WO2016100593A1 - Procédés et dosages de quantificaton et de normalisation de liaison de kinases et de ligands - Google Patents

Procédés et dosages de quantificaton et de normalisation de liaison de kinases et de ligands Download PDF

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WO2016100593A1
WO2016100593A1 PCT/US2015/066244 US2015066244W WO2016100593A1 WO 2016100593 A1 WO2016100593 A1 WO 2016100593A1 US 2015066244 W US2015066244 W US 2015066244W WO 2016100593 A1 WO2016100593 A1 WO 2016100593A1
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target
btk
occupancy
assay
antibody
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PCT/US2015/066244
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English (en)
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Stella CHANG
Tarikere L. GURURAJA
Betty Y. CHANG
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Pharmacyclics Llc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases

Definitions

  • the present invention relates generally to a method and assay system for determination of the quantity of a target or enzyme, particularly a kinase, including a Tec family kinase, particularly Bruton's tyrosine kinase (BTK), and the relative amount of available target or enzyme associated with a target ligand or enzyme binder, particularly wherein the target ligand or enzyme binder is a covalent or irreversible kinase inhibitor, particularly including irreversible BTK inhibitor.
  • the invention also relates to assays for determining target binding or occupancy, normalized to total quantified target in a biological system or clinical sample, wherein target is quantified, normalized and occupancy determined using the same or a common assay format.
  • Protein kinases are a large family of conserved enzymes that transfer a ⁇ -phosphate group from ATP to amino acid residues, including tyrosine, serine and threonine, in a ubiquitous mechanism for cellular signal transduction.
  • Protein kinase inhibitors PKIs
  • Irreversible inhibitors or covalent kinase inhibitors that react specifically with the enzyme and/or change it via covalent modification are attractive alternatives.
  • inhibitors target the highly nucleophilic thiol group of cysteine residues, which are found in and around the ATP -binding site.
  • a series of irreversible inhibitors have advanced to clinical trials that target Cys797 conserved in EGFR, Her2 and Her4.
  • a number of non-EGFR family kinases possess a cysteine at a comparable position to the Cys797 of EGFR. These include members of the Tec family of kinases (BMX, BTK (Btk), ITK, TEC and TXK), Scr family member BLK, MKKa7 kinase, and JAK3 kinase (Liu, Q et al (2013) Chemistry & Biology 20: 146-159).
  • Btk Bruton's tyrosine kinase
  • BCR cell surface B-cell receptor
  • Btk is expressed in all hematopoietic cells types except T lymphocytes and natural killer cells, and participates in a number of TLR and cytokine receptor signaling pathways including lipopoly saccharide (LPS) induced TNF-a production in macrophages, suggesting a general role for Btk in immune regulation.
  • LPS lipopoly saccharide
  • Btk contains an amino-terminal pleckstrin homology (PH) domain, followed by a Tec homology (TH) domain, regulatory Src homology (SH3, SH2) domains, and a C-terminal kinase (SHI) domain.
  • PH pleckstrin homology
  • TH Tec homology
  • SH3 regulatory Src homology
  • SHI C-terminal kinase
  • Membrane-associated Btk is then phosphorylated at Tyr 551 in the activation loop by Src family kinases. Subsequent Btk auto-phosphorylation at Tyr 223 stabilizes the active conformation and fully activates Btk kinase activity. Activated Btk phosphorylates phospholipase (PLCy), initiating calcium mobilization and generating diacylglycerol (DAG) as secondary signals, eventually leading to transcriptional activation and amplification of BCR stimulation.
  • PLCy phospholipase
  • DAG diacylglycerol
  • Small molecule Btk inhibitors have been developed with anticipated therapeutic benefit in the treatment of lymphoma and autoimmune diseases.
  • a potent irreversibly acting small molecule inhibitor of Btk, PCI-32765 has demonstrated promising clinical activity in an ongoing phase I study in patients with B-cell NHL.
  • PCI-32765 inhibits BCR signaling downstream of Btk, selectively blocks B-cell activation, and is efficacious in animal models of arthritis, lupus, and B-cell lymphoma (Honigberg LA et al. (2010) PNAS 107(20): 13075-13080).
  • Irreversible inhibitor PCI-32765 is one of a series of Btk inhibitors that bind covalently to cysteine residue Cys-481 in the active site leading to irreversible inhibition of Btk enzymatic activity (Pan Z et al (2007) Chem Med Chem 2:58-61).
  • Potent and selective kinase inhibitors are highly desirable as potential specific therapeutics and as probes to pharmacologically interrogate kinase biology. Assessing potency and inhibition of irreversible or covalent inhibitors necessitates an accurate and efficient determination of the relative amount of bound inhibitor to the total amount of enzyme target.
  • One approach has been to use target site occupancy assays to understand the optimal dosing regimen to achieve a desired therapeutic effect and minimize side effects.
  • assay methods and assay systems are provided for quantification of a protein or enzyme target and target occupancy, particularly for a covalent or irreversible inhibitor of the target.
  • a single, common format is utilized and serves as the basis for both quantification of target and for determination of target occupancy.
  • the methods and assays of the invention provide for use of a common set of reagents for determination and evaluation of target quantification and target occupancy.
  • the methods and assays of the invention allow for standardization and eliminate the need for multiple formats or evaluation protocols.
  • the invention provides a method or assay to determine target occupancy and quantitate total target of a kinase, particularly a Tec family kinase, wherein target occupancy is determined utilizing a target occupancy probe and total target is quantitated using an anti-target specific binding member, wherein the target occupancy probe and anti-target specific binding member are tagged with a moiety for capture by the same capture moiety binder.
  • a target containing sample is obtained and split or separated for each of (a) target occupancy determination and (b) total target determination.
  • each of (a) target occupancy determination and (b) total target determination comprising the steps of: (i) contacting a sample with target occupancy probe in (a) and anti-target specific binding member in (b), each tagged with a same capture moiety or a capture moiety that binds to the same capture binder, to form an occupancy probe-target complex in (a) and a specific binding member-target complex in (b); (ii) capturing the complex from (a) and (b) with a capture moiety binder coated solid support; and (iii) detecting the presence and amount of captured occupancy probe-target complex in (a) and specific binding member-target complex in
  • the invention provides a method or assay to determine target occupancy and quantitate total target of a kinase, particularly a non-receptor tyrosine kinase, particularly a Tec family kinase, comprising:
  • target is quantified by comparison to a standard curve generated using recombinant target protein, wherein the standard curve is obtained simultaneously or approximately the same time as the samples are analyzed.
  • the primary anti-target specific binding member is an antibody, particularly a labeled antibody.
  • the target complex bound solid support is contacted with a secondary tagged or labeled antibody that recognizes or binds the primary anti-target specific binding member for detection.
  • the solid support is washed between steps (iii) and (iv).
  • the solid support is washed in (iv) after primary anti-target specific binding member is contacted and prior to contacting with a secondary tagged or labeled antibody that recognizes or binds the primary anti-target specific binding member.
  • the sample is a cell lysate, particularly a lysate of PBMCs.
  • the samples for (a) target occupancy determination and (b) total target determination are processed side by side, simultaneously or in series.
  • the target is BTK
  • the target occupancy probe is a labeled or biotinylated BTK inhibitor
  • the anti-target specific binding member is an anti-BTK monoclonal antibody.
  • the capture moiety is biotin and the capture moiety binder is avidin, streptavidin or neutravidin.
  • the anti-BTK monoclonal antibody is a biotinylated anti-BTK antibody.
  • the invention provides a method or assay to determine BTK target occupancy and quantitate total target of BTK in a sample, comprising:
  • the BTK occupancy probe is a labeled covalent or irreversible inhibitor of BTK.
  • the BTK occupancy probe is labeled ibrutinib, particularly biotinylated ibrutinib, particularly having the structure of Formula IV.
  • the invention further provides a test kit for the demonstration of the quantity of target and target occupancy of inhibitor, particularly BTK target and BTK inhibitor comprising:
  • BTK-occupancy probe such as biotinylated ibrutinib and a predetermined amount of biotinylated anti-BTK antibody
  • FIGURE 1 depicts a sandwich ELISA for total BTK, utilizing a first coating antibody (for example goat anti-rabbit IgG), followed by a capture antibody (for example rabbit anti-target BTK). Sample is then applied, an anti-target antibody (for example mouse anti-BTK) is added, and then a secondary antibody which is tagged or detectable and recognizes or binds to the anti- target antibody (for example anti-mouse IgG with a TAG) is introduced for detection.
  • a first coating antibody for example goat anti-rabbit IgG
  • a capture antibody for example rabbit anti-target BTK
  • FIGURE 2A-2D depicts assay results using sandwich ELISA as depicted in Figure 1 for BTK in an MSD format. Results are tabulated in A and C and standard curves are shown in B and D. A and B provide results with 1 ⁇ g/ml capture antibody; C and D provide results with
  • Capture antibody was Rabbit anti-BTK monoclonal antibody
  • Detection antibody was anti-BTK monoclonal antibody (BD Bioscience Cat No 611117, N-term rec protein 2-172 aa). BTK protein was from
  • FIGURE 3A-3C provides sandwich ELISA assay results for recombinant BTK versus DoHH2 cell lysate protein.
  • A tabulates results with recombinant BTK.
  • B graphs recombinant BTK protein results.
  • Cell lysate results are tabulated in C.
  • Cap-Ab-1 refers to 1 ⁇ g/ml capture antibody;
  • Cap-Ab-2 refers to 0.5 ⁇ g/ml capture antibody.
  • FIGURE 4A-4C depicts sandwich ELISA MSD format for cell lysates (DoHH2 cells). Assay results using higher amounts of coating antibody 2 ⁇ g/ml and 4 ⁇ g/ml, capture antibody are shown in A and B respectively. C graphs results with 0.5 ⁇ g/ml, 1 ⁇ g/ml, 2 ⁇ g/ml, and 4 ⁇ g/ml coating antibody.
