Decrease in Heparan Sulphate Binding in Tropism-Retargeted Oncolytic Herpes Simplex Virus (ReHV) Delays Blood Clearance and Improves Systemic Anticancer Efficacy.

The role of the interaction with cell-surface glycosaminoglycans (GAGs) during in vivo HSV infection is currently unknown. The rationale of the current investigation was to improve the anticancer efficacy of systemically administered retargeted oHSVs (ReHVs) by decreasing their binding to GAGs, including those of endothelial cells, blood cells, and off-tumor tissues. As a proof-of-principle approach, we deleted seven amino acids critical for interacting with GAGs from the glycoprotein C (gC) of R-337 ReHV. The modification in the resulting R-399 recombinant prolonged the half-life in the blood of systemically administered R-399 and enhanced its biodistribution to tumor-positive lungs and to the tumor-negative liver. Ultimately, it greatly increased the R-399 efficacy against metastatic-like lung tumors upon IV administration but not against subcutaneous tumors upon IT administration. These results provide evidence that the increased efficacy seen upon R-399 systemic administration correlated with the slower clearance from the circulation. To our knowledge, this is the first in vivo evidence that the partial impairment of the gC interaction with GAGs resulted in a prolonged half-life of circulating ReHV, an increase in the amount of ReHV taken up by tissues and tumors, and, ultimately, an enhanced anticancer efficacy of systemically administered ReHV.

Efficacy of Systemically Administered Retargeted Oncolytic Herpes Simplex Viruses-Clearance and Biodistribution in Naïve and HSV-Preimmune Mice.

We investigated the anticancer efficacy, blood clearance, and tissue biodistribution of systemically administered retargeted oncolytic herpes simplex viruses (ReHVs) in HSV-naïve and HSV-preimmunized (HSV-IMM) mice. Efficacy was tested against lung tumors formed upon intravenous administration of cancer cells, a model of metastatic disease, and against subcutaneous distant tumors. In naïve mice, HER2- and hPSMA-retargeted viruses, both armed with mIL-12, were highly effective, even when administered to mice with well-developed tumors. Efficacy was higher for combination regimens with immune checkpoint inhibitors. A significant amount of infectious virus persisted in the blood for at least 1 h. Viral genomes, or fragments thereof, persisted in the blood and tissues for days. Remarkably, the only sites of viral replication were the lungs of tumor-positive mice and the subcutaneous tumors. No replication was detected in other tissues, strengthening the evidence of the high cancer specificity of ReHVs, a property that renders ReHVs suitable for systemic administration. In HSV-IMM mice, ReHVs administered at late times failed to exert anticancer efficacy, and the circulating virus was rapidly inactivated. Serum stability and in vivo whole blood stability assays highlighted neutralizing antibodies as the main factor in virus inactivation. Efforts to deplete mice of the neutralizing antibodies are ongoing.

Innovative retargeted oncolytic herpesvirus against nectin4-positive cancers.

Nectin4 is a recently discovered tumor associated antigen expressed in cancers that constitute relevant unmet clinical needs, including the undruggable triple negative breast cancer, pancreatic ductal carcinoma, bladder/urothelial cancer, cervical cancer, lung carcinoma and melanoma. So far, only one nectin4-specific drug-Enfortumab Vedotin-has been approved and the clinical trials that test novel therapeutics are only five. Here we engineered R-421, an innovative retargeted onco-immunotherapeutic herpesvirus highly specific for nectin4 and unable to infect through the natural herpes receptors, nectin1 or herpesvirus entry mediator. In vitro, R-421 infected and killed human nectin4-positive malignant cells and spared normal cells, e.g., human fibroblasts. Importantly from a safety viewpoint, R-421 failed to infect malignant cells that do not harbor nectin4 gene amplification/overexpression, whose expression level was moderate-to-low. In essence, there was a net threshold value below which cells were spared from infection, irrespective of whether they were malignant or normal; the only cells that R-421 targeted were the malignant overexpressing ones. In vivo, R-421 decreased or abolished the growth of murine tumors made transgenic for human nectin4 and conferred sensitivity to immune checkpoint inhibitors in combination therapies. Its efficacy was augmented by the cyclophosphamide immunomodulator and decreased by depletion of CD8-positive lymphocytes, arguing that it was in part T cell-mediated. R-421 elicited in-situ vaccination that protected from distant challenge tumors. This study provides proof-of-principle specificity and efficacy data justifying nectin4-retargeted onco-immunotherapeutic herpesvirus as an innovative approach against a number of difficult-to-drug clinical indications.

