**3. Properties of the Tropism-Retargeted oHSVs Generated in Our Laboratory**

*3.1. Three Generations of Tropism Retargeted oHSVs*

For heuristic reasons we divide the retargeted oHSVs generated in our laboratory into three groups (Table 1). In all, the retargeting was achieved by insertion of a single chain antibody (scFv) to the receptor of choice, while detargeting was achieved by deletion of appropriate portions in gD [27,41] [WO2009144755] (Table 1).


**Table 1.** 1st, 2nd and 3rd generation retargeted oHSV, genotypic modifications for retargeting and detargeting purposes.

The 1st generation recombinants carry the scFv in gD, in place of either aa 6-38 or aa 61–218. Such deletions eliminate the portions in gD responsible for interactions with HVEM and nectin1, and confer full detargeting. In different recombinants, the scFvs were addressed alternatively to HER2 (human epithelial growth factor receptor 2), EGFR (epithelial growth factor receptor), EGFRVIII (EGFR variant III) or PSMA (prostate specific membrane antigen) [27,42] and WO2009144755.

The 2nd generation recombinants carried the scFv to HER2 or to EGFR in either gH or gB. This recombinant group explored the possibility that glycoproteins essential for HSV entry, other than gD, serve as vector for the scFv. They carry the ∆6–38 in gD [43,44] and WO201612849.

The 3rd generation recombinants simultaneously carry two retargeting moieties. The rationale is detailed below (see, paragraph 3.3). One moiety is the anti-HER2 scFv inserted in gD for cancer cell retargeting. The other moiety is the GCN4 peptide engineered alternatively in gD, gH or gB for retargeting to an ad hoc producer cell line. For detargeting purposes, the 3rd generation recombinants contained one of the following deletions in gD: aa 6–38, two single amino acids—∆D30 and ∆Y38, or deletions in the nectin binding site encompassing aa 214–223 [44–47] [WO2017211941, WO2017211944, WO2017211945].
