Noncompetitive Antibody Neutralization of IL-10 Revealed by Protein Engineering and X-Ray Crystallography

Josephson, Kristopher; Jones, Brandi C.; Walter, Leigh J.; DiGiacomo, Ruth; Indelicato, Stephen R.; Walter, Mark R.

doi:10.1016/s0969-2126(02)00791-8

Cited by 25 publications

(14 citation statements)

References 23 publications

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“…Detailed analysis of the scFv-IL-1␤ interface and comparison with other antibody-antigen structures are summarized in Table 1 and supplemental Table 2. The nature of the interface, in particular the high content of aromatic and non-polar residues and the size of the buried surface area, are consistent with a representative sample of previously reported antibody-target protein complexes (35)(36)(37)(38)(39)(40)(41)(42). Of particular importance for the design and humanization of therapeutic antibodies is a knowledge of which residues from the antibody make interactions with the target protein.…”

Section: Discussionsupporting

confidence: 85%

High Resolution NMR-based Model for the Structure of a scFv-IL-1β Complex

Wilkinson

Hall

Veverka

et al. 2009

Journal of Biological Chemistry

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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-1␤. 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.The ability of antibodies to bind to an almost unlimited number of target proteins with high specificity makes them one of the fastest growing classes of therapeutics in the biological drugs market (1). Since the first description of monoclonal antibodies (2), dramatic progress has been made in the expression, engineering, humanization, and applications of antibodies as therapeutics. A wide variety of antibody fragments have been evaluated as potential therapeutics including the well characterized antigen binding fragment (Fab), which contains the light chain (V L and C L domains) and N-terminal portion of the heavy chain (V H and C H domains). The smallest fragment to retain full binding activity has also attracted considerable interest, with the so-called single chain variable fragment (scFv) 3 (3) consisting of the two variable domains joined by a short peptide.A detailed understanding of the interactions between candidate therapeutic antibodies and target proteins is key to further progress in rational design and humanization. Currently, identification of the binding sites for antibodies on target proteins is achieved via one or a combination of indirect methods such as protease protection, peptide scanning, site-directed mutagenesis, or analysis of backbone amide exchange (4 -6). Although providing valuable information, each of these approaches has drawbacks; in particular, they may not detect discontinuous epitopes and do not provide information on the spatial organization of ep...

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Section: Discussionsupporting

confidence: 85%

High Resolution NMR-based Model for the Structure of a scFv-IL-1β Complex

Wilkinson

Hall

Veverka

et al. 2009

Journal of Biological Chemistry

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“…1C). As previously described, cIL-10M1 is biologically active, and its structure is identical to one subunit of the cIL-10 dimer (10,22,29). Thus, cIL-10M1 provides an important tool for determining accurate binding parameters for the cIL-10⅐IL-10R1 site 1 interface and for mapping the IL-10R2 binding site.…”

Section: Experimental Strategy To Identify the Il-10r2 Binding Site-mentioning

confidence: 84%

Conformational Changes Mediate Interleukin-10 Receptor 2 (IL-10R2) Binding to IL-10 and Assembly of the Signaling Complex

Yoon

Logsdon

Sheikh

et al. 2006

Journal of Biological Chemistry

116

102

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“…To overcome the problem, Josephson et al [44] extended loop DE between Asn116 and Lys117 by insertion of an additional six amino acids (GlyGlyGlySerGlyGly) and showed that the expressed protein folded as a monomer. The crystal structure of monomeric IL-10 [45] confirmed that it is very similar to a single domain of the IL-10 dimer. Further analysis showed that it was able to form a 1:1 complex with IL-10 receptor 1 (IL-10R1); although affinity was 60-fold reduced but the protein still retained biological activity, which was 10-fold lower as compared with the dimeric wild-type IL-10.…”

Section: Monomeric Il-10mentioning

confidence: 65%