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Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates

Abstract

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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Figure 1: The site of conjugation modulates the therapeutic activity of antibody conjugates.
Figure 2: The plasma stability of antibody conjugates is dependent on the conjugation sites but independent of linker, drug and antibody.
Figure 3: Proposed mechanism for the influence of the conjugation site on linker stability and therapeutic activity of antibody conjugates.
Figure 4: The conjugation site influences antibody-conjugate stability in vivo.

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Acknowledgements

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.

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B.-Q.S. and K.X. designed experiments developed the methodology, performed plasma stability studies and wrote the manuscript. L.L. and M.K. performed plasma stability and LC-MS analyses. H.R. conducted ADC conjugations and S.B. generated THIOMAB constructs and performed in vitro potency studies. K.L.P.-R., J.T. and S.-F.Y. performed in vivo efficacy studies. E.M., D.L. and J.T. conducted pharmacokinetic studies. J.B. and O.M.S. quantified free MMAE levels in the plasma. S.J.S. performed internalization studies. P.J.M. and P.E.H. purified antibodies. C.E. performed antibody structural analysis. T.N., W.A.S., R.N.F. and K.M.F. designed and conducted preclinical safety studies. D.P. and S.D.S. provided project management support. L.A.K., A.E., W.L.W., R.V., S.K., M.X.S. and R.H.S. provided direction and guidance. P.P. provided direction, guidance and assisted in writing the manuscript. J.R.J. led the overall conjugation site-dependent antibody conjugate program, generated trastuzumab THIOMAB constructs, designed experiments and wrote the manuscript.

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Correspondence to Jagath R Junutula.

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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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