*3.3. Cultivation of Tropism-Retargeted oHSVs in Non-Cancerous Producer Cells*

A critical feature of the retargeted oHSVs generated in our laboratory is that they are strictly dependent on the targeted cancer receptor for infection, including infection of the producer cells. Often, the target receptor is an oncogene, i.e., it contributes to the oncogenic potential of the cancer cells. While the retargeted oHSVs can be readily cultivated in human cancer cells lines positive for the targeted receptor, approval of clinical grade virus production in cancer cells by competent authorities might likely imply specific motivations. A goal in the design of the 3rd generation retargeted oHSVs was to generate a non-cancerous producer cell line for the in vitro growth of the retargeted oHSVs. To this end, as mentioned above, we designed recombinants that simultaneously carry two retargeting moieties (Figures 1 and 2A). The scFv retargets HSV to the cancer receptor (HER2, in our case). The second one consists of a small high affinity ligand (GCN4 peptide) engineered in one of the entry glycoproteins—gD, gH or gB (Figure 2A) [45–47]. The producer cell line is a Vero cell derivative, named Vero-GCN4R-HER2, which expresses an artificial receptor for the GCN4 peptide, along with human HER2.

In subsequent sections of this review, we shall focus on the two most advanced recombinants from the 3rd generation group, R-335 and R-337. They carry (i) the insertion of the GCN4 peptide in gB between residues 81 and 82; (ii) the deletion of only two amino acids in gD—D30 and Y38—for HVEM and nectin1 detargeting; (iii) the insertion in gD of scFv to HER2 in place of Y38. R-335 and R-337 carry mIL-12 in the US1/US2 intergenic region, a site that enables a high expression level. While R-335 carried the natural form of mIL-12, made of p40 and p35 subunits, R-337 carried the fusion form, in which the two subunits are held together by a linker to form a single peptide. In cultured cells, the fusion form was produced at 50 to 100-fold higher amounts than the dimeric form and possibly was more stable. R-335 and R-337 genotypes are depicted in Figure 2A. The properties of OVs, and particularly oHSVs expressing IL-12 are reviewed in [53].

Preliminarily, we quantified the ability of the two recombinants to grow in Vero-GCN4R-HER2 and in the human HER2-positive cancer cell line SK-OV-3. The yields of R-335 and R-337 in Vero-GCN4R-HER2 are shown in Figure 2B, which also shows the yields of R-LM5 (an essentially wt HSV carrying EGFP [27]) and of the 1st generation recombinant R-115. R-335 and R-337 infect these cells through both the GCN4R and HER2 receptors, whereas R-115 infects only through HER2. Figure 2C shows the fold-increase of the yields relative to that of R-115. Two features emerged. R-335 and R-337 replicated to seven to eight-fold higher yields than R-115. As expected, R-335 and R-337 replicated to fivefold lower yields than R-LM5; this is a common feature for recombinant viruses and accounts for different receptor usage, in that R-LM5 infection occurs through the simian orthologs of the natural receptors nectin1 and HVEM, which ensure the best possible interaction for HSV entry into the cells.

**Figure 2.** Growth kinetics of 1st and 3rd generation HER2-retargeted oHSVs in cancerous and non-cancerous producer cells. (**A**) Schematic representation of genomes of wt HSV named R-LM5 (carrying GFP), R-115 (1st generation), R-335 and R-337 (3rd generation) retargeted oHSVs. Indicated are the genetic loci of gB, gD, the insertion site of mIL-12 in the US1 and US2 intergenic locus. 1st and 3rd generation recombinant viruses carry the insertion of scFv anti-HER2 for the retargeting to HER2-positive cells, and the deletion of indicated portions of gD for the detargeting from HSV-1 natural receptors HVEM and nectin1. The 3rd generation R-335 and R-337 viruses carry the GCN4 peptide in gB between aa 81 and 82, and were engineered as detailed in [17,54]. (**B**–**E**) Yields of the wt HSV named R-LM5 (carrying GFP), R-115, R-335 and R-337 in VERO-GCN4R-HER2 (**B**,**C**) and in SK-OV-3 (**D**,**E**) cells at 24, 48 and 72 h after infection. Replicate cultures of each cell line were infected with the indicated viruses at 0.1 PFU/cell as titrated in SK-OV-3 cell line. Progeny virus was titrated in SK-OV-3 cells. In panels C and E, yields are expressed relative to that of R-115. The data represent the average of at least five independent experiments ± SD. (**F**) Plaque size of the indicated viruses five days after infection. For each virus-infected culture 20 plaque pictures were taken, expressed as arbitrary units and plotted ± SD. (**B**–**F**) Statistical significance was calculated by One Way ANOVA test and expressed as \* = *p*-value < 0.05; \*\* = *p*-value < 0.01; \*\*\* = *p*-value < 0.001.

In our experience the highest yields for both wt-HSV and HER2-reatargeted oHSVs are obtained in SK-OV-3. Figure 2D,E show that the wt R-LM5 grew somewhat better in SK-OV-3 than in Vero-GCN4R-HER2 cells, as expected. The growth of R-335 and R-337 could not be differentiated from that of R-LM5 and was about three-fold higher than that of R-115. In addition, R-335 and R-337 plaques in SK-OV-3 cells were doubled in size relative to those from R-115, in agreement with the virus yields results (Figure 2F). The higher replication of R-335 and R-337 relative to that of R-115 was surprising in that SK-OV-3 cells lack the receptor for GCN4 peptide. We interpret these results to indicate that the smaller deletion in gD improved the glycoprotein performance, and that the GCN-4 insertion in gB somehow activated gB or a combination of these effects.
