Biparatopic CAR-T cells targeting full-length MUC1 and truncated MUC1β circumvent MUC1α shedding in ovarian cancer

Background MUC1 is a transmembrane protein consisting of α and β subunits and is overexpressed in many cancers. Most MUC1-targeted CAR-T strategies focus on the shed-prone MUC1α subunit, making them potentially vulnerable to epitope loss after MUC1α shedding. Meanwhile, neoepitopes on the membrane-retained truncated MUC1β exposed after MUC1α shedding have received relatively limited attention. Here, we report the first biparatopic CAR targeting both full-length MUC1 and truncated MUC1β. Methods We screened alpaca immune antibody libraries to isolate an antibody targeting truncated MUC1β. We performed multiplex immunohistochemistry with two antibodies recognizing truncated MUC1β and tumor-associated MUC1α glycoforms respectively in ovarian cancer tissues, to define MUC1 forms in situ. We then engineered MUC1 biparatopic CAR-T cells targeting both full-length MUC1 and truncated MUC1β, and examined their functionality in ovarian cancer cell lines in vitro, in vivo xenograft models, and patient-derived tumor-like cell clusters. We also evaluated the impact of soluble MUC1α on MUC1 biparatopic CAR-T cells. Results We found that MUC1 commonly exists in a partially shed state, with co-occurrence of full-length MUC1 and truncated MUC1β in ovarian cancer tissue. In vitro, MUC1 biparatopic CAR-T cells showed broader target coverage, efficiently recognizing and killing MUC1-positive ovarian cancer cells regardless of whether MUC1 was present as full-length MUC1 alone, truncated MUC1β alone, or both, whereas monospecific CAR-T cell killed only target cells displaying its corresponding epitope. MUC1 biparatopic CAR-T cells also showed higher cytotoxicity and cytokine release against targets co-expressing full-length MUC1 and truncated MUC1β. Notably, soluble CA153 did not impair the killing ability of MUC1 biparatopic CAR-T cells and instead induced CAR-T activation. MUC1 biparatopic CAR-T cells exhibited superior tumor control in ovarian tumor xenografts and enhanced activity against ovarian cancer patient-derived tumor-like cell clusters. Conclusions Our study establishes partial MUC1α shedding as a prevalent and exploitable feature of ovarian cancer, and supports biparatopic CAR targeting full-length MUC1 and shedding-exposed truncated MUC1β as a promising strategy to circumvent antigen shedding–mediated escape in MUC1-positive ovarian cancer.