Assays to determine receptor occupancy in vivo

Abstract

The present invention relates to an assay for monitoring and measuring the binding of a drug of interest to a receptor. In this assay, a blood sample collected from a subject to whom a compound of interest is administered is incubated with a lysing solution containing a quencher. The drug-bound and quencher-bound receptors are then separated from the lysed blood sample. The separated drug-bound and quencher-bound receptors are digested to produce surrogate drug-bound and quencher-bound peptides. The amount of the surrogate peptide is determined. Receptor occupancy can be determined by comparing the amount of drug-bound surrogate peptide to the total amount of drug-bound and quencher-bound surrogate peptide.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Serial No. 62 / 669,442, filed May 10, 2018, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to methods for directly measuring real-time in vivo receptor occupancy of covalently bound compounds in blood lysates. More specifically, the present invention relates to real-time in vivo BTK receptor occupancy measurement as a pharmacodynamic biomarker for clinical drug development. Background Art

[0004] Drug discovery and development is a long and high-risk process. Due to the failure of countless candidate drugs in the early discovery and development process, the average cost of developing a successful new drug is estimated to be as high as $2.6 billion, and the overall survival rate of candidate drugs entering clinical research is estimated to be less than 12% (PhRMA report, "Biopharmaceutical Research & Development: The Process Behind New Medicines", 2015). The main problem leading to the failure of these candidate drugs is related to the lack of clinical efficacy or lack thereof during the late development period, especially during the proof-of-concept (Phase II) clinical trial (Kola, I., et al., J. Nature Reviews. Drug Discovery 2004, 3, 711-715; Arrowsmith, J., Nature Reviews. Drug Discovery 2011, 10, 87; Morgan, P., et al., Drug Discovery Today 2012, 17, 419-424).

[0005] Many small molecule drugs and biologics in development have been designed to mediate the immune system. As such, these drug candidates often require very low dose levels to trigger the desired response. Therefore, early understanding of the pharmacokinetic (PK) and pharmacodynamic (PD) properties of these drug candidates is crucial for determining the appropriate dose range for clinical trials (Topalian, SL, et al., The New England Journal of Medicine 2012, 366, 2443-2454; Brahmer, JR, et al., The New England Journal of Medicine 2012, 366, 2455-2465; Tolcher, AW, et al., Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology 2009, 27, 5800-5807; Hua, F., et al., Journal of Clinical Pharmacology 2014, 54, 14-22; Rutgeerts, PJ, et al., Gut 2013, 62, 1122-1130). In order to improve clinical efficiency and reduce costs during the drug development stage, quantitative measurement of PD curves is as important as PK curves for the rational design of clinical trials. This is especially true when the PD curve varies with multiple doses.

[0006] Receptor occupancy (RO) assay measures the binding of a molecule to its receptor protein (or target) and provides quantitative data that can be used to generate a PD curve (Liang, M., et al., Cytometry B Clin Cytom 2016, 90, 117-127). In preclinical animal models and clinical studies, the measurement of RO is a key assay to associate efficacy with mechanism. In practice, RO is particularly useful in making dose escalation decisions in first-in-human (FIH) studies.

[0007] Bruton's tyrosine kinase (BTK) plays a key role in signal transduction and activation via B cell receptor, Fc receptor and RANKL pathways (Seiler, T., et al., Expert Opin Investig Drugs 2017, 26, 909-915; Whang, JA, et al., Drug Discovery Today 2014, 19, 1200-1204). For antibody-based drugs, RO is usually monitored by flow cytometry using antibodies that compete with target molecules to measure free receptors and using non-competing antibodies to measure total receptors (Liang, M, et al., Cytometry B Clin Cytom 2016, 90, 117-127; Woska, JR, Jr., et al., Journal of immunological methods 2003, 277, 101-115). However, for small molecule antagonists, there is no antibody reagent for directly detecting occupied receptors by flow cytometry. Previously, the BTK RO of ibrutinib in peripheral blood mononuclear cells (PBMC) was determined by using a fluorescent affinity probe that binds to the active occupancy site of free (unoccupied) BTK, followed by detection using SDS / PAGE and fluorescent gel scanning (Honigberg, LA; J. Proceedings of the National Academy of Sciences of the United States of America 2010, 107, 13075-13080). The determination based on the fluorescent affinity probe can only determine the amount of free BTK, so a different determination using protein blot is needed to measure total BTK. Since a large number of samples are generated in clinical studies, it is impractical to use two determination platforms. In addition, the method lacks direct comparability between unbound measurements and total measurements, resulting in higher determination variability.

[0008] Recently, ELISA-based BTK RO assays using biotinylated covalent probes have been reported and have been used in acalabrutinib and CC-292 clinical studies (Barf, T., et al., The Journal of pharmacology and experimental therapeutics 2017; Evans, EK, et al., The Journal of pharmacology and experimental therapeutics 2013, 346, 219-22). In this method, when drug-bound BTK (DB-BTK) is washed away, only free BTK is detected. The total BTK concentration is determined using the sample before administration, where it is assumed that the total BTK level between samples remains consistent.

[0009] Alternatively, the BTK occupancy in peripheral blood mononuclear cells (PBMCs) can be estimated using separate time-resolved fluorescence resonance energy transfer (TR-FRET)-based assays for free and total BTK (Lutz, JDN, et al., Poster presentation at the 7th American Conference on Pharmacometrics (ACoP7) in Bellevue, Washington. October 23-26, 2016.2016). However, free and total BTK levels are measured independently rather than simultaneously in the same sample, and it is found that total BTK levels vary significantly between samples (Honigberg, LA, supra). Therefore, methods based on ELISA or TR-FRET are limited by inherent variability caused by method selection.

[0010] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) assays show potential in protein quantification due to their excellent assay selectivity and multiplexing capabilities (Neubert, H., et al., Bioanalysis 2014, 6, 1731-1733). In particular, the "hybrid" LBA-LC-MS assay, which combines immunocapture enrichment and then LC-MS / MS detection, has become a powerful technology platform that measures protein biomarkers or therapeutic agents with extremely high detection selectivity (Stevenson, L., et al., Bioanalysis 2013, 5, 2903-2918). The main benefit of LC-MS assays is that they can simultaneously quantify both DB-BTK and free BTK in the same sample. Therefore, RO assays are much less sensitive to sample or run changes.

[0011] However, the development of LC-MS assays presents a number of challenges. First, blood samples from animals or humans that have been given a drug can contain excess drug, which can react with free receptors ex vivo to form drug-bound receptors, leading to an overestimation of RO. Second, LC-MS / MS is generally not as sensitive as ELISA assays. Summary of the invention

[0012] The present invention relates to an assay for monitoring and measuring the binding of a drug of interest to a receptor. In this assay, a blood sample collected from a subject to whom a compound of interest is administered is incubated with a lysing solution containing a quencher. The drug-bound and quencher-bound receptors are then separated from the lysed blood sample. The separated drug-bound and quencher-bound receptors are digested to produce surrogate drug-bound and quencher-bound peptides. The amount of the surrogate peptide is determined. Receptor occupancy can be determined by comparing the amount of drug-bound surrogate peptide to the total amount of drug-bound and quencher-bound surrogate peptide.

[0013] In one embodiment of the invention, the compound of interest is covalently bound to the receptor.

[0014] In another embodiment of the invention, the quencher binds irreversibly to the receptor in competition with the compound of interest at the same site as the compound of interest binds.

[0015] In another embodiment of the invention, the quencher is added to the lysis buffer at a molar excess level to the compound of interest. In another embodiment of the invention, the quencher is added to the lysis buffer at an excess of 105 to 1000 times based on the endogenous receptor concentration.

