Homogeneous BTK Occupancy Assay for Pharmacodynamic Assessment of Tirabrutinib Target Engagement.

Research Highlights Life Science

A duplex assay for drug-bound and total BTK suitable for Phase I/II clinical studies

Expert opinion

The quest for potent new drugs is a very long process, with many different stages and milestones for the compound candidate. After years of thorough research during the drug discovery process, the entry of a potent compound in Phase I, then Phase II clinical trials is already a great achievement, but that’s really just the beginning. Will the compound offer real efficacy for patients without any major side effects in healthy volunteers? The answer to that question is critical – and mandatory – to obtain approval from health authorities and launch the compound on the market as a new drug.
Problems of drug selectivity, sourcing, and processing of clinical samples are among the major hurdles that researchers need to cope with during clinical stages. The demand for robust and reliable assays to overcome these issues is high. That is exactly what Helen Yu details in this article. She explains a strategy to easily assess the potency of a new-generation inhibitor, Tirabrutinib, on a well-known target in oncology, Bruton’s Tyrosine Kinase (BTK).
The compound is an irreversible ATP-competitive inhibitor that binds to BTK with high affinity but can also cross-react with other kinases belonging to the same family (ITK, BMX) or on EGFR. To fine-tune appropriate drug dosage for patient administration, namely the pharmacodynamic (PD) parameters, she designed an-vitro assay to fully characterize Tirabrutinib binding on BTK.
She developed a smart assay to quantify the fraction of BTK bound to the drug, as that induces the biological effect. The accuracy of target occupancy measurement, or target engagement, depends on quantification of both total and drug-bound targets. The duplex assay was developed in-vitro using recombinant BTK and checked on Ramos (BTK positive cells) and Jurkat (BTK negative cells). Once fully optimized, the assays were used on clinical samples: PBMC, whole-blood or bone marrow preparations from either healthy volunteers or patients suffering from chronic lymphocytic leukemia (CLL). This set-up provides her with a reliable assay, named PD biomarker assay, that will be used throughout Phase I and Phase II clinical studies for Tirabrutinib.
Technologies and assays used during early stage drug discovery programs can now be transposed to clinical study stages. A way to get trusted data over the entire drug discovery process!


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 derived from lymph node and bone marrow samples from patients with CLL and DLBCL. Our TR-FRET-based BTK occupancy assay provides accurate, quantitative assessment of BTK occupancy in the clinical trial program for tirabrutinib and is in use in ongoing clinical studies.


SLAS Discovery, 2018 Oct;23(9):919-929.

Read on PUBMED