Wawrzynczak E J, Cumber A J, Henry R V, May J, Newell D R, Parnell G D, Worrell N R, Forrester J A
Drug Targeting Laboratory, Institute of Cancer Research, Sutton, Surrey, England.
Cancer Res. 1990 Dec 1;50(23):7519-26.
A panel of immunotoxins was constructed by chemically attaching the ribosome-inactivating proteins abrin A chain, ricin A chain, gelonin, and momordin to the monoclonal mouse IgG2a antibody Fib75 by means of a disulfide linkage. All the immunotoxins were toxic in tissue culture to the EJ human bladder carcinoma cell line expressing the antigen recognized by Fib75, inhibiting the incorporation of [3H]leucine by 50% at concentrations between 1 x 10(-10) M and 8 x 10(-10) M. The pharmacokinetics of the immunotoxins in the normal Wistar rat was determined following i.v. administration. The concentrations of intact immunotoxin in serum samples taken at various intervals after injection for up to 24 h were measured by solid-phase enzyme-linked immunosorbent assays specific for each of the four different ribosome-inactivating proteins. The Fib75 immunotoxins were cleared from the circulation with comparable, but not identical, biphasic kinetics best described by a two compartment open pharmacokinetic model. The alpha-phase half-lives of the panel, between 0.35 and 0.71 h, were similar. The beta-phase half-life of Fib75-abrin A chain, 13.3 h, was significantly longer than the beta-phase half-lives of Fib75-ricin A chain, Fib75-gelonin, and Fib75-momordin, between 7.5 and 8.6 h. Fib75-abrin A chain was found to be about 3- to 4-fold more resistant than the other immunotoxins to breakdown by reduction of the disulfide linkage between the A chain and the antibody with glutathione in vitro. This suggests that the longer serum half-life of Fib75-abrin A chain may have been due to greater stability against reduction in vivo. Analysis of serum samples obtained up to 24 h after injection of Fib75-abrin A chain revealed that the chemically intact immunotoxin present in the circulation retained full cytotoxic activity. An abrin A chain immunotoxin made with a different monoclonal mouse IgG2a antibody was also found to be more stable against reduction by glutathione in vitro than an analogous ricin A chain immunotoxin. Thus, abrin A chain may posses unique molecular properties that endow immunotoxins made with this A chain with greater stability in vivo than immunotoxins made with ricin A chain or other ribosome-inactivating proteins.
通过二硫键将核糖体失活蛋白相思子毒素A链、蓖麻毒素A链、去甲氧基浸麻毒素和苦瓜素与单克隆小鼠IgG2a抗体Fib75化学连接，构建了一组免疫毒素。所有免疫毒素在组织培养中对表达Fib75识别抗原的EJ人膀胱癌细胞系均具有毒性，在1×10⁻¹⁰ M至8×10⁻¹⁰ M的浓度下可抑制[³H]亮氨酸掺入50%。静脉注射后测定了免疫毒素在正常Wistar大鼠体内的药代动力学。通过针对四种不同核糖体失活蛋白各自的固相酶联免疫吸附测定法，测量了注射后长达24小时内不同时间采集的血清样本中完整免疫毒素的浓度。Fib75免疫毒素以类似但不完全相同的双相动力学从循环中清除，最好用二室开放药代动力学模型描述。该组免疫毒素的α相半衰期在0.35至0.71小时之间，相似。Fib75-相思子毒素A链的β相半衰期为13.3小时，明显长于Fib75-蓖麻毒素A链、Fib75-去甲氧基浸麻毒素和Fib75-苦瓜素的β相半衰期，后者在7.5至8.6小时之间。发现Fib75-相思子毒素A链在体外比其他免疫毒素对谷胱甘肽还原A链与抗体之间的二硫键导致的分解具有约3至4倍的抗性。这表明Fib75-相思子毒素A链较长的血清半衰期可能是由于其在体内对还原具有更高的稳定性。对注射Fib75-相思子毒素A链后长达24小时采集的血清样本分析表明，循环中存在的化学完整免疫毒素保留了全部细胞毒性活性。还发现用不同的单克隆小鼠IgG2a抗体制备的相思子毒素A链免疫毒素在体外比类似的蓖麻毒素A链免疫毒素对谷胱甘肽还原更稳定。因此，相思子毒素A链可能具有独特的分子特性，使以此A链制备的免疫毒素在体内比用蓖麻毒素A链或其他核糖体失活蛋白制备的免疫毒素具有更高的稳定性。
