Oncolytic viruses, which preferentially lyse cancer cells and stimulate an antitumor immune response, represent a promising approach to the treatment of cancer. However, how they evade the antiviral immune response and their selective delivery to, and replication in, tumor over normal tissue has not been investigated in humans. Here,we treated patients with a single cycle of intravenous reovirus before planned surgery to resect colorectal cancer metastases in the liver. Tracking the viral genome in the circulation showed that reovirus could be detected in plasma and blood mononuclear, granulocyte, and platelet cell compartments after infusion. Despite the presence of neutralizing antibodies before viral infusion in all patients, replication-competent reovirus that retained cytotoxicity was recovered from blood cells but not plasma, suggesting that transport by cells could protect virus for potential delivery to tumors. Analysis of surgical specimens demonstrated greater, preferential expression of reovirus protein in malignant cells compared to either tumor stroma or surrounding normal liver tissue. There was evidence of viral factories within tumor, and recovery of replicating virus from tumor (but not normal liver)was achieved in all four patients from whom fresh tissue was available. Hence, reovirus could be protected from neutralizing antibodies after systemic administration by immune cell carriage, which delivered reovirus to tumor.These findings suggest new preclinical and clinical scheduling and treatment combination strategies to enhance in vivo immune evasion and effective intravenous delivery of oncolytic viruses to patients in vivo.

Purpose Reovirus type 3 Dearing (RT3D) replicates preferentially in Ras-activated cancers. RT3D shows synergistic in vitro cytotoxicity in combination with platins and taxanes. The purpose of this phase I/II study was to assess RT3D combined with carboplatin/paclitaxel in patients with advanced cancers. Experimental Design Patients were initially treated in a dose-escalating, phase I trial with intravenous RT3D days 1 to 5, carboplatin [area under curve (AUC) 5, day 1] and paclitaxel (175 mg/m2, day 1) 3-weekly. RT3D was escalated through three dose levels: 3 × 109, 1 × 1010, and 3 × 1010 TCID50 in cohorts of three. Primary endpoints were to define the maximum tolerated dose and dose-limiting toxicity and to recommend a dose for phase II studies. Secondary endpoints included pharmacokinetics, immune response, and antitumor activity. A subsequent phase II study using the 3 × 1010 TCID50 dose characterized the response rate in patients with head and neck cancer. Results Thirty-one heavily pretreated patients received study therapy. There were no dose-limiting toxicities during dose-escalation and most toxicities were grade I/II. Overall effectiveness rates were as follows: one patient had a complete response (3.8%), six patients (23.1%) had partial response, two patients (7.6%) had major clinical responses clinically evaluated in radiation pretreated lesions which are not evaluable by Response Evaluation Criteria in Solid Tumors (RECIST), nine patients (34.6%) had stable disease, and eight patients (30.8%) had disease progression. Viral shedding was minimal and antiviral immune responses were attenuated compared with previous single-agent data for RT3D. Conclusions The combination of RT3D plus carboplatin/paclitaxel is well tolerated with evidence of activity in cancer of the head and neck. A randomized phase III study is currently open for recruitment.

Purpose REOLYSIN (Oncolytics Biotech) consists of a wild-type oncolytic reovirus, which has selective cytotoxicity for tumor cells while sparing normal cells. In a phase I study as a single agent, repeated infusions of reovirus were safe with evidence of antitumor activity. Preclinical studies indicate potential for synergy between reovirus and chemotherapeutic agents. A multicenter, phase I dose escalation study was designed to assess the safety of combining reovirus with docetaxel chemotherapy in patients with advanced cancer. Experimental Design Patients received 75 mg/m2 docetaxel (day 1) and escalating doses of reovirus up to 3 × 1010 TCID50 (days 1-5) every 3 weeks. Results Twenty-five patients were enrolled, and 24 patients were exposed to treatment, with 23 completing at least one cycle and 16 suitable for response assessment. Dose-limiting toxicity of grade 4 neutropenia was seen in one patient, but the maximum tolerated dose was not reached. Antitumor activity was seen with one complete response and three partial responses. A disease control rate (combined complete response, partial response, and stable disease) of 88% was observed. Immunohistochemical analysis of reovirus protein expression was observed in posttreatment tumor biopsies from three patients. Conclusion The combination of reovirus and docetaxel is safe, with evidence of objective disease response, and warrants further evaluation in a phase II study at a recommended schedule of docetaxel (75 mg/m2, three times weekly) and reovirus (3 × 1010 TCID50, days 1-5, every 3 weeks).

