Background: HER3-DXd is an antibody drug conjugate (ADC) comprised of a fully human anti-HER3 IgG1 monoclonal antibody (patritumab), attached to a topoisomerase 1 inhibitor via a tetrapeptide-based cleavable linker that has shown promising efficacy in pts with HER3-expressing MBC (Krop, 2022). This 3-part Phase 2 study examines efficacy of HER3-DXd across MBC subsets and defines the pt population likely to derive greater benefit (NCT04699630). Methods: Part A is reported here (Data cutoff 6Sep2022). Pts had HER2 negative MBC with measurable disease per RECIST v1.1, 0-2 prior chemo and prior endocrine therapy (ET) + CDK4/6 inhibitor for hormone receptor (HR)+ BC, or 1-3 prior chemo for triple negative BC (TNBC). Prior treatment (tx) with anti-HER3 agents and ADCs with exatecan derivatives were prohibited. Pts provided pre-tx tissue to evaluate the association of biomarker expression with progression free survival at 6 months (PFS_6months). Primary endpoints for Part A are objective response rate (ORR) and PFS_6months. Secondary endpoints are safety/tolerability, duration of response (DOR), and clinical benefit rate (CBR) (CR, PR or SD ≥180 days). Results: 60 pts were treated in Part A: median age 57.5 y, 98.3% female; median 5 prior lines of therapy (range 1-15). 32% had TNBC. 48% were HR+. 48% had liver and 32% had lung metastases. HER3 membrane expression was evaluated by overall % membrane positivity at 10X. 47/60 (78%) pts provided evaluable samples at baseline. Among evaluable pts, 64% (30/47) had HER3 expression ≥75%, 28% (13/47) had 25-74% expression and 8% (4/47) had <25% expression. The median tx duration was 5.2 mos and 21 pts remained on tx at data cut-off. All pts experienced a tx emergent adverse event and 93% of pts experienced a tx related AE (TRAE) with Gr ≥3 TRAE in 19 pts (32%). The most common (≥ 25%) any grade TRAEs were nausea (50%), fatigue (45%), diarrhea (37%), vomiting (32%), alopecia and anemia (30% each). 7 pts (12%) experienced a serious AE (SAE), including 4 pts (7%) with a related SAE (interstitial lung disease, nausea/vomiting, pneumonitis, thrombocytopenia). 15% of pts experienced a dose reduction and 23% experienced a dose interruption due to an AE. 3 pts died while on tx, 2 unrelated to tx 1 cause unknown. ORR was 35% (95% CIs 23.1, 48.1) for all pts, and the CBR was 48% (95% CIs 35.2, 61.6). Pts with ≥75% HER3 expression had an ORR of 33% and CBR of 50%, pts with HER3 25-74% expression had an ORR of 46% and CBR of 54%. There were 4 pts with HER3 < 25% expression, limiting efficacy assessment. The median DOR was 10.0 mos (95% CIs 5.5, NA). The PFS_6months was 60% for all pts, 50% for pts with HER3 ≥75%, and 70% for pts with HER3 25-74%. Conclusions: HER3-DXd had an acceptable safety profile, and the data further confirm the clinical activity in MBC in heavy pre-tx MBC across the broad range of HER3 expression levels. Parts B and Z are ongoing and data from this report support the potential entry of HER3-DXd into the therapeutic paradigm in MBC. Clinical trial information: NCT04699630.