PREPARATION AND CHARACTERIZATION OF INTERLEUKIN-2-GELONIN CONJUGATES MADE USING DIFFERENT CROSS-LINKING REAGENTS

Conjugates of IL-2 with the ribosome-inactivating protein gelonin were prepared using heterobifunctional reagents to link the proteins via d isulfide, acid-labile, and noncleavable linkers. In each case, one pro tein was modified using 2-iminothiolane. The sulfhydryl groups so intr oduced were then reacted either with 2-nitro-5-dithiobenzoate groups o r with iodoacetamido groups which had been introduced into the second protein. In the case of the acid-labile linkage, a reagent which forms a labile bond upon reaction with amino groups, 4-(iodoacetamido)-l-cy clohexene-1,2-dicarboxylic acid anhydride (its synthesis is described in this paper) was used to modify the toxin. The conjugates were separ ated from nonconjugated proteins by gel filtration on Sephadex G100 (S F). Each was analyzed with respect to its ribosome-inactivating activi ty, its ability to bind to the IL-2 receptor, and its in vitro cytotox icity. The ribosome-inactivating activity of gelonin was unaffected by modification with 2-iminothiolane and was retained in conjugates prep ared using this ragent. Modification of the toxin with 4-(iodoacetamid o)-1-cyclohexene-1,2-dicarboxylic acid anhydride to form the acid-labi le link drastically reduced the activity of the toxin. However, the ac tivity of the toxin was recovered following acid treatment to release the native protein. Conjugates containing each type of linkage exhibit ed both specific binding and selective cytotoxicity toward cells expre ssing the IL-2 receptor. The most potent of these toxins, that contain ing the disulfide linkage, exhibited a cytotoxicity which was 2 orders of magnitude greater than that of unconjugated gelonin.