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.
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.
Lee
Yie Chia
YC
Department of Biochemistry and Molecular Biology, Royal Free and University College Medical School, University College London, Gower Street, London WC1E 6BT, UK.
Boehm
Mark K
MK
Chester
Kerry A
KA
Begent
Richard H J
RH
Perkins
Stephen J
SJ
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
D000970:Antineoplastic Agents; D007128:Immunoglobulin Fragments; C451184:MFE-23 protein, human; D011993:Recombinant Fusion Proteins