NADPH oxidase-mediated reactive oxygen species production activates hypoxia-inducible factor-1 (HIF-1) via the ERK pathway after hyperthermia treatment.

Hyperthermia (HT) is a strong adjuvant treatment with radiotherapy and chemotherapy because it causes tumor reoxygenation. However, the detailed molecular mechanisms of how HT enhances tumor oxygenation have not been elucidated. Here we report that 1 h of HT activates hypoxia-inducible factor-1 (HIF-1) in tumors and its downstream targets, vascular endothelial growth factor (VEGF) and pyruvate dehydrogenase kinase 1 (PDK1). Consistent with HIF-1 activation and up-regulation of its downstream genes, HT also enhances tumor perfusion/vascularization and decreases oxygen consumption. As a result, tumor hypoxia is reduced after HT, suggesting that these physiological changes contribute to HT-induced tumor reoxygenation. Because HIF-1 is a potent regulator of tumor vascularization and metabolism, our findings suggest that HIF-1 plays a role in HT-induced tumor reoxygenation by transactivating its downstream targets. We demonstrate that NADPH oxidase-mediated reactive oxygen species production, as a mechanism, up-regulates HIF-1 after HT. Furthermore, we determine that this pathway is initiated by increased transcription of NADPH oxidase-1 through the ERK pathway. In conclusion, this study determines that, although HIF-1 is a good therapeutic target, the timing of its inhibition needs to be optimized to achieve the most beneficial outcome when it is combined with other treatments of HT, radiation, and chemotherapy.

Fc-mOX40L fusion protein produces complete remission and enhanced survival in 2 murine tumor models.

OX40L is a member of the tumor necrosis factor superfamily that provides a costimulatory signal to CD4+ and CD8+ T cells while inhibiting the effects of suppressive CD4+ CD25+ regulatory T cells. Because of this dual activity, OX40L may provide significant antitumor immunity in tumor-bearing mice. To study its clinical potential, a fusion protein consisting of mOX40L linked to the C-terminus of the Fc fragment of immunoglobulin was genetically engineered. After demonstrating its potency in vitro, several assays were performed to evaluate its antitumor effect in comparison to the OX40 agonist antibody OX86. Dosing studies in Colon 26-bearing and renal cell carcinoma (RENCA)-bearing mice showed that although OX86 produced modest tumor regression, Fc-mOX40L produced complete remission in both tumor models. Survival studies confirmed these results and showed that Fc-mOX40L treatment produced lasting responses throughout the 5-month observation period. Flow cytometric analysis of treated and untreated tumors and tumor-draining lymph nodes identified a qualitative difference in the activity of Fc-mOX40L compared with OX86 treatment as evidenced by differences in lymphoid and macrophage populations. These studies reflect the profound therapeutic potential of Fc-mOX40L, which substantially exceeds the agonist antibody OX86 in ability to produce complete tumor remissions and promote long-term survival in solid tumor models.

Construction and preclinical characterization of Fc-mGITRL for the immunotherapy of cancer.

PURPOSE: To provide proper costimulation required for effective cancer T-cell immunity, Fc-GITRL fusion proteins were generated for use in immunotherapy protocols. EXPERIMENTAL DESIGN: Soluble fusion proteins consisting of the Fc fragment of immunoglobulin and the murine glucocorticoid-induced tumor necrosis factor-related receptor ligand (mGITRL) connected with different linkers were genetically engineered and tested for their potency in two BALB/c solid tumor models. RESULTS: In vivo, construct #178-14 (-5aa, -linker) showed the best activity (>90% tumor reduction) at doses ranging from 5 to 25 microg and was found to be intact by gel electrophoresis. Similar doses used with construct #175-2 (-linker) produced good but not as high tumor regression. Construct #5-1 (+linker), which was found to be relatively unstable by SDS gel electrophoresis, produced <60% tumor regression and required a higher dose (100 microg) to produce optimal results. Survival curves showed that Fc-mGITRL treatment extended the life of 80% of tumor-bearing mice to >3 months compared with controls that died by day 40. T-cell depletion studies showed that CD8(+) T cells play a major role in Fc-mGITRL immunotherapy, and tumors removed from Fc-mGITRL- and DTA-1-treated mice showed a significant influx of granzyme B(+) lymphocytes compared with controls. Finally, T regulatory (Treg) cell assays showed that, unlike other Fc fusion proteins, all three Fc-mGITRL constructs profoundly suppressed Treg activity. CONCLUSIONS: These studies suggest that a stable, intact Fc-mGITRL fusion protein can provide missing costimulation for the immunotherapy of solid tumors. In addition, Fc-mGITRL may alter Treg activity to enhance its effectiveness for tumor immunotherapy.

