Journal: Slas Discovery

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

doi: 10.1177/2472555218786165

Figure Lengend Snippet: ( A ) TR-FRET-based detection of free and total BTK. The distinct, nonoverlapping emission spectra of Tb allows it to serve as a common fluorescence donor for two spectrally distinct acceptors: G2-streptavidin (SA)-bound biotinylated tirabrutinib (to detect free BTK) and D2-coupled anti-BTK antibody that binds to a different BTK epitope (to detect total BTK). TR-FRET readout values are the ratio of acceptor to donor emissions for free BTK (520 nm/615 nm) and total BTK (665 nm/615 nm). This multiplexed assay allows detection of both free and total BTK simultaneously in the same sample. ( B ) Principle of the BTK occupancy assay. In the absence of tirabrutinib, 100% of BTK in the sample is unbound. The free BTK is captured by the biotinylated tirabrutinib probe and measured by the Tb-to-G2 TR-FRET readout. In the presence of a saturating dose of tirabrutinib, all BTK in the sample is drug bound. The lack of TR-FRET signal for free BTK indicates 100% BTK occupancy.

Article Snippet: Briefly, one 5 mm stainless steel bead (QIAGEN) and 600 µL of 0.34× lysis buffer (Invitrogen, cat. FNN0021) + 2× blocking reagent from the Total BTK kit (Cisbio) were added to each tube containing lymph node tissue.

Techniques: Fluorescence

Journal: Slas Discovery

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

doi: 10.1177/2472555218786165

Figure Lengend Snippet: ( A ) Assay range of quantitation. A set of six experiments (in quadruplicate) using full-length purified rBTK protein was used to generate independent standard curves for free and total BTK. The lower limit of quantitation of the assay was 12 ng/mL, and the upper limit was 166 ng/mL. The BTK concentration in cell samples in subsequent experiments was interpolated from these standard curves. ( B ) Assay accuracy and precision. Low percent CV (solid lines) in interassay performance demonstrated precision of the assay, and percent recovery (dotted lines) after spikes of 110, 8, and 0 nM tirabrutinib demonstrated accuracy of the assay. Data represent mean values from four independent experiments in quadruplicate. ( C ) Specificity of the assay for BTK inhibition was demonstrated by detection of free and total BTK in titrated lysates of Ramos B cells (light purple dots) but not Jurkat T cells (pink dots). BTK expression was lower in PBMCs (dark purple dots) than in Ramos cells. The decrease in TR-FRET at high Ramos cell concentration reflects analyte saturation. Representative data are shown from three or more experiments.

Article Snippet: Briefly, one 5 mm stainless steel bead (QIAGEN) and 600 µL of 0.34× lysis buffer (Invitrogen, cat. FNN0021) + 2× blocking reagent from the Total BTK kit (Cisbio) were added to each tube containing lymph node tissue.

Techniques: Quantitation Assay, Purification, Concentration Assay, Inhibition, Expressing

Journal: Slas Discovery

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

doi: 10.1177/2472555218786165

Figure Lengend Snippet: Inhibition of BTK by tirabrutinib. The TR-FRET assay was used to detect free and total BTK using rBTK, Ramos B cells, purified human PBMCs, and whole blood (WB) samples preincubated for 2 h with increasing concentrations of tirabrutinib. Values were normalized to vehicle-treated samples to obtain percent BTK occupancy. The top left panel shows the standard dose–response curve generated using recombinant BTK. In Ramos B cells, purified human PBMCs, and WB samples, BTK binding to the biotinylated tirabrutinib probe was competitively inhibited in a dose-dependent manner by tirabrutinib. The EC 50 of tirabrutinib as measured by the TR-FRET assay is shown in the table (mean ± SD of n = 3 experiments, each in quadruplicate).

Article Snippet: Briefly, one 5 mm stainless steel bead (QIAGEN) and 600 µL of 0.34× lysis buffer (Invitrogen, cat. FNN0021) + 2× blocking reagent from the Total BTK kit (Cisbio) were added to each tube containing lymph node tissue.

Techniques: Inhibition, Purification, Generated, Recombinant, Binding Assay

Journal: Slas Discovery

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

doi: 10.1177/2472555218786165

Figure Lengend Snippet: Assay utility in CLL and DLBCL patient samples: ( A ) PBMCs, ( B ) lymph nodes, and ( C ) bone marrow. ( A ) The concentration of total BTK in PBMCs from nine patients with CLL ranged from 33 to 174 ng/mL. The average total BTK expression in PBMCs from four healthy volunteers (HV, gray bar) was 30 ng/mL. Data represent the average and standard error of quadruplicate samples from each subject or from four healthy donors tested in quadruplicate. ( B ) The TR-FRET assay detected robust levels of free (light blue bars) and total (dark blue bars) BTK in lymph node (LN) tissue lysates from two patients with DLBCL. Data represent the average and standard error from samples tested in triplicate. ( C ) Matched BMMCs and PBMCs from two patients with CLL were treated by spiking 110, 8, and 0.1 nM tirabrutinib with a final DMSO concentration of 0.1%. The TR-FRET assay showed dose-dependent BTK binding after ex vivo treatment with tirabrutinib. BTK occupancy reached nearly 100% at a dose of 110 nM tirabrutinib. Data represent the average and standard error from triplicate samples.

Article Snippet: Briefly, one 5 mm stainless steel bead (QIAGEN) and 600 µL of 0.34× lysis buffer (Invitrogen, cat. FNN0021) + 2× blocking reagent from the Total BTK kit (Cisbio) were added to each tube containing lymph node tissue.

