Evaluation of adenoviral vector-mediated gene therapy strategies in a mouse model of breast cancer

A receptor tyrosine kinase EphA2 is overexpressed in breast cancer and is regarded as a potential target for breast cancer therapeutics. Human adenovirus vector serotype 5 (HAd5)-mediated EphrinA1, the EphA2 ligand, expression inhibited growth of breast cancer cells in vitro and a single intratumoral (i.t.) inoculation partially reduced tumorigenicity in vivo. Recombinant HAd vectors, in general, are widely used gene delivery vehicles for pre-clinical investigations due to the ease of inserting transgenes into their genome coupled with a relatively high gene expression and purified vector manufacturing capability. However, the presence of pre-existing immunity in human population limits the use of HAd5 vectors for clinical applications. To circumvent this limitation, a bovine adenovirus vector (BAd3) has been developed and it retains most of the attributes of HAd5. Furthermore, no pre-existing immunity against BAd3 is prevalent in human population and BAd3 or HAd5 immune responses are not cross-neutralizing. To determine the usability of these vectors for breast cancer therapy after i.t. inoculations we investigated in an immunocompetent FVB/n mouse model of breast cancer the following; 1) a comparative biodistribution, persistence, transgene (green fluorescent protein/GFP) expression and safety of BAd3 and HAd5 vectors in the absence or presence of a pre-existing immune response, 2) in vitro mechanism/s of EphrinA1-mediated inhibition of EphA2 overexpressing breast cancer cells using an HAd5 vector expressing EphrinA1 (HAd-EphrinA1-Fc), and 3) in vivo antitumor effects of multiple inoculations of HAd-EphrinA1-Fc alone or in combination with an immunostimulatory molecule, FMS-like tyrosine kinase ligand (Flt3L). First, after i.t. inoculations, the BAd3 vector incorporating the GFP transgene (BAd-GFP) was not affected by the presence of transgene and efficiently transduced the tumors and persisted at least for 16 days. The vector also spread to the spleen, kidneys, liver, lungs and heart but revealed an altered distribution and gene expression pattern compared to HAd-GFP. The HAd-GFP vector immunity did not hinder the BAd-GFP persistence and transgene expression in the tumors and vice versa; however, the homologous vector immunity was associated with a rapid clearance and reduction in transgene expression. The histopathological alterations were comparable with HAd-GFP and BAd-GFP vectors with the exception of a transient rise in hepatic enzymes with BAd-GFP, however, with repeat inoculations, HAd-GFP treated mice revealed enhanced inflammatory changes compared to BAd-GFP or control groups. Secondly, infection of human or murine breast cancer cells with HAd-EphrinA1-Fc was observed to be cytotoxic as it reduced the breast cancer cell viability, caused cellular rounding, nuclear condensation and induced apoptotic cell death as evidenced by an increase in Annexin V staining, enhanced tumor necrosis factor gene expression and reduction in Bcl2 family protein expression. The apoptotic cell death was confirmed in vitro and in vivo by the cleavage of executioner caspase 3 of apoptotic pathway and cleavage of DNA damage repair enzyme poly ADP ribose polymerase. Thirdly, we tested the anti-tumor effects of multiple inoculations of HAd-EphrinA1-Fc alone or along with HAd-Flt3L in pre-established (non-palpable) or established (palpable) tumors and compared it with single i.t. inoculation in the mouse model. A significant reduction in tumor progression was evident with three inoculation of HAd-EphrinA1-Fc in the pre-established tumors. The combination of HAd-EphrinA1-Fc with HAd-Flt3L further reduced but did not completely eliminate the progression of the established tumors. This effect correlated with the development of an adaptive immune response in the responding mice. Taken together, our results support further development of BAd3 vector for breast cancer gene therapy to overcome pre-existing immune response. It seems EphA2-EphrinA1 interaction accompanied by immunotherapy to enhance anti-tumor responses is a promising approach for breast cancer therapeutics