WO2019132517A1

WO2019132517A1 - Method and apparatus for analyzing intracellular or intercellular protein-protein interaction

Info

Publication number: WO2019132517A1
Application number: PCT/KR2018/016675
Authority: WO
Inventors: 이홍원; 나유진; 최병산; 이대희
Original assignee: 주식회사 프로티나
Priority date: 2017-12-26
Filing date: 2018-12-26
Publication date: 2019-07-04

Abstract

The present invention relates to a method for analyzing an activation state of a signaling pathway in a cell or tissue and/or between cells or tissues through protein-protein interaction analysis, and medicine selection using the same, and/or a method for monitoring efficacy of a medicine or a method for drug candidate screening, and an apparatus for use therein.

Description

【Specification】

Title of the Invention

Method and apparatus for analyzing intracellular or intercellular protein-protein interactions

A method for analyzing the activation state of a signaling pathway between a cell or tissue and / or a cell or tissue through analysis of protein-protein interaction, a method for selecting a therapeutic agent and / or a method for monitoring the efficacy of a therapeutic agent, And a device for use therein. BACKGROUND ART [0002]

Recently, disease diagnosis, prognosis prediction and treatment have been focused on gene analysis (genomics profiling). However, the cause of a specific disease, including cancer, is due to the abnormal activity of the cells constituting the body, more specifically, the abnormal interaction between various proteins that constitute and regulate the cell. There is a difficult problem. Indeed, profiling of these gene mutations also leads to different susceptibility to target chemotherapy and to prognosis for patients with the same genetic characteristics. Thus, an accurate analysis of protein-protein interactions, rather than individual protein analyzes, will help to understand how the intracellular and / or intercellular signaling networks change, And information on the characteristics and methods of selecting and treating the therapeutic agents.

DETAILED DESCRIPTION OF THE INVENTION

[Technical Problem]

One example includes measuring protein-protein interactions between a first protein and a second protein,

Wherein the first protein is a protein involved in a signaling pathway between cells or tissues and / or between cells or tissues, and the second protein is a protein involved in the signal transduction pathway in the cell or tissue and / A protein that interacts with the first protein (e.g., a sub protein of the first protein, a ligand of the first protein, the same cell as the first protein, an allogeneic cell or a heterologous cell, &Lt; RTI ID = 0.0 > and / or < 2019/132517 1 »(: 1 ^ {2018/016675

Measuring (or identifying or determining or determining) the signal transduction pathway in a cell or tissue and / or between a cell or tissue; A method for predicting the reactivity of said cell or tissue, or an individual from which said cell or tissue is derived, to a drug targeting said first protein, And a method for screening a subject suitable for treatment targeting the first protein or a method for providing information to screening.

When two or more of the first proteins are used, the drug targeting the first protein may be a drug targeting at least one of the two or more first proteins, The target treatment may be a treatment targeting one or more of the two or more kinds of the first protein. The treatment may include prescribing and / or administering a drug targeting the first protein. (E.g., cancer) associated with the first protein-mediated signal transduction or interactions between the first protein and the second protein (i. E., An individual that is at risk of contracting An individual requiring the prevention and / or treatment of diseases associated with signal transduction or interactions between the first protein and the second protein). An individual suitable for treatment targeting the first protein is one in which the drug targeting the first protein has a desired effect (e. G., Signal transduction mediated by the first protein or interaction between the first protein and the second protein, Or prevention and / or treatment and / or relief of related diseases (anticancer effect when the disease is cancer), and the like.

Another example is a first protein as a target suitable for application to the cell or tissue, or to an individual from which the cell or tissue is derived, wherein the step of measuring the protein-protein interaction is performed on two or more first proteins, A method for screening a drug that targets the drug, or a method for providing information to screening.

Another example is a cell or tissue treated with a drug that targets the first protein or a cell or tissue isolated from a subject to which the drug is administered and / - measuring the protein or protein interaction, or a drug targeting said first protein of said subject and / or a signal transduction or first signal transduction involving said first protein and low 12 protein, A method for monitoring the response to a therapeutic agent of a disease associated with protein task 2 protein interactions or 2019/132517 1 »(: 1 ^ {2018/016675

Provides a way to provide information to monitoring. In another example, a candidate protein targeted to a first protein is isolated from a separate cell or tissue that has been treated, or within a cell or tissue separated from the subject to which the drug is administered, and / or between cells or tissue, Wherein the first protein-mediated drug and / or the first protein-second protein-mediated signal transduction involving the step of measuring liver protein-protein interactions or the interaction between the first protein and the second protein A method for screening a therapeutic agent for a disease associated with the action. In one embodiment, there is provided a method of measuring activation of a signal transduction pathway in a cell or tissue and / or between a cell or tissue, comprising the steps of:

(1) preparing a substrate to which a first protein is immobilized by adding a test sample containing a first protein to a substrate including a substance that specifically binds to the first protein on a surface thereof;

(2) adding a low-level protein to which a labeling substance is bound to a substrate on which the prepared first protein is immobilized; And

(3) immediately determining a signal from the reactant obtained in step (2).

In addition to steps (1) to (3) above,

(4) obtaining a signal value of the unit amount of the first protein in the test sample added in step (1) using the signal measured in step (3), or

(4-1) obtaining a signal value of a unit amount of the test sample added in step (1) using the signal measured in step (3); And

(4-2) obtaining a value for the unit amount of the first protein contained in the test sample using the signal value of the unit amount of the test sample obtained in the step (4-1)

May be further included.

remind

The value for the unit amount of the first protein contained in the test sample obtained in the step (4) or (4-2) may be a value obtained by summing the values obtained for the respective first protein and the respective second proteins.

