Significance A chemoenzymatic method was developed that permits site-selective Fab and Fc glycan remodeling of intact antibodies. Thus, homogeneous glycoforms of cetuximab were generated in which the immunogenic Fab N-glycans and the less active core-fucosylated Fc N-glycans were precisely remodeled to a desired sialylated N-glycan and an optimal nonfucosylated complex N-glycan, respectively. The glycoengineered cetuximab showed increased affinity for the FcγIIIa receptor and significantly enhanced antibody-dependent cell-mediated cytotoxicity activity. This approach opens a new avenue to manipulating antibody glycosylations to produce specific homogeneous glycoforms useful for functional studies and therapeutic applications.

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.

Monoclonal antibodies are an important class of therapeutic proteins widely used for the treatment of cancer and autoimmune and infectious diseases (1, 2). IgG antibodies contain two heavy chains and two light chains that form three major domains: two identical Fab domains responsible for antigen binding and an Fc domain that is engaged in Fc receptor-mediated effector functions. All IgG1 antibodies contain N-glycans at the highly conserved N297 glycosylation sites (3). It has been shown that the fine structures of the Fc N-glycans (e.g., the status of core fucosylation or terminal sialylation) are critical in defining an antibody’s effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antiinflammatory activities (2, 4⇓⇓⇓–8). On the other hand, Fab glycosylation, which is present in some monoclonal antibodies and about 20% of circulating i.v. immunoglobulins, can play an important role for immunity, antigen recognition, and serum half-life of antibodies (9, 10). Thus, a method to selectively manipulate Fc and Fab glycosylation is highly desirable for better understanding the roles of antibody glycosylation and for developing more efficient antibody-based therapeutics. Unfortunately, the conventional genetic method has been unable to selectively alter the nature of glycans at different sites of a multiply glycosylated protein.

We and others have previously described a chemoenzymatic method for glycan remodeling of glycoproteins (including antibodies) that uses an endoglycosidase and endoglycosynthase pair for deglycosylation and subsequent glycosylation with defined N-glycans (11⇓⇓⇓⇓⇓⇓⇓–19). In particular, we have shown that the Endo-S and Endo-S2 enzymes and their corresponding glycosynthase mutants, including Endo-S D233Q and Endo-S2 D184M, are efficient and highly specific for antibody Fc glycosylation (13⇓–15), whereas the Endo-F3 and its glycosynthase mutant (such as Endo-F3 D165A) demonstrated more relaxed substrate specificity but required core-fucosylated GlcNAc as an acceptor (16, 20). Recently, we have shown that Endo-F3 mutant could achieve certain site-selectivity in enzymatic glycan remodeling of human erythropoietin that carries N-glycans at three distinct sites (20). Despite these progresses, discriminative and site-selective glycan modification of antibodies carrying both Fc and Fab glycosylation remains to be achieved.

Here, we report an efficient chemoenzymatic method that permits highly site-selective enzymatic glycoengineering of both Fc and Fab glycans of cetuximab, a chimeric mouse-human anti-epidermal growth factor receptor (anti-EGFR) therapeutic antibody used for the treatment of colorectal cancer and squamous-cell carcinoma of the head and neck (21). Cetuximab is glycosylated in both Fab and Fc domains at the N88 and N297 sites of the heavy chain, respectively, with tremendous heterogeneity in the N-glycan structures (22, 23). The majority of the Fc N-glycans are core fucosylated, which has been shown to have much lower binding affinity to FcγIIIa receptor (FcγRIIIa) and lower ADCC activity than the corresponding afucosylated antibody. Two glycoengineered anti-EGFR antibodies with low fucose (Fuc) content, imgatuzumab (GA201) and tomuzotuximab (CetuGEX), showed enhanced ADCC activity and improved therapeutic efficacy in clinical trials for treatment of head and neck cancer (24⇓–26). On the other hand, about 30% of the N-glycans in cetuximab are terminated with one or two α1,3Gal epitopes that turn out to be the major cause of anaphylaxis observed in some patients with cetuximab treatment (21, 27). Thus, we sought to develop a method enabling independent glycoengineering of the Fab and Fc glycans to provide a homogeneous glycoform of cetuximab with enhanced ADCC activity and without the allergic αGal epitope. By taking advantage of the substrate specificity of several endoglycosidases and their glycosynthase mutants, together with an unexpected site-selectivity of a bacterial α1,6-fucosidase, we were able to independently remodel the Fc and Fab glycans of cetuximab into a homogeneous glycoform carrying a uniformed sialylated N-glycan at the Fab domain and a nonfucosylated galactosylated N-glycan at the Fc domain that showed significantly enhanced ADCC activity.

Conclusion An efficient chemoenzymatic approach to site-specific glycoengineering of both Fab and Fc glycans of an intact monoclonal antibody is described. This was exemplified by independent Fab and Fc glycan remodeling of the monoclonal antibody cetuximab by taking advantage of the substrate specificity of three endoglycosidases (Endo-S, Endo-S2, and Endo-F3) and their glycosynthase mutants, as well as an unexpected substrate site-selectivity of the AlfC. The resulting 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 replaced with a nonfucosylated N-glycan, demonstrates increased affinity for FcγRIIIa and significantly enhanced ADCC activity. This method opens a new avenue to discriminately manipulate the Fc and Fab glycosylations of monoclonal antibodies to generate biobetter version of antibodies. It also provides a method to construct a library of selectively glycosylated antibody glycoforms for probing the structure–function relationship of antibody glycosylation.

Methods Endo-S and the glycosynthase mutant Endo-S D233Q were expressed and purified as described previously (13). Endo-F3 and the glycosynthase mutant Endo-F3 D165A were expressed and purified as described previously (16). The SCT oxazoline and the asialylated complex type glycan oxazoline were synthesized following previously described procedures (33). The AlfC gene (GenBank CAQ67984.1) was codon optimized for E. coli expression and subcloned into the expression plasmid pCPD-L (34). The enzyme activity was confirmed by assaying with p-nitrophenyl-α-l-fucopyranoside following the previously reported procedures (35). The binding affinity of cetuximab and remodeled cetuximab to FcγRIIIa was determined by SPR. EGRF binding affinity and ADCC activity was measured using the EGFR+ epidermoid carcinoma cell line A431. All glycoengineered intermediates and final products were characterized using LC-MS analysis to confirm glycan removal or transfer. Detailed materials and methods are in SI Appendix, including materials, plasmid construction, enzyme expression and purification, antibody purification and glycoengineering, EGRF binding and ADCC assay, LC-MS analysis of antibodies, and MALDI-TOF MS analysis of released glycans.

Acknowledgments We thank David Knorr (The Rockefeller University) for his assistance in preparation of the manuscript. This work was supported, in part, by the National Institutes of Health Grants R01 GM096973 (to L.-X.W.) and P01 CA190174 and R35 CA196620 (to J.V.R.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Footnotes Author contributions: J.P.G., J.V.L., D.J.D., J.V.R., and L.-X.W. designed research; J.P.G., J.V.L., and D.J.D. performed research; J.P.G., J.V.L., D.J.D., J.V.R., and L.-X.W. analyzed data; and J.P.G., J.V.L., D.J.D., J.V.R., and L.-X.W. wrote the paper.

Reviewers: S.G.W., University of British Columbia; and C.-H.W., Academia Sinica.

The authors declare no conflict of interest.

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