Fourth-generation chimeric antigen receptor T cells targeting mucin 1 for enhanced anti-tumor activity against breast cancer

Breast cancer (BC) is the leading cause of cancer-related mortality worldwide, and current treatments are unmet clinical needs. Although chimeric antigen receptor (CAR) T cells have been approved for hematological malignancies, their advancements in solid tumors are challenging. Mucin 1 (MUC1), particularly its hypoglycosylated tumor-associated form that is largely absent from normal tissues, is an attractive target for T cell-based therapies. Analysis of the cancer genome atlas (TCGA) data revealed that high MUC1 expression in BC is associated with reduced overall survival and disease‑free survival. Here, we showed that 57 of 59 (96.6%) BC samples were MUC1-positive using immunohistochemistry staining. High MUC1 expression was also confirmed in BC cell lines. To characterize and validate efficacy of the fourth‑generation anti‑MUC1 CAR T cells (αM.CAR4), we compared their phenotypes and anti-tumor functions with traditional second‑ and third‑generation CARs in BC models. At baseline, αM.CAR4 T cells displayed immunophenotypes comparable to the traditional CARs. Following antigen exposure, αM.CAR4 T cells demonstrated significantly highest proliferation but lowest exhausted phenotype ( p < 0.05). In addition, they markedly required lower doses to exert higher cytotoxicity ( p < 0.01), produced significantly higher levels of cytotoxic effector proteins, and induced rapid spheroid destruction ( p < 0.05). In summary, MUC1 expression is a potential prognostic biomarker in BC, and αM.CAR4 T cells represent a promising alternative strategy for BC immunotherapy. These findings support further development and evaluation of MUC1-targeting CAR T cells in pre-clinical and clinical studies.