Crystal structure of the anti-(carcinoembryonic antigen) single-chain Fv antibody MFE-23 and a model for antigen binding based on intermolecular contacts

MFE-23 is the first single-chain Fv antibody molecule to be used in patient s and is used to target colorectal cancer through its high affinity for car cinoembryonic antigen (CEA), a cell-surface member of the immunoglobulin su perfamily. MFE-23 contains an N-terminal variable heavy-chain domain joined by a (Gly(4)Ser)(3) linker to a variable light-chain (V-L) domain (kappa c hain) with an 11-residue C-terminal Myc-tag. Its crystal structure was dete rmined at 2.4 Angstrom resolution by molecular replacement with an R-cryst of 19.0 %. Five of the six antigen-binding loops, L1, L2, L3, H1 and H2, co nformed to known canonical structures. The sixth loop, H3, displayed a uniq ue structure, with a beta-hairpin loop and a bifurcated apex characterized by a buried Thr residue. In the crystal lattice, two MFE-23 molecules were associated back-to-back in a manner not seen before. The antigen-binding si te displayed a large acidic region located mainly within the H2 loop and a large hydrophobic region within the H3 loop. Even though this structure is unliganded within the crystal, there is an unusually large region of contac t between the H1, H2 and H3 loops and the beta-sheet of the V-L domain of a n adjacent molecule (strands DEBA) as a result of intermolecular packing. T hese interactions exhibited remarkably high surface and electrostatic compl ementarity. Of seven MFE-23 residues predicted to make contact with antigen , five participated in these lattice contacts, and this model for antigen b inding is consistent with previously reported site-specific mutagenesis of MFE-23 and its effect on CEA binding.