Reovirus exerts potent oncolytic effects in head and neck cancer cell lines that are independent of signalling in the EGFR pathway

Background: reovirus exploits aberrant signalling downstream of Ras to mediate tumor-specific oncolysis. Since ~90% squamous cell carcinomas of the head and neck (SCCHN) over-express EGFR and SCCHN cell lines are sensitive to oncolytic reovirus, we conducted a detailed analysis of the effects of reovirus in 15 head and neck cancer cell lines. Both pre- and post-entry events were studied in an attempt to define biomarkers predictive of sensitivity/resistance to reovirus. In particular, we analysed the role of EGFR/Ras signalling in determining virus-mediated cytotoxicity in SCCHN. Methods: to test whether EGFR pathway activity was predictive of increased sensitivity to reovirus, correlative analyses between reoviral IC50 by MTT assay and EGFR levels by western blot and FACS were conducted. Inhibition or stimulation of EGFR signalling were analysed for their effect on reoviral oncolysis by MTT assay, and viral growth by TCID50 assay. We next analysed the effects of inhibiting signalling downstream of Ras, by specific inhibitors of p38MAPK, PI3-K or MEK, on reoviral killing examined by MTT assay. The role of PKR in reoviral killing was also determined by blockade of PKR using 2-aminopurine and assaying for cell survival by MTT assay. The apoptotic response of SCCHN to reovirus was examined by western blot analysis of caspase 3 cleavage. Results: correlative analyses between reoviral sensitivity and EGFR levels revealed no association. Intermediate sub-viral and core particles showed the same infectivity/cytotoxicity as intact reovirus. Therefore, sensitivity was not determined by cell entry. In 4 cell lines, oncolysis and viral growth were both unaffected by inhibition or stimulation of EGFR signalling. Inhibition of signalling downstream of Ras did not abrogate reoviral oncolysis and, in addition, modulation of PKR using 2-aminopurine did not alter reovirus sensitivity in resistant cell lines. Caspase 3 cleavage was not detected in infected cells and oncolysis was observed in pan-caspase inhibited cells. Conclusions: in summary, reovirus is potently oncolytic in a broad panel of SCCHN cell lines. Attempts to define sensitivity/resistance by analysis of the EGFR/Ras/MAPK pathway have failed to provide a clear predictive biomarker of response. Further analysis of material from in vitro and clinical studies is ongoing in an attempt to shed further light on this issue

The role of killer immunoglobulin-like receptors in HTLV-1 infection

Human T-lymphotropic virus type 1 (HTLV-1) causes the debilitating neuroinflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in a minority of infected individuals, while the majority are lifelong asymptomatic carriers (ACs). The effectiveness of the CD8+ T-cell response to HTLV-1 is a major determinant of the HTLV-1 proviral load (PVL) and the associated HAM/TSP risk. Host genotype, particularly of HLA class I, strongly influences CD8+ T-cell effectiveness against HTLV-1. Furthermore, possession of the KIR gene 2DL2 was recently shown to enhance protective and detrimental HLA class I-associated immunity to HTLV-1 and HCV (Seich al Basatena et al., 2011). The proposed mechanism explaining this effect is a KIR expression-induced increase in activation thresholds, thus promoting survival. I investigated KIR expression and function on T-cells and NK cells in HTLV-1 infection, focussing on CD8+ T-cells and using new anti-KIR mAbs to analyse 2DL2 expression alongside four other KIRs. This thesis presents evidence supporting the genetic findings and the KIR expression-induced CD8+ T-cell survival hypothesis. AC status was associated with expanded 2DL2+CD8+ T EM cell populations and 2DL2+ and 2DL3+CD8+ T EM cell frequencies were inversely correlated with PVL, consistent with a protective role for 2DL2 (and potentially 2DL3) expression on CD8+ T-cells in HTLV-1 infection. Rather than a generalised reduction in CD8+ T-cell function, specific KIR expression was associated with a shift in effector function profile towards a cytotoxic phenotype without cytokine production. This is consistent with raised activation thresholds and may suggest fine-tuning of CD8+ T-cell effector functions by KIRs, preserving killing without causing inflammation. I further speculate that 2DL2/3 could be a context-dependent modulator of CD8+ T-cell function, optimally tuning responses and contributing to CD8+ T-cell effectiveness. Together with the longevity of 2DL2/3+CD8+ T-cells in vivo, this hypothesis requires further testing, since it may be relevant to other persistent viral infections.Open Acces

A phase I study of the combination of intravenous reovirus type 3 Dearing and gemcitabine in patients with advanced cancer

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 x 10(10) TCID50. 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 x 10(10) TCID50 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

REO-10: A Phase I Study of Intravenous Reovirus and Docetaxel in Patients with Advanced Cancer

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/m(2) docetaxel (day 1) and escalating doses of reovirus up to 3 × 10(10) TCID(50) (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/m(2), three times weekly) and reovirus (3 × 10(10) TCID(50), days 1-5, every 3 weeks)

Inhibitory killer cell immunoglobulin-like receptors strengthen CD8+ T cell-mediated control of HIV-1, HCV, and HTLV-1.

Killer cell immunoglobulin-like receptors (KIRs) are expressed predominantly on natural killer cells, where they play a key role in the regulation of innate immune responses. Recent studies show that inhibitory KIRs can also affect adaptive T cell-mediated immunity. In mice and in human T cells in vitro, inhibitory KIR ligation enhanced CD8+ T cell survival. To investigate the clinical relevance of these observations, we conducted an extensive immunogenetic analysis of multiple independent cohorts of HIV-1-, hepatitis C virus (HCV)-, and human T cell leukemia virus type 1 (HTLV-1)-infected individuals in conjunction with in vitro assays of T cell survival, analysis of ex vivo KIR expression, and mathematical modeling of host-virus dynamics. Our data suggest that functional engagement of inhibitory KIRs enhances the CD8+ T cell response against HIV-1, HCV, and HTLV-1 and is a significant determinant of clinical outcome in all three viral infections

Phase I/II trial of carboplatin and paclitaxel chemotherapy in combination with intravenous oncolytic reovirus in patients with advanced malignancies

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