A CROSS-LINKED MONOCLONAL-ANTIBODY FRAGMENT FOR IMPROVED TUMOR TARGETING

Cross-linked F(ab')(2) fragments derived from PR1A3, a murine monoclon al antibody used in radioimmunoscintigraphy of colorectal tumors, were produced using the bifunctional reagent bismaleimidohexane (BMH) as f ollows: Digestion of PR1A3 with pepsin gave F(ab')(2) fragments which were purified by ion-exchange chromatography. Fab' was produced by red uction of F(ab')(2) with cysteine. Following reaction with BMH, cross- linked F(ab')(2) fragments, XL-F(ab')(2), were isolated by preparative size-exclusion HPLC. Analysis by HPLC and SDS-PAGE demonstrated the p resence of a molecule of similar to 100 kDa containing a nonreducible 50 000 MWt chain. Competitive and direct radioligand binding assays de monstrated that the XL-F(ab')(2) had a capacity to bind to antigen sim ilar to that of unmodified F(ab')(2). The biodistribution of I-125-lab eled XL-F(ab')(2) and unmodified F(ab')(2) was compared in a nude mous e human tumor xenograft model at 4, 24, and 48 h after injection. Diff erences between the two preparations were most significant after 24 or 48 h. Tumor uptake of the XL-F(ab')(2) was greater and normal tissue retention less than with the unmodified fragment. Tumor to normal tiss ue ratios at 48 h ranged from 6.2 to 35.2 for XL-F(ab')(2) while for t he normal F(ab')(2) they ranged from 1.5 to 14.2. These results sugges t that cross-linked antibody fragments may produce better tumor target ing in clinical application.