Efficacy, Tolerability, and Pharmacokinetic Studies of Antibody-Drug Conjugates Containing a Low-Potency Pyrrolobenzodiazepine Dimer.

Antibody-drug conjugate (ADC) research has typically focused on the release of highly potent cytotoxic agents to achieve antitumor efficacy. However, recently approved ADCs trastuzumab deruxtecan and sacituzumab govitecan release lower-potency topoisomerase inhibitors. This has prompted interest in ADCs that release lower-potency cytotoxic drugs to potentially enhance therapeutic index and reduce unwanted toxicity. Pyrrolobenzodiazepine (PBD) dimer ADCs have been widely investigated in human clinical trials, which have focused on high-potency PBDs. In this study, we evaluated five ADCs that release the low-potency PBD dimer SG3650. The relatively low clogD for this agent facilitated higher drug-to-antibody ratio (DAR) conjugation without the need for antibody engineering or functionalization of the drug. The rank order of potency for DAR 2 site-specific ADCs (conjugated at the C239i position) matched the order for the corresponding free drugs in vitro. Despite free drug SG3650 being inactive in vivo, the DAR 2 ADCs derived from the corresponding drug-linker SG3584 showed antitumor efficacy in solid (anti-HER2) and hematologic (anti-CD22) xenograft models. Antitumor activity could be enhanced by conjugating SG3584 to trastuzumab at higher DARs of 4 and 8 and by adjusting dosing and schedule. Higher-DAR conjugates were stable and displayed good rat pharmacokinetic profiles as measured by ELISA and LC/MS-MS. A single intravenous dose of isotype control SG3584 DAR 2 ADC resulted in no mortality in rats or monkeys at doses of up to 25 and 30 mg/kg, respectively. These findings suggest that further investigations of low-potency PBD dimers in ADCs that target hematologic and solid tumors are warranted.

TR1801-ADC: a highly potent cMet antibody-drug conjugate with high activity in patient-derived xenograft models of solid tumors.

cMet is a well-characterized oncogene that is the target of many drugs including small molecule and biologic pathway inhibitors, and, more recently, antibody-drug conjugates (ADCs). However, the clinical benefit from cMet-targeted therapy has been limited. We developed a novel cMet-targeted 'third-generation' ADC, TR1801-ADC, that was optimized at different levels including specificity, stability, toxin-linker, conjugation site, and in vivo efficacy. Our nonagonistic cMet antibody was site-specifically conjugated to the pyrrolobenzodiazepine (PBD) toxin-linker tesirine and has picomolar activity in cancer cell lines derived from different solid tumors including lung, colorectal, and gastric cancers. The potency of our cMet ADC is independent of MET gene copy number, and its antitumor activity was high not only in high cMet-expressing cell lines but also in medium-to-low cMet cell lines (40 000-90 000 cMet/cell) in which a cMet ADC with tubulin inhibitor payload was considerably less potent. In vivo xenografts with low-medium cMet expression were also very responsive to TR1801-ADC at a single dose, while a cMet ADC using a tubulin inhibitor showed a substantially reduced efficacy. Furthermore, TR1801-ADC had excellent efficacy with significant antitumor activity in 90% of tested patient-derived xenograft models of gastric, colorectal, and head and neck cancers: 7 of 10 gastric models, 4 of 10 colorectal cancer models, and 3 of 10 head and neck cancer models showed complete tumor regression after a single-dose administration. Altogether, TR1801-ADC is a new generation cMet ADC with best-in-class preclinical efficacy and good tolerability in rats.

A Reactive Antibody Platform for One-Step Production of Antibody-Drug Conjugates through a Diels-Alder Reaction with Maleimide.

The normal electron-demand Diels-Alder (DA) cycloaddition is a classic transformation routinely used in synthesis; however, applications in biological systems are limited. Here, we report a spiro[2.4]hepta-4,6-diene-containing noncanonical amino acid (SCpHK) capable of efficient incorporation into antibodies and subsequent coupling with maleimide via a DA reaction. SCpHK was stable throughout protein expression in mammalian cells and enabled covalent attachment of maleimide drug-linkers yielding DA antibody-drug conjugates (DA-ADCs) with nearly quantitative conversion in a one-step process. The uncatalyzed DA reaction between SCpHK and maleimide in aqueous buffer was rapid (1.8-5.4 M-1 s-1), and the antibody-drug adduct was stable in rat serum for at least 1 week at 37 °C. Anti-EphA2 DA-ADCs containing AZ1508 or SG3249 maleimide drug-linkers were potent inhibitors of tumor growth in PC3 tumor models in vivo. The DA bioconjugation strategy described here represents a simple method to produce site-specific and stable ADCs with maleimide drug-linkers.

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.

Fractionated Dosing Improves Preclinical Therapeutic Index of Pyrrolobenzodiazepine-Containing Antibody Drug Conjugates.

Purpose: To use preclinical models to identify a dosing schedule that improves tolerability of highly potent pyrrolobenzodiazepine dimers (PBDs) antibody drug conjugates (ADCs) without compromising antitumor activity.Experimental Design: A series of dose-fractionation studies were conducted to investigate the pharmacokinetic drivers of safety and efficacy of PBD ADCs in animal models. The exposure-activity relationship was investigated in mouse xenograft models of human prostate cancer, breast cancer, and gastric cancer by comparing antitumor activity after single and fractionated dosing with tumor-targeting ADCs conjugated to SG3249, a potent PBD dimer. The exposure-tolerability relationship was similarly investigated in rat and monkey toxicology studies by comparing tolerability, as assessed by survival, body weight, and organ-specific toxicities, after single and fractionated dosing with ADCs conjugated to SG3249 (rats) or SG3400, a structurally related PBD (monkeys).Results: Observations of similar antitumor activity in mice treated with single or fractionated dosing suggests that antitumor activity of PBD ADCs is more closely related to total exposure (AUC) than peak drug concentrations (Cmax). In contrast, improved survival and reduced toxicity in rats and monkeys treated with a fractionated dosing schedule suggests that tolerability of PBD ADCs is more closely associated with Cmax than AUC.Conclusions: We provide the first evidence that fractionated dosing can improve preclinical tolerability of at least some PBD ADCs without compromising efficacy. These findings suggest that preclinical exploration of dosing schedule could be an important clinical strategy to improve the therapeutic window of highly potent ADCs and should be investigated further. Clin Cancer Res; 23(19); 5858-68. ©2017 AACR.

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.