title: Where Did the Linker-Payload Go? A Quantitative Investigation on the Destination of the Released Linker-Payload from an Antibody-Drug Conjugate with a Maleimide Linker in Plasma.
abstract: The reactive thiol of cysteine is often used for coupling maleimide-containing linker-payloads to antibodies resulting in the generation of antibody drug conjugates (ADCs). Currently, a numbers of ADCs in drug development are made by coupling a linker-payload to native or engineered cysteine residues on the antibody. An ADC conjugated via hinge-cysteines to an auristatin payload was used as a model in this study to understand the impact of the maleimide linkers on ADC stability. The payload was conjugated to trastuzumab by a protease-cleavable linker, maleimido-caproyl-valine-citruline-p-amino-benzyloxy carbonyl (mcVC-PABC). In plasma stability assays, when the ADC (Trastuzumab-mcVC-PABC-Auristatin-0101) was incubated with plasma over a 144-h time-course, a discrepancy was observed between the measured released free payload concentration and the measured loss of drug-to-antibody ratio (DAR), as measured by liquid chromatography-mass spectrometry (LC-MS). We found that an enzymatic release of payload from ADC-depleted human plasma at 144 h was able to account for almost 100% of the DAR loss. Intact protein mass analysis showed that at the 144 h time point, the mass of the major protein in ADC-depleted human plasma had an additional 1347 Da over the native albumin extracted from human plasma, exactly matching the mass of the linker-payload. In addition, protein gel electrophoresis showed that there was only one enriched protein in the 144 h ADC-depleted and antipayload immunoprecipitated plasma sample, as compared to the 0 h plasma immunoprecipitated sample, and the mass of this enriched protein was slightly heavier than the mass of serum albumin. Furthermore, the albumin adduct was also identified in 96 h and 168 h postdose in vivo cynomolgus monkey plasma. These results strongly suggest that the majority of the deconjugated mc-VC-PABC-auristatin ultimately is transferred to serum albumin, forming a long-lived albumin-linker-payload adduct. To our knowledge, this is the first report quantitatively characterizing the extent of linker-payload transfer to serum albumin and the first clear example of in vivo formation of an albumin-linker-payload adduct.
authors.affiliation: Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc. , Eastern Point Road, Groton, Connecticut 06340, United States.
authors.forename: Cong
authors.initials: C
authors.lastname: Wei
authors.affiliation: Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc. , Eastern Point Road, Groton, Connecticut 06340, United States.
authors.forename: Guodong
authors.initials: G
authors.lastname: Zhang
authors.affiliation: Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc. , Eastern Point Road, Groton, Connecticut 06340, United States.
authors.forename: Tracey
authors.initials: T
authors.lastname: Clark
authors.affiliation: Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc. , Eastern Point Road, Groton, Connecticut 06340, United States.
authors.forename: Frank
authors.initials: F
authors.lastname: Barletta
authors.affiliation: Worldwide Medicinal Chemistry, Pfizer Inc. , Eastern Point Road, Groton, Connecticut 06340, United States.
authors.forename: L Nathan
authors.initials: LN
authors.lastname: Tumey
authors.affiliation: Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc. , Eastern Point Road, Groton, Connecticut 06340, United States.
authors.forename: Brian
authors.initials: B
authors.lastname: Rago
authors.affiliation: Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc. , Eastern Point Road, Groton, Connecticut 06340, United States.
authors.forename: Steven
authors.initials: S
authors.lastname: Hansel
authors.affiliation: Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc. , Eastern Point Road, Groton, Connecticut 06340, United States.
authors.forename: Xiaogang
authors.initials: X
authors.lastname: Han
mesh_terms: D062365:Aminobenzoates / Q000097:blood / Q000737:chemistry*; D000818:Animals; D006801:Humans; D008252:Macaca fascicularis; D008301:Maleimides / Q000097:blood / Q000737:chemistry*; D009842:Oligopeptides / Q000097:blood / Q000737:chemistry*; D051381:Rats; D000068878:Trastuzumab / Q000097:blood / Q000737:chemistry*
chemical_list: D062365:Aminobenzoates; D008301:Maleimides; D009842:Oligopeptides; C543533:auristatin; C043592:maleimide; D000068878:Trastuzumab