Methods for predicting the responsiveness of drugs targeting BCL2 family proteins

Abstract

The present invention provides a method for predicting the responsiveness of a drug targeting a BCL2 family protein, and a method for selecting a subject suitable for treatment with a drug targeting a BCL2 family protein.

Description

Technical Field

[0001] The present invention provides a method for predicting the responsiveness of a BCL2 family protein targeted drug, and a method for selecting a subject suitable for treatment with the BCL2 family protein targeted drug. Background Art

[0002] BCL2 (B-cell lymphoma 2) family proteins regulate apoptosis by either induction (pro-apoptotic activity) or inhibition (anti-apoptotic activity) of apoptosis.

[0003] BCL2 family proteins can cause a variety of cancers, including melanoma, breast cancer, prostate cancer, chronic lymphocytic leukemia, and lung cancer, and are known to be factors that induce resistance to cancer treatment. It has also been suggested that BCL2 family proteins may contribute to schizophrenia and autoimmune diseases. For these reasons, BCL2 family proteins may be promising targets for anticancer drugs, and various inhibitors targeting BCL2 family proteins have been developed.

[0004] In order to effectively utilize BCL2 family protein-targeted drugs as cancer therapeutics, it is necessary to develop a means to quantitatively analyze the apoptosis regulatory signals induced by BCL2 family proteins. Summary of the invention

[0005] Technical issues

[0006] The present invention provides a personalized treatment technology in which protein interactions that mediate signal transduction are quantitatively analyzed, thereby allowing the use of BCL2 family protein-targeted drugs to treat cancer (e.g., blood cancer) in patients.

[0007] In one aspect, a method for predicting the responsiveness of a subject to a BCL2 family protein targeted drug or for predicting the medicinal efficacy of a BCL2 family protein targeted drug in a subject or a method for providing information for prediction is provided.

[0008] On the other hand, a method for selecting (identifying) a subject responsive to a BCL2 family protein targeted drug is provided, wherein the BCL2 family protein targeted drug shows pharmaceutical efficacy, or the BCL2 family protein targeted drug is appropriately suitable for the subject, or a method for providing information for selection (identification).

[0009] The method may further comprise the step of administering the drug to a subject responsive to or showing efficacy of the drug or a subject suitable for treatment with the drug.

[0010] On the other hand, a composition is provided, which comprises a BCL2 family protein targeted drug for use in the treatment of cancer in a subject responsive to the BCL2 family protein targeted drug, wherein the BCL2 family protein targeted drug shows pharmaceutical efficacy; or in the treatment of cancer in a subject to which the BCL2 family protein targeted drug or treatment thereof is applicable.

[0011] On the other hand, a method for treating cancer is provided, which comprises the step of administering to a subject a pharmaceutically effective amount of a BCL2 family protein targeted drug confirmed by the method, wherein the subject is responsive to the BCL2 family protein targeted drug, wherein the BCL2 family protein targeted drug shows pharmaceutical efficacy; or is suitable for the BCL2 family protein targeted drug or its treatment.

[0012] A method for treating cancer is provided, comprising the step of administering a pharmaceutically effective amount of a BCL2 family protein targeting drug.

[0013] The BCL2 family protein targeted drug may target a BCL2 family protein (eg, a BCL2 protein inhibitor or an MCL1 protein inhibitor).

[0014] Technical Solution

[0015] A method for predicting a subject's responsiveness to a BCL2 family protein targeted drug or for predicting the pharmaceutical efficacy of a BCL2 family protein targeted drug in a subject, or a method for providing information for prediction; and / or a method for selecting (identifying) a subject responsive to a BCL2 family protein targeted drug, wherein the BCL2 family protein targeted drug exhibits pharmaceutical efficacy, or the BCL2 family protein targeted drug is suitable for the subject; or a method for providing information for selection (identification), are all provided herein, and the method comprises the following steps: (a1) measuring the interaction between apoptosis-inhibiting (anti-apoptotic) proteins and apoptosis-inducing (pro-apoptotic) proteins in a sample (e.g., the following steps (i) and / or (ii)); and / or (a2) measuring the levels of apoptosis-inducing (pro-apoptotic) proteins and / or apoptosis-inhibiting (anti-apoptotic) proteins in a sample (e.g., the following steps (iii)).

[0016] In one embodiment, the methods provided herein may include at least one (e.g., one, two, or three) selected from the following steps:

[0017] (i) a step of measuring the interaction between a first protein in a sample and a protein interacting with the first protein, wherein the protein interacting with the first protein is externally provided (first protein-protein interaction measurement step);

[0018] (ii) a step of measuring the interaction between a first protein endogenous to the sample and a protein interacting with the first protein, wherein the protein interacting with the first protein is endogenous to the sample (a second protein-protein interaction measurement step); and

[0019] (iii) a step of measuring the level of the first protein, a protein that interacts with the first protein, or both in the sample.

[0020] In one embodiment, the method provided herein may include step (i), step (ii) or step (iii). In another embodiment, the method provided herein may include a combination of step (i) and step (ii), a combination of step (i) and step (iii), a combination of step (ii) and step (iii), or a combination of step (i), step (ii) and step (iii).

[0021] The first protein and the protein interacting with the first protein (hereinafter, "protein interacting with the first protein" is also referred to as "first protein interacting protein") can be selected from anti-apoptotic subtype proteins and cell survival inducing (pro-survival) subtype proteins.

[0022] In one embodiment, the first protein in steps (i) to (iii) may be at least one of the “pro-survival or anti-apoptotic subtype proteins” (hereinafter referred to as “anti-apoptotic proteins”) selected from the BCL2 family proteins. For example, the first proteins in steps (i) to (iii) may be the same or different, and each may independently be at least one (e.g., one, two or three) selected from the group consisting of BCL2, BCLxL, BCLw, BFL1 and MCL1. The protein interacting with the first protein in steps (i) to (iii) may be at least one selected from the pro-apoptotic subtype proteins (hereinafter referred to as “pro-apoptotic subtype proteins”) of the BCL2 family proteins. In one embodiment, the first protein-interacting proteins in steps (i) to (iii) may be the same or different, and each may independently be at least one selected from the group consisting of the BH3 subtype (BH3 only) protein (second protein) and the effector subtype protein (third protein) of the pro-apoptotic proteins. For example, the second protein can be at least one (e.g., one, two, or three) selected from the group consisting of BAD, BIM, PUMA, BMF, BID, tBID, BIK, HRK, and NOXA, and the third protein can be at least one (e.g., one or two) selected from the group consisting of BAX, BOK, and BAK. In one embodiment, the first protein-interacting protein can be at least one (e.g., one, two, three, four, or five) selected from the group consisting of BAD, BIM, PUMA, BMF, BID, tBID, BIK, HRK, NOXA, BAX, BOK, and BAK. When the method includes two or more of steps (i) to (iii), the first protein-interacting proteins in each step can be the same or different.

[0023] In another embodiment, the first protein in steps (i) to (iii) may be at least one of the pro-apoptotic proteins selected from the BCL2 family proteins. In one embodiment, the first protein in steps (i) to (iii) may be the same or different, and may be at least one of the group consisting of BH3 subtype (BH3 only) proteins and effector subtype proteins selected from the pro-apoptotic proteins of the BCL2 family proteins. For example, the BH3 subtype (BH3 only) protein is at least one (e.g., one, two or three) selected from the group consisting of BAD, BIM, PUMA, BMF, tBID, BIK, HRK and NOXA, and the effector subtype protein may be at least one (e.g., one or two) selected from the group consisting of BAX, BOK and BAK. The first protein-interacting protein in steps (i) to (iii) may be at least one of the anti-apoptotic proteins selected from the BCL2 family proteins. For example, the first protein-interacting proteins in steps (i) to (iii) can be the same or different, and each can be independently selected from at least one (e.g., one, two or three) of the group consisting of BCL2, BCLxL, BCLw, BFL-1 and MCL1.

[0024] When the method comprises two or more of steps (i) to (iii), the first protein and the first protein-interacting protein in each step may be the same or different.

[0025] As used herein, the term "sample" (biological sample) is intended to include cells or cell lysates isolated from a subject (e.g., human). As used herein, the term "subject" refers to an animal in need of treatment for a disease (e.g., cancer), a subject to be administered a BCL2 family protein targeted drug, a subject to be treated with a BCL2 family protein targeted drug, or a subject to be determined for administration of a BCL2 family protein targeted drug and for treatment with a BCL2 family protein targeted drug. The medicinal efficacy (drug response) of the subject. In one embodiment, the subject can be a cancer patient (e.g., human), and the cell can be a cancer cell isolated from the subject. For example, the subject can be a human patient suffering from a blood cancer, and the sample can be at least one selected from the group consisting of leukocytes (e.g., granulocytes, lymphocytes and / or monocytes (e.g., peripheral blood mononuclear cells (PBMCs)), bone marrow cells (e.g., hematopoietic stem cells), blood, bone marrow, and buffy coat (each layer containing cells and cell lysates) isolated from a blood cancer patient. The sample obtained from the subject selected or identified by the methods provided herein can be represented as a "test sample" to distinguish it from a control sample for comparison (e.g., a positive control sample or a negative control sample for selecting a standard value for comparison, as described below).

[0026] As used herein, the term "externally provided protein" (e.g., first protein-interacting protein) refers to a protein that is not endogenous to the sample but is added to the sample from the outside (exogenous). In one embodiment, the externally provided protein can be labeled with a typical probe (e.g., a fluorescent substance, etc.), but is not limited thereto.

[0027] In one embodiment, step (i) may include measuring the interaction between a BCL2 family protein (first protein) in a sample and an externally provided (exogenous) BCL2 family protein (first protein-interacting protein) (first protein-protein interaction measurement step), wherein the probe-labeled exogenous first protein-interacting protein is brought into contact with the first protein immobilized on a substrate (protein in the sample), and then the protein-protein interaction between the first protein (protein in the sample) and the protein interacting therewith (exogenous protein) is measured (for example, the level of a complex formed between the first protein and the first protein-interacting protein is measured).

[0028] In one embodiment, step (ii) may include measuring the interaction between endogenous BCL2 family proteins in the sample (a second protein-protein interaction measurement step), wherein a sample containing a first protein is contacted with a substrate comprising a substance that can bind to the first protein (e.g., an antibody that binds to the first protein, etc.), and then the protein-protein interaction between the first protein (endogenous protein in the sample) and the first protein-interacting protein (endogenous protein in the sample) is measured (e.g., measuring the level of a complex formed between the first protein and the first protein-interacting protein).

[0029] In one embodiment, in steps (i) and / or (ii), the level of the complex formed between the first protein and the protein interacting therewith can be measured by the signal intensity generated by the probe bound to the first protein-interacting protein (step (i)) or bound to the substrate bound to the first protein-interacting protein (step (ii)), but is not limited thereto.

[0030] In one embodiment, step (iii) may include a step of measuring the total expression level of any one or both of the first protein and the first protein-interacting protein in the sample. The level in the sample can be the expression level (e.g., protein concentration) of the protein in the sample, the expression level (e.g., activated protein concentration) of the activated protein, the expression level (e.g., activated protein concentration) of the phosphorylated protein, or a combination thereof.

[0031] In one embodiment, step (i) (first protein-protein interaction measurement step) may include at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) selected from the following sub-steps:

[0032] (i-1) measuring the protein-protein interaction between BCL2 protein in the sample and externally provided BAD protein;

[0033] (i-2) measuring the protein-protein interaction between BCL2 protein in the sample and externally provided BIM protein;

[0034] (i-3) measuring the protein-protein interaction between BCL2 protein in the sample and externally provided BAX protein;

[0035] (i-4) measuring the protein-protein interaction between BCL2 protein in the sample and externally provided BAK protein;

[0036] (i-5) measuring the protein-protein interaction between BCLxl protein in the sample and externally provided BAD protein;

[0037] (i-6) measuring the protein-protein interaction between BCLxl protein in the sample and externally provided BIM protein;

[0038] (i-7) measuring the protein-protein interaction between BCLxl protein in the sample and externally provided BAX protein;

[0039] (i-8) measuring the protein-protein interaction between BCLxl protein in the sample and externally provided BAK protein;

[0040] (i-9) measuring the protein-protein interaction between the MCL1 protein in the sample and the externally provided NOXA protein;

[0041] (i-10) measuring the protein-protein interaction between MCL1 protein in the sample and externally provided BIM protein;

[0042] (i-11) measuring the protein-protein interaction between the MCL1 protein in the sample and the externally provided BAX protein; and

[0043] (i-12) Measuring the protein-protein interaction between the MCL1 protein in the sample and the externally provided BAK protein.

[0044] In one embodiment, each sub-step of measuring protein-protein interaction in step (i) (first protein-protein interaction measurement step) may be performed by or include the following steps:

[0045] (1) providing a sample to a substrate, wherein the substrate includes a substance that binds to a first protein, so as to fix the first protein to the substrate;

[0046] (2-1) providing a probe-labeled protein that interacts with the first protein to the outside of the substrate to allow reaction with the first protein on the substrate; and

[0047] (3) The signal generated by the probe is measured in the near-field region of the substrate surface using total internal reflection fluorescence (TIRF) microscopy.

[0048] In the first protein-protein interaction measurement step, the protein (first protein interaction protein) provided externally can be a protein labeled (combined) with a probe. In the first protein-protein interaction measurement step, the measurement of protein-protein interaction can be carried out by measuring the level of the complex formed between the first protein (sample endogenous protein) and the protein (exogenous protein) interacting with it. In one embodiment, the level of the complex can be measured based on the signal generated by the probe connected to the first protein interaction protein.

[0049] In one embodiment, step (ii) (second protein-protein interaction measurement step) may include at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11) selected from the following sub-steps:

[0050] (ii-1) measuring a protein-protein interaction between a BCL2 protein in a sample and a BIM protein in a sample (e.g., the level of a complex formed thereby);

[0051] (ii-2) measuring a protein-protein interaction between a BCL2 protein in the sample and a BAX protein in the sample (e.g., the level of a complex formed thereby);

[0052] (ii-3) measuring a protein-protein interaction between a BCL2 protein in the sample and a BAK protein in the sample (eg, the level of a complex formed thereby);

[0053] (ii-4) measuring the protein-protein interaction between the BCLxl protein in the sample and the BAD protein in the sample (e.g., the level of the complex formed thereby);

[0054] (ii-5) measuring the protein-protein interaction between the BCLxl protein in the sample and the BIM protein in the sample (e.g., the level of the complex formed thereby);

[0055] (ii-6) measuring the protein-protein interaction between the BCLxl protein in the sample and the BAX protein in the sample (e.g., the level of the complex formed thereby);

[0056] (ii-7) measuring the protein-protein interaction between the BCLxl protein in the sample and the BAK protein in the sample (e.g., the level of the complex formed thereby);

[0057] (ii-8) measuring the protein-protein interaction between the MCL1 protein in the sample and the NOXA protein in the sample (eg, the level of a complex formed thereby);

[0058] (ii-9) measuring the protein-protein interaction between the MCL1 protein in the sample and the BIM protein in the sample (e.g., the level of the complex formed thereby);

[0059] (ii-10) measuring a protein-protein interaction between an MCL1 protein in the sample and a BAX protein in the sample (eg, the level of a complex formed thereby); and

[0060] (ii-11) Measuring the protein-protein interaction between the MCL1 protein in the sample and the BAK protein in the sample (eg, the level of the complex formed thereby).