Towards a Precision Medicine Approach and In Situ Vaccination against Prostate Cancer by PSMA-Retargeted oHSV.

Prostate specific membrane antigen (PSMA) is a specific high frequency cell surface marker of prostate cancers. Theranostic approaches targeting PSMA show no major adverse effects and rule out off-tumor toxicity. A PSMA-retargeted oHSV (R-405) was generated which both infected and was cytotoxic exclusively for PSMA-positive cells, including human prostate cancer LNCaP and 22Rv1 cells, and spared PSMA-negative cells. R-405 in vivo efficacy against LLC1-PSMA and Renca-PSMA tumors consisted of inhibiting primary tumor growth, establishing long-term T immune response, immune heating of the microenvironment, de-repression of the anti-tumor immune phenotype, and sensitization to checkpoint blockade. The in situ vaccination protected from distant challenge tumors, both PSMA-positive and PSMA-negative, implying that it was addressed also to LLC1 tumor antigens. PSMA-retargeted oHSVs are a precision medicine tool worth being additionally investigated in the immunotherapeutic and in situ vaccination landscape against prostate cancers.

Specificity, Safety, Efficacy of EGFRvIII-Retargeted Oncolytic HSV for Xenotransplanted Human Glioblastoma.

Glioblastoma is a lethal primary brain tumor lacking effective therapy. The secluded onset site, combined with the infiltrative properties of this tumor, require novel targeted therapies. In this scenario, the use of oncolytic viruses retargeted to glioblastoma cells and able to spread across the tumor cells represent an intriguing treatment strategy. Here, we tested the specificity, safety and efficacy of R-613, the first oncolytic HSV fully retargeted to EGFRvIII, a variant of the epidermal growth factor receptor carrying a mutation typically found in glioblastoma. An early treatment with R-613 on orthotopically transplanted EGFRvIII-expressing human glioblastoma significantly increased the median survival time of mice. In this setting, the growth of human glioblastoma xenotransplants was monitored by a secreted luciferase reporter and showed that R-613 is able to substantially delay the development of the tumor masses. When administered as late treatment to a well-established glioblastomas, R-613 appeared to be less effective. Notably the uninfected tumor cells derived from the explanted tumor masses were still susceptible to R-613 infection ex vivo, thus suggesting that multiple treatments could enhance R-613 therapeutic efficacy, making R-613 a promising oncolytic HSV candidate for glioblastoma treatment.

Genotype of Immunologically Hot or Cold Tumors Determines the Antitumor Immune Response and Efficacy by Fully Virulent Retargeted oHSV.

We report on the efficacy of the non-attenuated HER2-retargeted oHSV named R-337 against the immunologically hot CT26-HER2 tumor, and an insight into the basis of the immune protection. Preliminarily, we conducted an RNA immune profiling and immune cell content characterization of CT26-HER2 tumor in comparison to the immunologically cold LLC1-HER2 tumor. CT26-HER2 tumor was implanted into HER2-transgenic BALB/c mice. Hallmarks of R-337 effects were the protection from primary tumor, long-term adaptive vaccination directed to both HER2 and CT26-wt cell neoantigens. The latter effect differentiated R-337 from OncoVEXGM-CSF. As to the basis of the immune protection, R-337 orchestrated several changes to the tumor immune profile, which cumulatively reversed the immunosuppression typical of this tumor (graphical abstract). Thus, Ido1 (inhibitor of T cell anticancer immunity) levels and T regulatory cell infiltration were decreased; Cd40 and Cd27 co-immunostimulatory markers were increased; the IFNγ cascade was activated. Of note was the dampening of IFN-I response, which we attribute to the fact that R-337 is fully equipped with genes that contrast the host innate response. The IFN-I shut-down likely favored viral replication and the expression of the mIL-12 payload, which, in turn, boosted the antitumor response. The results call for a characterization of tumor immune markers to employ oncolytic herpesviruses more precisely.

