*Review* **Immunotherapeutic Efficacy of Retargeted oHSVs Designed for Propagation in an Ad Hoc Cell Line**

**Andrea Vannini 1,\*, Valerio Leoni <sup>1</sup> , Mara Sanapo <sup>1</sup> , Tatiana Gianni <sup>1</sup> , Giorgia Giordani <sup>2</sup> , Valentina Gatta <sup>1</sup> , Catia Barboni <sup>3</sup> , Anna Zaghini <sup>3</sup> and Gabriella Campadelli-Fiume 1,\***


**Simple Summary:** The onco-immunotherapeutic viruses, among which stand the onco-immunotherapeutic herpes simplex viruses, have gained renewed interest due to their ability to unlock the potential of checkpoint inhibitors in preclinical and clinical settings. In prior decades, safety concerns led to the generation of overall safe, partially or highly attenuated oncolytic viruses. Current focus is on more efficacious onco-immunotherapeutic viruses with limited ability to cause off-tumor and off-target infections and the capability to subvert the tumor microenvironment immunosuppression hence to potentiate checkpoint inhibitors. These viruses might serve as potential partners of Tcell therapies.

**Abstract:** Our laboratory has pursued the generation of cancer-specific oncolytic herpes simplex viruses (oHSVs) which ensure high efficacy while maintaining a high safety profile. Their blueprint included retargeting to a Tumor-Associated Antigen, e.g., HER2, coupled to detargeting from natural receptors to avoid off-target and off-tumor infections and preservation of the full complement of unmodified viral genes. These oHSVs are "fully virulent in their target cancer cells". The 3rd generation retargeted oHSVs carry two distinct retargeting moieties, which enable infection of a producer cell line and of the target cancer cells, respectively. They can be propagated in an ad hoc Vero cell derivative at about tenfold higher yields than 1st generation recombinants, and more effectively replicate in human cancer cell lines. The R-335 and R-337 prototypes were armed with murine IL-12. Intratumorally-administered R-337 conferred almost complete protection from LLC-1- HER2 primary tumors, unleashed the tumor microenvironment immunosuppression, synergized with the checkpoint blockade and conferred long-term vaccination against distant challenge tumors. In summary, the problem intrinsic to the propagation of retargeted oHSVs—which strictly require cells positive for targeted receptors—was solved in 3rd generation viruses. They are effective as immunotherapeutic agents against primary tumors and as antigen-agnostic vaccines.

**Keywords:** oncolytic virus; herpes simplex virus; retargeted virus; tropism retargeting; tumor; immunotherapy; checkpoint inhibitor; vaccination; antigen-agnostic vaccination; HER2
