CAR T-cell Efficacy in Solid Tumors Is Affected by N-glycosylation

The ability to form a lytic immune synapse underlies CAR therapeutic potential, with antigen density and accessibility playing a major role in this process. Aberrant glycosylation on tumor cells can disrupt the ability to form these immune synapses, but preclinical evaluation of CAR efficacy has yet to take this feature into account. Therefore, Greco and colleagues knocked out expression of MGAT5, which encodes for N-acetylglucosaminyltransferase-V, to generate N-glycosylation–defective pancreatic cancer cells and assessed CAR T-cell tumor targeting using CD44v6 as the CAR antigen (termed 44v6.28ζ CARs). Reduction of branched N-glycans was found to increase antitumor efficacy of the CARs, leading to enhanced cytolytic activity and secretion of interferon-γ and TNF-α. Furthermore, the immunologic synapse engaged between 44v6.28ζ CARs and MGAT5 knockout pancreatic cancer cells was superior with a greater degree of T-cell activation. To pharmacologically inhibit glycosylation, 2DG was used, as it has been previously reported to block N-linked glycosylation. Pancreatic cancer cells treated with 2DG were more efficiently killed by 44v6.28ζ CARs, and the addition of exogenous mannose confirmed this was due to N-glycosylation inhibition as it blocked the effects of 2DG exposure. Using in vivo models of either low or high tumor burden indicated tumor control occurred in the context of low tumor burden regardless of 2DG treatment, but in the high tumor burden models, growth was more effectively controlled in mice treated with both 44v6.28ζ CARs and 2DG. Specifically, 44v6.28ζ CARs exhibited a reduced exhaustion profile as well as a reduction in inhibitory receptors like PD-1. Additionally, cotreatment regimens were more efficacious at reducing tumor burden as compared to both individual treatments and improved the survival of tumor-bearing mice with similar effects seen in bladder, lung, and ovarian tumors as well as when the CAR antigen was altered to carcinoembryonic antigen. This study supports the role of N-glycans in reducing the efficacy of CAR T-cell therapy and suggests a new direction for the use of this therapy in solid tumors. ■

Greco B, Malacarne V, De Girardi F, Scotti GM, Manfredi F, Angelino E, et al. Disrupting N-glycan expression on tumor cells boosts chimeric antigen receptor T cell efficacy against solid malignancies. Sci Transl Med 2022;14:eabg3072.

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