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A Strategy for Cultivation of Retargeted Oncolytic Herpes Simplex Viruses in Non-cancer Cells.

ABSTRACT: The oncolytic herpes simplex virus (HSV) that has been approved for clinical practice and those HSVs in clinical trials are attenuated viruses, often with the neurovirulence gene ?134.5 and additional genes deleted. One strategy to engineer nonattenuated oncolytic HSVs consists of retargeting the viral tropism to a cancer-specific receptor of choice, exemplified by HER2 (human epidermal growth factor receptor 2), which is present in breast, ovary, and other cancers, and in detargeting from the natural receptors. Because the HER2-retargeted HSVs strictly depend on this receptor for infection, the viruses employed in preclinical studies were cultivated in HER2-positive cancer cells. The production of clinical-grade viruses destined for humans should avoid the use of cancer cells. Here, we engineered the R-213 recombinant, by insertion of a 20-amino-acid (aa) short peptide (named GCN4) in the gH of R-LM113; this recombinant was retargeted to HER2 through insertion in gD of a single-chain antibody (scFv) to HER2. Next, we generated a Vero cell line expressing an artificial receptor (GCN4R) whose N terminus consists of an scFv to GCN4 and therefore is capable of interacting with GCN4 present in gH of R-213. R-213 replicated as well as R-LM113 in SK-OV-3 cells, implying that addition of the GCN4 peptide was not detrimental to gH. R-213 grew to relatively high titers in Vero-GCN4R cells, efficiently spread from cell to cell, and killed both Vero-GCN4R and SK-OV-3 cells, as expected for an oncolytic virus. Altogether, Vero-GCN4R cells represent an efficient system for cultivation of retargeted oncolytic HSVs in non-cancer cells.IMPORTANCE There is growing interest in viruses as oncolytic agents, which can be administered in combination with immunotherapeutic compounds, including immune checkpoint inhibitors. The oncolytic HSV approved for clinical practice and those in clinical trials are attenuated viruses. An alternative to attenuation is a cancer specificity achieved by tropism retargeting to selected cancer receptors. However, the retargeted oncolytic HSVs strictly depend on cancer receptors for infection. Here, we devised a strategy for in vitro cultivation of retargeted HSVs in non-cancer cells. The strategy envisions a double-retargeting approach: one retargeting is via gD to the cancer receptor, and the second retargeting is via gH to an artificial receptor expressed in Vero cells. The double-retargeted HSV uses alternatively the two receptors to infect cancer cells or producer cells. A universal non-cancer cell line for growth of clinical-grade retargeted HSVs represents a step forward in the translational phase.

SUBMITTER: Leoni V

PROVIDER: S-EPMC5411604 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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A Strategy for Cultivation of Retargeted Oncolytic Herpes Simplex Viruses in Non-cancer Cells.

Leoni Valerio V Gatta Valentina V Casiraghi Costanza C Nicosia Alfredo A Petrovic Biljana B Campadelli-Fiume Gabriella G

Journal of virology 20170428 10

The oncolytic herpes simplex virus (HSV) that has been approved for clinical practice and those HSVs in clinical trials are attenuated viruses, often with the neurovirulence gene γ<sub>1</sub>34.5 and additional genes deleted. One strategy to engineer nonattenuated oncolytic HSVs consists of retargeting the viral tropism to a cancer-specific receptor of choice, exemplified by HER2 (human epidermal growth factor receptor 2), which is present in breast, ovary, and other cancers, and in detargeting ...[more]

PMID: 28250120

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Project description:Insertion of a single-chain variable-fragment 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 to highly specific nonattenuated oncolytic agents. Clinical-grade virus production cannot rely on cancer cells. Recently, we developed a double-retargeting strategy whereby gH carries the GCN4 peptide for retargeting to the noncancer 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 amino acids (aa) 35 to 39, 214 to 223, or 219 to 223 and replacement of the deleted sequences with one of the two ligands. The last 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 nonretargeted 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 effective in vitro and in vivoIMPORTANCE There is increasing interest in oncolytic viruses following the FDA and European Medicines Agency (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 noncancer 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 and one for retargeting to the HER2 cancer receptor. These insertions were combined with novel, minimally disadvantageous detargeting modifications. The current and accompanying studies indicate how to best achieve the clinical-grade cultivation of retargeted oncolytics.

Project description:Oncolytic viruses are emerging as a potential new way of treating cancers. They are selectively replication-competent viruses that propagate only in actively dividing tumor cells but not in normal cells and, as a result, destroy the tumor cells by consequence of lytic infection. At least six different oncolytic herpes simplex viruses (oHSVs) have undergone clinical trials worldwide to date, and they have demonstrated an excellent safety profile and intimations of efficacy. The first pivotal Phase III trial with an oHSV, talimogene laherparepvec (T-Vec [OncoVex(GM-CSF)]), is almost complete, with extremely positive early results reported. Intuitively, therapeutically beneficial interactions between oHSV and chemotherapeutic and targeted therapeutic drugs would be limited as the virus requires actively dividing cells for maximum replication efficiency and most anticancer agents are cytotoxic or cytostatic. However, combinations of such agents display a range of responses, with antagonistic, additive, or, perhaps most surprisingly, synergistic enhancement of antitumor activity. When synergistic interactions in cancer cell killing are observed, chemotherapy dose reductions that achieve the same overall efficacy may be possible, resulting in a valuable reduction of adverse side effects. Therefore, the combination of an oHSV with "standard-of-care" drugs makes a logical and reasonable approach to improved therapy, and the addition of a targeted oncolytic therapy with "standard-of-care" drugs merits further investigation, both preclinically and in the clinic. Numerous publications report such studies of oncolytic HSV in combination with other drugs, and we review their findings here. Viral interactions with cellular hosts are complex and frequently involve intracellular signaling networks, thus creating diverse opportunities for synergistic or additive combinations with many anticancer drugs. We discuss potential mechanisms that may lead to synergistic interactions.

Dataset Information

A Strategy for Cultivation of Retargeted Oncolytic Herpes Simplex Viruses in Non-cancer Cells.

Publications

A Strategy for Cultivation of Retargeted Oncolytic Herpes Simplex Viruses in Non-cancer Cells.

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