Trabectedin Enhances Oncolytic Virotherapy by Reducing Barriers to Virus Spread and Cytotoxic Immunity in Preclinical Pediatric Bone Sarcoma.

We previously reported that the DNA alkylator and transcriptional-blocking chemotherapeutic agent trabectedin enhances oncolytic herpes simplex viroimmunotherapy in human sarcoma xenograft models, though the mechanism remained to be elucidated. Here we report trabectedin disrupts the intrinsic cellular anti-viral response which increases viral transcript spread throughout the human tumor cells. We also extended our synergy findings to syngeneic murine sarcoma models, which are poorly susceptible to virus infection. In the absence of robust virus replication, we found trabectedin enhanced viroimmunotherapy efficacy by reducing immunosuppressive macrophages and stimulating granzyme expression in infiltrating T and NK cells to cause immune-mediated tumor regressions. Thus, trabectedin enhances both the direct virus-mediated killing of tumor cells and the viral-induced activation of cytotoxic effector lymphocytes to cause tumor regressions across models. Our data provide a strong rationale for clinical translation as both mechanisms should be simultaneously active in human patients.

Opportunities and challenges of combining adoptive cellular therapy with oncolytic virotherapy.

The use of oncolytic viruses (OVs) and adoptive cell therapies (ACT) have independently emerged as promising approaches for cancer immunotherapy. More recently, the combination of such agents to obtain a synergistic anticancer effect has gained attention, particularly in solid tumors, where immune-suppressive barriers of the microenvironment remain a challenge for desirable therapeutic efficacy. While adoptive cell monotherapies may be restricted by an immunologically cold or suppressive tumor microenvironment (TME), OVs can serve to prime the TME by eliciting a wave of cancer-specific immunogenic cell death and inducing enhanced antitumor immunity. While OV/ACT synergy is an attractive approach, immune-suppressive barriers remain, and methods should be considered to optimize approaches for such combination therapy. In this review, we summarize current approaches that aim to overcome these barriers to enable optimal synergistic antitumor effects.

Leveraging gene therapy to achieve long-term continuous or controllable expression of biotherapeutics.

T cells redirected to cancer cells either via a chimeric antigen receptor (CAR-T) or a bispecific molecule have been breakthrough technologies; however, CAR-T cells require individualized manufacturing and bispecifics generally require continuous infusions. We created an off-the-shelf, single-dose solution for achieving prolonged systemic serum levels of protein immunotherapeutics via adeno-associated virus (AAV) gene transfer. We demonstrate proof of principle in a CD19+ lymphoma xenograft model using a single intravenous dose of AAV expressing a secreted version of blinatumomab, which could serve as a universal alternative for CD19 CAR-T cell therapy. In addition, we created an inducible version using an exon skipping strategy and achieved repeated, on-demand expression up to at least 36 weeks after AAV injection. Our system could be considered for short-term and/or repeated expression of other transgenes of interest for noncancer applications.

Endogenous retrovirus envelope as a tumor-associated immunotherapeutic target in murine osteosarcoma.

Osteosarcoma remains one of the deadliest cancers in pediatrics and young adults. We administered two types of immunotherapies, oncolytic virotherapy and immune checkpoint inhibition, to two murine osteosarcoma models and observed divergent results. Mice bearing F420 showed no response, whereas those with K7M2 showed prolonged survival in response to combination therapy. K7M2 had higher expression of immune-related genes and higher baseline immune cell infiltrates, but there were no significant differences in tumor mutational burden or predicted MHC class I binding of nonsynonymous mutations. Instead, we found several mouse endogenous retrovirus sequences highly expressed in K7M2 compared with F420. T cell tetramer staining for one of them, gp70, was detected in mice with K7M2 but not F420, suggesting that endogenous retrovirus proteins are targets for the anti-tumor immune reaction. Given prior observations of endogenous retrovirus expression in human osteosarcomas, our findings may be translatable to human disease.

Engineered oncolytic virus for the treatment of cholesteatoma: A pilot in vivo study.