After step (4) or (4-2) above,

(5) comparing the result obtained in the step (4) or (4-2) with the result obtained in the reference sample,

The reference sample may be a normal cell, a cell in which the degree of activation of the signal transduction pathway involved in the first protein is confirmed, or a signal transduction pathway in which the first protein is involved 2019/132517 1 »(: 1 ^ {2018/016675

The degree of activation may be that of cells isolated from the identified individual. Another embodiment is a method of manufacturing a semiconductor device comprising steps (1), (2) and (3), or (1), (2), (3), and (4) , (4-1) and (4-2), and in addition thereto,

(5) comparing the results obtained in the above step, wherein the first protein is at least two selected from among proteins involved in a cell or tissue and / or a signal transduction pathway between cells or tissues, A protein is a protein that interacts with the aforementioned protein,

Step (5) comprises comparing the results obtained for the two or more first proteins with each other,

Provides a method of providing information to the screening of a target drug suitable for application to an individual.

In the above method, the test sample may be a lysate, a lysate, an extract, or a body fluid of a cell, a tissue, a cell or a tissue isolated from a mammalian body, and the lysate of the cancer cell, the cancer tissue, the cancer cell, , Lysates, or extracts.

In the above method, the labeling substance may be at least one selected from the group consisting of small molecule compounds, proteins, peptides, and nucleic acid molecules that generate signals measurable through enzyme reaction, fluorescence, luminescence, or radiation detection.

In this method, the labeling substance of step (2) is at least one selected from the group consisting of small molecule compounds, proteins, peptides, and nucleic acid molecules that generate fluorescence, and the step of measuring the signal of step (3) A microscope or a fluorescence camera, or both.

In the above method, the exposure time per frame of the fluorescent camera may be about 0.0 () 1 second to about 1 second.

In another embodiment,

(I) treating the candidate compound with the test sample, and

(II) the protein-protein interaction, the interaction level, and the activation level are measured for each of the test sample treated with the candidate compound and the untreated test sample, and then the candidate compound Lt; RTI ID = 0.0 > of:

As a result of the comparison, the protein-protein interaction, the protein-protein interaction level, or the activation level of the test sample to which the candidate compound has been treated, If the compound is lower than in the untreated test sample, the candidate compound may be further selected as a candidate drug targeting the first protein.

In another embodiment,

A multi-well comprising a substance for capturing a first protein that specifically binds to a first protein, and

Any signal detection means

/ RTI >

Wherein the multiwell includes a plurality of tubes whose one surface is open or a plurality of non-through holes spaced apart from the support plate,

A sample injecting portion located on one open side of the tube or non-through hole, a reaction portion where protein-protein interaction occurs in the tube or non-through hole, and

A capturing portion of a first protein capable of immobilizing or immobilizing a substance capable of capturing a first protein on a surface of at least a part of the inner wall in contact with the inside of the tube or the non-penetrating hole,

Wherein the surface of the capturing portion of the first protein is surface-treated with a compound containing a functional group selected from the group consisting of an aldehyde group, a carboxyl group and an amine group.

To provide an apparatus for analyzing protein-protein interactions within cells or tissues and / or between cells or tissues.

In the analyzer,

The surface treatment may be performed using biotin, biotin-conjugated bovine serum albumin, polyethylene glycol (PEG), polyethylene glycol-biotin, or polysorbate It can be done.

The surface treatment may further include at least one selected from the group consisting of neutravidin, streptavidin, and avidin.

The multiwall may further comprise at least one second protein that interacts with the first protein on the signal transduction pathway, labeled with a labeling substance that generates a detectable signal.

The multi-well may be a well-known method in which interaction occurs between the first protein and the second protein One or more wells including a reaction part (first reaction part) and a reaction part (second reaction part) in which a first protein and a detection substance binding to the low U protein bind to each other .

In the above method and apparatus, it is preferable that, in the method for measuring activation of a signal transduction pathway in the cell or tissue and / or between cells or tissues, the first protein and the second protein are independently in cells or tissues and / And the second protein is a protein that interacts with the first protein in the signal transduction pathway (for example, a protein involved in a lower pathway than the first protein, A ligand of a protein, a protein which is located on the same cell as the first protein, on the surface of allogeneic cells or xenogeneic cells, or secreted in a different cell and interacts with the first protein, etc.). The signal transfer may be understood to mean a signal transfer within the cell, a signal transfer from the outside of the cell to the inside of the cell, or a signal transfer between two or more cells of the same or different type (for example, immune signal transmission between immune checkpoints) . The first protein may be a plasma membrane protein. The first protein may be selected from the group consisting of receptor tyrosine kinase, toll-like receptors, G-protein coupled receptors (GPCR), transferrin receptors, low density lipoprotein , R0S1; BCR-Abl l fusion protein; Non-drinking body type kaineise; GTPases (GTPases); hormone receptors; Anti-apoptotic protein; And immune checkpoint proteins, and the like.

In one example, one of PD-L1 and egg-1 as the first protein, and the other as the second protein may be selected and applied to the method and apparatus. In another example, a heterodimer of HER2 and HER3 is selected as the first protein and a sub-protein of HER2 and / or HER3 is selected as the second protein in the cell or tissue and / Applicable to devices.

