Neutral BackboneModifications Enhance the Pharmacokineticsand Biodistribution of Antibody–Oligonucleotide Conjugateswith High Drug-to-Antibody Ratios

Abstract Antibody–oligonucleotide conjugates (AOCs) enable targeted delivery of small interfering RNAs (siRNAs) by coupling them to antibodies for receptor-mediated uptake, enhancing tissue specificity and therapeutic potential. A key determinant of AOC performance is the drug-to-antibody ratio (DAR), which influences pharmacokinetics, biodistribution, and knockdown efficiency. Prior studies with transferrin receptor 1 (TfR1)-targeted AOCs revealed that DAR ≥2 constructs exhibit rapid plasma clearance, preferential hepatic uptake, and reduced muscle delivery compared with DAR1. We hypothesized that increased negative charge from multiple phosphodiester and phosphorothioate linkages drives nonspecific plasma protein binding and scavenger receptor-mediated clearance of DAR2 AOCs. To test this, we investigated siRNA modifications designed to reduce charge and phosphorothioate content, including phosphorothioate removal and incorporation of neutral backbone chemistries such as phosphoryl guanidine (PG), methoxypropylphosphonate, and triester linkages. PG-modified DAR2 AOCs improved pharmacokinetics and tissue distribution in mice while maintaining activity. These findings lay the groundwork for optimizing high-DAR AOCs.