Homogeneous BTK Occupancy Assay for Pharmacodynamic Assessment of Tirabrutinib (GS-4059/ONO-4059) Target Engagement

SLAS Discovery
Helen YuNikos Pagratis

Abstract

Bruton's tyrosine kinase (BTK) is a clinically validated target for B-cell leukemias and lymphomas with FDA-approved small-molecule inhibitors ibrutinib and acalabrutinib. Tirabrutinib (GS-4059/ONO-4059, Gilead Sciences, Inc., Foster City, CA) is a second-generation, potent, selective, irreversible BTK inhibitor in clinical development for lymphoid malignancies, including chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL). An accurate pharmacodynamic assay to assess tirabrutinib target coverage in phase 1/2 clinical studies will inform dose and schedule selection for advanced clinical evaluation. We developed a novel duplex homogeneous BTK occupancy assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) to measure free and total BTK levels in a multiplexed format. The dual-wavelength emission property of terbium-conjugated anti-BTK antibody served as the energy donor for two fluorescent energy acceptors with distinct excitation and emission spectra. The assay was characterized and qualified using full-length purified recombinant human BTK protein and peripheral blood mononuclear cells derived from healthy volunteers and patients with CLL. We demonstrated assay utility using cells...Continue Reading

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Citations

Feb 6, 2020·Journal of Medicinal Chemistry·Chiara BorsariMaria P Costi
Apr 4, 2021·Cancers·Philipp von Hundelshausen, Wolfgang Siess

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Methods Mentioned

BETA
fluorescence resonance
dissection
Assay
FRET
biopsies
biopsy
xenograft
blood draw

Software Mentioned

GraphPad Prism

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