RA8, a human anti-CD25 antibody against human Treg cells.

Although anti-CD25 antibodies exist for clinical use in patients, there is a need for the development of a human Treg antibody that will abrogate the immunosuppressive function of this small but critical T cell subtype. Based upon mounting evidence that the level of Treg cells in the tumor microenvironment correlates with clinical prognosis and stage in man, it appears that Treg cells play an important role in the tumor's ability to overcome host immune responses. In mice, the rat anti-mouse CD25 antibody PC61 causes depletion of CD25-bearing Treg cells both peripherally in lymphatic tissues and in the tumor microenvironment, without inducing symptoms of autoimmunity. A similar antibody, though with the ability to delete Treg cells specifically, would be an important new tool for reversing tumor escape associated with Treg immunosuppression in man. To begin to generate such a reagent, we now describe the development of a human anti-CD25 antibody using a novel yeast display library. The target antigen CD25-Fc was constructed and used for five rounds of selection using a non-immune yeast display library that contained as many as 10(9) single chain variable fragments (scFv). Two unique clones with low K(D) values (RA4 and RA8) were then selected to construct fully human anti-CD25 antibodies (IgG1/kappa) for stable expression. One antibody, RA8, showed excellent binding to human CD25(+) cell lines and to human Treg cells and appears to be an excellent candidate for the generation of a human reagent that may be used in man for the immunotherapy of cancer.

Targeted and untargeted CD137L fusion proteins for the immunotherapy of experimental solid tumors.

INTRODUCTION: CD137L is a member of the tumor necrosis factor superfamily that provides a costimulatory signal to T cells. In this study, two novel CD137L fusion proteins were produced and compared with the CD137 agonist antibody 2A. MATERIALS AND METHODS: Murine CD137L was linked to the COOH terminus of either the Fc fragment of immunoglobulin (untargeted version) or TNT-3 (targeted version), an antibody that binds to necrotic regions of tumors. Groups of mice bearing established Colon 26 tumors were then treated daily x 5 with each fusion protein or 2A to determine their immunotherapeutic potential. RESULTS: Both fusion proteins retained CD137L activity in vitro and TNT-3/CD137L showed tumor-binding activity by biodistribution analysis in tumor-bearing mice. The fusion proteins also produced similar responses in vivo at the 1 nmol per dose range and showed a 60% (TNT-3/CD137L) or 40% (Fc/CD137L) survival of treated mice at 150 days after tumor implantation, similar to the effects of 2A. Morphologic and immunohistochemical analyses showed massive central necrosis and infiltration of granzyme B-positive cells in necrotic areas and viable peripheral regions of treated tumors. Finally, cell depletion studies showed that CD137L-mediated tumor regression was CD8(+) T cell dependent. CONCLUSIONS: From these studies, it was determined that both targeted and untargeted CD137L fusion proteins showed effective antitumor activity, but that the targeted version was more potent. Therefore, the use of the natural CD137 ligand is a promising approach to the treatment of solid tumors by virtue of its ability to produce physiologic costimulation within the tumor, limiting side effects often seen with agonist antibody therapies.