An example includes measuring protein-protein interactions between a first protein and a second protein, wherein said first protein is HER2 and HER3, said HER2 and HER3 forming a heterodimer, HER2, HER3, or HER2 in a cell or tissue, which is a sub-protein of HER2, HER3, or both in a tissue and / or in a signal transduction pathway between cells or tissues. (Or confirmation or determination or analysis) of the signal transduction pathway in which both (e. G., HER2-HER3 heterodimer forms) are involved. Another example provides a method of measuring (or identifying or determining or determining) the degree of activation of the HER2-HER3 heterodimer, comprising measuring the degree of phosphorylation of HER2 and / or HER3 in the HER2-HER3 heterodimer. In this case, the second protein may be selected from proteins that bind to the phosphorylating group of the HER2-HER3 heterodimer (e.g., phosphorylated tyrosine). .

Another example provides an apparatus for use in the method described above.

In another example,

1 by supplying a labeled substance-bound egg-1 to a substrate to which PD-L1 is attached; And

Determining a signal (A) generated in the reacting step,

The reactants of PD-L1 and PD-1 form clusters,

Provides a method for measuring the interaction between PD-L1 and PD-1. The method may further include the step of supplying PC-L1 to a substrate to which a substance binding to the PD-L1 is attached to the surface, and attaching the PD-L1 to the substrate before the step of reacting. A step of supplying a PC-L1 in which a labeling substance is bound to a substrate to which a substance binding to the PD-L1 has been attached, and a step of measuring a signal (generated by the PD-L1 and the substance- . (A) value (i.e., [signal (A) value] / [signal (B) value] for the signal) and quantifying the signal. In the above-mentioned step of reacting, a step of adding a substance competing with PD-L1 or PD-1 to the binding between PD-L1 and PD-1 is performed to perform a step of determining a reaction Chicago signal. In the method, the signal (A) or () may be measured in the near-field region. In the above method, the substance binding to the PD-L1 attached to the substrate may be a substance binding to the end of the PD-L1.

In another example,

1 mixture of the candidate substance, egg-Ll, and the labeled substance bound thereto

Supplying and reacting a substance that binds with PD-L1 to a substrate having a surface attached thereto; And

The signal obtained in the step of reacting (measuring the time Lt; RTI ID = 0.0 > PD-1. &Lt; / RTI > In this method, a PC-L1 having a label attached to a substrate to which a substance binding to PD-L1 is attached is supplied, and a signal (produced by the reaction between PD-L1 and the substance on the surface of the substrate is measured The signal A may be measured in the near-field region. The method may include measuring the value of the signal A for the signal (i.e., (A) value] / [signal (B) value]) of the candidate substance, and quantifying the candidate substance. When the signal (A) value or the signal (A) value for the signal (A) value at the treatment group (for example, after the candidate substance treatment) decreases, the candidate substance is selected as an inhibitor of interaction between PD-L1 and PD- Step may be further included.

In another example,

Introducing a first receptor tyrosine kinase or a gene encoding the first receptor tyrosine kinase into a second receptor tyrosine kinase-expressing cell;

Introducing a tyrosine phosphorylase ligand into the cell at a first demand to form a duplex between the first receptor tyrosine phosphorylase and the second receptor tyrosine phosphorylase; And

Treating the cell on which the dimer is formed with a cholesterol-like detergent

Lt; / RTI >

The first receptor tyrosine phosphorylase and the second receptor tyrosine phosphorylase are different kinds,

Lt; RTI ID = 0.0 > in vitro, < / RTI >

And a method for producing an activated receptor tyrosine kinase dimer. The prepared receptor tyrosine kinase duplex may be in an activated form, as compared to the case without the method. In this method, the first receptor tyrosine phosphorylase may be HER3 and the second receptor tyrosine phosphorylase may be HER2. In addition, the cholesterol-like detergent may be at least one selected from 0.05 to 5% (DGTN) and 0.003 to 2% (GDNO) of glycolic acid.

[Technical Solution]

This will be described in more detail below. As used herein, the term " protein-protein interaction (PPI) " may refer to a physical and / or chemical association or complex formation between a low U protein and a second protein, , Binding frequency, binding strength (intensity), and binding time. In addition, the interaction (binding) between the first protein and the second protein includes not only direct interaction (binding) between them, but also other proteins (proteins located between the first protein and the second protein in the signal transduction pathway) It can be applied indirectly to the impression (bond) for indirect impression. The protein-protein interaction in the present specification may be a single-molecule reaction (reaction between one molecule of the first protein and one molecule of the second protein). Cell or tissue interactions may refer to all interactions taking place between two or more homologous cells or allogeneic tissues, or surface proteins of the same / homogeneous / heterogeneous cell or homologous / heterologous / heterogeneous tissue.

In the present specification, the first protein and the second protein are each independently at least one selected from proteins involved in signal transduction pathways of cells or tissues of eukaryotic organisms (for example, multicellular animals, multicellular plants, etc.) Receptor and the other is a ligand, or may be _one or more selected from the surface proteins of xenogeneic cells _.

In one example, one or more of the first and second proteins may be phosphorylated (e.g., one or more of the first and second proteins may comprise phosphorylated tyrosine (p-Tyr) ). Thus, when at least one of the first protein and the second protein is phosphorylated, the interaction between the first protein and the second protein may be dependent on the degree of phosphorylation (p-Tyr level). In another example, one or more of the first and second proteins may have a surface variation that affects interactions between them. The mutation is not dependent on the presence or level of the protein of interest to interact with on the signal path (e.g., if the first protein is a receptor, irrespective of the presence or level of the interacting ligand or subprotein) (E. G., Exon 19 and / or exon 21 mutation (deletion) of EGFR, etc.).

