The effects of aromatic and aliphatic maleimide crosslinkers on anti-CD5 ricin immunotoxins

Dorothea E. Myers, Fatih M. Uckun, Steven E. Swaim, Daniel A. Vallera

Research output: Contribution to journalArticle

13 Scopus citations

Abstract

The aromatic maleimide crosslinkers m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), succinimidyl 4-(p-maleimidophenyl) butyrate (S-SMPB) and m-maleimidobenzoylsulfosuccinimide ester (S-MBS) and the aliphatic crosslinker N-γ-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.

Original languageEnglish (US)
Pages (from-to)129-142
Number of pages14
JournalJournal of Immunological Methods
Volume121
Issue number1
DOIs
StatePublished - Jul 6 1989

Keywords

  • Aliphatic crosslinker
  • Aromatic crosslinker
  • Bone marrow transplantation
  • Immunotoxin

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