  • FIGURE 5 depicts an alternative sandwich ELISA format without coating antibody (goat anti-rabbit IgG).
  • the capture antibody (rabbit or mouse anti-BTK antibody) on the plates recognizes target (e.g. BTK).
  • target e.g. BTK
  • Sample is then applied, followed by mouse or rabbit anti-target (e.g. BTK) antibody, and then secondary anti-mouse or rabbit IgG with a tag or HRP as signal for detection.
  • FIGURE 7 depicts an experimental protocol for a total BTK quantification ELISA using a capture antibody only, as shown in Figure 5.
  • Each box depicts a step in the protocol.
  • Primary anti-BTK antibody is added in 1%BSA (100 ⁇ ) for 2 hours at RT, followed by secondary antibody in 1%
  • FIGURE 8A-8D depicts results of the capture antibody ELISA method in standard
  • a and C provide results with recombinant BTK protein
  • C and D provide results with cell lysates.
  • the capture antibody utilized was anti-BTK rabbit monoclonal antibody (CST Cat No 8547) and detection antibody was anti-BTK mouse monoclonal antibody (BD Cat No 611117).
  • FIGURE 9A-9D depicts results of the capture antibody ELISA method in standard (STD) (A and C) and meso-scale discovery (MSD) (B and D) formats.
  • a and B provide results with recombinant BTK protein
  • C and D provide results with cell lysates.
  • the capture antibody utilized was anti-BTK rabbit polyclonal antibody (Abeam Cat No 8547) and detection antibody was anti-BTK mouse monoclonal antibody (BD Cat No 611117). No BTK protein was captured and quantified in the cell lysates.
  • FIGURE 10A-10D depicts results of the capture antibody ELISA method in standard (STD) (A and C) and meso-scale discovery (MSD) (B and D) formats.
  • a and B provide results with recombinant BTK protein
  • C and D provide results with cell lysates.
  • the capture antibody utilized was anti-BTK mouse monoclonal antibody (BD Cat No 611117) and detection antibody was anti-BTK rabbit polyclonal antibody (Abeam Cat No 8547). Almost no BTK protein was captured and quantified in the cell lysates.
  • FIGURE 11 A-l ID depicts results of the capture antibody ELISA method in standard (STD) (A and C) and meso-scale discovery (MSD) (B and D) formats.
  • a and B provide results with recombinant BTK protein
  • C and D provide results with cell lysates.
  • the capture antibody utilized was anti-BTK mouse monoclonal antibody (BD Cat No 611117) and detection antibody was biotinylated anti-BTK rabbit monoclonal antibody (CST Cat No 12624S).
  • CST Cat No 12624S biotinylated anti-BTK rabbit monoclonal antibody
  • FIGURE 12 depicts a novel and simplified sandwich ELISA format without coating or capture antibody.
  • sample is mixed with biotinylated anti-target antibody (e.g. biotinylated mouse or rabbit anti-BTK antibody) allowed to bind to plates and then detected using anti-target antibody (e.g. mouse or rabbit anti-BTK antibody) and a secondary labeled or tagged antibody (e.g. anti-mouse or anti-rabbit antibody with tag or HRP).
  • biotinylated anti-target antibody e.g. biotinylated mouse or rabbit anti-BTK antibody
  • anti-target antibody e.g. mouse or rabbit anti-BTK antibody
  • secondary labeled or tagged antibody e.g. anti-mouse or anti-rabbit antibody with tag or HRP
  • FIGURE 13 depicts an experimental protocol for a total BTK quantification ELISA using a biotinylated anti-target antibody, as shown in Figure 12. Each box depicts a step in the protocol.
  • the assay provides target (e.g. BTK) occupancy data (normalized to total target (BTK)) and quantification of total target (BTK).
  • the assay is suitable for recombinant target (BTK) or cell lysate samples.
  • a sample for analysis is split and a portion is run through a target occupancy assay using biotinylated target occupancy probe (biotin-probe), while the other portion is used for a total target (BTK) assay using biotinylated anti-target antibody (e.g. biotin- anti-BTK).
  • biotin-probe complex target occupancy probe-target complex
  • biotin- anti-BTK complex anti-target specific binding member-target complex
  • FIGURE 14A-14B provides results of standard (STD) format ELISA for target (BTK) occupancy using (A) recombinant BTK (Rec-BTK) and (B) cell lysates using biotinylated BTK target occupancy probe (biotin ibrutinib/41025). Protein concentrations for the cell lysate studies range from 0 to 1500 ⁇ g/ml.
  • FIGURE 15A-15D provides results of standard (STD) format ELISA for total target (BTK) using (A) and (C) recombinant BTK (Rec-BTK) and (B) and (D) cell lysates using biotinylated anti-target (anti-BTK) antibody (biotinylated rabbit anti-BTK monoclonal antibody, Cell Signal Technology, Cat No 12624S). Protein concentrations for the cell lysate studies range from 0 to 1500 ⁇ g/ml (B) and 0 to 200 ⁇ g/ml (D).
  • FIGURE 16A-16B provides results of MSD format ELISA for target (BTK) occupancy using (A) recombinant BTK (Rec-BTK) and (B) cell lysates using biotinylated BTK target probe (biotin ibrutinib/41025). Protein concentrations for the cell lysate studies range from O to 1500 ⁇ g/ml.
  • FIGURE 17A-17D provides results of MSD format ELISA for total target (BTK) using (A) and (C) recombinant BTK (Rec-BTK) and (B) and (D) cell lysates using biotinylated anti-target (anti-BTK) antibody (biotinylated rabbit anti-BTK monoclonal antibody, Cell Signal Technology, Cat No 12624S). Protein concentrations for the cell lysate studies range from 0 to 1500 ⁇ g/ml (B) and 0 to 200 ⁇ g/ml (D).
  • FIGURE 18A-18B provides results of STD format ELISA for target (BTK) occupancy using (A) recombinant BTK (Rec-BTK) and (B) cell lysates using biotinylated BTK target occupancy probe (biotin ibrutinib/41025). Protein concentrations for the cell lysate studies range from 0 to 4000 ⁇ g/ml. The results of each assay are tabulated below the graphs.
  • FIGURE 19A-19B provides results of MSD format ELISA for target (BTK) occupancy using (A) recombinant BTK (Rec-BTK) and (B) cell lysates using biotinylated BTK target occupancy probe (biotin ibrutinib/41025). Protein concentrations for the cell lysate studies range from 0 to 4000 ⁇ g/ml. The results of each assay are tabulated below the graphs.
  • FIGURE 20 provides results of total target (BTK) ELISA STD format using biotinylated anti-target (anti-BTK) antibody for (A) recombinant BTK (Rec-BTK) and (B) cell lysates. Protein concentrations for the cell lysate studies range from 0 to 4000 ⁇ g/ml. The results of each assay are tabulated below the graphs.
  • FIGURE 21 provides results of total target (BTK) ELISA MSD format using biotinylated anti-target (anti-BTK) antibody for (A) recombinant BTK (Rec-BTK) and (B) cell lysates. Protein concentrations for the cell lysate studies range from 0 to 4000 ⁇ g/ml. The results of each assay are tabulated below the graphs.
  • FIGURE 22 depicts BTK quantification strategies and three methods denoted Method 1, Method 2, and Method 3.
  • Method 1 corresponds to the present novel method.
  • Cell lysates are split initially and side by side assays run for BTK occupancy using a target occupancy probe (e.g. Biotin-probe) and for total BTK determination using an anti-target specific binding member (e.g. Biotin anti-BTK antibody).
  • target occupancy probe e.g. Biotin-probe
  • an anti-target specific binding member e.g. Biotin anti-BTK antibody
  • Alternative Method 2 and Method 3 are depicted wherein lysates are screened for target occupancy (e.g. BTK), the supernatant collected, transferred, and assessed for total target using anti-target antibody (e.g. anti-BTK).
  • anti-target antibody e.g. anti-BTK
  • FIGURE 23 depicts the steps of Method 1.
  • FIGURE 24 provides assay results and BTK occupancy, free BTK and standard curve for patient #1 with Method 1.
  • Patient was administered inhibitor (ibrutinib; PCI-32765) 420 mg daily dose and BTK target occupancy was determined on Day 1 (vehicle dose), Day 2, Day 8, Day 15, Day 29 and at the end of the study.
  • inhibitor ibrutinib; PCI-32765
  • FIGURE 25 provides assay results and BTK occupancy, free BTK and standard curve for patient #2 with Method 1.
  • Patient was administered inhibitor (ibrutinib; PCI-32765) 420 mg daily dose and BTK target occupancy was determined on Day 1 (vehicle dose), Day 8, Day 15 and Day 29.
  • inhibitor ibrutinib; PCI-32765
  • FIGURE 26 provides assay results and BTK occupancy, free BTK and standard curve for patient #3 with Method 1.
  • Patient was administered inhibitor (ibrutinib; PCI-32765) 420 mg daily dose and BTK target occupancy was determined on Day 1 (vehicle dose), Day 2, Day 8, Day 15 and Day 29.
  • inhibitor ibrutinib; PCI-32765
  • FIGURE 27 provides assay results and BTK occupancy, free BTK and standard curve for patient #4 with Method 1.
  • Patient was administered inhibitor (ibrutinib; PCI-32765) 420 mg daily dose and BTK target occupancy was determined on Day 1 (vehicle dose), Day 2, Day 8, Day 15, Day 29 and at the end of the study.
  • FIGURE 28 depicts the steps of Method 2. Lysates are screened for BTK occupancy and the supernatants transferred for separate analysis to determine total BTK in the recovered supernatants.
  • FIGURE 29 provides assay results and BTK occupancy, free BTK and standard curve for patient #1 with Method 2.
