title: Site-Dependent Degradation of a Non-Cleavable Auristatin-Based Linker-Payload in Rodent Plasma and Its Effect on ADC Efficacy.
abstract: 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.
authors.affiliation: Rinat Laboratories, Pfizer Inc., 230 East Grand Avenue, South San Francisco, CA, 94080, United States of America.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Dorywalska
authors.affiliation: Rinat Laboratories, Pfizer Inc., 230 East Grand Avenue, South San Francisco, CA, 94080, United States of America.
authors.forename: Pavel
authors.initials: P
authors.lastname: Strop
authors.affiliation: Rinat Laboratories, Pfizer Inc., 230 East Grand Avenue, South San Francisco, CA, 94080, United States of America.
authors.forename: Jody A
authors.initials: JA
authors.lastname: Melton-Witt
authors.affiliation: Rinat Laboratories, Pfizer Inc., 230 East Grand Avenue, South San Francisco, CA, 94080, United States of America.
authors.forename: Adela
authors.initials: A
authors.lastname: Hasa-Moreno
authors.affiliation: Rinat Laboratories, Pfizer Inc., 230 East Grand Avenue, South San Francisco, CA, 94080, United States of America.
authors.forename: Santiago E
authors.initials: SE
authors.lastname: Farias
authors.affiliation: Rinat Laboratories, Pfizer Inc., 230 East Grand Avenue, South San Francisco, CA, 94080, United States of America.
authors.forename: Meritxell
authors.initials: M
authors.lastname: Galindo Casas
authors.affiliation: Rinat Laboratories, Pfizer Inc., 230 East Grand Avenue, South San Francisco, CA, 94080, United States of America.
authors.forename: Kathy
authors.initials: K
authors.lastname: Delaria
authors.affiliation: Rinat Laboratories, Pfizer Inc., 230 East Grand Avenue, South San Francisco, CA, 94080, United States of America.
authors.forename: Victor
authors.initials: V
authors.lastname: Lui
authors.affiliation: Rinat Laboratories, Pfizer Inc., 230 East Grand Avenue, South San Francisco, CA, 94080, United States of America.
authors.forename: Kris
authors.initials: K
authors.lastname: Poulsen
authors.affiliation: Rinat Laboratories, Pfizer Inc., 230 East Grand Avenue, South San Francisco, CA, 94080, United States of America.
authors.forename: Janette
authors.initials: J
authors.lastname: Sutton
authors.affiliation: Rinat Laboratories, Pfizer Inc., 230 East Grand Avenue, South San Francisco, CA, 94080, United States of America.
authors.forename: Gary
authors.initials: G
authors.lastname: Bolton
authors.affiliation: Worldwide Medicinal Chemistry, Pfizer Inc., 445 Eastern Point Road, Groton, CT, 06340, United States of America.
authors.forename: Dahui
authors.initials: D
authors.lastname: Zhou
authors.affiliation: Worldwide Medicinal Chemistry, Pfizer Inc., 445 Eastern Point Road, Groton, CT, 06340, United States of America.
authors.forename: Ludivine
authors.initials: L
authors.lastname: Moine
authors.affiliation: Worldwide Medicinal Chemistry, Pfizer Inc., 445 Eastern Point Road, Groton, CT, 06340, United States of America.
authors.forename: Russell
authors.initials: R
authors.lastname: Dushin
authors.affiliation: Rinat Laboratories, Pfizer Inc., 230 East Grand Avenue, South San Francisco, CA, 94080, United States of America.
authors.forename: Thomas-Toan
authors.initials: TT
authors.lastname: Tran
authors.affiliation: Rinat Laboratories, Pfizer Inc., 230 East Grand Avenue, South San Francisco, CA, 94080, United States of America.
authors.forename: Shu-Hui
authors.initials: SH
authors.lastname: Liu
authors.affiliation: Rinat Laboratories, Pfizer Inc., 230 East Grand Avenue, South San Francisco, CA, 94080, United States of America.
authors.forename: Mathias
authors.initials: M
authors.lastname: Rickert
authors.affiliation: Rinat Laboratories, Pfizer Inc., 230 East Grand Avenue, South San Francisco, CA, 94080, United States of America.
authors.forename: Davide
authors.initials: D
authors.lastname: Foletti
authors.affiliation: Rinat Laboratories, Pfizer Inc., 230 East Grand Avenue, South San Francisco, CA, 94080, United States of America.
authors.forename: David L
authors.initials: DL
authors.lastname: Shelton
authors.affiliation: Rinat Laboratories, Pfizer Inc., 230 East Grand Avenue, South San Francisco, CA, 94080, United States of America.
authors.forename: Jaume
authors.initials: J
authors.lastname: Pons
authors.affiliation: Rinat Laboratories, Pfizer Inc., 230 East Grand Avenue, South San Francisco, CA, 94080, United States of America.
authors.forename: Arvind
authors.initials: A
authors.lastname: Rajpal
mesh_terms: D062365:Aminobenzoates / Q000008:administration & dosage / Q000737:chemistry / Q000493:pharmacokinetics*; D000818:Animals; D000906:Antibodies / Q000008:administration & dosage; D004337:Drug Carriers / Q000008:administration & dosage / Q000737:chemistry / Q000493:pharmacokinetics*; D004355:Drug Stability; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D008252:Macaca fascicularis; D016513:Mice, SCID; D009842:Oligopeptides / Q000008:administration & dosage / Q000737:chemistry / Q000493:pharmacokinetics*; D051381:Rats
chemical_list: D062365:Aminobenzoates; D000906:Antibodies; D004337:Drug Carriers; D009842:Oligopeptides; C543533:auristatin