**1. Cancer-Selective Oncolytic Herpes Simplex Viruses and Synergism with Check Point Blockade**

Herpes simplex viruses (HSVs) were among the first viruses taken into consideration as candidate oncolytic viruses (OVs) [1–4] and still rank high in the list of OVs in clinical trials [5]. The early approaches to generate OVs, including oHSVs, were rather conservative.

**Citation:** Vannini, A.; Leoni, V.; Sanapo, M.; Gianni, T.; Giordani, G.; Gatta, V.; Barboni, C.; Zaghini, A.; Campadelli-Fiume, G. Immunotherapeutic Efficacy of Retargeted oHSVs Designed for Propagation in an Ad Hoc Cell Line. *Cancers* **2021**, *13*, 266. https://doi.org/10.3390/ cancers13020266

Received: 30 October 2020 Accepted: 8 January 2021 Published: 12 January 2021

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Safety was a major concern, probably because scientists wanted to avoid the problems which characterized the initial approaches to gene therapy, and because of the frailty of patients. Most of the OVs that entered the clinical trials were attenuated, or over-attenuated. In reality, safety proved not to be a major clinical issue. oHSVs, and OVs in general, are well tolerated in clinical applications with very limited description of serious adverse effects [6,7]. However, the efficacy in humans did not keep up to the expectations raised by animal experimentation. For most OVs, and particularly for oHSVs, attenuation has been the prerequisite for cancer selectivity, and hence for safety. Cancer cells exhibit varying defects in innate responses and weakly contrast viral replication. Attenuated viruses exploit such defects to target cancers cells, which sustain the replication of both wt and attenuated viruses, and to spare non-cancer cells, in which the replication of attenuated viruses is contrasted, but not fully abolished, by the antiviral innate response. One such example is the attenuation conferred by the deletion of the γ34.5 virulence gene [2–4]. Since attenuation also weakens virus replication in cancer cells, additional modifications were introduced in the ∆γ34.5 oHSVs to rescue replication and virulence. The α47 open reading frame was deleted to augment antigen presentation. The deletion modified the expression profile of the late US11 gene. Immunomodulatory genes were engineered in the viral genome. This is essentially the genotype of the approved OncovexGM-CSF, also known as T-VEC or Imlygic® [2,8].

The interest in OVs, including oHSVs, was boosted by check point inhibitors (CPIs) [9]. These molecules disable the breaks imposed by some tumors on the anti-cancer immune machinery and unleash the T-cell response against tumors. In humans, CPIs are limited by the fact that they target a restricted range of tumors—typically those with high mutational load, high tumor-specific T-cell infiltration and low T-cell activity due to the checkpoint brakes—and are effective only against a fraction of patients. The rationale for combining oncolytic viruses, like OncovexGM-CSF, with checkpoint inhibitors is compelling [10–13]. OVs inflame tumors, overcome the tumor microenvironment (TME) immunosuppression, and unlock the potential of checkpoint inhibitors across many cancer types. The underlying mechanism rests on the direct oncolysis induced by virus replication and consequent increase in both total and cancer-specific antigenic load in the TME, on the ability to recruit the immune cells to TME and to induce the expression of pro-inflammatory molecules. In turn, the latter activate immune effector cells and cause tumor inflammation. Given the heterogeneity in cancer genotypes and phenotypes, it is foreseen that even more complex combinations of immunomodulatory agents may be required to obtain consistent and durable therapeutic responses against a broad spectrum of cancers [14,15]. oHSVs are well equipped to do this job, since their large genome has space for extra genes. GM-CSF is a potent pro-inflammatory cytokine, most frequently employed as a payload in oHSVs [5]; it targets mainly the myeloid lineage, activates the dendritic cells, and enhances anticancer effects; it is present in OncovexGM-CSF. IL-12 (interleukin-12) [16] is another highly effective pro-inflammatory cytokine transgenically expressed by OVs, since it orchestrates the innate and the adaptive immune response against cancer and pathogens [17]. It is present in the oHSV named M032, currently in clinical trial [18,19]. Additional payloads recently engineered in oHSVs include the ligands to co-stimulatory immune receptors, CPI or combinations thereof [20,21].