  • Patient was administered inhibitor (ibrutinib; PCI-32765) 420 mg daily dose and BTK target occupancy determined on Day 1 (vehicle), Day 2, Day 8, Day 15, Day 29 and at the end of the study.
  • inhibitor ibrutinib; PCI-32765
  • FIGURE 30 provides assay results and BTK occupancy, free BTK and standard curve for patient #2 with Method 2.
  • Patient was administered inhibitor (ibrutinib; PCI-32765) 420 mg daily dose and BTK target occupancy was determined on Day 1 (vehicle), Day 8, Day 15 and Day 29.
  • inhibitor ibrutinib; PCI-32765
  • FIGURE 31 provides assay results and BTK occupancy, free BTK and standard curve for patient #3 with Method 2.
  • Patient was administered inhibitor (ibrutinib; PCI-32765) 420 mg daily dose and BTK target occupancy determined on Day 1 (vehicle), Day 2, Day 8, Day 15 and Day 29.
  • inhibitor ibrutinib; PCI-32765
  • FIGURE 32 provides assay results and BTK occupancy, free BTK and standard curve for patient #4 with Method 2.
  • Patient was administered inhibitor (ibrutinib; PCI-32765) 420 mg daily dose and BTK target occupancy determined on Day 1 (vehicle), Day 2, Day 8, Day 15, Day 29 and at the end of the study.
  • inhibitor ibrutinib; PCI-32765
  • FIGURE 33 depicts the steps of Method 3. Lysates are screened for BTK occupancy and the supernatants transferred for separate analysis to determine total BTK in the recovered supernatants using anti-BTK pre-coated plates.
  • FIGURE 34 provides assay results and BTK occupancy, free BTK and standard curve for patient #1 with Method 3.
  • Patient was administered inhibitor (ibrutinib; PCI-32765) 420 mg daily dose and BTK target occupancy determined on Day 1 (vehicle), Day 2, Day 8, Day 15, Day 29 and at the end of the study.
  • inhibitor ibrutinib; PCI-32765
  • FIGURE 35 provides assay results and BTK occupancy, free BTK and standard curve for patient #2 with Method 3.
  • Patient was administered inhibitor (ibrutinib; PCI-32765) 420 mg daily dose and BTK target occupancy determined on Day 1 (vehicle), Day 8, Day 15and Day 29.
  • FIGURE 36 provides assay results and BTK occupancy, free BTK and standard curve for patient #3 with Method 3.
  • Patient was administered inhibitor (ibrutinib; PCI-32765) 420 mg daily dose and BTK target occupancy determined on Day 1 (vehicle), Day 2, Day 8, Day 15 and Day 29.
  • FIGURE 37 provides assay results and BTK occupancy, free BTK and standard curve for patient #4 with Method 3.
  • Patient was administered inhibitor (ibrutinib; PCI-32765) 420 mg daily dose and BTK target occupancy determined on Day 1 (vehicle), Day 2, Day 8, Day 15, Day
  • FIGURE 38 provides a comparison of patient results across Methods 1, 2, and 3 and compares same to BTK occupancy evaluated in a separate study with patients administered an 820 mg ibrutinib dose and where occupancy was determined by relative amount of free unbound BTK in a time course study.
  • BTK Bruton's tyrosine kinase
  • Btk a nonreceptor tyrosine kinase, which is a member of the Tec family of non-receptor tyrosine kinases.
  • BTK plays a role in the B-cell signaling pathway, particularly linking cell surface B-cell receptor (BCR) stimulation to downstream intracellular responses.
  • Tec family kinases are a family of related kinases which are non-receptor tyrosine kinases characterized by a pleckstrin homology (PH) domain and a proline rich region.
  • Kinases that are recognized members of the Tec family include particularly BTK (Bruton's tyrosine kinase), BMX (BMX non-receptor tyrosine kinase), ITK (also denoted EMT, TSK; IL-2 inducible T cell kinase), TEC (tyrosine protein kinase) and TXK (also denoted RLK; tyrosine protein kinase).
  • BTK Brun's tyrosine kinase
  • BMX BMX non-receptor tyrosine kinase
  • ITK also denoted EMT, TSK; IL-2 inducible T cell kinase
  • TEC tyros
  • An "irreversible binder” refers to a ligand or binder which associates with a target or receptor includes wherein noncovalent binding is so tight that the ligand or binder does not dissociate for a very long period of time (sometimes days). In such cases, the association is effectively irreversible and does not reach equilibrium (or equal binding and dissociation) within the relevant time frame.
  • An "irreversible inhibitor” is an inhibitor that binds to an enzyme or target, usually reacting with the enzyme and causing chemical changes to the active sites of enzymes.
  • An irreversible inhibitor's effects cannot be reversed.
  • a main role of irreversible inhibitors includes modifying key amino acid residues needed for enzymatic activity.
  • Irreversible inhibitors are covalently or noncovalently bound to the target enzyme and dissociate very slowly from the enzyme. Irreversible inhibitors often form strong covalent bonds with an enzyme or target. These inhibitors may act at, near, or remote from the active site and they may not be displaced by the addition of excess substrate.
  • Irreversible inhibitors can modify the basic structure of the enzyme/target such that the enzyme activity is impaired or the enzyme ceases to work.
  • a "covalent inhibitor” refers to an inhibitor that directly and covalently binds to its target or enzyme.
  • a covalent inhibitor is an inhibitor that acts via a covalent mechanism of action.
  • a covalent inhibitor achieves a sustained duration of inhibition.
  • a "reversible binder” In contrast to an irreversible or covalent binder, a "reversible binder” binds through a non-covalent binding process, wherein the ligand can both bind to and dissociate from the receptor. Equilibrium is reached when the time following mixing the binder and receptor or target to which it binds is long compared to the time for 50% binding and dissociation.
  • a "reversible inhibitor” Reversible inhibitors can bind to enzymes through weak non-covalent interactions such as ionic bonds, hydrophobic interactions, and hydrogen bonds. Because reversible inhibitors do not form any chemical bonds or reactions with the enzyme, they are formed rapidly and can be easily removed; thus the enzyme and inhibitor complex is rapidly dissociated in contrast to irreversible inhibition.
  • target occupancy relates to what percentage of target, such as enzymes, receptors, ion channels, transporters are occupied by a drug, ligand, binder etc.
  • target occupancy it is necessary to relate (i) the amount of a target that is interacting with or bound by a target binder, including a drug, ligand, inhibitor to (ii) the total amount of a target in a given milieu, sample, physiological system.
  • capture moiety refers to a part or group of a molecule that is capable of being specifically bound by another molecule or entity, particularly wherein the another molecule or entity is attached or linked to a solid support.
  • useful capture moieties include affinity labels for which specific and selective ligands are available (e.g., biotin with avidin or streptavidin, glutathione with GST), haptens and proteins for which antisera or monoclonal antibodies are available, nucleic acid molecules with a sequence complementary to a target, and peptides for which specific and selective ligands are available (e.g., histidine tag with Ni).
  • the solid support can be, for example, a filter, a plate, a membrane, a chromatographic resin, or a bead.
  • the term "specific binding member” refers to a member of a pair of molecules which have binding specificity for one another.
  • the members of a specific binding pair may be naturally derived or wholly or partially synthetically produced.
  • types of specific binding pairs are antigen-antibody, hormone-hormone receptor, receptor-ligand, enzyme-substrate. This application is particularly concerned with antigen-antibody type binding pairs.
  • a specific binding pair is an antibody, binding fragment thereof, or a ligand that binds a molecule without altering the molecule.
  • an “antibody” is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope.
  • the term “antibody” describes an immunoglobulin whether natural or partly or wholly synthetically produced.
  • the term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain.
  • CDR grafted antibodies are also contemplated by this term.
  • the term encompasses polyclonal, monoclonal, and chimeric antibodies, the last mentioned are described in further detail in U.S. Patent Nos. 4,816,397 and 4,816,567.
  • antibody(ies) includes a wild type immunoglobulin (Ig) molecule, generally comprising four full length polypeptide chains, two heavy (H) chains and two light (L) chains, or an equivalent Ig homologue thereof (e.g., a camelid nanobody, which comprises only a heavy chain); including full length functional mutants, variants, or derivatives thereof, which retain the essential epitope binding features of an Ig molecule, and including dual specific, bispecific, multispecific, and dual variable domain antibodies; Immunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2). Also included within the meaning of the term “antibody” are any "antibody fragment".
  • an "antibody fragment” means a molecule comprising at least one polypeptide chain that is not full length, including (i) a Fab fragment, which is a monovalent fragment consisting of the variable light (VL), variable heavy (VH), constant light (CL) and constant heavy 1 (CHI) domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a heavy chain portion of an Fab (Fd) fragment, which consists of the VH and CHI domains; (iv) a variable fragment (Fv) fragment, which consists of the VL and VH domains of a single arm of an antibody, (v) a domain antibody
  • CDR Single Chain Fv Fragment wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site
  • a diabody which is a bivalent, bispecific antibody in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementarity domains of another chain and creating two antigen binding sites (WO94/13804; P. Holliger et al
  • a minibody which is a bivalent molecule comprised of scFv fused to constant immunoglobulin domains, CH3 or CH4, wherein the constant CH3 or CH4 domains serve as dimerization domains (Olafsen T et al (2004) Prot Eng Des Sel 17(4):315-323; Hollinger P and
  • antibody should be construed as covering any specific binding member or substance having a binding domain with the required specificity.
  • this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023 and U.S. Patent Nos. 4,816,397 and 4,816,567.
  • an "antibody combining site” is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen.
  • antibody molecule in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule.
  • Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope, including those portions known in the art as Fab, Fab', F(ab') 2 and F(v), which portions are preferred for use in the therapeutic methods described herein.
