Abstract 4016: Homogeneous BTK occupancy assay for pharmacodynamic assessment of GS-4059 target engagement
Authors: Yu Helen , Truong Hoa , Mitchell Scott A. , Gosink John J. , Lin Julie , Feng Joy Y. , Jürgensmeier Juliane M. , Billin Andrew N. , Xu Ren , Mitra Siddhartha 
Published: July 2017

Abstract
Burton’s Tyrosine Kinase (BTK) plays an important role in B cell signaling, cell proliferation and survival. Hence, it is an attractive target for therapeutic intervention in B cell malignancies and autoimmune disorders. GS-4059 (ONO-4059) is a covalent, potent and selective inhibitor of BTK in clinical development for CLL, NHL, and rheumatoid arthritis. Accurate measurement of target coverage in early stage clinical studies is critical to informing dose selection for GS-4059 in more advanced clinical studies. We developed a novel duplex homogeneous BTK occupancy assay to enable quantitative measurement of GS-4059 binding to human BTK for assessing target coverage in the clinic. The assay is based on Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) and simultaneously measures the levels of free BTK (BTK that is not bound to GS-4059 in treated human PBMCs ex vivo or PBMCs from patients treated with GS-4059 in clinical studies) as well as the levels of total BTK protein. We took advantage of the dual wavelength emission property of Terbium (Tb) to serve as energy donor for 2 fluorescent energy acceptors with distinct excitation and emission spectra. We designed and synthesized a GS-4059 analog with biotin conjugate which showed equal potency to GS-4059, suggesting that it binds in the same BTK pocket. To detect free BTK, a Tb-coupled anti-BTK antibody was used as the FRET energy donor and G2-strepavidin-bound biotinylated GS-4059 as the energy acceptor. Total BTK was detected in the same well with a second D2-coupled anti-BTK antibody that binds to a different BTK epitope as the FRET energy acceptor. The use of a common detection reagent in this multiplexed format allows measurement of free and total BTK levels in the same well data. The assay was characterized and qualified using full length purified recombinant human BTK protein and PBMCs derived from healthy volunteers and CLL patients. Preliminary data indicate the assay is also suitable for assessing BTK occupancy in bone marrow derived cells. Using this assay, we evaluated the BTK synthesis rate following a 1 hour high dose (5 μM) treatment of GS-4059 in BTK inhibitor resistant ABC-DLBCL and GCB-DLBCL tumor cells. While negligible new BTK synthesis was observed in BTK inhibitor sensitive DLBCL cells, the free BTK level in the inhibitor resistant tumor cells recovered rapidly post-treatment. In summary, we have established a homogeneous TR-FRET based BTK occupancy assay that simultaneously measures free and total BTK levels in the same well, yielding quantitative BTK occupancy data to support GS-4059 clinical development.
Citation Format: Helen Yu, Hoa Truong, Scott A. Mitchell, John J. Gosink, Julie Lin, Joy Y. Feng, Juliane M. Jürgensmeier, Andrew N. Billin, Ren Xu, Siddhartha Mitra. Homogeneous BTK occupancy assay for pharmacodynamic assessment of GS-4059 target engagement [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4016. doi:10.1158/1538-7445.AM2017-4016