Synergistic cytotoxicity of oncolytic reovirus in combination with cisplatin-paclitaxel doublet chemotherapy.

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.

Inflammatory tumour cell killing by oncolytic reovirus for the treatment of melanoma.

Reovirus is a promising unmodified double-stranded RNA (dsRNA) anti-cancer oncolytic virus, which is thought to specifically target cells with activated Ras. Although reovirus has been tested in a wide range of preclinical models and has entered early clinical trials, it has not previously been tested for the treatment of human melanoma. Here, we show that reovirus effectively kills and replicates in both human melanoma cell lines and freshly resected tumour; intratumoural injection also causes regression of melanoma in a xenograft in vivo model. Reovirus-induced melanoma death is blocked by caspase inhibition and is dependent on constituents of the Ras/RalGEF/p38 pathway. Reovirus melanoma killing is more potent than, and distinct from, chemotherapy or radiotherapy-induced cell death; a range of inflammatory cytokines and chemokines are released by infected tumour cells, while IL-10 secretion is abrogated. Furthermore, the inflammatory response generated by reovirus-infected tumour cells causes bystander toxicity against reovirus-resistant tumour cells and activates human myeloid dendritic cells (DC) in vitro. Hence, reovirus is suitable for clinical testing in melanoma, and may provide a useful danger signal to reverse the immunologically suppressive environment characteristic of this tumour.

Characterization of the adaptive and innate immune response to intravenous oncolytic reovirus (Dearing type 3) during a phase I clinical trial.

There is an emerging realization from animal models that the immune response may have both detrimental and beneficial therapeutic effects during cancer virotherapy. However, there is a dearth of clinical data on the immune response to viral agents in patients. During a recently completed phase I trial of intravenous reovirus type 3 Dearing (RT3D), heavily pretreated patients with advanced cancers received RT3D at doses escalating from 1 x 10(8) tissue culture infectious dose-50 (TCID(50)) on day 1 to 3 x 10(10) TCID(50) on 5 consecutive days of a 4 weekly cycle. A detailed analysis of the immune effects was conducted by collecting serial clinical samples for analysis of neutralizing anti-reoviral antibodies (NARA), peripheral blood mononuclear cells (PBMC) and cytokines. Significant increases in NARA were seen with peak endpoint titres >1/10 000 in all but one patient. The median fold increase was 250, with a range of 9-6437. PBMC subset analysis showed marked heterogeneity. At baseline, CD3+CD4+ T cells were reduced in most patients, but after RT3D therapy their numbers increased in 47.6% of patients. In contrast, most patients had high baseline CD3+CD8+ T-cell levels, with 33% showing incremental increases after therapy. In some patients, there was increased cytotoxic T-cell activation post-therapy, as shown by increased CD8+perforin/granzyme+ T-cell numbers. Most patients had high numbers of circulating CD3-CD56+ NK cells before therapy and in 28.6% this increased with treatment. Regulatory (CD3+CD4+CD25+) T cells were largely unaffected by the therapy. Combined Th1 and Th2 cytokine expression increased in 38% of patients. These data confirm that even heavily pretreated patients are capable of mounting dynamic immune responses during treatment with RT3D, although these responses are not clearly related to the administered virus dose. These data will provide the basis for future studies aiming to modulate the immune response during virotherapy.

Escherichia coli nitroreductase plus CB1954 enhances the effect of radiotherapy in vitro and in vivo.

Escherichia coli nitroreductase (NTR) converts the prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) into a bifunctional alkylating agent that causes DNA crosslinks. In this study, the ability of NTR to enhance the combined effects of CB1954 and radiation has been tested in vitro and in vivo. Stably transduced ovarian cancer cells (SKOV3-NTR) that are sensitive to CB1954 (IC(50)=0.35 muM) demonstrated enhanced cytotoxicity when treated with CB1954 and single-fraction irradiation. The NTR-CB1954 system mediated a bystander effect in combination with radiation on transfer of conditioned medium from SKOV3-NTR, but not SKOV3, cells to SW480 target cells. The ability of CB1954 to enhance radiation-induced cytotoxicity in SKOV3-NTR (but not SKOV3) cells was also demonstrated by fluorescence-activated cell sorting (FACS) with dual staining for propidium iodide/fluorescein diacetate, 4',6-diamidino-2-phenylindole dichloride staining of apoptotic cells and measurement of double-stranded DNA breaks by FACS and confocal microscopy for gammaH2AX foci. Adenoviral delivery of NTR, under constitutive cytomegalovirus or tissue-specific CTP1 promoters, increased the in vitro cytotoxicity of CB1954 plus radiation in MTT and clonogenic assays. Finally, adenoviral delivery of NTR plus CB1954 enhanced the effect of fractionated radiotherapy (12 Gy in four fractions) in SW480 xenograft tumours in nude mice.