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

Abstract

Despite the strong in vitro activity of some immunotoxins (ITs), clinical application did not result in complete cure. The outcome of therapy may be improved by combining ITs with ITcytotoxicity enhancing agents. We studied the effect of various agents that influence the intracellular routing of ITs 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-and 382-fold, respectively, against the cell line Daudi, and 81-and 318-fold, respectively, against the cell line Ramos. IT activity to Daudi and Ramos was enhanced to a lesser extent by the lysosomotropic amines chloroquine (14-and 11-fold, respectively) and NH 4 Cl (nine-and 10-fold, respectively). However, the combination of NH 4 Cl and chloroquine induced more than an additive effect (145-and 107-fold, respectively). Cytotoxicity was not influenced by brefeldin A, all-trans retinoic acid (ATRA), verapamil and perhexiline maleate. Bacitracin enhanced the IT cytotoxicity in contrast to the other protease inhibitors aprotinin, leupeptin and soybean trypsin inhibitor, albeit enhancement was weak (two-fold). The enhancers exerted only a negligible effect on bone marrow progenitor cells. We recently developed a flow cytometric cytotoxicity assay in which cell elimination can be assessed. In order to detect enhancement in this assay, we used 5 × 10 2.06 log). 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 partially restored the activity of CD22-rec ricin A in the presence of human serum. We conclude that monensin, nigericin and the combination of NH 4 Cl and chloroquine can be used instead of NH 4 Cl to potentiate CD22-rec ricin A activity in purging autologous bone marrow transplants contaminated with malignant B cells. Chloroquine might be a promising enhancer of CD22-rec ricin A for treating patients in vivo.

Potent and specific antitumor effects of an anti-CD22–targeted cytotoxic ribonuclease: potential for the treatment of non-Hodgkin lymphoma

“…43 Apparently, onconase can also enter the cytosol through a disrupted Golgi when internalized by LL2 suggesting that the pathways of onconase and LL2-onconase converge at some point after internalization. All together, the in vitro potency and specificity of LL2-onconase on Daudi cells (IC 50 s 10 to 100 pM) is comparable to anti-CD22 immunotoxin conjugates constructed with plant, eg, anti-CD22 ricin A-chain (IC 50 s 1 to 30 pM) 11,44 and bacterial toxins, eg, anti-CD22 Pseudomonas exotoxin derivatives (IC 50 s 1 to 40 pM). 16,45 Conjugation to LL2 decreased systemic toxicity of onconase to mice in addition to enhancing the potency and specificity.…”

Ricin and Ricin-Containing Immunotoxins: Insights into Intracellular Transport and Mechanism of action in Vitro

“…Compounds known to sensitize cells to some of the toxins may also potentate the action of immunotoxins [38]. Horssen et al reported that the action of a recombinant ricin A-chain conjugate is potentated by chloroquine, and proposed that this could also be applied in vivo [39].…”

NEW EMBO MEMBERS' REVIEW: Entry of ricin and Shiga toxin into cells: molecular mechanisms and medical perspectives

“…Interestingly, compounds known to sensitize cells to some of the toxins may also potentiate the action of immunotoxins (Sandvig and van Deurs, 1996). An example is found in a recent article by van Horssen et al . (1999), who report that the action of a recombinant ricin A‐chain conjugate is potentiated by chloroquine, and propose that this could also be applied in vivo .…”