**3. Parvovirus-Based Combinatorial Immunotherapy against Glioblastoma**

Glioblastoma multiforme (GBM) is the most common and aggressive human primary brain tumor. Similar to PDAC, GBM patients experience a very poor outcome. The 5-year overall survival rate is very low, around 5.1% [43]. GBM treatment faces a unique challenge: the presence of the blood–brain barrier (BBB), which largely prevents drugs, including small-molecule ones, from entering the central nervous system [44]. Current therapeutic approaches therefore include surgical resection of the tumor—to the largest extent feasible and safe—followed by radiotherapy and concomitant chemotherapy [45]. Unfortunately, despite all clinical efforts, tumor progression and recurrence typically occur, calling for alternative therapeutic solutions [46].

Based on the so far unmet need for novel, more efficient treatments, GBM was among the preclinical tumor models most extensively studied in our laboratory. H-1PV capacity for selectively killing glioma cells through cytosolic activation of lysosomal proteases was first demonstrated in vitro [47]. These results were validated in animal models, namely in immunocompetent rats bearing orthotopic autologous RG-2 tumors and in immunodeficient rats bearing xeno-transplanted human U87 gliomas. In these models, tumor regression after local, intravenous or intranasal virus administration was observed [47–49]. H-1PV treatment was not associated with any significant off-target toxicities; accordingly, virus transcription and NS1 protein accumulation could be detected in regressing tumor remnants and not in the surrounding normal tissues [48]. Interestingly, the therapeutic effect was potentiated in the presence of an intact host immune system. T cell depletion impaired H-1PV-induced glioma suppression; conversely, the presence of T cell only, in the absence of PV treatment, was not sufficient to inhibit tumor growth [11]. These preclinical observations provided the first hints of host T cell response involvement in PV-mediated glioma regression, hence the rationale for the development of PV-based immunotherapies against glioblastoma.

Pursuant to the above-described preclinical findings, the ParvOryx01 trial (NCT01301430) in recurrent glioblastoma patients delivered the first clinical proof-of-concept for tumorinfiltrating lymphocytes (TILs) playing substantial role in H-1PV-mediated immunomodulation of GBM TME. Although ParvOryx01 primary objectives were to determine virus safety, tolerability, pharmacokinetics, shedding and maximum tolerated dose, the analysis of post-virus-treatment resected tumor tissues revealed the presence of prominent immune cell infiltrates [50]. These infiltrates were comprised of CD45+CD3+CD4+ and CD45+CD3+CD8+ TILs. The latter contained both perforin and granzyme B-positive secretory granules, which is indicative of CTL cytolytic activity. TILs proved, in addition, to be CD25 (IL2 receptor alpha chain)-positive. Only a minor fraction of these cells expressed FOXP3, indicating the scarcity of Treg cells within the intra-tumoral immune infiltrates. Intra-tumoral production of proinflammatory cytokines (IFN-γ, IL-2) was also detected, together with inducible nitric oxide synthase (iNOS) expression in CD68+ tumorassociated microglia/macrophage cells [50,51]. Interestingly, tumor cells expressed the CD40 ligand (CD40L), a positive prognostic factor in glioblastoma [52]. Co-expression of CD40L and CD40, considered as a negative prognostic factor, was not seen [50,51]. Taken together, these first clinical findings indicated that H-1PV has the capacity to exert immunostimulating effects on glioblastoma TME. This makes the virus a worthwhile partner in therapeutic combinations, which aim at warming up the intrinsically immunosuppressive and immune-evasive environment of brain tumors.
