The simultaneous insertion of two ligands in gD for the cultivation of oncolytic HSVs in non-cancer cells and the retargeting to cancer receptors

Insertion of a single chain antibody (scFv) to HER2 (human epidermal growth factor receptor 2) in gD, gH, or gB gives rise to herpes simplex viruses (HSVs) specifically retargeted to HER2-positive cancer cells, hence in highly specific non-attenuated oncolytic agents. Clinical grade virus production can not rely on cancer cells. Recently, we developed a double retargeting strategy whereby gH carries the GCN4 peptide for retargeting to the non-cancer producer Vero-GCN4R cell line, and gD carries the scFv to HER2 for cancer retargeting. Here, we engineered double retargeted recombinants, which carry both the GCN4 peptide and the scFv to HER2 in gD. Novel, more advantageous detargeting strategies were devised, so as to optimize the cultivation of the double-retargeted recombinants. Nectin1 detargeting was achieved by deletion of aa 35-39, 214-223, or 219-223, and replacement of the deleted sequences with one of the two ligands. The latter two deletions were not attempted before. All recombinants exhibited the double retargeting to HER2 and to the Vero-GCN4R cells, as well as detargeting from the natural receptors HVEM and nectin1. Of note, some recombinants grew to higher yields than others. The best performing recombinants carried a gD deletion as small as 5 amino acids, and grew to titers similar to those exhibited by the singly retargeted R-LM113, and by the non-retargeted R-LM5. This study shows that double retargeting through insertion of two ligands in gD is feasible and, when combined with appropriate detargeting modifications, can result in recombinants highly effectivein vitroandin vivo.IMPORTANCEThere is increasing interest in oncolytic viruses, following FDA and EMA approval of the oncolytic HSV OncovexGM-CSF, and, mainly, because they greatly boost the immune response to the tumor and can be combined with immunotherapeutic agents, particularly immune checkpoint inhibitors. A strategy to gain high cancer specificity and avoid virus attenuation is to retarget the virus tropism to cancer-specific receptors of choice. However, cultivation of retargeted oncolytics in cells expressing the cancer receptor may not be approvable by regulatory agencies. We devised a strategy for their cultivation in non-cancer cells. Here, we describe a double retargeting strategy, based on the simultaneous insertion of two ligands in gD, one for retargeting to a producer, universal Vero cell derivative, one for retargeting to the HER2 cancer receptor. These insertions were combined with novel, minimally-disadvantageous detargeting modifications. The current and accompanying studies teach how to best achieve the clinical-grade cultivation of retargeted oncolytics.