[0016] In another embodiment of the present invention, the lysing solution with the quencher is added immediately after the blood sample is collected. In another embodiment of the present invention, the lysing solution with the quencher is added within 5 minutes of the blood sample collection.

[0017] In another embodiment of the invention, an immunocapture step is used to separate drug-bound and quencher-bound receptors. In another embodiment of the invention, the receptor is specifically bound to a capture agent immobilized on a solid support (e.g., magnetic beads, agarose beads, or column packing material) and is thus separated from other endogenous proteins and peptides that are not very tightly bound to the capture antibody [Fung, EN et al., Bioanalysis 2016, 8, 847-856].

[0018] In another embodiment of the invention, the drug-bound and quencher-bound receptors are removed from the immunocapture substrate prior to the digestion step.

[0019] In another embodiment of the invention, the drug-bound and quencher-bound receptors are digested while still associated with the immunocapture substrate.

[0020] In another embodiment of the present invention, the protease used during the digestion step is selected from serine proteases, cysteine ​​proteases, threonine proteases, aspartic proteases, glutamic proteases and metalloproteases. Examples of proteases include trypsin, chymotrypsin, Glu-C protease, Lys-C protease, Lys-N protease, Asp-N protease, Arg-C protease.

[0021] In another embodiment of the invention, drug binding and quencher binding of surrogate peptides are measured simultaneously by a single LC-MS / MS run.

[0022] In another embodiment of the invention, receptor occupancy is calculated by comparing the amount of drug-bound surrogate peptide to the total amount of drug-bound and quencher-bound surrogate peptide.

[0023] In one embodiment of the invention, receptor occupancy is measured by: 1) collecting blood from a subject who is administered a compound of interest that irreversibly binds to the receptor, 2) immediately adding a lysis solution to the collected blood, wherein the lysis solution contains a quencher compound that irreversibly binds to the same site as the compound of interest, 3) separating the drug-bound and quencher-bound receptors from the lysed and quenched blood sample using an immunocapture step, 4) digesting the separated drug-bound and quencher-bound receptors while still bound to the immunocapture substrate, 5) simultaneously measuring the amount of drug-bound and quencher-bound surrogate peptides in a single LC-MS / MS run, and 6) determining receptor occupancy by comparing the amount of drug-bound surrogate peptide to the total amount of drug-bound and quencher-bound surrogate peptides.

[0024] In a preferred aspect of the above embodiment, the receptor is BTK, and the compound of interest is shown in Figure 1A and the corresponding quenchers are shown in Figure 1B middle.

[0025] In another preferred aspect of the above embodiment, the immunocapture substrate is streptavidin T1 capture beads.

[0026] In another preferred aspect of the above embodiment, mAb clone #MAB 5807 or mAb clone #MAB D3H5 is the anti-BTK antibody attached to the immunocapture substrate.

[0027] In another preferred aspect of the above embodiment, the separated quencher-bound and drug-bound BTK is trypsin-digested while still associated with the immunocapture beads.

[0028] In another preferred aspect of the above embodiment, BTK receptor occupancy is calculated by comparing the amount of drug-bound surrogate peptide to the total amount of drug-bound and quencher-bound surrogate peptide.

[0029] In one embodiment of the present invention, BTK receptor occupancy is measured by: 1) from a subject given Figure 1A 1) Collect blood from subjects containing the compound in the Figure 1B The invention relates to a method for preparing a lysate of drug-bound and quencher-bound BTK by removing a lysate solution of drug-bound and quencher compound from the lysate, 3) separating drug-bound and quencher-bound BTK using an immunocapture step, 4) digesting the separated drug-bound and quencher-bound BTK with trypsin while still associated with the immunocapture beads, 5) simultaneously measuring the amount of drug-bound and quencher-bound surrogate peptides in a single LC-MS / MS run, and 6) determining BTK receptor occupancy by comparing the amount of drug-bound surrogate peptide to the total amount of drug-bound and quencher-bound surrogate peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1A and 1B Shown are the structures of A) Compound A; B) the quencher compound.

[0031] Figure 2 Sequence alignments of BTK proteins from humans and monkeys (Rhesus / Cyno) are shown, respectively. Boxes indicate tryptic peptides used for analysis. Within the amino acid sequence, Cys 481 (marked by an arrow) is bound to Compound A (DB-BTK) or a quencher compound (QB-BTK). Figure 1A or 1B compound bound to tryptic peptide QRPIFIITEYMANG C LLNYLR (SEQ ID NO: 1, as shown in Table 1) was used for quantification.

[0032] Figure 3 Schematic diagram of BTK RO assay by immunocapture-LC-MS / MS is shown. The surrogate peptides used were drug-bound peptide (DB-QRP) and quencher-bound peptide (QB-QRP).

[0033] Figure 4 It was shown that capture antibodies from different suppliers (■mAb-D3H5, □mAb-MAB5807) had different immunocapture properties between endogenous BTK and recombinant BTK (used as calibrator) in human blood.

[0034] Figures 5A-5DThe effect of trypsin concentration and digestion time on the LC-MS / MS response of surrogate peptides generated from DB-BTK (5A and 5B) and QB-BTK (5C and 5D) at 50°C (5A and 5C) or 60°C (5B and 5D) is shown. TM Hybrid Quadrupole-Orbitrap TM LC-HRMS on a mass spectrometer measures the surrogate peptides DB-QRP or QB-QRP. For each condition, the experiment was performed in triplicate.

[0035] Figure 6 Comparison of the signal response at each charge stage of DB-BTK (■) and QB-BTK (□) is shown.

[0036] Figures 7A-7C The (7A) DB-QRP peptide obtained from a Sciex Triple Quad 5500 mass spectrometer shows the [M+3H] 3+ Electrospray positive ionization MS / MS product ion spectrum of (7B) QB-QRP peptide at m / z 962.5 [M+3H] 3+ Electrospray positive ion MS / MS product ion spectrum of (7C) SIL-QB-QRP peptide (internal standard) at m / z 965.9 [M+3H] 3+ Electrospray positive ion MS / MS product ion spectrum.

[0037] Figures 8A-8F Shown are multiple reaction monitoring (MRM) chromatograms of DB-QRP (surrogate peptide of DB-BTK) (8A-8C) and QB-QRP (surrogate peptide of QB-BTK) (8D-8F) and their internal standards (IS); (8A and 8D) chromatograms of analytes obtained from blank monkey blood lysate, where the influence of endogenous BTK was eliminated by pretreatment with quencher or drug alone; (8B and 8E) chromatograms of analytes obtained from monkey blood lysate containing analytes at LLOQ and their IS; (8C and 8F) chromatograms of IS obtained from monkey blood lysate containing only IS. (c and f) [ 13 C9, 15 N]-QB-QRP was used as the IS for DB-QRP and QB-QRP.

[0038] Figures 9A-9D Shown are the PK and PD results obtained from monkeys #5149, #5151 and #5169, which were given two different doses of 0.10 mg / kg or 0.5 mg / kg as shown in Figure 1ACompound (BMS-986195) in the 1st day. Blood samples were collected at 1, 3, 5, 7 and 24 hours for PK evaluation (data shown in 9A and 9B), and blood samples were collected before administration, 1, 3, 5, 7, 24, 48, 72, 144 and 168 hours for PD evaluation (data shown in 9C and 9D) (see Example 2). Citation of sequence listing submitted electronically via EFS-WEB

[0039] The contents of the sequence listing submitted electronically in the ASCII text file 12728WOPCT_ST25.txt (size: 16kb, created on April 8, 2019) submitted with this application are incorporated herein by reference in their entirety. DETAILED DESCRIPTION

[0040] The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention and the Examples included therein.

