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

Area of Science:

• Biochemistry
• Immunology
• Synthetic Biology

Background:

• Siglecs are immune cell receptors that bind sialic acids (Sia) and are key targets for immunotherapy.
• Current methods for synthesizing Sia derivatives are limited by enzyme substrate tolerance, hindering therapeutic applications.

Purpose of the Study:

• To engineer a CMP-sialic acid synthase (CSS) for improved activity with bulky Sia analogs.
• To develop a scalable synthesis platform for diverse sialoside analogs.
• To apply engineered cells for targeted cancer immunotherapy.

Main Methods:

• Structure-guided engineering of Neisseria meningitidis CMP-Sia synthetase (NmCSS) for enhanced activity with sterically demanding substrates.
• One-pot two-enzyme (OPTE) synthesis of sialoside analogs using engineered NmCSS and a sialyltransferase.
• Glycoengineering of natural killer (NK)-92MI cells with tailored Siglec ligands.
• In vitro assessment of engineered NK cell cytotoxicity against B-cell lymphoma and acute myeloid leukemia models.

Main Results:

• An optimized NmCSS variant demonstrated enhanced activity with bulky Sia substrates.
• The OPTE system enabled scalable synthesis of diverse sialoside analogs with selective Siglec binding profiles.
• Engineered NK-92MI cells displayed potent cytotoxicity against Siglec-2+ and Siglec-3+ cancer models.
• Enhanced NK cell functionality included increased granzyme release and cytokine production with maintained viability.

Conclusions:

• A modular platform was established for enzymatic synthesis of modified sialosides and their display on therapeutic cells.
• This approach overcomes limitations in synthesizing and utilizing Sia analogs for immunotherapy.
• The engineered NK cells show promise for next-generation immunotherapies targeting the Sia-Siglec axis with improved specificity and efficacy.