Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA.
J Biol Chem. 2011 Feb 11;286(6):4165-72. doi: 10.1074/jbc.M110.186973. Epub 2010 Dec 7.
Gelonin-based immunotoxins vary widely in their cytotoxic potency as a function of antigen density, target cell internalization and trafficking kinetics, and conjugate properties. We have synthesized novel gelonin immunotoxins using two different binding scaffold types (single-chain antibody variable fragments and fibronectin domains) targeting two different tumor antigens (carcinoembryonic antigen and EGF receptor). Constructs were characterized using an antigen-negative cell line (HT-1080), cell lines positive for each antigen (HT-1080(CEA) for carcinoembryonic antigen and A431 for EGF receptor), and a cell line positive for both antigens (HT-29). Immunotoxins exhibited K(d) values between 8 and 15 nm and showed 20-2000-fold enhanced cytotoxicity compared with gelonin (IC(50) ∼ 0.25-30 nM versus 500 nM). Using quantitative fluorescence flow cytometry, we measured internalization of gelonin (via pinocytosis) and gelonin-based immunotoxins (via antigen-dependent, receptor-mediated endocytosis). Results were matched with cytotoxicity measurements made at equivalent concentration and exposures. Unexpectedly, when matched internalization and cytotoxicity data were combined, a conserved internalized cytotoxicity curve was generated that was common across experimental conditions. Considerable variations in antigen expression, trafficking kinetics, extracellular immunotoxin concentration, and exposure time were all found to collapse to a single potency curve on the basis of internalized immunotoxin. Fifty percent cytotoxicity occurred when ∼ 5 × 10(6) toxin molecules were internalized regardless of the mechanism of uptake. Cytotoxicity observed at a threshold internalization was consistent with the hypothesis that endosomal escape is a common, highly inefficient, rate-limiting step following internalization by any means tested. Methods designed to enhance endosomal escape might be utilized to improve the potency of gelonin-based immunotoxins.
基于蓖麻毒素的免疫毒素的细胞毒性效力因其抗原密度、靶细胞内化和转运动力学以及缀合物特性而有很大差异。我们使用两种不同的结合支架类型（单链抗体可变片段和纤连蛋白结构域）针对两种不同的肿瘤抗原（癌胚抗原和表皮生长因子受体）合成了新型的蓖麻毒素免疫毒素。使用阴性抗原细胞系（HT-1080）、每种抗原阳性的细胞系（针对癌胚抗原的 HT-1080(CEA)和针对表皮生长因子受体的 A431）和同时针对两种抗原阳性的细胞系（HT-29）对构建体进行了表征。免疫毒素的 K(d) 值在 8 至 15nm 之间，与蓖麻毒素相比，显示出 20 至 2000 倍的增强细胞毒性（IC(50)∼0.25-30nm 与 500nm）。通过定量荧光流式细胞术，我们测量了蓖麻毒素（通过胞饮作用）和基于蓖麻毒素的免疫毒素（通过抗原依赖性、受体介导的内吞作用）的内化。结果与在等效浓度和暴露条件下进行的细胞毒性测量相匹配。出乎意料的是，当匹配内化和细胞毒性数据时，生成了一个保守的内化细胞毒性曲线，该曲线在所有实验条件下都是通用的。在基于内化免疫毒素的基础上，发现抗原表达、转运动力学、细胞外免疫毒素浓度和暴露时间的差异很大，都可以归为单一效力曲线。当内化约 5×10(6)个毒素分子时，会发生 50%的细胞毒性，而与摄取机制无关。在阈值内化时观察到的细胞毒性与内体逃逸是任何测试方法内化后常见的、效率极低的限速步骤的假设一致。可以利用旨在增强内体逃逸的方法来提高基于蓖麻毒素的免疫毒素的效力。
