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Background HER2-positive breast cancer is characterized by aggressive growth and high metastatic potential. Although various therapeutic agents have significantly improved clinical outcomes for patients with HER2-positive tumors, both primary and acquired resistance still remain major clinical challenges. In particular, aberrant activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway occurs in a substantial proportion of HER2-positive breast cancers and is strongly associated with resistance to anti-tumor therapies. 1 Targeting the PI3K pathway is therefore a promising strategy to enhance anti-tumor efficacy and mitigate resistance.2 PTEN, a key negative regulator of the PI3K pathway, is frequently lost or suppressed in resistant tumors, contributing to immune evasion and reduced therapeutic response.3 Thus, the development of a formulation capable of modulating PTEN expression in a cancer cell-selective manner to enhance anti-tumor immune responses is a technically demanding but clinically valuable strategy.Methods Using click chemistry, we conjugated trastuzumab to lipid nanoparticles (LNPs). We evaluated the selectivity of this conjugate toward HER2-overexpressing tumor cells in comparison to a conventional Moderna LNP formulation. Flow cytometry analysis was performed using multiple cell lines, including HER2-positive cell lines (SKBR3 and HCC1954), as well as control lines (HEK293 and HUVEC). To assess the therapeutic synergy of antibody targeting and PTEN restoration, we formulated trastuzumab-conjugated LNPs encapsulating PTEN mRNA. Their cytotoxic effects on tumor cells were evaluated in vitro. Furthermore, anti-tumor efficacy was assessed in an HCC1954 tumor-bearing mouse model. Tumor growth inhibition and serum-based evaluation of hepatic and renal toxicity were performed to assess therapeutic efficacy and off-target safety.Results Compared with the conventional LNP formulation, our trastuzumab-conjugated LNPs exhibited enhanced gene delivery in HER2-positive cancer cell lines (SKBR3 and HCC1954), while significantly reducing expression in non-target control lines (HEK293 and HUVEC). Furthermore, trastuzumab-conjugated PTEN LNPs induced potent cytotoxic effects not only in SKBR3 cells, which are sensitive to trastuzumab, but also in HCC1954 cells, which are known to exhibit resistance to trastuzumab. In vivo, treatment with trastuzumab-conjugated PTEN LNPs in HCC1954 tumor-bearing mice led to a significant reduction in tumor size compared to either conventional PTEN LNPs or trastuzumab alone, without increasing hepatic or renal toxicity.Conclusions We have developed a novel trastuzumab-conjugated LNP system encapsulating PTEN mRNA, which enhances both the efficacy and selectivity of the immune response in HER2-positive tumor cells. By leveraging the synergistic therapeutic effects of antibody-mediated activity and PTEN modulation, this platform offers significant potential for overcoming resistance and improving outcomes in targeted cancer immunotherapy.References Rasti AR, Guimaraes-Young A, Datko F, Borges VF, Aisner DL, Shagisultanova E. PIK3CA mutations drive therapeutic resistance in human epidermal growth factor receptor 2-positive breast cancer. JCO Precis Oncol. 2022;6: e2100370.Wright SCE, Vasilevski N, Serra V, Rodon J, Eichhorn PJA. Mechanisms of resistance to pi3k inhibitors in cancer: adaptive responses, drug tolerance and cellular plasticity. Cancers (Basel.) 2021;13(7).Chalhoub N, Baker SJ. PTEN and the PI3-kinase pathway in cancer. Annu Rev Pathol. 2009;4:127–50.