Phage display techniques identified a scFv, 15-9, which binds to murine
laminin-1 and accumulated selectively in
tumors. In this study, a covalent diabody was constructed by changing the
amino acid residues at positions VH44 and VL100 to
cysteine residues so that the diabody form could be stabilized via a
disulfide bond. The covalent diabody was expressed in Pichia pastoris and purified by affinity chromatography. The binding properties were measured by surface plasmon resonance and solid phase binding of (125)I diabody and scFv. Data from the plasmon resonance method yielded calculated K(D)s of 4.4 x 10(-10) M for the covalent diabody and 9.9 x 10(-8) M for the scFv. K(D)s calculated from solid phase binding of radioiodinated
proteins were 1.7-2.1 x 10(-10) M and 2.1-2.4 x 10(-8) M respectively. The rate of dissociation of (125)I scFv from solid phase
laminin was independent of
laminin concentration; however, the dissociation of the (125)I diabody was dependent both on the concentration of
laminin and on the concentration of the diabody. Specifically, high concentrations of
laminin yielded very slow rates of diabody dissociation indicating that bivalent attachments had formed. When higher amounts of diabody were used that essentially saturated the
laminin sites with univalent binding, the dissociation rate was similar to that for the scFv indicating univalent binding. Biodistribution studies in
tumor-bearing SCID mice showed that the covalent diabody improved the ratio of
tumor/muscle 2 fold over that obtained with the scFv, although the absolute amount of
protein bound to the
tumor site was not significantly different for the two forms. The data also showed that retention of the diabody in the
tumor and kidney, sites where
laminin is present in high concentration, was much longer compared to that of scFv. These data are consistent with the hypothesis that both scFv and diabody forms bind to available
laminin in vivo with similar association kinetics, but that in situations of high target concentration, the diabody can bind bivalently and is thus retained at the binding site much longer than the scFv.