*2.1. Tropism Retargeting*

The notion of tropism retargeting was pioneered by Glorioso and co-workers, and by Roizman and Zhou, and has been reviewed [22–25]. It entails the genetic engineering of a ligand to a selected cancer receptor into one of the viral glycoproteins that mediate HSV entry into the cells, most frequently gD [24,26,27]. Crucial to this development has been the elucidation of the molecular events that govern HSV entry. It requires four essential glycoproteins, gD, gH, gL and gB, which are activated in a cascade fashion, and two major alternative receptors HVEM (herpesvirus entry mediator) and nectin1 which interact with gD and activate it; they serve as major tropism determinants. Thereafter, the receptoractivated gD and additional integrin receptors activate the heterodimer gH/gL, and finally gB. The latter executes the fusion between the virion envelope and the cell membranes plasma membranes or endocytic membranes [28–32]. The presence of cell surface receptors is a requirement for HSV entry [33].

The strategy developed in our laboratory to generate cancer-specific oHSVs and increase their efficacy entails (a) the retargeting of the virus tropism to Tumor-Associated Antigens (TAAs), i.e., to molecules that are selectively present at cancer cell surfaces; (b) the detargeting of the virus tropism from the natural receptors HVEM and nectin, to avoid off-target and off-tumor infections. When combined, retargeting and detargeting provide specificity and safety; (c) preservation of the full spectrum of viral genes, to enable a robust anti-tumor response [26,27]. Since the retargeted oHSVs do not carry any genetic modification in virulence or other genes, they are "fully virulent viruses in their target cancer cells" (Figure 1). Clearly, the extent of cancer-specificity reflects the specificity of the target to which the oHSVs are addressed. Some TAAs—such as EGFRVIII (epidermal growth factor receptor Variant III), IL-13 Receptor 2-α and HER2 (human epidermal growth factor receptor 2) are more specific than others. A further improvement was introduced by Glorioso and coworkers through point mutations in gD that impair the binging of neutralizing antibodies, and thus make the oHSV stealth to anti-HSV antibodies present in the human population [34].

Bench and preclinical studies foresee the following advantages for the tropism retargeted oHSVs. In contrast to the ∆γ34.5 oHSVs that can replicate solely in cancer cells that carry defects in certain pathways of the innate response and may potentially replicate in non-cancerous cells defective in innate responses, the retargeted oHSVs infect cancer cells irrespective of the status of their innate response and are designed to prevent off-target infections. The extent of infections in tissues with low level expression of the targeted receptors remains to be verified in humans. Moreover, once the retargeted oHSVs infect the tumor cells, they are essentially wt-viruses, they blunt the cell innate response and promote high viral replication [35–38]. Lastly, in the tumor bed, the retargeted oHSVs exclusively infect the cancer cells, whereas the ∆γ34.5 and the attenuated viruses additionally infect immune cells, with unclear effects on the global immune response.

**Figure 1.** Schematic view of HSV tropism retargeting and immunotherapy induced by armed oHSVs. (**A**–**D**). (**A**) wt-HSV infects normal tissues, i.e., its natural target cells through natural major receptors HVEM and nectin1 (blue). (**B**) 1st and 2nd generation tropism retargeted oHSVs infect cancer cells expressing the target Tumor Associated Antigen (TAA, violet) and fails to infect its natural targets. The scFv to TAA which mediated the virus retargeting to cancer cells is engineered in the following virion glycoproteins: gD (1st generation), either gB or gH (2nd generation). (**C**) 3rd generation tropism retargeted oHSV, can infect both the cancer cells that express the target TAA (violet) and the producer cells that express an artificial receptor (orange), and fails to infect its natural targets. (**D**) The IL-12-armed retargeted oHSV specifically infects tumors cells, causes immunogenic cell death of cancer cells and elicits immuno-therapeutic response, that result in inhibition of tumor growth, sensitization to checkpoint blockade and antigen-agnostic vaccination.
