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.

Microfluidic Mobility Shift Assay for Real-Time Analysis of Peptide N-Palmitoylation.

The Hedgehog pathway is a key developmental signaling pathway but is also implicated in many types of cancer. The extracellular signaling protein Sonic hedgehog (Shh) requires dual lipidation for functional signaling, whereby N-terminal palmitoylation is performed by the enzyme Hedgehog acyltransferase (Hhat). Hhat is an attractive target for small-molecule inhibition to arrest Hedgehog signaling, and methods for assaying Hhat activity are central to understanding its function. However, all existing assays to quantify lipidation of peptides suffer limitations, such as safety hazards, high costs, extensive manual handling, restriction to stopped-assay measurements, or indirect assessment of lipidation. To address these limitations, we developed a microfluidic mobility shift assay (MSA) to analyze Shh palmitoylation. MSA allowed separation of fluorescently labeled Shh amine-substrate and palmitoylated Shh amide-product peptides based on differences in charge and hydrodynamic radius, coupled with online fluorescence intensity measurements for quantification. The MSA format was employed to study Hhat-catalyzed reactions, investigate Hhat kinetics, and determine small-molecule inhibitor IC50 values. Both real-time and stopped assays were performed, with the latter achieved via addition of excess unlabeled Shh peptide. The MSA format therefore allows direct and real-time fluorescence-based measurement of acylation and represents a powerful alternative technique in the study of N-lipidation.

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.