A Comparative Mechanistic Study of Live-Cell Glycocalyx Engineering: Improving Adoptive Cell Therapies Against B Lymphoma

Adoptive cell therapies (ACTs) have achieved remarkable clinical success in treating cancers; however, their broader application is greatly impeded by high cost and restricted antigen specificity. Recently, engineering the glycocalyx has provided a convenient transgene-free means to design ACTs with high-avidity glycan ligands to target CD22, offering a new avenue for B lymphoma immunotherapy. In this work, we perform a comparative analysis of the molecular profiles involved in metabolic or chemoenzymatic glycocalyx engineering and explore their multiplexing capability. The glycoproteomic results revealed content-dependent customization of the NK-92MI glycocalyx. Compared with metabolic engineering, exogenous chemoenzymatic engineering has comparable or even superior ligand-loading efficiency, with some immune synapse components modified to facilitate their spatial recognition against target cells. Next, we tested the orthogonal creation of ligands on natural killer (NK)-92MI cells by further engineering α2,3-sialylated N -acetyllactosamine moieties to produce selectin ligands that are essential for better in vivo eradication of mouse xenograft B lymphoma. Finally, we demonstrate that analogous engineering of CD19-targeted chimeric antigen receptor T (CAR-T) cells to produce CD19/CD22 bitargeted therapy can enhance antigen targeting and tumor cell killing, offering an alternative cost-efficient agent for treating cancer relapse with decreased levels of CD19 antigens. These findings establish a mechanistic foundation for glycocalyx engineering and support the rational design of next-generation ACTs against B lymphoma.