van Horssen P J, van Oosterhout Y V, Evers S, Backus H H, van Oijen M G, Bongaerts R, de Witte T, Preijers F W
Central Hematology Laboratory, University Hospital St Radboud, Nijmegen, The Netherlands.
Leukemia. 1999 Feb;13(2):241-9. doi: 10.1038/sj.leu.2401262.
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 IT-cytotoxicity 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 NH4Cl (nine- and 10-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-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 x 10(-11) M IT (approximately the 50% protein synthesis inhibiting dose (ID50)). This concentration killed 41% of the Daudi cells and 42% of the Ramos cells. In the presence of 10 nM monensin the IT killed 74% and 99% and in the presence of 10 nM nigericin 96% and 99% of the Daudi and Ramos cells, respectively. At 10(-8) M, CD22-rec ricin A eliminated malignant cells originating from three patients with B-CLL (0.42 log) and two with B-ALL (0.19 log) patients. Cytotoxicity to malignant cells was enhanced by NH4Cl, chloroquine, monensin and nigericin. The combination of NH4Cl and chloroquine enhanced the activity most effectively (up to 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 NH4Cl and chloroquine can be used instead of NH4Cl 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.
尽管某些免疫毒素（ITs）在体外具有很强的活性，但临床应用并未带来完全治愈的效果。将ITs与增强IT细胞毒性的药物联合使用可能会改善治疗结果。我们研究了各种影响ITs细胞内转运的药物对抗B细胞IT CD22重组（rec）蓖麻毒素A活性的影响。在蛋白质合成抑制试验中，羧酸离子载体莫能菌素和尼日利亚菌素分别使IT对Daudi细胞系的活性增强了117倍和382倍，对Ramos细胞系的活性分别增强了81倍和318倍。溶酶体促渗胺氯喹（分别为14倍和11倍）和NH4Cl（分别为9倍和10倍）对Daudi和Ramos细胞的IT活性增强程度较小。然而，NH4Cl和氯喹的联合使用诱导了超过相加效应（分别为145倍和107倍）。布雷菲德菌素A、全反式维甲酸（ATRA）、维拉帕米和马来酸哌克昔林对细胞毒性没有影响。杆菌肽增强了IT的细胞毒性，这与其他蛋白酶抑制剂抑肽酶、亮抑肽酶和大豆胰蛋白酶抑制剂不同，尽管增强作用较弱（两倍）。这些增强剂对骨髓祖细胞的影响微乎其微。我们最近开发了一种流式细胞术细胞毒性试验，可用于评估细胞清除情况。为了在该试验中检测增强作用，我们使用了5×10^(-11) M的IT（约为50%蛋白质合成抑制剂量（ID50））。该浓度杀死了41%的Daudi细胞和42%的Ramos细胞。在存在10 nM莫能菌素的情况下，IT分别杀死了74%和99%的Daudi细胞和Ramos细胞，在存在10 nM尼日利亚菌素的情况下，分别杀死了96%和99%的Daudi细胞和Ramos细胞。在10^(-8) M时，CD22-rec蓖麻毒素A清除了来自3例B细胞慢性淋巴细胞白血病（B-CLL）患者（0.42对数）和2例B细胞急性淋巴细胞白血病（B-ALL）患者（0.19对数）的恶性细胞。NH4Cl、氯喹、莫能菌素和尼日利亚菌素增强了对恶性细胞的细胞毒性。NH4Cl和氯喹的联合使用最有效地增强了活性（高达2.06对数）。为了确定IT与增强剂联合在体内的适用性，我们研究了人血清的影响。人血清抑制了IT活性，由于血清对这些增强剂的完全抑制，莫能菌素和尼日利亚菌素无法恢复其活性。相比之下，氯喹在人血清存在的情况下部分恢复了CD22-rec蓖麻毒素A的活性。我们得出结论，莫能菌素、尼日利亚菌素以及NH4Cl和氯喹的组合可用于替代NH4Cl，以增强CD22-rec蓖麻毒素A在清除被恶性B细胞污染的自体骨髓移植中的活性。氯喹可能是一种有前途的CD22-rec蓖麻毒素A体内治疗患者的增强剂。
