Comprehensive analyses of the intracellular and in vivo disposition of Fab-siRNA conjugate to identify key issues to improve its in vivo activity.

Comprehensive analyses of intracellular disposition and in vivo pharmacokinetics were performed for small interfering RNA (siRNA) conjugated with the Fab fragment of Panitumumab, a fully humanized anti-epidermal growth factor receptor (EGFR) monoclonal antibody. The Fab-siRNA conjugate was internalized into EGFR-expressing cancer cells in an antigen-dependent manner. Intracellular disposition was quantitatively evaluated using fluorescent-labeled Panitumumab and confocal microscopy. The majority of internalized Panitumumab was suggested to be transferred into lysosomes. In vivo pharmacokinetics were evaluated in EGFR-expressing tumor-bearing mice. Intact Fab-siRNA was measured by immunoprecipitation using anti-Fab antibody followed by quantitative polymerase chain reaction. The Fab portion was measured by a ligand binding assay. Intact Fab-siRNA concentrations rapidly decreased in the plasma and tumor, although the Fab portion concentration remained high, suggesting extensive degradation in linker-siRNA portion. After incubation of Fab-siRNA in mouse plasma, samples were digested with proteinase K, and extracted siRNA tagged with Fab-derived peptide was subjected to an ion-pair reversed-phase liquid chromatography with mass spectrometry analysis. Results suggested that hydrolysis from the 3'-end of the antisense strand of siRNA is the major metabolizing pathway. Based on these findings, endosomal escape and stability in lysosomes, blood and tumor are key factors to improve to achieve efficient target gene knockdown in tumors and stabilizing the 3'-end of the antisense strand was suggested to be most efficient. Our approaches clearly identified the key issues of Fab-siRNA from a pharmacokinetics aspect, which will be useful for improving the in vivo activity of siRNA conjugated with not only Fab but also other immunoproteins. Significance Statement The intracellular and in vivo disposition of Fab-siRNA conjugate was comprehensively investigated using various approaches, including newly developed analytical methods. This study clearly shows that improvements in siRNA stability in lysosomes, blood and tumor are needed for target gene knockdown in tumors. The major metabolic pathway of Fab-siRNA is 3' exonuclease degradation, suggesting that optimization of the conjugation site to Fab might help improve stability.