Il-Kyu Choi

Enhanced Antitumor Effect of Oncolytic Adenovirus Expressing Interleukin-12 and B7-1 in an Immunocompetent Murine Model

Purpose: We investigated whether an armed viral platform, where lytic property of a viral infection is coupled to viral-mediated delivery of therapeutic genes, could increase the therapeutic potential of a viral-based therapy. Experimental Design: We generated interleukin (IL)-12-expressing oncolytic adenovirus (YKL-IL-12) and IL-12- and B7-1-expressing (YKL-IL12/B7) oncolytic adenovirus. Therapeutic efficacy of these newly engineered adenoviruses was then evaluated in vivo using an immunocompetent mouse bearing murine melanoma B16-F10 tumors. Overall survival was assessed with the Kaplan-Meier method. The induction of immune cell cytotoxicity was assessed by CTL assay, IFN-γ enzyme-linked immunospot assay, and immunohistochemical studies. Results: YKL-IL12/B7 oncolytic adenovirus, expressing both IL-12 and B7-1, showed a higher incidence of complete tumor regression compared with the analogous oncolytic adenovirus, YKL-1, or IL-12-expressing, YKL-IL12. Significant survival advantage was also seen in response to YKL-IL12/B7. Moreover, IL-12 and IFN-γ levels produced in tumors treated with YKL-IL12/B7 was significantly greater than those treated with YKL-IL12. The enhanced survival advantage was mediated by the induction of immune cell cytotoxicity. In agreement with these results, massive infiltration of CD4+ and CD8+ T cells into tissues surrounding the necrotic area of the tumor was observed following in situ delivery of YKL-IL12/B7. Conclusion: Combination of oncolysis and the enhancement of antitumor immune response by oncolytic adenovirus expressing both IL-12 and B7-1 elicits potent antitumor effect and survival advantage.

Therapeutic and Tumor-specific Immunity Induced by Combination of Dendritic Cells and Oncolytic Adenovirus Expressing IL-12 and 4-1BBL

Recently, gene-based cytokine treatment has been actively pursued as a new promising approach in treating cancer. In an effort to augment the efficiency of antitumor effect by cytokine-mediated immunotherapy, we selected both interleukin (IL)-12 and 4-1BB ligand (4-1BBL) as suitable cytokines to fully activate the type-1 immune response. Coexpression of IL-12 and 4-1BBL mediated by oncolytic adenovirus (Ad) greatly enhanced the antitumor effect. Further, synergistic enhancement in interferon (IFN)-γ levels were seen in mice treated with oncolytic Ad expressing both IL-12 and 4-1BBL. Next, to improve the overall antitumor immune response, we coadministered IL-12- and 4-1BBL-coexpressing oncolytic Ad with dendritic cells (DCs). Combination treatment of IL-12- and 4-1BBL-coexpressing oncolytic Ad and DCs elicited greater antitumor and antimetastatic effects than either treatment alone. Moreover, enhanced type-1 antitumor immune response and higher migratory abilities of DCs in tumors were also observed in the combination arms. The nature of the enhanced antitumor immune response seems to be mediated through the enhanced cytolytic activity of cytotoxic T lymphocytes (CTLs) and IFN-γ-releasing immune cells. Taken together, these data highlight the potential therapeutic benefit of combining IL-12- and 4-1BBL-coexpressing oncolytic Ad with DCs and warrants further evaluation in the clinic.Recently, gene-based cytokine treatment has been actively pursued as a new promising approach in treating cancer. In an effort to augment the efficiency of antitumor effect by cytokine-mediated immunotherapy, we selected both interleukin (IL)-12 and 4-1BB ligand (4-1BBL) as suitable cytokines to fully activate the type-1 immune response. Coexpression of IL-12 and 4-1BBL mediated by oncolytic adenovirus (Ad) greatly enhanced the antitumor effect. Further, synergistic enhancement in interferon (IFN)-γ levels were seen in mice treated with oncolytic Ad expressing both IL-12 and 4-1BBL. Next, to improve the overall antitumor immune response, we coadministered IL-12- and 4-1BBL-coexpressing oncolytic Ad with dendritic cells (DCs). Combination treatment of IL-12- and 4-1BBL-coexpressing oncolytic Ad and DCs elicited greater antitumor and antimetastatic effects than either treatment alone. Moreover, enhanced type-1 antitumor immune response and higher migratory abilities of DCs in tumors were also observed in the combination arms. The nature of the enhanced antitumor immune response seems to be mediated through the enhanced cytolytic activity of cytotoxic T lymphocytes (CTLs) and IFN-γ-releasing immune cells. Taken together, these data highlight the potential therapeutic benefit of combining IL-12- and 4-1BBL-coexpressing oncolytic Ad with DCs and warrants further evaluation in the clinic.

