Urothelial bladder cancer is estimated to cause 165,084 annual deaths worldwide including over 15,960 in the United States. Outcomes for metastatic urothelial cancer remain poor with an overall 5-year survival rate of 15%, despite modern chemotherapy regimens. Recently, five antiPD-1/PD-L1 antibodies were approved between 2016 and 2017, which reignited immunotherapeutic development in bladder cancer. Despite the impressive activity of these drugs, including many patients with complete responses, the objective response rates remain only 15- 20%. Thus, the majority of patients with bladder cancer still fail to respond to immunotherapy.

Responsiveness to immune checkpoint inhibitors such as anti-PD-1/PD-L1 therapy is dependent on the presence of a preexisting immune infiltrate, also referred to as the T cell-inflamed phenotype. Our recent analysis of muscle invasive bladder cancer has indicated that the majority of bladder cancers lack a T cell-inflamed tumor microenvironment, as measured by immune gene expression profiling and immunohistochemistry. The development of an anti-tumor T cell response is driven by the presence of type I interferon (IFN. Recent data have shown that the production of type I IFN in dendritic cells occurs though cytosolic sensing of tumor-derived DNA through activation of the Stimulator of Interferon Genes (STING) pathway. Therapeutic development of synthetic cyclic dinucleotides is underway and the first drugs are in clinical testing. In bladder cancer, the relevance of this pathway to immune priming and the development of an anti-tumor immune response has not previously been studied. Thus, I proposed to characterize the role of innate immune activation in the development of a T cell inflamed tumor microenvironment, and determine whether activation of STING may have therapeutic efficacy in localized bladder cancer.

Prior to the start of CRF funding, my lab had preliminary data showing that treatment with DMXAA, a STING agonist, delayed tumor progression in a subcutaneous transplantable tumor model, and prolonged survival when administered in an intravesicular fashion to treat MB49 bladder cancers. During the course of the initial year of CRF funding, we have now replicated intravesical experiments, and developed a new model to monitor, in real time, the growth of bladder tumors using intravital imaging via luciferace expression by tumor cells. We have used this model to evaluate growth curve data after STING agonist treatment. These experiments have confirmed efficacy of this approach, which showed a reduction in tumor growth over time compared with treatment with PBS control. We also have analyzed the tumors and found that STING agonist treatment led to a significant increase in the number of antigen-specific T lymphocytes compared with control, thus supporting the original hypothesis that non-T cellinflamed tumors could be converted into T cell-inflamed tumors. The immune-mediated mechanism of action is additionally supported by IFN gamma ELISPOT assays performed on splenocytes demonstrated that circulating antigen-specific T lymphocytes are also increased with DMXAA treatment compared to controls. Depletion experiments and STING knockout experiments have now indicated that the mechanism of action is both CD8 and STING-dependent.

Ongoing Experiments: Current work underway is aimed at further delineating the mechanism of action using additional knock out mice and depletion experiments. Alternate dosing schedules will be employed (i.e. high dose of 500 micrograms instilled once versus repeated dosing at varying concentrations) to determine the optimal strategy to devise a clinical trial in patients. The differences in immune microenvironment with the different dosing strategies will also be evaluated. Finally, rechallenge experiments are planned to evaluate the immune memory and determine if subsequent tumors will be rejected after successful rejection of tumors via STING agonist treatment.

Expansion Plans: Preliminary data from this project was used to secure a pre-clinical as well as clinical collaboration with Audro Biotech, through which I plan to explore the clinical-grade cyclic dinucleotides in these mouse models. This will further establish the optimal dosing schedule for these STING agonists that will, in parallel to ongoing pre-clinical mechanistic experiments, be written into a clinical protocol. In particular, I will evaluate these drugs as monotherapy, but also in combination with anti-PD-1 immunotherapy to see if the combination of innate immune activation via STING and reversing adaptive immune resistant will be a synergistic combination. The goal would be to yield long term durable responses. I expect to open a clinical trial by the end of the second year of CRF funding.