[0061] In one embodiment, each sub-step of measuring the protein-protein interaction (e.g., the level of the complex formed thereby) in step (ii) (the second protein-protein interaction measurement step) may be performed by or include the following steps:

[0062] (1) providing (adding) a sample to a substrate, wherein the substrate includes a substance that binds to a first protein, so as to fix the first protein to the substrate;

[0063] (2-2) providing a probe-labeled substance that binds to a first protein-interacting protein to a substrate to allow reaction with the first protein-interacting protein in a complex formed between the first protein (endogenous to the sample) and the first protein-interacting protein (endogenous to the sample);

[0064] (3) The signal generated by the probe is measured in the near-field region of the substrate surface using total internal reflection fluorescence (TIRF) microscopy.

[0065] In another embodiment, each sub-step of measuring the protein-protein interaction (e.g., the level of the complex formed thereby) in step (ii) (the second protein-protein interaction measurement step) may be performed by or include the following steps:

[0066] (1) providing (adding) a sample to a substrate, wherein the substrate includes a substance that binds to a first protein-interacting protein, so as to fix the first protein-interacting protein to the substrate;

[0067] (2-2) providing a substance labeled with a probe that binds to a first protein to a substrate to allow reaction with the first protein in a complex formed between the first protein (endogenous to the sample) and a first protein-interacting protein (endogenous to the sample); and

[0068] (3) The signal generated by the probe is measured in the near-field region of the substrate surface using total internal reflection fluorescence (TIRF) microscopy.

[0069] In the second protein-protein interaction measurement step, complex is endogenously formed between the first protein endogenous to the sample and the first protein-interaction protein endogenous to the sample. In the second protein-protein interaction measurement step, the measurement of protein-protein interaction can be carried out by measuring the level of the complex formed between the first protein (sample endogenous) and the protein (sample endogenous) interacting with it. In one embodiment, the level of the complex can be measured based on the signal generated by the probe, and the probe is bound to the substance bound to the first protein-interaction protein or the first protein.

[0070] In one embodiment, step (iii) may include at least one of the following sub-steps:

[0071] (iii-1) measuring the level of BCL2 protein in the sample;

[0072] (iii-2) measuring the level of BCLxl protein in the sample;

[0073] (iii-3) measuring the level of MCL1 protein in the sample;

[0074] (iii-4) measuring the level of BAX protein in the sample; and

[0075] (iii-5) Measuring the level of BAK protein in the sample.

[0076] Each level measurement sub-step in step (iii) may be performed by or include the following steps:

[0077] - providing a sample to a substrate, the substrate comprising either or both of a substance that binds to the first protein (capture substance for fixing the first protein to the substrate) and a substance that binds to the first protein-interacting protein (capture substance for fixing the first protein-interacting protein), so as to fix either or both of the first protein and the first protein-interacting protein to the substrate;

[0078] - providing a probe-labeled substance that binds to the first protein (a detector for detecting the first protein in the case where the first protein is immobilized to a substrate), a probe-labeled substance that binds to the first protein-interacting protein (a detector for detecting the first protein-interacting protein in the case where the first protein-interacting protein is immobilized to a substrate), or immobilizing both of them (in the case where both the first protein and the first protein-interacting protein are immobilized to a substrate) to a substrate to allow a reaction; and

[0079] - The signal generated by the probe is measured in the near-field region of the substrate surface using total internal reflection fluorescence (TIRF) microscopy.

[0080] The capture substance and the detector that bind to different sites of the same target protein can each be independently selected from any binding substance (eg, an antibody) whose binding sites do not completely overlap with each other.

[0081] The substance used in step (ii) and / or (iii) that binds to the first protein-binding substance or the first protein-interacting protein is an adhesive (e.g., an antibody, an antigen-binding fragment, an antibody analog, an aptamer, a small molecule compound, etc.) coupled to the first protein or the first protein-interacting protein, and may be labeled with a probe. In this case, the sites of the first protein-interacting protein to which the binding substance and the first protein respectively bind to the first protein-interacting protein may be different from each other.

[0082] In one embodiment, in step (i), (ii) or (iii), the substance bound to the first protein for fixing (pulling down) the first protein to the substrate, in step (ii), the substance bound to the first protein-interacting protein for detecting the complex formed between the first protein and the first protein-interacting protein, and / or the substance bound to the first protein or the first protein-interacting protein for detecting the first protein or the first protein-interacting protein in step (iii) can be selected from at least one of the typical available antibodies against the target protein. When two or more of steps (i) to (iii) are performed and the same first protein and / or the same first protein-interacting protein are generally used in two of the steps, the binding substances used for fixing (pulling down) and / or detecting the same protein in the corresponding steps can be the same and different.

[0083] The substance (eg, antibody) that binds to the first protein and the first protein-interacting protein may be as follows:

[0084] Anti-apoptotic proteins

[0085] BCL2: ab32124, ab182858, ab238041, ab692 (all from Abcam), 3498S, 15071S, #4223 (all from CST), BSM-52304R, BSM-10846M, BSM-33411M, BSM-52022M (all from Bioss), NB100-56098 (Novus), 12789-1-AP (Proteintech), LS-C159170-400 (LSBio), 13-8800 (ThermoFisher Scientific), 610538 (BD), sc-509, sc-23960, sc-7382 (all from Santa Clara, CA), Cruz), TA806591, CF806589, UM800117, CF806639 (all from OriGene), etc.;

[0086] BCL-XL: 2764S, 2762S, 2870, 3498S (all from CST), ab77571, ab59348, ab32370, ab178844 (all from Abcam), MA5-11950, MA5-28637 (all from Invitrogen), MA5-15142 (ThermoFisher Scientific), BSM-51229M (Bioss), sc-56021, sc-136207, sc-271121, sc-70417, sc-23958 (all from Santa Clara, CA), Cruz), ALX-804-660-C100 (Enzo), 66020-1-IG (Proteintech), NBP2-25240, NBP2-32810, NBP2-44416, NBP1-47665 (all from Novus), TA506188 (OriGene), H00000598-M01A (Abnova), etc.;

[0087] MCL1: ab32087 (Abcam), 5453S, 39224S, #94296 (all from CST), MA5-32060, 14-6701-82, 14-9047-82, MA5-15236, 14-6701-82, 14-9047-82 (all from Invitrogen), SP2170P, TA500986, TA500990 (all from OriGene), 16225-1-AP (Proteintech), LS-C475-0.1 (LSBio), MAB8825 (Abnova), sc-12756, sc-74437, sc-74436, sc-377487, sc-69839, sc-69840, sc-53951 (all from Santa Cruz), etc.;

[0088] BCLw: MA5-15076 (Invitrogen), #2724 (CST), sc-293236, sc-130701 (all from Santa Cruz), NBP2-61706 (Novus), etc.;

[0089] BFL1: NBP2-67563, NBP2-46568CST (both from Novus), #14093 (CST), MA5-11757 (Invitrogen), sc-166943 (Santa Cruz), etc.;

[0090] Pro-apoptotic proteins

[0091] BAX: ab32503, ab182733, ab81083 (all from Abcam), #5023, 2774S, 2772S (all from CST), MA5-14003, MA5-16322, 35-9700 (all from Invitrogen), BSM-52316R, BSM-33279M (all from Bioss), 50599-2-Ig (Proteintech), SAB4502546 (Merck), NB100-56096 (Novus), 610982, 610983 (all from BD), sc-7480, sc-23959, sc-70408 (all from Santa Clara, CA), Cruz), CF810335, TA810334 (all from OriGene), #633602 (BioLegend), LS-C357719-20 (LSBio), etc.;

[0092] BAK: ab238515, ab32371, ab69404, ab137646 (all from Abcam), 6947S, 12105S (all from CST), B5897 (Merck), 06-536, AM03-100UGCN, SAB1305656 (all from Sigma), H00000578-M01A, MAB8753 (Abnova), sc-517390, sc-518110 (all from Santa Cruz), MA5-36225 (Invitrogen), GTX10808 (GeneTex), etc.;

[0093] BAD: 9296S (CST), sc-8044 (Santa Cruz), MA5-11117, MA5-15230 (all from Invitrogen), etc.;

[0094] NOXA: ab68523 (Abcam), sc-515840, sc-56169, sc-398873 (all from Santa Cruz), etc.; and

[0095] BIM: MA5-15763, MA5-15764 (both from Invitrogen), sc-374358 (Santa Cruz), CF812441 (OriGene), etc.

[0096] The above antibodies are for illustration only and are not intended to be limiting. As long as the antibody is known to have binding activity to a specific protein, any antibody may be used and is expected to exhibit substantially the same effect.

[0097] The substance used herein that binds to the first protein-interacting protein or the first protein to detect the first protein-interacting protein or the first protein in the endogenous protein-protein interaction measurement step (step (ii)) can be labeled with a probe. When the binding substance is an antibody, the antibody or a secondary antibody of the antibody can be bound to the probe.

[0098] In addition to the step (a1) of measuring the interaction between BCL2 family proteins and / or the step (a) of measuring the level of BCL2 family proteins in a sample, the method provided herein may further include a step (b): comparing the measured value of the interaction between BCL2 family proteins in step (a1) and / or the measured value of the level of BCL2 family proteins in the sample in step (a2) with a reference value (b1); and / or identifying (determining or deciding) patients whose samples or samples isolated from subjects are responsive to BCL2 family protein targeted drugs and / or whether BCL2 family protein targeted drugs exhibit medicinal efficacy in samples or samples isolated from subjects; and / or selecting (identifying, determining and deciding) subjects who are suitable for treatment targets using BCL2 family protein targeted drugs (b2).

[0099] After step (b) (more specifically, step (b2)), the methods provided herein may further include the step of administering a BCL2 family protein targeted drug to the subject identified in step (b) (more specifically, step (b2)) to produce a response to the BCL2 family protein targeted drug to allow the drug to exert its medicinal effect therein, or to perform appropriate treatment with the drug.

[0100] As used herein, the term "responsiveness to a BCL2 family protein targeting drug" or "pharmaceutical efficacy of a drug" refers to the pathological effect obtained as a result of drug targeting (e.g., inhibiting) a target protein (i.e., BCL2 protein), and is intended to cover anti-cancer effects (alleviating, reducing, alleviating, removing and treating cancer (or cancer cells), and / or inhibiting or delaying the progression and / or metastasis of cancer, etc.), more specifically, the anti-cancer effect on blood cancer.

[0101] As used herein, the term "subject suitable for administration of BCL2 family protein targeted drugs or treatment with drugs" refers to patients in whom BCL2 family protein targeted drugs can show therapeutic effects on diseases. For example, the subject can be a patient in whom administration of BCL2 family protein targeted drugs or therapeutic treatment using BCL2 family protein targeted drugs can show anti-cancer effects (mitigation, reduction, alleviation, removal and / or treatment of cancer or cancer cells).

[0102] The reference value in step (b) (specifically, step (b1)) can be established by the methods provided herein based on information obtained from pre-existing cancer patients (e.g., blood cancer patients). In one embodiment, the reference value can be 1) a measurement value obtained by applying the methods provided herein (steps (a1) and / or (a2)) to a patient or patient group that can show a therapeutic effect using a BCL2 family protein targeted drug (a patient or patient group is responsive to a BCL2 family protein targeted drug), or to a sample separated from a patient or patient group (a sample in an effective group), or 2) a measurement value obtained by applying the methods provided herein (steps (a1) and / or (a2)) to a patient or patient group that cannot show a therapeutic effect using a BCL2 family protein targeted drug (a patient or patient group is unresponsive to a BCL2 family protein targeted drug), or to a sample separated from a patient or patient group (a sample in a non-effective group).

[0103] In an embodiment in which a reference value is obtained from a sample in an effective group, if the measured value obtained in the step of measuring the interaction between BCL2 family proteins in the test sample (a1) and / or the step of measuring the level of BCL2 family proteins in the test sample (a2) is up to or greater than the reference value (a value range surrounding the reference value (e.g., the reference value itself or an error range of 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less) or a value exceeding the reference value), the sample or the sample obtained from the subject can be selected as a patient responsive to a BCL2 family protein targeted drug (or a patient suitable for treatment with a BCL2 family protein targeted drug), or can be identified (determined or determined) as a patient in whom a BCL2 family protein targeted drug can exhibit a medicinal efficacy (step (b2)).

[0104] In another embodiment, in the case where the reference value is obtained from a sample in a non-effective group, if the measured value obtained in the step of measuring the interaction between BCL2 family proteins in the test sample (a1) and / or the step of measuring the level of BCL2 family proteins in the test sample (a2) is within a range around the reference value (for example, the reference value itself or the error range is 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less) or is less than the reference value, the sample or the sample obtained from the subject can be selected as a patient who is unresponsive to the BCL2 family protein targeted drug (or a patient who does not show pharmaceutical efficacy after treatment with (administration of) the BCL2 family protein targeted drug), or can be identified (determined or determined) as a patient for whom the BCL2 family protein targeted drug cannot show pharmaceutical efficacy (step (b2)).

[0105] In another embodiment, the reference value can be determined as the minimum, median or average of the measured values ​​obtained in the step (a1) of measuring the interaction between BCL2 family proteins in the effective group sample and / or in the step (a2) of measuring the level of BCL2 family proteins in the effective group sample. The reference value can be adjusted by adding patient information or according to equations or algorithms derived from various analytical values. In step (b) of the method provided herein, when the measured value obtained in step (a1) and / or step (a2) is higher than (similar to and / or exceeds) the corresponding reference value, the test sample or the sample obtained from the subject can be selected as a patient who has a therapeutic response to a BCL2 family protein targeted drug, or a patient suitable for treatment with a BCL2 family protein targeted drug.

[0106] In another embodiment, the reference value can be determined as the maximum, median or average of the measured values ​​obtained in the step (a1) of measuring the interaction between BCL2 family proteins in the sample of the non-effective group and / or in the step (a2) of measuring the level of BCL2 family proteins in the sample of the non-effective group. The reference value can be adjusted by adding patient information or according to equations or algorithms derived from various analytical values. In step (b) of the method provided herein, when the measured value obtained in step (a1) and / or step (a2) is lower than (similar to and / or lower than) the corresponding reference value, the test sample or the sample obtained from the subject can be selected as a patient for whom the BCL2 family protein targeted drug does not have a therapeutic effect.

[0107] In another embodiment, the reference value can be the degree of interaction between the comparison protein in the sample and the protein that interacts with the comparison protein and / or the level (expression level or concentration) of the comparison protein in the sample and / or the protein that interacts with the comparison protein. The comparison protein and the protein that interacts with the comparison protein can be selected from anti-apoptotic proteins and pro-apoptotic proteins. The comparison protein and the protein that interacts with the comparison protein can be the same or different from the above-mentioned first protein and the first protein-interacting protein, respectively. In one embodiment, the comparison protein can be different from the first protein, and the protein that interacts with the comparison protein can be the same or different from the first protein-interacting protein.

[0108] In this case, the method provided herein may further include the following steps (a1') and / or (a2') before step (b):

[0109] (a1') measuring the interaction between the comparison protein and the protein interacting with the comparison protein in the sample and / or (a2') measuring the level of the comparison protein and / or the protein interacting with the comparison protein in the sample.