Strategies to Increase Drug Penetration in Solid Tumors

Despite significant improvement in modalities for treatment of cancer that led to a longer survival period, the death rate of patients with solid tumors has not changed during the last decades. Emerging studies have identified several physical barriers that limit the therapeutic efficacy of cancer therapeutic agents such as monoclonal antibodies, chemotherapeutic agents, anti-tumor immune cells, and gene therapeutics. Most solid tumors are of epithelial origin and, although malignant cells are de-differentiated, they maintain intercellular junctions, a key feature of epithelial cells, both in the primary tumor as well as in metastatic lesions. Furthermore, nests of malignant epithelial tumor cells are shielded by layers of extracellular matrix (ECM) proteins (e.g., collagen, elastin, fibronectin, laminin) whereby tumor vasculature rarely penetrates into the tumor nests. In this chapter, we will review potential strategies to modulate the ECM and epithelial junctions to enhance the intratumoral diffusion and/or to remove physical masking of target receptors on malignant cells. We will focus on peptides that bind to the junction protein desmoglein 2 and trigger intracellular signaling, resulting in the transient opening of intercellular junctions. Intravenous injection of these junction openers increased the efficacy and safety of therapies with monoclonal antibodies, chemotherapeutics, and T cells in mouse tumor models and was safe in non-human primates. Furthermore, we will summarize approaches to transiently degrade ECM proteins or downregulate their expression. Among these approaches is the intratumoral expression of relaxin or decorin after adenovirus- or stem cell-mediated gene transfer. We will provide examples that relaxin-based approaches increase the anti-tumor efficacy of oncolytic viruses, monoclonal antibodies, and T cells.

Optimizing DC Vaccination by Combination With Oncolytic Adenovirus Coexpressing IL-12 and GM-CSF

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 interleukin (IL)-12- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-coexpressing oncolytic adenovirus (Ad-ΔB7/IL12/GMCSF) as suitable therapeutic adjuvant to eliminate immune suppression and promote DC function. By treating tumors with Ad-ΔB7/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 CC-chemokine ligand 21 (CCL21+) lymphatics in tumors treated with Ad-ΔB7/IL12/GMCSF. Moreover, the proportion of CD4+CD25+ T-cells and vascular endothelial growth factor (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-ΔB7/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.

Effect of decorin on overcoming the extracellular matrix barrier for oncolytic virotherapy

The pressing challenge for contemporary gene therapy is to deliver enough therapeutic genes to enough cancer cells in vivo. With the aim of improving viral distribution and tumor penetration, we explored the use of decorin to enhance viral spreading and tumor tissue penetration. We generated decorin-expressing replication-incompetent (dl-LacZ-DCNG, dl-LacZ-DCNQ and dl-LacZ-DCNK) and replication-competent (Ad-ΔE1B-DCNG, Ad-ΔE1B-DCNQ and Ad-ΔE1B-DCNK) adenoviruses (Ads). Point mutants of decorin gene (DCNG), DCNK and DCNQ, have a negative and moderate binding affinity to type-I collagen fibril, respectively. In both tumor spheroids and established solid tumors in vivo, tissue penetration potency of dl-LacZ-DCNG was greatly enhanced than those of dl-LacZ, dl-LacZ-DCNQ and dl-LacZ-DCNK, and this enhanced tissue penetration effect derived from decorin-expressing Ad was dependent on the binding affinity of decorin to collagen fibril. Expression of DCNG enhanced viral spread of replicating Ad, leading to improved tumor reduction and survival benefit. Moreover, the tumoricidal effects of Ad-ΔE1B-DCNQ and Ad-ΔE1B-DCNK were lessened, as the binding affinity to collagen was decreased, showing that the increased cancer cell cytotoxicity was driven by the action of decorin on extracellular matrix (ECM). Furthermore, Ad-ΔE1B-DCNG substantially decreased ECM components within the tumor tissue. Finally, intratumoral injection of Ad-ΔE1B-DCNG in primary tumor site greatly reduced the formation of B16BL6 melanoma cell pulmonary metastases in mice. Taken together, these data show the utility of decorin as a dispersion agent and highlight its utility and potential in improving the efficacy of replicating Ad-mediated cancer gene therapy.

