Stabilization of cysteine-linked antibody drug conjugates with N-aryl maleimides

J Control Release. 2015 Dec 28;220(Pt B):660-70. doi: 10.1016/j.jconrel.2015.09.032. Epub 2015 Sep 24.


Maleimides are often used to covalently attach drugs to cysteine thiols for production of antibody-drug conjugates (ADCs). However, ADCs formed with traditional N-alkyl maleimides have variable stability in the bloodstream leading to loss of drug. Here, we report that N-aryl maleimides form stable antibody conjugates under very mild conditions while also maintaining high conjugation efficiency. Thiol-maleimide coupling and ADC stabilization via thiosuccinimide hydrolysis were accelerated by addition of N-phenyl or N-fluorophenyl groups to the ring-head nitrogen. Cysteine-linked ADCs prepared with N-aryl maleimides exhibited less than 20% deconjugation in both thiol-containing buffer and serum when incubated at 37 °C over a period of 7 days, whereas the analogous ADCs prepared with N-alkyl maleimides showed 35-67% deconjugation under the same conditions. ADCs prepared with the anticancer drug N-phenyl maleimide monomethyl-auristatin-E (MMAE) maintained high cytotoxicity following long-term exposure to serum whereas the N-alkyl maleimide MMAE ADC lost potency over time. These data demonstrate that N-aryl maleimides are a convenient and flexible platform to improve the stability of ADCs through manipulation of functional groups attached to the maleimide ring-head nitrogen.

Keywords: Antibody-drug conjugate; Maleimide; Retro-Michael reaction; Serum stability; Thiol conjugation; Thiosuccinimide hydrolysis.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Monoclonal / blood
  • Antibodies, Monoclonal / chemistry*
  • Antineoplastic Agents / chemistry*
  • Chemistry, Pharmaceutical
  • Cross-Linking Reagents / chemistry*
  • Cysteine
  • Drug Stability
  • Hydrolysis
  • Immunoconjugates / blood
  • Immunoconjugates / chemistry*
  • Kinetics
  • Maleimides / blood
  • Maleimides / chemistry*
  • Models, Chemical
  • Models, Molecular
  • Oligopeptides / chemistry*
  • Protein Conformation
  • Protein Stability
  • Sulfhydryl Compounds / chemistry*


  • Antibodies, Monoclonal
  • Antineoplastic Agents
  • Cross-Linking Reagents
  • Immunoconjugates
  • Maleimides
  • Oligopeptides
  • Sulfhydryl Compounds
  • N-phenylmaleimide
  • Cysteine
  • monomethyl auristatin E