Valentina Gatta

αVβ3-integrin routes herpes simplex virus to an entry pathway dependent on cholesterol-rich lipid rafts and dynamin2

HSVs enter cells in a receptor-dependent [nectin1 or herpesviruses entry mediator (HVEM)] fashion by fusion of the viral envelope with plasma membrane (neutral pH compartment), by endocytosis into neutral or acidic compartments, or by macropinocytosis/phagocytosis. The cellular determinants of the route of entry are unknown. Here, we asked what cellular factors determine the pathway of HSV entry. CHO cells lack β3-integrin and the respective α-subunits’ heterodimers. We report that, in the absence of αVβ3-integrin, HSV enters CHO-nectin1 cells through a pathway independent of cholesterol-rich rafts and dynamin2. In the presence of αVβ3-integrin, HSV enters CHO-nectin1 cells through a pathway dependent on cholesterol-rich rafts and dynamin2. HSV enters J-nectin1 and 293T cells through a neutral compartment independent of cholesterol-rich rafts and dynamin2. αVβ3-integrin overexpression in these cells modifies the route of entry to an acidic compartment dependent on cholesterol-rich rafts and dynamin2, hence similar to that in αVβ3-integrin–positive CHO-nectin1 cells. In some cells, the diversion of entry from an integrin- and raft-independent pathway to an acidic compartment requiring cholesterol-rich lipids rafts and dynamin2 is irreversible. Indeed, HSV cannot infect CHO-nectin1-αVβ3 cells through any compartment when the αvβ3-integrin–dependent pathway is blocked by anti-integrin antibody, anti-dynamin2, or anti-acidification drugs. We conclude that the αvβ3-integrin is a determinant in the choice of HSV entry pathway into cells. Because the pathway dictated by αvβ3-integrin is through lipid rafts, the platforms for a number of Toll-like receptors, current findings raise the possibility that αvβ3-integrin acts as a sentinel of innate immunity.

Inhibition of human tumor growth in mice by an oncolytic herpes simplex virus designed to target solely HER-2-positive cells

Oncolytic virotherapy exploits the ability of viruses to infect, replicate into, and kill tumor cells. Among the viruses that entered clinical trials are HSVs. HSVs can be engineered to become tumor-specific by deletion of selected genes or retargeting to tumor-specific receptors. A clinically relevant surface molecule is HER-2, hyperexpressed in one fourth of mammary and ovary carcinomas, and associated with high metastatic ability. As a previously undescribed strategy to generate HSV recombinants retargeted to HER-2 and detargeted from natural receptors, we replaced the Ig-folded core in the receptor-binding virion glycoprotein gD with anti-HER-2 single-chain antibody. The recombinant entered cells solely via HER-2 and lysed HER-2–positive cancer cells. Because of the high specificity, its safety profile in i.p. injected mice was very high, with a LD50 >5 × 108 pfu, a figure at least 10,000-fold higher than that of corresponding WT-gD carrying virus (LD50 ≈ 5 × 104 pfu). When administered intratumorally to nude mice bearing HER-2–hyperexpressing human tumors, it strongly inhibited progressive tumor growth. The results provide a generally applicable strategy to engineer HSV recombinants retargeted to a wide range of receptors for which a single-chain antibody is available, and show the potential for retargeted HSV to exert target-specific inhibition of human tumor growth. Therapy with HER-2-retargeted oncolytic HSV could be effective in combined or sequential protocols with monoclonal antibodies and small inhibitors, particularly in patients resistant to HER-2-targeted therapy because of alterations in HER-2 signaling pathway, or against brain metastases inaccessible to anti-HER-2 antibodies.

Rethinking herpes simplex virus: the way to oncolytic agents

Oncolytic viruses infect, replicate in and kill cancer cells. HSV has emerged as a most promising candidate because it exerts a generally moderate pathogenicity in humans; it is amenable to attenuation and tropism retargeting; the ample genome provides space for heterologous genes; specific antiviral therapy is available in a worst case scenario. The first strategy to convert HSV into an oncolytic agent consisted in deletion of the γ134.5 gene which counteracts the protein kinase R (PKR) response, and of the UL39 gene which encodes the large ribonucleotide reductase subunit. Tumor specificity resided in low PKR activity, and high deoxyribonucleotides content of cancer cells. These highly attenuated viruses have been and presently are in clinical trials with encouraging results. The preferred route of administration has been intratumor or in tissues adjacent to resected tumors. Although the general population has a high seroprevalence of antibodies to HSV, studies in animals and humans demonstrate that prior immunity is not an obstacle to systemic routes of administration, and that oncolytic HSV (o‐HSVs) do populate tumors. As the attenuated viruses undergo clinical experimentation, the research pipeline is developing novel, more potent and highly tumor‐specific o‐HSVs. These include viruses which overcome tumor heterogeneity in PKR level by insertion of anti‐PKR genes, viruses which reinforce the host tumor clearance capacity by encoding immune cytokines (IL‐12 or granulocyte‐macrophage colony‐stimulating factor), and non‐attenuated viruses fully retargeted to tumor specific receptors. A strategy to generate o‐HSVs fully retargeted to human epidermal growth factor receptor‐2 (HER‐2) or other cancer‐specific surface receptors is detailed. Copyright

