HEK293-6E cells were cultured in Gibco FreeStyle F17 expression medium supplemented with 4 mM L-glutamine,0.1% (w/v) pluronic acid and 50 ug/mL geneticin at 37°C under an atmosphere containing 5% CO2 and 60% humidity, 5% CO2 with 100 rpm shaking. When a density of 1.5–2 9 10e6 cells/mL was achieved, cells were transfected using PEIpro (Polyplus-transfection SA, Illkirch, France). DNA and PEIpro were mixed 1:1 (100 lg each), vortexed three times and incubated for 3 min at room temperature before adding to cells.

Human IgG1 and IgG3 antibodies (Abs) can mediate Ab-dependent cellular cytotoxicity (ADCC), and engineering of the Ab Fc (point mutation; defucosylation) has been shown to affect ADCC by modulating affinity for FcRgammaIIIa. In the absence of a CH 1 domain, many camelid heavy-chain Abs (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 Abs and characterized their affinities for recombinant EGFR and FcRgammaIIIa, 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 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 for four similarly engineered chimeric HCAbs directed against distinct EGFR epitopes. These findings demonstrate that the ADCC of some HCAbs can be modulated simply by varying the length of the Ab 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 Abs for cancer.