Disrupting N-glycan expression on tumor cells boosts chimeric antigen receptor T cell efficacy against solid malignancies

TLDR

CAR‑T cell therapy has been highly effective in B‑cell malignancies but shows limited efficacy in solid tumors due to unique obstacles. The study aims to identify tumor‑derived determinants of CAR‑T recognition and develop strategies to overcome resistance. By genetically deleting MGAT5 and pharmacologically inhibiting N‑glycan synthesis with 2‑deoxy‑d‑glucose, the authors disrupted the tumor N‑glycan shield and PD‑1–PD‑L1 signaling to enhance CAR‑T synapse formation and function. Disruption of tumor N‑glycosylation—via MGAT5 knockout or 2‑deoxy‑d‑glucose treatment—improved CAR‑T cytotoxicity, reduced exhaustion, and achieved tumor regression across multiple solid‑tumor models, demonstrating that N‑glycans regulate CAR‑T efficacy.

Abstract

Immunotherapy with chimeric antigen receptor (CAR)–engineered T cells showed exceptional successes in patients with refractory B cell malignancies. However, first-in-human studies in solid tumors revealed unique hurdles contributing to poor demonstration of efficacy. Understanding the determinants of tumor recognition by CAR T cells should translate into the design of strategies that can overcome resistance. Here, we show that multiple carcinomas express extracellular N-glycans, whose abundance negatively correlates with CAR T cell killing. By knocking out mannoside acetyl-glucosaminyltransferase 5 ( MGAT5 ) in pancreatic adenocarcinoma (PAC), we showed that N-glycans protect tumors from CAR T cell killing by interfering with proper immunological synapse formation and reducing transcriptional activation, cytokine production, and cytotoxicity. To overcome this barrier, we exploited the high metabolic demand of tumors to safely inhibit N-glycans synthesis with the glucose/mannose analog 2-deoxy- d -glucose (2DG). Treatment with 2DG disrupts the N-glycan cover on tumor cells and results in enhanced CAR T cell activity in different xenograft mouse models of PAC. Moreover, 2DG treatment interferes with the PD-1–PD-L1 axis and results in a reduced exhaustion profile of tumor-infiltrating CAR T cells in vivo. The combined 2DG and CAR T cell therapy was successful against multiple carcinomas besides PAC, including those arising from the lung, ovary, and bladder, and with different clinically relevant CAR specificities, such as CD44v6 and CEA. Overall, our results indicate that tumor N-glycosylation regulates the quality and magnitude of CAR T cell responses, paving the way for the rational design of improved therapies against solid malignancies.