(1), (2), and (3), or (1), (2), (3), and (4) in the protein-protein interaction measurement in all methods provided herein, or (1), (2), (3) and (4-1), or (1), (2), (3), (4-1) 2019/132517 1 »(: 1 ^ {2018/016675

( ₃ ) interaction between the receptor (first protein) and the ligand (second protein) (in this case,

1 change measurement possible),

(1) the interaction between the first protein and the second protein when at least one of the first protein and the second protein has a phosphorylated tyrosine residue (in this case, the degree of phosphorylation of the tyrosine predicts the degree of activation of the first protein and / Available), and

( ₀ ) Interaction between the first protein and the second protein of the low protein in the signal transduction pathway involved in the first protein

, &Lt; / RTI > two or more, or all three.

As used herein, the first protein may refer to one or more (e.g., one to ten or two or ten) proteins involved in the signal transduction pathway. The second protein is a protein that interacts with (binds to) the first protein. The second protein may be one or more proteins selected from the proteins involved in the lower pathway of the signal transduction pathway involved in the first protein, Ten to ten or two to ten species) proteins. When the first protein is two or more kinds, the second protein may be independently selected for each of the first proteins, and the second protein selected for each first protein may be different, partially or completely It can be duplicated.

In the present specification, the first protein is selected from among proteins associated with pathological conditions (e.g., cancer, inflammation, other immune diseases, etc.), so that the pathological conditions (e.g., cancer, inflammation, (And / or < / RTI > improve and / or alleviate) the disease. In this respect, the first protein may be a protein that is a therapeutic target of the disease to be treated. Specifically, the first protein may be a target therapeutic agent for a disease to be treated or a target protein of a therapeutic agent to be tested. Accordingly, the first protein can be appropriately selected depending on the therapeutic agent to be sought for the disease or effect to be treated.

In one example, the first protein is a protein involved in the mediator pathway in the biological signal transduction pathway of a cell or a tissue, and a cell surface protein or a protein secreted (released) by xenogeneic cells (for example, exo And the like), and can be advantageously used as a therapeutic target of the drug In order to function, one or more of the cell membrane proteins having a domain exposed to an extracelular environment (for example, an aqueous environment) and present in the cell membrane may be selected. For example, the first protein may be various receptors present in cell membranes, structural proteins associated with microfilaments, cell adhesion molecules, membrane enzymes, membrane receptors, And at least one selected from the group consisting of all kinds of cell membrane proteins, such as lipid-anchored proteins, carrier proteins, chamel proteins, transport proteins, lipid-anchored proteins, .

In one embodiment, the first protein is selected from the group consisting of receptor tyrosine kinase (RTK) (e.g., epidermal growth factor receptor (EGFR; ErbBl), human epidermal growth factor receptor 2 protein (HER2) ErbB2), HER3 (Human Epidermal Growth Factor Receptor 3 protein: ErbB3), hepatocyte growth factor receptor (HGFR): MET, platelet-derived growth factor receptors (PDGFR, VGFR1, VEGFR2, VEGFR3, etc.), Insulin-like Growth Factor 1 Receptor (IGF1R, VEGFR, EGFR receptors, fibroblast growth factor receptors (FGFRs such as FGFR1 and FGFR2), insulin-like growth factor receptors (e.g., 1 ike Growth Factor Receptor; IGFR, such as IGF1R), c-KIT, RET receptor tyrosine kinase, Anaplastic lymphoma kinase (ALK), etc.); Toll-like receptors (Toll like receptor; e. G., TLR1, TLR2, TLR3, TLR4 , TLR5, · TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, TLR13): G_ protein connection receiving stay (G-protein- coupled receptors (GPCRs); transferrin receptors; Low-Density Lipoprotein (LDL) receptors; R0S1; BCR-Abll fusion protein; (Eg, BRAF, mitogen-activated protein kinase kinase (MEK), Src, PI3K (Phosphoinositide 3-kinase), CDK cyclin-dependent kinases such as CDK4, CDK6, etc.); GTPases (GTPases) (e.g., KRAS and the like); The hormone receptor (e.g., Estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR), etc.); Anti- apoptotic proteins (such as BCL2 (B-cel 1 lymphoma 2), BIM (Bcl-2-1 ike protein 11), immune checkpoint proteins, (For example, CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4), PD-1 (programmed death 1), PD-L1 (programmed death ligand 1) But is not limited thereto.

The c-Met protein may be derived from any species and includes, for example, a human c-Met (c-Met) (E. G., NP_000236.2), a c-Met (e. G., Macaca mulatta,

NPJ301162100), or rodent-derived ones such as mouse c-Met (e.g., NP_032617.2), rat c-Met (e.g., NP_113705.1), and the like. Such a protein includes, for example, a polypeptide encoded by the nucleotide sequence provided in GenBank Ace Session Number __00 (crime 45.3, or a protein comprising the amino acid sequence provided in GenBank Aceession Number NP_000236.2, or an extracellular domain thereof. The receptor tyrosine kinase c-Met is involved in various mechanisms such as, for example, cancer development, cancer metastasis, cancer cell migration, cancer cell infiltration, and neovascularization process.

