Incorporation of Peptides Targeting EGFR and FGFR1 into the Adenoviral Fiber Knob Domain and Their Evaluation as Targeted Cancer Therapies

Abstract

Oncolytic virotherapies based on adenovirus 5 (Ad5) hold promise as adjunctive cancer therapies; however, their efficacy when delivered systemically is hampered by poor target cell specificity and preexisting anti-Ad5 immunity. Ovarian cancer represents a promising target for virotherapy, since the virus can be delivered locally into the peritoneal cavity. Both epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor 1 (FGFR1) are overexpressed in the majority of human tumors, including ovarian cancer. To generate adenoviral vectors with improved tumor specificity, we generated a panel of Ad5 vectors with altered tropism for EGFR and FGFR, rather than the natural Ad5 receptor, hCAR. We have included mutations within AB loop of the viral fiber knob (KO1 mutation) to preclude interaction with hCAR, combined with insertions in the HI loop to incorporate peptides that bind either EGFR (peptide YHWYGYTPQNVI, GE11) or FGFR1 (peptides MQLPLAT, M*, and LSPPRYP, LS). Viruses were produced to high titers, and the integrity of the fiber protein was validated by Western blotting. The KO1 mutation efficiently ablated hCAR interactions, and significantly increased transduction was observed in hCARlow/EGFRhigh cell lines using Ad5.GE11, while transduction levels using Ad5.M* or Ad5.LS were not increased. In the presence of physiological concentrations of human blood clotting factor X (hFX), significantly increased levels of transduction via the hFX-mediated pathway were observed in cell lines, but not in primary tumor cells derived from epithelial ovarian cancer (EOC) ascites samples. Ad5-mediated transduction of EOC cells was completely abolished by the presence of 2.5% serum from patients, while, surprisingly, incorporation of the GE11 peptide resulted in significant evasion of neutralization in the same samples. We thus speculate that incorporation of the YHWYGYTPQNVI dodecapeptide within the fiber knob domain may provide a novel means of circumventing preexisting Ad5 immunity that warrants further investigation.

Ad5NULL-A20: A Tropism-Modified, αvβ6 Integrin-Selective Oncolytic Adenovirus for Epithelial Ovarian Cancer Therapies

“…Genetic modifications were carried out by AdZ homologous recombineering methods (29) as described previously (30). Viruses were produced in T-REx-293 or HEK293-b6 cells (for A20-modified viruses) and purified as described previously (30,31). A triply detargeted vector genome, Ad5 NULL , was generated by introducing mutations in key genes encoding of each of the major capsid proteins to preclude cellular uptake by all known native Ad5 pathways.…”

“…Primary EOC cells from ascites were obtained through Wales Cancer Bank under existing ethical permissions (WCB 14/004). Cells were processed and subcultured as described previously (30,31), and tested regularly for Mycoplasma infection by commercially available PCR-based methods.…”

“…Antigen specificity of the antibodies was assessed by Western blot. Transduction efficiency was assessed by standard luciferase assays, described previously (30,31). Animal experiments were approved by Institutional Care and Use Committee (IACUC) and performed at Mayo Clinic, Rochester, MN.…”

Identification of folate receptor α (FRα) binding oligopeptides and their evaluation for targeted virotherapy applications

“…HAdV-C5 is the most commonly used HAdV vector for gene and virotherapy applications; however, the clinical utility of HAdV-C5 is severely hampered by a lack of tumour specificity, neutralisation by pre-existing antibodies and interactions with blood coagulation factors that sequester adenovirus to the liver [24,[32][33][34][35][36][37][38][39][40]. To improve the specificity of HAdV vectors for ovarian cancer cells, we developed a panel of vectors retargeted to the FRα by genetically incorporating peptides identified to bind FRα from phage biopanning.…”

“…In contrast to antibody-based agents, small cancer-targeting peptides have the advantage of their small size, potentially permitting easy penetration into tissue, reduced immunogenicity and easy synthesis and scale-up [18]. Peptides identified from phage libraries that have been used for HAdV retargeting to the tumour vasculature include the RGD-binding cellular integrins [22], NGR peptide-binding aminopeptidease N (APN) [23] and human epidermal growth factor (EGFR) GE11 peptide [24]. HAdV-based virotherapies can be readily genetically modified to incorporate peptides targeting cancer-restricted receptors within permissive regions of the adenoviral fibre knob protein; however, further refinements are often necessary to overcome limitations in clinical efficacy associated with off-target uptake [25].…”