Abstract Introduction: Antibody-based therapy is one of the most successful strategies for treating patients with cancers. This is also true for breast cancers as trastuzumab (a monoclonal antibody against Her2) is used as the first line therapy for Her2+ breast cancer. Despite substantial progress, there remains a notable gap in effective targeted treatments for TNBC. In the landscape of cancer treatment, chemotherapy is the common treatment option for TNBC. However, resistance and tumor relapse remain the major obstacles hindering the effectiveness of chemotherapeutic agents in cancer patients. Therefore, the development of new therapeutics, especially targeted approaches like antibody-based therapies, holds immense significance. This study aims to identify and test the role of secretory protein/s that may support the growth and progression of TNBC cells. Additionally, it seeks to determine if targeting these oncogenic secretory proteins with antibodies can provide a novel therapeutic approach for TNBC patients. Our study identified secretory protein “Signal peptide CUB domain EGF-like 3 (SCUBE3)” as a critical factor that promotes survival and therapy resistance of TNBC cells. Importantly, using antibody-development platform, our study presents a novel therapeutic approach targeting SCUBE3 using anti-SCUBE3 monoclonal antibody, offering new hope for TNBC patients with limited treatment options. Methods: Unbiased high-throughput loss of function genomic screen was performed on TNBC cells in the presence or absence of doxorubicin. TNBC cells were transfected either with SCUBE3 overexpression plasmid or shRNA specific to SCUBE3 to perform functional assay, in vivo tumor xenograft assays, RNA sequencing and Mass spectrometry (MS). Syngeneic mouse model was used to study the effect of SCUBE3 in shaping tumor immune microenvironment of TNBCs. Hybridoma culture technique was used to generate anti-SCUBE3 monoclonal antibodies. Screening of top anti-SCUBE3 antibody producing clone was done based on ELISA, long-term and short-term colony formation assay. Therapeutic efficacy of antibodies was evaluated in tumor xenograft models. Result: Through a loss-of-function genomic screening approach, we identified secretory SCUBE3 as a critical player that promotes the survival, therapy resistance and metastasis of TNBC cells. Our MS data shows that SCUBE3 interacts with several oncogenic proteins including EFGR, TGFβR and mutant CALR. Transcriptomic analysis demonstrates that SCUBE3 depletion leads to inhibition of pro-tumorigenic signaling as well as activation of anti-tumor immunity pathways. Our results reveal that SCUBE3 regulates the expression of FOXR2 which in turn stabilizes Myc and recruits DNMT to form a repressor complex on the promoter of IRF1. Inactivation of IRF1 gene suppresses the expression of MHC class I and class II genes which leads to immune suppression. Further, our syngeneic mouse model study shows that suppression of SCUBE3 leads to increased infiltration of CD8+ T cells and NK cells into the tumor. Importantly, using multiple pre-clinical models including PDX models, we show that antibody targeting SCUBE3 blocks tumor growth. Conclusion: This study will set the stage for a new paradigm of treating TNBCs by inhibiting SCUBE3 activity. Our study is first to identify SCUBE3 as a novel ligand of EGFR1 and CALRmut. Notably, our study shows that SCUBE3 promotes pro-tumorigenic signaling cascade while inhibiting anti-tumor immunity to promote TNBC growth and render them resistant to therapy. Importantly, we provide compelling evidence that antibody targeting SCUBE3 may serve as a potent therapeutic for treating TNBCs. Citation Format: D. Singh, B. Onyeagucha, D. Medina, P. Subbarayalu, R. Mojidra, P. Venkata, J. Huang, J. Prochnau, P. Do, M. Rao. Antibody Inhibiting SCUBE3 for the Treatment of Triple Negative Breast Cancer abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS4-04-29.
Singh et al. (Tue,) studied this question.