Reovirus, a replication competent RNA virus, has preclinical activity against melanoma lines and xenografts. We conducted a phase II trial of reovirus in metastatic melanoma patients. Patients received 3 × 10(10) TCID50 on days 1-5 of each 28 day cycle, administered intravenously. Twenty-one eligible patients were enrolled. Treatment was well tolerated without any dose reductions having to be implemented. Post-treatment biopsy samples were obtained in 15 patients, 13/15 contained adequate tumor for correlative analysis. In two patients, productive reoviral replication (viral antigen coexpression with tubulin) was demonstrated, despite increase in neutralizing antibody titers. There were no objective responses although 75-90% tumor necrosis, consistent with treatment effect, was observed in one patient who had metastatic lesions surgically removed. Median time to progression and survival were 45 days (range 13-96 days) and 165 days (range 15 days-15.8 months) respectively. In conclusion, reovirus treatment was well tolerated in metastatic melanoma patients; viral replication was demonstrated in biopsy samples. Based on preclinical data showing synergy with taxane and platinum compounds, a phase II combination trial in metastatic melanoma patients is ongoing.

Purpose: This study combined systemic administration of the oncolytic reovirus type 3 Dearing (reovirus) with chemotherapy in human subjects. We aimed to determine the safety and feasibility of combining reovirus administration with gemcitabine and to describe the effects of gemcitabine on the antireoviral immune response.Experimental Design: Patients received reovirus in various doses, initially we dosed for five consecutive days but this was poorly tolerated. We amended the protocol to administer a single dose and administered up to 3 Â 10 10 TCID 50 . Toxicity was assessed by monitoring of clinical and laboratory measurements. We assessed antibody response by cytotoxicity neutralization assay.Results: Sixteen patients received 47 cycles of reovirus. The two initial patients and one patient in the final cohort experienced dose limiting toxicity (DLT). The DLTs consisted of two asymptomatic grade 3 liver enzyme rises and one asymptomatic grade 3 troponin I rise. Common toxicities consisted of known reovirus and gemcitabine associated side effects. Further analysis showed a potential interaction between reovirus and gemcitabine in causing liver enzyme rises. Grade 3 rises in liver enzymes were associated with concomitant aminocetophen use. Importantly, the duration of the liver enzyme rise was short and reversible. Neutralizing antibody responses to reovirus were attenuated both in time-to-occurrence and peak height of the response.Conclusions: Reovirus at the dose of 1 Â 10 10 TCID 50 can be safely combined with full dose gemcitabine.Combination of reovirus with gemcitabine affects the neutralizing antibody response and this could impact both safety and efficacy of this treatment schedule.