Oncolytic adenovirus co-expressing IL-12 and IL-18 improves tumor-specific immunity via differentiation of T cells expressing IL-12Rβ 2 or IL-18Rα

The oncolytic adenovirus (Ad) is currently being advanced as a promising antitumor remedy as it selectively replicates in tumor cells and can transfer and amplify therapeutic genes. Interleukin (IL)-12 induces a potent antitumor effect by promoting natural killer (NK) cell and cytotoxic T cell activities. IL-18 also augments cytotoxicity of NK cells and proliferation of T cells. This effect further enhances the function of IL-12 in a synergistic manner. Therefore, we investigated for the first time an effective cancer immunogene therapy of syngeneic tumors via intratumoral administration of oncolytic Ad co-expressing IL-12 and IL-18, RdB/IL-12/IL-18. Intratumoral administration of RdB/IL-12/IL-18 improved antitumor effects, as well as increased survival, in B16-F10 murine melanoma model. The ratio of T-helper type 1/2 cytokine as well as the levels of IL-12, IL-18, interferon-γ and granulocyte–macrophage colony-stimulating factor was markedly elevated in RdB/IL-12/IL-18-treated tumors. Mice injected with RdB/IL-12/IL-18 also showed enhanced cytotoxicity of tumor-specific immune cells. Consistent with these results, immense necrosis and infiltration of NK cells, as well as CD4+ and CD8+ T cells, were observed in RdB/IL-12/IL-18-treated tumor tissues. Importantly, tumors treated with RdB/IL-12/IL-18 showed an elevated number of T cells expressing IL-12Rβ2 or IL-18Rα. These results provide a new insight into therapeutic mechanisms of IL-12 plus IL-18 and provide a potential clinical cancer immunotherapeutic agent for improved antitumor immunity.

Strengthening of antitumor immune memory and prevention of thymic atrophy mediated by adenovirus expressing IL-12 and GM-CSF

Interleukin (IL)-12 and granulocyte-monocyte colony-stimulating factor (GM-CSF) have recently been used as immunotherapeutic agents in cancer gene therapy. IL-12 and GM-CSF have differential roles in the antitumor immune response, as IL-12 targets T, NK and natural killer T (NKT) cells and GM-CSF principally targets antigen-presenting cells (APCs). To strengthen the therapeutic efficacy of these two cytokines, we generated an oncolytic adenovirus (Ad), Ad-ΔB7/IL12/GMCSF, coexpressing IL-12 and GM-CSF. Using a murine B16-F10 syngeneic tumor model, we show that Ad-ΔB7/IL12/GMCSF promoted antitumor responses and increased survival compared with an oncolytic Ad expressing IL-12 or GM-CSF alone (Ad-ΔB7/IL12 or Ad-ΔB7/GMCSF, respectively). By measuring cytotoxic T lymphocyte activity and interferon-γ production, we show that the enhanced therapeutic effect was mediated by the induction of immune cell cytotoxicity. In situ delivery of Ad-ΔB7/IL12/GMCSF resulted in massive infiltration of CD4+ T cells, CD8+ T cells, NK cells and CD86+ APCs into the tissue surrounding the necrotic area of the tumor. Moreover, GM-CSF effectively promoted antitumor immune memory, which was significantly augmented by IL-12. Lastly, IL12-expressing oncolytic Ads prevented tumor-induced thymic atrophy and was associated with reduced apoptosis and increased proliferation in the thymus. Taken together, these data demonstrate that an oncolytic Ad coexpressing IL-12 and GM-CSF is a potential therapeutic tool for the treatment of cancer.

