Site-selective chemoenzymatic glycoengineering of Fab and Fc glycans of a therapeutic antibody

Abstract

The N-glycans attached to the Fab and Fc domains play distinct roles in modulating the functions of antibodies. However, posttranslational site-selective modifications of glycans in antibodies and other multiply glycosylated proteins remain a challenging task. Here, we report a chemoenzymatic method that permits independent manipulation of the Fab and Fc N-glycans, using cetuximab as a model therapeutic monoclonal antibody. Taking advantage of the substrate specificity of three endoglycosidases (Endo-S, Endo-S2, and Endo-F3) and their glycosynthase mutants, together with an unexpected substrate site-selectivity of a bacterial α1,6-fucosidase from Lactobacillus casei (AlfC), we were able to synthesize an optimal homogeneous glycoform of cetuximab in which the heterogeneous and immunogenic Fab N-glycans were replaced with a single sialylated N-glycan, and the core-fucosylated Fc N-glycans were remodeled with a nonfucosylated and fully galactosylated N-glycan. The glycoengineered cetuximab demonstrated increased affinity for the FcγIIIa receptor and significantly enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity.

Unique Microbial Catabolic Pathway for the Human Core N -Glycan Constituent Fucosyl-α-1,6- N -Acetylglucosamine-Asparagine

“…We previously characterized the ␣-L-fucosidase AlfC from L. casei (45) and demonstrated that it displays high regiospecific transglycosylation activity that produces 6=FN disaccharide (47). This enzyme constitutes the only characterized bacterial ␣-Lfucosidase acting on ␣-1,6 linkages in core fucosylation structures (core fucosidase), and it was recently employed as a tool for IgG glycoengineering to obtain defucosylated immunoglobulins with enhanced antibody cell-mediated toxicity (51). Core fucosylation of N-glycopeptides and N-glycoproteins has also been attained using L. casei AlfC mutant enzymes (64).…”

“…The permease AlfH might also transport more complex substrates, like N-glycosylated peptides derived from the proteolysis of host-and food-derived proteins. Furthermore, the fact that AlfC is able to release L-fucose from core fucosylation of the Fc fragment from immunoglobulins (51) shows that this enzyme has the capacity to hydrolyze ␣-1,6 linkages in polypeptidic substrates and could act on core-fucosylated N-glycopeptides. In this case, after the release of L-fucose by AlfC, amino acids would be removed by PepV to liberate GlcNAc-Asn (Fig.…”

Glycoengineering of Antibodies for Modulating Functions

“…Hitherto, it had been a difficult task to selectively modify Fc or Fab glycans of an intact antibody or a multiply glycosylated protein. Recently, Wang and coworkers (101) reported efficient chemoenzymatic synthesis that allows discriminative glycoengineering of both Fc and Fab glycans of cetuximab, a therapeutic antibody. Cetuximab is a chimeric mouse-human anti-EGFR monoclonal antibody that is widely used for the treatment of colorectal, head, and neck cancers (102).…”

New Approaches to Targeting B Cells for Myasthenia Gravis Therapy

“…The mechanism involves preferential binding of Fc to activating-FcγRIIIa (relative to inhibitory-FcγR) on effector cells, as determined by in vitro natural killer cell-based assays. Site-selective glycoengineering of both the Fc and Fab domains of a chimeric anti-epidermal growth factor receptor (EGFR) mAb has recently been shown to lead to increased ADCC activity (76). Future strategies for targeted B cell therapies may further exploit these technological advances by producing engineered mAbs that can potentially enhance B cell killing function and therapeutic efficacy.…”