Background: For patients with BTC that has progressed after first-line therapy, prognosis is poor with a median OS of 6-9 months with subsequent chemotherapy. Zanidatamab is a HER2-targeted bispecific antibody that binds to two non-overlapping HER2 domains and crosslinks neighboring HER2 proteins. In the primary analysis of the phase 2b HERIZON-BTC-01 trial, after a median follow-up of 12.4 months (data cutoff: October 10, 2022), zanidatamab showed encouraging antitumor activity (41% confirmed objective response rate [cORR]) with rapid and durable responses and a manageable safety profile in patients with previously treated HER2+ BTC. OS data were immature at that time. Here, we report updated analyses, including OS. Methods: HERIZON-BTC-01 (NCT04466891) is an ongoing phase 2b trial assessing zanidatamab (20 mg/kg IV Q2W) in patients with HER2/ERBB2 gene amplification and immunohistochemistry (IHC) 2+ or 3+ (Cohort 1; HER2+); or 0 or 1+ (Cohort 2) locally advanced, unresectable, or metastatic BTC (gallbladder cancer, intra/extrahepatic cholangiocarcinoma) who received prior gemcitabine-containing treatment. The primary endpoint was cORR. Select secondary endpoints included duration of response (DoR), OS, and frequency and severity of adverse events (AEs). Updated efficacy analyses include only Cohort 1; safety analyses include Cohorts 1 and 2. Results: As of the data cutoff (July 28, 2023), median (range) follow-up was 21.9 (16-34) months. In the 80 patients in Cohort 1 (HER2+), treatment was ongoing for 9 (11%) patients and 20 (25%) patients remained on the study. cORR was unchanged from the primary analysis (n = 33; 41%) with 1 additional complete response (n = 2; 2.5%). The median DoR (95% CI) increased approximately 2 months to 14.9 (7.4, not reached) months. Median OS (95% CI) was 15.5 (10.4, 18.5) months; 12-month OS (95% CI) was 56.2% (44.3, 66.5). Among all 87 patients (Cohort 1 and Cohort 2), 18 (21%) experienced grade ≥3 treatment-related AEs (TRAEs). The most common grade 3 TRAEs (occurring in > 2 patients) were diarrhea (4 [5%]); anemia (3 [3%]), and ejection fraction decreased (3 [3%]). Only one patient had a grade 4 TRAE (aspartate aminotransferase increased). There were no deaths due to TRAEs. Serious AEs occurred in 8 (9%) patients; 2 (2%) patients discontinued treatment due to an AE (pneumonitis and ejection fraction decreased; 1 patient each). Conclusions: With close to 2 years of median follow-up, zanidatamab demonstrated a median OS of 15.5 months and a median duration of response of 14.9 months (an increase compared with the initial report) in previously-treated patients with HER2+ BTC; a patient population with significant unmet need. With additional follow-up, safety remained manageable. A phase 3 trial of zanidatamab in the first-line setting for patients with metastatic BTC is planned. Clinical trial information: NCT04466891.