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Publication Detail
Reversible dimer formation and stability of the anti-tumour single-chain Fv antibody MFE-23 by neutron scattering, analytical ultracentrifugation, and NMR and FT-IR spectroscopy
  • Publication Type:
    Journal article
  • Publication Sub Type:
  • Authors:
    Lee YC, Boehm MK, Chester KA, Begent RHJ, Perkins SJ
  • Publication date:
  • Pagination:
    107, 127
  • Journal:
    Journal of Molecular Biology
  • Volume:
  • Issue:
  • Print ISSN:
  • Keywords:
    Antibodies, Antibody, dimer formation, FTIR, Fv antibody, neutron scattering, NMR, NMR spectroscopy, SPECTROSCOPY, stability, ultrcentrifugation, AFFINITIES, affinity, analytical, analytical ultracentrifugation, analytical ultracentrifuge, anti-tumour, antibodies, ANTIGEN, As, Association, Back, C terminal, cancer, Carcinoembryonic Antigen, CEA, CHAIN, Chromatography, Colorectal, Colorectal Cancer, COLORECTAL-CANCER, Concentration, CREATION, CRYSTAL, CRYSTAL STRUCTURE, crystal structures, CRYSTAL-STRUCTURE, CRYSTAL-STRUCTURES, CURVE-FITTING PROCEDURE, domain, DOMAINS, EGG-WHITE LYSOZYME, English, equilibrium, experiments, Form, formation, FORMS, H-1 NMR spectroscopy, H1, HIGH-AFFINITY, M1, MARKER, MED, model, modelling, Molecule, MOLECULES, MONOCLONAL-ANTIBODY, Monomer, nature, Neutron Scattering, Other, PHAGE DISPLAY LIBRARIES, PRESS, PROTEIN SECONDARY STRUCTURE, RD, RELEVANT, reversible, rights, Royal, S, scattering, scFv, Science, sedimentation, SEDIMENTATION-VELOCITY, self-association, SINGLE, SINGLE-CHAIN FV, single-chain Fv antibody, size-exclusion chromatography, spectroscopy, ST, starting, Structure, SURFACE, target, TERM, thermal, TIME, TRANSFORM INFRARED-SPECTROSCOPY, Tumour, Ultracentrifugation, VALUES, VELOCITY, WEIGHT, X-ray
  • Notes:
    Journal English Article ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD JUN 28 569NZ LONDON Perkins SJ Royal Free & Univ Coll Med Sch, Dept Biochem & Mol Biol, Gower St, London WC1E 6BT, England J MOL BIOL 24-28 OVAL RD, LONDON NW1 7DX, ENGLAND
MFE-23 is a single chain Fv (scFv) antibody molecule used to target colorectal cancer through its high affinity for the tumour marker carcinoembryonic antigen (CEA). ScFv molecules are formed from peptide-linked antibody V-H and V-L domains, and many of these form dimers. Our recent crystal structure for MFE-23 showed that this formed an unusual symmetric back-to- back association of two monomers that is consistent with a domain-swapped diabody structure. Neutron scattering and modelling fits showed that MFE-23 existed as compact V-H-V-L- Iinked monomers at therapeutically relevant concentrations below 1 mg/ml. Size-exclusion gel chromatography showed that the monomeric and dimeric forms of MFE-23 could be separated, and that the proportions of these two forms depended on the starting MFE-23 concentration. Sedimentation equilibrium experiments by analytical ultracentrifugation at nine concentrations of MFE-23 indicated a reversible monomer-dimer self-association equilibrium with an association constant of 1.9 x 10(3)-2.2 x 10(3) M-1. Sedimentation velocity experiments using the time derivative g(s*) method showed that MFE-23-His has a concentration-dependent weight average sedimentation coefficient that increased from 1.8 S for the monomer to about 3-6 S for the dimer. Both values agreed with those calculated from the MFE-23 crystal structure. In relation to the thermal stability of MFE-23, denaturation experiments by H-1 NMR and FT-IR spectroscopy showed that the molecule is stable up to 47degreesC, after which denaturation was irreversible. MFE-23 dimerisation is discussed in terms of a new model for diabody structures, in which the V-H and V-L domains in the monomer are able to dissociate and reassociate to form a dimer, or diabody, but in which symmetric back-to-back contacts between the two monomers are formed. This dimerisation in solution is attributed to the complementary nature of the C-terminal surface of the MFE-23 monomer. Crystal structures for seven other scFv molecules have shown that, while the contact residues for symmetric back-to-back dimer formation in MFE-23 are not fully conserved, in principle, back-to-back contacts can be formed in these too. This offers possibilities for the creation of other forms of scFv molecules. (C) 2002 Elsevier Science Ltd. All rights reserved
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