Targeted Therapy for CD13‐Overexpressing Triple‐Negative Breast Cancers Using Apoptosis‐Inducing Protein Cage Nanoparticles

Triple-negative breast cancer (TNBC) remains one of the most challenging breast cancer subtypes to treat due to the lack of well-defined molecular targets. Cluster of differentiation 13 (CD13), a cell surface aminopeptidase, is highly expressed in various tumors and play critical roles in promoting angiogenesis, aberrant proliferation, invasion, and metastasis. In this study, we investigated CD13 as a potential therapeutic target in TNBC cell lines to enable targeted therapy. Accordingly, we employed a protein cage nanoparticle, AaLS/TRAIL/aCD13Nb, which simultaneously displays CD13-binding nanobodies (aCD13Nb) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via the SpyCatcher/SpyTag protein ligation system. This dual-ligand nanoparticle exhibited enhanced and specific binding to CD13-overexpressing TNBC cell lines, including HCC1937, MDA-MB-468, and BT-549 cells. aCD13Nb-mediated tight binding facilitated sustained interaction of TRAIL with death receptors, resulting in robust activation of apoptotic signaling cascades and significantly enhanced therapeutic efficacy in CD13-overexpressing TNBC cell lines. Moreover, systemic administration of AaLS/TRAIL/aCD13Nb via intravenous injection markedly suppressed tumor growth in an HCC1937 xenograft mouse model, without evidence of systemic toxicity. These findings validate CD13 as a promising therapeutic target in TNBC and underscore the potential of dual-ligand protein cage nanoparticles as an effective platform for targeted cancer therapy.