[0041] As used herein, the term "irreversibly binds" refers to a class of compounds that permanently bind to the receptor by forming a covalent bond with the active site or, alternatively, by simply binding to the active site so tightly that the dissociation rate is effectively zero on a relevant time scale.

[0042] As used herein, the terms "compound of interest" and "drug" are used interchangeably and refer to a molecule that binds to a target of interest.

[0043] As used herein, the terms "target" and "receptor" are used interchangeably when referring to the focus of a RO assay.

[0044] As used herein, when referring to the moment when the lysis solution containing the quencher molecule is added to the blood sample, the term "immediately" means as soon as possible after the blood sample is collected. Alternatively, the lysis solution containing the quencher molecule is added within the first 5 minutes after the blood sample is collected.

[0045] The present invention describes the development of an in vivo receptor occupancy assay, and more specifically, describes the development of an in vivo Bruton's tyrosine kinase (BTK) receptor occupancy (RO) assay. Assay sensitivity is key to determining RO at very high or very low occupancy levels (bound or free target may be less than 5%).

[0046] The present invention is a method comprising the steps of: a) collecting a blood sample from a subject treated with a compound of interest, b) adding a lysis solution containing a receptor-specific quencher to the blood sample, c) isolating the receptor of interest, d) digesting the isolated receptor to produce a surrogate peptide, and e) measuring the amount of the surrogate peptide and determining receptor occupancy by comparing the amount of drug-bound surrogate peptide to the total amount of drug-bound and quencher-bound surrogate peptide.

[0047] Blood samples are collected from subjects to whom the compound of interest is administered.

[0048] In one embodiment of the present invention, the compound of interest binds irreversibly to at least one amino acid of the receptor. An example of a compound of interest is shown in Figure 1A A compound in which the compound irreversibly binds to BTK.

[0049] Add lysis solution containing receptor-specific quencher to blood sample

[0050] There are many challenges that affect the selectivity and sensitivity of receptor occupancy (RO) assay development. In particular, the presence of free drug and free receptor in blood samples can generate drug-bound receptors ex vivo during the lysis step, which will lead to an overestimation of RO. It was found that the addition of a receptor-specific quencher during the lysis step can rapidly convert free receptors to quencher-bound (QB) receptors, thereby blocking the ex vivo formation of drug-bound receptors.

[0051] In one embodiment, the RO assay comprises lysing a blood sample in the presence of a quencher to convert endogenous receptors to quencher-bound receptors, thereby preventing the formation of endogenous drug-bound receptors.

[0052] In one embodiment of the invention, the quencher irreversibly binds to the same amino acid as the target compound. Preferably, the quencher competes with the target compound for binding to the receptor. In another embodiment of the invention, the quencher is added to the lysis solution in a molar excess over the target compound. In another embodiment of the invention, the lysis solution containing the quencher is added immediately after the blood sample is collected.

[0053] Those skilled in the art are aware of cell lysis solutions. Commercially available cell lysis buffers include NP-40 lysis buffer (TheromFisher Scientific), RIPA lysis buffer (Abbexa), ACK lysis buffer (TheromFisher Scientific), cell lysis buffer (10x) (Catalog No.: 9803, Cell Signaling Technology). Lysis buffer is used to lyse cells under non-denaturing conditions.

[0054] An example of a quencher compound is shown in Figure 1B A compound in which the compound irreversibly binds to BTK.

[0055] Separation of drug-bound and quencher-bound receptors from cell lysate

[0056] Immunocapture is a highly selective sample cleanup method that exploits the unique immunoaffinity of the target protein and the capture agent (Stevenson, L., et al., Bioanalysis 2013, 5, 2903-2918). Selecting a suitable capture antibody with high immunocapture efficiency for the endogenous target is key to a successful assay. If the specific antibody is not commercially available, those skilled in the art will know how to generate an antibody specific for the target protein.

[0057] To evaluate the immunocapture efficiency, antibodies were screened using drug-bound and quencher-bound targets. Antibodies were selected based on high affinity for both drug-bound and quencher-bound targets. For example, to select antibodies specific for BTK, the immunocapture capabilities of commercially available mAb clone #MAB5807 and mAb clone #D3H5 were evaluated. Unexpectedly, although mAb clone #MAB5807 showed high affinity for recombinant BTK, DB-BTK or QB-BTK, the clone was unable to detect endogenous BTK, DB-BTK or QB-BTK with the desired sensitivity. Figure 4 As shown, the LC-MS response from endogenous BTK was too low when mAb clone #5807 was used, while another clone mAb clone #D3H5 gave a moderate response to drug-bound and quencher-bound recombinant BTK and a greater response to drug-bound and quencher-bound endogenous BTK. Since the ultimate goal was to determine drug-bound and quencher-bound endogenous BTK in human blood, clone #D3H5 was selected for the BTKRO assay.

[0058] Generation of drug- and quencher-bound receptor surrogate peptides

[0059] The receptor surrogate peptides generated for RO assays must contain the binding site for the compound of interest. In addition, the surrogate peptides must be sensitive to LC-MS detection.

[0060] In one embodiment of the invention, the separated drug-bound and quencher-bound receptors are digested with one or more proteases selected from serine proteases, cysteine ​​proteases, threonine proteases, aspartic proteases, glutamic proteases and metalloproteases. Examples of acceptable proteases include trypsin, chymotrypsin, Glu-C protease, Lys-C protease, Lys-N protease, Asp-N protease, Arg-C protease.

[0061] Table 1 describes the alternative peptides generated when BTK is digested with various proteases.

[0062] Table 1. BTK peptides generated when treated with various proteases

[0063]

[0064] Since quantification of drug-bound and quencher-bound targets is based on the MS responses of representative surrogate peptides, the digestion yields of these surrogate peptides can have a significant impact on detection sensitivity.

[0065] In one embodiment of the invention, the digestion step is performed before or after eluting the drug-bound and quencher-bound receptors from the immunocapture substrate. Preferably, the digestion step is performed while the target is bound to the immunocapture substrate. For example, the drug-bound and quencher-bound BTK is treated by trypsin digestion while still bound by the immunocapture beads.

[0066] Measuring the amount of surrogate peptide

[0067] In one embodiment of the invention, receptor occupancy can be determined by simultaneously measuring quencher-bound and drug-bound surrogate peptides in a single LC-MS / MS run.

[0068] During initial method development, DB-BTK and QB-BTK produced by the reaction were analyzed using Q Exactive HF high resolution mass spectrometry (HRMS). All surrogate peptides were identified using the accurate masses expected from the surrogate peptides. The signal abundance for each charge phase is shown in Figure 6 The data show that DB-BTK and QB-BTK give the highest response in the three charge stages, so [M+3H] 3+ The ion was used for both DB-QRP (surrogate peptide of DB-BTK) and QB-QRP (surrogate peptide of QB-BTK) as the precursor ion for MRM detection. Figures 7A-7C As shown, several product ions are shown in the MS / MS spectrum. Product ions corresponding to the y2 ion (m / z about 288) and the b6 ion (m / z about 755 for DB-QRP and QB-QRP, and m / z about 765 for IS) show better intensity for MRM detection, wherein the y2 ion is better than the b6 ion. Since the y2 ion with an m / z of about 288 has an interfering peak shown in the MRM channel of DB-QRP and QB-QRP, the b6 ion is used as the product ion for MRM detection.

