Background: Checkpoint inhibitors (CPIs) are standard therapy for pts with mUC after PLT-based regimens, with limited long-term disease control. SG is an antibody-drug conjugate composed of an anti-trophoblast cell-surface antigen 2 (Trop-2) antibody coupled to SN-38 (a topoisomerase-I inhibitor) via a proprietary hydrolyzable linker. In the TROPHY-U-01 registrational phase 2 trial, SG monotherapy demonstrated significant activity and manageable safety in pts with mUC who progressed after prior PLT-based chemotherapy and CPI, with 27% objective response rate (ORR) and median overall survival of 11 months (Tagawa, et al. J Clin Oncol. 2021). Here, we present interim efficacy and safety results of combining SG with Pembro as 2nd-line therapy in CPI-naive pts with mUC who progressed after PLT-based chemotherapy (cohort 3). Methods: TROPHY-U-01 is a multicohort, open-label, global phase 2 trial. Eligible pts had measurable disease, Eastern Cooperative Oncology Group performance status (ECOG PS) 0–1, and creatinine clearance ≥30 mL/min. The recommended phase 2 dose (RP2D) was determined during a 10-pt safety lead-in, and additional pts were enrolled at the RP2D in a Simon 2-stage design. Primary endpoint: ORR by blinded independent central review per Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1). Key secondary endpoints: investigator-assessed ORR, clinical benefit rate [CBR; complete response (CR) + partial response (PR) + stable disease], progression-free survival (PFS), and safety. Results: At the time of data cutoff, 41 pts received at least a dose of SG at the RP2D (10 mg/kg). Of these 41 pts, median (range) age was 67y (46–86), 83% men, 61% ECOG PS 1, 76% had ≥1 Bellmunt risk factor, and median (range) number of prior anticancer regimens was 1 (1–3). At a median follow-up of 5.8 mo, the investigator-assessed ORR was 34% (95% CI, 20.1–50.6; 1 CR; 13 PR); CBR was 44% (95% CI, 28.5–60.3); 6-mo PFS rate was 47%. Median time to response was 2.0 mo (95% CI, 1.3–2.8). Most common treatment-emergent adverse events (TEAEs) were diarrhea (76%), nausea (59%), anemia (56%), neutropenia (44%), and asthenia (41%). Treatment-related grade ≥3 AEs occurred in 59% of pts. Key grade ≥3 TEAEs of any cause included diarrhea (24%), anemia (20%), febrile neutropenia (10%), fatigue (7%), and asthenia (5%). Two pts discontinued treatment due to treatment-related AEs. No treatment-related death occurred. Conclusions: SG in combination with Pembro demonstrated encouraging ORR and CBR, with an overall manageable safety profile with no new safety signal in CPI-naive pts who progressed after prior PLT-based chemotherapy. The data support further evaluation of SG plus CPI in mUC. Limitations: small sample size, short follow-up, and lack of randomization. Biomarker evaluation is ongoing. Clinical trial information: NCT03547973.