Abstract
Carboxylesterase 1c (CES1c) is responsible for linker-drug instability and poor pharmacokinetics (PK) of several antibody-drug conjugates (ADC) in mice, but not in monkeys or humans. Preclinical development of these ADCs could be improved if the PK in mice would more closely resemble that of humans and is not affected by an enzyme that is irrelevant for humans. SYD985, a HER2-targeting ADC based on trastuzumab and linker-drug vc-seco-DUBA, is also sensitive to CES1c. In the present studies, we first focused on the interaction between CES1c and SYD985 by size- exclusion chromatography, Western blotting, and LC/MS-MS analysis, using recombinant CES1c and plasma samples. Intriguingly, CES1c activity not only results in release of the active toxin DUBA but also in formation of a covalent bond between CES1c and the linker of vc-seco-DUBA. Mass spectrometric studies enabled identification of the CES1c cleavage site on the linker-drug and the structure of the CES1c adduct. To assess the in vivo impact, CES1c-/- SCID mice were generated that showed stable PK for SYD985, comparable to that in monkeys and humans. Patient-derived xenograft (PDX) studies in these mice showed enhanced efficacy compared with PDX studies in CES1c+/+ mice and provided a more accurate prediction of clinical efficacy of SYD985, hence delivering better quality data. It seems reasonable to assume that CES1c-/- SCID mice can increase quality in ADC development much broader for all ADCs that carry linker-drugs susceptible to CES1c, without the need of chemically modifying the linker-drug to specifically increase PK in mice. Mol Cancer Ther; 17(11); 2389-98. ©2018 AACR.
Highlights
Stability of the linker-drug in plasma has been, and still is, a major challenge in the development of antibody–drug conjugates (ADC)
SYD985, a HER2-targeting ADC based on trastuzumab and vc-seco-DUBA, a cleavable linker-duocarmycin payload, was found to be stable in human and monkey plasma, but unstable in mouse and rat plasma [13, 14]
carboxylesterase 1c (CES1c) expressed in rodent plasma cleaves vc-seco-DUBA In vitro plasma stability studies, as well as in vivo PK studies, showed that SYD985 conjugated antibody levels rapidly decrease in mouse and rat plasma, but are stable in monkey www.aacrjournals.org
Summary
Stability of the linker-drug in plasma has been, and still is, a major challenge in the development of antibody–drug conjugates (ADC). Linkers should be stable enough to prevent release of the active toxin in plasma, whereas on the other hand, linkers should allow for an efficient release of active toxin once the ADC targets a tumor or tumor cell. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Other possible causes of instability in plasma include maleimide exchange of the cysteineconjugated linker-drugs to other thiol-containing molecules circulating in plasma, such as serum albumin [9] and enzymatic instability by circulating enzymes in plasma [10, 11], which can cleave functional groups like esters, amides, lactones, lactams, carbamides, sulfonamides, and peptide mimetics [12]
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