Exploring the Potentials of Antibody–siRNA Conjugates in Tumor Cell Gene Silencing without Cationic Assistance

Antibody–siRNA conjugates (ARCs) are a type of promising drug modalities for cancer therapy. However, initial reports of ARCs present the gene knockdown effect only in limited tissues (e.g., muscles) or tumors of special targets (e.g., TenB2 and BCMA receptors). To expand the scope to more targets, herein we built structurally defined DAR2 ARCs and examined their effect on targeted gene silencing of tumor cells without cationic assistance. We first evaluated the impact of linker structures and Cys-engineered sites on conjugation efficiency, revealing that the ThioMab conjugation of siRNA is favored by the rigid sulfo-SMCC linker coupling at the HC-A118C site. With an additional anion exchange purification process, reactions of this condition can yield a homogeneous ARC product with DAR close to 2 (DAR: 1.9). We found that ARCs, despite a lack of endosome-disrupting ability, can induce effective gene silencing in multiple types of tumor cells by free uptake. The RNAi potency of ARCs is largely affected by siRNA stability and the time interval of uptake. The maximal mRNA knockdown (70–80%) can be achieved on the fourth day after the uptake of ARC bearing the fully modified siRNAs (Adv ESC mode). The delayed silencing activity, together with the restriction to the stable siRNA, indicates that ARC escape from endosomes is a slow and rate-limiting step, suggesting the importance of siRNA stability and resistance to endosomal degradation in ARC activity. In vivo, the anti-HER2 ARC displays a much-compromised circulatory half-life ( t 1/2 ∼ 8 h) in mice but can induce gene silencing in HCC1954 xenograft tumors and retard the tumor growth. This study demonstrates the potential of ARCs to treat cancers and other extrahepatic diseases.