Directing CAR NK Cells via the Metabolic Incorporation of CAR Ligands into Malignant Cell Glycans.

The abundance of sialic acid-containing glycans in the glycocalyx of malignant cells enables immune evasion. Here, we leverage the biosynthetic pathways that permit pervasive sialylation to incorporate a chimeric antigen receptor (CAR) ligand into malignant cell glycans, and demonstrate that this increases the susceptibility of malignant cells to the cytolytic activity of CAR-expressing natural killer (NK) cells. Specifically, we applied a C-9-functionalized nonnatural sialic acid [i.e., fluorescein sialic acid (FL-SA)] to modify malignant cell glycans. We confirm the metabolic incorporation of FL-SA into plasma membrane-associated glycans. The preparation of anti-fluorescein CAR NK cells permitted studies demonstrating that treating malignant cells with FL-SA increased susceptibility to CAR NK cell-mediated cytolysis. Furthermore, we observed that the specificity of the anti-fluorescein CAR NK cells is enhanced for fluorescein-labeled cells, and an increased release of cytokines from the CAR NK cells upon incubation with FL-SA-treated cells. The results arising from this study demonstrate that CAR ligands can be metabolically incorporated into malignant cells, and we reason that such strategies could be leveraged to tackle the issue of antigen heterogeneity that limits the clinical efficacy of CAR T/NK cell therapies.