Transductional retargeting of human adenovirus type 5 to ανβ6 integrin for cancer gene therapy
A key impediment to successful cancer therapy with adenoviral vectors is the inefficient transduction of malignant tissue in vivo. Compounding this problem is the lack of cancer-specific targets, coupled with a shortage of corresponding high efficiency ligands, permitting selective retargeting. The epithelial specific integrin ανβ6 represents an attractive target for directed therapy since generally it is not expressed on normal epithelium, but is upregulated in numerous carcinomas where it plays a role in tumour progression. We previously have characterised a high affinity, ανβ6-selective peptide (A20FMDV2), derived from VP1 of Foot and Mouth Disease Virus. In an attempt to subvert the tropism of Ad5 to ανβ6, A20FMDV2 was incorporated genetically into the HI loop of the Ad5 fibre protein to generate Ad5-EGFPA20. Furthermore, the native CAR-binding and putative FIX/C4BP-binding capacity of Ad5-EGFPA20 was ablated to generate Ad5-477dlTAYTA20. In vitro, these A20-retargeted vectors displayed up to 50- fold increases in CAR-independent transduction, and up to 480-fold increased cytotoxicity on a panel of ανβ6-positive human carcinoma lines compared with Ad5- EGFPWT. In vivo, both Ad5-EGFPA20 and Ad5-477dlTAYTA20 improved tumour targeting (~2- fold/3-fold) following systemic delivery in immunodeficient mice, when compared with Ad5-EGFPWT. Furthermore, ~5-fold fewer Ad5-EGFPA20/Ad5-477dlTAYTA20 genomes were detected in the liver, which correlated with reduced serum transaminase levels and minimal E1A expression. Warfarin pre-treatment, to deplete coagulation factors, did not improve tumour uptake significantly with either virus, but did further reduce liver sequestration and hepatic toxicity. The unexpected reduction in liver tropism and toxicity we observed was supported by similar results obtained in immunocompetent mice. Furthermore, in immunocompetent mice, Ad5-EGFPWT induced dramatic elevations in serum cytokines/chemokines in addition to inducing acute thrombocytopenia. These effects were attenuated substantially in Ad5-EGFPA20/Ad5- 477dlTAYTA20 treated cohorts. In summary, we have found that in vivo retargeting Ad5 to ανβ6 in a murine model results in improved tumour delivery, limited hepatotropism/toxicity and a reduced induction of anti-viral innate immune responses.
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