[0110] According to another embodiment, a method for treating cancer (e.g., blood cancer) is provided, the method comprising the step of administering a BCL2 family protein targeted drug to the following subjects; and a pharmaceutical composition comprising the BCL2 family protein targeted drug for treating cancer (e.g., blood cancer) in the following subjects by application (administration):

[0111] A subject in which the interaction between BCL2 family proteins in a sample obtained (isolated) from the subject (e.g., measured in (i) and / or (ii) of step (a1)), and / or a subject in which the level of BCL2 family proteins in the subject (e.g., measured in step (a2)) is similar to or exceeds a reference value (e.g., obtained from an effective group) (e.g., a subject identified as responsive to a BCL2 family protein targeted drug or allowing measurement of the pharmaceutical efficacy of the drug by the methods provided herein, for predicting responsiveness to a BCL2 family protein targeted drug and / or predicting the pharmaceutical efficacy of the drug; and / or a subject identified as suitable for administration of a BCL2 family protein targeted drug or suitable for treatment with a BCL2 family protein targeted drug as measured by a method for selecting a subject suitable for administration of a BCL2 family protein targeted drug or treatment with a BCL2 family protein targeted drug).

[0112] BCL2 family proteins generally refer to proteins with a common BCL-2 homology domain (BH; BH1, BH2, BH3), which function to regulate cell apoptosis (eg, mitochondrial apoptosis).

[0113] As used herein, the first protein is a BCL2 family protein, referring to an anti-apoptotic protein that prevents apoptosis, interacting (binding) with an apoptotic protein and / or a pro-apoptotic protein to interfere with the function of an apoptotic protein and / or a pro-apoptotic protein. The first protein may be contained in a biological sample separated from a subject, or may be present in a form separated from the sample. In one embodiment, the first protein may be a protein derived from a mammal such as humans. For example, the first protein may be selected from at least one of the groups consisting of BCL2 (e.g., NCBI reference sequence NM_000633.2, etc.), BCLxL (e.g., NCBI reference sequence NM_138578.1, etc.), MCL1 (e.g., NCBI reference sequence NM_021960.3, etc.), BCLw (e.g., NCBI reference sequence NM_001199839.2, etc.), and BFL1 (e.g., NCBI reference sequence NM_001114735.2, etc.).

[0114] The first protein-interacting protein may be at least one selected from the group consisting of a BH3 isoform (BH3 only) protein (second protein); and a pro-apoptotic effector protein (third protein).

[0115] As used herein, the second protein (BH3 subtype protein) consists only of the BH3 domain and is therefore referred to as a BH3-only protein. For example, the second protein can be at least one selected from the group consisting of: BIM (e.g., NCBI reference sequence B033694), BID (e.g., NCBI reference sequence NM_001196.2), tBID (e.g., N-terminal truncated form of NCBI reference sequence NM_001196.2, etc.), BAD (e.g., NCBI reference sequence NM_004322.2, etc.), NOXA (e.g., NCBI reference sequence NM_001382616.1, etc.), PUMA (e.g., NCBI reference sequence AAO16862.1, etc.), BMF (e.g., NCBI reference sequence NM_001003940.2, etc.), BIK (e.g., NCBI reference sequence 1.NM_001197.5, etc.) and HRK (e.g., NCBI reference sequence 1.NM_003806.4, etc.).

[0116] As used herein, the third protein is a pro-apoptotic protein comprising multiple BH domains, and may be at least one selected from the group consisting of, for example, BAX (e.g., NCBI reference sequence NM_138761.3, etc.), BAK (e.g., NCBI reference sequence NP_001179.1, etc.) and BOK (e.g., NCBI reference sequence NM_032515.5, etc.).

[0117] In another embodiment, the first protein can be at least one selected from the group consisting of BH3-only proteins (second proteins) and pro-apoptotic effector proteins (third proteins) as described above, and the first protein-interacting protein can be at least one selected from the anti-apoptotic proteins as described above.

[0118] Here, the first protein-interacting protein provided externally can be a wild-type protein, or can be a variant protein having 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity or homology with the wild-type protein, provided that it can interact with the first protein. In addition, the first protein-interacting protein can be derived from humans or can be a foreign protein that can interact with the first protein derived from humans.

[0119] The subject may be a mammal, such as a human being, and may be, for example, a cancer patient (blood cancer, solid cancer), more specifically, a blood cancer patient. In order to determine the reference value, the subject may need to be administered a BCL2 family protein targeted drug or confirm the medicinal efficacy of the drug therein.

[0120] Here, the blood cancer can be selected from the group consisting of leukemia (e.g., acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, etc.), lymphoma (e.g., malignant lymphoma, B-cell lymphoma, small lymphocytic lymphoma (SLL), etc.), multiple myeloma and myelodysplastic syndrome.

[0121] The sample is a biological sample separated (derived) from a patient and may include cells, cell lysates, tissues or cultures thereof, or established cell lines. For example, the cell or tissue may be a cancer cell or cancer tissue. Specifically, the cancer may be a blood cancer or a solid cancer (e.g., breast cancer, lung cancer (e.g., lung adenocarcinoma, etc.), etc.). In one embodiment, the subject may be a human patient suffering from a blood cancer, and the sample may be at least one selected from the group consisting of white blood cells (e.g., granulocytes, lymphocytes and / or monocytes) and bone marrow cells (hematopoietic stem cells, etc.), blood, bone marrow, and a yellow layer of blood clots containing cells, cancer cells, cancer tissues, and their lysates, wherein the white blood cells and bone marrow cells are separated from patients suffering from blood cancer or solid cancer (e.g., breast cancer or lung cancer (e.g., lung adenocarcinoma, etc.).

[0122] In one embodiment, the sample can be a lysate of at least one selected from the group consisting of leukocytes (e.g., granulocytes, lymphocytes, and / or monocytes) and bone marrow cells (hematopoietic stem cells, etc.), blood, bone marrow, and buffy coat containing cells, cancer cells, and cancer tissue, wherein both leukocytes and bone marrow cells are isolated from patients with blood cancer or solid cancer (e.g., breast cancer or lung cancer (e.g., lung adenocarcinoma, etc.). The lysate can be obtained using a solvent comprising at least one selected from the group consisting of HEPES, NaCl, glycerol, a cholesterol-based detergent (hereinafter referred to as a "surfactant"), and a phosphatase inhibitor mixture. In this regard, cholesterol-based detergents (e.g., glycol diosgenin (GDN), digitonin, etc.), PEG-based detergents (e.g., Triton x-100, etc.), etc. can be used. The detergents that can be used in the present invention may include GDN, digitonin, Triton x-100 (TX-100) or a combination thereof. For example, based on the total weight of the solvent, the amount of GDN used may be 0.001-10%, 0.005-5%, 0.008-1.2%, 0.01-10%, 0.05-5%, 0.08-1.2%, 0.1-10%, 0.5-5%, 0.8-1.2% or about 1% (weight to weight). Before use, the lysate may be diluted to an appropriate concentration. Compared to other types of detergents, cholesterol-based detergents and / or PEG-based detergents can stably maintain cell function and activity and / or three-dimensional protein structure in vitro in a manner similar to that in vivo, and allow proteins to maintain relatively intact structure and / or function in a cellular environment, even in vitro. The inherent protein properties of cells can be advantageously observed.

[0123] In step (i), the externally provided (exogenous) first protein-interacting protein labeled with a probe can be used in the form of a cell lysate. Cell lysates can be obtained by treating recombinant cells expressing the first protein-interacting protein labeled with a probe with a suitable solvent (e.g., a solvent including TX-100 or a cholesterol-based detergent). For example, the content of TX-100 can be 0.001%-1% or 0.2-5% (weight to weight), especially 0.5-2%, more especially 0.8-1.2%, and even more especially 1%, based on the total weight of the solvent. The cell lysate thus obtained is diluted before being applied to the substrate. In this regard, the cell lysate can be adjusted to contain 0.02-0.04% TX-100. When containing TX-100 at a concentration greater than the upper limit, the solvent can reduce the nonspecific binding signal appearing on the PEG-coated substrate, but reduce the measurement value (signal value) observed from the specific interaction between the first protein and the first protein-interacting protein, making protein analysis difficult. When the amount of TX-100 used is less than the lower limit, the dissolution of the first protein-interacting protein becomes unstable because the concentration of TX-100 present in the solvent is lower than the critical micelle concentration (CMC). Therefore, the nonspecific binding signal generated by the first protein-interacting protein on the PEG-coated substrate increases, and the measurement of invalid signals makes protein analysis difficult. The critical micelle concentration also applies to cholesterol-based detergents.

[0124] The concentration of the first protein-interacting protein in the cell lysate can be 10 -2 Up to 10 3 nM or 10 -1 Up to 10 2 It is within the nM range, but can be adjusted appropriately and is not limited.

[0125] Here, the BCL2 family protein targeted drug can be at least one substance selected from the group consisting of free proteins (e.g., antibodies, analogs thereof, etc.), nucleic acid molecules (e.g., siRNA, etc.) and small molecule compounds, each of which recognizes and / or inhibits BCL2 family proteins (e.g., anti-apoptotic proteins), and can be an apoptosis control drug. In one embodiment, the BCL2 family protein targeted drug can exhibit an anti-cancer effect on, for example, blood cancer. Examples of BCL2 targeted drugs (e.g., BCL2 inhibitors) include anti-BCL2 antibodies, venetoclax, and ABT737. As BCLxL targeted drugs (e.g., BCLxL inhibitors), for example, anti-BCLxL antibodies, ABT263, etc. can be used. MCL-1 targeted drugs (e.g., MCL-1 inhibitors) can be exemplified by anti-MCL-1 antibodies, AMG-176, AZD5991, and Maritoclax.

[0126] venetoclax ( or ) can be used to treat acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), etc.

[0127] Hereinafter, a detailed description of the present invention will be given.

[0128] The embodiments are explained with reference to venetoclax, which represents a BCL2 family protein targeted drug.

[0129] This paper proposes a diagnostic method for patients with blood cancer, which is based on quantifying the ability of BCL2 family proteins to inhibit apoptosis using the above-mentioned single-molecule protein interaction measurement method. The key technologies are as follows: (1) The observation of protein-protein interactions takes into account the various states of BCL2 family proteins present in cells, and (2) through the multifaceted observation of protein-protein interactions and the quantitative comparison of protein level observations, it is possible to select cell lines and patients that are sensitive to BCL2 family protein targeted drugs (such as venetoclax, AMG176 or AZD5991).

[0130] (1) Capturing BCL2 family proteins onto substrates by maintaining their intracellular state

[0131] Here, endogenous BCL2 family proteins are captured onto substrates while retaining their intracellular state.

[0132] In one embodiment, the typical blood cancer cell lines HL60 (acute myeloid leukemia) or OCI-LY3 (B-cell lymphoma) cell lines were cultured with venetoclax to induce apoptosis. Then, a test was performed to measure the apoptotic protein BAX with a displayed (exposed) N-terminus, which explains its activated conformation. It was observed that the level of BAX with a displayed N-terminus increased with the treatment of venetoclax. In contrast, after artificially exposing the N-terminus of the BAX protein by treating the cell lysates with TX-100, it was observed that detergent-activated BAX remained at similar levels as measured by the same test. It can be understood from the test that treatment with venetoclax increased the proportion of the activated conformation of BAX with an exposed N-terminus, although without changing the expression level of BAX (see Fig.17 ).

[0133] The results showed that the method proposed in this paper can reflect the complete intracellular conformation / PTM (post-translational modification) of BCL2 family proteins.

[0134] (2) Identification of protein-protein interactions (PPIs) that explain changes in the status of BCL2 family proteins in cells

[0135] Here, we demonstrate that changes in the status of BCL2 family proteins within cells can be explained by PPI measurements.

[0136] In one embodiment, OCI-LY3 or HL60 cells are treated with the BCL2 inhibitor venetoclax or the MCL1 inhibitor AZD5991, and then the changes in the interaction between the BCL2 family proteins (PPI) are compared. When applied to cells, inhibitors are expected to induce state changes (e.g., complex formation) in intracellular BCL2 family proteins because they selectively inhibit the interaction of BCL2 and MCL1 proteins with other proteins. In fact, cell lysates were prepared from OCI-LY3 cells after 24 hours of culture with or without venetoclax (BCL2 inhibitor) or AZD5991 (MCL-1 inhibitor). Then, BCL2 and MCL1 proteins were extracted from the cell lines and analyzed for their interactions with BAD and NOXA (both marked with GFP). The BCL2-BAD interaction (PPI) of the cell lysates treated with venetoclax was higher than that of the cell lysates not treated with any inhibitor (non-treated group). In addition, the interaction level between MCL1 protein and NOXA (Tag) from AZD5991-treated cell lysates was detected to be higher than that between MCL1 protein and NOXA (Tag) from the untreated group. In contrast, venetoclax treatment had no effect on the MCL1-NOXA interaction (PPI) and AZD5991 treatment had no effect on the BCL2-BAD interaction (PPI). The study also demonstrated that under the conditions used in this experiment, treatment with each drug did not cause significant changes in the expression levels of BCL2 and MCL1.

[0137] That is, the identification of specific proteins that undergo a change of state in response to a specific drug and / or proteins that do not undergo a change of state in response to a drug other than the specific drug demonstrates that the methods provided herein can analyze the exact state change caused by drug treatment.

[0138] Traditional studies have all been directed toward measuring the expression level of BCL2 protein to assess the intracellular apoptotic activity of BCL2. However, the present invention is characterized in that apoptosis inhibition is quantitatively analyzed by observing PPI (including the molecular characteristics of the protein).

[0139] (3) Predicting sensitivity to BCL2 family protein-targeted drugs by observing multiple aspects of protein-protein interactions and protein levels

[0140] Regarding the effects of pro-apoptotic and anti-apoptotic proteins on apoptosis of cells or patient samples, the interactions between endogenous and exogenous proteins in the sample (first protein-protein interactions), the interactions between proteins (both of which are endogenous to the sample (second protein-protein interactions)), and / or protein expression levels are quantitatively measured to analyze apoptotic signals from multiple aspects. Using the analytical data, the sensitivity to drugs targeting BCL2 family proteins (such as venetoclax) is determined.

[0141] The measurement of the interaction between endogenous and exogenous proteins is based on the interaction between the endogenous anti-apoptotic proteins of the sample (such as BCL2, BCLxL, MCL1, etc.) and the pro-apoptotic proteins (such as BIM, BAD, NOXA, BAX, BAK, etc.) provided externally (exogenous to the sample) (first protein-protein interaction, also called "first PPI"), and is used to quantitatively analyze the correlation with the responsiveness to BCL2 targeted inhibitors (such as venetoclax) or MCL1 targeted inhibitors (such as AMG 176). To this end, the pro-apoptotic proteins provided externally are labeled with probes.

[0142] The measurement of interactions between endogenous proteins (e.g., protein complex formation) is based on the interaction between anti-apoptotic proteins (such as BCL2, BCLxL, MCL1, etc.) and pro-apoptotic proteins (such as BAX, BAK, BIM, BAD, NOXA, etc.), both of which are endogenous to the same sample (second protein-protein interaction, also called second PPI), and is used to quantitatively analyze the correlation with sensitivity to BCL2 targeted inhibitors (such as venetoclax) or MCL1 targeted inhibitors (such as AMG 176).

