*2.1. H-1PV + Nucleoside Analogues (Gemcitabine)*

As gemcitabine is currently considered the gold chemotherapeutic standard in PDAC clinical management, the therapeutic efficacy of gemcitabine in combination with H-1PV was tested in a rat syngeneic orthotopic PDAC model. H-1PV administration to gemcitabine-pretreated animals led to significant tumor suppression and survival prolongation in comparison with the mock-infected or gemcitabine-only treated groups [23]. These in vivo findings could not be straightforwardly ascribed to synergistic tumor cell death enhancement only. Indeed, in vitro studies showed that the cytotoxic effects of the combination, while allowing effective dose reduction for both agents, did not result in complete PDAC culture elimination. This prompted the investigation of the immunological effects exerted by the H-1PV + gemcitabine combination as an added value to direct tumor cell killing. Markers of ICD induction were analyzed in various PDAC cell lines, treated with either virus (or gemcitabine) alone or with H-1PV + gemcitabine. It was demonstrated that the release of high-mobility group box 1 protein (HMGB1) is a strikingly robust feature of H-1PV-infected PDAC cells [24]. Furthermore, H-1PV-triggered HMGB1 release did not require lytic infection, in line with the above-described PBMC activation by non-productively infected PDAC cells [17]. Gemcitabine alone was unable to induce HMGB1 secretion, yet H-1PV-induced HMGB1 release remained unaffected in gemcitabine-treated cells. Gemcitabine, on the other hand, was able to induce—albeit not in all cell lines tested—mature interleukin 1-beta (IL-1ß) accumulation in culture supernatants. Taken together, these data show that H-1PV and gemcitabine complement each other in the induction of immunogenic signals. The compatibility of H-1PV-induced alarmin (HMGB1) secretion with other (ICD-inducing) chemotherapeutic regimens warrants the consideration of PV inclusion into various multimodal anti-PDAC treatment protocols [24]. The therapeutic promise of H-1PV administration in gemcitabine-treated pancreatic cancer patients is further supported by several reports in the literature showing that, unlike most nucleoside analogues, gemcitabine is lacking immunosuppressive properties. On the contrary, gemcitabine may be beneficial not only to the cytocidal but also to the pro-immune outcome of H-1PV infection, as assumed from the findings below.

• One study conducted in gemcitabine-treated PDAC patients revealed the ability of the drug to enhance T cell-mediated and DC-dependent host immune responses [25].


Based on favorable preclinical data hinting at the potentiation of OV-induced antitumor effects in the presence of gemcitabine, a clinical trial, ParvOryx02 (NCT02653313), was designed and conducted with the aim to provide a clinical proof-of-principle of the safety (and efficacy) of H-1PV + gemcitabine co-treatment. Patients with inoperable metastatic (at least one hepatic metastasis) pancreatic cancer were treated with H-1PV. The virus was first administered intravenously (40% of the total virus dose on four consecutive days), and the remaining virus dose was then given intra-metastatically as single hepatic injection, followed by gemcitabine treatment [31]. Partial response and extended overall survival were observed in two out of seven trial patients, and immunological signatures most likely contributed to this improved outcome. The ParvOryx02 study therefore provided the first clinical indication that immune mechanisms underlie PV-mediated tumor suppression [32].
