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Background Antibody-drug conjugates (ADCs) targeting the human epidermal growth factor receptor 2 (HER2), such as Enhertu (T-DXd), have created a new standard in targeted cancer treatment. T-DXd’s robust activity against HER2-overexpressing tumors is linked to its TOPO I inhibitor payload, which is well-tolerated and effectively penetrates tissues, facilitating the bystander killing effect. Simultaneously, a notably elevated drug-to-antibody ratio (DAR=8) enhances the effective transport of a significant therapeutic payload into cells. Still, the advantages remain restricted within the HER2-low breast cancer population. We hypothesize that an ADC incorporating a highly permeable payload and a greater DAR will improve payload delivery in HER2-low tumors, thus enhancing tumor suppression.Methods We used an alternative TOPO I inhibitor, Exatecan, known for its enhanced membrane permeability. To improve drug loading and reduce surface hydrophobicity, we engineered a unique super-hydrophilic hyperbranched polymer as a drug carrier. Exatecan was first attached to the polymer’s core. Subsequently, this complex was conjugated explicitly to the hinge area of trastuzumab in a site-specific manner to form the final ADC (TPE). TPE’s biochemical and biological properties were evaluated through detailed in vitro and in vivo tests on breast cancer with low HER2 levels.Results The polymer carrier supported a roughly 20 DAR while still ensuring effective HER2 targeting by Trastuzumab. TPE exhibited similar levels of cytotoxicity in HER2-high breast cancer cell lines compared to T-DXd, while showing 3 to 20 times greater effectiveness in HER2-moderate and HER2-low breast cancer cell lines. In the bystander killing assay, TPE inhibited the proliferation of HER2-negative cells more effectively than T-DXd. The super-hydrophilicity of the polymer successfully concealed the hydrophobic traits of Exatecan in vivo, facilitating improved systemic circulation and tumor accumulation as opposed to T-DXd. TPE yielded better treatment outcomes in murine models with HER2-low breast tumors than T-DXd.Conclusions The advanced ADC, utilizing Exatecan and a super-hydrophilic polymer carrier, achieved ultra-high drug loading, improved bystander killing effects, extended plasma half-life, and boosted tumor retention, resulting in more potent inhibition of HER2-low breast tumors compared to T-DXd. This tactic is promising for HER2-low breast cancer and could extend its benefits to other cancers with low antigen expression, benefiting a broader population of cancer patients.Ethics Approval This study was approved by the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham, APN 223018.