[0143] Comparative analysis of the level of interaction between endogenous and exogenous proteins (first PPI) and / or interaction between endogenous proteins (second PPI) allows analysis of the correlation with sensitivity to BCL2 targeted inhibitors (such as venetoclax) or MCL1 targeted inhibitors (such as AMG 176). By comparative analysis, the state of endogenous anti-apoptotic proteins (such as BCL2, BCLxL, MCL1, etc.) in cells can be determined. For example, the degree of exposure of the active site where anti-apoptotic proteins interact with pro-apoptotic proteins can be measured. By measuring, the degree of binding and binding affinity between proteins, remaining binding sites, drug sensitivity, etc. can be indirectly estimated.

[0144] Here, the levels of endogenous anti-apoptotic proteins (such as BCL2, BCLxL, MCL1, etc.) or pro-apoptotic proteins (such as BAX, BAK, etc.) in the same cell are measured to determine the correlation with sensitivity to BCL2 targeted inhibitors (such as venetoclax) or MCL1 targeted inhibitors (such as AMG 176). The protein level can be the expression level of a protein, an activated protein, or a phosphorylated protein.

[0145] Measurements of protein-protein interactions provided herein can be based on single molecule pull-downs and / or co-immunoprecipitation (co-IP).

[0146] As used herein, the term "protein-protein interaction" (PPI) refers to the physical and / or chemical association between a first protein and a first protein-interacting protein, or the formation of a complex therebetween, and can be quantified according to at least one factor of, for example, binding frequency, binding strength (affinity) and binding time. In addition, the term "interaction (binding) between a first protein and a first protein-interacting protein" is intended to include interactions (binding) mediated by different proteins (between a first protein and a first protein-interacting protein in a signaling pathway) and direct interactions (binding) therebetween. Here, a protein-protein interaction can be a unimolecular reaction (a reaction between a molecule of the first protein and a molecule of the first protein-interacting protein).

[0147] As described above, the first protein as a BCL2 family protein (anti-apoptotic protein) can be BCL-2 (e.g., NCBI reference sequence NM_000633.2, etc.), BCLxL (e.g., NCBI reference sequence NM_138578.1, etc.) or MCL-1 (e.g., NCBI reference sequence NM_021960.3, etc.). The first protein-interacting protein that interacts with the first protein can be at least one selected from the group consisting of BIM (e.g., NCBI reference sequence B033694), BID (e.g., NCBI reference sequence NM_001196.2) or tBID (e.g., NCBI reference sequence NM_001196.2 N-terminal truncated form, etc.), BAD (e.g., NCBI reference sequence NM_004322.2, etc.), BAX (e.g., NCBI reference sequence NM_138761.3, etc.), NOXA (e.g., NCBI reference sequence NM_001382616.1) and BAK (e.g., NCBI reference sequence NP_001179.1, etc.).

[0148] In the methods provided herein, the step (a1; (i) and / or (ii)) of measuring the interaction between the first protein and the first protein-interacting protein can be performed ex vivo or in vitro on isolated cells or tissues.

[0149] The measurement of the present invention will be explained step by step in detail.

[0150] Step (1): Preparation of the first protein immobilization substrate (same as steps (i) and (ii))

[0151] In step (1), a substrate on which the first protein is immobilized is prepared by adding the first protein or a test sample containing the first protein to the substrate, and a substance capable of specifically binding to the first protein is anchored on the surface of the substrate.

[0152] The sample can be a cell or tissue isolated from a subject (patient), a lysate, homogenate or extract of a cell or tissue, or a body fluid (e.g., blood (whole blood, plasma or serum), saliva, etc.) from a subject. The patient may be a cancer patient, such as a blood cancer patient. The sample may contain cancer cells (or blood cancer cells). Specific examples are as described above.

[0153] Any material and / or structure can be applied to the substrate (crystalline or non-crystalline materials can be used) as long as it allows the first protein to be fixed to the surface of the substrate. In one embodiment, the substrate can be made of a material with a refractive index as high as or higher than the refractive index of water (about 1.3), because water accounts for the majority of biological substances. In one embodiment, the thickness of the substrate ranges from about 0.1 to about 1 mm, about 0.1 to about 0.5 mm, about 0.1 to about 0.25 mm, or about 0.13 to about 0.21 mm, and the refractive index is about 1.3 to about 2, about 1.3 to about 1.8, about 1.3 to about 1.6, or about 1.5 to about 1.54. The substrate can be made of any material that meets the refractive index range. For example, the substrate can be made of a material selected from the group consisting of glass (refractive index: about 1.52) and quartz, but is not limited thereto. The substrate can be in any form commonly used for observing biological samples, and can be a well type, a slide type, a channel type, an array type, a microfluidic chip type, or a microtube (capillary) type, but is not limited thereto. For microscopic observation, a cover glass may be mounted on the substrate to which the sample is applied. The material for the cover glass is as described above for the substrate, and may be as thin as or thinner than the substrate (eg, refractive index 1.52, thickness 0.17 mm, but not limited thereto).

[0154] The substance that specifically binds to the first protein can be any substance that can bind to the first protein, i.e., BCL2, BCLxL, MCL1, or all of them. For example, the substance that specifically binds to the first protein can be selected from at least one of an antibody, an antigen-binding fragment thereof (e.g., scFv, (scFv)2, scFv-Fc, Fab, Fab', F(ab')2, etc.), an aptamer (protein or nucleic acid molecule), and a small molecule compound, each of which specifically binds to BCL2, BCLxL, MCL1, or all of them. In this regard, the substance that specifically binds to the first protein (BCL2, BCLxL, MCL1, or all of them) can act on a site that does not interfere with the interaction between the first protein and the first protein-interacting protein, i.e., a site other than the site at which the first protein interacts (binds) with the first protein-interacting protein.

[0155] In one embodiment, the surface of the substrate can be appropriately modified to include (anchoring) biological substances (such as antibodies, etc.) that specifically bind to the first protein thereon, or the substrate can have a surface to which the substance that specifically binds to the first protein is anchored. For surface modification, one side of the substrate can be treated (for example, coated) with any compound having a functional group, which can maintain the biological substance (for example, antibodies, etc.) that specifically binds to the first protein. For example, a compound treatment substrate containing a functional group selected from the group consisting of an aldehyde group, a carboxyl group, and an amine group can be used. In one embodiment, a compound containing a functional group selected from the group consisting of an aldehyde group, a carboxyl group, and an amine group can be selected from at least one of the groups consisting of biotin, bovine serum albumin (BSA), biotinylated bovine serum albumin, polyethylene glycol (PEG), biotinylated PEG (PEG-biotin) and polysorbate (for example, Tween 20), but is not limited thereto. The surface-treated substrate can be further treated (for example, coated) with at least one selected from the group consisting of neutravidin, streptavidin, and avidin.

[0156] Step (2): reacting the first protein with the first protein-interacting protein

[0157] Step (2) can be performed by the following steps (2-1) and / or step (2-2).

[0158] Step (2-1): Interaction between endogenous protein and exogenous protein in the sample (the first step of step (i) Protein-protein interactions

[0159] In step (2-1), a probe-labeled (attached) first protein-interacting protein (exogenous protein) is added to the substrate (e.g., prepared in step (1)) with the pulled-down first protein (endogenous protein) to allow reaction between them. The signal generated by the probe can be measured to determine the level of complex formed between the endogenous protein and the exogenous protein, which indicates the level of interaction between the endogenous protein and the exogenous protein.

[0160] The labeled first protein-interacting protein refers to a form labeled with a probe that produces a detectable signal (chemical (e.g., covalent or non-covalent), recombinant, or physically coupled to the probe) or is bound to a label to which the probe can bind. The detectable signal can be any signal that can be measured by detecting a typical enzyme reaction, fluorescence, brightness, and / or radiation (e.g., light, radioactive rays, etc.). The probe can be any one selected from the group consisting of small molecule compounds, proteins, peptides, and nucleic acid molecules, all of which can produce a signal. For example, the probe can be at least one selected from the group consisting of fluorescent dyes (small molecule compounds; cyanine (Cyanine), Alex, Dylight, Fluoprobes, etc.) and fluorescent proteins (e.g., green fluorescent protein (GFP, enhanced GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), cherry fluorescent protein (mcherry), etc.). Any label, such as His-tag / Ni-NTA, can be used as long as it is generally available in the art. In order to allow accurate and easy detection without generating noise, the concentration of the probe can be appropriately determined in the range of up to about 1 μM, for example, 1 nM to 1000 nM, 1 nM to 500 nM, 1 nM to 100 nM, 10 nM to 1000 nM, 10 nM to 500 nM or 10 nM to 100 nM, but not limited thereto. The signal generated by the probe can be measured by any common detector (e.g., fluorescence microscope, fluorescence camera, fluorescence intensity measurement (quantification) device, etc.).

[0161] Step (2-2): Interaction between endogenous proteins (second protein-protein interaction in step (ii))

[0162] In step (2-2), a substance that binds to the first protein-interacting protein (a binding substance labeled with a probe) is added to the substrate with the pulled-down first protein (e.g., prepared in step (1)) so that the binding substance reacts with the first protein-interacting protein of the complex formed between the first protein (endogenous) and the first protein-interacting protein (endogenous). The signal generated by the probe can be measured to determine the level of the complex in the sample, which indicates the level of endogenous protein-protein interaction.

[0163] The substance that binds to the first protein-interaction protein refers to a probe that produces a detectable signal (chemically (e.g., covalently or non-covalently), recombinantly or physically coupled to the first protein-interaction protein) or can be bound to a label. The detectable signal can be any signal that can be measured by detecting a typical enzyme reaction, fluorescence, brightness and / or radiation (e.g., light, radioactive rays, etc.). The probe can be any one selected from the group consisting of small molecule compounds, proteins, peptides and nucleic acid molecules, all of which can produce a signal. For example, the probe can be at least one selected from the group consisting of fluorescent dyes (small molecule compounds; cyanine (Cyanine), Alex, Dylight, Fluoprobes, etc.) and fluorescent proteins (e.g., green fluorescent protein (GFP, enhanced GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), cherry fluorescent protein (mcherry), etc.). As long as it is generally available in the art, any label can be used, such as His-tag / Ni-NTA. In order to allow accurate and easy detection without generating noise, the concentration of the probe can be appropriately determined in the range of up to about 1 μM, for example, 1 nM to 1000 nM, 1 nM to 500 nM, 1 nM to 100 nM, 10 nM to 1000 nM, 10 nM to 500 nM or 10 nM to 100 nM, but not limited thereto. The signal generated by the probe can be measured by any common detector (e.g., fluorescence microscope, fluorescence camera, fluorescence intensity measurement (quantification) device, etc.).

[0164] In order to more accurately measure the protein-protein interaction, the method may further include a washing step of the reacted substrate between the reaction step (step (2)) and the subsequent protein-protein interaction measurement step (step (3)).

[0165] Step (3): Protein-protein interaction measurement

[0166] In step (3), a signal is measured from the reaction mixture in step (2) (i.e., (2-1) and / or (2-2)). Signal measurement can be performed using any means capable of detecting (measuring or identifying) a signal generated by the probe used in step (2) (e.g., a generally detectable signal such as an enzyme reaction, fluorescence, brightness, or radiation).

[0167] In one embodiment, the measurement of protein-protein interaction in step (3) can be based on real-time analysis.

[0168] In one embodiment, when the signal is a fluorescent signal, signal detection can be achieved by imaging and / or quantifying the fluorescent signal generated by the probe that provides absorbable light using a fluorescent microscope, a fluorescent (measurement) camera and / or a fluorescence intensity measurement (quantification) device.

[0169] When the signal is a fluorescent signal, step (3) (protein-protein interaction measurement step) may include the following sub-steps:

[0170] (i) providing a light beam from a light source to the reaction mixture of step (2); and

[0171] (ii) detecting a fluorescent signal generated in response to providing a light beam from a light source.

[0172] In the sub-step (i) of providing a light beam from a light source, in step (2), a light beam from a light source is provided to the reaction mixture of the first protein and the first protein-interacting protein. As long as this goal is achieved, the time for providing the light beam from the light source is not limited. For example, the light beam may be provided continuously for a period of time before step (1), simultaneously with step (1), or after step (1) until after step (2), or the light beam may be provided for a predetermined period of time before step (2), simultaneously with step (2), or just after step (2), but is not limited thereto.

[0173] Any light source may be used as long as it emits a wavelength corresponding to a fluorescent signal, and a laser, a halogen lamp, etc. may be exemplified.

[0174] In one embodiment, the step (3) of measuring protein-protein interaction can be performed by providing a light beam using a total internal reflection fluorescence (TIRF) microscope or a confocal microscope and observing the fluorescence signal. In another embodiment, the total internal reflection fluorescence microscope can be equipped with a fluorescence (measurement) camera for signal imaging, such as an EMCCD (electron multiplying charge coupled device) or a CMOS (complementary metal oxide semiconductor), and can be used to provide a light beam, an image and / or quantify the fluorescence signal.

[0175] The step (3) of measuring protein-protein interactions will be described in more detail below with reference to an example using a total internal reflection fluorescence microscope and a fluorescence camera, as follows:

[0176] A) mounting the substrate of step (1) or (2) on a TRIF microscope. In a TRIF microscope, regardless of the position of the light source, when the lens is positioned, the light source is usually directed toward the substrate to be observed;

[0177] B) The light source may be a laser having an intensity ranging from about 0.5 mW to about 5 mW, about 0.5 mW to about 4.5 mW, about 0.5 mW to about 4 mW, about 0.5 mW to about 3.5 mW, about 0.5 mW to about 3 mW, about 0.5 mW to about 2.5 mW, about 0.5 mW to about 2 mW, about 1 mW to about 5 mW, about 1 mW to about 4.5 mW, about 1 mW to about 4 mW, about 1 mW to about 5 mW, about 1 mW to about 5 mW, about 1 mW to about 4.5 mW, about 1 mW to about 4 mW, about 1 mW to about 5 mW. The wavelength of the light source may be about 3.5 mW, about 1 mW to about 3 mW, about 1 mW to about 2.5 mW, about 1 mW to about 2 mW, about 1.5 mW to about 5 mW, about 1.5 mW to about 4.5 mW, about 1.5 mW to about 4 mW, about 1.5 mW to about 3.5 mW, about 1.5 mW to about 3 mW, about 1.5 mW to about 2.5 mW, or about 1.5 mW to about 2 mW, and its intensity may be about 2 mW. As previously described, the wavelength of the light source may be appropriately selected according to the configuration of the fluorescent signal, the probe used, and / or the device (e.g., an attenuation filter allows the use of a high-intensity light source).

[0178] C) The fluorescence signal generated by providing a light beam from a light source can be captured, imaged and / or quantified by a fluorescence (measurement) camera.

[0179] Taking into account the retention time (luminescence time, lifetime) of the fluorescent signal generation of the probe, the capture (or imaging) of the fluorescent signal can be performed simultaneously with the provision of the light source, or can be performed within the retention time of the signal generation.

