Influence of cytotoxicity enhancers in combination with human serum on theactivity of CD22-recombinant ricin A against B cell lines, chronic and acute lymphocytic leukemia cells

Despite the strong in vitro activity of some immunotoxins (ITs), clinical a pplication did not result in complete cure. The outcome of therapy may be i mproved by combining ITs with IT-cytotoxicity enhancing agents. We studied the effect of various agents that influence the intracellular routing of IT s on the activity of the anti-B cell IT CD22-recombinant (rec) ricin A. In protein synthesis inhibition assays the carboxylic ionophores monensin and nigericin enhanced the activity of the IT 117- end 382-fold, respectively, against the cell line Daudi, and 81- and 318-fold, respectively, against th e cell line Ramos. IT activity to Daudi and Ramos was enhanced to a lesser extent by the lysosomotropic amines chloroquine (14- and 11-fold, respectiv ely) and NH4Cl (nine- and in-fold, respectively). However, the combination of NH4Cl and chloroquine induced more than an additive effect (145- and 107 -fold, respectively). Cytotoxicity was not influenced by brefeldin A, all-t rans retinoic acid (ATRA), verapamil and perhexiline maleate. Bacitracin en hanced the IT cytotoxicity in contrast to the other protease inhibitors apr otinin, leupeptin and soybean trypsin inhibitor, albeit enhancement was wea k (two-fold). The enhancers exerted only a negligible effect on bone marrow progenitor cells. We recently developed a flow cytometric cytotoxicity ass ay in which cell elimination can be assessed. In order to detect enhancemen t in this assay, we used 5x10(-11) M IT (approximately the 50% protein synt hesis inhibiting dose (ID50)). This concentration killed 41% of the Daudi c ells and 42% of the Ramos cells. In the presence of 10 nM monensin the IT k illed 74% and 99% and in the presence of in nM nigericin 96% and 99% of the Daudi and Ramos cells, respectively. At 10(-8) M, CD22-rec ricin A elimina ted malignant cells originating from three patients with B-CLL (0.42 log) a nd two with B-ALL (0.19 log) patients. Cytotoxicity to malignant cells was enhanced by NH4Cl, chloroquine, monensin and nigericin. The combination of NH4CI and chloroquine enhanced the activity most effectively (up to 2.06 lo g). To determine the applicability of the IT in combination with enhancers in vivo we investigated the effect of human serum. Human serum inhibited IT activity which could not be restored by monensin and nigericin because of complete inhibition of these enhancers by serum. In contrast, chloroquine p artially restored the activity of CD22-rec ricin A in the presence of human serum. We conclude that monensin, nigericin and the combination of NH4Cl a nd chloroquine can be used instead of NH4Cl to potentiate CD22-rec ricin A activity in purging autologous bone marrow transplants contaminated with ma lignant B cells. Chloroquine might be a promising enhancer of CD22-rec rici n A for treating patients in vivo.