  • Fab and F(ab') 2 portions of antibody molecules are prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibody molecules by methods that are well-known. See for example, U. S. Patent No.
  • Fab' antibody molecule portions are also well-known and are produced from F(ab') 2 portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide.
  • a reagent such as iodoacetamide.
  • the phrase "monoclonal antibody” in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular antigen.
  • a monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts.
  • a monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen; e.g., a bispecific (chimeric) monoclonal antibody.
  • alkyl by itself or as part of another molecule means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof.
  • the alkyl chain is fully saturated, mono- or polyunsaturated.
  • the alkyl chain includes di- and multivalent radicals, having the number of carbon atoms designated (i.e. Ci-Cio means one to ten carbons).
  • examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n- propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.
  • An unsaturated alkyl group is one having one or more double bonds or triple bonds.
  • examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers.
  • alkyl unless otherwise noted, includes those derivatives of alkyl defined in more detail herein, such as “heteroalkyl", “haloalkyl” and "homoalkyl”.
  • carbonyl refers to a group containing a moiety selected from the group consisting of -C(O)-, -S(O)-, -S(0) 2 -, and -C(S)-, including, but not limited to, groups containing a least one ketone group, and/or at least one aldehyde group, and/or at least one ester group, and/or at least one carboxylic acid group, and/or at least one thioester group.
  • Such carbonyl groups include ketones, aldehydes, carboxylic acids, esters, and thioesters. In some embodiments, such groups are a part of linear, branched, or cyclic molecules.
  • chemiluminescent group refers to a group which emits light as a result of a chemical reaction without the addition of heat.
  • luminol 5-amino-2,3-dihydro-l,4-phthalazinedione
  • oxidants like hydrogen peroxide ( ⁇ 2 0 2 ) in the presence of a base and a metal catalyst to produce an excited state product (3- aminophthalate, 3-APA).
  • chromophore refers to a molecule which absorbs light of visible wavelengths, UV wavelengths or IR wavelengths.
  • detectable label refers to a label which is observable using analytical techniques including, but not limited to, fluorescence, chemiluminescence, electron- spin resonance, ultraviolet/visible absorbance spectroscopy, mass spectrometry, nuclear magnetic resonance, magnetic resonance, and electrochemical methods.
  • die refers to a soluble, coloring substance which contains a chromophore.
  • fluorophore refers to a molecule which upon excitation emits photons and is thereby fluorescent.
  • label refers to a substance which is incorporated into a compound and is readily detected, whereby its physical distribution is detected and/or monitored.
  • subject or "patient” as used herein, refers to an animal which is the object of treatment, observation or experiment.
  • the subject or patient is a mammal including, but not limited to, a human.
  • probe refers to a compound or molecule for the detection of a target.
  • the probe may comprise an agent, a linker, a label, or any combination thereof.
  • the probe may comprise an agent and a linker, or an agent and a label, or a label.
  • the probe comprises an agent and a label.
  • substituted substituents also referred to as “non-interfering substituents” refers to groups which are used to replace another group on a molecule.
  • groups include, but are not limited to, halo, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C1-C10 alkoxy, C5-C12 aralkyl, C3-C12 cycloalkyl, C4-C12 cycloalkenyl, phenyl, substituted phenyl, toluolyl, xylenyl, biphenyl, C2-C12 alkoxyalkyl, C5-C12 alkoxyaryl, C5-C12 aryloxyalkyl, C7-C12 oxyaryl, Ci-C 6 alkyl sulfinyl, C1-C10 alkylsulfonyl, -(CH 2 ) m
  • each R group in the preceding list includes, but is not limited to, H, alkyl or substituted alkyl, aryl or substituted aryl, or alkaryl.
  • substituent groups are specified by their conventional chemical formulas, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left; for example, -CH 2 0- is equivalent to -OCH 2 -.
  • each R group in the preceding list includes, but is not limited to, hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, including but not limited to, aryl substituted with 1- 3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or aralkyl groups.
  • -NR 2 includes, but is not be limited to, 1-pyrrolidinyl and 4-morpholinyl.
  • the assay is performed on a sample obtained from a subject that has been administered a TEC family kinase inhibitor, particularly a BTK kinase inhibitor.
  • the sample is obtained about 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 3 days, 4, days, 5 days, 6 days, 1 week, 2 weeks or longer after administration of the TEC family kinase inhibitor or the BTK inhibitor.
  • a "replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.
  • a "vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • a "DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double- stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes.
  • linear DNA molecules e.g., restriction fragments
  • viruses e.g., plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
  • An "origin of replication" refers to those DNA sequences that participate in DNA synthesis.
  • a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus.
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences.
  • a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • a "promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site
  • Eukaryotic promoters (consensus sequences) responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain "TATA” boxes and “CAT” boxes.
  • Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.
  • An "expression control sequence” is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence.
  • a coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.
  • oligonucleotide as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.
  • primer refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH.
  • the primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent.
  • the exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method.
  • the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
  • the primers herein are selected to be “substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product. [000100] As used herein, the terms “restriction endonucleases” and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
  • a cell has been "transformed” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • the transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the transforming DNA may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA.
  • a "clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a "cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • Two DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al, supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.
  • Two amino acid sequences are "substantially homologous" when at least about 70% of the amino acid residues (preferably at least about 80%, and most preferably at least about 90 or 95%)) are identical, or represent conservative substitutions.
  • a "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature.
  • the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism.
  • Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
  • phrases “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • standard hybridization conditions refers to salt and temperature conditions substantially equivalent to 5 x SSC and 65°C for both hybridization and wash.
  • standard hybridization conditions are dependent on particular conditions including the concentration of sodium and magnesium in the buffer, nucleotide sequence length and concentration, percent mismatch, percent formamide, and the like.
  • Also important in the determination of “standard hybridization conditions” is whether the two sequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA.
  • standard hybridization conditions are easily determined by one skilled in the art according to well known formulae, wherein hybridization is typically 10-20 N C below the predicted or determined T m with washes of higher stringency, if desired.
  • pg means picogram
  • ng means nanogram
  • ug means nanogram
  • ug means nanogram
  • ug means nanogram
  • ug means nanogram
  • ug means nanogram
  • ug means nanogram
  • ug means nanogram
  • ug means nanogram
  • ug means nanogram
  • ug means nanogram
  • ug means nanogram
  • ug means nanogram
  • ug means nanogram
  • ug means nanogram
  • ug means microgram
  • mg means milligram
  • ul or “ ⁇ ” mean microliter
  • ml means milliliter
  • 1 means liter.
  • Target occupancy can help assure proper dosing and targeting of compounds in preclinical and clinical drug development as well as in basic research.
  • Target occupancy generalizations can be especially important in establishing initial dosing recommendations for the many new drug targets provided by genomic and proteomic initiatives, where little data is available on their functional responses.
  • PET Positron Emission Tomography
  • SPECT Single Photon Emission Computed Tomography
  • liquid scintillation spectroscopy which have relied on the use of radioisotopically labeled tracers, have also been utilized to drive the analysis of structure-activity-relationship within a drug-discovery effort.
  • BTK activity probes comprising a BTK inhibitor moiety, a reporter moiety, and a linker moiety linking the inhibitor moiety to the reporter moiety.
  • BTK inhibitors have been evaluated using signal based relative determinations wherein more or less probe is bound or detected, with relative amounts of probe occupied target, unoccupied target or drug occupied target altering the overall amount of signal in a relative fashion, but not using quantitative determination.
  • Chang et al WO2014/059368 describes Tec family drug efficacy or occupancy determinations based on increasing or decreasing signal intensity.
  • quantitative or normalized target occupancy is not determined in a single, comparable format.
  • the methods and assay systems of the present invention enable quantitative determination of total target and target occupancy in a single and simple format, replacing relative occupancy determinations and also costly and time consuming tests and formats, particularly multiple formats which cannot be cross-compared.
  • the present methods provide simple and standardized alternatives to cumbersome methods that have, for example, utilized sandwich assays (such as ELISA) combined with Western blotting and manual quantification (Pan, Z et al (2007) Chem Med Chem 2:58-61; De Rooji, MF et al (2012) Blood 119:2590-2594; Honigberg, LA et al ((2007) Blood 110:Abstract 1592).
  • the present invention relates to a novel method and assay system for determining both the amount of a given target and the relative amount of ligand bound to target, or target occupied by a ligand.
  • each determination is conducted on a sample utilizing a same or comparable format, wherein standard curves and standardized reagents are utilized in both determinations, and occupied target can be directly normalized to quantitated target.
  • Utilizing the same or comparable format permits direct comparison of the results and quantitative determinations and avoids application of multiple techniques or formats and extrapolation or assumptions across distinct formats.
  • the assay is completed using a single format and assay basis.
  • target occupancy and total target are evaluated and determined in a single comparable format.
  • the basis for the single and comparable format is utilization of a same capture moiety on both the target occupancy probe and on the anti-target specific binding member. Utilization of the same capture moiety enables cross comparison and same format reagents.
  • the invention provides a method or assay to determine target occupancy and quantitate total target of a kinase, particularly a non-receptor tyrosine kinase, particularly a Tec family kinase, wherein target occupancy is determined utilizing a target occupancy probe and total target is quantitated using an anti-target specific binding member, wherein the target occupancy probe and anti-target specific binding member are tagged with a moiety for capture by the same capture moiety binder.
  • a target containing sample is obtained and split or aliquoted for each of (a) target occupancy determination and (b) total target determination.
  • each of (a) target occupancy determination and (b) total target determination comprising the steps of: (i) contacting a sample with target occupancy probe in (a) and anti-target specific binding member in (b), each tagged with a same capture moiety or a capture moiety that binds to the same capture binder, to form an occupancy probe-target complex in (a) and a specific binding member-target complex in (b); (ii) capturing the complex from (a) and (b) with a capture moiety binder coated solid support; and (iii) detecting the presence and amount of captured occupancy probe-target complex in (a) and specific binding member-target complex in (b).
