A novel hepatic-targeting system for therapeutic cytokines that delivers to the hepatic asialoglycoprotein receptor, but avoids receptor-mediated endocytosis

Purpose. To demonstrate the utilities of a synthetic low-affinity ligand ((Gal)(3)) for the asialoglycoprotein receptor (ASGP-R) as a hepatic targeting device for therapeutic cytokines. Methods. The site-specific incorporation of (Gal)(3) or a typical high-affinity ligand (GalNAc)(3) into IL-2 was catalyzed by microbial transglutaminase. The anti-tumor activities, pharmacokinetic profiles and receptor-mediated endocytosis in hepatocytes of the ligand-IL-2 conjugates were examined in mouse. Results. The (Gal)(3) has approximately 50 times lower affinity to ASGP-R than (GalNAc)(3). Nevertheless, the antitumor effects were in the order of (Gal)(3)-IL-2 > unmodified IL-2 > (GalNAc)(3)-IL-2. The systemic elimination and the hepatic uptake of (GalNAc)(3)-IL- 2 were more rapid than (Gal)(3)-IL-2. The ratio of the rate constant representing dissociation from the cell-surface receptor (k(off)) to that representing endocytosis of the ligand (k(int)) was greater for (Gal)(3)-IL-2 than (GalNAc)(3)-IL-2, suggesting that (Gal)(3)-IL-2 preferably avoids internalization due to its lower affinity to the receptor. The simulation studies demonstrated that (Gal)(3)-IL-2 was present in the hepatic extracellular space for a longer period than (GalNAc)(3)IL-2. Conclusions. The (Gal)(3) ligand increases the therapeutic efficacy of IL-2 by enhancing its exposure to the cell-surface. The k(off)/k(int) affects the targeting efficacy of the conjugates to ASGP-R.