Dual-Targeting Strategy to Repurpose Cetuximab with HFn Nanoconjugates for Immunotherapy of Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is a highly aggressive and treatment-resistant malignancy, characterized by the absence of targeted therapies and poor clinical outcomes. Cetuximab (CTX), a monoclonal antibody targeting the epidermal growth factor receptor (EGFR), has shown potential in several cancers, including TNBC. However, its clinical efficacy is often compromised by poor tumor penetration and resistance mechanisms. To overcome these limitations, we developed a novel strategy by conjugating Cetuximab to H-ferritin (HFn), a nanoparticle targeting transferrin receptor 1 (TfR1), for potential immunotherapy in CTX-resistant tumors. The HFn-CTX nanoconjugate exhibited favorable biophysical properties, including a hydrodynamic size of <30 nm. This dual-targeting approach aims to increase drug internalization and effectiveness within the tumor. Following ethical guidelines, we first assessed the interaction capacity of the nanoconjugate in 2D TNBC systems. To refine our approach and avoid animal testing, we then utilized 3D TNBC spheroids, which more accurately replicate the tumor microenvironment and provide a relevant platform for studying drug penetration and internalization. Our study is further supported by employing CellProfiler, a software that enables automated, high-throughput image analysis, allowing us to assess the penetration and distribution of the HFn-CTX nanoconjugates, thereby maximizing data accuracy. Our findings demonstrated that HFn-CTX conjugates significantly improved drug uptake and penetration into the inner layers of tumor spheroids compared to Cetuximab alone, highlighting the potential of HFn as a carrier to enhance therapeutic delivery. Furthermore, the enhanced internalization of the nanoconjugates also facilitated stronger antibody-dependent cellular cytotoxicity (ADCC). In contrast, glioblastoma spheroids did not exhibit a similar response. This difference was associated with elevated EGFR expression on the cell surface and the retention of the nanoconjugate at the plasma membrane in TNBC cells, which promoted more effective immune activation. These results underline the potential of HFn-CTX nanoconjugates to repurpose CTX for refractory cancers that express high levels of EGFR, such as TNBC, leveraging dual-receptor targeting to amplify immune-mediated cytotoxicity and overcome resistance. Improving the reliability and translatability of our findings, this animal-free approach contributes to the development of more ethical and efficient preclinical cancer therapies in accordance with 3Rs principles.