Site-Dependent Degradation of a Non-Cleavable Auristatin-Based Linker-Payload in Rodent Plasma and Its Effect on ADC Efficacy

PLoS One. 2015 Jul 10;10(7):e0132282. doi: 10.1371/journal.pone.0132282. eCollection 2015.


The efficacy of an antibody-drug conjugate (ADC) is dependent on the properties of its linker-payload which must remain stable while in systemic circulation but undergo efficient processing upon internalization into target cells. Here, we examine the stability of a non-cleavable Amino-PEG6-based linker bearing the monomethyl auristatin D (MMAD) payload site-specifically conjugated at multiple positions on an antibody. Enzymatic conjugation with transglutaminase allows us to create a stable amide linkage that remains intact across all tested conjugation sites on the antibody, and provides us with an opportunity to examine the stability of the auristatin payload itself. We report a position-dependent degradation of the C terminus of MMAD in rodent plasma that has a detrimental effect on its potency. The MMAD cleavage can be eliminated by either modifying the C terminus of the toxin, or by selection of conjugation site. Both approaches result in improved stability and potency in vitro and in vivo. Furthermore, we show that the MMAD metabolism in mouse plasma is likely mediated by a serine-based hydrolase, appears much less pronounced in rat, and was not detected in cynomolgus monkey or human plasma. Clarifying these species differences and controlling toxin degradation to optimize ADC stability in rodents is essential to make the best ADC selection from preclinical models. The data presented here demonstrate that site selection and toxin susceptibility to mouse plasma degradation are important considerations in the design of non-cleavable ADCs, and further highlight the benefits of site-specific conjugation methods.

MeSH terms

  • Aminobenzoates / administration & dosage
  • Aminobenzoates / chemistry
  • Aminobenzoates / pharmacokinetics*
  • Animals
  • Antibodies / administration & dosage
  • Drug Carriers / administration & dosage
  • Drug Carriers / chemistry
  • Drug Carriers / pharmacokinetics*
  • Drug Stability
  • Female
  • HEK293 Cells
  • Humans
  • Macaca fascicularis
  • Mice, SCID
  • Oligopeptides / administration & dosage
  • Oligopeptides / chemistry
  • Oligopeptides / pharmacokinetics*
  • Rats


  • Aminobenzoates
  • Antibodies
  • Drug Carriers
  • Oligopeptides
  • auristatin

Grant support

Pfizer Inc. provided support in the form of salaries for authors M.D., P.S., J.A.M.-W., A.H.-M., S.E.F., M.G.C., K.D., V.L., K.P., J.S., G.B., D.Z., L.M., R.D., T.- T.T., S.-H.L., M.R., D.F., D.L.S., J.P., and A.R., but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.