Background Oncolytic viruses preferentially replicate in tumors as compared to normal tissue and promote immunogenic cell death and induction of host systemic anti-tumor immunity. HSV-1 was chosen for further development as an oncolytic immunotherapy in this study as it is highly lytic, infects human tumor cells broadly, kills mainly by necrosis and is a potent activator of both innate and adaptive immunity. HSV-1 also has a large capacity for the insertion of additional, potentially therapeutic, exogenous genes. Finally, HSV-1 has a proven safety and efficacy profile in patients with cancer, talimogene laherparepvec (T-VEC), an oncolytic HSV-1 which expresses GM-CSF, being the only oncolytic immunotherapy approach that has received FDA approval. As the clinical efficacy of oncolytic immunotherapy has been shown to be further enhanced by combination with immune checkpoint inhibitors, developing improved oncolytic platforms which can synergize with other existing immunotherapies is a high priority. In this study we sought to further optimize HSV-1 based oncolytic immunotherapy through multiple approaches to maximize: (i) the extent of tumor cell killing, augmenting the release of tumor antigens and danger-associated molecular pattern (DAMP) factors; (ii) the immunogenicity of tumor cell death; and (iii) the resulting systemic anti-tumor immune response. Methods To sample the wide diversity amongst clinical strains of HSV-1, twenty nine new clinical strains isolated from cold sores from otherwise healthy volunteers were screened across a panel of human tumor cell lines to identify the strain with the most potent tumor cell killing ability, which was then used for further development. Following deletion of the genes encoding ICP34.5 and ICP47 to provide tumor selectivity, the extent of cell killing and the immunogenicity of cell death was enhanced through insertion of a gene encoding a truncated, constitutively highly fusogenic form of the envelope glycoprotein of gibbon ape leukemia virus (GALV-GP-R − ). A number of further armed derivatives of this virus were then constructed intended to further enhance the anti-tumor immune response which was generated following fusion-enhanced, oncolytic virus replication-mediated cell death. These viruses expressed GMCSF, an anti-CTLA-4 antibody-like molecule, CD40L, OX40L and/or 4-1BB, each of which is expected to act predominantly at the site and time of immune response initiation. Expression of these proteins was confirmed by ELISA and/or western blotting. Immunogenic cell death was assessed by measuring the levels of HMGB1 and ATP from cell free supernatants from treated cells, and by measuring the surface expression of calreticulin. GALV-GP-R − mediated cell to cell fusion and killing was tested in a range of tumor cell lines in vitro. Finally, the in vivo therapeutic potential of these viruses was tested using human A549 (lung cancer) and MDA-MB-231(breast cancer) tumor ...

Purpose To determine the safety and feasibility of combining intratumoral reovirus and radiotherapy in patients with advanced cancer and to assess viral biodistribution, reoviral replication in tumors, and antiviral immune responses. Experimental Design Patients with measurable disease amenable to palliative radiotherapy were enrolled. In the first stage, patients received radiotherapy (20 Gy in five fractions) plus two intratumoral injections of RT3D at doses between 1 × 108 and 1 × 1010 TCID50. In the second stage, the radiotherapy dose was increased (36 Gy in 12 fractions) and patients received two, four, or six doses of RT3D at 1 × 1010 TCID50. End points were safety, viral replication, immunogenicity, and antitumoral activity. Results Twenty-three patients with various solid tumors were treated. Dose-limiting toxicity was not seen. The most common toxicities were grade 2 (or lower) pyrexia, influenza-like symptoms, vomiting, asymptomatic lymphopenia, and neutropenia. There was no exacerbation of the acute radiation reaction. Reverse transcription-PCR (RT-PCR) studies of blood, urine, stool, and sputum were negative for viral shedding. In the low-dose (20 Gy in five fractions) radiation group, two of seven evaluable patients had a partial response and five had stable disease. In the high-dose (36 Gy in 12 fractions) radiation group, five of seven evaluable patients had partial response and two stable disease. Conclusions The combination of intratumoral RT3D and radiotherapy was well tolerated. The favorable toxicity profile and lack of vector shedding means that this combination should be evaluated in newly diagnosed patients receiving radiotherapy with curative intent.

Oncolytic reovirus is currently under active investigation in a range of tumour types. Early phase studies have shown that this agent has modest monotherapy efficacy and its future development is likely to focus on combination regimens with cytotoxic chemotherapy. Indeed, phase I/II clinical trials have confirmed that reovirus can be safely combined with cytotoxic drugs, including a platin—taxane doublet regimen, which is currently being tested in a phase III clinical trial in patients with relapsed/metastatic head and neck cancer. Therefore, we have tested this triple (reovirus, cisplatin, paclitaxel) combination therapy in a panel of four head and neck cancer cell lines. Using the combination index (CI) method, the triple therapy demonstrated synergistic cytotoxicity in vitro in both malignant and non-malignant cell lines. In head and neck cancer cell lines, this was associated with enhanced caspase 3 and 7 cleavage, but no increase in viral replication. In vitro analyses confirmed colocalisation of markers of reovirus infection and caspase 3. Triple therapy was significantly more effective than reovirus or cisplatin—paclitaxel in athymic nude mice. These data suggest that the combination of reovirus plus platin—taxane doublet chemotherapy has significant activity in head and neck cancer and underpin the current phase III study in this indication.