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Cancer immunotherapy denotes a strategy for activating a host's immune response for the treatment of malignancy. Although cancer cells are less immunogenic, immune system is capable of recognizing and eliminating cancer cells. Oncolytic adenoviral vectors are currently being developed as biologic anti-tumor agents, and coupling the lytic function of an oncolytic adenovirus with its ability as a transgene delivery system represents a powerful extension of this methodology. IL-12 is reported to exhibit potent anti-tumor effect by promoting NK cell and cytotoxic T cell activities. IL-18 is also have shown that augments cytotoxicity of NK cells and proliferation of T cells. It stimulates Th1 cells to produce IL-2 and IFN-|[gamma]|. This effect augmented in a synergistic manner IL-12. To increase the potential anti-tumor effect of the oncolytic adenovirus, we have generated an E1B deleted and E1A mutated oncolytic adenoviruses, Ad-|[Delta]|B7-IL12, Ad-|[Delta]|B7-IL18, or Ad-|[Delta]|B7/IL12-IL18 that expresses IL-12, IL-18, or IL-12 plus IL-18, respectively. The therapeutic efficacy of these oncolytic adenoviruses was then evaluated in immunocompetent C57BL/6 mice bearing murine melanoma B16- F10 tumor. The results showed significant inhibition of tumor growth following Ad-|[Delta]|B7/IL12-IL18 treatment compared to Ad-|[Delta]|B7, Ad- |[Delta]|B7-IL18, or Ad-|[Delta]|B7-IL12 treated tumors. Moreover, the oncolytic adenovirus expressing both IL-12 and IL-18 demonstrated enhanced anti-tumor effect and higher incidences of complete tumor regression compared to Ad-|[Delta]|B7, Ad-|[Delta]|B7-IL18, or Ad-|[Delta]|B7-IL12. To establish that the observed anti-tumor effect is associated with the generation of a tumor-specific immune response, we examined the cytolytic activity by IFN-|[gamma]| ELISpot assay and CTL assay. We observed that Ad-|[Delta]|B7/IL12-IL18 induced significantly higher T cell-mediated anti- tumor effect than its cognate controls, Ad-|[Delta]|B7, Ad-|[Delta]|B7-IL12, and Ad-|[Delta]|B7-IL18. Furthermore, immunohistochemical studies demonstrated robust CD4+ and CD8+ T-cell infiltration in these mice compared to the Ad-|[Delta]|B7, Ad-|[Delta]|B7-IL18, or Ad-|[Delta]|B7-IL12- treated subjects. These data indicate that oncolytic adenovirus-mediated IL-12 and IL-18 gene transfer provides a potential therapeutic strategy for the management of neoplasia.

To combine the advantages of the prodrug/suicide gene approach with that of replicating adenoviruses, we have developed replicating adenoviral vectors harboring HSV-TK, generating Ad-|[Delta]|E1B19-TK, Ad-|[Delta]|E1B55-TK, Ad-|[Delta]|E1B19/55-TK adenoviruses. Infection with adenoviruses expressing HSV-TK increased the sensitivity of cells to GCV. Cell killing assay was used to compare the combined therapy with the two single therapy modalities and non-treated controls in human cancer cell line. The cytotoxicity of the combination therapy with replicating adenovirus and GCV was dramatically increased compared with replicating adenoviruses alone or replication incompetent adenovirus expressing TK. In contrast, the cytotoxicity of the combination therapy with Ad-|[Delta]|E1B19-TK and GCV was reduced when compared to Ad-|[Delta]|E1B19-TK alone. This result suggests that the inhibition of viral replication and oncolysis by the HSV-TK/GCV system counterbalance the potent cytotoxicity of Ad-|[Delta]|E1B19-TK. Animals treated with Ad-|[Delta]|E1-TK with GCV had some reduction in tumor growth compared to animals injected with PBS only. All replicating adenoviral vectors were more efficacious than Ad-|[Delta]|E1-TK with GCV treatment.

Cytokine immunogene therapy is a promising strategy for cancer treatment. Interleukin (IL)-12 boosts potent antitumor immunity by inducing T helper 1 cell differentiation and stimulating cytotoxic T lymphocyte and natural killer cell cytotoxicity. IL-23 has been proposed to have similar but not overlapping functions with IL-12 in inducing Th1 cell differentiation and antitumor immunity. However, the therapeutic effects of intratumoral co-expression of IL-12 and IL-23 in a cancer model have yet to be investigated. Therefore, we investigated for the first time an effective cancer immunogene therapy of syngeneic tumors via intratumoral inoculation of oncolytic adenovirus co-expressing IL-23 and p35, RdB/IL23/p35. Intratumoral administration of RdB/IL23/p35 elicited strong antitumor effects and increased survival in a murine B16-F10 syngeneic tumor model. The levels of IL-12, IL-23, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) were elevated in RdB/IL23/p35-treated tumors. Moreover, the proportion of regulatory T cells was markedly decreased in mice treated with RdB/IL23/p35. Consistent with these data, mice injected with RdB/IL23/p35 showed massive infiltration of CD4(+) and CD8(+) T cells into the tumor as well as enhanced induction of tumor-specific immunity. Importantly, therapeutic mechanism of antitumor immunity mediated by RdB/IL23/p35 is associated with the generation and recruitment of IFN-gamma- and TNF-alpha-co-producing T cells in tumor microenvironment. These results provide a new insight into therapeutic mechanisms of IL-12 plus IL-23 and provide a potential clinical cancer immunotherapeutic agent for improved antitumor immunity.

