GLV-1h68 vaccinia virus therapy of neuroendocrine tumors CURRENT STATUS: UNDER REVIEW

Background: Oncolytic virotherapy is an upcoming treatment option for many tumor entities. But so far, a first oncolytic virus only was approved for advanced stages of malignant melanomas. Neuroendocrine tumors (NETs) constitute a heterogenous group of tumors arising from the neuroendocrine system at diverse anatomic sites. Due to often slow growth rates and (in most cases) endocrine non-functionality, NETs are often detected only in a progressed metastatic situation, where therapy options are still severely limited. So far, immunotherapies and especially immunovirotherapies are not established as novel treatment modalities for NETs. Methods: In this immunovirotherapy study, pancreatic NET (BON-1, QGP-1), lung NET (H727, UMC-11), as well as neuroendocrine carcinoma (NEC) cell lines (HROC-57, NEC-DUE1) were employed. The well characterized genetically engineered vaccinia virus GLV-1h68, which has already been investigated in various clinical trials, was chosen as virotherapeutical treatment modality. Results: Profound oncolytic efficiencies were found for NET/NEC tumor cells. Besides, NET/NEC tumor cell bound expression of GLV-1h68-encoded marker genes was observed also. Further more, a highly efficient production of viral progenies was detected by sequential virus quantifications. More over, prospects of a combinatorial treatment of GLV-1h68 with the mTOR inhibitor everolimus, licensed for treatment of metastatic NETs, were assessed successfully. Conclusions: In summary, the oncolytic vaccinia virus GLV-1h68 was found to exhibit promising antitumoral activities, replication capacities and a potential for future combinatorial approaches. This has been shown in a widespread spectrum of cancers in a preclinical setting and now has to be further evaluated for treatment of metastatic neuroendocrine cancer.

galactosidase, as well as the Ruc-GFP marker gene) inserted into a vaccinia virus (VACV) backbone derived from the Lister strain which has demonstrated its safety throughout years serving as a major smallpox vaccine. These triple insertions reduce the replication of GLV-1h68 in healthy cells and favor its replication in tumor cells [11,12]; beyond they also allow the monitoring of virus activities in cancer patients [13]. As this oncolytic virus is not targeted to a specific type of tumor, oncolytic activity has already been detected in a broad spectrum of tumor entities in preclinical models as well as in several clinical trials [13][14][15][16]. Moreover, combinatorial approaches with chemotherapy, radiation or targeted therapies have displayed synergistic antitumor activities [17][18][19][20][21].
Currently, there are three active clinical studies (NCT02759588, NCT02714374, NCT01766739) which employ GLV-1h68/GL-ONC1. Virus delivery pathways include intraperitoneal, intrapleural, and intravenous delivery. Notably, early virus clearance constitutes a problem, especially when GLV-1h68 is applied systemically/intravenously. As complement inhibition seems to play a crucial role in virus depletion following intravenous application [22], a new strategy is the application of an anti-C5-antibody (eculizumab) prior to virotherapy [NCT02714374]. Another recent approach to prevent intravascular virus clearance is to administer virus loaded cells as a carrier system for viral particles [23,24]. Reasonable options for NENs constitute intravenous administrations as well as direct virus injections into the hepatic artery in case of liver involvement (NCT02749331; [9]). Further, intratumoral virus administrations or surgically guided administrations into the resection beds can be considered.
In this work, we now additionally have studied the combination of GLV-1h68 with molecular targeted therapy (MTT). The mTOR inhibitor everolimus is approved as a treatment for advanced lung, pancreatic and intestinal NETs. This situation would be suitable for virotherapy to enter the clinical development in NEN therapy. Another option 5 for MTT is the multi-kinase inhibitor sunitinib, which is approved for pancreatic NETs.
However, recent studies show significantly longer progression free survival with everolimus used as a first line MTT in pancreatic NETs compared to sunitinib. Also, everolimus MTT was found to be significantly more efficient in non-pancreatic NETs, which is why the combinatorial treatment of GLV-1h68 with everolimus was investigated here in a preferred way [25][26][27].
In this study, tumor cell lines originating from pancreatic NETs, lung NETs and intestinal NECs were evaluated for their susceptibility to vaccinia virus-mediated virotherapy. For this purpose, the lytic activity of GLV-1h68 was measured, viral gene expression was visualized and virus replication was quantified. Beyond that, also a combinatorial treatment regimen being set up for the conjoint usage of GLV-1h68 and everolimus was studied for its ability to deplete NEN tumor cells; besides, possible interactions between everolimus and replication of the oncolytic virus GLV-1h68 were investigated also.