  • the determined amount of occupancy probe-target complex can be normalized to the amount of specific binding member-target complex to provide quantitative determination of target occupancy.
  • the determined target occupancy amount can be a direct occupancy value, for example wherein the target inhibitor being evaluated is used as the target occupancy probe and is directly labeled with the capture moiety.
  • the determined occupancy amount can be an indirect occupancy value, for example wherein a target inhibitor to be evaluated is combined with target prior to step (i) and then a sample is contacted with target occupancy probe, wherein the amount of captured occupancy probe-target complex indicates the remaining target which is unoccupied by the target inhibitor being evaluated.
  • target occupancy of the target inhibitor being evaluated is the inverse of the amount of captured occupancy probe-target complex, for example the amount of target that is not captured corresponds to the target occupancy of the inhibitor being evaluated.
  • target site specific target occupancy probe allows for the screening, identification, and comparison of alternative potential or lead target site specific inhibitors.
  • the availability of a common format and quantitative target occupancy evaluations permits the comparison of candidate target site specific inhibitors using a standardized and quantitative system.
  • the invention provides a method or assay to determine target occupancy and quantitate total target of a kinase, particularly a non-receptor tyrosine kinase, particularly a Tec family kinase, comprising:
  • target is quantified by comparison to a standard curve generated using recombinant target protein, wherein the standard curve is obtained simultaneously or approximately the same time as the samples are analyzed.
  • the primary anti-target specific binding member is an antibody, particularly a labeled antibody.
  • the target complex bound solid support is contacted with a secondary tagged or labeled antibody that recognizes or binds the primary anti-target specific binding member for detection.
  • the solid support is washed between steps (iii) and (iv).
  • the solid support is washed in (iv) after primary anti-target specific binding member is contacted and prior to contacting with a secondary tagged or labeled antibody that recognizes or binds the primary anti-target specific binding member.
  • the target occupancy probe and anti-target specific binding member are directly captured to a solid support, for example by direct binding to a plate, filter, or bead.
  • the plate, filter, or bead is preferably coated or pre-coated with capture binder.
  • the plate, filter, or bead is coated or pre-coated with streptavidin.
  • the bead may be a magnetic bead.
  • the methods, kits, and compositions disclosed herein comprise a solid support.
  • a solid support comprises any solid platform to which a probe or antibody can be attached.
  • a solid support may comprise a bead, plate, an array or a bead attached to a plate. Examples of plates include, but are not limited to, MSD multi-array plates, MSD Multi-Spot® plates, microplate, ProteOn microplate, AlphaPlate, DELFIA plate, IsoPlate, and LumaPlate.
  • beads examples include streptavidin beads, agarose beads, magnetic beads, Dynabeads®, MACS® microbeads, antibody conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A or G conjugated beads, protein L conjugated beads, oligo- dT conjugated beads, silica beads, silica-like beads, anti-biotin microbead, anti-fluorochrome microbead, and BcMagTM CarboxyTerminated Magnetic Beads.
  • Suitable samples for any of the methods and assays provided herein comprise, but are not limited to, a whole blood sample, peripheral blood sample, lymph sample, tissue sample, tumor biopsy sample, bone marrow sample, or other bodily fluid sample.
  • the sample may contain one or more cell types, or a lysate thereof, derived from a whole blood sample, peripheral blood sample, peripheral blood mononuclear cell (PBMC) sample, lymph sample, tissue sample, tumor biopsy sample, bone marrow sample, or other bodily fluid sample.
  • PBMC peripheral blood mononuclear cell
  • bodily fluids include, but are not limited to, smears, sputum, biopsies, secretions, cerebrospinal fluid, bile, blood, lymph fluid, saliva, and urine.
  • Cells of the sample may be isolated from other components of the sample prior to use in the methods provided. Particular cell types of the sample may be isolated from other cell types of the sample prior to use in the methods provided.
  • peripheral blood mononuclear cells PBMCs, e.g., lymphocytes, monocytes and macrophages
  • lymphocytes e.g., B cells, T cells or K cells
  • B cells of the sample may be isolated from other cell types of the sample prior to use in the methods provided.
  • Cells of the sample may be lysed prior to use in the methods provided.
  • cancer cells may be isolated from normal cells of the sample prior to use in the methods provided.
  • a sample may comprise complex populations of cells, which can be assayed as a population, or separated into sub-populations. Such cellular and acellular samples can be separated by centrifugation, elutriation, density gradient separation, apheresis, affinity selection, panning, FACS, filtration, centrifugation with
  • Hypaque using antibodies specific for markers identified with particular cell types.
  • a heterogeneous cell population can be used. Once a sample is obtained, it can be used directly, frozen, or maintained in appropriate culture medium for short periods of time. Methods to isolate one or more cells for use according to the methods of this invention are performed according to standard techniques and protocols well-established in the art.
  • the target is an enzyme, particularly a kinase, particularly an EGFR or non-EGFR kinase.
  • the target is particularly a non-EGFR kinase and may be a member of the Tec family of kinases (BMX, BTK (Btk), ITK, TEC and TXK), Scr family member BLK, MKKa7 kinase, or a JAK3 kinase.
  • target is a Tec family kinase and is BMX, BTK, ITK, TEC or TXK.
  • target is BTK.
  • Target inhibitor is particularly a specific inhibitor, particularly inhibiting a kinase family, such as particularly a Tec family kinase inhibitor, and may be a non specific Tec family inhibitor, a preferential inhibitor against one or more member of the Tec family of kinases or may be a specific kinase inhibitor.
  • the target inhibitor may be a BMX specific inhibitor, a BTK specific inhibitor, an ITK specific inhibitor, a TEC specific inhibitor or a TXK specific inhibitor.
  • the target inhibitor is particularly a BTK inhibitor.
  • the target inhibitor is particularly a covalent inhibitor.
  • the target inhibitor is particularly an irreversible inhibitor.
  • the target inhibitor and/or target occupancy probe is directed against a cysteine (Cys) conserved in the ATP -binding site of the target kinase.
  • the target inhibitor is directed against Cys481 in BTK, or the comparable Cys in one or more of the Tec family kinases.
  • the target inhibitor is utilized as target occupancy probe.
  • the target inhibitor or target occupancy probe is a Tec family kinase inhibitor.
  • the target inhibitor or target occupancy probe is a Tec family kinase inhibitor, and is capable of inhibiting one or more of BMX, BTK, ITK, TEC and TXK.
  • the target inhibitor or target occupancy probe is a BTK inhibitor.
  • the target inhibitor or target occupancy probe is a BMX inhibitor.
  • the target inhibitor or target occupancy probe is an ITK inhibitor.
  • the target inhibitor or target occupancy probe is a TEC inhibitor.
  • the target inhibitor or target occupancy probe is a TXK inhibitor.
  • BTK inhibitors particularly irreversible and covalent inhibitors are in development and being evaluated clinically for various indications, including chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), and other relapsed or refractory B cell lymphomas (D'Cruz OJ and Uckun, FM (2013) OncoTargets Ther 6: 161-176).
  • ibrutinib/PCI- 32765 (l-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperidin-l- yl]prop-2-en-l-one), which is an orally active, small-molecule inhibitor that forms an irreversible bond with cysteine-481 in the active site of BTK and inhibits BTK phosphorylation on Tyr223 (Honigsberg LA et al (2010) PNAS 107(29): 13075-13080;).
  • BTK inhibitors include AVL-101 and AVL-294 (Avila Therapeutics), Dasatinib/Sprycel or Dasatinib plus fludarabine, LFM-A13, ONO-WG-307, GDC-0834 (D'Cruz OJ and Uckun FM (2013) OncoTargets and Therapy 6: 161-176).
  • the target occupancy probe is an irreversible inhibitor and is a Tec family inhibitor.
  • the target occupancy probe is a BTK inhibitor.
  • the target occupancy probe may comprise an inhibitor portion or moiety and a reporter or capture moiety, wherein the reporter or capture moiety may particularly correspond to the capture moiety.
  • Tec kinase family activity probes, particularly BTK activity probes which may be suitable as components of the target occupancy probe are described in US2008214501 and in WO2008054827, incorporated herein by reference.
  • the target occupancy probe comprises a probe of Formula (I) comprising:
  • A is a Tec family inhibitor moiety, particularly a Btk inhibitor moiety
  • B is absent or is a linker moiety
  • C is a reporter or capture moiety
  • R a is hydrogen or alkyl.
  • the B linker moiety is absent and the reporter or capture moiety may be directly linked to the inhibitor moiety.
  • the C is a capture moiety.
  • the moiety comprising an irreversible inhibitor is derived from an irreversible inhibitor of Btk.
  • such irreversible inhibitors of Btk should possess at least one of the following characteristics: potency, selectively and cell permeability.
  • such irreversible inhibitors of Btk possess at least two of the aforementioned characteristics, and in further embodiments, at least all of the aforementioned characteristics.
  • the target occupancy probe comprises a Btk inhibitor moiety is derived from a Btk inhibitor havin the structure of Formula (II):
  • L a is CH 2 , O, NH or S
  • Ar is a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl
  • Y is an optionally substituted group selected from among alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.
  • L a is CH 2 , O, or NH. In other embodiments, L a is O or NH. In yet other embodiments, L a is O.
  • Ar is a substituted or unsubstituted aryl. In yet other embodiments, Ar is a 6-membered aryl or heteroaryl. In some other embodiments, Ar is phenyl.
  • Y is an optionally substituted group selected from among alkyl, heteroalkyl, cycloalkyl, and heterocycloalkyl. In other embodiments, Y is an optionally substituted group selected from among Ci-Cealkyl, Ci-Ceheteroalkyl, 4-, 5-, 6-, or 7-membered cycloalkyl, and 4-, 5-, 6-, or 7-membered heterocycloalkyl.
