Dhanabal M, Fryxell D K, Ramakrishnan S
Department of Pharmacology, University of Minnesota, Minneapolis 55455, USA.
J Immunol Methods. 1995 Jun 9;182(2):165-75. doi: 10.1016/0022-1759(95)00036-a.
Monoclonal antibodies linked to toxin polypeptides (immunotoxins) are developed for clinical application against cancer and graft rejection. Immunotoxins prepared by many conventional methods often contain a trace amount of free antibody. Present studies describe a method to purify immunotoxins from free antibody in conjugation mixtures. Recombinant ricin A chain and a truncated form of diphtheria toxin (385 residues) containing ten consecutive histidine residues at the amino terminus were prepared. The modified toxin polypeptides retaining full biological activity were chemically linked to monoclonal antibodies (317G5 and 454C11) reactive to breast cancer cells. The high affinity of consecutive histidine residues for nickel-based resin (Ni-NTA) was exploited to purify immunotoxins from unreacted free antibodies. SDS-PAGE analysis of conjugates eluted from nickel column contained trace amounts of detectable free antibody whereas conjugates purified by other conventional methods using phenyl Sepharose or Cibacron blue Sepharose chromatography contained significant amounts of unconjugated antibody. Furthermore, the immunotoxin fraction containing predominantly two toxin molecules linked to one antibody can be separated from stoichiometric conjugates by Ni-NTA column. Cytotoxicity experiments showed that the complex of two toxin molecules linked to an antibody was more cytotoxic to tumor cells in vitro than the fraction enriched with immunotoxin containing equimolar stoichiometry.
与毒素多肽相连的单克隆抗体（免疫毒素）被开发用于临床对抗癌症和移植排斥反应。许多传统方法制备的免疫毒素通常含有微量的游离抗体。目前的研究描述了一种从偶联混合物中纯化免疫毒素以去除游离抗体的方法。制备了重组蓖麻毒素A链和一种截短形式的白喉毒素（385个残基），其在氨基末端含有十个连续的组氨酸残基。将保留完整生物活性的修饰毒素多肽化学连接到对乳腺癌细胞有反应性的单克隆抗体（317G5和454C11）上。利用连续组氨酸残基对镍基树脂（Ni-NTA）的高亲和力从未反应的游离抗体中纯化免疫毒素。对从镍柱洗脱的偶联物进行SDS-PAGE分析，结果显示含有痕量可检测的游离抗体，而通过使用苯基琼脂糖或Cibacron blue琼脂糖色谱的其他传统方法纯化的偶联物含有大量未偶联的抗体。此外，主要含有与一个抗体相连的两个毒素分子的免疫毒素组分可以通过Ni-NTA柱与化学计量的偶联物分离。细胞毒性实验表明，与抗体相连的两个毒素分子的复合物在体外对肿瘤细胞的细胞毒性比富含等摩尔化学计量免疫毒素的组分更强。
