CRYSTAL STRUCTURE OF MFE-23, A CLINICALLY IMPORTANT ANTI-CEA SCFV ANTIBODY FRAGMENT, AT 0.28 NM RESOLUTION. M. K. Boehm¹, A. L. Corper², T. Wan², M. Sohi², B. J. Sutton², J. D. Thornton¹, P. A. Keep¹, R. H. J. Begent¹, S. J. Perkins¹, ¹Depts. Biochemistry Molecular Biology, and Clinical Oncology, Royal Free Hospital Sch. Med., Rowland Hill St., London NW3 2PF, UK, ²Randall Institute, King's College London, 26-29 Drury Lane, London WC2B 5RL, UK

MFE-23 is a single-chain Fv (scFv) antibody fragment that was selected from a phage-display library for high affinity binding to carcinoembryonic antigen (CEA), and has important clinical uses in the detection, monitoring and targetting of colon cancer. CEA is a seven-domain cell adhesion molecule which is expressed at high levels on colon carcinoma cells. MFE-23 (M_r 27,150; 256 residues) consists of an N-terminal V_H domain, a 15-residue linker, a V_L domain and a 12-residue tag. MFE-23 was expressed in E. coli and purified on CEA coupled to Sepharose. Crystals were produced by the hanging-drop method. MFE-23 at 2 mg/ml was mixed 1:1 with precipitant (100 mM Tris-HCI at pH 6.5 containing 45% saturated ammonium sulfate), and a 10 ul drop of this mixture was equilibrated against 0.5 ml of precipitant at 18deg.C. Well-formed crystals formed within several days, and diffracted to 0.2 nm resolution. Diffraction data were collected using an R-AXIS-IIC mounted on an RU200 rotating anode X-ray source. The crystal belongs to the trigonal space group P3₂21 and has unit cell dimensions of a = b = 6.170 nm and c = 12.794 nm. A single crystal was used for structure determination. Data to a resolution of 0.28 nm were processed using DENZO and the CCP4 program package. The structure was solved by molecular replacement using the Fv coordinates from a murine IgA Fab fragment (Brookhaven code 2FBJ). The antibody complementarily determining regions, the linker and the tag were omitted for the calculation of initial 2Fo-Fc electron density maps. Refinement of the MFE-23 structure is currently in progress, utilizing the rigid-body refinement and the positional refinement algorithms of X-PLOR and manual rebuilding of the model in O.