Targeted Delivery of IL-12 Adjuvants Immunotherapy by Oncolytic Viruses.

The great hopes raised by the discovery of the immunoregulatory cytokine interleukin 12 (IL-12) as an anticancer agent were marred during early clinical experimentation because of severe adverse effects, which prompted a search for alternative formulations and routes of administration. Onco-immunotherapeutic viruses (OIVs) are wild-type or genetically engineered viruses that exert antitumor activity by causing death of the tumor cells they infect and by overcoming a variety of immunosuppressive mechanisms put in place by the tumors. OIVs have renewed the interest in IL-12, as they offer the opportunity to encode the cytokine transgenically from the viral genome and to produce it at high concentrations in the tumor bed. A large body of evidence indicates that IL-12 serves as a potent adjuvant for the immunotherapeutic response elicited by OIVs in murine tumor models. The list of OIVs includes onco-immunotherapeutic herpes simplex, adeno, measles, Newcastle disease, and Maraba viruses, among others. The large increase in IL-12-mediated adjuvanticity was invariably observed for all the OIVs analyzed. Indirect evidence suggests that locally delivered IL-12 may also increase tumor antigenicity. Importantly, the OIV/IL-12 treatment was not accompanied by adverse effects and elicited a long-lasting immune response capable of halting the growth of distant tumors. Thus, OIVs provide an avenue for reducing the clinical toxicity associated with systemic IL-12 therapy, by concentrating the cytokine at the site of disease. The changes to the tumor microenvironment induced by the IL-12-armed OIVs primed the tumors to an improved response to the checkpoint blockade therapy, suggesting that the triple combination is worth pursuing in the future. The highly encouraging results in preclinical models have prompted translation to the clinic. How well the IL-12-OIV-checkpoint inhibitors' combination will perform in humans remains to be fully investigated.

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

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.

Retargeted and Multi-cytokine-Armed Herpes Virus Is a Potent Cancer Endovaccine for Local and Systemic Anti-tumor Treatment.

Oncolytic viruses (OVs) are novel anti-tumor agents with the ability to selectively infect and kill tumor cells while sparing normal tissue. Beyond tumor cytolysis, OVs are capable of priming an anti-tumor immune response via lysis and cross-presentation of locally expressed endogenous tumor antigens, acting as an "endovaccine." The effectiveness of OVs, similar to other immunotherapies, can be hampered by an immunosuppressive tumor microenvironment. In this study, we modified a previously generated oncolytic herpes simplex virus (oHSV) retargeted to the human HER2 (hHER2) tumor molecule and encoding murine interleukin-12 (mIL-12), by insertion of a second immunomodulatory molecule, murine granulocyte-macrophage colony-stimulating factor (mGM-CSF), to maximize therapeutic efficacy. We assessed the efficacy of this double-armed virus (R-123) compared to singly expressing GM-CSF and IL-12 oHSVs in tumor-bearing mice. While monotherapies were poorly effective, combination with α-PD1 enhanced the anti-tumor response, with the highest efficacy of 100% response rate achieved by the combination of R-123 and α-PD1. Efficacy was T cell-dependent, and the induced immunity was long lasting and able to reject a second contralateral tumor. Importantly, systemic delivery of R-123 combined with α-PD1 was effective in inhibiting the development of tumor metastasis. As such, this approach could have a significant therapeutic impact paving the way for further development of this platform in cancer immunotherapy.

Replicative conditioning of Herpes simplex type 1 virus by Survivin promoter, combined to ERBB2 retargeting, improves tumour cell-restricted oncolysis.

