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Protein Science (2004), 13:1476-1488. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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A novel and conserved pocket of human {kappa}-Fab fragments: Design, synthesis, and verification of directed affinity ligands

Enrique Carredano, Herbert Baumann, Anna Grönberg, Nils Norrman, Gunnar Glad, Jinyu Zou, Oguz Ersoy, Elles Steensma and Andreas Axén

Amersham Biosciences, Björkgatan 30, S-751 84 Uppsala, Sweden

(RECEIVED February 10, 2004; FINAL REVISION March 22, 2004; ACCEPTED March 22, 2004)



Abstract

Antibodies of type IgG may be divided into two classes, called {lambda}or {kappa}, depending on the type of light chain. We have identified a conserved pocket between the two domains CH1 and CL of human IgG {kappa}-Fab, which is not present in the {lambda} type. This pocket was used as a target docking site with the purpose of exploring the possibilities of designing affinity ligands that could function as such even after immobilization to gel. The idea of the design arose mainly from the results of the saturated transfer difference (STD-NMR) screening of 46 compounds identified by means of virtual docking of 60 K diverse compounds from the Available Chemicals Directory (ACD). Surface plasmon resonance (SPR) was used as an alternative method to monitor binding in solution. A total of 24 compounds belonging to a directed library were designed, synthesized, and screened in solution. They consist essentially of an amino acid condensed to a N,N'-methylated phenyl urea. STD-NMR results suggest that a small hydrophobic side chain in the condensed amino acid promotes binding, whereas a hydroxyl-group–containing side chain implies absence of STD-NMR signals. Three compounds of the directed library were immobilized and evaluated as chromatographic probes. In one case, using D-Pro as the condensed amino acid, columns packed with ligand-coupled Sepharose (Amersham Biosciences) retained two different monoclonal samples of {kappa}-Fab fragments with different variable regions, whereas a sample of monoclonal {lambda}-Fab fragments was not retained under similar chromatographic conditions.

Keywords: affinity; chromatography; Fab; IgG; ligand; separation; SPR; STD-NMR; structure-based design; virtual screening

Abbreviations: ACD, Available Chemicals Directory • CH1, first constant domain of the heavy chain • CIP, cleaning in place • CL, constant domain of the light chain • CV, column volume • DCM, dichloromethane • DMF, N,N-dimethylformamide • DMSO, dimethyl sulfoxide • LC-MS, liquid-chromatography mass spectroscopy • MAS-HR, magic angle spin high resolution • RP-HPLC, reversed-phase high-performance liquid chromatography • RU, response units • SPR, surface plasmon resonance • STD, saturation transfer difference • THF, tetrahydro furan • VH, variable domain of the heavy chain • VL, variable domain of the light chain

Reprint requests to: Andreas Axén, Amersham Biosciences R&D, Björkgatan 30, S-751 84 Uppsala, Sweden, e-mail: andreas.axen{at}amersham.com; fax: 46-18-612-18-44.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04687404.


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