Mutations of Two Lysine Residues in the CDR Loops of a Recombinant Immunotoxin That Reduce Its Sensitivity to Chemical Derivatization

Itai Benhar, Ulrich Brinkmann, Keith O. Webber, Ira Pastan

Research output: Contribution to journalArticlepeer-review

Abstract

B3(Fv)-PE38 is a recombinant single-chain immunotoxin in which the Fv region of monoclonal antibody B3 is connected to a truncated form of Pseudomonas exotoxin. It would be desirable to use the lysine residues of the molecule for chemical modification so that it can be derivatized with poly(ethylene glycol) to achieve reduced immunogenicity or with the Bolton-Hunter reagent for biodistribution studies. We found that derivatizing lysine residues of B3(Fv)-PE38 causes a marked loss of specific target cell cytotoxicity and/or immunoreactivity. Here we show that two lysine residues in the antibody-combining region of B3(Fv)-PE38 can be replaced with arginines, with only a small loss of cytotoxicity and no change in specificity. This mutant molecule is 3-fold more resistant to inactivation by derivatization with succinimidyl 4-(N-maleimidomethyl)cyclohexane 1-carboxylate (SMCC) or Bolton-Hunter reagent.

Original languageEnglish
Pages (from-to)321-326
Number of pages6
JournalBioconjugate Chemistry
Volume5
Issue number4
DOIs
StatePublished - 1 Jul 1994
Externally publishedYes

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