Oncolytic virotherapy is emerging as a promising therapeutic option for solid tumours. Several oncolytic vectors in clinical testing are based on attenuated viruses; thus, efforts are being taken to develop a new repertoire of oncolytic viruses, based on virulent viral genomes. This possibility, however, raises concerns dealing with the safety features of the virulent phenotypes. We generated a double regulated Herpes simplex type-1 virus (HSV-1), in which tumour cell restricted replicative potential was combined to selective entry via ERBB2 receptor retargeting. The transcriptional control of the viral alpha4 gene encoding for the infected cell protein-4 (ICP4) by the cellular Survivin/BIRC5 promoter conferred a tumour cell-restricted replicative potential to a virulent HSV-1 genome. The combination of the additional ERBB2 retargeting further improved the selectivity for tumour cells, conferring to the double regulated virus a very limited ability to infect and propagate in non-cancerous cells. Accordingly, a suitable replicative and cytotoxic potential was maintained in tumour cell lines, allowing the double regulated virus to synergize in vivo with immune checkpoint (anti-PD-1) blockade in immunocompetent mice. Thus, restricting the replicative spectrum and tropism of virulent HSV-1 genomes by combination of conditional replication and retargeting provides an improved safety, does not alter the oncolytic strength, and is exploitable for its therapeutic potential with immune checkpoint blockade in cancer.

oHSV Genome Editing by Means of galK Recombineering.

Since the cloning of the herpes simplex virus (HSV) genome as BAC (bacterial artificial chromosome), the genetic engineering of the viral genome has become readily feasible. The advantage is that the modification of the animal virus genome is carried out in bacteria, with no replication or production of viral progeny, and is separated from the reconstitution or regeneration of the recombinant virus in mammalian cells. This allows an easy engineering of essential genes, as well. Many technologies have been developed for herpesvirus BAC engineering. In our hands the most powerful is galK recombineering that exploits a single marker (galK) for positive and negative selection and PCR amplicons for seamless modification in the desired genome locus. Here we describe the engineering of the HSV recombinant BAC 115 by the insertion of a heterologous cassette for the expression of murine interleukin 12 (mIL12) in the intergenic sequence between US1 and US2 ORFs.

Rescue, Purification, and Characterization of a Recombinant HSV Expressing a Transgenic Protein.

In the previous chapter, we describe the engineering of a HSV-BAC genome by galK recombineering. Here we describe the procedures to reconstitute, or regenerate, the replicating recombinant virus, and the methods to purify it and characterize it for the correct expression of the transgene. We present the example of R-115, a recombinant expressing murine interleukin 12 (mIL12) from the US1-US2 intergenic region. A specific method for the production of highly purified virions by iodixanol gradient, suitable for in vivo applications, is also detailed.

αvß3-integrin regulates PD-L1 expression and is involved in cancer immune evasion.

Tumors utilize a number of effective strategies, including the programmed death 1/PD ligand 1 (PD-1/PD-L1) axis, to evade immune-mediated control of their growth. PD-L1 expression is mainly induced by IFN receptor signaling or constitutively induced. Integrins are an abundantly expressed class of proteins which play multiple deleterious roles in cancer and exert proangiogenic and prosurvival activities. We asked whether αvß3-integrin positively regulates PD-L1 expression and the anticancer immune response. We report that αvß3-integrin regulated constitutive and IFN-induced PD-L1 expression in human and murine cancerous and noncancerous cells. αvß3-integrin targeted STAT1 through its signaling C tail. The implantation of ß3-integrin-depleted tumor cells led to a dramatic decrease in the growth of primary tumors, which exhibited reduced PD-L1 expression and became immunologically hot, with increased IFNγ content and CD8+ cell infiltration. In addition, the implantation of ß3-integrin-depleted tumors elicited an abscopal immunotherapeutic effect measured as protection from the challenge tumor and durable splenocyte and serum reactivity to B16 cell antigens. These modifications to the immunosuppressive microenvironment primed cells for checkpoint (CP) blockade. When combined with anti-PD-1, ß3-integrin depletion led to durable therapy and elicited an abscopal immunotherapeutic effect. We conclude that in addition to its previously known roles, αvß3-integrin serves as a critical component of the cancer immune evasion strategy and can be an effective immunotherapy target.

Eradication of glioblastoma by immuno-virotherapy with a retargeted oncolytic HSV in a preclinical model.

