An antibody with Fab-constant domains exchanged for a pair of C(H)3 domains

We have designed a complete antibody-like construct where the C(H)1 and C(κ) domains are exchanged for a pair of the C(H)3 domains and efficient pairing of the heavy and light variable domain is achieved using “Knobs-into-Holes” strategy. This construct, composed of only naturally occurring immunoglobulin sequences without artificial linkers, expressed at a high level in mammalian cells, however exhibited low solubility. Rational mutagenesis aimed at the amino acid residues located at the interface of the variable domains and the exchanged C(H)3 domains was applied to improve the biophysical properties of the molecule. The domain-exchanged construct, including variable domains of the HER2/neu specific antibody trastuzumab, was able to bind to the surface of the strongly HER2/neu positive cell line SK-BR3 4-fold weaker than trastuzumab, but could nevertheless incite a more potent response in an antibody-dependent cell cytotoxicity (ADCC) reporter assay with FcγRIIIa-overexpressing T-cells. This could be explained with a stronger binding to the FcγRIIIa. Importantly, the novel construct could mediate a specific ADCC effect with natural killer cells similar to the parental antibody.