Myers D E, Uckun F M, Swaim S E, Vallera D A
Department of Therapeutic Radiology, University of Minnesota Hospital and Clinic, Minneapolis 55455.
J Immunol Methods. 1989 Jul 6;121(1):129-42. doi: 10.1016/0022-1759(89)90428-6.
The aromatic maleimide crosslinkers m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfosuccinimidyl 4-(p-maleimidophenyl) butyrate (S-SMPB) and m-maleimidobenzoylsulfosuccinimide ester (S-MBS) and the aliphatic crosslinker N-gamma-maleimidobutyryloxysuccinimide ester (GMBS) were used to make anti-CD5 intact ricin immunotoxins (IT). IT made with the various crosslinkers were compared under standard conjugation conditions for differences in yield, toxicity of the toxin moiety, binding of the antibody moiety, IT activity, and IT specificity. Our findings showed that IT yield was dramatically improved using crosslinkers with an aromatic, rather than an aliphatic configuration. Gel analysis showed that all IT were of similar, but not identical composition. Conjugation resulted in several IT species including antibody linked to one or two molecules of ricin. For MBS IT and S-SMPB IT, differences in amounts of IT in final fractions and IT in fractions after removal of IT species containing galactose binding sites showed that differences in yield may be attributable to the formation of IT species with obstructed galactose binding sites. All IT bound selectively by FACS analysis and blocking studies. The aliphatic GMBS crosslinker yielded the most toxic IT in cell-free translation assays as well as in shorter-term protein synthesis inhibition and mitogen assays. However, evaluation in the longer-term, more sensitive clonogenic assay showed that at 1000 ng/ml there were no differences in potency between any of the IT. We conclude that the yield of intact ricin IT can be improved using aromatic maleimide crosslinkers without sacrificing IT potency.
芳香族马来酰亚胺交联剂间马来酰亚胺苯甲酰 -N-羟基琥珀酰亚胺酯（MBS）、磺基琥珀酰亚胺基 4-（对 - 马来酰亚胺苯基）丁酸酯（S - SMPB）和间马来酰亚胺苯甲酰磺基琥珀酰亚胺酯（S - MBS）以及脂肪族交联剂 N-γ-马来酰亚胺丁酰氧基琥珀酰亚胺酯（GMBS）被用于制备抗 CD5 完整蓖麻毒素免疫毒素（IT）。在标准偶联条件下，比较了用不同交联剂制备的 IT 在产量、毒素部分的毒性、抗体部分的结合、IT 活性和 IT 特异性方面的差异。我们的研究结果表明，使用具有芳香族而非脂肪族构型的交联剂可显著提高 IT 的产量。凝胶分析表明，所有 IT 的组成相似但不完全相同。偶联产生了几种 IT 种类，包括与一分子或两分子蓖麻毒素相连的抗体。对于 MBS IT 和 S - SMPB IT，最终组分中 IT 的量以及去除含有半乳糖结合位点的 IT 种类后的组分中 IT 的量的差异表明，产量差异可能归因于具有受阻半乳糖结合位点的 IT 种类的形成。通过流式细胞术分析和封闭研究，所有 IT 均具有选择性结合。在无细胞翻译试验以及短期蛋白质合成抑制和丝裂原试验中，脂肪族 GMBS 交联剂产生的 IT 毒性最大。然而，在更长期、更敏感的克隆形成试验中的评估表明，在 1000 ng/ml 时，任何一种 IT 的效力之间没有差异。我们得出结论，使用芳香族马来酰亚胺交联剂可提高完整蓖麻毒素 IT 的产量，而不会牺牲 IT 的效力。
