Organic Carborane NHS Esters Enable Boron-Rich Antibody Conjugates for BNCT, Retaining Native Activity

Effective Boron Neutron Capture Therapy (BNCT), a binary cancer treatment, hinges on selectively delivering boron-containing drugs to tumor cells. Current methods for high-content boron incorporation into biologics are limited, hindering the development of potent BNCT agents. Therapeutic peptides and antibodies offer precise targeting capabilities, but require robust conjugation strategies to carry sufficient boron payloads without compromising their biological function. This research addresses the critical need for versatile methods to create boron-rich biologics for enhanced tumor selectivity in BNCT.

Researchers developed novel organic carborane reagents based on N-hydroxysuccinimide (NHS) esters to enable selective conjugation to amine-containing residues of small molecules and polypeptides. They synthesized bifunctional carborane reagents, which facilitated peptide-driven multimerization into carborane-peptide polymers. Therapeutic antibodies were then functionalized with these reagents, and the impact of linker length on loading efficiency was investigated. Mass spectrometry mapping was employed to identify specific lysine modification sites on IgG antibodies under native-like conditions, using NHS esters bearing hydrophobic (carborane) substituents.

The developed organic carborane NHS ester reagents proved to be a versatile platform for high-content boron incorporation. These reagents enabled the creation of novel carborane-peptide polymers through peptide-driven multimerization. Critically, therapeutic antibodies functionalized with these reagents yielded boron-rich conjugates containing up to 13 carboranes per antibody molecule. These highly loaded conjugates remarkably retained their native antibody activity, a crucial factor for therapeutic efficacy. Linker length was identified as a critical parameter for maximizing loading efficiency. Mass spectrometry mapping revealed up to 30 preferential lysine modification sites per antibody, with these sites predominantly located outside the complementarity-determining region(s) (CDRs), which are essential for antigen binding. This preferential modification pattern helps preserve antibody function. The study established a useful framework for the rational design and optimization of antibody-drug conjugates by providing prevalence-ranked lysine mapping of IgG antibodies. > The functionalized therapeutic antibodies successfully incorporated up to 13 carboranes per antibody molecule while fully retaining their native biological activity.