[0069] Typical MRM chromatograms of DB-QRP, QB-QRP, and IS from blank monkey blood lysate (blank monkey blood lysate spiked with DB-BTK and QB-BTK) are shown in Fig. 8A , 8B , 8D and 8E, where DB-BTK was at the LLOQ concentration of 0.250 nM and QB-BTK was 0.125 nM. 13 C9, 15 A typical MRM mass spectrum of [N]-QB-QRP is shown in Figure 8C and 8F In the analysis of blank control monkey blood lysate, no significant interfering peaks from blank monkey lysate were found in the retention time or ion channel of DB-QRP, QB-QRP or IS. DB-QRP, QB-QRP and their IS were eluted from the UHPLC column at retention times of 5.02, 4.87 and 4.87 min, respectively.

[0070] Lower limit of quantitation

[0071] The lower limits of quantification (LLOQ) for DB-QRP and QB-QRP were 0.250 nM and 0.125 nM, which were the lowest concentrations of the two analytes in the standard curve. Figure 8B and 8E As shown, at the LLOQ concentration level, the signal-to-noise ratio (S / N) of DB-QRP or QB-QRP is at least 3 or higher.

[0072] Calibration curve linearity and QC accuracy and precision

[0073] For method validation of quantifying BTK RO in blood lysates, all standard curves were fitted to a 1 / x weighted quadratic regression model, with a standard curve range of 0.250 to 12.5 nM for DB-BTK and a standard curve range of 0.125 to 12.5 nM for QB-BTK. In each run, the back-calculated concentration was within ±20.0% of its nominal value (±25.0% at the LLOQ level) for at least two-thirds of the calibration standards. As shown in Tables 2 to 3, for three accuracy and precision runs, the back-calculated concentration of DB-BTK was within ±11.1% of its nominal value, and the back-calculated concentration of QB-BTK was within ±19.8% of its nominal value. Accuracy and precision were evaluated using quality control samples.

[0074] Table 2. Summary of data obtained from the precision and accuracy of the standard curve samples used for the DB-BTK assay.

[0075]

[0076] Table 3. Summary of data obtained from the precision and accuracy of the standard curve samples used for the QB-BTK assay.

[0077]

[0078] Table 4. Assay Validation: Quality Control Data Obtained from Monkey Blood Lysates with 25%, 50%, and 90% BTK Occupancy

[0079]

[0080]

[0081] As shown in Table 4, the differences between the measured BTK RO% and the theoretical BTK RO% were within ±5% for all three runs of all QCs, except for one of the six QC_25% RO samples and one of the six QC_50% RO samples in Run 2 where the differences between the measured and theoretical values ​​were >5%.

[0082] The coefficient of variation % and bias % of all QCs in three runs were within 10% except for one QC_25% RO which had a bias % of -16.2% (Table 5). The results demonstrate that the method is accurate and precise for the analysis of BTK RO % in monkey blood lysate.

[0083] Table 5. Summary of quality control data obtained from monkey blood lysates with 25%, 50% and 90% BTK occupancy (n=6).

[0084]

[0085] Stability evaluation

[0086] The established stability of DB-BTK and QB-BTK in monkey blood lysate is summarized in Table 6. The results indicate that the stability of DB-BTK and QB-BTK has no or little effect on BTK RO%, as the difference between the measured BTK RO% and the theoretical BTK RO% is within ±5% for all QCs, regardless of the decrease in the absolute concentration of DB-BTK or QB-BTK after 2 freeze-thaw cycles at -80°C or after storage at room temperature for 24h or at -80°C for 224 days.

[0087] Table 6. Stability evaluation of quality control samples at BTK RO% of 25% and 90%

[0088]

[0089]

[0090] Uses of Immunocapture LC-MS / MS Receptor Occupancy Assays

[0091] Measurement of RO is a key assay for relating efficacy to mechanism in preclinical animal models and clinical studies. In practice, RO is particularly useful in making dose escalation decisions in first-in-human (FIH) studies.

[0092] Example 1

[0093] Preparation of drug-bound BTK (DB-BTK) and quencher-bound BTK (QB-BTK) standards and quality control samples

[0094] Reagents and materials

[0095] Shown in Figure 1A and 1B The drug and quencher compounds in the assay were synthesized by the R&D department of Bristol-Myers Squibb (Princeton, NJ). Biotinylated rabbit anti-BTK monoclonal antibody (Lot No. D3H5, Catalog No. 12624) and 10x lysis buffer (Catalog No. 9803) were purchased from Cell Signaling Technology (Denver, MA). Dynabeads Myone Streptavidin T1 beads (Catalog No. 65602) were purchased from ThermoFisher Scientific (Carlsbad, CA). Recombinant BTK protein (Catalog No. PV3587) was purchased from Life Technologies (Carlsbad, CA). Protease inhibitors (Catalog No. 539134) were purchased from Millipore Sigma (Billerica, MA). The internal standard (IS) used for the assay was [ 13 C9, 15 N]-QB-QRP (Table 8) was synthesized by the Chemical Synthesis Department of Bristol-Myers Squibb (BMS) (Princeton, NJ, USA). Monkey ACD-A whole blood was purchased from Bioreclamation, Inc. (Westbury, NY). All other reagents were of analytical grade. MagnaBot 96 Magnetic Separation Device (Catalog No.: V8151) was purchased from Promega (Madison, WI).

[0096] Preparation of reference standard of drug-bound BTK (DB-BTK)

[0097] Recombinant BTK (5.9 μM, 200 μL) was combined with 400 μL PBS and then spiked with 15 μL of a 1.0 mg / mL drug solution in acetonitrile (shown in Figure 1A The solution was then incubated at room temperature for 1 h, resulting in a DB-BTK concentration of 1919 nM. The DB-BTK concentration was based on the total amount of free BTK used in the reaction, since in the presence of excess drug, the conversion of free BTK to DB-BTK was approximately 100%.

[0098] Preparation of reference standard of quencher-bound BTK (QB-BTK)

[0099] Recombinant BTK (5.9 μM, 200 μL) was combined with 400 μL PBS and then spiked with 15 μL of a 1.0 mg / mL quencher solution in acetonitrile (shown in Figure 1B The solution was incubated at room temperature for 1 h, and the resulting QB-BTK concentration was 1919 nM. The QB-BTK concentration was based on the total amount of free BTK used in the reaction, because in the presence of excess quencher, the conversion of free BTK to QB-BTK was approximately 100%.

[0100] Prepare lysis buffer mixture containing quencher: 2x lysis buffer, and 0.0133x protease inhibitor and 1.5 μM quencher solution:

[0101] Mix a vial containing 15 mL of 10x lysis buffer with 1.0 mL of protease inhibitor and 80 μL of 0.5 mg / mL quencher solution (shown in Figure 1B The solution was then diluted with 59 mL of deionized water to a total volume of 75 mL.

[0102] Prepare lysis buffer mixture containing drug: 2x lysis buffer, as well as 0.0133x protease inhibitor and 1.5 μM drug solution

[0103] Combine a vial containing 15 mL of 10x lysis buffer with 1.0 mL of protease inhibitor and 83 μL of 0.5 mg / mL drug solution (shown in Figure 1A The solution was then diluted with 59 mL of deionized water to a total volume of 75 mL.

[0104] Lysate preparation from monkey blood +433 (100% QB-BTK)

[0105] Monkey ACD-A whole blood (3.5 mL) was mixed with 7 mL of 1.5 μM quencher (shown in Figure 1B The samples were shaken at room temperature for 1 hour.

[0106] Lysate preparation from monkey blood +195 (100% DB-BTK)

[0107] Monkey ACD-A whole blood (3.5 mL) was mixed with 7 mL of 1.5 μM drug (shown in Figure 1A The samples were shaken at room temperature for 1 hour.

