Date of Award

2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biochemistry & Molecular Biophysics

First Advisor

Andrew Tsourkas

Abstract

Antibodies have become powerful therapeutics and research tools because they can be developed to directly inhibit almost any protein, but their inaility to enter the cytosol limits inhibitory antibodies to extracellular targets. Given that roughly two-thirds of drug targets lie inside of cells and many targets lack binding targets for small-molecule drugs, developing a cytosolic antibody delivery system would dramatically expand the druggable proteome. Cytosolic antibodies also offer unique opportunities to directly inhibit and study intracellular protein function.

Here we demonstrate that immunoglobulin G (IgG) antibodies that are conjugated with anionic polypeptides (ApPs) can be complexed with cationic lipids through electrostatic interactions, enabling close to 90% cytosolic delivery efficiency with only 500 nM IgG. The ApP is fused to a small photoreactive antibody-binding domain (pAbBD) that can be site-specifically photocrosslinked to nearly all off-the-shelf IgGs without perturbing IgG binding affinity. Furthermore, the pAbBD can be functionalized with chemical moieties such as fluorophores at its C-terminus via proximity-based sortase-mediated ligation (PBSL), a chemoenzymatic bioconjugation approach that we have developed.

We show that cytosolically delivered IgGs can inhibit the drug efflux pump multidrug resistance-associated protein 1 (MRP1) and the transcription factor NFκB. This work establishes a new approach for using existing antibody collections to modulate intracellular protein function and provides the foundations for therapeutic cytosolic antibodies.

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