Replication-competent Herpes Simplex Virus Retargeted to HER2 as Therapy for High-grade Glioma

Oncolytic herpes simplex viruses (HSVs) represent a novel frontier against tumors resistant to standard therapies, like glioblastoma (GBM). The oncolytic HSVs that entered clinical trials so far showed encouraging results; however, they are marred by the fact that they are highly attenuated. We engineered HSVs that maintain unimpaired lytic efficacy and specifically target cells that express tumor-specific receptors, thus limiting the cytotoxicity only to cancer cells, and leaving unharmed the neighboring tissues. We report on the safety and efficacy in a high-grade glioma (HGG) model of R-LM113, an HSV recombinant retargeted to human epidermal growth factor receptor 2 (HER2), frequently expressed in GBMs. We demonstrated that R-LM113 is safe in vivo as it does not cause encephalitis when intracranially injected in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, extremely sensitive to wild-type HSV. The efficacy of R-LM113 was assessed in a platelet-derived growth factor (PDGF)-induced infiltrative glioma model engineered to express HER2 and transplanted intracranially in adult NOD/SCID mice. Mice injected with HER2-engineered glioma cells infected with R-LM113 showed a doubled survival time compared with mice injected with uninfected cells. A doubling in survival time from the beginning of treatment was obtained also when R-LM113 was administered into already established tumors. These data demonstrate the efficacy of R-LM113 in thwarting tumor growth.

Systemic delivery of HER2-retargeted oncolytic-HSV by mesenchymal stromal cells protects from lung and brain metastases

Fully retargeted oncolytic herpes simplex viruses (o-HSVs) gain cancer-specificity from redirection of tropism to cancer-specific receptors, and are non-attenuated. To overcome the hurdles of systemic delivery, and enable oncolytic viruses (o-viruses) to reach metastatic sites, carrier cells are being exploited. Mesenchymal stromal cells (MSCs) were never tested as carriers of retargeted o-viruses, given their scarse-null expression of the cancer-specific receptors. We report that MSCs from different sources can be forcedly infected with a HER2-retargeted oncolytic HSV. Progeny virus spread from MSCs to cancer cells in vitro and in vivo. We evaluated the organ distribution and therapeutic efficacy in two murine models of metastatic cancers, following a single i.v. injection of infected MSCs. As expected, the highest concentration of carrier-cells and of viral genomes was in the lungs. Viral genomes persisted throughout the body for at least two days. The growth of ovarian cancer lung metastases in nude mice was strongly inhibited, and the majority of treated mice appeared metastasis-free. The treatment significantly inhibited also breast cancer metastases to the brain in NSG mice, and reduced by more than one-half the metastatic burden in the brain.

Preclinical Therapy of Disseminated HER-2+ Ovarian and Breast Carcinomas with a HER-2-Retargeted Oncolytic Herpesvirus

Oncolytic viruses aim to specifically kill tumor cells. A major challenge is the effective targeting of disseminated tumors in vivo. We retargeted herpes simplex virus (HSV) tropism to HER-2 oncoprotein p185, overexpressed in ovary and breast cancers. The HER-2-retargeted R-LM249 exclusively infects and kills tumor cells expressing high levels of human HER-2. Here, we assessed the efficacy of systemically i.p. delivered R-LM249 against disseminated tumors in mouse models that recapitulate tumor spread to the peritoneum in women. The human ovarian carcinoma SK-OV-3 cells implanted intraperitoneally (i.p.) in immunodeficient Rag2−/−;Il2rg−/− mice gave rise to a progressive peritoneal carcinomatosis which mimics the fatal condition in advanced human patients. I.p. administration of R-LM249 strongly inhibited carcinomatosis, resulting in 60% of mice free from peritoneal diffusion, and 95% reduction in the total weight of neoplastic nodules. Intraperitoneal metastases are a common outcome in breast cancer: i.p. administration of R-LM249 strongly inhibited the growth of ovarian metastases of HER-2+ MDA-MB-453 breast cells. Brain metastases were also reduced. Cumulatively, upon i.p. administration the HER-2-redirected oncolytic HSV effectively reduced the growth of ovarian and breast carcinoma disseminated to the peritoneal cavity.