  • Y is an optionally substituted group selected from among Ci-Cealkyl, Ci-Ceheteroalkyl, 5- or 6- membered cycloalkyl, and 5- or 6-membered heterocycloalkyl containing 1 or 2 N atoms.
  • Y is a 5- or 6-membered cycloalkyl, or a 5- or 6-membered heterocycloalkyl containing 1 or 2 N atoms.
  • Y is a 4-, 5-, 6-, or 7- memebered cycloalkyl ring; or Y is a 4-, 5-, 6-, or 7-membered heterocycloalkyl ring.
  • Y is pyrrolidinyl, piperdinyl, or cyclohexyl.
  • the Btk inhibitor moiety is derived from a compound selected from among : 1 -(3 -(4-amino-3 -(4-phenoxyphenyl)- 1 H-pyrazolo [3 ,4-d]pyrimidin- 1 -yl)piperidin- 1 - yl)prop-2-en- 1 -one; (E)- 1 -(3 -(4-amino-3 -(4-phenoxyphenyl)- 1 H-pyrazolo [3 ,4-d]pyrimidin- 1 - yl)piperidin- 1 -yl)but-2-en- 1 -one; 1 -(3 -(4-amino-3 -(4-phenoxyphenyl)- lH-pyrazolo[3 ,4- d]pyrimidin- 1 -yl)piperidin- 1 -yl)sulfonylethene; 1 -(3 -
  • the linker moiety is selected from a bond, a polymer, a water soluble polymer, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted heterocycloalkylalkenyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocycloalkylalkenylalkyl.
  • the linker moiety is an optionally substituted heterocycle.
  • the heterocycle is selected from aziridine, oxirane, episulfide, azetidine, oxetane, pyrroline, tetrahydrofuran, tetrahydrothiophene, pyrrolidine, pyrazole, pyrrole, imidazole, triazole, tetrazole, oxazole, isoxazole, oxirene, thiazole, isothiazole, dithiolane, furan, thiophene, piperidine, tetrahydropyran, thiane, pyridine, pyran, thiapyrane, pyridazine, pyrimidine, pyrazine, piperazine, oxazine, thiazine, dithiane, and dioxane.
  • the heterocycle is piperazine.
  • the linker moiety is optionally substituted with halogen, CN, OH, N0 2 , alkyl, S(O), and S(0) 2 .
  • the water soluble polymer is a PEG group.
  • the target occupancy probe BTK inhibitor moiety is ibrutinib and has the formula l-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)pyrazolo[3,4-d]pyrimidin- l-yl]piperi in-l-yl]prop-2-en-l-one). Ibrutinib structure is as follows:
  • the target occupancy probe is ibrutinib linked to biotin.
  • the target occupancy probe comprises the following structure:
  • the target occupancy probe and anti-target specific binding member are each tagged with a same capture moiety or a capture moiety that binds to the same capture binder.
  • the capture moiety may be captured by binding or interacting directly or indirectly to a solid support.
  • the target probe and anti-target specific binding member are captured directly to a solid support, such as a plate, filter, or a bead.
  • the plate, filter, or bead may preferably be coated with the capture binder. Binding and affinity between the capture moiety and capture binder should be significant and stable, in order that capture can be efficient and sufficiently quantitative.
  • Small capture moieties are preferred, particularly wherein the capture moieties do not interfere or minimally interfere with the target inhibitor and/or anti-target specific binding member binding to the target.
  • the capture moiety needs to be suitable and applicable for attachment to small molecules, such as the inhibitor moieties described herein.
  • the capture moiety need be readily attachable, directly or via a linker, to such small molecules as ibrutinib, without interfering with or altering ibrutinib binding or association with target, e.g. BTK.
  • the capture moiety is biotin.
  • the capture binder is a biotin binder and may be avidin, streptavidin, or neutravidin.
  • Biotin is a vitamin (Vitamin H, Vitamin B7, Coenzyme R) that is present in small amounts in all living cells and is critical for a number of biological processes including cell growth and the citric acid cycle. Because biotin is relatively small, it can be conjugated to many proteins and other molecules without significantly altering their biological activity. The highly specific interaction of biotin-binding proteins with biotin make it a useful tool in assay systems designed to detect and target biological analytes. Many proteins, such as antibodies, can be labeled with several biotin tags, each able to be bound by a biotin-binding protein. An optimized biotin-to-probe ratio can greatly increase the signal output of a detection system making it possible to create very sensitive assays.
  • the bond formation between biotin and avidin is very rapid, and once formed, is unaffected by extremes of pH, temperature, organic solvents and other denaturing agents.
  • These features of biotin and avidin are useful for purifying or detecting proteins conjugated to either component of the interaction.
  • Streptavidin is a tetrameric biotin-binding protein that is isolated from Streptomyces avidinii and has a mass of 60,000 Daltons.
  • An alternative to streptavidin includes deglycosylated avidin, commercially available as NeutrAvidin (Thermo-Scientific).
  • Suitable anti-target specific binding members applicable in the instant methods and assays include antibodies and fragments thereof that bind to the target.
  • an anti-target specific binding member is an antibody or binding fragment thereof specifically directed against the target, and which does not react with or bind other targets, including related family targets, (e.g., having a binding affinity 3 ⁇ 4 > 1 ⁇ or > 10 ⁇ , or at least 100 fold or 1000 fold more binding affinity with the target that it specifically binds).
  • the anti-target specific binding member is an anti-BTK antibody that is specific for BTK, particularly human BTK, and that does not or does not significantly recognize or bind another Tec family member or kinase (e.g., having a binding affinity 3 ⁇ 4 > 1 ⁇ or > 10 ⁇ , or at least 100 fold or 1000 fold more binding affinity with BTK).
  • the anti-target specific binding member is an antibody specifically directed against a Tec kinase family member which is not BTK. In another embodiment of the invention the anti-target specific binding member is an antibody specifically directed against Tec kinase. In a further embodiment of the invention the anti-target specific binding member is an antibody specifically directed against ITK kinase. In another aspect of the invention the anti-target specific binding member is an antibody specifically directed against BMX kinase. In another embodiment of the invention the anti-target specific binding member is an antibody specifically directed against TXK kinase.
  • a monoclonal anti-BTK antibody is utilized in determining total target BTK.
  • the anti-BTK may be a commercially available BTK antibody, particularly a monoclonal antibody recognizing human BTK and not cross reacting with Tec kinase or other Tec kinase family members.
  • anti-BTK antibodies for use in the methods of the invention include anti-BTK monoclonal antibody (BD Bioscience Cat No 611117, N-term rec protein 2-172 aa), rabbit anti-BTK monoclonal antibody (Abeam Cat No. 32555, C-term peptide), anti-BTK rabbit mAb (CST Cat No. 12624S, N-term peptide).
  • Preferred anti-BTK antibody is monoclonal anti- BTK antibody.
  • Preferred anti-BTK antibody is biotinylated anti-BTK rabbit mAb (Cell Signal Technology (CST) Cat No. 12624S, N-term peptide).
  • CST Cell Signal Technology
  • a new or novel BTK antibody including monoclonal BTK antibody, may be generated utilizing methods and approaches well known and available to one of skill in the art.
  • the antibody or fragment thereof can be tagged directly or indirectly with the capture moiety.
  • the antibody or fragment is directly tagged with the capture moiety.
  • Antibodies tagged with capture moieties may be commercially available and utilized. As provided herein, for example, biotin labeled anti-BTK antibody for use in the invention may be commercially obtained.
  • Panels of monoclonal antibodies produced against target peptides, including Tec family member kinase peptides can be screened for various properties; i.e., isotype, epitope, affinity, etc. High affinity antibodies are also particularly useful.
  • the anti-target antibody used in the diagnostic methods of this invention is an affinity purified polyclonal antibody.
  • the antibody is a monoclonal antibody (mAb).
  • mAb monoclonal antibody
  • the anti-target antibody molecules used herein be in the form of Fab, Fab', F(ab') 2 or F(v) portions of whole antibody molecules.
  • a monoclonal antibody useful in practicing the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate antigen specificity.
  • the culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium.
  • the antibody-containing medium is then collected.
  • the antibody molecules can then be further isolated by well-known techniques.
  • Media useful for the preparation of these compositions are both well-known in the art and commercially available and include synthetic culture media, inbred mice and the like.
  • An exemplary synthetic medium is Dulbecco's minimal essential medium (DMEM) supplemented with 4.5 gm/1 glucose, 20 mm glutamine, and 20% fetal calf serum.
  • An exemplary inbred mouse strain is Balb/c.
  • Methods for producing monoclonal anti-target antibodies are also well-known in the art.
  • the present target or a peptide analog is used either alone or conjugated to an immunogenic carrier, as the immunogen in the before described procedure for producing anti- target monoclonal antibodies.
  • the hybridomas are screened for the ability to produce an antibody that immunoreacts with the target peptide and target kinase.
  • the presence of target in cells can be ascertained by the usual immunological procedures applicable to such determinations.
  • a number of useful procedures are known. Three such procedures which are especially useful utilize either the target labeled with a detectable label, antibody Abi labeled with a detectable label, or antibody Ab 2 labeled with a detectable label.
  • the procedures may be summarized by the following equations wherein the asterisk indicates that the particle is labeled, and " ⁇ " stands for the target:
  • Abi a characteristic property of Ab 2 is that it will react with Abi. This is because Abi raised in one mammalian species has been used in another species as an antigen to raise the antibody Ab 2 .
  • Ab 2 may be raised in goats using rabbit antibodies as antigens. Ab 2 therefore would be anti-rabbit antibody raised in goats.
  • Abi will be referred to as a primary or anti-target antibody, and Ab 2 will be referred to as a secondary or anti-Abi antibody.