[0108] Preparation of quality control samples (QC) using blood lysate containing endogenous BTK

[0109] BTK RO QC was prepared by mixing different percentages of Lysate + 433 and Lysate + 195. BTK RO% was prepared by mixing Lysate + 433 / Lysate + 195 at 2250 / 750, 1500 / 1500, 300 / 2700 (v / v) to prepare lower QC (LQC), medium QC (MQC) and high QC (HQC) with BTK RO% of 25%, 50% and 90%, as shown in Table 7.

[0110] Table 7. Preparation of quality control samples using monkey blood lysate containing endogenous BTK

[0111]

[0112] a By using a quencher solution ( Figure 1B ) pretreated monkey lysate to obtain lysate +433.

[0113] b By using drug solution ( Figure 1A ) pretreated monkey lysate to obtain lysate ++195.

[0114] Prepare DB-BTK (BTK+195) standard curve: Use monkey blood lysate +433

[0115] Calibration standards (STD) of 0.250, 0.500, 1.00, 2.00, 5.00, 10.0 and 12.5 nM of DB-BTK were prepared in monkey blood lysate containing 0% DB-BTK by independently diluting DB-BTK stock solution (1919 nM in buffer). During the preparation process, intermediate dilutions of 200 nM and 20 nM of DB-BTK were prepared by diluting 1919 nM DB-BTK stock solution with blood lysate + 433, and then further diluted to the final STD sample. The nominal concentration is based on the blood volume, and the actual concentration in the blood lysate is 1 / 3 of each concentration due to the dilution factor of 3.

[0116] Prepare QB-BTK (BTK++433) standard curve: Use monkey blood lysate ++195

[0117] Calibration standards (STD) of QB-BTK at 0.125, 0.250, 0.500, 1.00, 2.00, 5.00, 10.0 and 12.5 nM were prepared in monkey blood lysate containing 0% QB-BTK by independently diluting QB-BTK stock solution (1919 nM in buffer). During the preparation process, intermediate dilutions of 200 nM and 20 nM of QB-BTK were prepared by diluting 1919 nM QB-BTK stock solution with blood lysate ++ 195, and then further diluted to the final STD sample. The nominal concentration is based on the blood volume, and the actual concentration in the blood lysate is 1 / 3 of each concentration due to the dilution factor of 3.

[0118] Prepare Streptavidin T1 capture beads with anti-BTK biotinylated antibody. The concentration of Dynabeads Myone Streptavidin T1 beads used for immunoprecipitation is 10 mg / mL. The binding capacity of biotinylated mAb is 20 μg mAb / mg beads. A total of 25 mL of Streptavidin T1 beads was aliquoted into 10 tubes, 2.5 mL in each tube. Wash the samples with 3 mL of PBST solution. DynaMag TM -5 magnet is used to separate magnetic beads from liquid sample matrix. After discarding the last wash solution, the beads are resuspended in 2.5mL PBST in each tube, and then 0.500mL biotinylated anti-BTK antibody (1mg / mL, batch number: D3H5) is added. The samples in 10 test tubes (0.500mL) are incubated at room temperature for 1h. The beads are separated from the solution and the beads in each tube are washed with 3mL PBST. The beads in the test tube are resuspended in 2.5mL PBST. The final anti-BTK antibody concentration is 0.2μg / μL beads. The samples are stored at 4°C for future use.

[0119] Immunocapture was performed to enrich drug-bound BTK (DB-BTK) and quencher-bound BTK (QB-BTK) from blood lysates. For control monkey blood lysates, calibration standards, quality control samples or monkey research samples, aliquots of 3000 μL blood lysate samples were centrifuged at room temperature. The anti-BTK capture antibody (60 μL, 0.2 μg mAb / μL beads) on the beads was added to the supernatant of the centrifuged sample, and the sample was incubated at room temperature for 120 minutes and then centrifuged. The supernatant was removed and PBST was added to the bead sample. The sample was transferred to a LowBind 96-well plate on a TECAN liquid handler and placed on top of a MagnaBot 96 magnetic separation device. The beads were washed twice with 800 μL PBST and then washed with 500 μL 25mMNH4OAc and 0.05% Tween-20. After removing the wash buffer, the beads in the LowBind 96-well plate were used for trypsin digestion, as described below.

[0120] Trypsin digestion.Except for blank lysate (to which 100 μ L of 25 mM NH4OAc with 0.05% Tween-20 was added), after IS (100 μ L of 0.5 μ g / mL SIL-QB-QRP in 25 mM NH4OAc with 0.05% Tween-20) was added to the beads in 96-well plates, 25 μ L 100 mM NH4HCO3 was added to each sample.Samples were digested by adding 25 μ L of 160 ng / μ L freshly prepared trypsin solution in 100 mM NH4HCO3, and incubated at 50°C for 30 min, then quenched with 20 μ L 50% / 50% MeOH / formic acid (v / v).Samples were vortexed briefly, then placed on top of MagnaBot 96 devices, supernatants were transferred to new 96-well plates, then centrifuged at 4000 rpm for 2 minutes. Samples were analyzed by LC-MS / MS.

[0121] UHPLC-MS / MS Analysis For assay validation and sample analysis, an AB Sciex Triple Quad 5500 mass spectrometer from Sciex (Foster City, CA) was used for LC-MS / MS data acquisition.

[0122] The following LC-MS / MS conditions were used for assay validation and PD sample analysis. For each treated sample, a 50 μL volume of the digest was injected into an ultra-high performance liquid chromatography (UHPLC) system (model LC-30AD, Shimadzu Scientific Instruments, Columbia, MD). The UHPLC-MS / MS conditions described below were specifically used to quantify occupied and quenched BTK in monkey blood lysates. Waters CORTECS TMUPLC C18+ column (2.1×100 mm, 1.6 μm, Waters, Milford, MA) was used to analyze DB-QRP and QB-QRP peptides. Mobile phase A consisted of 0.1% formic acid in water; while mobile phase B was 0.1% formic acid in acetonitrile. UHPLC separation was performed using the following gradient elution: starting from 15% B and holding for 0.5 min, then % B changed from 15% to 30% in 2.5 min, then % B changed from 30% to 45% in 4.9 min, then % B increased from 45% to 90% in 0.1 min and held for 1 min, then % B decreased from 95% to 15% in 0.1 min, held for 1.0 min, and stopped at 10.0 min. The flow rate was 0.600 mL / min, and the column temperature was 60°C. The UHPLC eluent was introduced into a Triple Quad 5500 equipped with a turbo ion spray source obtained from AB Sciex (Foster City, CA) in a switching valve configuration with a time window of 3.0-6.5 min. TM in the mass spectrometer. The mass spectrometer was operated in positive electrospray ionization mode with the following settings: curtain gas, 30 units; CAD gas, 9 units; gas 1, 65 units; gas 2, 65 units; turbo ion spray voltage was positive 4000 V, and turbo probe temperature was 600 °C. For QB-QRP, DB-QRP, and SIL-QB-QRP (IS), the mass spectrometer was operated in MRM mode with m / z transitions of 962.493>755.40, 967.174>755.40, and 965.825>765.50, respectively (Table 8). The software (version 1.6.2, ABSCIEX) processes the raw data for chromatographic peak integration and exports the processed results to Watson LIMS TM (Version 7.4, ThermoFisher Scientific) to perform regression of calibration standards and concentration calculations for QC and other samples. TM Statistics on assay performance were calculated (ANOVA).

