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Abstract
Growing numbers of therapeutic antibodies offer excellent treatment strategies for many diseases. Elucidation of the interaction between a potential therapeutic antibody and its target protein by structural analysis reveals the mechanism of action and offers useful information for developing rational antibody designs for improved affinity. Here, we developed a rapid, high-yield cell-free system using dialysis mode to synthesize antibody fragments for the structural analysis of antibody–antigen complexes. Optimal synthesis conditions of fragments (Fv and Fab) of the anti-EGFR antibody 059–152 were rapidly determined in a day by using a 30-μl-scale unit. The concentration of supplemented disulfide isomerase, DsbC, was critical to obtaining soluble antibody fragments. The optimal conditions were directly applicable to a 9-ml-scale reaction, with linear scalable yields of more than 1 mg/ml. Analyses of purified 059-152-Fv and Fab showed that the cell-free synthesized antibody fragments were disulfide-bridged, with antigen binding activity comparable to that of clinical antibodies. Examination of the crystal structure of cell-free synthesized 059-152-Fv in complex with the extracellular domain of human EGFR revealed that the epitope of 059-152-Fv broadly covers the EGF binding surface on domain III, including residues that formed critical hydrogen bonds with EGF (Asp355EGFR, Gln384EGFR, H409EGFR, and Lys465EGFR), so that the antibody inhibited EGFR activation. We further demonstrated the application of the cell-free system to site-specific integration of non-natural amino acids for antibody engineering, which would expand the availability of therapeutic antibodies based on structural information and rational design. This cell-free system could be an ideal antibody-fragment production platform for functional and structural analysis of potential therapeutic antibodies and for engineered antibody development.
Highlights
Antibody therapy offers effective treatment strategies for many diseases with a highly specific binding of a monoclonal antibody to its target antigen protein, when its inactivation leads to the suppression of the disease [1]
We show that a dialysis-mode cell-free system based on Escherichia coli (E. coli) S30 extract is a powerful tool for producing antibody fragments for structural analysis, including by X-ray crystallography
Bacterial disulfide isomerase, DsbC was introduced to the cell-free reaction to reshuffle incorrectly formed disulfide bonds. 059-152-Fv was synthesized in the presence of three different concentrations of DsbC (0, 0.2, or 0.4 mg/ml). 059-152-VH and 059-152-VL were mostly soluble under all three conditions
Summary
Antibody therapy offers effective treatment strategies for many diseases with a highly specific binding of a monoclonal antibody to its target antigen protein, when its inactivation leads to the suppression of the disease [1]. Epidermal growth factor (EGF) binding to the ECD induces dimerization of EGFR, which leads to activation of the cytoplasmic tyrosine kinase domain and subsequent stimulation of cell growth and differentiation [4]. The antigen-binding fragment (Fab) of cetuximab or IMC-11F8 binds to the EGF-binding site on domain III of EGFR, thereby preventing EGF-induced activation [5, 6]. The matuzumab Fab fragment interacts with domain III of EGFR, where apart from the EGF-binding site, and it sterically blocks the domain rearrangement of EGFR to the EGF-binding conformation [7]. 059–152 IgG inhibits the binding of EGF to EGFR as well as the phosphorylation of EGFR, the molecular mechanism is not well understood
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