Impact of afucosylation strategy on antibody function: a comparative study of glycoengineered anti-CD20 antibodies Obinutuzumab and Obinutuzumab beta

Enhancing antibody-dependent cellular cytotoxicity (ADCC) via N-glycan afucosylation of Asn-297 is a validated strategy to improve the clinical efficacy of therapeutic antibodies. However, the impact of distinct glycoengineering approaches on the function of antibodies has not been systematically elucidated. Here, we experimentally compared two type II anti-CD20 antibodies, Obinutuzumab (Gazyva®) and Obinutuzumab beta (MIL62, Bejescin®), which share identical amino acid sequences but exhibit divergent glycosylation profiles. MIL62 was engineered with complete core afucosylation (fucose < 0.1%) lacking bisecting N-acetylglucosamine (GlcNAc) via knockout of the GDP-fucose transporter (GFT) in Chinese hamster ovary (CHO) cells used to produce the antibody. Conversely, Gazyva was produced in CHO cells that overexpress β-1,4-N-acetylglucosaminyltransferase III (GnT-III) and α-mannosidase II (α-ManII), resulting in ~50% fucose content and >80% bisecting GlcNAc occupancy. These distinct glycoengineering strategies led to disparate functional outcomes: afucosylated MIL62 showed improved FcγRIIIA binding and ADCC potency, while Gazyva with bisecting GlcNAc modifications exhibited higher glycoform heterogeneity and reduced thermal stability. Both antibodies displayed comparable FcRn binding, mannosylation, sialylation, and murine pharmacodynamics, mediating complete depletion of B cells in blood, lymph nodes, and spleen. Upon antigen rechallenge, MIL62 suppressed specific antibody titers, indicating memory B-cell eradication and profound potential to prevent autoimmune relapse. This study demonstrates that distinct glycoengineering strategies fundamentally reshape the antibody glycan profile beyond merely reducing fucose. These structural differences not only fine-tune ADCC potency, but also impact antibody stability. Particularly, complete afucosylation of MIL62 led to optimized ADCC potency and effectively suppressed pathogenic B cells repopulation in a delta-like ligand 3 (DLL3) re-challenge model, providing a critical framework for designing next-generation antibodies with superior therapeutic efficacy.