[0123] Table 8. MRM transitions used for LC-MS / MS detection of surrogate peptides and their stable isotope labeled internal standards.

[0124]

[0125]

[0126] aThe replacement peptides were named QRP based on the first three amino acid symbols of the 21 amino acids in the tryptic peptide. DB-QRP = drug-bound QRP peptide; QB-QRP = quencher-bound QRP peptide.

[0127] b The symbol of F* represents [ 13 C9, 15 N]-phenylalanine. The underlined peptide segments are tryptic peptides and were monitored in the LC-MS / MS assay.

[0128] Assay validation. Assay validation consists of three accuracy and precision runs, and additional stability evaluations (including freeze-thaw, RT and long-term stability of BTK RO QC). In all validation runs, the linearity of the method was assessed using seven DB-BTK (0.250nM-12.5nM) or eight QB-BTK (0.125nM-12.5nM) calibration curve points. Quantification was performed using a quadratic regression model weighted 1 / x. The acceptance criteria are as follows: In each run, for at least two-thirds of the calibration standards, the deviation of the back-calculated concentration from its nominal value was within ± 20.0% (± 25.0% at the LLOQ level). For BTKRO QC, the difference between the measured BTK RO% and the theoretical BTK RO% should be within ± 5% for at least 50% of the QCs at each level. For example, for BTK RO QC_25%, the measured BTK RO% should be within 25.0 ± 5.0%. Only blank blood lysate QC0 (blank lysate with IS only) was used to evaluate the selectivity of the assay.

[0129] Example 2

[0130] Application in monkey PD research.

[0131] To evaluate the utility and robustness of the BTK RO assay by LC-MS / MS compared to established ELISA-based assays, the Figure 1A A single ascending dose study of a compound in . Drug compound 1A was orally administered to cynomolgus monkeys at 0.1, 0.2 and 0.5 mg / kg. Blood samples were collected at 1, 3, 5, 7 and 24 h on day 1 for PK evaluation, and blood samples were collected at pre-dose, 1, 3, 5, 7, 24, 48, 72, 144 and 168 h for PD evaluation using the BTK RO assay performed by LC-MS / MS and the ligand binding assay (LBA) described in Example 3.

[0132] PD samples for BTK RO assay by LC-MS / MS.

[0133] Prior to PD sample collection, the following solution containing 2x lysis buffer with 0.0133x protease inhibitor and 1.5 μM quencher (shown in Figure 1B Lysis buffer mixture of compound 10x solution: Mix a vial containing 15 mL of 10x lysis buffer with 1.0 mL of protease inhibitor and 80 μL of 0.5 mg / mL quencher compound 1B (in DMSO). The solution is then diluted with 59 mL of deionized water for a total volume of 75 mL.

[0134] As described above, 2 mL blood samples were collected at set time points. 1.5 mL of collected blood was lysed with 3 mL of a lysis buffer mixture containing quencher compound 1B. The samples were shaken at room temperature for 1 hour immediately after mixing. The blood lysate samples were stored at -70°C or lower immediately after collection. The samples were used for immunocapture determined using LC-MS / MS.

[0135] Example 3

[0136] BTK RO assay by LBA

[0137] Whole blood from ACD-A treated monkeys was collected at set time points after administration and added to 96-well V-bottom 2 ml plates (Costar, catalog number 3960) and lysed with 2x lysis buffer (Cell Signaling, catalog number 9803) containing protease inhibitors (Calbiochem, catalog number 539134) and biotinylated probes (BMT-105186). The lysate was transferred to a streptavidin-coated plate (ThermoFisher Scientific, Catalog number 15128) and incubated at room temperature with shaking for 1 hour. After washing, anti-BTK antibody (Cell Signaling, catalog number 8547, diluted 1:1000 in PBS+0.05% Tween 20+0.5% BSA) was added and incubated at room temperature with shaking for 1 hour. The plate was washed and then goat anti-rabbit horseradish peroxidase secondary antibody (Invitrogen, G21234) was added at a dilution of 1:2,500 in PBS+0.05% Tween 20+0.5% BSA. ELISA was performed by adding tetramethylbenzidine (TMB) (Sigma, catalog number T0440), and color was developed, and ELISA was terminated by adding 2.0N sulfuric acid. The absorbance at 450 nm was read and the relative % of BTK inactivation of the study samples was calculated from a standard curve containing varying ratios of lysate from normal monkey blood mixed with quenched lysate from blood pretreated with 2 μM BMT-126867 (to allow complete BTK inactivation prior to lysis).

