UCL logo

UCL Discovery

UCL home » Library Services » Electronic resources » UCL Discovery

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

Lee, YC; Boehm, MK; Chester, KA; Begent, RHJ; Perkins, SJ; (2002) 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. J MOL BIOL , 320 (1) 107 - 127. 10.1016/S0022-2836(02)00403-5.

Full text not available from this repository.

Abstract

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.

Type: Article
Title: 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
DOI: 10.1016/S0022-2836(02)00403-5
Keywords: single-chain Fv antibody, size-exclusion chromatography, analytical ultracentrifuge, neutron scattering, H-1 NMR spectroscopy, PROTEIN SECONDARY STRUCTURE, TRANSFORM INFRARED-SPECTROSCOPY, PHAGE DISPLAY LIBRARIES, CURVE-FITTING PROCEDURE, EGG-WHITE LYSOZYME, X-RAY, CRYSTAL-STRUCTURE, SEDIMENTATION-VELOCITY, MONOCLONAL-ANTIBODY, EQUILIBRIUM ULTRACENTRIFUGATION
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute > Research Department of Oncology
URI: http://discovery.ucl.ac.uk/id/eprint/156000
Downloads since deposit
0Downloads
Download activity - last month
Download activity - last 12 months
Downloads by country - last 12 months

Archive Staff Only

View Item View Item