Funding: These funds were partially used to support a laboratory technician salary who assisted in conducting the in vitro and in vivo experiments. Additional funds were applied to supplies such as flow cytometry antibodies, ELISPOT reagents, anti-PD-L1 antibodies, and routine cell line maintenance. Finally, some of the funding was applied to cover the cost of core facility charges including the flow cytometry core and the bioinformatics core facility.

Urothelial bladder cancer is estimated to cause 165,084 annual deaths worldwide including over 15,960 in the United States. Outcomes for metastatic urothelial cancer remain poor with an overall 5-year survival rate of 15%, despite modern chemotherapy regimens. Recently, five anti- PD-1/PD-L1 antibodies were approved between 2016 and 2017, which reignited immunotherapeutic development in bladder cancer. Despite the impressive activity of these drugs, including many patients with complete responses, the objective response rates remain only 15- 20%. Thus, the majority of patients with bladder cancer still fail to respond to immunotherapy.
Responsiveness to immune checkpoint inhibitors such as anti-PD-1/PD-L1 therapy is dependent on the presence of a preexisting immune infiltrate, also referred to as the T cell-inflamed phenotype. Our recent analysis of muscle invasive bladder cancer has indicated that the majority of bladder cancers lack a T cell-inflamed tumor microenvironment, as measured by immune gene expression profiling and immunohistochemistry. The development of an anti-tumor T cell response is driven by the presence of type I interferon (IFN. Recent data have shown that the production of type I IFN in dendritic cells occurs though cytosolic sensing of tumor-derived DNA through activation of the Stimulator of Interferon Genes (STING) pathway. Therapeutic development of synthetic cyclic dinucleotides is underway, and the first drugs are in clinical testing. In bladder cancer, the relevance of this pathway to immune priming and the development of an anti-tumor immune response has not previously been studied. Thus, I proposed to characterize the role of innate immune activation in the development of a T cell inflamed tumor microenvironment and determine whether activation of STING may have therapeutic efficacy in localized bladder cancer.
During the course of CRF funding, we have now replicated intravesical experiments, and developed a new model to monitor, in real time, the growth of bladder tumors using intravital imaging via luciferase expression by tumor cells. We have used this model to evaluate growth curve data after STING agonist treatment. These experiments have confirmed efficacy of this approach, which showed a reduction in tumor growth over time compared with treatment with PBS control. Depletion experiments and STING knockout experiments were completed, and the data indicate that the mechanism of action is both CD8 and STING-dependent. We analyzed the tumors and found that STING agonist treatment led to a significant increase in the number of antigen-specific T lymphocytes compared with control, thus supporting the original hypothesis that non-T cell-inflamed tumors could be converted into T cell-inflamed tumors. The immune- mediated mechanism of action is additionally supported by IFN gamma ELISPOT assays performed on splenocytes demonstrated that circulating antigen-specific T lymphocytes are also increased with DMXAA treatment compared to controls.
Preclinical work supported by this project has now demonstrated the efficacy of this approach in bladder cancer. Thus, we have now focused on rapidly bringing this approach to clinical trials for patients. In parallel, we are currently contracting to test pharmaceutical grade STING agonists in this bladder cancer model, and a clinical trial for non-muscle invasive bladder cancer patients has been drafted and is undergoing revisions. Since the start of this project multiple STING agonists have progressed into phase I studies, which has facilitated the rapid translation of our data into patient testing. Existing studies employ direct injection into tumors or systemic administration of STING agonists. Our study will uniquely test whether intravesical treatment is effective. Our aim going forward is to open this clinical trial specifically for bladder cancer patients.
The findings achieved in this project could not have been accomplished without support from the Cancer Research Foundation. We are extremely grateful as the award provided the much- needed funding to support personnel costs, laboratory supplies, as well as core facility charges including use of intravital imaging necessary for the in vivo experiments. We look forward to advancing the care of bladder cancers patients through the results from this project.