Combining De-Glycosylating Agents with CAR-T Cells for Targeting Solid Tumors and Reducing Toxicity

Background: The adoptive transfer of CAR-T cells have shown impressive results against B-cell malignancies, but still limited efficacy against solid tumors. The discovery of the key factors regulating the activity of CAR-T cells is required to improve their antitumor potency and modulate toxicities. Since solid tumors display a wide range of glycosylation alterations, including increased N-glycan branching, we hypothesized that peptidic epitopes may be masked by glycans from CAR-T cell targeting, especially in richly glycosylated proteins. Results: To investigate if sugar chains may be sterically hulking for CAR-T cell targeting, we generated N-glycosylation-defective pancreatic tumor cell lines. This aim has been achieved by knocking-out the expression of the glycosyltransferase Mgat5, a key enzyme involved in the process of N-glycan branching, using the CRISPR-Cas9 technology. As model antigens for CAR targeting, we focused on CD44v6 and CEACAM-5 (CEA) since they are both heavily glycosylated proteins over-expressed on a wide variety of solid tumors, including pancreatic adenocarcinoma. Strikingly, the impairment of N-glycosylation resulted in a dramatic increase of tumor targeting by both CD44v6 (4-fold, p Conclusions: Our results indicate that i) the glycosylation status of tumor cells regulates the efficacy of CAR-T cells, especially when targeting highly glycosylated antigens, and ii) combining CAR-T cells with the de-glycosylation agent 2DG, which preferentially accumulates in tumor masses, may pave the way for a successful immunotherapy against solid tumors. Disclosures Bonini: Intellia Therapeutics: Research Funding. Bondanza:Novartis: Employment.