Modulation of antibody-dependent cellular cytotoxicity of EGFR-specific heavy-chain antibodies through hinge engineering.

Human IgG1 and IgG3 antibodies can mediate antibody-dependent cellular cytotoxicity (ADCC), and engineering of the antibody Fc (point mutation; defucosylation) has been shown to affect ADCC activity by modulating affinity for FcRγIIIa. In the absence of a CH 1 domain, many camelid heavy-chain antibodies (HCAbs) naturally bear very long and flexible hinge regions connecting their VH H and CH 2 domains. To better understand the influence of hinge length and structure on HCAb ADCC, we produced a series of hinge-engineered epidermal growth factor receptor (EGFR)-specific chimeric camelid VH H-human Fc antibodies and characterized their affinities for recombinant EGFR and FcRγIIIa, their binding to EGFR-positive tumor cells, and their ability to elicit ADCC. In the case of one chimeric HCAb (EG2-hFc), we found that variants bearing longer hinges (IgG3 or camelid hinge regions) showed dramatically improved ADCC activity in comparison with a variant bearing the human IgG1 hinge, in similar fashion to a variant with reduced CH 2 fucosylation. Conversely, an EG2-hFc variant bearing a truncated human IgG1 upper hinge region failed to elicit ADCC. However, there was no consistent association between hinge length and ADCC activity for four similarly-engineered chimeric HCAbs directed against distinct EGFR epitopes. These findings demonstrate that the ADCC activity of some HCAbs can be modulated simply by varying the length of the antibody hinge. Although this effect appears to be heavily epitope-dependent, this strategy may be useful to consider during the design of VH H-based therapeutic antibodies for cancer. This article is protected by copyright. All rights reserved.