The "Epidermal growth factor receptor "

EGFR), HER2 (human epidermal growth factor receptor 2 protein), and HER3 (human epidermal growth factor receptor 3 protein) are HER family members and receptor tyrosine kinases (RTKs) composed of EGFR (HERD, HER2, HER3 and HER4) Binding of the ligand to the extracellular domain of EGFR, HER2, or HER3 induces receptor homo- or hetero-dimerization with other ErbB receptors, leading to intracellular autophosphorylation of specific tyrosine residues EGFR autophosphorylation leads to downstream signaling networks, including MAPK and PI3K / Akt activation, that affect cell proliferation, angiogenesis and metastasis EGFR, HER2, and / or HER3 overexpression, gene amplification, The sequences are frequently observed in many types of human malignant tumors and are associated with poor prognosis of cancer treatment and poor clinical outcome. For this reason, these EGFR, HER2, and / or HER3 EGFR, HER2, or HER3 may be a primate such as human, monkey, mouse, And may be derived from mammals such as rodents such as rats. For example, the EGFR can be obtained from GenBank Accession Nos. JQ739160, JQ739161, JQ739162, JQ739163, JQ739164, JQ739165, JQ739166, JQ739167, NM_005228.3, NM_201284.1,

NM_201282.1, or a polypeptide encoded by the nucleotide sequence (A) provided in NM_201283.1 or the like. For example, the HER2 may be derived from GenBank Accession No. < / RTI > Or may be a polypeptide encoded by a nucleotide sequence (mRNA) provided in X03363.1 or the like. For example, the HER3 can be obtained from GenBank Accession No. < / RTI > And may be a polypeptide encoded by a nucleotide sequence (mRNA) provided in NM_001982 or the like.

The "Vascular Endothelial Growth Factor Receptor "

Growth Factor Receptor (VEGFR) "exists in normal vascular endothelial growth factor cells. In particular, it binds to vascular endothelial growth factor (VEGF) secreted from cancer cells to induce angiogenesis and supply necessary nutrients to cancer cells. Overexpression of VEGFR is a cause of various diseases, and it is involved not only in the development of cancer but also after bad cases such as invasion and metastasis. For this reason, VEGF is an important target in chemotherapy. The VEGFR may be derived from mammals such as primates such as humans and monkeys, and rodents such as mice and rats. For example, the VEGFR may be a polypeptide encoded by a nucleotide sequence (mRNA) provided in GenBank Accession Number AF063657.2.

The above-mentioned " platelet-derived growth factor receptors (PDGFR) "is one of the surface receptor tyrosine kinases, and is associated with many diseases that cause cell growth, cell differentiation, cell growth, The PDGFR may be derived from mammals such as primates such as humans and monkeys, and rodents such as mice and rats. For example, the PDGFR can be obtained from GenBank Accession Nos. NM_006206.4 (PDGFR-A), NM_002609.3 (PDGFR-B),

(SEQ ID NO: 1), or a nucleotide sequence (mRNA) provided in SEQ ID NO.

The "Insulin-i Ike Growth Factor 1 Receptor (IGF1R) ¹ " is one of the receptor tyrosine kinases and is a transmembrane receptor that is activated by insulin-like growth factor 1 (IGF-1) . The IGF1R may be derived from a mammal such as a primate such as a human, a monkey, a rodent such as a mouse or a rat, etc. For example, the IGF1R may be derived from a nucleotide sequence encoded by nucleotide sequence (A) provided in GenBank Accession No. NM - 2019/132517 1 »(: 1/10/06 018/016675

Lt; / RTI > polypeptide.

The above-mentioned "ephrins receptors" are one of the surface receptor tyrosine kinases, which regulate the axon guidance, the format ion of the ssue boundaries, the cell migrat ion, and the segmentation ion. The ephrin receptor may be derived from mammals such as primates such as humans and monkeys, and rodents such as mice and rats. For example, the ephrin receptor may be derived from GenBank Accession Nos. NM_004440.3, NM_004438.3, NM_004431.3,

NM_004442.6, NM_017449.3, NM_004093.3, NM_004441.4, NM_182472.2,

NM-005232.4, NM_005233.5, NM_173641.2, NM_001099439.1, NM_001080448.2, NM_001080448.2, NM_004443.3, NM_182689.1, NM_004428.2, NM_004439.5,

NH001962.2, NM_004429.4, NM_182644.2, NM_004952.4, NM_173655.2,

Or may be a polypeptide encoded by a nucleotide sequence (mRNA) provided in NM_182690.2, NM_020526.3, NM_001406.3, NM_005227.2, NM_182685.1, and the like.

The "transferrin receptors" are carrier proteins of transferrin, which participate in the intracellular transport of iron through receptor-mediated endocytosis and regulate intracellular iron concentrations. The transferrin receptor may be derived from a mammal such as a rat, a primate such as a monkey, or a rodent such as a mouse or a rat. For example, the transferrin receptor may be derived from GenBank Accession Nos. NM_001128148.1, NM_003234.2, NM_001206855.1,

NM_003227.3, B₃ 01188.1, M11507.1, and the like.

The " low-density lipoprotein (LDL) receptor "is a carrier protein of transferrin, which participates in the intracellular transport of iron through receptor-mediated endocytosis and regulates intracellular iron concentration. The LDL receptor may be derived from mammals such as primates such as humans and monkeys, and rodents such as mice and rats. For example, the transferrin receptor may be a GenBank

Accession Nos. NM_000527.4, NM_001195802.1, NM_001195799.1,

Or may be a polypeptide encoded by a nucleotide sequence (mRNA) provided in NM_001195803.1, NM_001195800.1, NM_001195798.1, and the like.

The above-mentioned "cluster of diatoms (CD)" is a protein that functions as a receptor or a ligand in various manners, _and about 350 kinds of proteins are known in humans. Cel l signal ing, and cell adhesion. The surface-differentiating antigens may be derived from primates such as humans and monkeys, and mammals such as rodents such as mice and rats. For example, the surface-differentiating antigens may be of any CD sequence, and in particular may be CD44, CD147, or their antigens associated with cancer metastasis, and more particularly GenBank Accession Nos.