  • Detection means and labels are employed in the methods and assays of the invention so as to detect the target complexes and may include radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others.
  • a number of fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow.
  • a particular detecting material is anti- rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.
  • the radioactive label can be detected by any of the currently available counting procedures.
  • the preferred isotope may be selected from 3 H, 14 C, 32 P, 35 S, 36 C1, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, and 186 Re.
  • Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques.
  • the enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized.
  • peroxidase B-glucuronidase, B-D-glucosidase, B-D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase.
  • labels include, but are not limited to, chemical, biochemical, biological, colorimetric, enzymatic, fluorescent, luminescent labels, chemiluminescent labels, and electrochemiluminescent labels.
  • the label may be a dye, a photocrosslinker, a cytotoxic compound, a drug, an affinity label, a photoaffmity label, a reactive compound, an antibody or antibody fragment, a biomaterial, a nanoparticle, a spin label, a fluorophore, a metal-containing moiety, a radioactive moiety, a novel functional group, a group that covalently or noncovalently interacts with other molecules, a photocaged moiety, an actinic radiation excitable moiety, a ligand, a photoisomerizable moiety, biotin, a biotin analogue, a moiety incorporating a heavy atom, a chemically cleavable group, a photocleavable group, a redox-active agent
  • the label may be a chemical label.
  • chemical labels can include, but are not limited to, biotin and radioisotopes (e.g., iodine, carbon, phosphate, hydrogen).
  • the methods, assays and kits disclosed herein comprise a biological label.
  • Biological labels comprise metabolic labels, including, but not limited to, bioorthogonal azide-modified amino acids, sugars, and other compounds.
  • Enzymatic labels can include, but are not limited to horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, and b-galactosidase.
  • the enzymatic label is luciferase.
  • a fluorescent label may be an organic dye (e.g., FITC), biological fluorophore (e.g., green fluorescent protein), or quantum dot.
  • FITC fluorescein isothiocyante
  • DyLight Fluors fluorescein, rhodamine (tetramethyl rhodamine isothiocyanate, TRITC), coumarin, Lucifer Yellow, and BODIPY.
  • the label is a fluorophore.
  • fluorophores include, but are not limited to, indocarbocyanine (C3), indodicarbocyanine (C5), Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Texas Red, Pacific Blue, Oregon Green 488, Alexa Fluor®-355, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor-555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, JOE, Lissamine, Rhodamine Green, BODIPY, fluorescein isothiocyanate (FITC), carboxy-fluorescein (FAM), phycoerythrin, rhodamine, dichlororhodamine (dRhodamine), carboxy tetramethylrhodamine (TAMRA), carboxy- Xrhodamine (ROXTM), LIZTM, VICTM, NEDTM, PETTM, S
  • the fluorescent label may be a green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein, phycobiliproteins (e.g., allophycocyanin, phycocyanin, phycoerythrin, and phycoerythrocyanin) .
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • phycobiliproteins e.g., allophycocyanin, phycocyanin, phycoerythrin, and phycoerythrocyanin
  • the method and assay determines the target occupancy of the Tec family kinase inhibitor in the target occupancy probe or the probe.
  • the method and assay determines the target occupancy of a compound other than the probe, such as an unlabeled compound that is suspected of occupying the target when in contact with the target.
  • the compound can be in contact with the target in the sample prior to the sample being in contact with the probe.
  • the compound may occupy the target after administration, and the occupancy can be determined by the methods and assays described herein.
  • the contacting of the target in the sample with the compound may occur after obtaining the sample by, e.g., adding the compound to the sample.
  • a compound may be added in vitro to a sample containing a recombinant kinase before the sample is contacted with the probe.
  • the target occupancy probe When subjecting such samples to the methods and assays described above, the target occupancy probe will bind to target sites not already bound by the compound and the target site occupancy of the compound is indirectly measured. With more compound occupying the target, less target occupancy probe is bound to target and captured and detected in the assay. In such an instance, target occupancy of the compound being assessed is inversely proportional to the amount of labeled target probe detected. Less target occupancy probe indicates that more compound is bound and occupying target.
  • a method or assay is provided to determine target occupancy by a compound suspected of occupying the target, and quantitate total target of a Tec family kinase target in a sample, comprising:
  • the capture moiety tagged target occupancy probe, capture moiety tagged anti-target specific binding member, capture moiety binder coated solid support, primary anti-target specific binding member, secondary tagged or labeled antibody, assay and determination methods, etc. are as described in details herein.
  • the compound suspected of occupying the target can be any compound of interest for the determination, such as compounds in a screening experiment for determining potential binding with one or more targets, compounds in in vivo experiments, or clinical uses for determining their target occupancy when administered to a subject.
  • the compound is a known BTK inhibitor, such as those described in US Patents 7,514,444, 7,732,454, 7,825, 118, 7,960,396, 8,236,812, 8,377,946, 8,501,724, 8,563,568, 8,883,435, 8,940,725, 9,073,947, 9, 133, 134, and 9, 138,436, US Patent Applications US2015/0094319, US2014/0107151, US2014/0144099, US2014/0155385, US2014/0155406, US2014/0221333, US2014/0221398, US/2014/0303161, US2014/0364410, US2015/0174128, and US US2015/0158865, and International Patent Applications WO2014/022390, WO2015/048689, WO2015/134210, WO2015/095099, WO2015/095102, and WO2015/002894, each and all incorporated herein by reference.
  • the compound is ibrutinib, ACP-196, AVL-101, AVL-294, BGB-3111, Dasatinib/Sprycel or Dasatinib plus fludarabine, LFM-A13, ONO-WG-307, or GDC-0834.
  • kits suitable for use by a medical specialist may be prepared to determine the quantity of target, quantity of available target, or target occupancy prior to, during, or after treatment with a kinase inhibitor, particularly an irreversible Tec kinase inhibitor, particularly an irreversible BTK inhibitor.
  • a kinase inhibitor particularly an irreversible Tec kinase inhibitor, particularly an irreversible BTK inhibitor.
  • kits will contain at least the tagged anti-target specific binding member, for instance an antibody specific thereto, and directions, of course, depending upon the method selected, e.g., ELISA and standard or MSD format, and the like.
  • the kits may also contain peripheral reagents such as buffers, stabilizers, etc.
  • test kit may be prepared for the demonstration of the quantity of target and target occupancy of inhibitor, particularly BTK and BTK inhibitor comprising:
  • BTK-occupancy probe such as biotinylated ibrutinib and a predetermined amount of biotinylated anti-BTK antibody
  • kits further comprises one or more other reagents such as buffers, stabilizers, etc., used in assays such as ELISA.
  • the MSD format assay was evaluated using 1 ⁇ g/ml capture Ab and 0.5 ⁇ g/ml capture Ab. The tabulated results and standard curves are shown in FIGURE 2. The assay was repeated using BTK protein from cell lysates and compared (FIGURE 3). The assay, however, failed to quantitate BTK protein in DoHH2 cell lysates.
  • Sandwich ELISA was next evaluated on both standard and MSD formats for total BTK determination. In this instance, coating Ab goat anti-rabbit IgG was eliminated and anti- BTK Rabbit or mouse antibody (Ab) was used as capture Ab.
  • the assay design is depicted in FIGURE 5. A series of commercially available BTK recombinant proteins were evaluated.
  • FIGURE 7 Briefly, 96 well high protein binding plates (Pierce/MSD) are utilized. Capture antibody at 2 ⁇ g/ml is coated onto the plates in PBS overnight at 4°C. The plates are then washed and blocked in 3%BSA (250 ⁇ ) for 1-2 hours at room temperature (RT). Either recombinant BTK (R-BTK) (Invitrogen) or cell lysates are added in 1%BSA (100 ⁇ ) and incubated overnight at 4°C. Primary anti-BTK antibody is added in 1%BSA (100 ⁇ ) for 2 hours at RT, followed by secondary antibody in 1% BSA (100 ⁇ ) for 1 hour at RT. Detection reagent
  • DoHH2 and Jurkat cells were evaluated, as was recombinant BTK in both a standard (STD) and meso-scale discovery (MSD) format.
  • BTK in the cell lysates peaked and flattened at a maximum luminescence.
  • the novel assay method is suitable for either standard (STD) or the Meso-Scale Discovery (MSD) formats.
  • STD standard
  • MSD Meso-Scale Discovery
  • the principles are depicted visually in FIGURE 12.
  • the use of both a coating antibody and a capture antibody were eliminated from the assay.
  • the sample is mixed with tagged anti-BTK antibody (for example biotinylated anti-BTK antibody) wherein the tagged BTK antibody is tagged with a capture moiety that facilitates direct attachment to the plates via the capture moiety (tag).
  • biotin is used for capture moiety and facilitates binding to streptavidin coated plates.
  • bound BTK - which is bound by virtue of direct association of the tagged or labeled anti- BTK antibody with the assay plate or surface - is detected.
  • Detection is accomplished for example by addition of another anti-BTK antibody, which recognizes and binds to the bound BTK on the assay plate or surface.
  • the detection antibody is mouse anti-BTK antibody.
  • Detection can be direct, wherein the detection antibody is directly labeled, or facilitated by a secondary antibody that recognizes the detection antibody, for example anti-mouse antibody with a tag or HRP or other direct label.
  • FIGURE 13 The experimental strategy for a method and assay system to quantitate total target and target occupancy is presented in FIGURE 13.
  • the strategy is shown for BTK, particularly for determining total BTK quantification in absolute amounts and BTK target occupancy data normalized to total BTK.
  • the strategy permits determination of total BTK and BTK target occupancy using a single simple system.
  • Biotin is utilized as a capture moiety on both the anti- BTK antibody used for total BTK quantification, and on the target occupancy probe used to determine BTK target occupancy.
