[PMC free article] [PubMed] [Google Scholar] 37. a double-retargeting strategy has been developed. Here we show that several sites in the N terminus of gB are suitable to harbor the 20-amino-acid (aa)-long GCN4 peptide, which readdresses HSV tropism to Vero cells expressing the artificial GCN4 receptor and thus enables virus cultivation in the producer noncancer Vero-GCN4R cell line. The gB modifications can be combined with a minimal detargeting modification in gD, consisting in the deletion of two residues, aa 30 and 38, and replacement of aa 38 with the scFv to human epidermal growth factor receptor 2 (HER2), for retargeting to the cancer receptor. The panel of recombinants was analyzed comparatively in terms of virus growth, cell-to-cell Zidebactam sodium salt spread, cytotoxicity, and antitumor efficacy to define the best double-retargeting strategy. IMPORTANCE There is increasing interest in oncolytic viruses, following FDA and the European Medicines Agency (EMA) approval of HSV OncovexGM-CSF, and, mainly, because they greatly boost the immune response to the tumor and can be combined with immunotherapeutic agents, particularly checkpoint inhibitors. A strategy to gain cancer specificity and avoid virus attenuation is to retarget the virus tropism to cancer-specific receptors of choice. Cultivation of fully retargeted viruses is challenging, since they require cells that express the cancer receptor. We devised a strategy for their cultivation in producer noncancer Vero cell derivatives. Here, we developed a double-retargeting strategy, based on insertion of one ligand in gB for retargeting to a Vero cell derivative and of anti-HER2 ligand in gD for cancer retargeting. Zidebactam sodium salt These modifications were combined with a minimally destructive detargeting strategy. This study and its companion paper explain the clinical-grade cultivation of retargeted oncolytic HSVs and promote their translation to the clinic. cultivation in noncancer cells; one such modification was combined with a gD detargeting strategy based on the deletion of Rabbit Polyclonal to CDH11 two single amino acids (residues 30 and 38) and replacement of aa 38 with the scFv to HER2 for retargeting to the cancer receptor. RESULTS Insertion of ligands in gB and in gD for the simultaneous retargeting to two different targets. We generated four recombinants, R-313, R-315, R-317, and R-319, carrying the GCN4 peptide in gB at one of four sites, i.e., between aa 43 and 44, 81 and 82, 76 and 77, and 95 and 96, and carrying the scFv to HER2 in gD, in place of aa 6 to 38 (Fig. 1 and Table 1). A description of these viruses is given in European patent application PCT/EP2017/063944 (M. G. Campadelli and B. Petrovic, 14 December 2017). The tropism of the recombinants was evaluated in the HER2-positive SK-OV-3 cancer cells, in the Vero-GCN4R, in wt Vero cells, and in derivatives of the receptor-negative J cells, transgenically expressing a single receptor, e.g., HER2, nectin1, or HVEM (20, 36). R-LM113, retargeted to HER2 but not to GCN4R, was included as a control. Figure 2A to ?toDD shows that the recombinant R-313, R-315, R-317, and R-319 viruses were retargeted to GCN4R, as indicated by the ability to infect Vero-GCN4R cells, in the presence of the anti-HER2 monoclonal antibody (MAb) trastuzumab. All recombinants were retargeted to HER2, as indicated by ability to infect J-HER2 and SK-OV-3 cells in a trastuzumab-dependent fashion. This property is shared with R-LM113 (Fig. 2E). Consistent with the deletion of aa 6 to 38 (6C38) in gD and replacement of the deleted sequences with the scFv to HER2 (22), all recombinants failed to infect J-HVEM and J-nectin1 cells, i.e., they were detargeted from natural gD receptors. They infected the wt Vero cells in a trastuzumab-inhibited fashion, very likely through the simian orthologue of HER2. Indeed, the whole-genome sequence of Vero cells is incomplete, Zidebactam sodium salt and so far, there is no documentation of a HER2 homologue in this cell line. Nonetheless, Vero Zidebactam sodium salt cells were isolated from an African green monkey (sp.), and the sequence of the genome contains the HER2 homologue (test: *, 0.05; **, 0.01; ***, 0.001. This experiment is the same as that shown in Fig. 7 of the companion paper (37). DISCUSSION.