Single Versus Repeated Intravenous Oncolytic Virus Infusions: Implications for Rationalised Scheduling of Therapy in Hepatocellular Carcinoma.

Background: Oncolytic virus (OV) immunotherapy stands to widely improve patient outcomes in multiple solid malignancies. However, to date, the scheduling of OV therapy, with single versus multiple infusions on consecutive days, has not been correlated with immunological or clinical response. In hepatocellular carcinoma (HCC) patients, where the background liver is frequently chronically injured, repeated dosing may have deleterious implications, resulting in off-target immune-mediated damage, thereby tipping the balance between favourable clinical response and hepatotoxicity. As such, elucidation of the optimum dosing regime is necessary to ensure therapy, whilst limiting damage to the background liver. Methods: Herein, we expand upon our previous experience in neoadjuvant OV therapy to compare the immunological response from single versus repeated doses of reovirus in cancer patients. The impact of OV immunotherapy on HCC outcomes was examined in vivo following a high fat diet or induced liver fibrosis in the context of an abnormal background liver. Furthermore, we assess, in a syngeneic model of HCC, the potential immune-mediated toxicity of single versus multiple virus infusions in combination with PD-1 and PD-L1 blockade. Results: Clinical trial data indicate that a single dose of reovirus is equivalent or superior to repeat doses in achieving: (a) induction of an inflammatory cytokine/chemokine response; (b) peripheral blood immune cell activation; and (c) migration of activated CD8+ CTLs. Repeated doses on consecutive days do not improve the amplitude or duration of the immune response following virus infusion. Furthermore, repeated viral dosing leads to an unwanted influx of activated T cells into background liver. An increase in PD-L1 expression on T cells resulting from a single virus dose was observed. Combination therapy of reovirus plus anti-PD-L1, but not anti-PD-1, limited tumour growth and extended survival in vivo. Conclusions: A single dose of oncolytic virus is equivalent or superior to multiple consecutive doses in inducing an anti-tumour immune response. Combination immune checkpoint blockade with anti-PD-L1 holds potential to maximise the beneficial effects of a single dose, whilst simultaneously avoiding undesirable toxicities in the background liver in the context of HCC.