(719e) Glycosylation of Full-Length Antibodies in E. coli Equipped with a Bacterial Single-Subunit Oligosaccharyltransferase

Asparagine-linked (N-linked) glycosylation is a complex post-translational modification found in most major therapeutic proteins including monoclonal antibodies (mAbs) and fragment crystallizable (Fc) domain fusion proteins. This modification plays a critical role in shaping the glycan structures that determine the structural and functional properties of the therapeutic drug including half-life, Fc effector functions, and anti-inflammatory activity. Typically, full length mAbs are produced in mammalian cell lines capable of achieving complex human-like N-glycosylation. However, using mammalian cell lines for mAbs production is costly, complex, and susceptible to viral contamination. Therefore, there is a need for developing simpler processes that boost productivity and provide control over the glycosylation of mAbs. As an alternative, new protein glycosylation systems have arisen to understand, control, and customize the production of glycoproteins. In bacteria, the glycosylation machinery from Campylobacter jejuni has been successfully transferred to Escherichia coli, including the oligosaccharyltransferase (OST) known as PglB in bacteria. PglB is responsible for transferring the N-glycan from lipid-linked oligosaccharide (LLO) donors to asparagine residues of an acceptor protein. However, despite the remarkable advancements in our understanding of N-glycosylation processes in E. coli, the replication of eukaryotic-like glycosylation in bacteria continues to be a significant challenge in part to the lack of available PglB enzymes that can install N-linked glycans within the QYNST sequon of the Fc domain of mAbs. The archetypal PglB from C. jejuni requires an extended sequon, (D/E_-2-X_-1-N-X₊₁-S/T₊₂ where X≠P), in the acceptor protein to catalyze glycosylation. This requirement renders this enzyme incompatible with proteins such as mAbs that lack this more specific acceptor sequence. To address this challenge, we previously mined the genomes of diverse bacterial species for PglB homologs with relaxed sequon specificity. While several enzymes with relaxed sequon specificity were uncovered, most notably PglB from Desulfovibrio gigas, these enzymes exhibited low efficiency (<2%) against the QYNST sequon. Here, we hypothesized that PglB homologs from other Desulfovibrio species might exhibit increased catalytic efficiency for non-extended sequons, thereby enabling glycosylation of broad substrates including mAbs. To test this hypothesis, we assembled a collection of 19 PglB candidates from Desulfovibrio species. Among them, we identified a previously uncharacterized PglB from the bacterium Desulfovibrio marinus that exhibited greatly relaxed substrate specificity. DmPglB was the only enzyme able to efficiently glycosylate any sequon in multiple acceptor protein targets regardless of the residue at the -2 position. Estimation of the acceptor-site preferences of DmPglB using a high-throughput genetic assay for N-glycosylation activity indicated that this enzyme prefers the X_-2-X_-1-N-S₊₁-T₊₂ sequon, which captures the more specific QYNST sequon found in all mAbs. As a result, for the first time, a bacterial enzyme has successfully catalyzed glycosylation of a native QYNST sequon in the context of both a hinge-Fc fragment and a full-length mAbs expressed in E. coli. Although the attached glycans attached in the E. coli-derived hinge-Fc were bacterial in origin, these glycans were trimmed and subsequently remodeled in vitro by enzymatic transglycosylation, creating a recombinant product bearing homogeneous asialo complex-type biantennary N-glycan (G2). Importantly, the resulting G2-hinge-Fc exhibited strong binding to human FcγRIIIa (CD16a), one of the most potent receptors for eliciting antibody-dependent cellular cytotoxicity (ADCC). Taken together, the discovery of DmPglB provides previously unavailable biocatalytic capabilities to the bacterial glycoprotein engineering toolbox and opens the door to using E. coli for the production and glycoengineering of human mAbs and fragments derived thereof.