Techniques: Concentration Assay, Expressing, Binding Assay, Ex Vivo

Journal: Slas Discovery

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

doi: 10.1177/2472555218786165

Figure Lengend Snippet: ( A ) TR-FRET-based detection of free and total BTK. The distinct, nonoverlapping emission spectra of Tb allows it to serve as a common fluorescence donor for two spectrally distinct acceptors: G2-streptavidin (SA)-bound biotinylated tirabrutinib (to detect free BTK) and D2-coupled anti-BTK antibody that binds to a different BTK epitope (to detect total BTK). TR-FRET readout values are the ratio of acceptor to donor emissions for free BTK (520 nm/615 nm) and total BTK (665 nm/615 nm). This multiplexed assay allows detection of both free and total BTK simultaneously in the same sample. ( B ) Principle of the BTK occupancy assay. In the absence of tirabrutinib, 100% of BTK in the sample is unbound. The free BTK is captured by the biotinylated tirabrutinib probe and measured by the Tb-to-G2 TR-FRET readout. In the presence of a saturating dose of tirabrutinib, all BTK in the sample is drug bound. The lack of TR-FRET signal for free BTK indicates 100% BTK occupancy.

Article Snippet: A half volume of DPBS and 2× lysis mixture (0.68× lysis buffer [Invitrogen, Carlsbad, CA] + 4× blocking reagent from Total BTK kit [Cisbio]) were added to the cells at a concentration of 1.25 × 10 7 cells/mL.

Techniques: Fluorescence

Journal: Slas Discovery

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

doi: 10.1177/2472555218786165

Figure Lengend Snippet: ( A ) Assay range of quantitation. A set of six experiments (in quadruplicate) using full-length purified rBTK protein was used to generate independent standard curves for free and total BTK. The lower limit of quantitation of the assay was 12 ng/mL, and the upper limit was 166 ng/mL. The BTK concentration in cell samples in subsequent experiments was interpolated from these standard curves. ( B ) Assay accuracy and precision. Low percent CV (solid lines) in interassay performance demonstrated precision of the assay, and percent recovery (dotted lines) after spikes of 110, 8, and 0 nM tirabrutinib demonstrated accuracy of the assay. Data represent mean values from four independent experiments in quadruplicate. ( C ) Specificity of the assay for BTK inhibition was demonstrated by detection of free and total BTK in titrated lysates of Ramos B cells (light purple dots) but not Jurkat T cells (pink dots). BTK expression was lower in PBMCs (dark purple dots) than in Ramos cells. The decrease in TR-FRET at high Ramos cell concentration reflects analyte saturation. Representative data are shown from three or more experiments.

Article Snippet: A half volume of DPBS and 2× lysis mixture (0.68× lysis buffer [Invitrogen, Carlsbad, CA] + 4× blocking reagent from Total BTK kit [Cisbio]) were added to the cells at a concentration of 1.25 × 10 7 cells/mL.

Techniques: Quantitation Assay, Purification, Concentration Assay, Inhibition, Expressing

Journal: Slas Discovery

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

doi: 10.1177/2472555218786165

Figure Lengend Snippet: Inhibition of BTK by tirabrutinib. The TR-FRET assay was used to detect free and total BTK using rBTK, Ramos B cells, purified human PBMCs, and whole blood (WB) samples preincubated for 2 h with increasing concentrations of tirabrutinib. Values were normalized to vehicle-treated samples to obtain percent BTK occupancy. The top left panel shows the standard dose–response curve generated using recombinant BTK. In Ramos B cells, purified human PBMCs, and WB samples, BTK binding to the biotinylated tirabrutinib probe was competitively inhibited in a dose-dependent manner by tirabrutinib. The EC 50 of tirabrutinib as measured by the TR-FRET assay is shown in the table (mean ± SD of n = 3 experiments, each in quadruplicate).

Article Snippet: A half volume of DPBS and 2× lysis mixture (0.68× lysis buffer [Invitrogen, Carlsbad, CA] + 4× blocking reagent from Total BTK kit [Cisbio]) were added to the cells at a concentration of 1.25 × 10 7 cells/mL.

Techniques: Inhibition, Purification, Generated, Recombinant, Binding Assay

Journal: Slas Discovery

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

doi: 10.1177/2472555218786165

Figure Lengend Snippet: Assay utility in CLL and DLBCL patient samples: ( A ) PBMCs, ( B ) lymph nodes, and ( C ) bone marrow. ( A ) The concentration of total BTK in PBMCs from nine patients with CLL ranged from 33 to 174 ng/mL. The average total BTK expression in PBMCs from four healthy volunteers (HV, gray bar) was 30 ng/mL. Data represent the average and standard error of quadruplicate samples from each subject or from four healthy donors tested in quadruplicate. ( B ) The TR-FRET assay detected robust levels of free (light blue bars) and total (dark blue bars) BTK in lymph node (LN) tissue lysates from two patients with DLBCL. Data represent the average and standard error from samples tested in triplicate. ( C ) Matched BMMCs and PBMCs from two patients with CLL were treated by spiking 110, 8, and 0.1 nM tirabrutinib with a final DMSO concentration of 0.1%. The TR-FRET assay showed dose-dependent BTK binding after ex vivo treatment with tirabrutinib. BTK occupancy reached nearly 100% at a dose of 110 nM tirabrutinib. Data represent the average and standard error from triplicate samples.

Article Snippet: A half volume of DPBS and 2× lysis mixture (0.68× lysis buffer [Invitrogen, Carlsbad, CA] + 4× blocking reagent from Total BTK kit [Cisbio]) were added to the cells at a concentration of 1.25 × 10 7 cells/mL.

Techniques: Concentration Assay, Expressing, Binding Assay, Ex Vivo