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Abstract
Recent studies have demonstrated that IgG-Fc fragments (Fcabs) can be engineered to form antigen-binding sites with antibody properties. Thus they may serve as an attractive alternative to conventional antibodies in therapeutic applications. The critical influence of Fc glycosylation on effector functions of IgGs is well documented; however, whether this applies to Fcabs is not known. Here we used human cells, wild type, and glycoengineered plants to generate four different glycoforms of H10-03-6, an Fcab with engineered HER2/neu-binding sites. Plant-derived H10-03-6 differed in the presence/absence of single oligosaccharide residues, i.e., core fucose and xylose, and terminal galactose. All of the glycoforms had similar binding to HER2/neu expressed on human tumor cells. By contrast, glycoforms that lacked core oligosaccharide modifications (i.e., core α1,3-fucose and β1,2-xylose) showed significantly enhanced binding to the Fcγ receptor IIIa, irrespective of whether plant or human expression systems were used. Consistent with this finding, plant-derived H10-03-6 glycoforms lacking core N-glycan residues mediated higher antibody-dependent cellular cytotoxicity against human tumor cells. No alteration in γ-receptor binding and antibody-dependent cellular cytotoxicity activity was observed upon decoration of N-glycans by terminal galactose. The results point to a significant impact of distinct N-glycan residues on effector functions of Fcabs. Moreover, the outcomes imply that the effector functions mediated by H10-03-6 can be optimized by altering the N-glycosylation profile. Biasing vaccine-induced immune responses toward optimal Fc glycosylation patterns could result in improved vaccine efficacy.
Highlights
Fcabs are small antibody-like molecules that medicate effector functions
The outcomes imply that the effector functions mediated by H10-03-6 can be optimized by altering the N-glycosylation profile
Expression of H10-03-6 in N. benthamiana WT and ⌬XTFT— In this study we used the viral-based magnICON pro-vector system [12] for efficient transient expression of the Fc-based antibody-like molecule with human epidermal growth factor receptor 2 (HER2)/neu-binding sites H10-03-6 [5] in N. benthamiana, a tobacco-related plant species commonly used for recombinant protein expression
Summary
Fcabs are small antibody-like molecules that medicate effector functions. Results: Single N-glycan residues on Fcabs significantly contribute to therapeutically relevant effector functions. Antigen-binding Fc fragments (Fcab) that carry engineered HER2/neu-binding sites in structural loops of Fc fragments were described [5] One of these Fcabs (H10-03-6) showed features of full-length IgG antibodies such as antigen binding, effector functions, and a long half-life [5], suggesting that the Fc-based antigen-binding molecules could serve as an attractive alternative small-sized antibody format. Because of the large endogeneous glycosylation repertoire of mammalian cells, the system commonly used to produce mAbs, it is currently difficult to generate mAbs with homogeneous profiles, which hampers investigation of the possible biological impacts of specific N-glycan residues Despite these shortcomings, clinical trials using therapeutic glycoengineered mAbs are underway, and. These results highlight the importance of N-glycan composition present on Fcabs
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