Project Summary/Abstract Checkpoint blockade immunotherapy (ICB) improves overall survival in a subset of patients with metastatic urothelial cancer. Atezolizumab, a programmed death ligand-1 (PD-L1) targeting agent, was FDA-approved in 2017 for cisplatin-ineligible patients with urothelial cancer based on a response rate of 24% and a median survival of 15.9 months. However, only a minority of patients responds, and some develop resistance after an initial period of response. Though myriad studies exploring the long-term clinical benefits of immune modulation in these cancers are ongoing or under development, a mechanistic rationale for novel therapeutic combinations in metastatic urothelial cancer is lacking. Research Project 3 (RP-3) seeks to determine, within the context of a prospective, randomized, multicenter study, whether the addition of antiangiogenic therapy to anti-PD-L1 therapy improves survival in patients who are ineligible for cisplatin-based chemotherapy. That trial is based on the hypothesis that co-treatment with PD-L1 and VEGF-targeting antibodies will have antitumor effects by altering the tumor microenvironment, in part by depleting immunosuppressive cell types such as myeloid-derived suppressor cells (MDSCs). Changes in the tumor microenvironment (T-cell receptor [TCR] clonality, MDSC levels) and intrinsic tumor factors (tumor mutation load, neoantigen load and clonality, PD-L1 staining, etc.) correlate with clinical benefit from ICB, but a unified model for optimal clinical decision making is lacking. We propose a systematic approach employing a prospective clinical trial, large-scale analysis of blood and tumor samples from ICB-treated patients with diverse clinical outcomes, large-scale dissection of molecular determinants, and characterization of microenvironmental changes that occur from treatment. The objectives of this proposal are to 1) assess tumor and blood immune markers to predict ICB response; 2) characterize adaptive changes in the tumor microenvironment on treatment; and 3) identify mechanisms of acquired resistance to ICB. The Specific Aims of RP-3 are to compare the efficacy of atezolizumab plus bevacizumab as compared to atezolizumab alone, while studying biomarkers of response and resistance in this context (Aim 1); examine treatment-induced somatic and microenvironmental adaptations in the tumor to discover disease-specific targets for combination therapy (Aim 2); and dissect mechanisms of acquired resistance in patients on this trial and patients receiving standard-of-care ICB (Aim 3). The goal of these analyses will be to develop more robust biomarkers of immunotherapy response, identify rational targets for effective combinatorial therapies, and understand acquired resistance to ICB in patients with urothelial cancer.