[0138] Pharmacokinetic analysis by LC-MS

[0139] At the designated time points, 0.5 mL of monkey blood was collected for PK evaluation. Each blood sample was centrifuged to separate plasma. 10 μL of 125 μM quencher solution (shown in Figure 1B The compound in ( ) was immediately added to 200 μL of plasma. After mixing, the samples were stored at -20°C and used for LC-MS / MS analysis. Sequence Listing <110> Bristol-Myers Squibb <120> Assays to determine receptor occupancy in vivo <130> 12728-WO-PCT <150> 62 / 669442 <151> 2018-05-10 <160> 10 <170> PatentIn Version 3.5 <210> 1 <211> twenty one <212> PRT <213> Homo sapiens <220> <221> Peptides <222> (1)..(21) <223> Cys at position 15 is the binding site for the irreversible quencher or target compound <400> 1 Gln Arg Pro Ile Phe Ile Ile Thr Glu Tyr Met Ala Asn Gly Cys Leu 1 5 10 15 Asn Tyr Asn Arg 20 <210> 2 <211> 9 <212> PRT <213> Homo sapiens <220> <221> Peptides <222> (1)..(9) <223> The Cys at position 5 is the binding site for the irreversible quencher or target compound <400> 2 Met Ala Asn Gly Cys Leu Leu Asn Tyr 1 5 <210> 3 <211> 13 <212> PRT <213> Homo sapiens <220> <221> Peptides <222> (1)..(13) <223> Cys at position 6 is the binding site for the irreversible quencher or target compound <400> 3 Tyr Met Ala Asn Gly Cys Leu Leu Asn Tyr Leu Arg Glu 1 5 10 <210> 4 <211> 37 <212> PRT <213> Homo sapiens <220> <221> Peptides <222> (1)..(37) <223> Cys at position 15 is the binding site for the irreversible quencher or target compound <400> 4 Gln Arg Pro Ile Phe Ile Ile Thr Glu Tyr Met Ala Asn Gly Cys Leu 1 5 10 15 Leu Asn Tyr Leu Arg Glu Met Arg His Arg Phe Gln Thr Gln Gln Leu 20 25 30 Leu Glu Met Cys Lys 35 <210> 5 <211> 37 <212> PRT <213> Homo sapiens <220> <221> Peptides <222> (1)..(37) <223> Cys at position 16 is the binding site for the irreversible quencher or target compound <400> 5 Lys Gln Arg Pro Ile Phe Ile Ile Thr Glu Tyr Met Ala Asn Gly Cys 1 5 10 15 Leu Leu Asn Tyr Leu Arg Glu Met Arg His Arg Phe Gln Thr Gln Gln 20 25 30 Leu Leu Glu Met Cys 35 <210> 6 <211> 64 <212> PRT <213> Homo sapiens <220> <221> Peptides <222> (1)..(64) <223> Cys at position 42 is the binding site for the irreversible quencher or target compound <400> 6 Asp Glu Phe Ile Glu Glu Ala Lys Val Met Met Asn Leu Ser His Glu 1 5 10 15 Lys Leu Val Gln Leu Tyr Gly Val Cys Thr Lys Gln Arg Pro Ile Phe 20 25 30 Ile Ile Thr Glu Tyr Met Ala Asn Gly Cys Leu Leu Asn Tyr Leu Arg 35 40 45 Glu Met Arg His Arg Phe Gln Thr Gln Gln Leu Leu Glu Met Cys Lys 50 55 60 <210> 7 <211> 65 <212> PRT <213> Homo sapiens <220> <221> Peptides <222> (1)..(65) <223> Cys at position 59 is the binding site for the irreversible quencher or target compound <400> 7 Gly Gln Tyr Asp Val Ala Ile Lys Met Ile Lys Glu Gly Ser Met Ser 1 5 10 15 Glu Asp Glu Phe Ile Glu Glu Ala Lys Val Met Met Asn Leu Ser His 20 25 30 Glu Lys Leu Val Gln Leu Tyr Gly Val Cys Thr Lys Gln Arg Pro Ile 35 40 45 Phe Ile Ile Thr Glu Tyr Met Ala Asn Gly Cys Leu Leu Asn Tyr Leu 50 55 60 Arg 65 <210> 8 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Phe at position 7 is [13C9,15N]-phenylalanine; Cys at position 17 is Binding site of irreversible quencher <400> 8 Thr Arg Gln Arg Pro Ile Phe Ile Ile Thr Glu Tyr Met Ala Asn Gly 1 5 10 15 Cys Leu Leu Asn Tyr Leu Arg Glu Ala 20 25 <210> 9 <211> 659 <212> PRT <213> Homo sapiens <400> 9 Met Ala Ala Val Ile Leu Glu Ser Ile Phe Leu Lys Arg Ser Gln Gln 1 5 10 15 Lys Lys Lys Thr Ser Pro Leu Asn Phe Lys Lys Arg Leu Phe Leu Leu 20 25 30 Thr Val His Lys Leu Ser Tyr Tyr Glu Tyr Asp Phe Glu Arg Gly Arg 35 40 45 Arg Gly Ser Lys Lys Gly Ser Ile Asp Val Glu Lys Ile Thr Cys Val 50 55 60 Glu Thr Val Val Pro Glu Lys Asn Pro Pro Pro Glu Arg Gln Ile Pro 65 70 75 80 Arg Arg Gly Glu Glu Ser Ser Glu Met Glu Gln Ile Ser Ile Ile Glu 85 90 95 Arg Phe Pro Tyr Pro Phe Gln Val Val Tyr Asp Glu Gly Pro Leu Tyr 100 105 110 Val Phe Ser Pro Thr Glu Glu Leu Arg Lys Arg Trp Ile His Gln Leu 115 120 125 Lys Asn Val Ile Arg Tyr Asn Ser Asp Leu Val Gln Lys Tyr His Pro 130 135 140 Cys Phe Trp Ile Asp Gly Gln Tyr Leu Cys Cys Ser Gln Thr Ala Lys 145 150 155 160 Asn Ala Met Gly Cys Gln Ile Leu Glu Asn Arg Asn Gly Ser Leu Lys 165 170 175 Pro Gly Ser Ser His Arg Lys Thr Lys Lys Pro Leu Pro Pro Thr Pro 180 185 190 Glu Glu Asp Gln Ile Leu Lys Lys Pro Leu Pro Pro Glu Pro Ala Ala 195 200 205 Ala Pro Val Ser Thr Ser Glu Leu Lys Lys Val Val Ala Leu Tyr Asp 210 215 220 Tyr Met Pro Met Asn Ala Asn Asp Leu Gln Leu Arg Lys Gly Asp Glu 225 230 235 240 Tyr Phe Ile Leu Glu Glu Ser Asn Leu Pro Trp Trp Arg Ala Arg Asp 245 250 255 Lys Asn Gly Gln Glu Gly Tyr Ile Pro Ser Asn Tyr Val Thr Glu Ala 260 265 270 Glu Asp Ser Ile Glu Met Tyr Glu Trp Tyr Ser Lys His Met Thr Arg 275 280 285 Ser Gln Ala Glu Gln Leu Leu Lys Gln Glu Gly Lys Glu Gly Gly Phe 290 295 300 Ile Val Arg Asp Ser Ser Lys Ala Gly Lys Tyr Thr Val Ser Val Phe 305 310 315 320 Ala Lys Ser Thr Gly Asp Pro Gln Gly Val Ile Arg His Tyr Val Val 325 330 335 Cys Ser Thr Pro Gln Ser Gln Tyr Tyr Leu Ala Glu Lys His Leu Phe 340 345 350 Ser Thr Ile Pro Glu Leu Ile Asn Tyr His Gln His Asn Ser Ala Gly 355 360 365 Leu Ile Ser Arg Leu Lys Tyr Pro Val Ser Gln Gln Asn Lys Asn Ala 370 375 380 Pro Ser Thr Ala Gly Leu Gly Tyr Gly Ser Trp Glu Ile Asp Pro Lys 385 390 395 400 Asp Leu Thr Phe Leu Lys Glu Leu Gly Thr Gly Gln Phe Gly Val Val 405 410 415 Lys Tyr Gly Lys Trp Arg Gly Gln Tyr Asp Val Ala Ile Lys Met Ile 420 425 430 Lys Glu Gly Ser Met Ser Glu Asp Glu Phe Ile Glu Glu Ala Lys Val 435 440 445 Met Met Asn Leu Ser His Glu Lys Leu Val Gln Leu Tyr Gly Val Cys 450 455 460 Thr Lys Gln Arg Pro Ile Phe Ile Ile Thr Glu Tyr Met Ala Asn Gly 465 470 475 480 Cys Leu Leu Asn Tyr Leu Arg Glu Met Arg His Arg Phe Gln Thr Gln 485 490 495 Gln Leu Leu Glu Met Cys Lys Asp Val Cys Glu Ala Met Glu Tyr Leu 500 505 510 Glu Ser Lys Gln Phe Leu His Arg Asp Leu Ala Ala Arg Asn Cys Leu 515 520 525 Val Asn Asp Gln Gly Val Val Lys Val Ser Asp Phe Gly Leu Ser Arg 530 535 540 Tyr Val Leu Asp Asp Glu Tyr Thr Ser Ser Val Gly Ser Lys Phe Pro 545 550 555 560 Val Arg Trp Ser Pro Pro Glu Val Leu Met Tyr Ser Lys Phe Ser Ser 565 570 575 Lys Ser Asp Ile Trp Ala Phe Gly Val Leu Met Trp Glu Ile Tyr Ser 580 585 590 Leu Gly Lys Met Pro Tyr Glu Arg Phe Thr Asn Ser Glu Thr Ala Glu 595 600 605 His Ile Ala Gln Gly Leu Arg Leu Tyr Arg Pro His Leu Ala Ser Glu 610 615 620 Lys Val Tyr Thr Ile Met Tyr Ser Cys Trp His Glu Lys Ala Asp Glu 625 630 635 640 Arg Pro Thr Phe Lys Ile Leu Leu Ser Asn Ile Leu Asp Val Met Asp 645 650 655 Glu Glu Ser <210> 10 <211> 659 <212> PRT <213> Rhesus cytomegalovirus <400> 10 Met Ala Ala Val Ile Leu Glu Ser Ile Phe Leu Lys Arg Ser Gln Gln 1 5 10 15 Lys Lys Lys Thr Ser Pro Leu Asn Phe Lys Lys Arg Leu Phe Leu Leu 20 25 30 Thr Val His Lys Leu Ser Tyr Tyr Glu Tyr Asp Phe Glu Arg Gly Arg 35 40 45 Arg Gly Ser Lys Lys Gly Ser Ile Asp Val Glu Lys Ile Thr Cys Val 50 55 60 Glu Thr Val Val Pro Glu Lys Asn Pro Pro Pro Glu Arg Gln Ile Pro 65 70 75 80 Arg Arg Gly Glu Glu Ser Ser Glu Met Glu Gln Ile Ser Ile Ile Glu 85 90 95 Arg Phe Pro Tyr Pro Phe Gln Val Val Tyr Asp Glu Gly Pro Leu Tyr 100 105 110 Val Phe Ser Pro Thr Glu Glu Leu Arg Lys Arg Trp Ile His Gln Leu 115 120 125 Lys Asn Val Ile Arg Tyr Asn Ser Asp Leu Val Gln Lys Tyr His Pro 130 135 140 Cys Phe Trp Ile Asp Gly Gln Tyr Leu Cys Cys Ser Gln Thr Ala Lys 145 150 155 160 Asn Ala Met Gly Cys Gln Ile Leu Glu Asn Arg Asn Gly Ser Leu Lys 165 170 175 Pro Gly Ser Ser His Arg Lys Thr Lys Lys Pro Leu Pro Pro Thr Pro 180 185 190 Glu Glu Asp Gln Ile Leu Lys Lys Pro Leu Pro Pro Glu Pro Ala Ala 195 200 205 Ala Pro Val Ser Thr Ser Glu Leu Lys Lys Val Val Ala Leu Tyr Asp 210 215 220 Tyr Met Pro Met Asn Ala Asn Asp Leu Gln Leu Arg Lys Gly Asp Glu 225 230 235 240 Tyr Phe Ile Leu Glu Glu Ser Asn Leu Pro Trp Trp Arg Ala Arg Asp 245 250 255 Lys Asn Gly Gln Glu Gly Tyr Ile Pro Ser Asn Tyr Val Thr Glu Ala 260 265 270 Glu Asp Ser Ile Glu Met Tyr Glu Trp Tyr Ser Lys His Met Thr Arg 275 280 285 Ser Gln Ala Glu Gln Leu Leu Lys Gln Glu Gly Lys Glu Gly Gly Phe 290 295 300 Ile Val Arg Asp Ser Ser Lys Ala Gly Lys Tyr Thr Val Ser Val Phe 305 310 315 320 Ala Lys Ser Thr Gly Asp Pro Gln Gly Val Ile Arg His Tyr Val Val 325 330 335 Cys Ser Thr Pro Gln Ser Gln Tyr Tyr Leu Ala Glu Lys His Leu Phe 340 345 350 Ser Thr Ile Pro Glu Leu Ile Asn Tyr His Gln His Asn Ser Ala Gly 355 360 365 Leu Ile Ser Arg Leu Lys Tyr Pro Val Ser Gln Gln Asn Lys Asn Ala 370 375 380 Pro Ser Thr Ala Gly Leu Gly Tyr Gly Ser Trp Glu Ile Asp Pro Lys 385 390 395 400 Asp Leu Thr Phe Leu Lys Glu Leu Gly Thr Gly Gln Phe Gly Val Val 405 410 415 Lys Tyr Gly Lys Trp Arg Gly Gln Tyr Asp Val Ala Ile Lys Met Ile 420 425 430 Lys Glu Gly Ser Met Ser Glu Asp Glu Phe Ile Glu Glu Ala Lys Val 435 440 445 Met Met Asn Leu Ser His Glu Lys Leu Val Gln Leu Tyr Gly Val Cys 450 455 460 Thr Lys Gln Arg Pro Ile Phe Ile Ile Thr Glu Tyr Met Ala Asn Gly 465 470 475 480 Cys Leu Leu Asn Tyr Leu Arg Glu Met Arg His Arg Phe Gln Thr Gln 485 490 495 Gln Leu Leu Glu Met Cys Lys Asp Val Cys Glu Ala Met Glu Tyr Leu 500 505 510 Glu Ser Lys Gln Phe Leu His Arg Asp Leu Ala Ala Arg Asn Cys Leu 515 520 525 Val Asn Asp Gln Gly Val Val Lys Val Ser Asp Phe Gly Leu Ser Arg 530 535 540 Tyr Val Leu Asp Asp Glu Tyr Thr Ser Ser Val Gly Ser Lys Phe Pro 545 550 555 560 Val Arg Trp Ser Pro Pro Glu Val Leu Met Tyr Ser Lys Phe Ser Ser 565 570 575 Lys Ser Asp Ile Trp Ala Phe Gly Val Leu Met Trp Glu Ile Tyr Ser 580 585 590 Leu Gly Lys Met Pro Tyr Glu Arg Phe Thr Asn Ser Glu Thr Ala Glu 595 600 605 His Ile Ala Gln Gly Leu Arg Leu Tyr Arg Pro His Leu Ala Ser Glu 610 615 620 Lys Val Tyr Thr Ile Met Tyr Ser Cys Trp His Glu Lys Ala Asp Glu 625 630 635 640 Arg Pro Thr Phe Lys Ile Leu Leu Ser Asn Ile Leu Asp Val Met Asp 645 650 655 Glu Glu Ser

