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. 2002 Jun 28;320(1):107-27. doi: 10.1016/S0022-2836(02)00403-5.

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)-linked 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 1.9x10(3)-2.2x10(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 (1)H NMR and FT-IR spectroscopy showed that the molecule is stable up to 47 degrees C, 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Antineoplastic Agents / chemistry
  • Antineoplastic Agents / metabolism*
  • Dimerization
  • Humans
  • Immunoglobulin Fragments / chemistry
  • Immunoglobulin Fragments / metabolism*
  • Magnetic Resonance Spectroscopy
  • Models, Molecular
  • Molecular Sequence Data
  • Neutrons
  • Protein Structure, Quaternary
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Sequence Alignment
  • Spectroscopy, Fourier Transform Infrared
  • Temperature
  • Ultracentrifugation

Substances

  • Antineoplastic Agents
  • Immunoglobulin Fragments
  • MFE-23 protein, human
  • Recombinant Fusion Proteins