TOXIN-TARGETED DESIGN FOR ANTICANCER THERAPY .2. PREPARATION AND BIOLOGICAL COMPARISON OF DIFFERENT CHEMICALLY LINKED GELONIN ANTIBODY CONJUGATES

To obtain more potent immunotoxins for anticancer therapy a gelonin-AR 3 antibody immunoconjugate was prepared with different new linkers and coupling procedures. The gelonin was derivatized with the heterobifun ctional thioimidate linkers ethyl-acetyl-3-mercaptopropionthioimidate (AMPT) and 3-(4-carboxamidophenyldithio)propionthioimidate (CDPT), and with the succinimidyl type reagents ccinimidyl-3-(4-carboxamidophenyl dithio)propionate (SCDP) and N-succinimidyl-S-acetyl thiolacetate (SAT A). The biological activity of gelonin modified with different linkers (AMPT, CDPT, SCDP, SATA) was determined by a rabbit reticulocyte assa y. We found that AMPT was the molecule of choice to derivatize the tox in, confirming the preferability of thioimidate linkers. The monoclona l antibody Mab was derivatized with CDPT and SCDP. Then the following immunoconjugates were prepared with different procedures: Mab-CDPT wit h gelonin-AMPT; Mab-CDPT with gelonin-CDPT; Mab-SCDP with gelonin-SATA . To verify whether selection of the most suitable coupling procedure could affect the antitumoral activity of the gelonin-AR3 immunoconjuga te, the three immunotoxins were tested on target HT-29 human colon car cinoma cells versus nontarget MeWo cells. The gelonin immunoconjugate linked via the AMPT-CDPT thioimidate reagents showed highest antitumor al activity as well as best selectivity for the target cells.