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

• Published in: Immunology and cell biology 2019-07, Vol.97 (6), p.526-537
• Main Authors: D'Eall, Calvin, Pon, Robert A, Rossotti, Martin A, Krahn, Natalie, Spearman, Maureen, Callaghan, Deborah, Faassen, Henk, Hussack, Greg, Stetefeld, Jörg, Butler, Michael, Durocher, Yves, Zhang, Jianbing, Henry, Kevin A, Tanha, Jamshid
• Format: Article
• Language: English
• Subjects: Adenocarcinoma - immunology; Adenocarcinoma - therapy; Animals; Antibodies; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - metabolism; antibody; Antibody Affinity; antibody engineering; Antibody-Dependent Cell Cytotoxicity; antibody‐dependent cellular cytotoxicity (ADCC); Breast Neoplasms - immunology; Breast Neoplasms - therapy; Camelidae; Cell Line, Tumor; Cytotoxicity; Epidermal growth factor; epidermal growth factor receptor (EGFR); Epidermal growth factor receptors; Epitopes; ErbB Receptors - immunology; ErbB Receptors - metabolism; Female; hinge; Humans; Immunoglobulin Fc Fragments - genetics; Immunoglobulin G; Immunoglobulin G - genetics; Immunoglobulin Heavy Chains - genetics; Immunoglobulin Variable Region - genetics; Immunotherapy - methods; Mutation - genetics; Point mutation; Protein Binding; Protein Engineering; Recombinant Fusion Proteins - genetics; single‐domain antibody; Tumor cells
• ISSN: 0818-9641; 1440-1711
• DOI: 10.1111/imcb.12238

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. The parameters governing antibody (Ab)‐dependent cellular cytotoxicity (ADCC) of heavy chain‐only antibodies (HCAbs) are poorly understood. We found that ADCC of chimeric HCAbs specific for the epidermal growth factor receptor was highly sensitive to hinge sequence, with some chimeric HCAbs bearing long hinges showing dramatically increased ADCC compared with a conventional human IgG1 hinge. However, the effect of hinge engineering was epitope‐dependent.