Abstract 230: Antibody-drug conjugates (ADCs) with indolinobenzodiazepine dimer (IGN) payloads: DNA-binding mechanism of IGN catabolites in target cancer cells
Authors: Singh Rajeeva , Harris Luke , Salomon Paulin , Reid Emily E. , Miller Michael L. , Chari Ravi V. , Keating Thomas A. 
Published: August 2019

A DNA-interacting indolinobenzodiazepine dimer (IGN) payload was designed with a single reactive imine group towards the goal of eliminating DNA cross-linking and avoiding related toxicities, while conferring a strong binding of the IGN scaffold to duplex DNA. Several IGN ADCs, wherein the payload is linked via a peptide or hindered disulfide, are currently being evaluated in the clinic. In contrast to our lead IGNs, DNA-interacting pyrrolobenzodiazepine (PBD)-based ADC payloads, such as talirine and tesirine, contain two reactive imine groups that can cross-link DNA.

Here, we investigated the mechanism of binding of IGN catabolites with DNA in target cancer cells, and with model duplex DNA or hairpin oligonucleotides. Hairpin and duplex oligonucleotides, designed for high melting temperatures (around 50-60 °C), were custom synthesized with labels. Model IGN catabolites bearing a single imine (mono-imine) or two imine groups (di-imine) were synthesized with a biotin label. Sensitive assays were developed to measure IGN-DNA binding in cells at sub-cytotoxic concentrations (lower than IC50) to allow studies of DNA adduct stability and repair.

The mono-imine IGN molecules bind readily to oligonucleotides, generating stable adducts as determined by gel filtration and reversed phase HPLC analysis. To investigate the binding of unconjugated IGNs with cellular DNA, cancer cells were incubated with mono- and di-imine IGNs for a short-term, followed by wash and further incubation in fresh media. Both mono-and di-imine IGN molecules remained bound to genomic DNA even at 2 days, suggesting a potent interaction with cellular DNA. The time course of binding of IGN to DNA in cells was slower than that observed with model oligonucleotides, as expected because the tightly coiled cellular DNA presumably binds IGN only after the unwinding of DNA during cell cycle or transcription.

Upon DNA cleavage by an added nuclease, free IGN was released from IGN adducts of model oligonucleotides and from genomic DNA of cells that had been treated with unconjugated IGN or IGN ADC. This dissociation of IGN from IGN-DNA adducts only upon cleavage with nuclease suggests that a strong non-covalent interaction between IGN and duplex DNA stabilizes the adduct. The amount of free IGN released from cellular DNA adduct upon nuclease treatment was about 2-fold greater for mono-imine IGN than di-imine IGN, presumably because di-imine IGN was partly cross-linked to cellular DNA. Mono-imine IGN-DNA adducts could potentially be repaired by cellular endonucleases via a DNA cleavage mechanism. In conclusion, the mono-imine IGN payload molecules form highly stable adducts with DNA, which dissociate upon DNA cleavage at physiological temperature.

Citation Format: Rajeeva Singh, Luke Harris, Paulin Salomon, Emily E. Reid, Michael L. Miller, Ravi V. Chari, Thomas A. Keating. Antibody-drug conjugates (ADCs) with indolinobenzodiazepine dimer (IGN) payloads: DNA-binding mechanism of IGN catabolites in target cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 230.