MicroRNA-296 is enriched in cancer cells and downregulates p21WAF1 mRNA expression via interaction with its 3′ untranslated region

MicroRNAs (miRNAs) are a class of noncoding small RNAs that act as negative regulators of gene expression. To identify miRNAs that may regulate human cell immortalization and carcinogenesis, we performed comparative miRNA array profiling of human normal and SV40-T antigen immortalized cells. We found that miR-296 was upregulated in immortalized cells that also had activation of telomerase. By an independent experiment on genomic analysis of cancer cells we found that chromosome region (20q13.32), where miR-296 is located, was amplified in 28/36 cell lines, and most of these showed enriched miR-296 expression. Overexpression of miR-296 in human cancer cells, with and without telomerase activity, had no effect on their telomerase function. Instead, it suppressed p53 function that is frequently downregulated during human cell immortalization and carcinogenesis. By monitoring the activity of a luciferase reporter connected to p53 and p21WAF1 (p21) untranslated regions (UTRs), we demonstrate that miR-296 interacts with the p21-3′UTR, and the Hu binding site of p21-3′UTR was identified as a potential miR-296 target site. We demonstrate for the first time that miR-296 is frequently upregulated during immortalization of human cells and contributes to carcinogenesis by downregulation of p53-p21WAF1 pathway.

Recent developments in oncolytic adenovirus-based immunotherapeutic agents for use against metastatic cancers

Recurrent or metastatic cancer in most cases remains an incurable disease, and thus alternative treatment strategies, such as oncolytic virotherapy, are of great interest for clinical application. Oncolytic adenoviruses (Ads) have many advantages as virotherapeutic agents and have been safely employed in the clinics. However, the efficacy of oncolytic Ads is insufficient to eradicate tumors and current clinical applications are restricted to local administration against primary tumors because of immunological obstacles and poor tumor-cell targeting. Thus, alternative viable approaches are needed to establish therapies based on oncolytic Ad that will eliminate both primary and metastatic cancers. To this end, rational design of oncolytic Ads that express immunostimulatory genes has been employed. Even when restricted to local viral delivery, these oncolytic Ad-based immunotherapeutics have been shown to exert systemic antitumor immunity and result in eradication of both primary and metastatic cancers. Moreover, oncolytic Ad-based immunotherapeutics in combination with either dendritic cell-based vaccine or radiotherapy further strengthen the systemic tumor-specific immunity, resulting in complete suppression of both local and distant tumor metastatic growth. This review will focus on the most recent updates in strategies to develop potent oncolytic Ad-based immunotherapeutics for use in cancer gene therapy.

Proproliferative Functions of Drosophila Small Mitochondrial Heat Shock Protein 22 in Human Cells

Aging is a complex process accompanied by a decreased capacity of cells to cope with random damages induced by reactive oxygen species, the natural by-products of energy metabolism, leading to protein aggregation in various components of the cell. Chaperones are important players in the aging process as they prevent protein misfolding and aggregation. Small chaperones, such as small heat shock proteins, are involved in the refolding and/or disposal of protein aggregates, a feature of many age-associated diseases. In Drosophila melanogaster, mitochondrial Hsp22 (DmHsp22), is localized in the mitochondrial matrix and is preferentially up-regulated during aging. Its overexpression results in an extension of life span (>30%) (Morrow, G., Samson, M., Michaud, S., and Tanguay, R. M. (2004) FASEB J. 18, 598–599 and Morrow, G., Battistini, S., Zhang, P., and Tanguay, R. M. (2004) J. Biol. Chem. 279, 43382–43385). Long lived flies expressing Hsp22 also have an increased resistance to oxidative stress and maintain locomotor activity longer. In the present study, the cross-species effects of Hsp22 expression were tested. DmHsp22 was found to be functionally active in human cells. It extended the life span of normal fibroblasts, slowing the aging process as evidenced by a lower level of the senescence associated β-galactosidase. DmHsp22 expression in human cancer cells increased their malignant properties including anchorage-independent growth, tumor formation in nude mice, and resistance to a variety of anticancer drugs. We report that the DmHsp22 interacts and inactivates wild type tumor suppressor protein p53, which may be one possible way of its functioning in human cells.