Regulatory T cells (Tregs) are key mediators of immune tolerance and feature prominently in cancer. Depletion of CD25+ FoxP3+ Tregs in vivo may promote T cell cancer immunosurveillance, but no strategy to do so in humans while preserving immunity and preventing autoimmunity has been validated. We evaluated the Food and Drug Administration–approved CD25-blocking monoclonal antibody daclizumab with regard to human Treg survival and function. In vitro, daclizumab did not mediate antibody-dependent or complement-mediated cytotoxicity but rather resulted in the down-regulation of FoxP3 selectively among CD25high CD45RAneg Tregs. Moreover, daclizumab-treated CD45RAneg Tregs lost suppressive function and regained the ability to produce interferon-γ, consistent with reprogramming. To understand the impact of daclizumab on Tregs in vivo, we performed a clinical trial of daclizumab in combination with an experimental cancer vaccine in patients with metastatic breast cancer. Daclizumab administration led to a marked and prolonged decrease in Tregs in patients. Robust CD8 and CD4 T cell priming and boosting to all vaccine antigens were observed in the absence of autoimmunity. We conclude that CD25 blockade depletes and selectively reprograms Tregs in concert with active immune therapy in cancer patients. These results suggest a mechanism to target cancer-associated Tregs while avoiding autoimmunity.

Malignant pleural mesothelioma is a fatal neoplasm that is unresponsive to standard modalities of cancer therapy. We conducted a phase I dose-escalation clinical trial of adenoviral (Ad)-mediated intrapleural herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) gene therapy in patients with mesothelioma as a model for treatment of a localized malignancy. The goals of this phase I trial were to assess the safety, toxicity, and maximally tolerated dose of intrapleural Ad.HSVtk, to examine patient inflammatory response to the viral vector, and to evaluate the efficiency of intratumoral gene transfer. Twenty-one previously untreated patients were enrolled in this single-arm, dose-escalation study with viral doses ranging from 1 x 10(9) plaque-forming units (pfu) to 1 x 10(12) pfu. A replication-incompetent recombinant adenoviral vector containing the HSVtk gene under control of the Rous sarcoma virus (RSV) promoter-enhancer was introduced into the pleural cavity of patients with malignant mesothelioma followed by 2 weeks of systemic therapy with GCV at a dose of 5 mg/kg twice a day. The initial 15 patients underwent thoracoscopic pleural biopsy prior to, and 3 days after, vector delivery. The last six patients underwent only the post-vector instillation biopsy. Dose-limiting toxicity was not reached. Side effects were minimal and included fever, anemia, transient liver enzyme elevations, and bullous skin eruptions, as well as a temporary systemic inflammatory response in those receiving the highest dose. Strong intrapleural and intratumoral immune responses were generated. Using RNA PCR, in situ hybridization, immunohistochemistry, and immunoblotting, HSVtk gene transfer was documented in 11 of 20 evaluable patients in a dose-related fashion. This study demonstrates that intrapleural administration of an adenoviral vector containing the HSVtk gene is well tolerated and results in detectable gene transfer when delivered at high doses. Further development of therapeutic trials for treatment of localized malignancy using this vector is thus warranted.

Purpose: This phase 1 dose escalation study evaluated the safety and feasibility of single-dose intrapleural IFN-β gene transfer using an adenoviral vector (Ad.IFN-β) in patients with malignant pleural mesothelioma (MPM) and metastatic pleural effusions (MPE). Experimental Design: Ad.IFN-β was administered through an indwelling pleural catheter in doses ranging from 9 × 1011 to 3 × 1012 viral particles (vp) in two cohorts of patients with MPM (7 patients) and MPE (3 patients). Subjects were evaluated for (a) toxicity, (b) gene transfer, (c) humoral, cellular, and cytokine-mediated immune responses, and (d) tumor responses via 18-fluorodeoxyglucose-positron emission tomography scans and chest computed tomography scans. Results: Intrapleural Ad.IFN-β was generally well tolerated with transient lymphopenia as the most common side effect. The maximally tolerated dose achieved was 9 × 1011 vp secondary to idiosyncratic dose-limiting toxicities (hypoxia and liver function abnormalities) in two patients treated at 3 × 1012 vp. The presence of the vector did not elicit a marked cellular infiltrate in the pleural space. Intrapleural levels of cytokines were highly variable at baseline and after response to gene transfer. Gene transfer was documented in 7 of the 10 patients by demonstration of IFN-β message or protein. Antitumor immune responses were elicited in 7 of the 10 patients and included the detection of cytotoxic T cells (1 patient), activation of circulating natural killer cells (2 patients), and humoral responses to known (Simian virus 40 large T antigen and mesothelin) and unknown tumor antigens (7 patients). Four of 10 patients showed meaningful clinical responses defined as disease stability and/or regression on 18-fluorodeoxyglucose-positron emission tomography and computed tomography scans at day 60 after vector infusion. Conclusions: Intrapleural instillation of Ad.IFN-β is a potentially useful approach for the generation of antitumor immune responses in MPM and MPE patients and should be investigated further for overall clinical efficacy.