Dendritic cell (DC)-based vaccination is a promising strategy for cancer immunotherapy. However, clinical trials have indicated that immunosuppressive microenvironments induced by tumors profoundly suppress antitumor immunity and inhibit vaccine efficacy, resulting in insufficient reduction of tumor burdens. To overcome these obstacles and enhance the efficiency of DC vaccination, we generated IL-12- and GM-CSF-coexpressing oncolytic adenovirus (Ad-αB7/IL12/GMCSF) as suitable therapeutic adjuvant to eliminate immune suppression and promote DC function. By treating tumors with Ad-αB7/IL12/GMCSF prior to DC vaccination, DCs elicited greater antitumor effects than in response to either treatment alone. DC migration to draining lymph nodes (DLNs) dramatically increased in mice treated with the combination therapy. This result was associated with upregulation of CCL21+ lymphatics in tumors treated with Ad-αB7/IL12/GMCSF. Moreover, the proportion of CD4+CD25+ T cells and VEGF expression was decreased in mice treated with the combination therapy. Furthermore, combination therapy using immature DCs also showed effective antitumor effects when combined with Ad-αB7/IL12/GMCSF. The combination therapy had a remarkable therapeutic efficacy on large tumors. Taken together, oncolytic adenovirus coexpressing IL-12 and GM-CSF in combination with DC vaccination has synergistic antitumor effects and can act as a potent adjuvant for promoting and optimizing DC vaccination. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4393. doi:1538-7445.AM2012-4393

Alfa-feto protein (AFP) protein is expressed in 70-80% of patients with hepatocellular carcinomas but not in normal adult tissues. In this study, we evaluated the liver cancer-specific oncolytic potential of E1B 19kDa-deleted replicating adenoviruses, Ad-hAFP|[Delta]|19 and Ad-rAFP|[Delta]|19, which express E1A gene controlled by human AFP promoter and rat AFP promoter, respectively. Transient transfection study using luciferase indicated the selective activation of the AFP promoter only in human liver cancer cells secreting AFP. Next, we evaluated in vitro efficacy and specificity by comparing viral replication and cytopathic effect in AFP-positive (Huh7, HepG2, and Hep3B), AFP-negative (HeLa, A549, U343, and SK-Hep1) cancer cells and normal (BJ, Chang, IMR90) cells. Our results show that the viral replication and cytopathic effects of Ad-hAFP|[Delta]|19 and Ad-rAFP|[Delta]|19 were substantially lower in AFP-negative cancer and normal cells compared to that of AFP-positive cancer cells. Ad-hAFP|[Delta]|19 and Ad-rAFP|[Delta]|19 also replicated in and cytolyzed the AFP-positive cancer cells in a dose-dependent manner. More importantly, Ad-hAFP|[Delta]|19 and Ad-rAFP|[Delta]|19 significantly inhibited the growth of established human hepatocellular carcinoma tumors. Taken together, these lines of evidence demonstrate that Ad-hAFP|[Delta]|19 and Ad-rAFP|[Delta]|19 hold a significant promise as an oncolytic agent in AFP-positive tumors.

Replication-competent adenoviruses (Ad) are a promising new modality for the treatment of cancer. Selective replication of viral agents in tumor may lead to improved efficacy over non-replicating adenoviruses due to their inherent ability to multiply, lyse and spread to surrounding cells. We have previously shown that the E1B 55kDa-deleted adenovirus (YKL-1) exhibits tumor specific replication and cell lysis, but its cytolytic effects were reduced in comparison to the wild type adenovirus. To increase the oncolytic potency of YKL-1, we have reintroduced the adenovirus death protein (ADP) gene under the control of either the CMV or the MLP promoter at the E3 region of YKL-1, generating YKL-cADP and YKL-mADP adenovirus, respectively. ADP is an 11.6kDa protein encoded by the E3 transcription unit, and is required to efficiently kill adenovirus-infected cells. However to date, the mechanism by which ADP mediates cell death has not been clearly defined. In this study, we report that ADP-overexpressing Ad markedly enhanced cytolytic effect (up to 100-fold) against all tumor cell lines tested, but did not increase CPE in normal skin fibroblast, BJ. Moreover, plaque size formed by YKL-cADP was substantially larger than that of YKL-1, indicating enhancement in cell lysis. TUNEL assay and Annexin-V/PI double staining indicate that ADP-mediated cytotoxicity was largely driven by apoptosis. Finally, YKL-cADP adenovirus also showed superior antitumor effect than YKL-1 and YKL-mADP in C33A cervical and Hep3B hepatoma xenograft tumor models. Taken together, these lines of evidence demonstrate that the newly generated adenovirus expressing ADP under the CMV promoter induces efficient but tumor selective cell lysis, which is critical for adding therapeutic value to replicating adenovirus for cancer gene therapy.