OBJECTIVE: Determine if oncolytic herpes simplex virus (oHSV) can eradicate cholesteatoma (CHST) in a gerbil model. METHODS: An in vivo model of CHST was developed in Mongolian gerbils by combining Pseudomonas aeruginosa inoculation with double ligation of the external auditory canal (EAC). CHST size and bone thickness were measured using morphometric and volumetric quantification techniques via micro-computed tomography (micro-CT). The CHST induction and quantification techniques were then used in an additional group of 10 gerbils (n = 20 ears) to determine the within-group treatment efficacy of oHSV against CHST in vivo. Treated animals received either one, two, or three intrabullar injections of oHSV between 2 and 6 weeks postinduction of CHST. RESULTS: The P. aeruginosa inoculation plus double EAC ligation technique successfully induced a range of CHST growth in 100% of the ears in the model-development group. Osteolytic effects of CHST were observed in 6% of ears whereas osteoblastic effects were observed in 31% of ears. CHST volume decreased by 50% or more in 12 of the 20 ears in the oHSV-treatment groups. An apparent reversal of osteoblastic effects was also observed in three out of four ears 6 weeks following the third oHSV injection. CONCLUSIONS: P. aeruginosa inoculation plus double EAC ligation reliably induces CHST formation in gerbil. CT-based volumetric measures are significantly more accurate than single-slice morphometric area measures for quantification of CHST size. Treatment with oHSV appears to be efficacious for reducing CHST volume by as much as 77% with as few as one treatment. LEVEL OF EVIDENCE: NA.

First-in-Human Intravenous Seprehvir in Young Cancer Patients: A Phase 1 Clinical Trial.

Seprehvir (HSV1716) is an oncolytic herpes simplex virus-1 (HSV-1) previously demonstrated to be well tolerated in pediatric patients when administered intratumorally. To determine the safety of administering Seprehvir systemically, we conducted the first-in-human phase I trial of intravenous injection in young patients with relapsed or refractory extra-cranial solid cancers. We delivered a single dose of 5 × 104 infectious units (iu)/kg (maximum dose of 2 × 106) or 2.5 × 105 iu/kg (maximum dose of 1 × 107 iu) of Seprehvir via the peripheral vein, monitored adverse events, and measured tumor responses by imaging. We monitored HSV-1 serology as well as viremia and shedding by PCR and culture. We administered a single dose of Seprehvir to seven patients and multiple doses to two patients. We did not observe any dose-limiting toxicities. All five HSV-1 seronegative patients seroconverted by day 28. Four of nine patients had detectable HSV-1 genomes in peripheral blood appearing on day +4 consistent with de novo virus replication. Two patients had stable disease in response to Seprehvir. Intravenous Seprehvir is well tolerated without viral shedding in children and young adults with late-stage cancer. Viremia consistent with virus replication holds promise for future Seprehvir studies at higher doses and/or in combination with other anti-neoplastic therapies.

A Cationic Nanomicellar Complex of the Quaternary Amphiphilic Amine RC16+ with Fenretinide as a New Multitasking System for Antitumor Therapy.

OBJECTIVES: This study investigated the antitumor effect of a new nanomicellar complex obtained by combining the antitumor agent fenretinide with a quaternary amphiphilic amine RC16+ also endowed with antitumor activity. METHODS: The complex (Fen-RC16+) strongly improved the aqueous solubility of fenretinide (from 1,71 ± 0.08 µg/ml, pure fenretinide to 1500 ± 164 µg /ml, Fen-RC16+ complex) and provided a cytotoxic effect on SH-SY5Y neuroblastoma cell lines resulting from the intrinsic activity of both the complex components. Moreover, the mean size of the nanomicellar complex (ranging from 20 ± 1.97 nm to 40 ± 3.05 nm) was suitable for accumulation to the tumor site by the enhanced permeability and retention effect and the positive charge provided by the quaternary RC16+ induced adsorption of the complex on the tumor cell surface improving the intracellular concentration of fenretinide. RESULTS: All these characteristics made the Fen-RC16+ complex a multitasking system for antitumor therapy. CONCLUSION: Indeed its in vivo activity, evaluated on SH-SY5Y xenografts, was strong, and the tumor growth did not resume after the treatment withdrawal.

Myelolytic Treatments Enhance Oncolytic Herpes Virotherapy in Models of Ewing Sarcoma by Modulating the Immune Microenvironment.

