Development of a low-seroprevalence, αvß6 integrin-selective virotherapy based on human adenovirus type 10.

Oncolytic virotherapies (OV) hold immense clinical potential. OV based on human adenoviruses (HAdV) derived from HAdV with naturally low rates of pre-existing immunity will be beneficial for future clinical translation. We generated a low-seroprevalence HAdV-D10 serotype vector incorporating an αvß6 integrin-selective peptide, A20, to target αvß6-positive tumor cell types. HAdV-D10 has limited natural tropism. Structural and biological studies of HAdV-D10 knob protein highlighted low-affinity engagement with native adenoviral receptors CAR and sialic acid. HAdV-D10 fails to engage blood coagulation factor X, potentially eliminating "off-target" hepatic sequestration in vivo. We engineered an A20 peptide that selectively binds αvß6 integrin into the DG loop of HAdV-D10 fiber knob. Assays in αvß6+ cancer cell lines demonstrated significantly increased transduction mediated by αvß6-targeted variants compared with controls, confirmed microscopically. HAdV-D10.A20 resisted neutralization by neutralizing HAdV-C5 sera. Systemic delivery of HAdV-D10.A20 resulted in significantly increased GFP expression in BT20 tumors. Replication-competent HAdV-D10.A20 demonstrated αvß6 integrin-selective cell killing in vitro and in vivo. HAdV-D10 possesses characteristics of a promising virotherapy, combining low seroprevalence, weak receptor interactions, and reduced off-target uptake. Incorporation of an αvß6 integrin-selective peptide resulted in HAdV-D10.A20, with significant potential for clinical translation.

Efficient Intravenous Tumor Targeting Using the αvß6 Integrin-Selective Precision Virotherapy Ad5NULL-A20.

We previously developed a refined, tumor-selective adenovirus, Ad5NULL-A20, harboring tropism ablating mutations in each major capsid protein, to ablate all native means of infection. We incorporated a 20-mer peptide (A20) in the fiber knob for selective infection via αvß6 integrin, a marker of aggressive epithelial cancers. Methods: To ascertain the selectivity of Ad5NULL-A20 for αvß6-positive tumor cell lines of pancreatic and breast cancer origin, we performed reporter gene and cell viability assays. Biodistribution of viral vectors in mice harboring xenografts with low, medium, and high αvß6 levels was quantified by qPCR for viral genomes 48 h post intravenous administration. Results: Ad5NULL-A20 vector transduced cells in an αvß6-selective manner, whilst cell killing mediated by oncolytic Ad5NULL-A20 was αvß6-selective. Biodistribution analysis following intravenous administration into mice bearing breast cancer xenografts demonstrated that Ad5NULL-A20 resulted in significantly reduced liver accumulation coupled with increased tumor accumulation compared to Ad5 in all three models, with tumor-to-liver ratios improved as a function of αvß6 expression. Conclusions: Ad5NULL-A20-based virotherapies efficiently target αvß6-integrin-positive tumors following intravenous administration, validating the potential of Ad5NULL-A20 for systemic applications, enabling tumor-selective overexpression of virally encoded therapeutic transgenes.