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

Cross-linked F(ab')(2) fragments derived from PR1A3, a murine monoclonal antibody used in radioimmunoscintigraphy of colorectal tumors, were produced using the bifunctional reagent bismaleimidohexane (BMH) as follows: Digestion of PR1A3 with pepsin gave F(ab')(2) fragments which were purified by ion-exchange chromatography. Fab' was produced by reduction 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 presence of a molecule of similar to 100 kDa containing a nonreducible 50 000 MWt chain. Competitive and direct radioligand binding assays demonstrated that the XL-F(ab')(2) had a capacity to bind to antigen similar to that of unmodified F(ab')(2). The biodistribution of I-125-labeled XL-F(ab')(2) and unmodified F(ab')(2) was compared in a nude mouse human tumor xenograft model at 4, 24, and 48 h after injection. Differences 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 tissue ratios at 48 h ranged from 6.2 to 35.2 for XL-F(ab')(2) while for the normal F(ab')(2) they ranged from 1.5 to 14.2. These results suggest that cross-linked antibody fragments may produce better tumor targeting in clinical application.