title: Next generation maleimides enable the controlled assembly of antibody-drug conjugates via native disulfide bond bridging.
abstract: The advent of Adcetris™ and Kadcyla™, two recently FDA-approved antibody-drug conjugates (ADCs), in the clinic has had a major impact on the treatment of lymphoma and breast cancer patients, respectively, worldwide. Despite these successes many new ADCs fail at various stages of development, often due to shortcomings in the methods used for their assembly. To address this problem we have developed next generation maleimides (NGMs), which specifically re-bridge reduced interchain disulfide bonds and allow the efficient conjugation of small molecules to antibodies, without the need for engineering of the target antibody. The method is site-specific and generates near homogeneous products in good yields. Moreover, adjustment of the reaction conditions allows control of the conjugation in terms of stoichiometry (drug-loading) and site selectivity. Using this method we prepared a series of ADCs from trastuzumab and doxorubicin (DOX) with a controlled drug-to-antibody ratio (DAR) of 1, 2, 3 and 4. All of these constructs were fully active by ELISA and had more than 90% of re-bridged disulfide bonds by CE-SDS when compared to clinical grade antibody. Furthermore, digest experiments of the DAR 2 material revealed that almost all of the drug had been targeted to the Fab arms of the antibody. Thus, NGMs offer a flexible and simple platform for the controlled assembly of ADCs from an antibody.
authors.affiliation: Department of Chemistry, University College London, 20 Gordon Street, London, WC1H 0AJ, UK. s.caddick@ucl.ac.uk j.r.baker@ucl.ac.uk.
authors.forename: Felix F
authors.initials: FF
authors.lastname: Schumacher
authors.forename: João P M
authors.initials: JP
authors.lastname: Nunes
authors.forename: Antoine
authors.initials: A
authors.lastname: Maruani
authors.forename: Vijay
authors.initials: V
authors.lastname: Chudasama
authors.forename: Mark E B
authors.initials: ME
authors.lastname: Smith
authors.forename: Kerry A
authors.initials: KA
authors.lastname: Chester
authors.forename: James R
authors.initials: JR
authors.lastname: Baker
authors.forename: Stephen
authors.initials: S
authors.lastname: Caddick
mesh_terms: D000906:Antibodies / Q000737:chemistry*; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D004317:Doxorubicin / Q000737:chemistry*; D008301:Maleimides / Q000138:chemical synthesis* / Q000737:chemistry; D015394:Molecular Structure; D000068878:Trastuzumab
chemical_list: D000906:Antibodies; D061067:Antibodies, Monoclonal, Humanized; D004220:Disulfides; D008301:Maleimides; D004317:Doxorubicin; D000068878:Trastuzumab
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