**5. Conclusions**

The main features to emerge from current review on retargeted oHSVs are as follows. A major argument that may be raised against tropism retargeted oHSVs is that they are not anti-pan-tumor agents. This argument does not take into account that any selected TAA is expressed across a number of cancer types, and therefore any retargeted oHSV can potentially be employed against a variety of indications. Moreover, clinical practice indicates that, due to cancer heterogeneity, the therapeutic effects of potentially pan-tumor OVs is not uniformly exerted on any type of tumor, or of cancer patient. The current trend in anti-cancer therapy is to develop therapeutic agents tailored on the patient characteristic, since it is futile and even counterproductive to administer a therapy to a patient who will not benefit of it (see reference [67] in this issue). The retargeted oHSVs meet this need. It is interesting to note that the restriction to predefined targets is shared with CAR-Ts (chimeric antigen receptor T cells) to solid tumors, which are essentially addressed to the same targets as the retargeted oHSVs [68,69]. The retargeted oHSVs and CAR-Ts could potentially be combined, and the combination could overcome some of the limits encountered by CAR-Ts, e.g., the difficulty to populate the tumors and to overcome the TME immunosuppression, essentially hostile to lymphocyte proliferation and activation [70].

A second Con is that the retargeted oHSVs strictly depend on the presence of the targeted receptor for infection, also for infection of producer cells. This property greatly limits the repertoire of cells which can be employed for production. Inasmuch as it advisable to make use of non-cancerous cells for clinical grade virus production, we set-up a strategy for virus growth, based on a double retargeting. Finally, it has long been debated that a disadvantage of employing human viruses as OVs is the prior immunity, which can block virions, particularly if they are administered by systemic routes. This notion should be reassessed in light of the interesting discovery that prior immunity, or an unrelated immune response, can actually contribute to unleashing the TME immunosuppression [71]. Indeed, the high efficacy deployed by the retargeted oHSVs enabled a systemic delivery, even in mice carrying a prior immunity to HSV [21]. In the field of vaccines to infectious agents, an analogous potentiation of the innate response by immunologically unrelated priming was also reported [72]. Whether such effects apply to humans treated with HSVbased OVs remains to be determined.

On the Pro side, the IL-12-positive R-337 was highly effective in eradicating the primary tumor, modifying the TME, and eliciting an antigen-agnostic long-term anti-tumor

vaccination. How do we explain the high abscopal efficacy and antigen-agnostic vaccination? It is well known that wt-HSVs cause immunogenic cell death and are inducers of innate response, which they blunt later in infection [35–38]. Typical features of the innate response to HSV are the secretion of IFN-α, -β, and -γ—through STING and other sensors—and of additional pro-inflammatory cytokines, infiltration by and activation of NK cells, of CD4+ and CD8+ lymphocytes, and of dendritic cells (DCs). The high levels of IFN-γ and the recruitment and activation of DCs, T and NK cells are hallmarks of the early phases of innate and adaptive responses to the tumor. Thus, the innate antiviral response may well serve as the switch that turns on the anti-tumor innate response; the latter then evolves into the adaptive response [73]. The LLC-1-HER2 tumor employed in current studies exemplifies a tumor insensitive to anti-PD-1. Treatment with R-337 efficiently unleashed the resistance, such that the efficacy of both R-337 and anti-PD-1 were dramatically increased in a combination regimen and resulted in high protection. Thus, the retargeted oHSVs appear to serve well the function of augmenting the tumor sensitivity to CPIs.

In mice there was no detectable off-tumor and off-target infections, even though the potential of spread to tissues with low level expression of the targeted receptor (HER-2, in our case) remains to be verified in humans for any given retargeted oHSV.

In conclusion, major difficulties facing the clinical translation of retargeted oHSVs to the clinic have been tackled and solutions have been found. The stage is ready for further developments and for verification at the bedside of how well this class of therapeutic agents will hold promise.

**Author Contributions:** Conceptualization and writing original draft G.C.-F. and A.V.; review and editing, all; Data curation, Methodology, A.V., V.L., M.S., T.G., C.B., G.G., V.G.; Supervision, G.C.-F. and A.Z.; Animal experimentation responsibility A.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** The work was funded by European Research Council (ERC) Advanced Grant number 340060 to G.C.-F., by RFO (University of Bologna) and Pallotti Legacy to T.G. The authors received funds for covering the costs to publish in open access (ERC grant).

**Institutional Review Board Statement:** Animal experiments were performed according to European directive 2010/63/UE, Italian law 26/2014. The experimental protocols were reviewed and approved by the University of Bologna Animal Care and Use Committee ("Comitato per il Benessere degli Animali", COBA), and approved by the Italian Ministry of Health. C57BL/6 mice transgenic for and tolerant to HER2 (B6.Cg-Pds5bTg(Wap-ERBB2)229Wzw/J) were obtained from Wayne State University through The Jackson Laboratories, and bred in the facility of the Department of Veterinary Medical Sciences, University of Bologna.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within current article or previous publications.

**Conflicts of Interest:** G.C.-F. is a minor shareholder in Nouscom.