(MRNA) provided in the nucleotide sequences (NM_000610.3, NM_001728.3, X55150.1) and the like.

The ⁿ G-protein coupled receptor (GPCR) is a transmembrane receptor protein that activates signal transduct ion pathway and cell response, and is involved in various diseases. GPCRs can be divided into six classes based on sequence homology and functional similarity as a large family of proteins: Class A or Class 1 (Rhodops in-1 ike receptors); Class B or Class 2 (Secret in receptor familys) ; Class C or Class 3 (Metabotropic glutamate / pheromone); Class D or Class 4 (Fungal matting pheromone receptors); Class E or Class 5

(Cycl ic AMP receptors); and Class F or Class 6

(Fr zled / Smoothened). The GPCR may be derived from mammals such as primates such as human, primate, rodent such as mouse and rat. For example, GPCRs can be chemokine receptors (Rhodopsin-1 ike receptor subfamily) associated with cancer metastasis, such as CXC chemokine receptors, CC chemokine receptors, CX3C chemokine receptors, etc., and more specifically GenBank Accession Nos. And may be a polypeptide encoded by a nucleotide sequence (mRNA) provided in NM_001123041.2, NM_005508.4, NM_005201.3, NM_016602.2, and the like.

The second protein may be any protein that interacts with the first protein selected as described above, for example, a protein involved in a sub-pathway of a biological signal transduction pathway of a cell or tissue involved in the first protein, A ligand, a protein which is present on the surface of a xenogeneic cell or released from a xenogeneic cell, and the like, or may be selected from two or more (for example, one to ten or two to ten) species. Cells or tissues, such as the signaling pathways and the proteins involved in human cells or tissues, ligands for specific receptors, xenogeneic cell surface proteins interacting with specific proteins present on the cell surface, Relatively well established (Untangling the ErbB signaling network, Nat. Rev. Mol. Cell Biol. 2, 127 (2001); Cel l 141, 1117 (2010)). When the first protein is selected, the selection of a protein involved in the downstream signal transduction pathway is not limited to the conventional method of the present invention. It is clear to those who have knowledge.

Also, it is well known that one protein participates in various bio-signal transmission pathways, and a plurality of signal transmission pathways can form a network. Thus, when two or more first proteins are present, one or more of the second proteins for any one first protein may overlap one or more of the second proteins for the other first protein (s) (i.e., 1 < / RTI > proteins, the second proteins for these two or more first proteins may be different, or some or all, the same).

In one example, the first protein may be a complex of two or more different proteins (first protein-first protein complex). In another example, two or more first protein-second protein complexes formed by interaction between a first protein and a second protein are linked (e.g., a bond between a second protein of a neighboring first protein-second protein complex) , And can be a cluster.

In one embodiment, the first protein may be at least one selected from the group consisting of EGFR, MET, HER2, and HER3, and the second proteins for each first protein may be the same or different, , Phospholipase C (PLC) (e.g., PLC-gamma (PLC-gamma 1) (e.g., GenBank Accession No. NP_002651.2, NP_877963.1, NP_037319.1, etc.) or its SH2 domain (including at least one of Src homology 2 domain: N-terminal SH2 domain and C-terminal SH domain; For example, amino acid sequence regions 545 to 765 in NP_037319.1 or amino acid sequence regions 540 or 545 to 765 in NP_002651.2), growth factor receptor-binding protein Grb (e.g., Grb2 (see, for example, GenBank Accession No. 2). NP_002077.1, NP_987102.1, etc.) or a portion thereof (e.g., the SH2 domain (NP_002077.1

(Amino acid sequence positions 57 to 155), SH3_N-SH2 domain (the first to 154th amino acid sequence regions of NP_002077.1), SH2- SH3_C domain (the 57th to 217th amino acids of NPJ302077.1 Amino acid sequence Site)), phosphatidylinositol 3-kinase modulating subunit

(For example, GenBank Accession No. NP_001229395.1, NP_852556.2, NP_852664.1, NP_852665.1, P26450.2, etc.) or its SH2 (SEQ ID NO: (Amino acid sequence region from 333rd to 428th), SH2_C domain (amino acid sequence region from 624th to 718th), or tandem SH2 domain (from the 333rd to the 428th amino acid sequence region) of NP_852664.1 (human p85a) (Amino acid sequence region from position 624 to position 718) or SH2_N domain (from position 333 to position 428) of P26450 (mouse p85a), SH2_C domain (amino acid sequence region from position 624 to 718), or tandem SH2 domain Amino acid sequence region from position 333 to position 718)), and the like, but the present invention is not limited thereto.

In the examples provided herein, the combination of EGFR, MET, HER2, and HER3 as the first protein and PLC-gamma 1, Grb2, and p85_alpha as the second common proteins of these first proteins In the case of breast cancer, a combination of HER2 and HER3 is used as the first protein and PLC-gamma 1, Grb2, and p85-alpha are used as common second proteins of these first proteins. The present invention is not limited thereto and can be appropriately selected according to the therapeutic agent to be sought based on the above-described diseases or effects to be treated, and it is obvious to those skilled in the art to which the present invention belongs.

In another embodiment, the first protein may be at least one selected from the group consisting of EGFR, MET, HER2, and HER3, and the second protein to the first protein may be selected from among ligands binding to the respective first protein (Eg, EGFR epidermal growth factor (EGF), MET hepatocyte growth factor (HGF), HER2 and HER3 Neuregul ins (NRGs)).

In one example, the first protein is any one selected from PD-L1 and egg-1, and the second protein is the other. In one embodiment, the first protein is egg-1 and the second protein is PD-Ll. In another embodiment, the first protein is PD-L1 and the second protein is PD-1.

