Abstract
Monoclonal antibodies have recently started to deliver on their promise as highly specific and active drugs; however, a more effective, knowledge-based approach to the selection, design, and optimization of potential therapeutic antibodies is currently limited by the surprising lack of detailed structural information for complexes formed with target proteins. Here we show that complexes formed with minimal antigen binding single chain variable fragments (scFv) reliably reflect all the features of the binding interface present in larger Fab fragments, which are commonly used as therapeutics, and report the development of a robust, reliable, and relatively rapid approach to the determination of high resolution models for scFv-target protein complexes. This NMR spectroscopy-based approach combines experimental determination of the interaction surfaces and relative orientations of the scFv and target protein, with NMR restraint-driven, semiflexible docking of the proteins to produce a reliable and highly informative model of the complex. Experience with scFvs and Fabs targeted at a number of secreted regulatory proteins suggests that the approach will be applicable to many therapeutic antibodies targeted at proteins, and its application is illustrated for a potential therapeutic antibody targeted at the cytokine IL-1beta. The detailed structural information that can be obtained by this approach has the potential to have a major impact on the rational design and development of an increasingly important class of biological pharmaceuticals.
Highlights
Removal of over 50% of the RDC data collected resulted in significant variability between the clusters of scFv-IL-1 complexes produced by docking, with no reliable criteria on which to select the correct complex structure
The work reported here clearly demonstrates that an NMR restraint-driven docking approach can be successfully used to determine a reliable and informative model for the structure of a scFv-target protein complex
One limitation of the approach described is the lack of direct experimental data to define the conformations of amino acid side chains in scFvtarget protein interfaces, which in the model reported here are a best computational solution to optimize the interactions of side chains involved in the interface
Summary
Protein Expression and Purification—The scFv, Fab, and IL-1 were expressed as soluble proteins in Escherichia coli and purified using a combination of affinity and size-exclusion chromatography. Backbone amide RDC values were derived from the differences between the 15N-1H scalar couplings for isotropic and partially aligned samples using 15N/1H HSQC and TROSY spectra (13), with acquisition times/spectral widths of 60 ms/14 ppm in F2 (1H) and 50 ms/36 ppm in F1 (15N), and collected for ϳ10 h. Sequence-specific resonance assignments (N, HN, C␣, C, and CЈ) were obtained for the scFv-IL-1 complex from the identification of intra- and inter-residue connectivities in TROSY-based three-dimensional triple-resonance spectra, with additional supporting evidence provided by sequential NOEs observed in 15N/1H NOESY-HSQC spectra. Calculation of a Reliable Model for the scFv-IL-1 Complex—The structure of the scFv-IL-1 complex was determined by NMR restraint-driven docking of IL-1 and the scFv using HADDOCK (25), in which residues involved in interaction sites were defined as semiflexible. Using the programs MOLMOL (30) and PyMOL (DeLano Scientific LLC)
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