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 1.9×10³–2.2×10³ 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 ¹H NMR and FT-IR spectroscopy showed that the molecule is stable up to 47 °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.

Introduction

During the past decade, the development and use of antibody-based therapeutics have been increasing steadily. With the advent of phage display technology and human antibody libraries,1., 2., 3., 4., 5. many clinically useful antibodies have been generated against well-defined molecular structures such as receptors and antigens. Several antibodies have reached clinical trials, with a substantial number used in the application of cancer diagnosis, imaging and therapy.6., 7., 8., 9. An example of a small two-domain anti-tumour antibody that has shown promising colorectal cancer targeting properties is the single-chain Fv (scFv) molecule MFE-23. MFE-23 is reactive against the tumour marker carcinoembryonic antigen (CEA), thus it has been used in clinical trials for radio-immunoimaging purposes.7., 9. It is under investigation as a fusion protein with carboxypeptidase G2 as a therapeutic for ADEPT (antibody-dependent enzyme prodrug therapy) as well as in other fusion proteins.5., 10.

scFv molecules consist of a variable heavy (V_H) domain and a variable light (V_L) domain typically joined by a flexible 15–18 amino acid residue peptide to form the smallest antibody fragment with antigen-binding activity.11., 12., 13. The linker ensures the equal expression of both domains, while stabilising the association of the V_H and V_L domains.14., 15. When compared to full-sized 12-domain antibody molecules, scFv molecules have enhanced tumour penetration with rapid blood clearance, together with a lower immunogenicity as the result of the removal of the effector four-domain Fc fragment.16., 17., 18. MFE-23 is formed from an N-terminal V_H domain linked by a (Gly₄Ser)₃ peptide to a C-terminal V_L domain, and has a high affinity for CEA $\text{(K}{}_{\mathrm{<mtext>d</mtext>}}\text{=2.5(±1.3)}\text{nM}\text{).}$¹⁹ In vivo biodistribution studies of MFE-23 showed superior localisation to CEA-producing tumour xenografts compared to another anti-CEA scFv molecule A5-SC.²⁰ The crystal structure of MFE-23 at 2.4 Å resolution clarified its anti-tumour properties.²¹ While five of its six antigen-binding loops (H1, H2, L1, L2 and L3) had conformations that could be predicted from canonical loop structures, the sixth loop, H3, possessed an atypical non-bulged, bifurcated structure. This crystal structure resulted in the prediction of a molecular model for the complex between MFE-23 and CEA that satisfactorily accounted for its known properties.22., 23. In addition, the comparison of the murine MFE-23 crystal structure with known human antibody crystal structures has provided a rational strategy for the humanisation of MFE-23 to reduce its immunogenicity.

scFv molecules form oligomers, and this is a significant factor in their practical applications.24., 25. The crystal structure of MFE-23 is of great interest, in that it revealed a symmetric V_H–V_L back-to-back association between two scFv molecules.²¹ The lack of electron density for the (Gly₄Ser)₃ linker made it difficult to determine whether this corresponds to two two-domain monomers or a new type of symmetric diabody structure (Figure 1). Diabodies are formed from the association of two scFv molecules when the linker peptide is shortened to 12 residues or less. The short linker prevents the usual interaction of the V_H and V_L domains within one molecule, whereupon the V_H and V_L domains are unassociated, as exemplified by monomer 2 in Figure 1, and reassociate to form the diabody.¹⁴ As suggested by the crystal structure of the diabody L5MK16,²⁶ diabodies are often considered to be formed by the association of the back of two V_H domains and not the V_L domains, and are stabilised by two linkers (see Figure 1 of Hudson & Kortt¹⁴ and Figure 3 of Wörn & Plückthun²⁷). Frequently, dimeric scFv molecules are discussed in terms of this stable diabody structure formed by short linkers, and the possibility that dimers may form by other mechanisms has not been considered in detail. Up to now, the oligomeric properties of MFE-23 in solution have not been characterised. If a stable MFE-23 dimer can be formed, this can provide extra avidity for its antigen and improve its retention in tumours, as exemplified by the 40-fold greater affinity of the C6.5 scFv diabody compared to its monomer.⁸ Here, information on the solution properties of MFE-23 is provided by a multidisciplinary structural approach based on neutron scattering, size-exclusion gel chromatography, and analytical ultracentrifugation. We show that MFE-23 exists as a reversible equilibrium of monomer and dimer forms in solution. Spectroscopic methods enabled its conformational stability to be characterised. Comparisons with crystal structures and sequences for other scFv molecules enabled us to discuss a novel model of MFE-23 dimerisation through the formation of a dimer that is stabilised by symmetric back-to-back contacts and which we name as a symmetric diabody.