Oncolytic Virus
The oncolytic vaccinia virus GLV-h168 was kindly provided by Genelux Corporation (San Diego, CA, USA). GLV-1h68 is a genetically engineered OV originating from the vaccinia Lister strain and also known under the proprietary name GL-ONC1 [11]. It was genetically modified by inserting three transgenes allowing therapeutic monitoring in its genome; RUC-GFP is employed for monitoring via fluorescence microscopy in this study.

Virus plaque assays
Plaque assays were conducted in order to determine the concentration of viral particles in cell cultures as described previously [11]. H727 and BON-1 cells were seeded in 6-well plates and infected with MOIs which led to approx. 50 % reduction of tumor cell densities.
One hour after virus infection, plates were carefully washed with PBS to remove all extracellular viral particles; then culture medium was added. Every 24 hours and at 1 hpi, infected cells and medium were harvested by scraping them into the culture medium. Combinatorial treatment with everolimus Next, a combinatorial treatment with the mTOR inhibitor everolimus was evaluated by comparing a combinatorial approach (GLV-1h68 + everolimus) to GLV-1h68 mono therapy.
In this purpose, SRB viability assay and virus quantification were conducted.
Oncolysis with GLV-1h68 and everolimus SRB viability assays were carried out using the lung NET cell line H727 and the NEC cell As a result, the addition of GLV-1h68 to sole everolimus treatment was found to be able to further reduce the remaining tumor cell count. This was observed in both cell lines tested and with both MOIs employed in each cell line. Interestingly, the benefit of the combinatorial therapy appeared to be more pronounced in NEC-DUE1 cells. However, the extent of this effect was limited in its extent, thereby not representing any additive mechanism of action.

Virus titer quantification
To investigate whether everolimus has any impact on virus replication, virus titers were assessed when GLV-1h68 was employed in a combinatorial setting with everolimus ( Figure   6, dotted lines). In both NET cell lines (H727, BON-1), where virus replication was determined previously, everolimus did not affect the production of viral progeny in any way.
Taking the results from both assays into account, the final benefit of the combinatorial therapy after 96 hours is visible but only small. Everolimus did not limit virus replication in a particular way. Given that evidence base, the combinatorial therapy of GLV-1h68 with everolimus was not found to be inferior to either monotherapy and can be regarded as a possible future combinatorial treatment option for metastatic neuroendocrine cancer. mechanism for tumor selectivity of VACVs have been described [36]. By selecting the most efficient virus strain and inserting several genes in different replication cassettes, GLV-1h68 was modified to be attenuated in healthy cells and its replication was found to be mainly selective to tumor cells. In line with the basic characteristics of VACVs, GLV-1h68 has the advantage of a stable cytoplasmic replication which avoids further virus-driven mutations in cancer cells or healthy cells [37]. In addition, the excellent safety profile of these VACVs is marked with years of clinical experience serving as smallpox vaccines as well as a preclinically well-established replication cycle [38]. Further, VACVs have no natural pathogenic potential in humans.