[0180] For capturing (or imaging) fluorescent signals using a fluorescent camera (e.g., an EMCCD camera), the exposure time, laser power, camera gain value, total imaging frames, etc. can be appropriately controlled. For example, a shorter exposure time per frame accumulates the signal over one frame to a smaller extent. To offset this, the laser power or the sensitivity of the fluorescent camera can be increased. In one embodiment, the exposure time of each frame can be set to about 0.001sec to about 5sec, about 0.001sec to about 3sec, about 0.001sec to about 2sec, about 0.001sec to about 1sec, about 0.001sec to about 0.5sec, about 0.001sec to about 0.3sec, about 0.001sec to about 0.1sec, about 0.01sec to about 5sec, about 0.01sec to about 3sec, about 0.01sec to about 2sec, about 0.01sec to about 1sec, about 0.01sec to about 0.5sec, about 0.01sec to about 0.3sec, about 0.01sec to about 0.1sec, about 0.05sec to about 5sec, about 0.05sec to about 3sec, about 0.05sec to about 0. About 2sec, about 0.05sec to about 1sec, about 0.05sec to about 0.5sec, about 0.05sec to about 0.3sec, about 0.05sec to about 0.1sec, about 0.07sec to about 5sec, about 0.07sec to about 3sec, about 0.07sec to about 2sec, about 0.07sec to about 1sec, about 0.07sec to about 0.5sec, about 0.07sec to about 0.3sec, about 0.07sec to about 0.1sec, about 0.1sec to about 5sec, about 0.1sec to about 3sec, about 0.1sec to about 2sec, about 0.1sec to about 1sec, about 0.1sec to about 0.5sec, or about 0.1sec to about 0.3sec, for example, about 0.1sec, but not limited to.

[0181] For example, when using an EMCCD camera, the photons generated from a probe (such as eGFP) are measured in the form of electrons converted by an EMCCD element (photoelectric effect). The number of electrons generated by each photon can be changed by a gain value. The higher the gain value is set, the more the number of electrons generated by each photon is, which causes the sensitivity of the EMCCD to increase, and the background noise also increases. Therefore, the signal-to-noise ratio may be very important. In one embodiment, the gain value may be set to fall within the following range, but is not limited to about 10 to about 100, about 10 to about 80, about 10 to about 60, about 10 to about 50, about 20 to about 100, about 20 to about 80, about 20 to about 60, about 20 to about 50, about 30 to about 100, about 30 to about 80, about 30 to about 60 or about 30 to about 50, such as about 40, to obtain a good signal-to-noise ratio. However, considering camera sensitivity and life, device configuration conditions, noise test conditions, etc., the gain value can be appropriately selected.

[0182] The total number of image frames is multiplied by the exposure time to obtain the total imaging time (exposure time x total number of frames = imaging time). Because the fluorescent signal disappears after the emission time (lifetime) of the fluorescent material, the total number of image frames and / or the exposure time can be adjusted to allow imaging within the emission time (lifetime).

[0183] In order to improve the accuracy of protein-protein interaction measurement, one or more (e.g., two or more, three or more, four or more, five or more, seven or more, or ten or more) substrates (the upper limit can be determined according to the size of the substrate and the area allowed for imaging) or one or more channels (each channel includes two or more substrates) are imaged. The fluorescent signal obtained can be quantified by measuring the number of signal expression points (also called PPI complexes), which is considered to be a method for quantifying protein-protein interactions.

[0184] In another embodiment, the quantification of protein-protein interaction can be achieved by digitizing the fluorescence intensity measured in step (3) with the aid of a typical device. For the accuracy of the data, the measured values ​​can be standardized with reference to the sample amount or the amount of protein in the sample.

[0185] When two or more different first proteins and / or first protein-interacting proteins are used, steps (1) to (3) may be performed for each of the combinations of a single first protein and a first protein-interacting protein (i.e., steps (1) to (3) may be repeated several times corresponding to the number of combinations of first proteins and first protein-interacting proteins).

[0186] The method provided herein may further comprise the following step (4).

[0187] Step (4): Comparison step (step (b), more specifically (b1))

[0188] In step (4), the result obtained in step (3) (the level of protein-protein interaction) is compared with the level of interaction between the first comparison protein and the first comparison protein interacting protein or with a reference value. The first comparison protein, the first comparison protein interacting protein and the reference value are as described above.

[0189] As used herein, the expression "targeting BCL2 family proteins" means recognizing (specifically binding to) BCL2 family proteins and / or inhibiting the activity of BCL2 family proteins. Inhibiting the activity of BCL2 family proteins means reducing or eliminating the inherent functions of BCL2 family proteins, such as anti-apoptotic functions, by binding to BCL2 family proteins and / or degrading and / or structurally modifying BCL2 family proteins.

[0190] The term "drug" as used herein refers to any substance having a pharmacological effect, for example, at least one selected from the group consisting of small molecule compounds, proteins (e.g., antibodies, antibody fragments, analogs thereof, etc.), peptides, nucleic acid molecules (e.g., DNA, RNA (i.e., siRNA, microRNA, shRNA, etc.), PNA (peptide nucleic acid), aptamers, etc.), plant extracts, animal extracts, and cell extracts.

[0191] Here, all externally provided proteins, such as first protein interaction proteins, fluorescent proteins as probes, antibodies as binding substances, etc., can be produced by recombination or chemical synthesis, but are not limited to this. In one embodiment, recombinant production may include transfecting a gene encoding a protein of interest or a vector carrying the gene into a mammalian cell (such as a HEK293 cell), an insect cell (such as SF9) or a bacterial cell (such as Escherichia coli), and then allowing the cell to express the protein. The expressed protein may undergo a further purification process, but this may not be necessary. The purified protein or cell lysate containing the expressed protein can be used as is for measuring protein-protein interactions.

[0192] As used herein, the terms "BCL2 family protein targeted therapy" and "treatment using a BCL2 family protein targeted drug" can be used interchangeably with each other, have the same meaning, and are intended to cover all medical and / or pharmaceutical actions that result in the inhibition of the activity of a first protein. For example, treatment may include prescribing and / or administering a drug that inhibits the activity of a BCL2 family protein to a subject in need. In more detail, "BCL2 family protein targeted therapy" may include prescribing and / or administering a drug that binds to, degrades and / or structurally modifies a BCL2 family protein to reduce or eliminate intrinsic functions, such as anti-apoptotic functions, to a subject in need.

[0193] Administration can be by oral or parenteral route. Parenteral administration can be carried out by intravenous, subcutaneous, intramuscular, intraperitoneal, intradermal, local, intranasal, intrapulmonary or rectal administration.

[0194] Examples of individuals, patients or subjects can include all mammals, for example, primates, such as humans, monkeys, etc., rodents, such as mice, rats, etc., and can be patients with a first protein-related disease. The first protein-related disease can be a disease associated with overexpression of the first protein or with activation of a signaling pathway, wherein the first protein is involved in cells or tissues, such as cancer and more specifically blood cancer. In one embodiment, the individual, patient or subject can be a cancer patient, especially a blood cancer patient.

[0195] As used herein, the term "responsiveness to a drug" refers to the degree to which a drug exhibits an effect in an individual to whom the drug is administered.

[0196] As used herein, the term "effect" or "efficacy" refers to the medical and / or pharmaceutical effect that a drug or therapy is intended to achieve on a treated subject, and may mean preventing and / or treating a subject's disease and / or reducing and / or alleviating disease symptoms. For example, when the drug, therapy, and subject are anticancer agents, anticancer treatments, and cancer patients, respectively, the effect may be an anticancer effect (preventive and / or therapeutic effect on cancer). Anticancer effects may include preventing migration, invasion, and / or metastasis of cancer, inhibiting cancer progression, and / or reducing or eliminating cancer resistance, and inhibiting the growth of cancer cells.

[0197] In another embodiment, an analysis device is provided for predicting the effect of a BCL2 family protein targeted drug and / or for the effect of treatment using a BCL2 family protein targeted drug.

[0198] The analytical device may include:

[0199] a reaction portion including the first protein or a substance for capturing the first protein by specifically binding to the first protein; and

[0200] Optionally, a reagent and / or signal detection device for detecting the first protein interaction protein. The reagent for detecting the first protein interaction protein comprises a substance that (specifically) binds to the first protein interaction protein. The binding substance is a binder (e.g., an antibody, an antigen binding fragment, an antibody analog, an aptamer, a small molecule compound, etc.) that binds to a site different from the first protein interaction protein and the first protein interaction (binding) and can be labeled with a probe.

[0201] The first protein, as described above, can be used as a purified protein or contained in a cell or cell lysate. The reaction portion can include the first protein, cell or cell lysate (each including the protein, cell or cell lysate), or a substrate having the protein, cell or cell lysate fixed thereon.

[0202] The analytical device may be adapted to qualitatively and / or quantitatively determine the interaction between the first protein and the first protein-interacting protein.

[0203] The reaction part may include a substrate on which a substance for capturing the first protein is fixed, or the first protein is fixed to the substrate directly or through a capturing substance. The substance for capturing the first protein may be a substance that specifically binds to the first protein, such as an antibody or an antigen binding fragment. The reaction part may be in a well (e.g., multi-well) form, a slide form, a channel form, an array form, a microfluidic chip form, or a microtube (capillary) form, but is not limited thereto.

[0204] The analytical device may be suitable for measuring the interaction between the first protein and the first protein-interaction protein. In this case, the analytical device may also include the first protein-interaction protein. In one embodiment, the first protein-interaction protein may be labeled with a probe that produces a detectable signal (e.g., chemistry (e.g., covalent or non-covalent), recombinant or physically coupled to a probe), or may have a label attached thereto that can be coupled to a probe.

[0205] In addition, the protein-protein interaction measuring device can also include a probe that is bonded to the first protein and a marker substance that can be coupled to the probe, so that the signal value measured by the signal detection device is standardized to the level of the first protein. The probe that is bonded to the first protein can be a biomolecule (such as an antibody), which is bonded to the first protein and is different from the position of the capture material contained in the aforementioned porous. The marker substance can be coupled to the probe that produces a detectable signal (for example, chemically (for example, covalently or non-covalently) is coupled to the probe), and can be a biomolecule (for example, an antibody) that can be coupled to the probe that is bonded to the first protein.

[0206] The signal detection device can be any signal detection device that is usually used according to the signal generated by the probe used. For example, the signal detection device may include a signal stimulation unit and a signal detection unit. In addition, a signal analysis unit for analyzing (e.g., quantifying or imaging) the measurement signal may be included. In one embodiment, signal stimulation, signal detection and signal analysis can be performed in various regions. Alternatively, at least two of signal stimulation, detection and analysis can be performed simultaneously or continuously in one region. In one embodiment, when the probe is a fluorescent material, the signal detection device can be selected from all devices capable of generating and detecting fluorescent signals, and may include, for example, a fluorescent signal stimulation unit (e.g., a light source) and a fluorescent signal detection unit, and / or a fluorescent signal analysis unit. In one embodiment, the signal detection device may include a total internal reflection fluorescence (TIRF) microscope or a confocal microscope (light source and fluorescent signal detection) and an optional fluorescent camera, for example, an electron multiplying charge coupled device (EMCCD) camera or a complementary metal oxide semiconductor (CMOS) camera, to provide a light source, an image and / or quantify the fluorescent signal. The wavelength and intensity of the light source and the measurement conditions of the fluorescent camera (e.g., exposure time per frame, laser power, camera gain value, total number of frames, etc.) are as described above.

[0207] The advantage of the protein-protein interaction measuring device provided herein is that, even with a small amount of sample, the interaction between the first protein and the first protein-interacting protein and / or the expression level of the first protein and the first protein-interacting protein can be accurately and effectively observed, analyzed, detected and / or measured. Therefore, even with a very small amount of biopsy (e.g., needle biopsy) sample, porous and porous analytical methods can be effectively applied.

[0208] Beneficial Effects of the Invention

[0209] BCL2 family protein targeted drugs, such as venetoclax, etc., can be used to treat various blood cancer patients. For example, venetoclax can be used to treat acute leukemia and bone marrow cancer. However, in order to reduce the burden on patients, a diagnostic technique for predicting medicinal efficacy is needed. Traditional methods for determining the expression level of a single BCL2 protein and the presence or absence of mutant genes cannot successfully predict the efficacy of BCL2 family protein targeted drugs (such as venetoclax, etc.). On the contrary, the method based on protein-protein interaction and / or protein expression provided herein can successfully predict whether BCL2 family protein targeted drugs, such as venetoclax, etc., are effective in individual patients with various blood cancers in a fast and specific manner, thereby allowing for fast and effective customized personal treatment for blood cancers.

[0210] BRIEF DESCRIPTION OF THE DRAWINGS

[0211] Figure 1a and Figure 1b The measurement of the interaction between individual antibodies immobilized on a substrate and a target protein is shown. Figure 1a Involvement of BCL2, BCLxL, and MCL1, Figure 1b Involvement of BCL-W and Bfl-1.

[0212] Figure 2a Shown are the PPI counts between endogenous proteins BCL2, MCL1, and BCLxL and exogenous proteins BIM, NOXA, and BAD. Figure 2b Shown are the PPI counts between the endogenous proteins BCL2 and BCL-XL and the exogenous protein tBID. Figure 2c The PPI counts between endogenous proteins BCL2, MCL1, and BCL-XL and exogenous proteins BAX and BAK are shown. Figure 2d Shown are the PPI counts between endogenous BCL2, BCL-XL, and MCL1, and the BH3-only domain of exogenous BIM protein as probe.

[0213] Figure 3 The figure shows the change in PPI count according to the concentration of the first protein-interacting protein by measuring the interaction between endogenous BCL2 and exogenous BAD.

[0214] Figure 4 It is shown that by measuring the interaction between endogenous BCL2 and exogenous BAD, the PPI count changes according to the type and concentration of detergent contained in the externally provided solution.

[0215] Figure 5 The graph shows changes in PPI counts according to the concentration of detergent Triton X-100 contained in an externally provided solution by measuring the interaction between endogenous BCL2 and exogenous BAD.

[0216] Figure 6 It is shown that when the exogenous BIM protein is mutated at amino acids that are highly conserved between species, the PPI counts between BIM variants and BCL-XL or BCL2 differ between variants.

[0217] Figure 7 Shown are counts of complexes between BCL2, MCL1 or BCL-XL and BIM, all of which are endogenous to the sample (HL60 and OCI-LY3).

[0218] Figure 8 Shown are complex counts between BCL2 and BAX, both endogenous to the sample (OCI-LY3).

[0219] Fig. 9Shown are counts of complexes between BCL2, BCL-XL or MCL1 and BAK, all of which are endogenous to the sample (OCI-LY3).

[0220] Fig.10 Shown are counts of complexes between BCL-XL and BAD, and between MCL1 and NOXA, all of which are endogenous to the sample (HL60, OCI-LY3, and SUDHL8).

[0221] Fig.11 It is shown that the first protein and the first protein-interacting protein are interchangeable with each other when measuring protein complex counts.

[0222] Fig.12a It is shown that two different types of BAK antibodies can both bind to BAK. Figure 12b Signal measurements obtained using the indicated pull-down antibodies (Abs) for cells (HEK293) co-expressing [MCL1, BAK], [BCL-XL, BAK], or [BCL2, BAK] are shown, demonstrating that the D4E4 cloned BAK antibody is able to bind to BAK and no longer bind to BCL2 family proteins when co-expressed. Fig.12c Shows the use of Fig.12a One of the antibodies used in the assay, AT38E2, detects the co-expressed BAK protein.

[0223] Fig.13 Shown are the expression levels of MCL1, BCL-XL, and BCL2, all of which are endogenous to the samples.

[0224] Fig.14 Shown are the expression levels of BAX and BAK, both endogenous to the samples.

