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Background CAR-T cell therapy has shown limited efficacy in solid tumors due to factors such as antigen heterogeneity, immunosuppressive microenvironments, and physical barriers. 1–7 The glycocalyx, a dense layer of membrane-bound mucins, including MUC1, can physically block CAR-T cell access to target antigens. In pancreatic cancer, the overexpression of truncated MUC1 (Tn-MUC1) and the Tn antigen results in a tumor-specific glycoform. While this glycoform offers a selective target, it also reinforces the mucin barrier and contributes to immune evasion.8–13 Methods CAR-T cells were engineered to express a non-signaling glycan-binding receptor, referred to here as a glyco-bridge, which binds to glycoproteins or glycans such as Tn-MUC1 and enhances CAR-T cell adhesion and activation. Initially, a Tn-MUC1 scFv was utilized, and we later developed tandem Helix pomatia agglutinin (HPA) lectins as a glyco-bridge to enable broader recognition of the Tn antigen ( figure 1). CAR-T cell function was evaluated both in vitro and in vivo using cell lines, the Capan-2 model, and a patient-derived xenograft (PDX) model of pancreatic cancer. Tissues were harvested to assess off-tumor T cell infiltration.Results The Tn-MUC1-based bridge not only facilitates CAR-T cell infiltration by >8-fold but also enhances avidity by 1.3-fold toward target cells. Additionally, it activates CAR signaling in a manner dependent on both the density of bridge-target antigens and the affinity of the antigen-binding domain. HPA-based CAR-T cells exhibited toxicity due to on-target/off-tumor binding. To mitigate this, we next incorporate HPA lectins into the glyco-bridge approach to enable selective targeting of universal Tn antigens on cancer cells. This toxicity was effectively mitigated by placing HPA within a non-signaling glyco-bridge. Using the same donors, glyco-bridge CAR-T cells showed improved tumor control and overall survival in the Capan-2 model, without systemic toxicity. In the PDX pancreatic cancer model, dual HPA glyco-bridge CAR-T cells exhibited significantly enhanced tumor regression (~46%; P = 0.00206) and survival compared to the control bridge.Conclusions Our study presents a proof-of-concept that equipping CAR-T cells with a Tn-MUC1 binder and a Tn antigen binder targeting the glycocalyx significantly enhances CAR-T efficacy in pancreatic cancer models in vitro and in vivo. To better understand the underlying mechanisms of enhanced CAR-T cell activity, we investigated how the glyco-bridge enhanced CAR-T cell efficacy using in vitro co-culture assays, acoustic force microscopy, and mouse xenograft models. Our data suggest that the glyco-bridge increased cell avidity and improved tumor penetration, offering a novel approach to engage the cancer cell glycocalyx without directly initiating cytotoxicity.Acknowledgements We thank the core facilities at the MGH Cancer Center: Flow Cytometry, Histopathology, and Blood Bank. S.P. is a Merck Fellow of the Damon Runyon Cancer Research Foundation (DRG-2529-24). DSB received funding from the CRIS Foundation Out-Back Fellowship Programme (outback2021_6) and from the Spanish Society of Medical Oncology (SEOM). FB received funding from the American-Italian Cancer Foundation (AICF) and the Italian Association for Cancer Research (AIRC). This work was funded by NIH R01 CA238268 (MVM).References Ghasempour S, Freeman SA. The glycocalyx and immune evasion in cancer. FEBS J. 2021 Oct 19.Öhlund D, Elyada E, Tuveson D. Fibroblast heterogeneity in the cancer wound. J Exp Med. 2014 Jul 28;211(8):1503–23.Connor AA, Gallinger S. 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