A Generic Approach for Miniaturized Unbiased High-Throughput Screens of Bispecific Antibodies and Biparatopic Antibody-Drug Conjugates.

The toolbox of modern antibody engineering allows the design of versatile novel functionalities exceeding nature's repertoire. Many bispecific antibodies comprise heterodimeric Fc portions recently validated through the approval of several bispecific biotherapeutics. While heterodimerization methodologies have been established for low-throughput large-scale production, few approaches exist to overcome the bottleneck of large combinatorial screening efforts that are essential for the identification of the best possible bispecific antibody. This report presents a novel, robust and miniaturized heterodimerization process based on controlled Fab-arm exchange (cFAE), which is applicable to a variety of heterodimeric formats and compatible with automated high-throughput screens. Proof of applicability was shown for two therapeutic molecule classes and two relevant functional screening read-outs. First, the miniaturized production of biparatopic anti-c-MET antibody-drug conjugates served as a proof of concept for their applicability in cytotoxic screenings on tumor cells with different target expression levels. Second, the automated workflow enabled a large unbiased combinatorial screening of biparatopic antibodies and the identification of hits mediating potent c-MET degradation. The presented workflow utilizes standard equipment and may serve as a facile, efficient and robust method for the discovery of innovative therapeutic agents in many laboratories worldwide.

Assessment of the potential of the MET inhibitor tepotinib to affect the pharmacokinetics of CYP3A4 and P-gp substrates.

Tepotinib is a highly selective, potent, mesenchymal-epithelial transition factor (MET) inhibitor, approved for the treatment of non-small cell lung cancer harboring MET exon 14 skipping alterations. The aims of this work were to investigate the potential for drug-drug interactions via cytochrome P450 (CYP) 3A4/5 or P-glycoprotein (P-gp) inhibition. In vitro studies were conducted in human liver microsomes, human hepatocyte cultures and Caco-2 cell monolayers to investigate whether tepotinib or its major metabolite (MSC2571109A) inhibited or induced CYP3A4/5 or inhibited P-gp. Two clinical studies were conducted to investigate the effect of multiple dose tepotinib (500 mg once daily orally) on the single dose pharmacokinetics of a sensitive CYP3A4 substrate (midazolam 7.5 mg orally) and a P-gp substrate (dabigatran etexilate 75 mg orally) in healthy participants. Tepotinib and MSC2571109A showed little evidence of direct or time-dependent CYP3A4/5 inhibition (IC50 > 15 µM) in vitro, although MSC2571109A did show mechanism-based CYP3A4/5 inhibition. Tepotinib did not induce CYP3A4/5 activity in vitro, although both tepotinib and MSC2571109A increased CYP3A4 mRNA. In clinical studies, tepotinib had no effect on the pharmacokinetics of midazolam or its metabolite 1'-hydroxymidazolam. Tepotinib increased dabigatran maximum concentration and area under the curve extrapolated to infinity by 38% and 51%, respectively. These changes were not considered to be clinically relevant. Tepotinib was considered safe and well tolerated in both studies. The potential of tepotinib to cause clinically relevant DDI with CYP3A4- or P-gp-dependent drugs at the clinical dose is considered low. Study 1 (midazolam): NCT03628339 (registered 14 August 2018). Study 2 (dabigatran): NCT03492437 (registered 10 April 2018).

Phase 1 study in healthy participants of the safety, pharmacokinetics, and pharmacodynamics of enpatoran (M5049), a dual antagonist of toll-like receptors 7 and 8.

This study evaluated the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of single and multiple oral doses of enpatoran (formerly named M5049), a new toll-like receptor (TLR) 7 and 8 dual antagonist, and the effect of food on a single dose in healthy participants. In this single phase 1, randomized (3:1), double-blind, placebo-controlled study, 96 participants received single and multiple ascending oral doses of enpatoran. Participants in single-dose cohorts received one dose of enpatoran (1, 3, 9, 25, 50, 100, or 200 mg) or placebo using a sentinel dosing strategy. Multiple-dose cohorts received enpatoran (9, 25, or 200 mg once daily, or 25 or 50 mg twice daily) or placebo for 14 days. Safety, tolerability, PK, and PD (ex vivo-stimulated cytokine secretion) were assessed in both parts. The effect of food was assessed in an open-label, one-way crossover study in the 25 mg single-dose cohort. Single- and multiple-oral doses of enpatoran up to 200 mg were well tolerated and no significant dose-limiting adverse events or safety signals were observed under fasting or fed conditions. PK parameters were linear and dose-proportional across the dose range evaluated, with a slightly delayed absorption and lower peak concentration observed at 25 mg with food. Exposure-dependent inhibition of ex vivo-stimulated interleukin-6 secretion was observed, with maximum inhibition at 200 mg. Enpatoran was well tolerated at doses up to 200 mg. Further investigation of enpatoran is warranted as a potential treatment for diseases driven by TLR7/8 overactivation, such as systemic lupus erythematosus and COVID-19 pneumonia.

Evobrutinib, a covalent Bruton's tyrosine kinase inhibitor: Mass balance, elimination route, and metabolism in healthy participants.

The highly selective, covalent Bruton's tyrosine kinase inhibitor evobrutinib is under investigation for treatment of patients with multiple sclerosis (MS). Early clinical studies in healthy participants and patients with relapsing MS indicated that evobrutinib is well-tolerated and effective. We undertook a mass balance study in six men who received a single 75-mg oral dose of evobrutinib containing ~ 3.6 MBq (100 µCi) 14 C-evobrutinib, to determine the absorption, metabolic pathways, and routes of excretion of evobrutinib. The primary objectives of this phase I study (NCT03725072) were to (1) determine the rates and routes of total radioactivity excretion, including the mass balance of total drug-related radioactivity in urine and feces, (2) assess the pharmacokinetics (PKs) of total radioactivity in blood and plasma, and (3) characterize the plasma PKs of evobrutinib. Exploratory end points included identifying and quantifying evobrutinib and its metabolites in plasma and excreta (urine and feces) and exploring key biotransformation pathways and clearance mechanisms. Evobrutinib was primarily eliminated in feces (arithmetic mean percentage, SD, 71.0, 2.1) and, to a lesser extent, in urine (20.6, 2.0), with most of the total radioactivity (85.3%) excreted in the first 72 h after administration. No unchanged evobrutinib was detected in excreta. Evobrutinib was rapidly absorbed and substantially metabolized upon absorption. Only one major metabolite M463-2 (MSC2430422) was identified in plasma above the 10% of total drug exposure threshold, which classifies M463-2 (MSC2430422) as a major metabolite according to the US Food and Drug Administration (FDA; metabolites in safety testing [MIST]) and the European Medicines Agency (EMA; International Conference on Harmonization [ICH] M3). These results support further development of evobrutinib and may help inform subsequent investigations.