CRYSTAL-STRUCTURE OF THE DISULFIDE-STABILIZED FV FRAGMENT OF ANTICANCER ANTIBODY B1 - CONFORMATIONAL INFLUENCE OF AN ENGINEERED DISULFIDE BOND

A recombinant Fv construct of the B1 monoclonal antibody that recogniz es the Lewis(Y)-related carbohydrate epitope on human carcinoma cells has been prepared, The Fv is composed of the polypeptide chains of the V-H and V-L domains expressed independently and isolated as inclusion bodies. The Fv is prepared by combining and refolding equimolar amoun ts of guanidine chloride solubilized inclusion bodies. The Fv is stabi lized by an engineered interchain disulfide bridge between residues V( L)100 and V(H)44. This construct has a similar binding affinity as tha t of the single-chain construct (Benhar and Pastan, Clin. Cancer Res. 1:1023-1029, 1995). The B1 disulfide-stabilized Fv (B1dsFv) crystalliz es in space group P6(1)22 with the unit cell parameters a = b = 80.1 A ngstrom, and c = 138.1 Angstrom. The crystal structure of the B1dsFv h as been determined at 2.1-Angstrom resolution using the molecular repl acement technique, The final structure has a crystallographic R-value of 0.187 with a root mean square deviation in bond distance of 0.014 A ngstrom and in bond angle of 2.74 degrees, Comparisons of the B1dsFv s tructure with known structures of Fv regions of other immunoglobulin f ragments shows closely related secondary and tertiary structures. The antigen combining site of B1dsFv is a deep depression 10-Angstrom wide and 17-Angstrom long with the walls of the depression composed of res idues, many of which are tyrosines, from complementarity determining r egions L1, L3, H1, H2, and H3. Model building studies indicate that th e Lewis(Y) tetrasaccharide, Fuc-Gal-Nag-Fuc, can be accommodated in th e antigen combining site in a manner consistent with the epitope predi cted in earlier biochemical studies (Pastan, Lovelace, Gallo, Rutherfo rd, Magnani, and Willingham, Cancer Res. 51:3781-3787, 1991). Thus, th e engineered disulfide bridge appears to cause little, if any, distort ion in the Fv structure, making it an effective substitute for the B1 Fab, Proteins 31:128-138, 1998. (C) 1998 Wiley-Liss, Inc.dagger