Discussion
However, the key mechanism of oncolytic virotherapy is thought to be a secondary immune response induced by the inflamed lytic tumor microenvironment. The release of tumor antigens and inflammatory cytokines disables immune evasion mechanisms of the tumor and facilitates profound antitumor immune responses [39]. This effect was observed earlier when it was found that not only VACV-injected melanoma metastases decreased in size, but also non-injected distant lesions responded to virotherapy with a granulocytemacrophage colony-stimulating factor (GM-CSF)-expressing vaccinia virus [40]. Both, a response of the innate immune system mediated by NK-cells, neutrophils and macrophages as well as an adaptive immunity facilitated by antigen-presenting cells and subsequent tumor-infiltrating CD8 + cells have been described after GLV-1h68 treatment [41]. Obviously, this secondary immune-mediated mechanism is complicated to mimic in an in vitro setting. However, since GLV-1h68 and other VACVs were reported to induce immunogenic cell death previously, the extent of direct tumor cell lysis can be regarded as a crucial factor in initiating an antitumor immunity [42,43].
In this work, the potential of GLV-1h68 to kill cells originating from neuroendocrine cancer has been demonstrated. GLV-1h68 exhibited stable cytotoxicity throughout neuroendocrine cancer cells from several anatomical origins (Fig. 1). Susceptibility to GLV-1h68 treatment was found to be dose dependent. Different responses of the variety of tumor cell lines was noted but could not be tracked back to a certain anatomical origin. In summary, two cell lines were found to be highly permissive, three were classified as permissive and one cell line as resistant to GLV-1h68 monotherapy.
It was shown earlier that cellular response to GLV-1h68 treatment depends on pleiotropic factors such as transcriptional patterns, cellular innate immunity pathways, efficiency of viral replication or proliferation rate [44]. Also, viral cytotoxicity was correlated with a strong transgene expression. Highly permissive cell lines (BON-1, HROC-57) displayed GFP expression even at very low MOIs, whereas transgene expression was only observed with high MOIs in the resistant cell line (UMC-11) (Fig. 2). For the representative NET cell line H727, a fast mechanism of action of GLV-1h68 therapy could be proven, resulting in a strong cytolytic response beginning as early as 36 hours after virus infection (Fig. 3).
Moreover, a strong virus replication was shown in both NET cell lines tested, reaching virus titers higher than 10 7 PFU/ml at 72 hpi (Fig. 4). Everolimus was tested for its effect on viral replication to exclude any restrictions on replication of GLV-1h68 in a combinatorial treatment regimen. It was found that everolimus does not influence GLV-1h68 replication in a negative way (Fig. 4). However, combinatorial treatment was not efficient in an additive manner, although not inferior to any single agent treatment (Fig. 5), which makes this treatment modality feasible for further investigations. Of note, previous studies regarding the combinatorial therapy of VACVs with the mTOR inhibitor rapamycin, had resulted in the detection of synergistic effect. Both, everolimus and rapamycin target and inhibit mTORC1. The synergistic effects were explained by the effect of mTORC1 inhibition on antiviral immunity. It was found that mTORC1 downstream signaling via p70S6K/4E-BP1 influences cellular type I IFN response.
Therefore, mTORC1 inhibition can make tumor cells more susceptible to VACV infection. In vivo, antiviral T-cell responses can be reduced by mTOR inhibitors, which also makes viral infections more effective [45][46][47]. These studies were conducted with malignant glioma models. In our study, these results could not be translated to neuroendocrine neoplasms, where the mTOR pathway might play another role in tumorigenesis. As both agents interfere with the immune system, further in vivo studies with immunocompetent animals have to be conducted to cover the whole range of mechanisms of action for this distinct combinatorial therapy.
Another possibility for combinatorial treatment with GLV-1h68 could be the usage of the multi-kinase inhibitor sunitinib, which was shown to exhibit synergistic effects together with VACV virotherapy recently. This is explained by multiple mechanisms such as suppression of viral resistance, increased leakiness of tumor vasculature and therefore more effective viral infection and increased CD8 + T-cell recruitment [48].

Conclusions
In summary, this study aimed to create the basis for the development of GLV-1h68 virotherapy in advanced neuroendocrine cancers. Its efficacy could be proven in a preclinical in vitro setting for the first time. Future research must be encouraged to further investigate VACV-mediated virotherapy for neuroendocrine cancer in preclinical in vitro and in vivo studies.

Ethics approval
None of the human cell lines used in this study required ethical approval.

Consent for publication
Not applicable.

Availability of data and materials
All datasets generated and/or analysed during this study are available from the corresponding author on reasonable request.

Competing interests
The authors report no conflicts of interest.

Supplementary Files
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