Ewing sarcoma is a highly aggressive cancer that promotes the infiltration and activation of pro-tumor M2-like macrophages. Oncolytic virotherapy that selectively infects and destroys cancer cells is a promising option for treating Ewing sarcoma. The effect of tumor macrophages on oncolytic virus therapy, however, is variable among solid tumors and is unknown in Ewing sarcoma. We tested the effects of macrophage reduction using liposomal clodronate (Clodrosome) and trabectedin on the antitumor efficacy of intratumoral oncolytic herpes simplex virus, rRp450, in two Ewing sarcoma xenograft models. Both agents enhanced antitumor efficacy without increasing virus replication. The most profound effects were in A673 with only a transient effect on response rates in TC71. Interestingly, A673 was more dependent than TC71 on macrophages for its tumorigenesis. We found Clodrosome and virus together induced expression of antitumorigenic genes and reduced expression of protumorigenic genes in both the tumor-associated macrophages and the overall tumor stroma. Trabectedin reduced intratumoral natural killer (NK) cells, myeloid-derived suppressor cells, and M2-like macrophages, and prevented their increase following virotherapy. Our data suggest that a combination of trabectedin and oncolytic herpes virotherapy warrants testing in the clinical setting.

Intratumoral Injection of HSV1716, an Oncolytic Herpes Virus, Is Safe and Shows Evidence of Immune Response and Viral Replication in Young Cancer Patients.

Purpose: HSV1716 is an oncolytic herpes simplex virus-1 (HSV-1) studied in adults via injection into the brain and superficial tumors. To determine the safety of administering HSV1716 to pediatric patients with cancer, we conducted a phase I trial of image-guided injection in young patients with relapsed or refractory extracranial cancers.Experimental Design: We delivered a single dose of 105 to 107 infectious units of HSV1716 via computed tomography-guided intratumoral injection and measured tumor responses by imaging. Patients were eligible for up to three more doses if they achieved stable disease. We monitored HSV-1 serum titers and shedding by PCR and culture.Results: We administered a single dose of HSV1716 to eight patients and two doses to one patient. We did not observe any dose-limiting toxicities. Adverse events attributed to virus included low-grade fever, chills, and mild cytopenias. Six of eight HSV-1 seronegative patients at baseline showed seroconversion on day 28. Six of nine patients had detectable HSV-1 genomes by PCR in peripheral blood appearing on day +4 consistent with de novo virus replication. Two patients had transient focal increases in metabolic activity on 18fluorine-deoxyglucose PET, consistent with inflammatory reactions. In one case, the same geographic region that flared later appeared necrotic on imaging. No patient had an objective response to HSV1716.Conclusions: Intratumoral HSV1716 is safe and well-tolerated without shedding in children and young adults with late-stage, aggressive cancer. Viremia consistent with virus replication and transient inflammatory reactions hold promise for future HSV1716 studies. Clin Cancer Res; 23(14); 3566-74. ©2017 AACR.

Identification of Cancer-Targeted Tropomyosin Inhibitors and Their Synergy with Microtubule Drugs.

Actin filaments, with their associated tropomyosin polymers, and microtubules are dynamic cytoskeletal systems regulating numerous cell functions. While antimicrotubule drugs are well-established, antiactin drugs have been more elusive. We previously targeted actin in cancer cells by inhibiting the function of a tropomyosin isoform enriched in cancer cells, Tpm3.1, using a first-in-class compound, TR100. Here, we screened over 200 other antitropomyosin analogues for anticancer and on-target activity using a series of in vitro cell-based and biochemical assays. ATM-3507 was selected as the new lead based on its ability to disable Tpm3.1-containing filaments, its cytotoxicity potency, and more favorable drug-like characteristics. We tested ATM-3507 and TR100 alone and in combination with antimicrotubule agents against neuroblastoma models in vitro and in vivo Both ATM-3507 and TR100 showed a high degree of synergy in vitro with vinca alkaloid and taxane antimicrotubule agents. In vivo, combination-treated animals bearing human neuroblastoma xenografts treated with antitropomyosin combined with vincristine showed minimal weight loss, a significant and profound regression of tumor growth and improved survival compared with control and either drug alone. Antitropomyosin combined with vincristine resulted in G2-M phase arrest, disruption of mitotic spindle formation, and cellular apoptosis. Our data suggest that small molecules targeting the actin cytoskeleton via tropomyosin sensitize cancer cells to antimicrotubule agents and are tolerated together in vivo This combination warrants further study. Mol Cancer Ther; 16(8); 1555-65. ©2017 AACR.

Aurora A kinase inhibition enhances oncolytic herpes virotherapy through cytotoxic synergy and innate cellular immune modulation.

