Kuan C T, Pai L H, Pastan I
Laboratory of Molecular Biology, Division of Basic Sciences, National Cancer Institute, NIH, Bethesda, Maryland 20892, USA.
Clin Cancer Res. 1995 Dec;1(12):1589-94.
Vascular leak syndrome (VLS) was originally found to be a major dose-limiting toxicity in humans with cancer treated with several immunotoxins (ITs) containing ricin A chain or blocked ricin. Recently, VLS has also been observed in patients treated with an IT containing the murine monoclonal antibody (MAb) B3 coupled to LysPE38, a recombinant truncated form of Pseudomonas exotoxin (PE) A. Antibody B3 (IgG1k) recognizes LewisY and related carbohydrate epitopes present on many human solid tumors, and B3-LysPE38 showed excellent antitumor activity in nude mice bearing tumors that express the B3 antigen. In the clinical trial, the development of VLS has prevented the administration of the amount of IT necessary to achieve blood levels required for good therapeutic responses. We have now investigated the effects of several PE-based ITs on different human endothelial cell lines to elucidate the mechanism of VLS induced by ITs containing PE. To assess the cytotoxic effect of IT on endothelial cells, various ITs were incubated with cells for 2 or 20 h, and the incorporation of [3H]leucine into protein was measured. The endothelial cells studied were human umbilical vein endothelial cells, human lung-derived microvascular endothelial cells (HUVECs), human adult dermal microvascular endothelial cells, human pulmonary artery endothelial cells, and human aortic endothelial cells. We found that both B3-LysPE38 (LMB-1), a chemical conjugate of MAb B3 with PE38, as well as B3(Fv)-PE38 (LMB-7), a recombinant single chain immunotoxin, inhibited protein synthesis, with 50% inhibitory concentrations between 600 and 1000 ng/ml for 20-h incubation in HUVECs, human lung-derived microvascular endothelial cells, and human adult dermal microvascular endothelial cells but not on human pulmonary artery endothelial cells. The cytotoxic effect was specific since PE38 itself or PE coupled to several other antibodies did not inhibit protein synthesis in these cells even at 10,000 ng/ml. Further evidence that the cytotoxicity of B3-containing ITs is due to specific B3 binding to endothelial cells comes from the fact that the cytotoxicity can be blocked by excess free MAb B3. HUVECs undergo overt morphological changes after treatment with B3-LysPE38 or B3(Fv)PE38. Gaps between the cells are formed after a 20-h exposure but not after 2 h. These studies suggest that VLS in patients is due to capillary damage caused by prolonged exposure to high concentrations of LMB-1.
血管渗漏综合征（VLS）最初被发现是接受几种含蓖麻毒素A链或去毒蓖麻毒素的免疫毒素（ITs）治疗的癌症患者的主要剂量限制性毒性反应。最近，在接受一种含与赖氨酸化铜绿假单胞菌外毒素（PE）A的重组截短形式LysPE38偶联的鼠单克隆抗体（MAb）B3的IT治疗的患者中也观察到了VLS。抗体B3（IgG1k）识别许多人类实体瘤上存在的LewisY及相关碳水化合物表位，并且B3-LysPE38在携带表达B3抗原肿瘤的裸鼠中显示出优异的抗肿瘤活性。在临床试验中，VLS的出现阻碍了给予达到良好治疗反应所需血药浓度所需的IT剂量。我们现在研究了几种基于PE的ITs对不同人内皮细胞系的影响，以阐明含PE的ITs诱导VLS的机制。为评估IT对内皮细胞的细胞毒性作用，将各种IT与细胞孵育2或20小时，并测量[3H]亮氨酸掺入蛋白质的情况。所研究的内皮细胞为人脐静脉内皮细胞、人肺微血管内皮细胞（HUVECs）、成人皮肤微血管内皮细胞、人肺动脉内皮细胞和人主动脉内皮细胞。我们发现，MAb B3与PE38的化学偶联物B3-LysPE38（LMB-1）以及重组单链免疫毒素B3（Fv）-PE38（LMB-7）均抑制蛋白质合成，在HUVECs、人肺微血管内皮细胞和成人皮肤微血管内皮细胞中20小时孵育的半数抑制浓度在600至1000 ng/ml之间，但对人肺动脉内皮细胞无此作用。细胞毒性作用具有特异性，因为即使在10,000 ng/ml时，PE38本身或与其他几种抗体偶联的PE也不抑制这些细胞中的蛋白质合成。含B3的ITs细胞毒性是由于B3与内皮细胞特异性结合的进一步证据来自于这样一个事实，即细胞毒性可被过量游离MAb B3阻断。用B3-LysPE38或B3（Fv）PE38处理后，HUVECs会发生明显的形态变化。20小时暴露后细胞间形成间隙，但2小时后不会。这些研究表明，患者中的VLS是由于长时间暴露于高浓度LMB-1导致的毛细血管损伤。
