Pietersz G A, Kanellos J, McKenzie I F
Department of Pathology, University of Melbourne, Parkville, Victoria, Australia.
Cancer Res. 1988 Aug 15;48(16):4469-76.
The use of tumor immunotherapy using whole ricin-antibody conjugates is complicated by the nonspecific lectin activity of the ricin B-chain which leads to toxic side effects. A novel method of coupling whole intact ricin to monoclonal antibody (MoAb) is described herein, where the nonspecific binding of the ricin B-chain is blocked. The coupling was done using the bifunctional reagents S-acetylmercaptosuccinic anhydride for antibody and succinimidyl 3-(2-pyridyldithio)propionate for ricin, and this resulted in the loss of B-chain binding activity, while impairing neither the toxic potential of the A-chain nor the activity of the MoAb. The purified immunotoxins could not bind to lactose-Sepharose and were equally cytotoxic in vitro to MoAb-reactive cell lines in the presence or absence of lactose. The coupling method was suitable for six different ricin-antibody conjugates and also using ricin deglycosylated by treatment with periodate. However, the blocking of the ricin B-chain was only effective with whole IgG molecules as F(ab')2-ricin immunotoxins could, like ricin, bind to lactose-Sepharose. Ricin-antibody conjugates reduced the [3H]leucine incorporation of appropriate target cells by 50% at a concentration of 6 to 45 ng/ml, whereas nonreactive antibody immunotoxins were not toxic to the target cells at concentrations as high as 10(4) ng/ml. The specific cytotoxicity of these immunotoxins could be inhibited by the addition of unconjugated reactive MoAb; the presence of lactose or a nonreactive MoAb did not significantly affect the observed cytotoxicity. Thus, whole ricin-antibody conjugates produced in this way do not bind nonspecifically to target cells, the most important implication being that such immunotoxins should be more potent that ricin A-chain conjugates and capable of being used in vivo.
使用完整的蓖麻毒素 - 抗体偶联物进行肿瘤免疫治疗会因蓖麻毒素B链的非特异性凝集素活性而变得复杂，这会导致毒性副作用。本文描述了一种将完整的蓖麻毒素偶联至单克隆抗体（MoAb）的新方法，其中蓖麻毒素B链的非特异性结合被阻断。偶联是使用双功能试剂进行的，抗体用S - 乙酰巯基琥珀酸酐，蓖麻毒素用N - 琥珀酰亚胺基 - 3 -（2 - 吡啶基二硫代）丙酸酯，这导致B链结合活性丧失，同时既不损害A链的毒性潜力也不损害MoAb的活性。纯化的免疫毒素不能与乳糖 - 琼脂糖结合，并且在有或没有乳糖存在的情况下，在体外对MoAb反应性细胞系具有同等的细胞毒性。该偶联方法适用于六种不同的蓖麻毒素 - 抗体偶联物，也适用于用过碘酸盐处理去糖基化的蓖麻毒素。然而，蓖麻毒素B链的阻断仅对完整的IgG分子有效，因为F(ab')2 - 蓖麻毒素免疫毒素能够像蓖麻毒素一样与乳糖 - 琼脂糖结合。蓖麻毒素 - 抗体偶联物在浓度为6至45 ng/ml时可使合适靶细胞的[3H]亮氨酸掺入减少50%，而无反应性的抗体免疫毒素在高达10(4) ng/ml的浓度下对靶细胞无毒。这些免疫毒素的特异性细胞毒性可通过添加未偶联的反应性MoAb来抑制；乳糖或无反应性MoAb的存在对观察到的细胞毒性没有显著影响。因此，以这种方式产生的完整蓖麻毒素 - 抗体偶联物不会非特异性地结合靶细胞，最重要的意义在于这种免疫毒素应该比蓖麻毒素A链偶联物更有效并且能够在体内使用。
