过继细胞疗法在癌症治疗中取得了显著的临床成效，但其广泛应用仍受限于治疗成本高昂和抗原特异性不足。近年来，糖萼工程化作为一种便捷且非转基因的细胞表面修饰策略，为过继细胞疗法的理性设计提供了新的可能。例如，装配有靶向CD22的高亲和力聚糖配体的过继细胞疗法，已开辟了B淋巴瘤免疫治疗的新途径。本研究系统比较了代谢工程与化学酶工程两种糖萼改造策略在分子层面的特征差异，并评估了其实现多重糖配体修饰的能力。在NK-92MI自然杀伤细胞模型中，糖蛋白组学分析表明，不同糖萼工程方式有赖于细胞表面糖萼的内在特性。相较于代谢工程，化学酶工程展现出相当甚至更优的糖配体负载效率，且对部分免疫突触组分的修饰有望增强细胞对靶细胞的空间识别能力。进一步，在唾液酸工程化修饰基础上，通过将α-2,3-唾液酸化氮-乙酰乳糖胺表位转变为选择素配体，这种糖配体的正交修饰对NK-92MI治疗小鼠异种移植B淋巴瘤至关重要。并且，对CD19靶向嵌合抗原受体T细胞进行唾液酸糖萼工程化，产生的CD19/CD22双靶向细胞展现了更好的抗原靶向和肿瘤杀伤能力，有望为CD19抗原下调的癌症复发提供经济高效的新疗法。综上，本研究不仅从机制层面阐明了不同糖萼工程策略的适用特性，还为B淋巴瘤下一代过继细胞疗法的设计与优化奠定了重要基础。

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 natural killer (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 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.