Oncolytic herpes simplex viruses are proving to be effective in clinical trials against a number of cancers. Here, R-115, an oncolytic herpes simplex virus retargeted to human erbB-2, fully virulent in its target cells, and armed with murine interleukin-12 was evaluated in a murine model of glioblastoma. We show that a single R-115 injection in established tumors resulted, in about 30% of animals, in the complete eradication of the tumor, otherwise invariably lethal. The treatment also induced a significant improvement in the overall median survival time of mice and a resistance to recurrence from the same neoplasia. Such a high degree of protection was unprecedented; it was not observed before following treatments with the commonly used, mutated/attenuated oncolytic viruses. This is the first study providing the evidence of benefits offered by a fully virulent, retargeted, and armed herpes simplex virus in the treatment of glioblastoma and paves the way for clinical translation.

A fully-virulent retargeted oncolytic HSV armed with IL-12 elicits local immunity and vaccine therapy towards distant tumors.

Oncolytic herpes simplex viruses (oHSVs) showed efficacy in clinical trials and practice. Most of them gain cancer-specificity from deletions/mutations in genes that counteract the host response, and grow selectively in cancer cells defective in anti-viral response. Because of the deletions/mutations, they are frequently attenuated or over-attenuated. We developed next-generation oHSVs, which carry no deletion/mutation, gain cancer-specificity from specific retargeting to tumor cell receptors-e.g. HER2 (human epidermal growth factor receptor 2)-hence are fully-virulent in the targeted cancer cells. The type of immunotherapy they elicit was not predictable, since non-attenuated HSVs induce and then dampen the innate response, whereas deleted/attenuated viruses fail to contrast it, and since the retargeted oHSVs replicate efficiently in tumor cells, but spare other cells in the tumor. We report on the first efficacy study of HER2-retargeted, fully-virulent oHSVs in immunocompetent mice. Their safety profile was very high. Both the unarmed R-LM113 and the IL-12-armed R-115 inhibited the growth of the primary HER2-Lewis lung carcinoma-1 (HER2-LLC1) tumor, R-115 being constantly more efficacious. All the mice that did not die because of the primary treated tumors, were protected from the growth of contralateral untreated tumors. The long-term survivors were protected from a second contralateral tumor, providing additional evidence for an abscopal immunotherapeutic effect. Analysis of the local response highlighted that particularly R-115 unleashed the immunosuppressive tumor microenvironment, i.e. induced immunomodulatory cytokines, including IFNγ, T-bet which promoted Th1 polarization. Some of the tumor infiltrating cells, e.g. CD4+, CD335+ cells were increased in the tumors of all responders mice, irrespective of which virus was employed, whereas CD8+, Foxp3+, CD141+ were increased and CD11b+ cells were decreased preferentially in R-115-treated mice. The durable response included a breakage of tolerance towards both HER2 and the wt tumor cells, and underscored a systemic immunotherapeutic vaccine response.

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

Previously, we engineered oncolytic herpes simplex viruses (o-HSVs) retargeted to the HER2 (epidermal growth factor receptor 2) tumor cell specific receptor by the insertion of a single chain antibody (scFv) to HER2 in gD, gH, or gB. Here, the insertion of scFvs to three additional cancer targets­EGFR (epidermal growth factor receptor), EGFRvIII, and PSMA (prostate specific membrane antigen)­in gD Δ6­38 enabled the generation of specifically retargeted o-HSVs. Viable recombinants resulted from the insertion of an scFv in place of aa 6­38, but not in place of aa 61­218. Hence, only the gD N-terminus accepted all tested scFv inserts. Additionally, the insertion of mIL12 in the US1-US2 intergenic region of the HER2- or EGFRvIII-retargeted o-HSVs, and the further insertion of Gaussia Luciferase, gave rise to viable recombinants capable of secreting the cytokine and the reporter. Lastly, we engineered two known mutations in gB; they increased the ability of an HER2-retargeted recombinant to spread among murine cells. Altogether, current data show that the o-HSV carrying the aa 6­38 deletion in gD serves as a platform for the specific retargeting of o-HSV tropism to a number of human cancer targets, and the retargeted o-HSVs serve as simultaneous vectors for two molecules.

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

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.

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

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.

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 αvß6 or αvß8 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.