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Antibody–Drug Conjugates with Indolinobenzodiazepine Dimer Payloads: DNA-Binding Mechanism of Indolinobenzodiazepine Dimer Catabolites in Target Cancer Cells
journal contribution
posted on 2019-12-03, 17:41 authored by Rajeeva Singh, Emily E. Reid, Luke Harris, Paulin L. Salomon, Michael L. Miller, Ravi V. J. Chari, Thomas A. KeatingDNA-targeting indolinobenzodiazepine dimer (IGN) payloads
are used
in several clinical-stage antibody–drug conjugates. IGN drugs
alkylate DNA through the single imine moiety present in the dimer
in contrast to the pyrrolobenzodiazepine dimer drugs, such as talirine
and tesirine, which contain two imine moieties per dimer and cross-link
DNA. This study explored the mechanism of binding of IGN to DNA in
cells and to synthetic duplex and hairpin oligonucleotides. New, highly
sensitive IGN–DNA binding enzyme-linked immunosorbent assay
methods were developed using biotinylated IGN analogues (monoimine,
diimine, and diamine IGNs) and digoxigenin-labeled duplex oligonucleotides,
which allowed the measurement of drug–DNA adducts in viable
cells at concentrations below IC50. Furthermore, the release
of free drug from the IGN–DNA adduct upon treatment with nuclease
ex vivo was tested under physiological conditions. The monoimine IGN
drug formed a highly stable adduct with DNA in cells, with stability
similar to that of the diimine drug analogue. Both monoimine and diimine
IGN–DNA adducts released free drugs upon DNA cleavage by nuclease
at 37 °C, although more free drug was released from the monoimine
compared to the diimine adduct, which presumably was partly cross-linked.
The strong binding of the monoimine IGN drug to duplex DNA results
from both the noncovalent IGN–DNA interaction and the covalent
bond formation between the 2-amino group of a guanine residue and
the imine moiety in IGN.
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Keywords
duplex DNA resultsdigoxigenin-labeled duplex oligonucleotidespyrrolobenzodiazepine dimer drugsadductbiotinylated IGN analoguesIGN drugs alkylate DNAIndolinobenzodiazepine Dimer Catabolitesmonoimine IGN drugcovalent bond formationTarget Cancer Cells DNA-targeting indolinobenzodiazepine dimerICIndolinobenzodiazepine Dimer Payloadsdiimine drug analogueimine moiety
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