**6. Conclusions**

Preclinical research and clinical experience have demonstrated the multimodal anticancer activity of the oncolytic parvovirus H-1PV. Two essential facets of H-1PV-induced tumor suppression consist of direct killing of malignant cells (oncolysis) and activation of cellular immune responses against the tumor. H-1PV infection, oncolysis and immune stimulation are interconnected, coordinated events, which cooperate towards multisided tumor elimination.

Glioblastoma and pancreatic adenocarcinoma are among the most devastating human malignancies, characterized by resistance to current therapies, tendency to recurrence and an overall poor outcome. H-1PV has undergone clinical testing in two recently conducted trials, ParvOryx01 in glioblastoma and ParvOryx02 in pancreatic carcinoma. Virus excellent safety and tolerability, together with the capacity for gentle TME immune landscape proinflammatory modulation, provide a strong impetus for further H-1PV clinical development. It should, however, be noted that in the clinical setting, various patientdependent factors may result in suboptimal antitumor effects. Large intra-tumoral tissue heterogeneity, emergence of tumor cells resistant to virus infection/killing, dominance of the highly immunosuppressive TME, hampered virus spreading, off-target infection and virus neutralization by antiviral antibodies are among the major barriers to efficient H-1PV-induced tumor elimination. While various other approaches (capsid modification, chimera generation, fitness mutant selection, armed vector construction) to H-1PV treatment optimization are currently under investigation, PV-based combinatorial therapies are considered as a particularly promising avenue that holds the potential of enhancing both oncolysis and immune-mediated tumor destruction. Combinations of the virus with other anticancer approaches, namely irradiation, chemotherapy (gemcitabine), epigenetic modulation (HDACi), angiogenesis regulation (bevacizumab) or immunotherapy (immune checkpoint blockade), were evaluated in both preclinical models and in cancer patients. The combinatorial H-1PV-based viro(immuno)therapeutic strategy was proven to achieve greater anticancer effects compared to individual agents alone. The synergistic boost was particularly pronounced in combinations including the HDACi VPA, bevacizumab or the PD-1 inhibitor nivolumab. Glioblastoma patients treated with this combination showed striking tumor remission and extended survival, notably after second or even third recurrence. These early clinical observations speak in favor of considering H-1PV inclusion into various immunotherapeutic protocols against glioblastoma and other poor-prognosis solid tumors (Figure 4). *Cancers* **2021**, *13*, x 12 of 15

**Figure 4.** H-1PV inclusion into combinatorial anticancer immunotherapy regimens. The development of H-1PV combinations with ionizing radiation, chemotherapeutics, histone deacetylase inhibitors (HDACis), angiogenesis inhibitors and immunomodulators holds significant promise for the future of poor-prognosis solid cancer treatment. **Figure 4.** H-1PV inclusion into combinatorial anticancer immunotherapy regimens. The development of H-1PV combinations with ionizing radiation, chemotherapeutics, histone deacetylase inhibitors (HDACis), angiogenesis inhibitors and immunomodulators holds significant promise for the future of poor-prognosis solid cancer treatment.

**Funding:** The parvovirus research described in this review was partially funded by research grants from the Luxembourg Cancer Foundation and Télévie to A.M., and from ORYX GmbH & Co. KG

**Acknowledgments:** The authors wish to thank all members and collaborators of the former Tumor Virology Division at the German Cancer Research Center, for their contribution to the work described in this review. We are deeply indebted to the clinical trial and compassionate use program participants and their families, for their contribution to gathering clinical experience and knowledge. We are grateful to K. Geletneky for helpful discussions of the glioblastoma compassion-

**Conflicts of Interest:** A.A., T.F., J.R. and A.M. are holders of patents or patent applications related to H-1PV use for cancer therapeutic purposes. The parvovirus clinical trials (ParvOryx01, ParvOryx02) and compassionate H-1PV uses were financially supported by ORYX GmbH & Co. KG (Baldham, Germany). The funders had no role in the writing of the manuscript or in the decision to

1. Bretscher, C.; Marchini, A. H-1 parvovirus as a cancer-killing agent: Past, present, and future. *Viruses* **2019**, *11*, 562,

2. Toolan, H.W.; Dalldore, G.; Barclay, M.; Chandra, S.; Moore, A.E. An unidentified, filtrable agent isolated from transplanted

3. Besselsen, D.G.; Besch-Williford, C.L.; Pintel, D.J.; Franklin, C.L.; Hook, R.R.; Riley, L.K. Detection of H-1 parvovirus and Kilham

4. Rommelaere, J.; Giese, N.; Cziepluch, C.; Cornelis, J.J. Parvoviruses as anticancer agents. In *Viral Therapy of Human Cancers*;

5. Rommelaere, J.; Geletneky, K.; Angelova, A.L.; Daeffler, L.; Dinsart, C.; Kiprianova, I.; Schlehofer, J.R.; Raykov, Z. Oncolytic parvoviruses as cancer therapeutics. *Cytokine Growth Factor Rev.* **2010**, *21*, 185–195, doi:10.1016/j.cytogfr.2010.02.011.

manuscript.

to A.A., J.R. and A.M.

ate parvovirus use program.

submit it for publication.

human tumors. *Proc. Natl. Acad. Sci. USA* **1960**, *46*, 1256–1258, doi:10.1073/pnas.46.9.1256.

rat virus by PCR. *J. Clin. Microbiol.* **1995**, *33*, 1699–1703, doi:10.1128/jcm.33.7.1699-1703.1995.

Sinkovics, J.G., Horvath, J.C., Eds.; Marcel Dekker: New York, NY, USA, 2005; pp. 627–675.

**References** 

doi:10.3390/v11060562.

**Author Contributions:** Writing—original draft preparation, A.A., T.F. and C.B.; writing—review and editing, A.A., J.R. and A.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** The parvovirus research described in this review was partially funded by research grants from the Luxembourg Cancer Foundation and Télévie to A.M., and from ORYX GmbH & Co. KG to A.A., J.R. and A.M.

**Acknowledgments:** The authors wish to thank all members and collaborators of the former Tumor Virology Division at the German Cancer Research Center, for their contribution to the work described in this review. We are deeply indebted to the clinical trial and compassionate use program participants and their families, for their contribution to gathering clinical experience and knowledge. We are grateful to K. Geletneky for helpful discussions of the glioblastoma compassionate parvovirus use program.

**Conflicts of Interest:** A.A., T.F., J.R. and A.M. are holders of patents or patent applications related to H-1PV use for cancer therapeutic purposes. The parvovirus clinical trials (ParvOryx01, ParvOryx02) and compassionate H-1PV uses were financially supported by ORYX GmbH & Co. KG (Baldham, Germany). The funders had no role in the writing of the manuscript or in the decision to submit it for publication.
