Background: Treatment options are limited for patients with unresectable, locally advanced or metastatic BTCs progressing after first line treatment. Standard second line chemotherapy yields objective response rates (ORR) of < 10% and median overall survival of these patients is < 6 months. Human epidermal growth factor receptor 2 (HER2) overexpression/ amplification is observed in 5–19% of BTCs. Zanidatamab is a bispecific HER2-targeted antibody that has demonstrated durable single agent activity with good tolerability in a range of HER2-overexpressing cancers. Methods: In the expansion cohort of this phase I study (NCT02892123), the primary objective is to characterize safety and tolerability of zanidatamab and secondary objectives include evaluation of anti-tumor activity. This cohort includes BTC patients with centrally confirmed HER2 overexpression (immunohistochemistry [IHC] 3+ or IHC 2+/ fluorescence in situ hybridization [FISH]+), disease progression after standard of care therapy, and measurable disease per RECIST 1.1. Zanidatamab is administered at the previously identified recommended dose of 20 mg/kg every 2 weeks (Q2W). Tumors are assessed every 8 weeks (response confirmed at ≥ 4 weeks). Results: As of the data cutoff date (Jul 28, 2020), 20 patients (median age: 63 years [range, 42–78]) with BTC (11 gallbladder cancers, 5 intra- and 4 extra-hepatic cholangiocarcinomas) have been treated with zanidatamab. The median number of prior systemic therapies was 2.5 (range, 1–8), including five patients who had received prior HER2-targeted therapy (trastuzumab). Fourteen (70%) patients experienced zanidatamab-related adverse events (AEs), all of which were grade 1 or 2 in severity. The most common (occurring in ≥ 20%) zanidatamab-related AEs were diarrhea (n = 9) and infusion-related reactions (n = 6). A single treatment-related serious AE of grade 2 fatigue was reported in one patient. Among patients evaluable for response (n = 17), the confirmed ORR was 47% (n = 8; 95% confidence interval [CI]: 23, 72), the disease control rate was 65% (n = 11; 95% CI: 38, 86) and the median duration of response was 6.6 months (95% CI: 3.2, not estimable). Conclusions: Zanidatamab is well tolerated with promising and durable anti-tumor activity in patients with HER2 overexpressing BTC. Based on these data, zanidatamab is now being evaluated in an ongoing global Phase 2b study in patients with advanced HER2+ BTC that have progressed after treatment with a gemcitabine-containing regimen (NCT04466891). Clinical trial information: NCT02892123