Background: ALT-P7 is an antibody-drug conjugate, in which two molecules of monomethyl auristatin E (MMAE) are site-specifically conjugated to a cysteine-containing peptide motif of trastuzumab variant. This is the first-in-human study evaluating safety and pharmacokinetics of ALT-P7 in patients with HER2-positive advanced breast cancer. Methods: This was an open label, 3+3 dose-escalation phase 1 trial. Eligible patients were with HER2-positive advanced breast cancer progressive to at least two kinds of prior anti-HER2 treatment. ALT-P7 at doses from 0.3mg/kg to 4.8mg/kg were intravenously administered once every 3 weeks. Dose limiting toxicities were evaluated over the first cycle of 21 days. Primary objective was to define the maximum tolerated dose. Results: ALT-P7 were administered in 27 patients (n=4 at 0.3mg/kg, n=3 at each of 0.6, 1.2, 2.4, 3.6, 4.2, 4.5 mg/kg, n=5 at 4.8mg/kg) between Jan 2018 and Feb 2020. The median number of previous line of systemic therapy was six, including median four prior anti-HER2 agents. The most common grade (G) 3/4 adverse event (AE) was neutropenia (n=4). The other common drug-related AEs of all grade were myalgia (n=9), fatigue (n=7), sensory neuropathy (n=6), alopecia (n=6), pruritus (n=6), and neutropenia (n=6). Dose limiting toxicities(DLTs) were observed in three patients at 4.8mg/kg (G4 febrile neutropenia, G4 thrombocytopenia, G4 hyperbilirubinemia, G3 myalgia, G4 hyponatremia). No DLTs have been observed up to 4.2mg/kg, and safety evaluation at 4.5mg/kg is currently ongoing. Toxicokinetic analysis for total antibody, drug-conjugated antibody, and free payload suggested that there were no accumulation of ALT-P7 upon repeated injection. In 22 patients with response evaluation, disease control rate at 6 weeks of ALT-P7 treatment was 77.3%(17/22) and partial response was achieved in two out of fifteen patients with measurable lesion. The median PFS at doses from 2.4 to 4.8mg/kg was 6.2 months (95% CI 2.5-9.9 months). Conclusions: ALT-P7 was well tolerated to a dose of 4.2mg/kg in heavily pretreated HER2-positive advanced breast cancer. DLTs were observed at 4.8mg/kg, and 4.5mg/kg is under evaluation. The observed clinical activity warrants further evaluation in a phase 2 trial. Clinical trial information: NCT03281824. Clinical trial information: NCT03281824.