Abstract Purpose: HER2-low breast cancers, which account for most metastatic cases, remain challenging to treat. Although trastuzumab-deruxtecan has shown clinical benefit, its use is limited by high toxicity and disease progression, underscoring the need for safer, more effective, and more accessible targeted options. These tumors disproportionately affect underserved populations, amplifying disparities in care. Building upon our previous development of a murine HER2-low model and NK/Trastuzumab (Tz) surface-engineering platform, we now evaluated the therapeutic potential and selectivity of SE-NK/Tz cells against HER2-low human and murine tumors, including 3D tumor spheroids. Methods: SE-NK cells were generated by embedding 25 µg of Tz onto the mouse-derived LNK cells or human NK92 cells using a hydrophobic linker, and conjugation efficiency was verified by flow cytometry and confocal imaging. HER2-specific cytotoxicity was evaluated by luciferase-based killing assays in 2D and 3D cultures of 4T1-HER2-low murine cells (generated in our lab) and HER2-low MDA-MB-175 human breast cancer cells. HER2-negative cells were used to assess specificity. Results: SE-LNK/Tz cells exhibited significantly enhanced cytotoxicity against 4T1-HER2-low targets (86 ± 1 % vs 55 ± 2 %, p 0.0001 at E:T 10:1), consistent across multiple effector-to-target ratios (20:1-2.5:1), compared with unmodified NK cells. SE-NK92/Tz cells similarly increased cytotoxicity against MDA-MB-175 targets (57 ± 1% vs 42 ± 3 %, p 0.01 at E:T 1:1). Importantly, SE-NK/Tz cells showed markedly lower killing of HER2-negative cells (mouse 6 ± 5% vs 69 ± 2%; human 42± 1% vs 57± 1 %, p 0.0001), confirming HER2-specific targeting. SE-LNK/Tz cells maintained activity in 3D spheroid assays, indicating effective tumor penetration. Conclusions: This study advances our surface-engineering NK platform from proof-of-concept to functional validation in both murine and human HER2-low systems, including 3D culture models. SE-NK/Tz cells display potent and selective cytotoxicity against HER2-low breast cancer, supporting their potential as a next-generation, low-toxicity immunotherapy for this underserved patient population. Ongoing work focuses on exploring mechanisms of immune activation and therapeutic efficacy in diverse in vivo HER2-low models. Citation Format: Maria Jose Godoy Calderon, Manali Patwardhan, Shruti Rodrigues, Saanvi Sethi, Eric Gauchat, Vk Gadi. Surface engineered-NK/trastuzumab cells as a novel immunotherapy for HER2-low breast cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 136.
Calderon et al. (Fri,) studied this question.