Malignant peripheral nerve sheath tumor (MPNST) and neuroblastoma models respond to the investigational small molecule Aurora A kinase inhibitor, alisertib. We previously reported that MPNST and neuroblastomas are also susceptible to oncolytic herpes virus (oHSV) therapy. Herein, we show that combination of alisertib and HSV1716, a virus derived from HSV-1 and attenuated by deletion of RL1, exhibits significantly increased antitumor efficacy compared to either monotherapy. Alisertib and HSV1716 reduced tumor growth and increased survival in two xenograft models of MPNST and neuroblastoma. We found the enhanced antitumor effect was due to multiple mechanisms that likely each contribute to the combination effect. First, oncolytic herpes virus increased the sensitivity of uninfected cells to alisertib cytotoxicity, a process we term virus-induced therapeutic adjuvant (VITA). Second, alisertib increased peak virus production and slowed virus clearance from tumors, both likely a consequence of it preventing virus-mediated increase of intratumoral NK cells. We also found that alisertib inhibited virus-induced accumulation of intratumoral myeloid derived suppressor cells, which normally are protumorigenic. Our data suggest that clinical trials of the combination of oHSV and alisertib are warranted in patients with neuroblastoma or MPNST.

Preparation and Evaluation of a Novel Class of Amphiphilic Amines as Antitumor Agents and Nanocarriers for Bioactive Molecules.

PURPOSE: We describe a novel class of antitumor amphiphilic amines (RCn) based on a tricyclic amine hydrophilic head and a hydrophobic linear alkyl tail of variable length. METHODS: We tested the lead compound, RC16, for cytotoxicity and mechanism of cell death in several cancer cell lines, anti tumor efficacy in mouse tumor models, and ability to encapsulate chemotherapy drugs. RESULTS: These compounds displayed strong cytotoxic activity against cell lines derived from both pediatric and adult cancers. The IC50 of the lead compound, RC16, for normal cells including human keratinocytes, human fibroblasts and human umbilical vein endothelial cells was tenfold higher than for tumor cells. RC16 exhibited significant antitumor effects in vivo using several human xenografts and a metastatic model of murine neuroblastoma by both intravenous and oral administration routes. The amphiphilic character of RC16 triggered a spontaneous molecular self-assembling in water with formation of micelles allowing complexation of Doxorubicin, Etoposide and Paclitaxel. These micelles significantly improved the in vitro antitumor activity of these drugs as the enhancement of their aqueous solubility also improved their biologic availability. CONCLUSIONS: RC16 and related amphiphilic amines may be useful as a novel cancer treatment.

Oncolytic HSV virotherapy in murine sarcomas differentially triggers an antitumor T-cell response in the absence of virus permissivity.

Multiple studies have indicated that in addition to direct oncolysis, virotherapy promotes an antitumor cytotoxic T cell response important for efficacy. To study this phenomenon further, we tested three syngeneic murine sarcoma models that displayed varied degrees of permissiveness to oncolytic herpes simplex virus replication and cytotoxicity in vitro, with the most permissive being comparable to some human sarcoma tumor lines. The in vivo antitumor effect ranged from no or modest response to complete tumor regression and protection from tumor rechallenge. The in vitro permissiveness to viral oncolysis was not predictive of the in vivo antitumor effect, as all three tumors showed intact interferon signaling and minimal permissiveness to virus in vivo. Tumor shrinkage was T-cell mediated with a tumor-specific antigen response required for maximal antitumor activity. Further analysis of the innate and adaptive immune microenvironment revealed potential correlates of susceptibility and resistance, including favorable and unfavorable cytokine profiles, differential composition of intratumoral myeloid cells, and baseline differences in tumor cell immunogenicity and tumor-infiltrating T-cell subsets. It is likely that a more complete understanding of the interplay between the immunologic immune microenvironment and virus infection will be necessary to fully leverage the antitumor effects of this therapeutic platform.

Phase 1 study of intratumoral Pexa-Vec (JX-594), an oncolytic and immunotherapeutic vaccinia virus, in pediatric cancer patients.

