Synthesis and evaluation of pyrrolobenzodiazepine dimer antibody-drug conjugates with dual ß-glucuronide and dipeptide triggers.

Antibody-drug conjugates (ADCs) containing pyrrolobenzodiazepine (PBD) dimers are currently being evaluated in human oncology clinical trials with encouraging results. To further improve the therapeutic window, next-generation PBD drug-linker design has focused on the inclusion of additional tumor-selective triggers and use of lower-potency PBDs. ß-Glucuronidase is a well-known target for discovery prodrugs due to increased presence in tumor cells and microenvironment. In this study, a ß-glucuronidase cleavable cap was investigated at the PBD N10-position and compared with corresponding free imine ADCs. SG3600 (glucuronide) ADCs showed in vitro and in vivo efficacy/tolerability comparable to SG3400 (imine) ADCs, and good 50% inhibitory concentration differentials were observed in vitro between control non-antigen-targeted ADCs and targeted ADCs. Dependence on ß-glucuronidase for SG3600 activity was demonstrated through CRISPRCas9 knockdown studies and addition of exogenous ß-glucuronidase. SG3600 showed better serum stability, improved conjugation efficiency and was able to reach high drug-to-antibody ratio without aggregation.

Potentiation of PBD Dimers by Lipophilicity Manipulation.

Background & Introduction: Pyrrolobenzodiazepine (PBD) dimers are highly potent DNA cross-linking agents used as warheads in Antibody Drug Conjugates (ADCs) for cancer therapy. We propose to investigate the correlation existing between the lipophilicity of those molecules and their activity (both in vitro and in vivo) as well as any effect observed during conjugation. MATERIALS AND METHODS: Reaction progress was monitored by Thin-Layer Chromatography (TLC) using Merck Kieselgel 60 F254 silica gel, with a fluorescent indicator on aluminium plates. Visualisation of TLC was achieved with UV light or iodine vapour unless otherwise stated. Flash chromatography was performed using Merck Kieselgel 60 F254 silica gel. RESULTS: We have successfully designed and synthesized a novel PBD warhead (SG3312) with enhanced physicochemical properties. The warhead also displayed increased potency in vitro. After overcoming some epimerization issues, the synthesis of enantiomerically pure payload was achieved (SG3259) and fulfilled our criteria for a simplified and more efficient conjugation. No addition of propylene glycol was required, and high DAR and excellent monomeric purity were achieved. CONCLUSION: The ADC (Herceptin-maia-SG3259) has been shown to release the active warhead (SG3312) upon exposure to Cathepsin B and demonstrated encouraging activity both in vitro and in vivo.

Synthesis and in vitro evaluation of SG3227, a pyrrolobenzodiazepine dimer antibody-drug conjugate payload based on sibiromycin.

A novel pyrrolobenzodiazepine dimer payload, SG3227, was rationally designed based on the naturally occurring antitumour compound sibiromycin. SG3227 was synthesized from a dimeric core in an efficient fashion. An unexpected room temperature Diels-Alder reaction occurred during the final step of the synthesis and was circumvented by use of an iodoacetamide conjugation moiety in place of a maleimide. The payload was successfully conjugated to trastuzumab and the resulting ADC exhibited potent activity against a HER2-expressing human cancer cell line in vitro.

Design and Synthesis of Tesirine, a Clinical Antibody-Drug Conjugate Pyrrolobenzodiazepine Dimer Payload.

Pyrrolobenzodiazepine dimers are an emerging class of warhead in the field of antibody-drug conjugates (ADCs). Tesirine (SG3249) was designed to combine potent antitumor activity with desirable physicochemical properties such as favorable hydrophobicity and improved conjugation characteristics. One of the reactive imines was capped with a cathepsin B-cleavable valine-alanine linker. A robust synthetic route was developed to allow the production of tesirine on clinical scale, employing a flexible, convergent strategy. Tesirine was evaluated in vitro both in stochastic and engineered ADC constructs and was confirmed as a potent and versatile payload. The conjugation of tesirine to anti-DLL3 rovalpituzumab has resulted in rovalpituzumab-tesirine (Rova-T), currently under evaluation for the treatment of small cell lung cancer.