[0225] Fig.15 Shown are BAX expression levels measured using detection antibodies after addition of therapeutic agents (Staurosporine and Venetoclax) in GDN-treated samples (a), Triton X-100-treated samples (b), GDN-treated samples (c), and Triton X-100-treated samples (b).

[0226] Fig.16 Shown are BAX expression levels of specific folding conformations in samples with respect to treatment time with venetoclax (a and c) and Triton X-100 (b and d).

[0227] Fig.17Shown are the PPI counts between endogenous BCL2 (HL60) and exogenous protein BAD in samples after treatment with venetoclax or AMG176 (a), and the PPI counts between endogenous BCL2 (OCI-LY3) and exogenous BAD in samples after treatment with venetoclax or AMG176.

[0228] Fig.18 Shown are the PPI counts between endogenous MCL1 (HL60) and exogenous NOXA in samples after treatment with venetoclax and AMG176 (a), and the PPI counts between endogenous MCL1 (OCI-LY3) and exogenous NOXA in samples after treatment with venetoclax or AMG176.

[0229] Fig.19 Shown are the PPI counts between endogenous BCL-XL (HL60) and exogenous BAD in samples after treatment with venetoclax and AMG176 (a), and the PPI counts between endogenous BCL-XL (OCI-LY3) and exogenous BAD in samples after treatment with venetoclax or AMG176.

[0230] Fig. 20 Shown are the expression levels of BCL2, MCL1, and BCL-XL in a sample (HL60) after treatment with venetoclax and AMG176 (a), and the expression levels of BCL2, MCL1, and BCL-XL in a sample (OCI-LY3) after treatment with venetoclax or AMG176 (b).

[0231] Fig.21 Shown are the complex counts between endogenous BCL2, MCL1 or BCL-XL and BIM in the sample (HL60) after treatment with venetoclax or AMG176 (a), and the complex counts between endogenous BCL2, MCL1 or BCL-XL and BIM in the sample (OCI-LY3) after treatment with venetoclax or AMG176.

[0232] Fig. 22 Shown are the complex counts between MCL1 and NOXA, both endogenous to the sample (HL60), after treatment with venetoclax or AMG176 (a), and the complex counts between MCL1 and NOXA, both endogenous to the sample (OCI-LY3), after treatment with venetoclax or AMG176.

[0233] Fig.23Shown are the complex counts between BCL-XL and BAD, both endogenous to the sample (HL60), after treatment with venetoclax or AMG176 (a), and the complex counts between BCL-XL and BAD, both endogenous to the sample (OCI-LY3), after treatment with venetoclax or AMG176.

[0234] Fig.24 Shown are the complex counts between BCL2 and BAX, both endogenous to the sample (HL60) (a), and the complex counts between BCL2 and BAX, both endogenous to the sample (OCI-LY3), in relation to the time of venetoclax treatment.

[0235] Fig.25 Shown are BAX expression levels in sample (HL60) (a) and sample (OCI-LY3) (b) in relation to venetoclax treatment time.

[0236] Fig.26 Shown are BAK expression levels in sample (HL60) (a) and sample (OCI-LY3) (b) in relation to venetoclax treatment time.

[0237] Fig. 27 PPI counts between endogenous BCL2 and exogenous BIM of samples (HL60) according to the dose of venetoclax or AZD5991 are shown.

[0238] Fig.28 PPI counts between endogenous MCL1 and exogenous BIM in samples (HL60) according to the dose of venetoclax or AZD5991 are shown.

[0239] Fig.29 is a heat map showing the Z-scores of the measured results of parameters (interaction (first PPI), complex (second interaction) and expression level) in samples of AML patients (n=32).

[0240] Fig.30 Shown is the quantitative measurement of BCL2 expression in samples from breast cancer patients (n=302).

[0241] Fig.31 Shown are the quantitative measurements of BCL2 expression in samples from lung cancer patients (n=15).

[0242] Fig.32Shown are PPI counts and complex counts between endogenous protein BCL2 and exogenous protein BIM in relation to treatment time of a sample (OCI-LY3) using venetoclax.

[0243] Fig.33 Shown are the PPI counts and complex counts between the endogenous protein MCL1 and the exogenous protein NOXA in relation to the treatment time of the sample (OCI-LY3) with AZD5991.

[0244] Fig.34 Shown are PPI counts between BCL2, MCL1, or BCL-XL, all endogenous to samples from patient 1, and the exogenous protein BIM, as measured in i) bone marrow obtained at diagnosis, ii) bone marrow obtained again after treatment with a FLT3-targeted anticancer agent (quizartinib), and iii) whole blood samples obtained after treatment with venetoclax and LDAC.

[0245] Fig.35 Shown are PPI counts between BCL2, MCL1, or BCL-XL, all endogenous to samples from patient 2, and exogenous BIM, as measured in i) bone marrow obtained at diagnosis, ii) bone marrow obtained again after treatment with a FLT3-targeted anticancer agent (quizartinib), and iii) whole blood samples obtained after treatment with venetoclas and LDAC.

[0246] Figures 36a to 36c The results of correlation analysis of responsiveness to venetoclax (vertical axis) in samples from AML patients (n=32) and 21 parameters including PPI (first PPI), complex (second PPI), expression level, etc. are shown.

[0247] Fig.37 Shown are the results of correlation analysis of responsiveness to venetoclax in samples from AML patients with parameters including the BCL2-BIM PPI (first PPI) and the BCL2-BIM complex (second PPI).

[0248] Figures 38a to 38c Predictions of responsiveness to venetoclax based on parameter combinations are shown. Fig.38a The correlation coefficients are shown when the number of parameters increases from 1 to 10. Figure 38b The results of the correlation analysis of the combination of two indicators compared with the single indicators are shown. Fig.38c Correlation analysis results for combinations of five parameters are shown.

[0249] Figures 39a to 39cThe results of correlation analysis of responsiveness to AZD5991 in samples from AML patients (n=26) and 21 parameters including PPI (first PPI), complex (second PPI), expression level, etc. are shown.

[0250] Fig.40 The prediction results of responsiveness to AZD5991 using a combination of four main indices are shown. DETAILED DESCRIPTION

[0251] Example

[0252] Hereinafter, the present invention will be described in detail by way of examples.

[0253] The following examples are intended only to illustrate the present invention and are not to be construed as limiting the present invention.

[0254] Example 1. Measurement of protein-protein interactions (PPI) between endogenous and exogenous proteins

[0255] 1.1. Preparation of analytical lysates (samples)

[0256] From bone marrow samples obtained from patients with various blood cancers such as chronic lymphocytic leukemia (CLL) and acute myeloid leukemia, only the buffy coat was selectively collected using a Ficoll gradient separation protocol (GE healthcare Ficoll-Paque plus 17-1440-03).

[0257] The separated blood clot yellow layer pellet was washed with dPBS and then separated with 500g dPBS for 5 minutes. This step was repeated twice to remove as many red blood cells and platelets as possible remaining in the blood clot yellow layer. 7 The cells were aliquoted and the aliquots were snap frozen in liquid nitrogen and stored at -80 °C until used in experiments.

[0258] Blood cancer cells (Korean Cell Line Bank and ATCC; HL-60 (CVCL_0002), OCI-LY3 (CVCL_8800)), Ramos (CVCL_0597), U937, THP1 and SUDHL8) and blood cancer cell pellets from the buffy coat of patients were suspended in lysis buffer (HEPES 50nM, NaCl 150nM, glycerol 10% (w / v) GDN (digitonin) 1% (w / v) and 2% (w / v) phosphatase inhibitor cocktail 2 / 3 (P5726, P0044)) and spun three times at regular intervals of 10 minutes. After 30 minutes, the lysis buffer containing the pellet homogenate was centrifuged at 15000g for 10 minutes. The supernatant thus formed was used as the analytical lysate and applied to the substrate in subsequent experiments.

[0259] 1.2. Preparation of probe-labeled first protein-interacting proteins

[0260] Probe-labeled first protein interacting proteins (probe proteins) were generated by transient transfection. Briefly, after digestion of pEGFP-C1 (Addgene: PT3028-5) with restriction endonucleases, genes encoding proteins BIM (Sino biological: HG13819-G), NOXA (Sino biological: HG16548-U) and BAD (Sino biological: HG10020-M) were inserted into the digested plasmids to construct GFP expression protein vectors. The expression vectors were transiently transfected into HEK293T cells (ATCC; CRL-3216) using polyethyleneimine to provide overexpressed GFP-tagged proteins.

[0261] HEK293T cells expressing GFP-tagged proteins were lysed in 1% TX100 lysis buffer. The cell lysate thus prepared was diluted to a concentration of 165 nM in 1% TX100 lysis buffer. Aliquots of the dilution were snap-frozen and thawed before the experiment. Finally, cell lysates containing the labeled first protein-interacting protein were prepared to have a concentration of 0.03% TX-100.

[0262] The method of the present invention was used to prepare BID, or a first protein-interacting protein labeled with only the BH3 domain of BIM, BAX or BAK. Table 1 below shows the first protein-interacting proteins (human derived) used in the following examples, as well as their Uniprot IDs and amino acid regions.

[0263] Table 1

[0264] first protein interacting protein Uniprot ID BAD Q92934 NOXA Q13794 BIM Q43521 BID P55957 BIM_BH3 Q43521(AA 134-166) BAX_BH3 Q07812(AA 59-73) BAK_BH3 Q16611(AA 74-88)

[0265] As a probe of the first protein-interacting protein, green fluorescent protein (hereinafter referred to as "GFP"; labeled using pEGFP-C1 vector (Cloning Technology)) was used to link eGFP to the C-terminus of the first protein-interacting protein.

[0266] 1.3. Substrate preparation

[0267] Glass coverslips with dimensions of 45 mm x 60 mm were coated with a 3:100 (w:w) mixture of biotin-PEG:mPEG.

[0268] To serve as a medium for pulling down BCL2 family proteins (primary protein) to the PEG-coated surface of the substrate, NTV (neutravidin; Thermo, A2666) and biotinylated goat anti-rabbit secondary antibody (111-065-046 Jackson Immunoresearch) were used.

[0269] After coupling NTV and secondary antibodies to the PEG-coated substrate, primary antibodies (see Table 2 below) that pull down the corresponding BCL2 family protein (first protein) are applied to the substrate.

[0270] A 10 μl amount of lysate of proprietary cells and cell lines (Korean Cell Line Bank and ATCC; HL-60, U937, THP1, OCI-LY3 and SUDHL8) was placed in each well coated with antibodies (see Table 2) to pull down the protein to be observed and incubated at room temperature for one hour, and then the excess lysate was washed with washing buffer (HEPES 50mM NaCl 150mM glycerol 1% GDN 0.03%).

[0271] Table 2

[0272]

[0273] The performance of the antibodies given in Table 2 was measured based on the extent to which the GFP-fused first protein was pulled down onto the substrate. Briefly, 1 nM of GFP-fused BCL2 protein was applied to the antibody-coupled substrate and incubated. After washing, the remaining GFP was quantified. The same experiment was performed for BCL-XL, MCL1, BCLw, and BFL1. The results thus obtained are shown in Table 2. Figure 1a and 1b As shown. Figure 1a and 1b As shown, it was observed that the antibodies given in Table 2 successfully pulled down the respective target proteins onto the substrate.

[0274] 1.4. Measurement of endogenous and exogenous proteins

[0275] The cell lysate containing the probe-labeled first protein-interacting protein prepared in Example 1.2 was added to the substrate prepared in Example 1.3. In short, 10 μl of the cell lysate of Example 1.2 was added to each well and incubated at room temperature for 15 minutes. Excess lysate was washed away with washing buffer (HEPES 50 mM NaCl 150 mM glycerol 1% GDN 0.03%). Then, protein-protein interactions were measured as follows:

[0276] The substrate was fixed on total internal reflection fluorescence and imaged to obtain data of protein-protein interaction (binding). In order to improve the accuracy of the results, images were acquired from a total of 10 different positions under the same conditions, and their average value was used as a representative value.

[0277] Fluorescence images are stored in 16-bit unsigned integer format. To observe the signal, give the laser a suitable wavelength (488 nm for GFP). Adjust the laser power to 2 mW to keep the light emission of eGFP for about 11 seconds.

[0278] In all frames (10 frames), early and mid-stage frames are discarded, and the average of three frames (frames 7 to 9) is taken to generate an image. This process is repeated at different positions in a well to obtain a total of ten images, and then the following steps are performed using these images. In this embodiment, an EMCCD (electron multiplying charge coupled device; Andor iXonUltra 897 EX2 (DU-897U-CS0-EXF)) camera is used to obtain a fluorescence image with an exposure time of 0.1 per frame and an EMCCD gain value of 40.

[0279] The signal from the fluorescent protein is detected as a local point spread function (PSF). The quantity of the PSF (physical value) is the PPI score (biological value) between the BCL2 family protein and the protein under test. The PSF value is converted to the PPI score (biological value) by the following procedure:

[0280] (a) Obtain the position of the local maximum (e.g., the pixel in the i-th row and j-th column). As mentioned above, the single-molecule fluorescence signal is located and formed at a specific position (about 5×5 pixel size under the current observation device, 1 pixel = 0.167 micron). Therefore, the discovery of the local maximum allows the selection of a single PSF. This can be obtained using the toolbox provided by MATLAB.

[0281] (b) A process is performed to determine whether the local maximum obtained in (a) is actually generated by the PSF. First, the minimum intensity value of the local maximum is defined. Analysis is performed only when the local maximum obtained as described above is greater than the minimum intensity value. The minimum intensity value used in this embodiment is 75, which may vary depending on the laser power / exposure time / established device state. Information of 5×5 pixels is retrieved based on the local maximum coordinates finally obtained, and the center of mass of the intensity is determined in the 5×5 pixels. Here, if the obtained center of mass of the intensity deviates from the existing local maximum coordinates by 0.5 pixels or more (if the 2D symmetry of the PSF pattern disappears), the fluorescence signal is determined to be abnormal and is excluded from the analysis.

[0282] (c) Only the PSFs that pass all test conditions are selected to determine the coordinates and total number. Here, the total number of PSFs obtained becomes the PPI count.

[0283] Each file taken under the same conditions is subjected to the above procedure to obtain the number of PSFs, which are then summed and used to calculate the mean and standard deviation. This value ultimately represents the PPI count under specific conditions. In this embodiment, the complex formed between the exogenous first protein and the exogenous first protein interacting protein can be estimated by PPI counting.

[0284] PPI counts are measured as Figure 2a (Results obtained based on the types of the first protein and the proteins that interact with the first protein), Figure 2b (Results based on cell line type), Figure 2c (results based on the type of first protein and cell line) and Figure 2d (Results according to the type of the first protein and cell line) are shown.

[0285] To determine factors that affect the data of the interaction between endogenous and exogenous proteins, (i) the PPI intensity (fluorescence intensity) according to the concentration of the analyzed lysate (sample) and the probe-labeled first protein-interacting protein ( Figure 3 ) and (ii) PPI counting according to the concentration of the analyzed lysate (sample) and the type of detergent contained in the cell lysate including the probe-labeled first protein-interacting protein ( Figure 4 ) and PPI counts according to detergent concentration ( Figure 5 ). In general, PPI counts increase with increasing concentrations of the analyzed lysate (sample) and the first protein-interacting protein.

[0286] In addition, PPI counts were measured based on changes in the first protein-interacting proteins, e.g. Figure 6 shown.

