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

2017 ◽  
Vol 92 (6) ◽  
Author(s):  
Biljana Petrovic ◽  
Valerio Leoni ◽  
Valentina Gatta ◽  
Anna Zaghini ◽  
Andrea Vannini ◽  
...  
Keyword(s):  
Herpes Simplex ◽  
Vero Cell ◽  
Cancer Cells ◽  
Cell Line ◽  
Host Response ◽  
Oncolytic Viruses ◽  
European Medicines Agency ◽  
Clinical Grade ◽  
Virus Growth ◽  
Herpes Simplex Viruses

ABSTRACTOncolytic 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, andin vivoantitumor efficacy to define the best double-retargeting strategy.IMPORTANCEThere 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.

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

2017 ◽  
Vol 92 (6) ◽  
Author(s):  
Valerio Leoni ◽  
Biljana Petrovic ◽  
Tatiana Gianni ◽  
Valentina Gatta ◽  
Gabriella Campadelli-Fiume
Keyword(s):  
Amino Acids ◽  
Herpes Simplex ◽  
Cancer Cells ◽  
Checkpoint Inhibitors ◽  
Oncolytic Viruses ◽  
European Medicines Agency ◽  
Clinical Grade ◽  
Single Chain ◽  
Her2 Positive ◽  
Herpes Simplex Viruses

ABSTRACTInsertion 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 effectivein vitroandin vivo.IMPORTANCEThere is increasing interest in oncolytic viruses following the FDA and European Medicines Agency (EMA) approval of the oncolytic HSV OncovexGM-CSFand, 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.

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

2017 ◽  
Vol 91 (10) ◽  
Author(s):  
Valerio Leoni ◽  
Valentina Gatta ◽  
Costanza Casiraghi ◽  
Alfredo Nicosia ◽  
Biljana Petrovic ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Clinical Practice ◽  
Herpes Simplex ◽  
Cancer Cells ◽  
Cell Line ◽  
Clinical Grade ◽  
Single Chain ◽  
Herpes Simplex Viruses ◽  
Artificial Receptor ◽  
Attenuated Viruses

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 γ134.5 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.

MP-2.08: Anti-tumor Effect on Bladder Cancer with Conditionally Replicating Oncolytic Viruses Derived from Herpes Simplex Viruses

Urology ◽  
2008 ◽  
Vol 72 (5) ◽  
pp. S69
Author(s):  
Y. Cho ◽  
K. Joo ◽  
H. Park ◽  
C. Kwon ◽  
J. Choi ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Herpes Simplex ◽  
Oncolytic Viruses ◽  
Herpes Simplex Viruses

scholarly journals Development of a selective biopharmaceutical from Herpes simplex virus type 1 glycoproteins E and I for blocking antibody mediated neutralization of oncolytic viruses

2010 ◽  
Vol 60 (4) ◽  
pp. 407-413
Author(s):  
Septimiu Bucurescu
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cancer Cells ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Oncolytic Viruses ◽  
Blocking Antibody ◽  
Simplex Virus

Development of a selective biopharmaceutical from Herpes simplex virus type 1 glycoproteins E and I for blocking antibody mediated neutralization of oncolytic virusesFuture cancer therapies will be molecular cures. They will correct, block or destroy cancer cells by targeting molecular changes that lead to carcinogenesis. Destroying cancer cells can be done using oncolytic viruses. By blocking antibody mediated neutralization of oncolytic viruses, Herpes simplex virus type 1 glycoproteins E and I could be used in the adjuvant treatment of cancer for improving the chances of oncolytic viruses to kill cancer cells in vivo.

scholarly journals Immunotherapeutic Efficacy of Retargeted oHSVs Designed for Propagation in an Ad Hoc Cell Line

Cancers ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 266
Author(s):  
Andrea Vannini ◽  
Valerio Leoni ◽  
Mara Sanapo ◽  
Tatiana Gianni ◽  
Giorgia Giordani ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Cell Line ◽  
Ad Hoc ◽  
Human Cancer ◽  
Primary Tumors ◽  
Herpes Simplex Viruses ◽  
Human Cancer Cell Lines ◽  
Tumor Associated Antigen ◽  
Full Complement ◽  
Target Cancer

Our laboratory has pursued the generation of cancer-specific oncolytic herpes simplex viruses (oHSVs) which ensure high efficacy while maintaining a high safety profile. Their blueprint included retargeting to a Tumor-Associated Antigen, e.g., HER2, coupled to detargeting from natural receptors to avoid off-target and off-tumor infections and preservation of the full complement of unmodified viral genes. These oHSVs are “fully virulent in their target cancer cells”. The 3rd generation retargeted oHSVs carry two distinct retargeting moieties, which enable infection of a producer cell line and of the target cancer cells, respectively. They can be propagated in an ad hoc Vero cell derivative at about tenfold higher yields than 1st generation recombinants, and more effectively replicate in human cancer cell lines. The R-335 and R-337 prototypes were armed with murine IL-12. Intratumorally-administered R-337 conferred almost complete protection from LLC-1-HER2 primary tumors, unleashed the tumor microenvironment immunosuppression, synergized with the checkpoint blockade and conferred long-term vaccination against distant challenge tumors. In summary, the problem intrinsic to the propagation of retargeted oHSVs—which strictly require cells positive for targeted receptors—was solved in 3rd generation viruses. They are effective as immunotherapeutic agents against primary tumors and as antigen-agnostic vaccines.

