46. Shielding of Ad5 by Minimal Geneti-Chemical Modification: Preventing Clearance While Preserving Infectivity
Authors: Lea Krutzke, Jan-Michael Prill, Tatjana Engler, Andrea Hoffmeister, Christoph Q. Schmidt, Zhili Xu, Andrew P. Byrnes, Stefan Kochanek, Florian Kreppel
Journal: Molecular Therapy (2015)

The clinical efficacy of adenovirus serotype 5-based vectors is limited by complex interactions of the vector with host blood components. These interactions trigger sequestration of systemically administered vector particles mainly by the reticuloendothelial system. In order to generate retargeted Ad vectors suitable for i.v. delivery it is mandatory to understand and manipulate these non-target interactions. In mice, it was shown that blood coagulation factor X bound to hexon reduces sequestration by shielding the virus from natural antibodies and macrophage uptake, but also mediates hepatocyte transduction. To generate Ad vectors deficient for hepatocyte transduction but shielded from natural antibodies, we employed a combination of point mutations ablating (i) CAR binding (Ad-ΔCAR), (ii) integrin binding, (iii) FX binding (Ad-ΔFX), and rendered the vectors amenable for position-specific PEGylation at hexon HVR1 (Ad-HVR1) to compensate the lack of a FX-mediated shielding. Surface plasmon resonance analysis confirmed complete ablation of FX binding by ΔFX mutation in combination with PEGylation. Upon i.v. delivery, unPEGylated Ad-HVR1-ΔCAR-ΔFX did not transduce hepatocytes in BALB/c mice, whereas robust transduction levels were observed in antibody-deficient JHD mice. In agreement with the literature, this suggested that the ΔFX vector particles became susceptible to natural antibodies. However and surprisingly, PEGylated Ad-HVR1-ΔCAR-ΔFX showed a strong FX-independent hepatocyte transduction in both BALB/c and JHD mice compared to unPEGylated Ad-HVR1-ΔCAR-ΔFX and wildtype capsid vectors. Preliminary results suggested that integrins were involved in this FX-independent transduction. Further, PEGylated Ad-HVR1-ΔCAR-ΔFX vectors displayed 20-fold increased blood persistence without being associated to blood cells in BALB/c mice. To analyze vector sequestration by macrophages more closely we performed in vitro assays measuring the uptake of vector particles by Raw264.7 cells in the presence of murine plasma. We found enhanced uptake of unPEGylated Ad-HVR1-ΔFX vectors compared to wildtype capsid vectors. Using antibody-deficient and heated plasma of Ad naïve mice confirmed a natural antibody- and complement-dependent uptake mechanism. This uptake was completely prevented by PEGylation of the vector particles, suggesting that position-specific PEGylation of hexon HVR1 interfered with natural antibody binding. Moreover, using hirudinized human whole blood we could show that a complement-dependent natural antibody-mediated binding of unPEGylated Ad-HVR1-ΔCAR-ΔFX vectors to erythrocytes could be prevented by PEGylation of hexon HVR1 with large PEG moieties. Thus, we conclude that a PEG-mediated evasion from sequestration by macrophages by shielding from natural antibodies or by a yet unknown mechanism maintained high vector concentrations in blood followed by enhanced hepatocyte transduction.