accumulate in tumours by entry through their leaky vasculature, the lack of site-directed uptake leaves room for off target events. To improve the targeting and toxicity of nanomedicines, tumour-targeting ligands, like antibodies, can be attached as a drug delivery system. This thesis describes the development of two ligation strategies to conjugate antibodies to AuNCs using strain-promoted azide-alkyne cycloadditions (SPAAC). SPAAC is a simple and robust bio-compatible conjugation approach, whereby a dibenzocyclooctyne (DBCO) bio-orthogonal functional group installed onto antibodies and can be ligated to azidated (N3)-AuNCs. Cysteine-maleimide ligation was used as a quick and effective means to install DBCO onto antibodies. N3-AuNCs were then ligated, and conjugation results were analyzed using SDS-PAGE, western blot, HPLC-SEC, flow-cytometry, and excitation-emission matrix measurements. Ongoing work includes a second strategy where DBCO is installed onto pre-made N-glycans to enable glyco-engineering by ENGase trans-glycosylation to antibodies. This work demonstrates the feasibility of conjugating antibodies to AuNCs and describes methods to characterize these products, thus enabling future work in examining Ab-AuNCs as cancer therapeutics.

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