Next generation maleimides enable the controlled assembly of antibody–drug conjugates via native disulfide bond bridging†

Abstract

Highly homogeneous ADCs are generated by the efficient bridging of interchain disulfide bonds in trastuzumab, using next generation maleimides.

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.

Notes

†Electronic supplementary information (ESI) available: Synthetic procedures, ¹H and ¹³C NMR spectra for synthesised compounds, additional SDS-PAGE gels, LCMS chromatograms, Ellman's analysis and other supplementary results. See DOI: 10.1039/c4ob01550a