IN VITRO ENZYMATIC POLYSIALYLATION OF THERAPEUTIC ANTIBODIES USING ENGINEERED GLYCOSYLTRANSFERASES
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Antibody-based immunotherapy is one of the most significant medical advancement of the 21st century. Monoclonal antibodies (mAb) and their conjugation variants are found to be effective in treating a myriad of conditions including autoimmune and cancer diseases. mAb is a complex biomolecule consisting of multiple polypeptides and post-translational modifications including disulfide bond and N-linked protein glycosylation. Such complexity contributes to a challenge in biomanufacturing, quality assessment, and storage of the mAb, which leads to a high cost of antibody therapy. Hence one of the major objectives of the mAb engineering is to increase its circulating half-life which will help to both reduce therapy cost and improve health care quality. In this work, we proposed using a cell-free glycoengineering strategy to enhance therapeutic antibody half-life. We employed engineered sialyltransferases derived from Campylobacter jejuni and Neisseria meningitidis to extend IgG-Fc N-glycans at the non-reducing end with the polysialic acid polymer. Using our optimized one-pot two-stage biosynthesis protocol, therapeutic antibody Herceptin modified with polysialic acid structure was efficiently produced and structurally validated. The ongoing works including a more extensive structural characterization as well as ex vivo functional assays will be discussed. Taken together, we anticipate cell-free glycoengineering technology producing polysialylated IgG will provide an attractive route for producing therapeutic mAb with enhanced stability and circulating half-life.