Another approach to improving oncolysis by replicative viruses involves the insertion of therapeutic transgenes into the viral genome. Such "armed" replication-competent viruses allow the action of therapeutic proteins to be combined with the anti-tumor properties of the viral infection. This approach embodies several possible advantages. First, as a result of the amplification of the virus within the tumor, transgene production and spread are also highly increased as compared with infection with the replication-defective vector counterparts. This concept was demonstrated for adenovirus using the marker gene Luciferase (57,58) as well as for HSV using the P-galactosidase genne (59-61). Marked increases in transgene expression were noted using the replication-competent compared with the replication-defective vectors. In addition, transgenes can be selected that have a nonoverlapping toxicity range with the viral-induced oncolytic effects in order to maximize their therapeutic benefit. Transgenes have been inserted that encode prodrug-converting enzymes, immunes stimulatory molecules, and apoptosis-enhancing proteins.
Several groups have constructed genetically engineered oncolytic viruses encoding a prodrug-converting enzyme. These enzymes convert nontoxic prodrugs into cytotoxic metabolites and are often soluble to allow for spreading within the tumor. Using this approach, a tumor-selective herpes virus was engineered encoding the rat cytochrome P450 (CYP2B1) transgene (62). This liver enzyme activates the prodrug cyclophosphamide into an active anti-cancer and immunosuppressive metabolite (63). Addition of cyclophosphamide potentiated oncolytic effects of this HSV mutant against cultured tumor cells and subcutaneous tumor xenografts established in athymic mice (62).
Recently, an ONYX-015-based adenovirus was described encoding the prodrug converting enzyme carboxylesterase (CE), which converts the camptothecin derivative CPT-11 to the much more potent chemotherapeutic SN-38. Survival of tumor-bearing mice was strongly enhanced with the CE-expressing virus in combination with CPT-11 compared with controls (64).
Multiple prodrug-activating gene therapies have also been used simultaneously in combination with oncolytic viruses. A replicating adenovirus with double enzyme/prodrug gene therapy containing the cytosine deaminase and HSV-Tk fusion gene markedly enhanced oncolysis relative to the isolated viral effect in cancer cells (65). The combination of HSV-TK and CYP21 gene transfer mediated by an oncolytic HSV provided anti-tumor effects that were more significant than all other treatment combinations (66).
The second type oftransgenes that have been inserted into the oncolytic viruses, were selected to boost the immune response to the infected tumor cells by stimulating localized inflammatory and/or immune responses. In the context of Adses, this was first demonstrated using a tumor-selective virus engineered to express interferon (IFN) which strongly enhanced anti-tumor activity compared to relevant control adenovirus in immune-deficient mice bearing breast carcinoma xenografts (9). Moreover, a number of replication-competent recombinant HSVs that encode immunostimulatory molecules have been constructed. Andreansky et al. (67) demonstrated that survival of immunocompetent mice bearing intracerebral tumors could be prolonged when treated with tumor-selective HSV encoding interleukin 4 (IL-4) compared to controls and immunohistochemical analysis demonstrated marked accumulation of inflammatory cells. Similarly, oncolytic HSV mutants expressing IL-12, IL-2, or the soluble B7-1 immunomodulatory molecule were found to produce survival benefit compared with control viruses in various tumor models including glioma in immunocompetent mice, by combining oncolytic and immunostimulatory effects (68- 71). Moreover, insertion of the potent immune stimulator granulocyte macrophage-colony stimulating factor (GM-CSF) into an oncolytic HSV backbone, improved shrinkage or clearance of tumors compared to control virus. These mice were also protected against re-challenge with tumour cells (72). This suggests not only that expression of immunomodulatory molecules can potentiate oncolysis but may also induce a level of anti-tumor immunity.
Finally, insertion of transgenes may improve the oncolytic potential of the replication-competent virus itself. Opportunities for enhancing the anti-tumor potential of oncolytic viruses are at the final stage of the reproductive cycle that involves the lysis of the host cell and release of viral progeny (73). Oncolysis of cancer cells when compared with lysis of the virus' natural host cells may be suboptimal as a result of cancer cell specific genetic alterations. These alterations mainly affect pro- and anti-apoptotic pathways that regulate the cell cycle. Coordinated and timely (over) expression ofkey players in these processes concomitant with the viral replicative cycle is expected to enhance the anti-tumor potential ofthe oncolytic virus. This concept was demonstrated using the AdA24 oncolytic adenovirus engineered to express p53 during late stages of viral replication and which exhibited up to >100-fold enhanced oncolytic potency on human cancer cell lines of various tissue origins (74). In another study, expression of a dominant-negative I-k B from a selectively replicating adenovirus sensitized tumor cells to recombinant human tumor necrosis factor a (TNF-a)-medi-ated apoptosis. Using this approach it could be demonstrated that induction of apoptosis during viral DNA replication compromised virus production, whereas apoptosis induced after virion assembly enhanced viral release from infected cells and dissemination (75).