Pexa-Vec (pexastimogene devacirepvec, JX-594) is an oncolytic and immunotherapeutic vaccinia virus designed to destroy cancer cells through viral lysis and induction of granulocyte-macrophage colony-stimulating factor (GM-CSF)-driven tumor-specific immunity. Pexa-Vec has undergone phase 1 and 2 testing alone and in combination with other therapies in adult patients, via both intratumoral and intravenous administration routes. We sought to determine the safety of intratumoral administration in pediatric patients. In a dose-escalation study using either 10(6) or 10(7) plaque-forming units per kilogram, we performed one-time injections in up to three tumor sites in five pediatric patients and two injections in one patient. Ages at study entry ranged from 4 to 21 years, and their cancer diagnoses included neuroblastoma, hepatocellular carcinoma, and Ewing sarcoma. All toxicities were ≤ grade 3. The most common side effects were sinus fever and sinus tachycardia. All three patients at the higher dose developed asymptomatic grade 1 treatment-related skin pustules that resolved within 3-4 weeks. One patient showed imaging evidence suggestive of antitumor biological activity. The two patients tested for cellular immunoreactivity to vaccinia antigens showed strong responses. Overall, our study suggests Pexa-Vec is safe to administer to pediatric patients by intratumoral administration and could be studied further in this patient population.

Preclinical evaluation of nanoparticle albumin-bound paclitaxel for treatment of pediatric bone sarcoma.

The combination of docetaxel and gemcitabine is frequently used to treat recurrent bone sarcoma. Nanoparticle albumin-bound paclitaxel (nab-paclitaxel) is less toxic and more active than docetaxel or paclitaxel for breast cancer patients. The combination of nab-paclitaxel and gemcitabine has preclinical synergy and is approved to treat pancreatic cancer. We observed growth inhibition and improved survival with nab-paclitaxel in a Ewing sarcoma xenograft, and activity was additive with gemcitabine in an osteosarcoma model. Primary Ewing sarcoma tumors expressed the transport protein SPARC, previously associated with nab-paclitaxel activity. These findings provide rationale for further evaluation of nab-paclitaxel with gemcitabine for bone sarcoma.

VEGF blockade enables oncolytic cancer virotherapy in part by modulating intratumoral myeloid cells.

Understanding the host response to oncolytic viruses is important to maximize their antitumor efficacy. Despite robust cytotoxicity and high virus production of an oncolytic herpes simplex virus (oHSV) in cultured human sarcoma cells, intratumoral (ITu) virus injection resulted in only mild antitumor effects in some xenograft models, prompting us to characterize the host inflammatory response. Virotherapy induced an acute neutrophilic infiltrate, a relative decrease of ITu macrophages, and a myeloid cell-dependent upregulation of host-derived vascular endothelial growth factor (VEGF). Anti-VEGF antibodies, bevacizumab and r84, the latter of which binds VEGF and selectively inhibits binding to VEGF receptor-2 (VEGFR2) but not VEGFR1, enhanced the antitumor effects of virotherapy, in part due to decreased angiogenesis but not increased virus production. Neither antibody affected neutrophilic infiltration but both partially mitigated virus-induced depletion of macrophages. Enhancement of virotherapy-mediated antitumor effects by anti-VEGF antibodies could largely be recapitulated by systemic depletion of CD11b(+) cells. These data suggest the combined effect of oHSV virotherapy and anti-VEGF antibodies is in part due to modulation of a host inflammatory reaction to virus. Our data provide strong preclinical support for combined oHSV and anti-VEGF antibody therapy and suggest that understanding and counteracting the innate host response may help enable the full antitumor potential of oncolytic virotherapy.

Cancer screening by systemic administration of a gene delivery vector encoding tumor-selective secretable biomarker expression.

Cancer biomarkers facilitate screening and early detection but are known for only a few cancer types. We demonstrated the principle of inducing tumors to secrete a serum biomarker using a systemically administered gene delivery vector that targets tumors for selective expression of an engineered cassette. We exploited tumor-selective replication of a conditionally replicative Herpes simplex virus (HSV) combined with a replication-dependent late viral promoter to achieve tumor-selective biomarker expression as an example gene delivery vector. Virus replication, cytotoxicity and biomarker production were low in quiescent normal human foreskin keratinocytes and high in cancer cells in vitro. Following intravenous injection of virus >90% of tumor-bearing mice exhibited higher levels of biomarker than non-tumor-bearing mice and upon necropsy, we detected virus exclusively in tumors. Our strategy of forcing tumors to secrete a serum biomarker could be useful for cancer screening in high-risk patients, and possibly for monitoring response to therapy. In addition, because oncolytic vectors for tumor specific gene delivery are cytotoxic, they may supplement our screening strategy as a "theragnostic" agent. The cancer screening approach presented in this work introduces a paradigm shift in the utility of gene delivery which we foresee being improved by alternative vectors targeting gene delivery and expression to tumors. Refining this approach will usher a new era for clinical cancer screening that may be implemented in the developed and undeveloped world.

