One‐Step Glycoengineering of NK Cells With High‐Affinity Siglec Ligands for Cancer Immunotherapy

ABSTRACT Siglecs, a family of sialic acid (Sia)‐binding immunomodulatory receptors selectively expressed on immune cells, are promising immunotherapeutic targets. While synthetic Sia derivatives can manipulate the Sia‐Siglec axis with high affinity, their therapeutic application has been hampered by the poor substrate tolerance of wild‐type CMP‐sialic acid synthase (CSS) for sterically demanding analogs. Here, we report a structure‐guided engineering strategy to evolve Neisseria meningitidis CMP‐Sia synthetase (NmCSS) for enhanced activity with bulky substrates. Coupling this optimized NmCSS variant with a sialyltransferase enabled a scalable “one‐pot two‐enzyme” (OPTE) synthesis of diverse sialoside analogs. Screening this library on glycan microarrays revealed novel high‐affinity ligands with selective Siglec binding profiles. Leveraging the OPTE system, we achieved single‐step glycoengineering of natural killer (NK)‐92MI cells with tailored Siglec‐2 or Siglec‐3 ligands, which exhibited potent cytotoxicity against B‐cell lymphoma (Siglec‐2 ⁺ ) and acute myeloid leukemia (Siglec‐3 ⁺ ) models. These engineered NK cells displayed significantly enhanced tumor killing capacity, mediated by enhanced granzyme release and cytokine production while maintaining excellent cell viability. This modular platform addresses critical limitations in enzymatic synthesis of modified sialosides and their efficient display on therapeutic cells. Our work establishes a versatile and practical platform for developing next‐generation immunotherapies that precisely target the Sia‐Siglec axis with improved specificity and functionality.