Journal Article

An antibody with Fab-constant domains exchanged for a pair of CH3 domains

Becker, Stefan, Gotsmy, Mathias, Rüker, Florian, Wozniak-Knopp, Gordana, Perthold, Jan Walther, Stadlbauer, Katharina, Stadlmayr, Gerhard

2018

Overview

We have designed a complete antibody-like construct where the CH1 and Cκ domains are exchanged for a pair of the CH3 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 CH3 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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Amino acids / Animals / Antibodies, Monoclonal - chemistry / Antibodies, Monoclonal - genetics / Antibodies, Monoclonal - immunology / Antibody-Dependent Cell Cytotoxicity / Antigens / Biology and Life Sciences / Biotechnology / Cell Line / Cytotoxicity / Engineering / ErbB-2 protein / Humans / Immunoglobulin Constant Regions - chemistry / Immunoglobulin Constant Regions - genetics / Immunoglobulin Constant Regions - immunology / Immunoglobulin Fab Fragments - chemistry / Immunoglobulin Fab Fragments - genetics / Immunoglobulin Fab Fragments - immunology / Immunoglobulin G - chemistry / Immunoglobulin G - genetics / Immunoglobulin G - immunology / Immunoglobulins / Immunotherapy / Killer cells / Killer Cells, Natural - immunology / Knobs / Laboratories / Life sciences / Lymphocytes T / Mammalian cells / Medicine and health sciences / Models, Molecular / Monoclonal antibodies / Mutagenesis / Mutagenesis, Site-Directed / Mutation / Natural killer cells / Natural resources / Physical Sciences / Protein Domains / Protein Engineering / Proteins / Receptor, ErbB-2 - immunology / Receptors, IgG - chemistry / Receptors, IgG - genetics / Receptors, IgG - immunology / Recombinant Proteins - chemistry / Recombinant Proteins - genetics / Recombinant Proteins - immunology / Research and Analysis Methods / Solubility / Toxicity / Trastuzumab / Trastuzumab - chemistry / Trastuzumab - genetics / Trastuzumab - immunology