Anti-Herpesvirus Activities of Pseudomonas sp. S-17 Rhamnolipid and its Complex with Alginate

2008 ◽  
Vol 63 (1-2) ◽  
pp. 75-81 ◽  
Author(s):  
Mimi Remichkova ◽  
Danka Galabova ◽  
Ivana Roeva ◽  
Elena Karpenko ◽  
Alexander Shulga ◽  
...  
Keyword(s):  
Herpes Simplex ◽  
Suppressive Effect ◽  
Pseudomonas Sp ◽  
Inhibitory Effects ◽  
Virus Growth ◽  
Herpes Simplex Viruses ◽  
Rhamnolipid Biosurfactant ◽  
Simplex Virus ◽  
Dose Dependent ◽  
Hsv 1

The rhamnolipid biosurfactant PS-17 and its complex with the polysaccharide alginate, both produced by the Pseudomonas sp. S-17 strain, were studied for their antiviral activity against herpes simplex virus (HSV) types 1 and 2. They significantly inhibited the herpesvirus cytopathic effect (CPE) in the Madin-Darby bovine kidney (MDBK) cell line. The investigations were carried out according to the CPE inhibition assay protocol. The suppressive effect of the compounds on HSV replication was dose-dependent and occurred at concentrations lower than the critical micelle concentration of the surfactant. The 50% inhibitory concentration (IC50) of rhamnolipid PS-17 was 14.5 μg/ml against HSV-1 and 13 μg/ml against HSV-2. The IC50 values of the complex were 435 μg/ml for HSV-1 and 482 μg/ml for HSV-2. The inhibitory effects of the substances were confirmed by measuring the infectious virus yields with the multicycle virus growth experimental design as well: ∆log CCID50 of 1.84−2.0 against the two types of herpes simplex viruses by rhamnolipid PS-17 (20 μg/ml), and a strong reduction of the HSV-2 virus yield under the effect of the alginate complex at a concentration of 450 μg/ml. The results indicate that rhamnolipid PS-17 and its alginate complex may be considered as promising substances for the development of anti-herpetic compounds.

The Important Role of Oncolytic Viruses in Common Cancer Treatments

2020 ◽  
Vol 16 (4) ◽  
pp. 292-305 ◽  
Author(s):  
Amir Mohamadi ◽  
Gilles Pagès ◽  
Mohammad S. Hashemzadeh
Keyword(s):  
Immune Response ◽  
Cell Death ◽  
Cancer Treatment ◽  
Cancer Cells ◽  
High Efficiency ◽  
Genetic Manipulation ◽  
Oncolytic Viruses ◽  
Tumor Cell Death ◽  
Cancer Treatments ◽  
Herpes Simplex Viruses

Oncolytic viruses (OV) are considered as promising tools in cancer treatment. In addition to direct cytolysis, the stimulation of both innate and adaptive immune responses is the most important mechanism in oncolytic virotherapy that finally leads to the long-standing tumor retardations in the advanced melanoma clinical trials. The OVs have become a worthy method in cancer treatment, due to their several biological advantages including (1) the selective replication in cancer cells without affecting normal cells; (2) the lack of resistance to the treatment; (3) cancer stem cell targeting; (4) the ability to be spread; and (5) the immune response induction against the tumors. Numerous types of viruses; for example, Herpes simplex viruses, Adenoviruses, Reoviruses, Poliovirus, and Newcastle disease virus have been studied as a possible cancer treatment strategy. Although some viruses have a natural orientation or tropism to cancer cells, several others need attenuation and genetic manipulation to increase the safety and tumor-specific replication activity. Two important mechanisms are involved in OV antitumor responses, which include the tumor cell death due to virus replication, and also induction of immunogenic cell death as a result of the immune system responses against the tumor cells. Furthermore, the high efficiency of OV on antitumor immune response stimulation can finally lead to a significant tumor shrinkage.

scholarly journals Generation of an Oncolytic Herpes Simplex Viral Vector Completely Retargeted to the GDNF Receptor GFRα1 for Specific Infection of Breast Cancer Cells

