Ad5-delta-24-RGD is currently the most clinically advanced recombinant adenovirus (rAd) for glioma therapy. We constructed a panel of fiber-modified rAds (Ad5RGD, Ad5/3, Ad5/35, Ad5/3RGD, and Ad5/35RGD, all harboring the delta-24 modification) and compared their infectivity, replication, reproduction, and cytolytic efficacy in human and rodent glioma cell lines and short-term cultures from primary gliomas. In human cells, both Ad5/35-delta-24 and Ad5/3-delta-24 displayed superior infectivity and cytolytic efficacy over Ad5-delta-24-RGD, while Ad5/3-delta-24-RGD and Ad5/35delta-24-RGD did not show further improvements in efficacy. The expression of the adenoviral receptors/coreceptors CAR, DSG2, and CD46 and the integrins aVb3/aVb5 did not predict the relative cytolytic efficacy of the fiber-modified rAds. The cytotoxicity of the fiber-modified rAds in human primary normal cultures of different origins and in primary glioma cultures was comparable, indicating that the delta-24 modification did not confer tumor cell selectivity. We also revealed that CT-2A and GL261 glioma cells might be used as murine cell models for the fiber chimeric rAds in vitro and in vivo. In GL261 tumor-bearing mice, Ad5/35-delta-24, armed with the immune costimulator OX40L as the E2A/DBP-p2A-mOX40L fusion, produced long-term survivors, which were able to reject tumor cells upon rechallenge. Our data underscore the potential of local Ad5/35-delta-24-based immunovirotherapy for glioblastoma treatment.

Two-cycle cesium chloride (2 × CsCl) gradient ultracentrifugation is a conventional approach for purifying recombinant adenoviruses (rAds) for research purposes (gene therapy, vaccines, and oncolytic vectors). However, rAds containing the RGD-4C peptide in the HI loop of the fiber knob domain tend to aggregate during 2 × CsCl gradient ultracentrifugation resulting in a low infectious titer yield or even purification failure. An iodixanol-based purification method preventing aggregation of the RGD4C-modified rAds has been proposed. However, the reason explaining aggregation of the RGD4C-modified rAds during 2 × CsCl but not iodixanol gradient ultracentrifugation has not been revealed. In the present study, we showed that rAds with the RGD-4C peptide in the HI loop but not at the C-terminus of the fiber knob domain were prone to aggregate during 2 × CsCl but not iodixanol gradient ultracentrifugation. The cysteine residues with free thiol groups after the RGD motif within the inserted RGD-4C peptide were responsible for formation of the interparticle disulfide bonds under atmospheric oxygen and aggregation of Ad5-delta-24-RGD4C-based rAds during 2 × CsCl gradient ultracentrifugation, which could be prevented using iodixanol gradient ultracentrifugation, most likely due to antioxidant properties of iodixanol. A cysteine-to-glycine substitution of the cysteine residues with free thiol groups (RGD-2C2G) prevented aggregation during 2 × CsCl gradient purification but in coxsackie and adenovirus receptor (CAR)-low/negative cancer cell lines of human and rodent origin, this reduced cytolytic efficacy to the levels observed for a fiber non-modified control vector. However, both Ad5-delta-24-RGD4C and Ad5-delta-24-RGD2C2G were equally effective in the murine immunocompetent CT-2A glioma model due to a primary role of antitumor immune responses in the therapeutic efficacy of oncolytic virotherapy.

Hepatotoxicity remains an as yet unsolved problem for adenovirus (Ad) cancer therapy. The toxic effects originate both from rapid Kupffer cell (KCs) death (early phase) and hepatocyte transduction (late phase). Several host factors and capsid components are known to contribute to hepatotoxicity, however, the complex interplay between Ad and liver cells is not fully understood. Here, by using intravital microscopy, we aimed to follow the infection and immune response in mouse liver from the first minutes up to 72 h post intravenous injection of three Ads carrying delta-24 modification (Ad5-RGD, Ad5/3, and Ad5/35). At 15–30 min following the infusion of Ad5-RGD and Ad5/3 (but not Ad5/35), the virus-bound macrophages demonstrated signs of zeiosis: the formation of long-extended protrusions and dynamic membrane blebbing with the virus release into the blood in the membrane-associated vesicles. Although real-time imaging revealed interactions between the neutrophils and virus-bound KCs within minutes after treatment, and long-term contacts of CD8+ T cells with transduced hepatocytes at 24–72 h, depletion of neutrophils and CD8+ T cells affected neither rate nor dynamics of liver infection. Ad5-RGD failed to complete replicative cycle in hepatocytes, and transduced cells remained impermeable for propidium iodide, with a small fraction undergoing spontaneous apoptosis. In Ad5-RGD-immune mice, the virus neither killed KCs nor transduced hepatocytes, while in the setting of hepatic regeneration, Ad5-RGD enhanced liver transduction. The clinical and biochemical signs of hepatotoxicity correlated well with KC death, but not hepatocyte transduction. Real-time in vivo tracking for dynamic interactions between virus and host cells provides a better understanding of mechanisms underlying Ad-related hepatotoxicity.