Optimizing Covalent Immune Recruiter Antibody Labelling Kinetics with Sulfur Fluoride Exchange Chemistry

Abstract

Covalent antibody recruiting molecules (cARMs) are synthetic chemical tools that direct antibodies naturally present in human serum to tumor receptors leading to immune recognition and elimination. cARMs have three general features: an antibody binding domain (ABD), an antibody labelling domain (ALD), and a tumor binding domain. Proximal to the ABD, the ALD contains an electrophilic group which is attacked by a nucleophilic amino acid residue on the target antibody upon cARM binding. Previous cARMs use an acyl-imidazole (AL) ALD to successfully covalently recruit anti-dinitrophenyl IgG validated via fluorescence SDS-PAGE,and form immune complexes with PSMA+ HEK cells in ADCP flow experiments.1 Through MS analysis, AL cARMs were demonstrated to target lysine-59 of antiDNP.1 Problematically, there are several properties associated with AL chemistry that limit the therapeutic potential of associated cARMs. These limitations include limited stability in vivo, single amino acid (lysine) selectivity, and modest intramolecular protein labelling rates (10-5 s-1)1. To target novel antibody residues and possibly improve the rate of antibody labelling, a second generation of cARMs has been developed using sulfur fluoride exchange (SuFEx) chemistry as the ALD. Sulfur fluorides have been broadly used in academic and industrial applications due to intrinsically high stability and broad amino acid reactivity.2 SuFEx cARMs are hypothesized to have enhanced hydrolytic stability compared to AL cARMs. This thesis explores the synthesis, stability, binding, and labeling kinetics of SuFEx cARMs to advance cARM design and development for in vivo applications. This research contributes collectively to the design and development of novel therapeutics in chemical biology and immune-oncology research.