[0287] Example 2: Measurement of interactions between endogenous proteins (measurement of complexes)

[0288] In this embodiment, the interaction between the first protein and the first protein-interacting protein is measured, and both proteins are endogenous proteins of the same sample. In short, the interaction is measured by detecting the first protein-first protein-interacting protein complex bound to the first protein-interacting protein in the analytical lysate. The first protein-first protein-interacting protein complex measured in this embodiment is shown in Table 3 below:

[0289] Table 3

[0290]

[0291] The data in the graph of Table 3 are obtained by measuring the complex formed between the first protein pulled down to the substrate and the labeled first protein-interacting protein by a substance binding to the labeled first protein-interacting protein. Alternatively, the complex count formed between the first protein-interacting protein immobilized on the substrate and the labeled first protein is measured using a substance binding to the labeled first protein (in this case, the antibody to the first protein-interacting protein and the antibody to the first protein are used as the pull-down antibody and the detection antibody, respectively) ( Fig.11 ).like Figures 7 to 11 As shown, the same detection effect can be obtained regardless of whether the first protein or the first protein-interacting protein is pulled down to the substrate.

[0292] By measuring the interaction between two first proteins and first protein interacting proteins, both of which are endogenous to the sample, the following results were obtained:

[0293] First, the analytical lysate prepared in the same manner as in Example 1.1 was added to the substrate prepared in Reference Example 1.3.

[0294] As binding substances for endogenous protein-protein complexes of the detection sample, a detection antibody against the primary protein-interacting protein was used. As a fluorescently labeled secondary antibody for probing the detection antibody, Cy3AffiniPure donkey anti-mouse IgG (Jackson ImmunoResearch: 715-165-151) was prepared in a diluted form.

[0295] The detection antibody is added to the prepared substrate. The detection antibody is added to each well in an amount of 10 μl and incubated at room temperature for 15 minutes, then, the excess antibody is washed away with a wash buffer (HEPES 50nM NaCl 150nM glycerol 1% GDN 0.03%). Then, the fluorescently labeled antibody Cy3 AffiniPure donkey anti-mouse IgG (Jackson ImmunoResearch: 715-165-151) is diluted 2000 times, 10 μl of diluent is added to each well, and incubated for 10 minutes. Excess antibody is washed away with a wash buffer (HEPES 50nM NaCl 150nM glycerol 1% GDN 0.03%). Protein-protein interactions (complexes) are measured. Measured in the same manner as in Example 1-4.

[0296] The information of the binding substances (antibodies) used in this example is summarized in Table 4 below:

[0297] Table 4

[0298]

[0299] As described above, the first protein and the first protein-interacting protein can be interchanged with each other. In addition, when the antibody used is a monoclonal antibody, any equivalent clone provided by any available manufacturer can be used.

[0300] Therefore, the list of antibodies is given only for illustrative purposes and should not be construed as limiting the present invention.

[0301] In the present invention, the antibody or binding substance specifically binds to the first protein or the first protein-interacting protein to form a complex together, and the use of the antibody or binding substance is not particularly limited. However, when a site known to be involved in the formation of a complex between two proteins is used as a recognition site of the antibody or binding substance, it is impossible to detect the complex. In one embodiment, in order to detect BCL2, BCL-XL and MCL1, each of which forms a complex with BAK, it is recommended that the antibody recognizes a site other than the BH3 domain of BAK (e.g., a site of non-transmembrane domain helix 1 to helix 4, etc.) ( Figures 12a to 12c ).

[0302] Example 3. Measurement of endogenous protein expression levels

[0303] In this example, the levels of specific proteins present in the assay lysate were quantitatively measured. The specific proteins included BCL2, BCL-XL, MCL1, BAX, and BAK.

[0304] In this regard, a cell lysate was prepared in the same manner as in Example 1-1 and added to a substrate prepared in the same manner as in Example 1-3. Then, a labeled detection antibody (labeled with Cy3AffiniPure donkey anti-mouse IgG (Jackson ImmunoResearch: 715-165-151)) prepared as in Example 2 was added. Then, the fluorescence signal was measured using the same method as in Example 1-4 to quantitatively analyze the interaction between the target protein in the lysate and its detection antibody.

[0305] In this example, the pull-down antibody used to immobilize the target protein to the substrate and the detection antibody used to detect the same protein should be identical in terms of antigen but different in terms of binding sites.

[0306] The following Table 5 summarizes the antibody information used in this example:

[0307] Table 5

[0308]

[0309] The first binding substance is an antibody that can be pulled down to the substrate, and the second binding substance is used to detect the target protein after adding the analytical lysate. Here, the first binding substance and the second binding substance can be used interchangeably. For example, even after the second binding substance is pulled down to the substrate, the same quantitative analysis can be performed, and the analytical lysate and the first binding substance can be provided to the substrate in this order. A given antibody does not limit the scope of the present invention. However, the first and second binding substances should avoid recognizing exactly the same epitope.

[0310] Example 4. Measurement of folded conformation-specific proteins

[0311] This example contemplates a method for detecting folding conformation-specific proteins present in a sample by using antibodies or binding substances that can specifically recognize proteins based on their folding conformation. In one embodiment, folding conformation-specific detection is applied to BAX and BAK.

[0312] It is reported that BAX protein can retain multiple folding conformations in cells. Among them, it is known that when BAX protein folds into a conformation that causes apoptosis, about 20 N-terminal amino acids are exposed (Yi-Te Hsu et al., J. Bio. Chem. 272, (1997), Michael A. Dengler et al., Cell Rep. 27 (2019)). In the process of migrating to the mitochondrial lipid membrane, BAK protein induces apoptosis in various folding conformations (Mark Xiang Li et al., PNAS 114 (2017)). The present invention relates to the use of an antibody or a binding substance that can recognize a domain that is exposed only in this specific folding conformation.

[0313] In one embodiment, an antibody known to recognize the N-terminus of the BAX protein and an antibody capable of recognizing a domain exposed in any folded conformation can be used to recognize the BAX protein in an active state that induces apoptosis in a sample. In another embodiment, an antibody that selectively recognizes the BH3 domain of the BAK protein and an antibody that recognizes the BAK domain exposed in any conformation can be used in pairs to detect the BAK protein in a folded conformation, thereby allowing BH3 to be exposed from a sample. In Table 6 below, antibody pairs used to implement the present invention are listed. However, antibodies do not limit the scope of the present invention. In addition, monoclonal antibodies can be used regardless of the manufacturer.

[0314] Table 6

[0315] Target protein Folding conformation Ab 1 Ab 2 BAX Exposed N-terminus Ab 32503 Sc-23959 BAK Exposed BH3 SC-517390 SAB1305656

[0316] In this table, Ab 1 and Ab 2 represent antibodies that are pulled down to the substrate and used for detection, respectively. Even if the two antibodies exchange roles, the same results may be obtained. For example, when Ab 2 is pulled down to the substrate and Ab 1 is used as the detection antibody, the same results can be expected.

[0317] For use in this method, analytical lysates can be prepared in the same manner as in Example 1.

[0318] The quantitative analysis of proteins in this embodiment can be performed in the same manner as described in Example 2 or 3. For example, the antibodies or protein-specific binding substances used can be suitable for binding to the same antigen or target protein. At the same time, the two proteins or protein-specific binding substances should bind to different sites of the target protein. In addition, when two antibodies are used, it is recommended that the hosts of the two antibodies are different from each other unless the antibodies are directly labeled with corresponding probes.

[0319] This embodiment can be performed with reference to the method set forth in Embodiment 2 or 3. For example, in order to detect a target protein in a specific folded conformation, Ab 1, a sample, and Ab 2 are added.

[0320] To implement this embodiment, a sample can be prepared by artificially inducing apoptosis. For example, apoptosis can be induced by using staurosporine for 1 hour or venetoclax for 24 hours. The sample thus prepared is lysed in a lysis buffer containing GDN to provide an analytical lysate, and then it is observed that when N-terminally exposed BAX is detected therefrom, it shows an increased signal ( Fig.14 ). For analytical lysates prepared from the same sample using a lysis buffer containing Triton X-100, the nonionic detergent Triton X-100 forced induction of N-terminal exposure. From this analytical lysate, N-terminally exposed BAX ( Fig.15 ). After analyzing lysates prepared from apoptosis-induced samples using GDN, the signal increased when measuring N-terminally exposed BAX. In contrast, when artificial N-terminal exposure was performed using Triton X-100, constant values ​​were obtained regardless of apoptosis induction.

[0321] In another embodiment, apoptosis is induced by culturing the AML cell line HL60 or the DLBCL cell line OCI-LY3 with venetoclax, and the ratio of N-terminal exposure of BAX is measured. After treatment with 100 nM venetoclax for 2, 4, 8, or 24 hours, the sample is lysed with a lysis buffer containing GDN to prepare analytical lysates. Then, the BAX detection signal changes with the venetoclax treatment time ( Fig.16). In contrast, after inducing N-terminal exposure by further mixing the same assay lysates with Triton X-100 (final concentration 1%), almost no change in the amount of BAX over time was observed.

[0322] As described above, the folding conformation-specific protein detection methods provided herein can provide indicators that are helpful for analyzing cell status, for example, the degree of apoptosis.

[0323] Example 5. Measurement of changes in intracellular BCL2 family signaling pathways after treatment with BCL2 family protein targeted inhibitors

[0324] This embodiment contemplates a method for measuring changes in intracellular signal transduction by treatment with a BCL2 family protein targeted inhibitor. More specifically, the present invention provides a method for comprehensively analyzing the strength of protein-protein interactions (complexes) between and within samples and changes in protein expression levels of various BCL2 family proteins (e.g., BCL2, BCL-XL, MCL1), wherein the proteins are treated with the BCL2 targeted inhibitor venetoclax or the MCL1 targeted inhibitor AZD5991.

[0325] To achieve the purpose of this embodiment, protein-protein interactions between samples, protein-protein interactions within samples, and changes in expression levels may be measured in the same manner as in Embodiments 1, 2, 3, and 4, respectively.

[0326] For example, the targeted inhibitors venetoclax or AZD5991 can be applied to cells at a concentration of 100 nM for 24 hours. For controls, dimethyl sulfoxide can be applied under the same conditions.

[0327] The analysis contents in this example are summarized in Tables 7 and 8 below:

[0328] Table 7

[0329] Protein-protein interaction analysis between samples

[0330]

[0331] (In Table 7, BIM* represents the GFP-tagged BH3 domain cleaved from BIM, the first protein being endogenous protein in the analyzed lysate and pulled down to the substrate surface)

[0332] Table 8

[0333] Measurement of protein complexes within samples

[0334]

[0335] In Table 8, both the first protein and the first protein-interacting (binding) protein (forming a complex) are included in the assay lysate. The same results may be obtained even if either of these two proteins is pulled down to the substrate surface. For example, the antibody pulled down to the substrate surface may be an antibody against the first protein or the first protein-interacting (binding) protein.

[0336] For example, the method proposed in this example can quantitatively measure changes in PPI intensity and complex formation before and after treatment with a BCL2 targeted inhibitor. To this end, HL60 or OCI-LY3 cells were treated with dimethyl sulfoxide, venetoclax 100nM (BCL2 targeted inhibitor) or AMG176 100nM (MCL1 targeted inhibitor) for 24 hours, and then lysates were prepared from the respective samples and the above indicators were measured. In both HL60 and OCI-LY3, it was observed that treatment with venetoclax increased the BCL2-BAD PPI intensity ( Fig.17 ). In contrast, treatment with the MCL1-targeted inhibitor AMG176 did not alter BCL2-BAD PPI. Results were not limited to venetoclax-BCL2. A selective increase in MCL1-NOXA PPI was observed after AMG176 treatment ( Fig.18 ). BCL-XL was not responsive to venetoclax / AMG176, and the PPI intensity changed according to the BCL-XL expression level. These data suggest that BCL2 and MCL1 targeted inhibitors selectively produce PPI intensity of the corresponding proteins ( Fig.19 and Fig. 20 ).

[0337] In another embodiment, changes in complex formation are measured in response to treatment with a BCL2 targeted inhibitor. The collected samples are analyzed under the same conditions as the PPI measurement to measure the BCL2 family BIM complex ( Fig.21 )、MCL1-NOXA complex ( Fig. 22 ) and the BCLXL-BAD complex ( Fig.23 ) formation changes. In addition, this method can be used to measure the BCL2-BAX complex ( Fig.24 ) and the expression levels of BAX and BAK ( Fig.25 and 26 ) changes.

[0338] This embodiment can be used to measure the direct relationship between the targeted inhibitor used and the BCL2 family proteins and related signaling proteins. To this end, BCL2 or MCL1 protein was used as the first protein, and BIM-GFP protein was used as the first protein interacting protein. When the first protein was pulled down to the substrate surface, different concentrations of the targeted inhibitor venetoclax or AZD5991 (MCL1 targeted inhibitor) and the first protein interacting protein were added to measure the change in PPI intensity. BCL2-BIMPPI intensity was only reduced in the presence of the BCL2 targeted inhibitor venetoclax, but was not affected by AZD5991 ( Fig. 27 In contrast, when MCL1 was used as the first protein, only AZD5991 reduced the MCL1-BIM PPI intensity ( Fig.28 ).

[0339] In summary, the results show that the method presented in this paper can quantitatively analyze the BCL2 family protein characteristics (PPI intensity, complex formation, expression level, etc.) that appear when BCL2 family targeted inhibitors are applied to samples. In addition, changes in PPI intensity were determined to be directly controlled by the application of the drug. Therefore, the method provided in this paper is intended to measure changes in BCL2 family proteins in response to treatment with BCL2 targeted inhibitors and can be used as a molecular diagnostic technique for samples.

[0340] Example 6. Practical analysis method for patient samples

[0341] The method described in this example is to measure the signature of BCL2 family proteins in a sample from a patient, such as acute myeloid leukemia (AML) or breast cancer.

[0342] For equivalent comparisons between patient samples, at least one method selected from Bradford assay, Lowry assay, and infrared absorption can be used to determine the total protein concentration. Under the condition of keeping the sample concentration added to the substrate constant, quantitative comparison between samples is allowed. For AML patients, samples can be isolated from blood or bone marrow. From solid cancer tissues, samples can be obtained by surgery or puncture biopsy.

[0343] All assays presented in Examples 1, 2 and 3 can be used with patient samples, but are not limited thereto. These assays are applicable to both blood cancers and solid cancers.

[0344] For example, based on the analysis results of samples obtained from 32 AML blood cancer patients, an analysis method for comprehensively measuring the PPI (Example 1), complex (Example 2) and expression level (see Example 3) of BCL2 family proteins can be provided ( Fig.29 ).

[0345] In another embodiment, analysis of samples obtained from 302 breast cancer patients can be used to quantify the expression level of endogenous BCL2 protein in the samples ( Fig.30 ).

[0346] In another embodiment, the method based on data from 15 lung adenocarcinoma patient samples can be applied to quantify the expression level of BCL2 protein ( Fig.31 ).

[0347] Example 7. Comparison of the interactive measurement of Example 1 and the complex measurement of Example 2

[0348] The two methods proposed herein, namely, measuring the PPI intensity by using the first protein-interacting protein (Example 1) and measuring the complex between the first protein-interacting protein and the first protein-binding protein (Example 2), can provide different information.