The Engineering of a Novel Ligand in gH Confers to HSV an Expanded Tropism Independent of gD Activation by Its Receptors

Herpes simplex virus (HSV) enters cells by means of four essential glycoproteins - gD, gH/gL, gB, activated in a cascade fashion by gD binding to one of its receptors, nectin1 and HVEM. We report that the engineering in gH of a heterologous ligand – a single-chain antibody (scFv) to the cancer-specific HER2 receptor – expands the HSV tropism to cells which express HER2 as the sole receptor. The significance of this finding is twofold. It impacts on our understanding of HSV entry mechanism and the design of retargeted oncolytic-HSVs. Specifically, entry of the recombinant viruses carrying the scFv-HER2–gH chimera into HER2+ cells occurred in the absence of gD receptors, or upon deletion of key residues in gD that constitute the nectin1/HVEM binding sites. In essence, the scFv in gH substituted for gD-mediated activation and rendered a functional gD non-essential for entry via HER2. The activation of the gH moiety in the chimera was carried out by the scFv in cis, not in trans as it occurs with wt-gD. With respect to the design of oncolytic-HSVs, previous retargeting strategies were based exclusively on insertion in gD of ligands to cancer-specific receptors. The current findings show that (i) gH accepts a heterologous ligand. The viruses retargeted via gH (ii) do not require the gD-dependent activation, and (iii) replicate and kill cells at high efficiency. Thus, gH represents an additional tool for the design of fully-virulent oncolytic-HSVs retargeted to cancer receptors and detargeted from gD receptors.

Insertion of a ligand to HER2 in gB retargets HSV tropism and obviates the need for activation of the other entry glycoproteins

Herpes simplex virus (HSV) entry into the cells requires glycoproteins gD, gH/gL and gB, activated in a cascade fashion by conformational modifications induced by cognate receptors and intermolecular signaling. The receptors are nectin1 and HVEM (Herpes virus entry mediator) for gD, and αvβ6 or αvβ8 integrin for gH. In earlier work, insertion of a single chain antibody (scFv) to the cancer receptor HER2 (human epidermal growth factor receptor 2) in gD, or in gH, resulted in HSVs specifically retargeted to the HER2-positive cancer cells, hence in highly specific non-attenuated oncolytic agents. Here, the scFv to HER2 was inserted in gB (gBHER2). The insertion re-targeted the virus tropism to the HER2-positive cancer cells. This was unexpected since gB is known to be a fusogenic glycoprotein, not a tropism determinant. The gB-retargeted recombinant offered the possibility to investigate how HER2 mediated entry. In contrast to wt-gB, the activation of the chimeric gBHER2 did not require the activation of the gD and of gH/gL by their respective receptors. Furthermore, a soluble form of HER2 could replace the membrane-bound HER2 in mediating virus entry, hinting that HER2 acted by inducing conformational changes to the chimeric gB. This study shows that (i) gB can be modified and become the major determinant of HSV tropism; (ii) the chimeric gBHER2 bypasses the requirement for receptor-mediated activation of other essential entry glycoproteins.

Efficacy of HER2 retargeted herpes simplex virus as therapy for high-grade glioma in immunocompetent mice.

Replication-competent oncolytic herpes simplex viruses (HSVs) are considered a promising therapeutic approach for treatment of high-grade gliomas (HGGs), which are usually resistant to all the available treatments. We previously demonstrated that R-LM113, a recombinant HSV-1 fully retargeted to the human epidermal growth factor receptor 2 (HER2), is safe and prolongs survival of immunodeficient NOD/SCID mice in an intracranial model of HGG. However, because the treatment is designed to be employed on immunocompetent patients, it is necessary to test whether the host immune system impairs the viral efficacy or triggers a potentially fatal reaction. Here we confirmed the safety of R-LM113 in the immunocompetent mouse strain BALB/c, where it does not trigger encephalitis when intracranially inoculated. Then, we set up a syngeneic HGG model expressing HER2 in adult BALB/c mice and evaluated R-LM113 therapeutic efficacy. We found that R-LM113 leads to a significant improvement in animal survival when administered at the time of tumor inoculation, as well as when injected into an already established tumor. This study suggests that the host immune defenses do not curtail the oncolytic antitumor activity of replication-competent HSV R-LM113, which results effective in counteracting tumor growth.

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 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.