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Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index

Abstract

Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogenous conjugates with relatively narrow therapeutic index (maximum tolerated dose/curative dose). Using leads from our previously described phage display–based method to predict suitable conjugation sites, we engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb immunoglobulin folding and assembly, or alter antigen binding. When conjugated to monomethyl auristatin E, an antibody against the ovarian cancer antigen MUC16 is as efficacious as a conventional conjugate in mouse xenograft models. Moreover, it is tolerated at higher doses in rats and cynomolgus monkeys than the same conjugate prepared by conventional approaches. The favorable in vivo properties of the near-homogenous composition of this conjugate suggest that our strategy offers a general approach to retaining the antitumor efficacy of antibody-drug conjugates, while minimizing their systemic toxicity.

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Figure 1: Characterization of THIOMABs.
Figure 2: THIOMAB drug conjugates retain interchain disulfide bonds with site-specific drug attachment.
Figure 3: In vivo efficacy is retained with the TDC format.
Figure 4: The TDC format is better tolerated in vivo.
Figure 5: In rats, a higher proportion of TDC is retained in circulation, compared with its ADC counterpart.

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Acknowledgements

The authors wish to thank our Genentech colleagues: Jennifer Speer for preparing trastuzumab THIOMAB DNA constructs; Mary Cole for insights into the OVCAR-3 intraperitoneal efficacy model; Elmer Wu, Darshana Patel, Mark Rowen and Anthony Delucchi for providing critical reagents; Natalia Gomez and George Dutina for large-scale transient transfection/fermentation; Fred Jacobson and Charity Bechtel for their help with analytical characterization of TDCs; and Allen Ebens for critical review of the manuscript. We thank Damon Meyer and his colleagues at Seattle Genetics for preparation of the early lots of anti-MUC16 ADC and TDC and for many helpful comments and suggestions. We thank employees of NPIL Pharma UK for their assistance with large-scale conjugations. We also thank the staff of Oncotest for conducting some of the efficacy studies described in this manuscript. Anti-MMAE mouse monoclonal antibody SG2.15 was generously provided by Seattle Genetics, Inc.

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Contributions

J.R.J. and W.M. led the overall program, designed experiments, performed in vitro studies, analyzed the data and wrote the manuscript. Y.C. and M.S.D. humanized the anti-MUC16 antibody. S.B. generated the anti-MUC16 THIOMAB DNA constructs and performed pilot expression studies. M.S., E.D. and J.G. performed larger-scale antibody production. H.R. established procedures for TDC conjugation and analytical characterization. C.C.L. carried out analytical characterization of TDCs. S.W., S.P.T., Y.L., Y.G.M., C.N. and J.Y. performed in vitro binding studies. S.C., R. Venook and S.R. performed in vivo efficacy studies. D.D.L. designed and analyzed pharmacokinetic studies. A.K., K.M. and K.F. designed and executed safety assessment studies. V.K., S.D.S., W.L.W., H.B.L., R. Vandlen, M.X.S., R.H.S. and P.P. provided direction and guidance for the various functional areas and assisted in writing the manuscript.

Corresponding authors

Correspondence to Jagath R Junutula or William Mallet.

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All authors are full-time employees of Genentech. Inc.

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Supplementary Figures 1–17, Tables 1–5, Methods (PDF 3037 kb)

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Junutula, J., Raab, H., Clark, S. et al. Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat Biotechnol 26, 925–932 (2008). https://doi.org/10.1038/nbt.1480

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