Claims

1. Use of a quencher in the preparation of a detection reagent, wherein the reagent is used in a method for determining drug receptor occupancy, the method comprising the following steps: a. collecting a blood sample from a subject treated with a drug of interest that covalently binds to the receptor, b. adding a lysis solution containing the quencher to the blood sample, wherein the quencher covalently binds to the same receptor site as the receptor site to which the target drug binds, c. Isolation of target receptors, d. digesting the isolated receptor to produce a replacement peptide, e. Measure the amount of surrogate peptide using LC-MS / MS and determine drug receptor occupancy by comparing the amount of drug-bound surrogate peptide to the total amount of drug-bound and quencher-bound surrogate peptide.

2. Use of a quencher in the preparation of a detection reagent, the reagent being used in a method for determining drug receptor occupancy in a subject administered with a target drug that covalently binds to a target receptor, wherein the quencher covalently binds to the same receptor site as the target drug, the method comprising the following steps: a. Collecting blood samples from subjects treated with the drug of interest b. adding a lysis solution containing the quencher to the blood sample, c. Separation of drug-bound and quencher-bound receptors by immunocapture, d. digesting the separated drug-bound and quencher-bound receptors to produce drug-bound and quencher-bound surrogate peptides, e. Measure the amount of surrogate peptide using LC-MS / MS and determine the amount of receptor bound to the drug by comparing the amount of drug-bound surrogate peptide to the total amount of drug-bound and quencher-bound surrogate peptide.

3. The use according to claim 2, wherein the target drug is 4. The use according to claim 2, wherein the quencher in step (b) is 5. The use according to claim 1 or 2, wherein the digestion step (d) is trypsin digestion.

6. Use of a quencher in the preparation of a detection reagent, the quencher being: The reagent is used in a method for determining BTK receptor occupancy in a subject administered a drug, wherein the drug is: The method comprises the following steps: a. collecting a blood sample from the subject to whom the drug is administered, b. adding a lysis solution containing the quencher to the blood sample, wherein the quencher covalently binds to the same receptor site as the drug, c. Separation of drug-bound and quencher-bound BTK using anti-BTK biotinylated antibody bound to streptavidin T1 beads, d. digesting the separated drug-bound and quencher-bound BTK-antibody complexes with trypsin to produce surrogate peptides, e. Measure the amount of surrogate peptide using LC-MS / MS chromatography and determine the amount of BTK bound to the drug by comparing the amount of drug-bound surrogate peptide to the total amount of drug-bound and quencher-bound surrogate peptide.

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