[0349] In short, these methods can detect differences in PPI intensity and complex formation in OCI-LY3 cells before and after incubation with Venetoclax 100nM or AZD5991 100nM. For example, it was observed that samples treated with venetoclax increased BCL2-BIM PPI counts but decreased BCL2-BIM complex counts ( Fig.32 ). In addition, samples treated with AZD5991 increased MCL1-NOXA PPI counts but decreased MCL1-NOXA complex counts ( Fig.33 ). It can be understood from the assay that the PPI count measurement achieved in Example 1 is substantially different from the complex count measurement achieved in Example 2.

[0350] Example 8. Determine the method of drug administration by tracking the patient's status by observing changes in the interaction between BCL2 family proteins Law

[0351] The methods provided herein can produce a method for tracking the molecular status of patients over time. In more detail, interaction analysis of bone marrow or blood samples collected from patients with blood cancer at different stages can be used to provide information that helps determine BCL2 family protein dependency and whether to administer corresponding targeted anticancer agents. In this regard, for patient 1, i) PPI determination between BCL2, MCL1, BCL-XL, and BIM-GFP was performed in the bone marrow extracted at diagnosis; ii) the same determination was performed in the bone marrow extracted after treatment with the FLT3 targeted anticancer agent quizartinib; and iii) based on the PPI information obtained in step ii), the same determination was performed on the whole blood sample obtained after treatment with venetoclax and low-dose cytarabine (LDAC) ( Fig.34). At the time of diagnosis, there was no sufficient reason to prescribe venetoclax because the patient showed a low BCL2-BIM PPI. However, after the FLT3-targeted inhibitor quizartinib was prescribed, the signaling reoccurred, thereby substantially increasing the BCL2-BIM PPI. Due to this phenomenon, when venetoclax and LDAC were used in combination, the patient's BCL2-BIM PPI was observed to decrease again. Clinically, it is believed that the white blood cell count increased sharply after quizartinib treatment, but the combination of venetoclax and LDAC reduced the white blood cell count to a stable level, indicating that venetoclax has medicinal efficacy even at the clinical level. Afterwards, cord blood stem cell transplantation brought the patient into complete remission.

[0352] In addition, a time series diagnosis was performed on patient 2 in the same manner as patient 1 ( Fig.35 ). In patient 2, the signaling pathway of BCL2 family proteins could not be controlled by prescribing FLT3 targeted inhibitors. However, a significant decrease in BCL2-BIM PPI was observed after combined treatment with venetoclax and LDAC. Consistent with this result, the white blood cell count test showed an approximately threefold increase in white blood cell count after quizartinib treatment. From the above results, it can be seen that quizartinib treatment could not bring any clinical effect.

[0353] Example 9. Development of a method for predicting the responsiveness of BCL2-targeted inhibitors

[0354] The method proposed in this article can predict the responsiveness of patients with acute myeloid leukemia to the BCL2-targeted inhibitor venetoclax.

[0355] In brief, among the BCL2 family proteins in samples (bone marrow or blood) extracted from AML patients as shown in Example 6, six PPI counts (Example 1), six expression levels (Example 3) and nine complex counts (Example 2) were measured (see Table 9 below).

[0356] Table 9

[0357]

[0358] To measure responsiveness to venetoclax, viable cells were counted by flow cytometry 24 hours after treatment of samples isolated from patients with 0, 30, 100, 300, or 1000 nM venetoclax. Dose-response curves of remaining viable cell counts were plotted. Individual patient responsiveness to venetoclax was quantified using the inverse value of the AUC (area under the curve).

[0359] The highest predictive index ( Figures 36a-36c For quantitative comparison, linear correlation coefficients (R, Pearson correlation coefficient) were obtained. Among a total of 21 indicators, the R values ​​of BCL2-BAD PPI, BCL2-BIM PPI, BCL2 expression level, and BCL2-BAX complex counts measured were 0.65 or greater ( Figures 36a-36c However, when establishing a venetoclax response prediction model, the selected indicators do not limit the scope of the present invention (e.g., various indicators involved in the simulation model).

[0360] To confirm that PPI counts and complex counts are distinct indicators from each other, BCL2-BIM PPI counts and BCL2-BIM complex counts can be compared to responsiveness to venetoclax, respectively ( Fig.37 ). Correlations as high as R = 0.74 were observed between BCL2-BIM PPI and venetoclax, but the correlation with BCL2-BIM complex counts dropped to R = 0.41. These results illustrate the use of PPI counts and complex counts as distinct indices, indicating that PPI counts can predict responsiveness to venetoclax with high accuracy.

[0361] The combination of measured values ​​can improve the accuracy of the prediction of responsiveness to venetoclax. Various methods including multiple linear regression, logistic regression, support vector machine, etc. can be used to achieve this combination of measurements. In this embodiment, multiple linear regression is used, but it does not limit the scope of the present invention. When a single parameter (indicator) is used, the highest value is detected at around R = 0.75, but it increases with the increase in the number of parameters ( Fig.38a ).

[0362] As many as 2,097,151 combinations can be derived from the 21 parameters (indicators). Various parameters such as biological plausibility, mathematical plausibility, etc. can be considered to search for the best model.

[0363] For example, to demonstrate that the combination of two or more indicators yielded better analytical results than a single indicator, the correlation between the BCL2-BAX or BCL2-BIM complex and the responsiveness to venetoclax, and the correlation between the combination of the BCL2-BAX or BCL2-BIM complex and the BCL2-BAD PPI and the responsiveness to venetoclax were presented in Figure 38b As described in the above and below figures. Figure 38bAs shown, the R values ​​of the BCL2-BAX and BCL2-BIM complexes were 0.67 and 0.41, respectively, but when the complexes were combined with the BCL2-BAD PPI, the R values ​​increased to 0.76 and 0.75, indicating that the combination of the two indices led to a greater improvement in the prediction results of drug responsiveness.

[0364] For example, in a model with a total of 5 parameter combinations, a linear correlation coefficient R of 0.85 ( Fig.38c ). In this regard, the parameters used are given in Table 10 below. Among the combinations of various parameters, this combination showed the most significant results.

[0365] Table 10

[0366]

[0367] Example 10. Establishment of a method for predicting responsiveness to MCL1 targeted inhibitors

[0368] A method for predicting responsiveness to a MCL1 targeted inhibitor can be provided in the same manner as in Example 9. For example, AZD5991 is used as a MCL1 targeted inhibitor, but any targeted inhibitor can be used as well as long as it retains a similar mechanism of action. The type of drug used does not limit the scope of the present invention.

[0369] As shown in Example 9, linear correlation analysis can be performed on individual indicators, revealing that indicators including MCL1-BIM PPI, BCLXL-BIM PPI, BAX expression level and BAK expression level are associated with responsiveness to AZD5991 ( Figures 39a-39c ).

[0370] In order to improve the accuracy of reactivity prediction, various combinations of parameters can be used, as shown in Example 9. For example, the correlation analysis of the reactivity of AZD5991 and the combination of parameters listed in Table 11 below yielded R = 0.78 ( Fig.40 ), the following Table 11 shows the most significant results among various parameter combinations.

[0371] Table 11

[0372]

[0373] Those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit and basic features of the present invention. Therefore, it should be understood that the above embodiments are illustrative in all aspects and not restrictive.

Claims

1. A method for providing information for predicting a subject's responsiveness to a BCL2 family protein targeted drug, the method comprising: (a) preparing a sample isolated from a subject, wherein the sample comprises cells or cell lysate; (b) obtaining a result by performing a combination of all of the following steps on the sample: (i) measuring the interaction between a first protein and a first protein-interacting protein in a sample, wherein the first protein-interacting protein is provided externally; (ii) measuring the interaction between a first protein endogenous to the sample and a first protein-interacting protein, wherein the first protein-interacting protein is an endogenous protein to the sample; and (iii) measuring the level of the first protein, the first protein interacting protein, or both in the sample, in, The first proteins in steps (i), (ii) and (iii) are the same or different from each other, and the first protein-interacting proteins in steps (i), (ii) and (iii) are the same or different from each other, Each of the first proteins in steps (i), (ii) and (iii) is at least one anti-apoptotic protein selected from the BCL2 family proteins, each of the first protein-interacting proteins in steps (i), (ii) and (iii) is at least one pro-apoptotic protein selected from the BCL2 family proteins, or, the first protein is at least one pro-apoptotic protein selected from the BCL2 family proteins, and the first protein-interacting protein is at least one anti-apoptotic protein selected from the BCL2 family proteins.

2. The method according to claim 1, wherein: Each of the first proteins in steps (i), (ii) and (iii) is at least one selected from the group consisting of BCL2, BCLxL, BCLw, BFL1 and MCL1, and each of the first protein-interacting proteins in steps (i), (ii) and (iii) is at least one selected from the group consisting of BAD, BIM, NOXA, PUMA, BMF, tBID, BIK, HRK, BAX, BAK and BOK, or Each of the first proteins in steps (i), (ii) and (iii) is at least one selected from the group consisting of BAD, BIM, NOXA, PUMA, BMF, tBID, BIK, HRK, BAX, BAK and BOK, and each of the first protein-interacting proteins in steps (i), (ii) and (iii) is at least one selected from the group consisting of BCL2, BCLxL, BCLw, BFL1 and MCL1.

3. The method according to claim 1, wherein: Step (i) comprises the sub-step of bringing the probe-labeled externally provided first protein-interacting protein into contact with the first protein immobilized on the substrate, and measuring the protein-protein interaction between the first protein in the sample and the externally provided first protein-interacting protein; Step (ii) comprises the sub-step of contacting the sample with a substrate comprising a substance that binds to a first protein, and measuring the level of a complex formed between the first protein endogenous to the sample and a first protein-interacting protein endogenous to the sample; and Step (iii) comprises the sub-step of measuring the total expression level of either or both of the first protein endogenous to the sample and the first protein-interacting protein endogenous to the sample.

4. The method according to claim 3, wherein: The sub-step of measuring protein-protein interactions in step (i) comprises: (1) providing a sample to a substrate, wherein the substrate includes a substance that binds to a first protein to fix the first protein to the substrate; (2) providing a probe-labeled first protein-interacting protein to the outside of the substrate to allow reaction with the first protein on the substrate; and (3) The signal generated by the probe is measured in the near-field region of the substrate surface using a total internal reflection fluorescence microscope.

5. The method according to claim 2, wherein: Step (i) comprises at least one selected from the following sub-steps: (i-1) measuring the protein-protein interaction between BCL2 protein in the sample and externally provided BAD protein; (i-2) measuring the protein-protein interaction between BCL2 protein in the sample and externally provided BIM protein; (i-3) measuring the protein-protein interaction between BCL2 protein in the sample and externally provided BAX protein; (i-4) measuring the protein-protein interaction between BCL2 protein in the sample and externally provided BAK protein; (i-5) measuring the protein-protein interaction between BCLxl protein in the sample and externally provided BAD protein; (i-6) measuring the protein-protein interaction between BCLxl protein in the sample and externally provided BIM protein; (i-7) measuring the protein-protein interaction between BCLxl protein in the sample and externally provided BAX protein; (i-8) measuring the protein-protein interaction between BCLxl protein in the sample and externally provided BAK protein; (i-9) measuring the protein-protein interaction between the MCL1 protein in the sample and the externally provided NOXA protein; (i-10) measuring the protein-protein interaction between MCL1 protein in the sample and externally provided BIM protein; (i-11) measuring the protein-protein interaction between MCL1 protein in the sample and externally provided BAX protein; and (i-12) Measuring the protein-protein interaction between the MCL1 protein in the sample and the externally provided BAK protein.

6. The method according to claim 3, wherein: The sub-step of measuring protein-protein interactions in step (ii) comprises: (1) providing a sample to a substrate, wherein the substrate includes a substance that binds to a first protein, so as to fix the first protein to the substrate; (2) providing a probe-labeled substance bound to the first protein-interacting protein to the substrate to allow reaction with the first protein-interacting protein of the complex formed between the endogenous first protein in the sample and the endogenous first protein-interacting protein in the sample; (3) using total internal reflection fluorescence microscopy to measure the signal generated by the probe in the near-field region of the substrate surface, or (1′) providing a sample to a substrate, wherein the substrate includes a substance that binds to a first protein-interaction protein, so as to fix the first protein-interaction protein to the substrate; (2') providing a probe-labeled substance bound to the first protein to the substrate to allow reaction with the first protein in a complex formed between the endogenous first protein in the sample and an endogenous first protein-interacting protein in the sample; and (3') The signal generated by the probe is measured in the near-field region of the substrate surface using total internal reflection fluorescence microscopy.

7. The method according to claim 2, wherein: Step (ii) comprises at least one selected from the following sub-steps: (ii-1) measuring the level of a complex formed between a BCL2 protein in a sample and a BIM protein in a sample; (ii-2) measuring the level of a complex formed between a BCL2 protein in the sample and a BAX protein in the sample; (ii-3) measuring the level of a complex formed between a BCL2 protein in the sample and a BAK protein in the sample; (ii-4) measuring the level of the complex formed between the BCLxl protein in the sample and the BAD protein in the sample; (ii-5) measuring the level of the complex formed between the BCLxl protein in the sample and the BIM protein in the sample; (ii-6) measuring the level of a complex formed between a BCLxl protein in the sample and a BAX protein in the sample; (ii-7) measuring the level of a complex formed between a BCLxl protein in the sample and a BAK protein in the sample; (ii-8) measuring the level of a complex formed between the MCL1 protein in the sample and the NOXA protein in the sample; (ii-9) measuring the level of the complex formed between the MCL1 protein in the sample and the BIM protein in the sample; (ii-10) measuring the level of a complex formed between the MCL1 protein in the sample and the BAX protein in the sample; and (ii-11) Measuring the level of the complex formed between the MCL1 protein in the sample and the BAK protein in the sample.

8. The method according to claim 3, wherein: The measuring sub-step in step (iii) comprises: providing a sample to a substrate, wherein the substrate includes either or both of a substance for capturing a first protein and a substance for capturing a first protein-interacting protein, so as to immobilize either or both of the first protein and the first protein-interacting protein on the substrate; providing either or both of a probe-labeled substance that binds to the first protein and a probe-labeled substance that binds to the first protein-interacting protein to allow a reaction; and The signal generated by the probe is measured in the near-field region of the substrate surface using total internal reflection fluorescence microscopy.

9. The method according to claim 2, wherein: Step (iii) comprises at least one of the following sub-steps: (iii-1) measuring the level of BCL2 protein in the sample; (iii-2) measuring the level of BCLxl protein in the sample; (iii-3) measuring the level of MCL1 protein in the sample; (iii-4) measuring the level of BAX protein in the sample; and (iii-5) Measuring the level of BAK protein in the sample.

10. The method according to claim 1, wherein: The BCL2 family protein targeted drug is a BCL2 inhibitor selected from the group consisting of venetoclax, ABT737 and anti-BCL2 antibodies.

11. The method according to claim 1, wherein: The BCL2 family protein targeted drug is a MCL1 inhibitor selected from the group consisting of anti-MCL-1 antibodies, AMG-176, AZD5991 and Maritoclax.

12. The method according to claim 1, wherein: The BCL2 family protein targeted drug is a BCLxL inhibitor selected from the group consisting of anti-BCLxL antibodies and ABT263.

13. The method according to claim 1, wherein: The subject is a cancer patient and the cell is a cancer cell isolated from the subject.

14. The method according to claim 13, wherein: The cancer is a blood cancer.

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