Immunogenicity of mRNA encoded MUC1 glycopeptides toward CAR-T cells

Vaccines and antibodies targeting hypoglycosylated glycoproteins have been focus of cancer immunotherapy. mRNA vaccines have shown success in inducing effective T cell immunity and antibody responses to viral glycoproteins and tumor antigens. However, the immunogenicity of mRNA-encoded cancer glycoproteins such as MUC1 remains unknown. In this study, we employed chimeric antigen receptor T cells as a caliber to measure the signaling strength of MUC1 glycopeptides encoded by mRNA vaccines. The results showed that glycopeptide within a single tandem repeat showed higher stimulation than glycopeptide bearing multiple tandem repeat sequence. Furthermore, more than five-fold higher stimulation was achieved when MUC1 glycopeptide was fused to GPI-anchor. Molecular mobility of glycopeptide antigens was measured by total internal reflection fluorescence microscopy, and glycosylphosphatidylinositol (GPI)-anchored MUC1 antigens showed five-fold faster diffusion than MUC1 glycopeptide with its own transmembrane domain. Our study reveals complex mechanisms by which the intrinsically disordered region of glycoproteins interacts with CAR-T cells. The combined used of mRNA vaccines targeting CAR-T cells with those targeting B cells, may be further studied to synergize the cellular and humoral arms of immunity. • mRNA vaccines' efficacy for cancer glycoproteins like MUC1 is explored using CAR-T cells as a measure of signaling strength. • Single tandem repeat MUC1 glycopeptides show higher stimulation than those with multiple tandem repeats. • MUC1 glycopeptides fused to GPI-anchored protein achieve over five-fold higher stimulation. • GPI-anchored MUC1 antigens have five-fold faster diffusion than those with original transmembrane domains.