MECHANISMS INVOLVED IN SERUM-DEPENDENT INACTIVATION OF THE IMMUNOTOXIN ENHANCERS MONENSIN AND CARRIER-PROTEIN-MONENSIN

The immunotoxin-enhancing properties of monensin and of human-serum-al bumin - monensin conjugates are severely impaired in the presence of h uman serum. In this study we have therefore investigated the interacti on between serum proteins and monensin leading to the inactivation of monensin function as immunotoxin potentiator. We found that the bindin g of monensin-specific mAb to thioether-cross-linked or disulfide-cros sl-linked protein-monensin conjugates is negatively affected by serum, as indicated by immunoenzymic (ELISA) and radioimmunobinding analysis . Size-exclusion chromatography of serum samples indicated that the gr eatest blocking effect is due to protein components of 40-90 kDa eluti ng as a broad peak (peak 4). Analysis of the proteins contained within peak 4 by ion-exchange chromatography followed by microsequencing rev ealed that the major components of peak no. 4 were transferrin, human serum albumin and immunoglobulin fragments. Investigations on the natu re of the interactions between serum proteins and monensin leading to monensin inactivation were conducted by affinity chromatography of ser um on immobilized human-serum-albumin-monensin conjugates, size-exclus ion chromatography, SDS/PAGE analysis of serum-treated human-serum-alb umin-monensin conjugates, and evaluation of the stability of immobiliz ed human-serum-albumin-bound I-125-monensin following treatment with s erum. Addition of esterase inhibitors (e.g. EDTA, 4-nitrophenyl phosph ate) or prior treatment of the serum at 56 degrees C partially reverse d the serum effects observed. We conclude that serum proteins block th e immunotoxin-enhancing effect of monensin and of human-serum-albumin- monensin conjugates by multiple mechanisms involving hydrophobic and c ovalent interactions and enzyme-mediated cleavage of protein-bound mon ensin.