Anti-EGFR VHH-armed death receptor ligand–engineered allogeneic stem cells have therapeutic efficacy in diverse brain metastatic breast cancers

Basal-like breast cancer (BLBC) shows brain metastatic (BM) capability and overexpresses EGFR and death-receptors 4/5 (DR4/5); however, the anatomical location of BM prohibits efficient drug-delivery to these targetable markers. In this study, we developed BLBC-BM mouse models featuring different patterns of BMs and explored the versatility of estem cell (SC)–mediated bi-functional EGFR and DR4/5-targeted treatment in these models. Most BLBC lines demonstrated a high sensitivity to EGFR and DR4/5 bi-targeting therapeutic protein, E_VDR_L [anti-EGFR VHH (E_V) fused to DR ligand (DR_L)]. Functional analyses using inhibitors and CRISPR-Cas9 knockouts revealed that the E_V domain facilitated in augmenting DR4/5-DR_L binding and enhancing DR_L-induced apoptosis. E_VDR_L secreting stem cells alleviated tumor-burden and significantly increased survival in mouse models of residual-tumor after macrometastasis resection, perivascular niche micrometastasis, and leptomeningeal metastasis. This study reports mechanism based simultaneous targeting of EGFR and DR4/5 in BLBC and defines a new treatment paradigm for treatment of BM.