Bio‐Orthogonal T Cell Targeting Strategy for Robustly Enhancing Cytotoxicity against Tumor Cells

T cells can kill tumor cells by cell surface immunological recognition, but low affinity for tumor‐associated antigens could lead to T cell off‐target effects. Herein, a universal T cell targeting strategy based on bio‐orthogonal chemistry and glycol‐metabolic engineering is introduced to enhance recognition and cytotoxicity of T cells in tumor immunotherapy. Three kinds of bicycle 6.1.0] nonyne (BCN)‐modified sugars are designed and synthesized, in which Ac₄ManN‐BCN shows efficient incorporation into wide tumor cells with a BCN motif on surface glycans. Meanwhile, activated T cells are treated with Ac₄GalNAz to introduce azide (N₃) on the cell surface, initiating specific tumor targeting through a bio‐orthogonal click reaction between N₃ and BCN. This artificial targeting strategy remarkably enhances recognition and migration of T cells to tumor cells, and increases the cytotoxicity 2 to 4 times for T cells against different kinds of tumor cells. Surprisingly, based on this strategy, the T cells even exhibit similar cytotoxicity with the chimeric antigen receptor T‐cell against Raji cells in vitro at the effector: target cell ratios (E:T) of 1:1. Such a universal bio‐orthogonal T cell‐targeting strategy might further broaden applications of T cell therapy against tumors and provide a new strategy for T cell modification.