Bisecting <i>N</i>‑Acetylglucosamineof the <i>N</i>‑Glycan of Immunoglobulin G Does NotAffect Binding to Fc Gamma Receptors

Monoclonal antibodies (mAb) produced in 1,4-mannosyl-glycoprotein 4-<i>N</i>-acetylglucosaminyltransferase (MGAT3) overexpressing cell lines have superior <i>in vitro</i> and <i>in vivo</i> activities. The <i>N</i>-glycan of the Fc-region of these mAbs have increased levels of bisecting <i>N</i>-acetylglucosamine (GlcNAc) and reduced core-fucosylation. Although a reduction in core-fucosylation will improve FcγRIIIa binding and antibody-dependent cellular cytotoxicity (ADCC) activity, the influence of bisecting GlcNAc on these activities has been difficult to probe. Here, we describe the preparation of a unique series of homogeneous glycoforms of trastuzumab (Herceptin) with and without core-fucose and with and without bisecting GlcNAc and examine binding to a comprehensive panel of Fcγ receptors. The glycoforms of trastuzumab were prepared by treatment with wild-type Endo-S2 to cleave the chitobiose core of the <i>N</i>-glycan to leave GlcNAc-Fuc that was exposed to an α-fucosidase to provide trastuzumab-GlcNAc. Glycan oxazolines with and without bisecting GlcNAc were prepared by enzymatic remodeling of a sialoglycopeptide isolated from egg yolk powder, which were employed in transglycosylations with trastuzumab-GlcNAc and trastuzumab-GlcNAc-Fuc catalyzed by Endo-S2 D184M resulting in well-defined glycoforms. As expected, core-fucosylation had a major effect on FcγRIIIa binding, which was not influenced by the presence of bisecting GlcNAc. It was found that an A2-glycan (GlcNAc₂Man₃GlcNAc₂) modified by bisecting GlcNAc cannot be core-fucosylated by FUT8. Thus, bisecting GlcNAc has only an indirect influence on FcγRIIIa binding and subsequent ADCC activity by inhibiting core-fucosylation. The results described here provide an understanding of the properties of therapeutic monoclonal antibodies.