The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a positively charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected positively by succinimide ring hydrolysis and negatively by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chemical and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.
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We thank our Genentech colleagues: J. Speer and E. Wu for reagent generation and inventory; D. Bumbaca for plasma stability study; J. Lau and I. Inigo for ADC efficacy studies; P. Carter and S. Panowski for critical review of the manuscript; H.B. Lowman, S. Kenkare-Mitra and I. Mellman for their insightful discussions. Anti-MMAE mouse monoclonal antibodies were a generous gift from Seattle Genetics.
All authors were employees of Genentech/Roche at the time this work was conducted.
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Shen, BQ., Xu, K., Liu, L. et al. Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates. Nat Biotechnol 30, 184–189 (2012). https://doi.org/10.1038/nbt.2108
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