PD-L1 (Programmed death-l igand 1) is a protein present on the surface of cancer cells. CD274 (cluster of diiferent ion 274) or B7_H1 (B7 homolog 1) and is a 40 kDa type 1 transmembrane protein. PD-L1 is present in subtypes of immune cells (Macrophage, etc.) as well as cancer cells. PD-L1 is known to play a major role in suppressing the immune system in various diseases such as tissue graft, immune diseases and hepatitis. The PD-L1 described in the present specification may be derived from a mammal such as a human, and examples thereof include human PD-L1 (eg, GenBank Accession No. NP_001254635.1 (gene: NM_014143.3), NP_001300958.1 NM_001314029.1),

NP_054862.1 (gene: NM_001267706.1), etc.).

PD-1 (Programmed cell death protein 1), also called CD279 (cluster of di fi erent iat ion 279), is a protein that is mainly present on the surface of activated T cells (immune cells) and is one of the immune checkpoints , Apoptosis in antigen-specific T cells (lymphocytes) in the lymph nodes, and apoptosis in regulatory T cells (lymphocytes) do. In addition, it is expressed on the surface of cancer cells and promotes tumor proliferation through signaling pathway such as mTOR. PD-1 described herein may be of mammalian origin, such as human, and may be, for example, human PD-1 (e.g., GenBank Accession Nos.

NP_005009.2 (gene: NM_005018.2), etc.).

When PD-L1 binds to the PD-1 protein on the T cell surface, T cells lose their ability to attack cancer cells.

PD-L1 and / or egg-1 may be important targets in the development of anti-cancer drugs.

As used herein, the first protein and / or the second protein may be used in a form contained in a cell, a tissue, or a lysate or an extract thereof, or in the form of a purified protein.

In another example, the first protein is HER2 (Human Epidermal Growth Factor Receptor 2 protein; ErbB2) and Human Epidermal Growth Factor

Receptor 3 protein; ErbB3), and HER2 and HER3 may be heterodimers (HER2-HER3 heterodimers)

HER2 and HER3 are members of the HER family receptor tyrosine kinases (RTKs), respectively. Binding of a ligand to the extracellular domain of HER2 or HER3 induces receptor homo- or hetero-dimerization with other ErbB receptors, which leads to intracellular autophosphorylation of specific tyrosine residues. HER2 and / or Autophosphorylation of HER3 leads to downstream signaling networks including MAPK and PI3K / Akt activation that affect cell proliferation, angiogenesis and metastasis. HER2 overexpression and gene amplification are frequently observed in breast and stomach cancer and are associated with poor prognosis of cancer treatment and poor clinical outcome. HER3, on the other hand, is well known as a resistance mechanism for several target drug therapies. For these reasons, these HER2 and / or HER3 are important targets in chemotherapy. HER2 and HER3 may be derived from mammals such as humans. For example, HER2 is GenBank Accession No. < RTI ID = 0.0 > NP_001005862.1, NP_001276865.1, NP_001276867.1, NP_004439.2, and the like, but is not limited thereto. HER3 was obtained from GenBank Accession No. NP_001005915.1, NP_001973.2, and the like, but are not limited thereto.

The second protein is a sub-protein of HER2 and / or HER3 (e.g., HER2-HER3 heterodimer) in various vital signal transduction pathways and interacts with HER2 and / or HER3 (e.g., HER2-HER3 heterodimer) ). &Lt; / RTI > In one example, the second protein may bind to a phosphorylating group (e.g., phosphorylated tyrosine) of HER2 and / or HER3 (e.g., HER2-HER3 heterodimer).

Measuring the protein-protein interaction between the first protein task 2 protein and the first protein task performed in the method provided in the present invention, measuring the degree of activation of the first protein or the first protein enzyme, Or in vitro or in vivo (in _{F / i} ro).

Measuring the protein-protein interaction between the first and second proteins may comprise the following steps:

(1) preparing a substrate to which a first protein is immobilized by adding a test sample containing a first protein to a substrate containing a substance that specifically binds to the first protein on the surface;

(2) adding a second protein labeled with the labeling substance to the substrate on which the prepared first protein is immobilized, and reacting; And

(3) immediately determining a signal from the reactant obtained in step (2)

₍ Protein-protein interaction measurement _).

Wherein measuring the protein-protein interaction between the first protein and the second protein comprises, in addition to the steps (1) to (3), (4) 2019/132517 1 »(: 1 ^ {2018/016675

Signal to measure the level of activation of the first protein.

The step (4) of measuring the activation level of the first protein using the signal measured in the step (3) includes the step of measuring the activation level of the first protein in the test sample added in step (1) And obtaining a signal value for the unit amount.

In one example, the step (4)

A step of obtaining a signal value for the first protein unit amount in the test sample added in step (1) using the signal measured in step (3)

(4-1) obtaining a signal value for the unit amount of the test sample added in the step (1) (measuring the protein-protein interaction level) using the signal measured in the step (3); And

(4-2) obtaining a value for the unit amount of the first protein contained in the test sample (signal level of activation level) using the signal value of the unit amount of the test sample obtained in the step (4-1)

.

In order to measure (confirm, determine or analyze) a desired substance by using the measured protein-protein interaction between the first protein task 2 protein, after the step (4)

(5) comparing the result obtained in the step (4) with the result obtained in the reference sample.

