Research: In recent years, intensive effort has focused on the investigation of biological approaches for the treatment of cancer, including immunotherapy utilizing cytokines, antibodies and/or vaccines, gene therapy as well as inhibition of tumor angiogenesis among others. Although immunotherapeutic approaches such as systemic administration of interleukin-2 (IL-2) have provided meaningful benefit to some patients with metastatic renal cell carcinoma or melanoma, many questions remain regarding the best approach to maximize the potential efficacy of biological therapy. Given the complexity of signals engaged during the host antitumor immune response, and the intricate network of interactions within the tumor microenvironment, it appears that much has occurred with the clinical evolution of combination chemotherapy, the full potential of biological therapies for cancer will most likely be realized using rationally-designed combinations of agents with complementary mechanisms of action. Laboratory studies: We investigate molecular mechanisms by which the host immune response may be engaged to induce alterations in the tumor microenvironment to effect disease regression (i.e. modulation of tumor neovascularization, induction of tumor and/or endothelial apoptosis, etc.), and also use these observations to facilitate the design of novel biologically-targeted treatment strategies for neuroblastoma and/or renal cell carcinoma. Much of our recent effort has focused on investigation of the antitumor activity of two cytokine-based combinations, IL-12/pulse IL-2 and IL-18/IL-2, and delineation of the respective mechanisms which mediate their therapeutic efficacy. In mice bearing well-established primary and/or metastatic neuroblastoma or renal cell carcinoma tumors, systemic administration of IL-12/pulse IL-2 can induce complete durable tumor regression in 80% or more of treated mice. Comparable responses are achieved after treatment with IL-18+/- IL-2. We have now defined several of the critical mechanisms by which these therapies can modulate the local tumor microenvironment to induce disease regression. Notably, IL-12/pulse IL-2 synergistically enhances IFN-gamma production, and induces the IFN-gamma-dependent expression of both Fas and Fas-L genes within the local tumor site. In turn, IL-12/pulse IL-2 induces rapid vascular endothelial injury with tumor and/or endothelial apoptosis, and inhibits tumor neovascularization and mediates overall tumor regression via mechanisms which share a common dependency on IFN-gamma and the Fas/ Fas-L apoptosis pathway. Collectively, these observations suggest a mechanism whereby CD8+ FAS-L+ T cells infiltrate the local tumor site and interact with Fas+ vascular endothelial and/or tumor cell populations to induce apoptosis, inhibition of angiogenesis, and ultimately, overall tumor regression. Recent studies also have now shown that the antitumor activity of IL-18/IL-2 is critically-dependent on CD8+ T cells, IFN-gamma , and the FAS/FAS-L pathway, but not IL-12. For future studies to specifically address the role of tumor versus host cell (i.e. endothelial) responsiveness to biological therapies such as IL-12/pulse IL-2 or IL-18/IL-2, N-myc transgenic mice are being bred with relevant knockout strains to establish N-myc transgenic-knockout murine hosts and/or novel derivative neuroblastoma cell lines with targeted disruption of the genes encoding the IFN-gamma receptor, and proapoptotic genes such as Fas among others. Using cDNA microarray, future studies will compare the patterns of gene expression induced induced in the local tumor site by IL-12 versus IL-18 and seek to identify novel molecular targets engaged by these cytokines in neuroblastoma-bearing mice. In other studies, we are investigating the angiogenic phenotype and basic mechanisms governing the neovascularization of neuroblastoma tumors. We have demonstrated marked constitutive expression of vascular endothelial growth factor (VEGF) and FLT-1/FLK-1, angiopoietin-1 and TIE-2, as well as the matrix metalloproteinases, MMP-2 and MMP-9 by both murine and human neuroblastoma tumors and cell lines. Further, although treatment of tumors with immune-based therapies such as IL-12/pulse IL-2 inhibits tumor neovascularization and induces local expression of antiangiogenic, IFN-gamma inducible CXC chemokines including IP-10 and MIG, there is no apparent impact on the expression of various proangiogenic mediators and/or their receptors. These observations suggest that targeted antagonists of proangiogenic mediators such as VEGF could play a role in the treatment of neuroblastoma , and that the efficacy of antiangiogenic immunoregulatory cytokines such as IL-12 might be expanded by combined administration with targeted antagonists of proangiogenic mediators such as VEGF for the treatment of neuroblastoma-a proposed immunoangiostatic approach. Clinical Investigation: Based on the potent efficacy of IL-12/pulse IL-2 in preclinical tumor models, we subsequently designed and executed a primate toxicology evaluation of the safety of this combination in cynomolgus macaques, and have now initiated a phase I investigation of IL-12/pulse IL-2 in adults with advanced solid tumors. Guided by our preclinical observations, hypothesis-driven translational studies have been incorporated into this clinical trial to investigate antitumor mechanisms engaged by the administration of IL-12/pulse IL-2 in humans. These include characterization of the functional immunoregulatory effects of IL-12/pulse IL-2 in vivo, prospective quantitative assessment of the antivascular activity of IL-12/pulse IL-2 utilizing dynamic-enhanced MRI (DEMRI) scans, as well as molecular and histopathologic investigation of antitumor mechanisms induced by IL-12/pulse IL-2 in the local tumor microenvironment. Additional studies are now planned using IL-12/pulse IL-2 as well as IL-18-based approaches in children with neuroblastoma and adults with solid tumors such as renal cell carcinoma.
