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The AAPS Journal

, Volume 19, Issue 4, pp 1123–1135 | Cite as

Site Selection: a Case Study in the Identification of Optimal Cysteine Engineered Antibody Drug Conjugates

  • L. Nathan Tumey
  • Fengping Li
  • Brian Rago
  • Xiaogang Han
  • Frank Loganzo
  • Sylvia Musto
  • Edmund I. Graziani
  • Sujiet Puthenveetil
  • Jeffrey Casavant
  • Kimberly Marquette
  • Tracey Clark
  • Jack Bikker
  • Eric M. Bennett
  • Frank Barletta
  • Nicole Piche-Nicholas
  • Amy Tam
  • Christopher J. O’Donnell
  • Hans Peter Gerber
  • Lioudmila Tchistiakova
Research Article

Abstract

As the antibody drug conjugate (ADC) community continues to shift towards site-specific conjugation technology, there is a growing need to understand how the site of conjugation impacts the biophysical and biological properties of an ADC. In order to address this need, we prepared a carefully selected series of engineered cysteine ADCs and proceeded to systematically evaluate their potency, stability, and PK exposure. The site of conjugation did not have a significant influence on the thermal stability and in vitro cytotoxicity of the ADCs. However, we demonstrate that the rate of cathepsin-mediated linker cleavage is heavily dependent upon site and is closely correlated with ADC hydrophobicity, thus confirming other recent reports of this phenomenon. Interestingly, conjugates with high rates of cathepsin-mediated linker cleavage did not exhibit decreased plasma stability. In fact, the major source of plasma instability was shown to be retro-Michael mediated deconjugation. This process is known to be impeded by succinimide hydrolysis, and thus, we undertook a series of mutational experiments demonstrating that basic residues located nearby the site of conjugation can be a significant driver of succinimide ring opening. Finally, we show that total antibody PK exposure in rat was loosely correlated with ADC hydrophobicity. It is our hope that these observations will help the ADC community to build “design rules” that will enable more efficient prosecution of next-generation ADC discovery programs.

KEY WORDS

antibody drug conjugate hydrophobicity linker stability PK exposure plasma stability 

Notes

Acknowledgements

We would like to gratefully acknowledge the work of Russell Dushin, Dahui Zhou, Matthew Doroski, Alex Porte, Hud Risley, Zecheng Chen, and Andreas Maderna for the preparation and identification of mcValCitPABC_06380101. Dosing of animals was performed by Judy Lucas and Christine Hosselet.

Supplementary material

12248_2017_83_MOESM1_ESM.docx (284 kb)
ESM 1 (DOCX 283 kb)

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Copyright information

© American Association of Pharmaceutical Scientists 2017

Authors and Affiliations

  • L. Nathan Tumey
    • 1
  • Fengping Li
    • 2
  • Brian Rago
    • 3
  • Xiaogang Han
    • 4
  • Frank Loganzo
    • 5
  • Sylvia Musto
    • 5
  • Edmund I. Graziani
    • 3
  • Sujiet Puthenveetil
    • 6
  • Jeffrey Casavant
    • 3
  • Kimberly Marquette
    • 2
  • Tracey Clark
    • 3
  • Jack Bikker
    • 7
  • Eric M. Bennett
    • 2
  • Frank Barletta
    • 3
  • Nicole Piche-Nicholas
    • 2
  • Amy Tam
    • 2
  • Christopher J. O’Donnell
    • 3
  • Hans Peter Gerber
    • 8
  • Lioudmila Tchistiakova
    • 2
  1. 1.Binghamton University, School of Pharmacy and Pharmaceutical SciencesBinghamtonUSA
  2. 2.Biomedicine Design, Pfizer, Inc.CambridgeUSA
  3. 3.Worldwide Research and Development, Pfizer, Inc.GrotonUSA
  4. 4.PKDM, Amgen, Inc.CambridgeUSA
  5. 5.Oncology Research and Development, Pfizer, Inc.Pearl RiverUSA
  6. 6.AbbVie, Inc.WorcesterUSA
  7. 7.International Flavors and FragrancesNew YorkUSA
  8. 8.Maverick Therapeutics, IncBrisbaneUSA

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