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.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Royal Free and University College Medical School, University College London, Gower Street, London WC1E 6BT, UK.
authors.forename: Yie Chia
authors.initials: YC
authors.lastname: Lee
authors.forename: Mark K
authors.initials: MK
authors.lastname: Boehm
authors.forename: Kerry A
authors.initials: KA
authors.lastname: Chester
authors.forename: Richard H J
authors.initials: RH
authors.lastname: Begent
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Perkins
mesh_terms: D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009502:Neutrons; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D014461:Ultracentrifugation
chemical_list: D000970:Antineoplastic Agents; D007128:Immunoglobulin Fragments; C451184:MFE-23 protein, human; D011993:Recombinant Fusion Proteins