Combination treatment with conventional therapies may offer a number of advantages. First, enhanced therapeutic efficacy of dual treatments will allow administration of lower viral doses to achieve a certain therapeutic effect, which is important given the fact that sufficient virus delivery to tumors remains one of the major hurdles in clinical viral (gene) therapy strategies. Furthermore, combined treatment allowing lower viral doses may also lower toxic side-effects.
The first studies to describe the effects of dual treatment with an oncolytic virus and conventional therapy were performed with the ONYX-015 Ad. The efficacy of this agent combined with cisplatin and 5-fluorouracil was significantly greater than either agent alone in nude mouse tumor xenografts (24). These results led to the design of a phase II trial using these agents in patients with squamous cell carcinoma of the head and neck (29). The results of this study mirrored the preclinical data, including the frequent occurrence of complete remissions in patients treated with combination therapy. Synergy with the chemotherapeutic agents paclitaxel and docetaxel was demonstrated with the prostate cancer-specific oncolytic Ad, CV706, in a xenograft model of prostate cancer (76).
Synergy with chemotherapeutic agents has also been described for HSV. The oncolytic effect of HSV-1716 in combination with mitomycin was synergistic in two of five nonsmall cell lung cancer cell lines in vitro and inhibited tumor growth more efficiently than either agent alone (77). Combination treatment of the HSV mutant G207 and vincristine led to strongly enhanced in vitro cytotoxicity without affecting infection efficiency and replication of G207 in rhabdomyosarcoma cells. In vivo combination treatment of alveolar rhabdomyosar-coma using intravenous G207 and vincristine resulted in complete tumor regression without evidence of regrowth in five of eight animals whereas none of the animals receiving either monotherapy were cured (78).
In the context of malignant brain tumors, the combination of oncolytic viruses with radiotherapy is perhaps more relevant than with chemotherapy considering the efficacy of standard treatments. Rogulski et al, (79) have studied the anti-tumor activity of ONYX-015 in combination with irradiation in colon carcinoma xenografts . ONYX-015 viral therapy combined with irradiation improved tumor control beyond that of either monotherapy. Studies with the prostate stimulating antigen (PSA) promoter-driven oncolytic Ad, CV706, in combination with radiotherapy demonstrated such an improvement in therapeutic response in prostate cancer xenografts without increasing toxicity, that a phase I study was initiated (80,81). In glioma, synergistic oncolytic activity of ONYX-015 with radiotherapy was demonstrated in subcutaneous xenografts (82). Also, the strong anti-tumor activity of Ad5-A24RGD, the integrin-targeted AdA24 variant, in malignant glioma could be further enhanced with low-dose irradiation such that the same therapeutic effect was achieved with a 10-fold lower viral dose (32).
Combination therapy with oncolytic HSV and irradiation has produced varying results. Whereas a potentiating effect of irradiation on G207 viral oncolysis in cervical and colorectal cancer xenografts was found (83,84), no enhancement of anti-tumor activity was seen when these treatment modalities were combined in subcutaneous tumor models of human and murine prostate cancer (85). Spear et al. (86) also found complementary toxicity between irradiation and the oncolytic HSV-1 mutant for the ICP6, regardless of cell type, time, MOI, irradiation dose, or culture conditions, without evidence of augmented apoptosis or viral replication. In human glioma xenografts on the other hand, dual treatment with the HSV-1 y34.5-mutant caused a significantly greater reduction in volume or total regression of tumors than either irradiation or infection alone. This enhanced oncolytic effect of the combined treatment correlated with two- to fivefold enhanced viral replication in irradiated tumor cells compared to tumors receiving virus only (87). These results were extended to a second study in mice bearing intracerebral tumors which received y34.5-mutant virus in combination with fractionated radiotherapy. Analysis of survival data revealed that the interaction between these treatment modalities was synergistic (88).
In conclusion, the results from studies into combination therapy, demonstrating enhanced therapeutic efficacy of oncolytic viral therapy over single modality treatment, are very encouraging. Currently, investigators are successfully combining the above-described strategies with armed therapeutic viruses. A trimodal approach (i.e., lytic virus, double enzym/prodrug gene therapy, and irradiation) was found to be superior to any other combination in carcinoma xenografts. Significant tumor regression and ultimately 100% tumor cure were reported (89).
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