Engineering tandem <scp>VHHs</scp> to target different epitopes to enhance antibody‐dependent cell‐mediated cytotoxicity

Engineering antibodies to elicit antibody-dependent cell-mediated cytotoxicity (ADCC) can be used to eliminate target cells. Here, we described how single domain VHH arms on a bispecific antibody (BsAb) format can be engineered to modulate cell binding and ADCC activity. The BsAbs, comprising two anti-epidermal growth factor receptor (EGFR) nanobodies, 7D12 and EGA1, were engineered onto a human IgG1 Fc domain in monovalent, bivalent, and tandem formats. While 7D12 had a stronger ADCC activity than EGA1, the tandem 7D12-EGA1 mediated a significantly stronger ADCC activity without significant changes in cell binding in numerous cancer cell lines. In addition, we present how the molecular design of the tandem 7D12 and EGA1 nanobodies could cross-link two different EGFR molecules to obtain stronger ADCC activity.