Dual Ligand Insertion in gB and gD of Oncolytic Herpes Simplex Viruses for Retargeting to a Producer Vero Cell Line and to Cancer Cells

Abstract

Oncolytic viruses gain cancer specificity in several ways. Like the majority of viruses, they grow better in cancer cells that are defective in mounting the host response to viruses. Often, they are attenuated by deletion or mutation of virulence genes that counteract the host response or are naturally occurring oncolytic mutants. In contrast, retargeted viruses are not attenuated or deleted; their cancer specificity rests on a modified, specific tropism for cancer receptors. For herpes simplex virus (HSV)-based oncolytics, the detargeting-retargeting strategies employed so far were based on genetic modifications of gD. Recently, we showed that even gH or gB can serve as retargeting tools. To enable the growth of retargeted HSVs in cells that can be used for clinical-grade virus production, 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 spread, cytotoxicity, and in vivo 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. 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.

Simultaneous Insertion of Two Ligands in gD for Cultivation of Oncolytic Herpes Simplex Viruses in Noncancer Cells and Retargeting to Cancer Receptors

“…In general, the murine cancer cells are not as permissive to HSV as the human cancer cells; hence, this model underestimates the antitumor efficacy, a property shared with the vast majority of murine cancer models for oncolytic HSVs ( 46 , 47 ). Here, the important result was that the antitumor efficacy of R-87 could not be differentiated from that of R-LM113 and of the gB recombinant R-317, described in the companion paper ( 45 ). Thus, the replication properties in cell cultures are recapitulated in the in vivo antitumor efficacy, and a recombinant carrying two retargeting moieties in gD is not at a disadvantage relative to R-LM113, which carries a single retargeting moiety.…”

“…at 3 to 4 days' intervals, with 1 × 10 8 PFU/injections, for a total of 4 treatments. As a comparison, we included in the experiment the prototypic R-LM113 and R-317 described in the companion paper ( 45 ). Figure 7A to C shows that the antitumor efficacy of R-87 was very similar to those of R-LM113 and of R-317, and the tumor size was significantly smaller than that in the untreated mice at 28 days ( Fig.…”

“…In a companion paper, we show that double retargeting is feasible also by insertion of the GCN4 peptide in gB and of the scFv in gD ( 45 ); even in that study ( 45 ), a novel gD detargeting strategy was developed. In both studies, the comparison of a number of recombinants led to optimization of double-retargeted recombinants.…”

Herpes Simplex Virus Vectors for Gene Transfer to the Central Nervous System

“…Using related ICP0+ vectors, we have developed promoter systems for regulated transgene expression in sensory nerves for the treatment of chronic pain models [ 114 ], but we do not yet know whether promoter regulation will be preserved in CNS neurons in the absence of ICP0. Over the years, significant efforts have been devoted to the retargeting of HSV vectors to achieve selective transduction of specific cell types, primarily cancer cells through growth factor receptors [ 115 , 116 , 117 , 118 , 119 ]. These technologies will be applied to our nontoxic CNS vectors.…”

HSV as A Platform for the Generation of Retargeted, Armed, and Reporter-Expressing Oncolytic Viruses

“…The availability of alternative retargeting strategies led to the construction of a non-cancer cell line for the in vitro cultivation of retargeted o-HSV [ 37 ]. In particular, the recombinants carrying the scFv to HER2 in gD and the GCN4 peptide in gB, gH, or gD infected and replicated in a Vero cell line derivative named Vero-GCN4R, which transgenically expresses an artificial receptor to GCN4, named GCN4R [ 37 , 38 , 39 ]. The double-retargeted recombinants alternatively infect the HER2-positive cancer cells via HER2, and the producer Vero-GCN4R cell line via the GCN4-GCN4R interaction.…”