  • Total BTK and BTK target occupancy probe-target complexes are captured using streptavidin plates and then detection proceeds, for example using streptavidin HRP or tag, for example using labeled detection antibody.
  • BTK target occupancy was determined in the assay ELISA using standard format.
  • the biotinylated probe used in this study for determining BTK occupancy is biotinylated irreversible BTK inhibitor ibrutinib/41025.
  • BTK occupancy is evaluated in cell lysates using biotin-ibrutinib as target occupancy probe (DoHH2 cells and BTK negative Jurkat cells as control) and also with recombinant BTK (Rec-BTK) from a commercial source, particularly Invitrogen BTK (FIGURE 14).
  • total BTK is determined in standard ELISA format using biotinylated anti-BTK antibody against recombinant BTK (Invitrogen) and cell lysates (DoHH2 and Jurkat cells as control) (FIGURE 15). BTK target occupancy and total BTK were similarly assessed using biotinylated-probe irutinib and biotinylated anti-BTK antibody in the MSD format (FIGURES 16 and 17).
  • BTK target occupancy studies evaluated direct occupancy of an inhibitor probe, wherein the biotin labeled target inhibitor served as target occupancy probe and was incubated and directly monitored and evaluated for occupancy.
  • the target occupancy probe can be added to a sample or incubated in cells wherein an unlabeled inhibitor is already present.
  • the target occupancy probe will bind to target sites not already bound by unlabeled inhibitor and the target site occupancy of unlabeled inhibitor is indirectly measured. With more unlabeled inhibitor occupying the target, less target occupancy probe is bound to target and captured and detected in the assay. In such an instance, target occupancy of the inhibitor being assessed is inversely proportional to the amount of labeled target probe detected. Less target occupancy probe indicates more unlabeled target inhibitor is bound and occupying target.
  • a simple assay system has been designed and developed to detect and quantify Tec family kinase, for example BTK, and to simultaneously or serially, using the same assay format and overlapping reagents, determine target occupancy of a target inhibitor or ligand.
  • This assay system eliminates the cumbersome current method that involves determination of inhibitor bound target using one format, such as ELISA, combined with an alternative and dissimilar format for quantification of total target, such as Western blotting and manual quantification of total target. Determination of both target (for example BTK) occupancy and its normalization to quantified total target (BTK) present in cellular samples or cell lysates can be done simultaneously using standard curves generated using recombinant BTK protein or cell lysate BTK.
  • the assay is reproducible, yields reliable data and the sensitivity/detection limit is ⁇ 1 ng/ml of total BTK.
  • the assay works in both standard (STD) and MSD format.
  • An exemplary detailed combined assay protocol for the occupancy and total BTK ELISA-based assay can be performed according to the following procedure. This procedure is provided for illustrative purposes and particular aspects or reagents may be altered or substituted by one of skill and knowledge in the art.
  • the protocol will determine the amount of BTK probe (e.g., 41025) binding to the target active site that is not already occupied by inhibitor (e.g. ibrutinib) and also to quantify the amount of total BTK present in the samples.
  • the BTK probe used in the exemplary assay is ibrutinib linked to biotin as shown above in Formula (IV).
  • test reagents are mostly from Meso Scale Discovery - Rockville, MD (denoted MSD))
  • SA plate 5-pack
  • MSD Standard Streptavidin plate
  • Blocking buffer 3% MSD Blocker A in lx Tris Wash buffer (3 g Blocker A + 100 ml lx Wash)
  • Dilution buffer 1% MSD blocker A in lx Tris Wash buffer
  • Wash buffer lx MSD Tris wash buffer (diluted from lOx stock in H 2 0; 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.02% Tween-20; PBS-T can also be used)
  • Read buffer lx MSD read buffer (diluted from lOx stock in H 2 0)
  • Probe 41025 Prepare 20X solution in dilution buffer from 100 uM stock (5ul/ml to give a final concentration of 25 nM)
  • Cell lysates prepare by repeated freeze-thaw of cell pellets resuspended in DPBS + protease inhibitors (PI); alternatively, cell lysates can be prepared by brief sonication.
  • PI protease inhibitors
  • Block SA plate w/ 250 ul/well blocking buffer, 1 hr RT, shaking
  • Blocking buffer 3% MSD Blocker A in lx Tris Wash buffer (3 g Blocker A + 100 ml lx Wash)
  • Dilution buffer 1% MSD blocker A in lx Tris Wash buffer
  • Wash buffer lx MSD Tris wash buffer (diluted from lOx stock in H 2 0; 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.02% Tween-20; PBS-T can also be used)
  • Read buffer lx MSD read buffer (diluted from lOx stock in H 2 0)
  • Cell lysates prepare by repeated freeze-thaw of cell pellets resuspended in DPBS + protease inhibitors (PI). Alternatively, cell lysates can be prepared by brief sonication
  • Block S A plate w/ 250 ul/well blocking buffer, 1 hr RT, shaking
  • BTK Occupancy Provides amount of BTK occupancy by the drug (based on protein normalization)
  • BTK ELISA Provides total BTK protein present in samples in terms of RLU (relative light units) which can be used to normalize the occupancy data to give accurate occupancy values.
  • Rec BTK Standard Curve Used to determine the total amount of BTK in terms of pg/ml in samples
  • Amount of BTK (pg/ml) Multiply the amount of BTK (pg/ml) found by linear equation with RLU of Occupancy and divide it by RLU of Total BTK
  • BTK Occupancy Data / Total BTK ELISA data Normalized BTK Occupancy by the drug
  • the new BTK Occupancy and Total BTK Assay was validated using clinical samples and compared to data derived from other methods.
  • the novel assay and method was evaluated on (human) clinical samples from patients treated with BTK inhibitor to determine BTK occupancy and Total BTK.
  • Patient samples were evaluated from a phase 2 clinical study trial (NCT01478581; Study of the Bruton's Tyrosine Kinase Inhibitor in Subjects with Relapsed or Relapsed and Refractory Multiple Myeloma).
  • the patients (Cohort 1) were administered ibrutinib (PCI-32765) 420 mg per day and evaluated.
  • the first method (Method 1) (FIGURE 22 and 23) corresponds to the novel method provided herein, wherein sample cell lysates are split and used directly to determine, on the one hand, ligand (BTK) occupancy using labeled probe and, on the other hand, total BTK using labeled anti-BTK antibody, with comparison against a BTK standard curve based on recombinant BTK protein.
  • the same format such as MSD ELISA, is used for all BTK determinations in Method 1.
  • Methods 2 and 3 cell ly sates are first incubated with labeled probe for BTK occupancy determination. The supernatant is then recovered, collected and transferred for separate analysis of total BTK using anti-BTK antibody (Method 3, FIGURE 22 and 33) or biotinylated anti-BTK antibody (Method 2, FIGURE 22 and 28).
  • PBMCs Peripheral blood mononuclear cell samples
  • patient #1, patient #2, patient #3 and patient #4 were evaluated to determine BTK occupancy and total BTK using Methods 1, 2 and 3.
  • Each patient received 420 mg ibrutinib daily and were evaluated on Day 1 (vehicle dose) and on each of inhibitor dosing Day 2, Day 8, Day 15 and Day 29, at the end of the study, as indicated in the figures.
  • the results of Method 1 (FIGURE 23) assays for Patient #1, #2, #3 and #4 are shown in FIGURES 24, 25, 26 and 27, respectively. Results are provided for linear standard curve, % BTK occupancy and amount of free BTK. Occupancy correlates against free BTK with Method 1.
  • Method 2 (FIGURE 28) assays for Patient #1, #2, #3 and #4 are shown in FIGURES 29, 30, 31 and 32, respectively.
  • the results of Method 3 (FIGURE 33) assays for Patient #1, #2, #3 and #4 are shown in FIGURES 34, 35, 36 and 37, respectively.
  • FIGURE 38 Patient sample data across the methods are compared in FIGURE 38. Also in FIGURE 38, patient sample results are shown for comparison from previous samples results from patients dosed with a single 840 mg dose of ibrutinib and followed for up to 8 days (200 hours) (Chaturvedi, S et al (2014, September 22) AACR Hematological Malignancies, Poster Session B, B18).
  • Figure 38 results demonstrate that the instant novel method results are reliable and sensitive and are consistent with other methods, without the requirement for multiple distinct formats, sample transfer between formats, and possible errors due to sample handling and transfer across formats or for separate assessments.

Abstract

L'invention concerne des procédés et des systèmes de dosage permettant de déterminer quantitativement à la fois une cible, en particulier une kinase, notamment une kinase de la famille Tec, en particulier la BTK, et une occupation cible, notamment la quantité relative de cible disponible associée à un inhibiteur ou ligand cible, l'inhibiteur ou le ligand cible étant en particulier un inhibiteur de kinase covalent ou irréversible, notamment un inhibiteur de BTK irréversible. L'invention concerne également des dosages permettant de déterminer une occupation cible, normalisée sur une cible quantifiée totale dans un système biologique ou dans un échantillon clinique, la cible étant quantifiée, normalisée et l'occupation déterminée au moyen du même format de dosage ou d'un format de dosage commun. Dans un aspect de l'invention, un anticorps anti-BTK biotinylé et une sonde d'inhibiteur anti-BTK biotinylé sont utilisés dans le format de dosage, sur des échantillons séparés pour une analyse simultanée ou proportionnelle.
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Publication number Priority date Publication date Assignee Title
WO2019217684A1 (fr) * 2018-05-10 2019-11-14 Bristol-Myers Squibb Company Dosage de détermination de l'occupation d'un récepteur in vivo
US20210156854A1 (en) * 2018-05-10 2021-05-27 Bristol-Myers Squibb Company Assay to determine in vivo receptor occupancy
CN113804873A (zh) * 2018-05-10 2021-12-17 百时美施贵宝公司 确定体内受体占有率的测定

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