title: Stabilization of cysteine-linked antibody drug conjugates with N-aryl maleimides.
abstract: 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.
authors.affiliation: Antibody Discovery and Protein Engineering, MedImmune, Gaithersburg, MD 20878, USA. Electronic address: christier@medimmune.com.
authors.forename: R James
authors.initials: RJ
authors.lastname: Christie
authors.affiliation: Antibody Discovery and Protein Engineering, MedImmune, Gaithersburg, MD 20878, USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: Fleming
authors.affiliation: Antibody Discovery and Protein Engineering, MedImmune, Gaithersburg, MD 20878, USA.
authors.forename: Binyam
authors.initials: B
authors.lastname: Bezabeh
authors.affiliation: Antibody Discovery and Protein Engineering, MedImmune, Gaithersburg, MD 20878, USA.
authors.forename: Rob
authors.initials: R
authors.lastname: Woods
authors.affiliation: Oncology Research, MedImmune, Gaithersburg, MD 20878, USA.
authors.forename: Shenlan
authors.initials: S
authors.lastname: Mao
authors.affiliation: Oncology Research, MedImmune, Gaithersburg, MD 20878, USA.
authors.forename: Jay
authors.initials: J
authors.lastname: Harper
authors.affiliation: SynChem, Inc., Elk Grove Village, IL 60007, USA.
authors.forename: Augustine
authors.initials: A
authors.lastname: Joseph
authors.affiliation: SynChem, Inc., Elk Grove Village, IL 60007, USA.
authors.forename: Qianli
authors.initials: Q
authors.lastname: Wang
authors.affiliation: SynChem, Inc., Elk Grove Village, IL 60007, USA.
authors.forename: Ze-Qi
authors.initials: ZQ
authors.lastname: Xu
authors.affiliation: Antibody Discovery and Protein Engineering, MedImmune, Gaithersburg, MD 20878, USA.
authors.forename: Herren
authors.initials: H
authors.lastname: Wu
authors.affiliation: Antibody Discovery and Protein Engineering, MedImmune, Gaithersburg, MD 20878, USA.
authors.forename: Changshou
authors.initials: C
authors.lastname: Gao
authors.affiliation: Antibody Discovery and Protein Engineering, MedImmune, Gaithersburg, MD 20878, USA. Electronic address: dimasin@medimmune.com.
authors.forename: Nazzareno
authors.initials: N
authors.lastname: Dimasi
mesh_terms: D000911:Antibodies, Monoclonal / Q000097:blood / Q000737:chemistry*; D000970:Antineoplastic Agents / Q000737:chemistry*; D002626:Chemistry, Pharmaceutical; D003432:Cross-Linking Reagents / Q000737:chemistry*; D003545:Cysteine; D004355:Drug Stability; D006868:Hydrolysis; D018796:Immunoconjugates / Q000097:blood / Q000737:chemistry*; D007700:Kinetics; D008301:Maleimides / Q000097:blood / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry*; D011487:Protein Conformation; D055550:Protein Stability; D013438:Sulfhydryl Compounds / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D003432:Cross-Linking Reagents; D018796:Immunoconjugates; D008301:Maleimides; D009842:Oligopeptides; D013438:Sulfhydryl Compounds; C028525:N-phenylmaleimide; D003545:Cysteine; C495575:monomethyl auristatin E
keywords: Antibody-drug conjugate; Maleimide; Retro-Michael reaction; Serum stability; Thiol conjugation; Thiosuccinimide hydrolysis