Modulating antibody‐dependent cellular cytotoxicity of epidermal growth factor receptor‐specific heavy‐chain antibodies through hinge engineering

Abstract

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 FcRγIIIa. In the absence of a CH1 domain, many camelid heavy‐chain Abs (HCAbs) naturally bear very long and flexible hinge regions connecting their VHH and CH2 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 VHH‐human Fc Abs 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 in comparison with a variant bearing the human IgG1 hinge, in similar fashion to a variant with reduced CH2 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 VHH‐based therapeutic Abs for cancer.

Hinge length contributes to the phagocytic activity of HIV-specific IgG1 and IgG3 antibodies

“…We have previously observed similar induction of NK-degranulation by VRC01 IgG1 and IgG3 [14]. In contrast, a recent study found that hinge extension improved ADCC for one monoclonal antibody, but that this effect was not generalizable across specificities that recognized different epitopes on the same antigen [83]. Recognizing that there are numerous means to assess ADCC, we tested VRC01 hinge variant activity using HIV-1 Infectious Molecular Clone-infected target cells and assessing the reduction in luciferase expression as readout for cellular cytotoxicity [84].…”

FcγR Binding and ADCC Activity of Human IgG Allotypes

“…Several studies have found that the length and flexibility of an antibody influences FcγR binding and ADCC activity (62)(63)(64)(65). However, there is no general consensus whether a short or a long hinge is beneficial for ADCC activity.…”

A highly expressing, soluble, and stable plant-made IgG fusion vaccine strategy enhances antigen immunogenicity in mice without adjuvant

“…For example, S239D and I332E mutations improved FcγRIII binding and ADCC activity of a CD37 antibody (Heider et al, 2011; Lazar et al, 2006). Replacing the IgG1 hinge region with the longer hinge region from IgG3 or from a camelid antibody increased ADCC of an epidermal growth factor receptor antibody (D’Eall et al, 2019), while an IgG1 with E345R, E430G, and S440Y mutations formed hexamers that had greatly enhanced C1q binding and complement activation (Diebolder et al, 2014). The mutations T437R and K248E also promoted multimerization and improved effector functions of an OX40 receptor antibody (Zhang et al, 2017).…”