Targeted drug delivery through the traceless release of tertiary and heteroaryl amines from antibody–drug conjugates

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The reversible attachment of a small-molecule drug to a carrier for targeted delivery can improve pharmacokinetics and the therapeutic index. Previous studies have reported the delivery of molecules that contain primary and secondary amines via an amide or carbamate bond; however, the ability to employ tertiary-amine-containing bioactive molecules has been elusive. Here we describe a bioreversible linkage based on a quaternary ammonium that can be used to connect a broad array of tertiary and heteroaryl amines to a carrier protein. Using a concise, protecting-group-free synthesis we demonstrate the chemoselective modification of 12 complex molecules that contain a range of reactive functional groups. We also show the utility of this connection with both protease-cleavable and reductively cleavable antibody–drug conjugates that were effective and stable in vitro and in vivo. Studies with a tertiary-amine-containing antibiotic show that the resulting antibody–antibiotic conjugate provided appropriate stability and release characteristics and led to an unexpected improvement in activity over the conjugates previously connected via a carbamate.

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Figure 1: Linker cleavage leads to traceless drug release.
Figure 2: Structures of drugs that show potential linkage sites.
Figure 3: A chemoselective two-step synthesis of a library of quaternary-ammonium-linked anticancer and antibacterial natural products and small molecules.
Figure 4: In vivo stability and efficacy of an antitumour agent conjugated to an antibody.
Figure 5: Synthesis of a disulfide-containing quaternary-ammonium-linked drug conjugate and the mechanism of reductive activation and drug release.
Figure 6: A quaternary-ammonium-linked antibiotic improves efficacy.

Change history

  • 03 November 2016

    In the version of this Article originally published, the structure of tubulysin was shown three times in Figure 5 and in each instance the five-membered ring was missing a nitrogen atom. This has been corrected in the online versions of this Article.


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We thank F. Fan, Z. Xu, J. Wai, H. Raab, B. Lin, K. Xu and A. Deese for helping in the preparation and analysis of the materials used in the study, and S. Spencer and R. Rowntree for coordinating the studies.

Author information




L.R.S., S.G.K., X.L., T.W., J.C. and T.H.P. designed and/or synthesized the linker–drug conjugates. S.M.L. designed, executed and analysed data for the in vitro antibiotic experiment. R.V. characterized cathepsin cleavage of linker–drug conjugates and ADCs. D.Z. characterized GSH reduction of the disulfide linker–drug conjugate. J.C. characterized the in vitro potency of cytotoxic ADCs. S.-F.Y. designed and analysed data for in vivo efficacy. C.N. characterized and analysed data for in vivo stability. J.G. characterized the potency of the free cytotoxic drugs. Y.L. confirmed the structure of linker–drug conjugates through NMR studies. A.F.-O. characterized the conjugate stability in whole blood. M.G. performed in vivo efficacy experiments. N.L.S. purified and characterized an antibiotic linker–drug conjugate. B.W. designed a peptidomimetic linker. G.D.L.P, K.X., K.R.K, S.M., J.A.F. and T.H.P. led groups on the project responsible for chemistry, biology and in vitro or in vivo characterization. T.H.P. conceived and initiated the project. T.H.P. wrote the manuscript with help and input from all of the authors.

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Correspondence to Thomas H. Pillow.

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All authors are full time employees of Genentech or WuXi AppTec.

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Staben, L., Koenig, S., Lehar, S. et al. Targeted drug delivery through the traceless release of tertiary and heteroaryl amines from antibody–drug conjugates. Nature Chem 8, 1112–1119 (2016).

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