Overexpression of the cellular DEK protein promotes epithelial transformation in vitro and in vivo.

High levels of expression of the human DEK gene have been correlated with numerous human malignancies. Intracellular DEK functions have been described in vitro and include DNA supercoiling, DNA replication, RNA splicing, and transcription. We have shown that DEK also suppresses cellular senescence, apoptosis, and differentiation, thus promoting cell growth and survival in monolayer and organotypic epithelial raft models. Such functions are likely to contribute to cancer, but direct evidence to implicate DEK as an oncogene has remained elusive. Here, we show that in line with an early role in tumorigenesis, murine papilloma formation in a classical chemical carcinogenesis model was reduced in DEK knockout mice. Additionally, human papillomavirus E6/E7, hRas, and DEK cooperated in the transformation of keratinocytes in soft agar and xenograft establishment, thus also implicating DEK in tumor promotion at later stages. Finally, adenoviral DEK depletion via short hairpin RNA expression resulted in cell death in human tumor cells in vitro and in vivo, but did not significantly affect differentiated epithelial cells. Taken together, our data uncover oncogenic DEK activities as postulated from its frequent up-regulation in human malignancies, and suggest that the targeted suppression of DEK may become a strategic approach to the treatment of cancer.

Efficacy and safety of the oncolytic herpes simplex virus rRp450 alone and combined with cyclophosphamide.

Oncolytic herpes simplex virus (oHSV) mutants are under development as anticancer therapeutics. One such vector, rRp450, is ICP6-deleted and expresses a prodrug enzyme for cyclophosphamide (CPA) (rat CYP2B1). Little is known about rRp450's toxicity profile, especially in combination with CPA. We tested rRp450/CPA for antitumor efficacy in an aggressive human xenograft sarcoma model, measured virus production in primary cells, and tested rRp450/CPA for safety in immunocompetent mice. CPA enhanced the antitumor efficacy of rRp450. Relative to wild-type HSV-1, rRp450 replication was attenuated approximately 10,000-fold in human primary hepatocytes, differentiated primary foreskin keratinocytes, and primary Schwann cells. In vivo, intravenous and intracranial (IC) rRp450 injection at the strength of 10(8) plaque-forming units (pfu) alone or followed 24 hours later by intraperitoneal (IP) CPA was well tolerated and had no significant effect clinically on blood counts or chemistries. By contrast, intravenous KOS was found to be uniformly neurotoxic at 10(5) and fatal at 10(6) pfu, and IC virus was fatal in most mice at 10(4) pfu. Low levels of virus DNA were detected in some organs following intravenous and IC virus injection, but were not significantly altered by CPA. HSV replication was not detected in reactivation studies of isolated organs. Our findings suggest rRp450/CPA is safe and warrants further study as a potential combination in anticancer therapeutics.

Oncolytic HSV and erlotinib inhibit tumor growth and angiogenesis in a novel malignant peripheral nerve sheath tumor xenograft model.

Malignant peripheral nerve sheath tumors (MPNSTs), driven in part by hyperactive Ras and epidermal growth factor receptor (EGFR) signaling, are often incurable. Testing of therapeutics for MPNST has been hampered by lack of adequate xenograft models. We previously documented that human MPNST cells are permissive for lytic infection by oncolytic herpes simplex viruses (oHSV). Herein we developed and characterized a xenograft model of human MPNST and evaluated the antitumor effects of oHSV mutants (G207 and hrR3) and the EGFR inhibitor, erlotinib. Additive cytotoxicity of these agents was found in human MPNST cell lines, suggesting that EGFR signaling is not critical for virus replication. Mice bearing human MPNST tumors treated with G207 or hrR3 by intraperitoneal or intratumoral injection showed tumor-selective virus biodistribution, virus replication, and reduced tumor burden. oHSV injection demonstrated more dramatic antitumor activity than erlotinib. Combination therapies showed a trend toward an increased antiproliferative effect. Both oHSV and erlotinib were antiangiogenic as measured by proangiogenic gene expression, effect on endothelial cells and xenograft vessel density. Overall, oHSVs showed highly potent antitumor effects against MPNST xenografts, an effect not diminished by EGFR inhibition. Our data suggest that inclusion of MPNSTs in clinical trials of oHSV is warranted.