--- Bacillus Calmette-Guerin (BCG) is the standard of care for intravesical therapy for carcinoma in situ and non-muscle invasive, non-metastatic human urothelial carcinoma. Although the responsiveness to this immunotherapeutic is believed to be linked with (a) a high number of somatic mutations and (b) a large number of tumor-infiltrating lymphocytes, recent findings of the roles that inhibitory immune receptors and their ligands play in tumor evasion may provide insights into the limitations of the effectiveness of BCG and offer new targets for immune-based therapy. In this study, an aggressive, bioluminescent orthotopic bladder cancer model, MB49 tumor cells transfected with luciferase (MB49(luc)), was used to study the antitumor effects of avelumab, an antibody to PD-L1. MB49(luc) murine tumor cells form multifocal tumors on the mucosal wall of the bladder reminiscent of non-muscle invasive, non-metastatic urothelial carcinomas. MB49(luc) bladder tumors are highly positive for the expression of PD-L1, and avelumab administration induced significant (P 0.05) antitumor effects. These antitumor effects were more dependent on the presence of CD4 than CD8 T cells, as determined by in vivo immune cell depletions. The findings suggest that in this bladder tumor model, interruption of the immune-suppressive PD-1/PD-L1 complex releases a local adaptive immune response that, in turn, reduces tumor growth. This bladder tumor model can be used to further identify host antitumor immune mechanisms and evaluate combinations of immune-based therapies for carcinoma in situ and non-muscle invasive, non-metastatic urothelial carcinoma, to provide the rationale for subsequent clinical studies. --- Targeted delivery of IL-12 might turn this cytokine into a safer, more effective cancer therapeutic. We describe a novel immunocytokine, NHS-IL12, consisting of two molecules of IL-12 fused to a tumor necrosis-targeting human IgG1 (NHS76). The addition of the human IgG1 moiety resulted in a longer plasma half-life of NHS-IL12 than recombinant IL-12, and a selective targeting to murine tumors in vivo. Data from both in vitro assays using human peripheral blood mononuclear cells (PBMCs) and in vivo primate studies showed that IFN-gamma production by immune cells is attenuated following treatment with the immunocytokine, suggesting an improved toxicity profile than seen with recombinant IL-12 alone. NHS-IL12 was superior to recombinant IL-12 when evaluated as an anti-tumor agent in three murine tumor models. Mechanistic studies utilizing immune cell subset-depleting antibodies, flow cytometric methods, and in vitro cytotoxicity and ELISA assays all indicated that the anti-tumor effects of NHS-IL12 were primarily CD8+ T cell-dependent and likely IL-12-mediated. Combining NHS-IL12 treatment with a cancer vaccine, radiation, or chemotherapy resulted in greater anti-tumor effects than each individual therapy alone. These preclinical findings provide a rationale for the clinical testing of this immunocytokine, both as a single agent and in combination with vaccines, radiation and chemotherapy.