2020 ◽  
Vol 21 (22) ◽  
pp. 8815 ◽  
Author(s):  
Bonnie L. Hall ◽  
Daniela Leronni ◽  
Yoshitaka Miyagawa ◽  
William F. Goins ◽  
Joseph C. Glorioso ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Herpes Simplex ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Viral Vector ◽  
Tumor Regression ◽  
Breast Cancers ◽  
Herpes Simplex Viruses ◽  
Viral Attachment

Oncolytic herpes simplex viruses (oHSV) are under development for the treatment of a variety of human cancers, including breast cancer, a leading cause of cancer mortality among women worldwide. Here we report the design of a fully retargeted oHSV for preferential infection of breast cancer cells through virus recognition of GFRα1, the cellular receptor for glial cell-derived neurotrophic factor (GDNF). GFRα1 displays a limited expression profile in normal adult tissue, but is upregulated in a subset of breast cancers. We generated a recombinant HSV expressing a completely retargeted glycoprotein D (gD), the viral attachment/entry protein, that incorporates pre-pro-GDNF in place of the signal peptide and HVEM binding domain of gD and contains a deletion of amino acid 38 to eliminate nectin-1 binding. We show that GFRα1 is necessary and sufficient for infection by the purified recombinant virus. Moreover, this virus enters and spreads in GFRα1-positive breast cancer cells in vitro and caused tumor regression upon intratumoral injection in vivo. Given the heterogeneity observed between and within individual breast cancers at the molecular level, these results expand our ability to deliver oHSV to specific tumors and suggest opportunities to enhance drug or viral treatments aimed at other receptors.

scholarly journals Herpes Simplex Virus and Pattern Recognition Receptors: An Arms Race

2021 ◽  
Vol 11 ◽  
Author(s):  
Jun Zhao ◽  
Chao Qin ◽  
Yongzhen Liu ◽  
Youliang Rao ◽  
Pinghui Feng
Keyword(s):  
Pattern Recognition ◽  
Herpes Simplex ◽  
Pattern Recognition Receptors ◽  
Oncolytic Viruses ◽  
Innate Immune Responses ◽  
Herpes Simplex Viruses ◽  
Host Interactions ◽  
Antiviral Therapies ◽  
Simplex Virus ◽  
Molecular Patterns

Herpes simplex viruses (HSVs) are experts in establishing persistent infection in immune-competent humans, in part by successfully evading immune activation through diverse strategies. Upon HSV infection, host deploys pattern recognition receptors (PRRs) to recognize various HSV-associated molecular patterns and mount antiviral innate immune responses. In this review, we describe recent advances in understanding the contributions of cytosolic PRRs to detect HSV and the direct manipulations on these receptors by HSV-encoded viral proteins as countermeasures. The continuous update and summarization of these mechanisms will deepen our understanding on HSV-host interactions in innate immunity for the development of novel antiviral therapies, vaccines and oncolytic viruses.

Synthesis and Characterization of Quinoline-3-Carboxamide Derivatives as Inhibitors of the ATM Kinase

2020 ◽  
Vol 20 (23) ◽  
pp. 2070-2079
Author(s):  
Srimadhavi Ravi ◽  
Sugata Barui ◽  
Sivapriya Kirubakaran ◽  
Parul Duhan ◽  
Kaushik Bhowmik
Keyword(s):  
Western Blot ◽  
Cancer Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cancer Cell Lines ◽  
Atm Kinase ◽  
Cytotoxicity Studies

Background: The importance of inhibiting the kinases of the DDR pathway for radiosensitizing cancer cells is well established. Cancer cells exploit these kinases for their survival, which leads to the development of resistance towards DNA damaging therapeutics. Objective: In this article, the focus is on targeting the key mediator of the DDR pathway, the ATM kinase. A new set of quinoline-3-carboxamides, as potential inhibitors of ATM, is reported. Methods: Quinoline-3-carboxamide derivatives were synthesized and cytotoxicity assay was performed to analyze the effect of molecules on different cancer cell lines like HCT116, MDA-MB-468, and MDA-MB-231. Results: Three of the synthesized compounds showed promising cytotoxicity towards a selected set of cancer cell lines. Western Blot analysis was also performed by pre-treating the cells with quercetin, a known ATM upregulator, by causing DNA double-strand breaks. SAR studies suggested the importance of the electron-donating nature of the R group for the molecule to be toxic. Finally, Western-Blot analysis confirmed the down-regulation of ATM in the cells. Additionally, the PTEN negative cell line, MDA-MB-468, was more sensitive towards the compounds in comparison with the PTEN positive cell line, MDA-MB-231. Cytotoxicity studies against 293T cells showed that the compounds were at least three times less toxic when compared with HCT116. Conclusion: In conclusion, these experiments will lay the groundwork for the evolution of potent and selective ATM inhibitors for the radio- and chemo-sensitization of cancer cells.

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