In one example, the first protein is any one selected from? ^- 11 and egg-1, the second protein is

And? ^ ^~ 1. In another example, the first protein may be a quadrivalent 2 - 1, 3 heterodimer. In this case, the step of measuring the degree of tyrosine kinase activation (e.g., phosphorylation of the tyrosine residue) of the other 1? 2 and / or 4? 3 in the 4 1? 2? 4 thymine 3 heterodimer may include the following steps :

(1-1) quaternary 2-hetero 3 dimer, or a sample comprising a 4 1 2-quaternary 3 heterodimer on a surface of a quartet 2 and / or other quaternary 3 To the included substrate

Preparing a substrate on which the heterodimer is immobilized;

(2-1) Suppression of the 2-depressant: 1? 3 heterodimer is phosphorylated and labeled Adding a phosphorylated Tyr-specific binding substance (e.g., an antibody) to which the substance is bound); And

(3-1) Step (tyrosine kinase activation assay) of immediately determining a signal from the reaction product obtained in the step (2-1).

In the step (2-1), the phosphorylating step may include treating the phosphorylating agent to the HER2-HER3 heterodimer immobilized on the substrate or the substrate. The phosphate reagent may be a HER2 and / or can be at least one selected from every substances capable of HER3 phosphorylation, e.g., ATP and magnesium (e.g., MgCl _2, etc.), without being limited thereto. The method of claim 1, wherein, before, after, or simultaneously with the phosphorylation, and before the activation measurement of step (3-1), at least one selected from cholesterol-like detergents including DGTN, GDN and the like is added to the HER2- The detergent may be treated with a CMC (cortical microcrystalline) concentration or higher to maintain the structural and / or functional activity of the HER2-HER3 heterodimer.

In the case where the first protein is a HER2-HER3 heterodimer, in addition to the above-described step (1) to (3) or (1-1) to (3-1), the following step (4) and / ).

Hereinafter, the above steps will be described in more detail:

Step (1) or (1-1): Substrate preparation step in which the first protein is immobilized

The step (1) is a step of preparing a substrate to which a first protein is immobilized by adding a test sample containing a first protein to a substrate containing a substance that specifically binds to the first protein on the surface.

The first protein is as described above.

The test sample may be any biological sample that can be used to test the response to a drug targeting the activating first protein in the cell or tissue and / or the signal transduction pathway between cells or tissues.

For example, the test sample may be a lysate, a lysate, or an extract of a cell, a tissue, a cell or a tissue isolated from an individual, a body fluid (e.g., blood (whole blood, plasma or serum), saliva, etc.). The subject is preferably selected from the group consisting of an activation test of a signal transduction pathway in a cell or tissue and / or a cell or tissue involved in the first protein, a reactivity test for a drug targeting the first protein, of the _crystal, the _first protein target therapeutic effect (For example, primates such as human, monkey, rodents such as mice, rats, etc.) which are to be used for monitoring and / or selecting effective first protein target therapeutic agents. In one example, the subject may be a patient having a disease associated with the first protein. The disease associated with the first protein may be a disease associated with overexpression of the first protein or activation of a signal transduction pathway in the cell or tissue and / or between the cell or tissue involved in the first protein, for example, the cancer. In one embodiment, the test sample is used to determine the degree of activation of the signal transduction pathway in a particular cancer patient (e. G., The cell or tissue involved by the first protein and / or between the cell or tissue, For example, a cancer cell, to determine whether it is an appropriate subject for a first protein target treatment, or to isolate a specific entity from which an effective first protein target therapeutic agent is to be selected). In the case of tissue, a size of at least 125 mm ³ is sufficient for grinding, and the amount of tissue sample required for one measurement may be in the range of about 1/50 to about 1/75 of the amount of tissues mentioned above (at least 125 mm ³ ) But is not limited thereto. In the case of cells (cancer cells), the amount of cellular sample required for one-time measurement of about 10 cel ls to 10 ¹⁰ cel ls, of about 10 cel ls to 10 ⁷ cel ls, of about 10 cel ls to 10 ⁵ cel ls, of about 10 ³ cel ls to 10 ¹⁰ cel ls, of about 10 ³ cel ls to 10 ⁷ cel ls, of about 10 ³ cel ls to 10 ⁵ cel ls, for example, may be an order of about 10 ⁴ + 50 cel ls-, it is not limited to, cell lines Which is a value that can be appropriately determined according to the type of the film.

In one example, in the case of prediction of responsiveness to a drug targeting the first protein and / or selection of a subject suitable for treatment of the first protein target, the test sample may comprise a treatment that targets the first protein (e.g., Administration of a drug targeting the protein) may be a cell or tissue that has not been subjected to an action, or a cell or tissue that has been separated from a subject in which the treatment (or administration of the drug) has not been performed, In the case of a method for monitoring the reactivity, the test sample may be a cell or a tissue on which the first protein-targeting treatment (for example, the administration of the drug targeting the first protein) has been performed or the treatment (or administration of the drug) Lt; RTI ID = 0.0 > isolated < / RTI >

The substrate may be of any material and / or of any structure capable of immobilizing the first protein on its surface (both crystalline and amorphous 2019/132517 1 »(: 1 ^ {2018/016675

Available). In one example, the substrate may be a material having a refractive index of water equal to or higher than that of water (about 1.3), which occupies a large portion of the biomaterial, taking into consideration the easiness of detecting a beacon signal. In one example, the substrate thickness reducible 0.1 to about 1 _, from about 0.1 to about 0.5 ■, 0.1 to about 0.25 _1®, or about 0.13 to about 0.21, and _■ be on the order of a refractive index of about 1.3 to about 2, about 1.3 About

1.8, about 1.3 to about 1.6, or about 1.5 to about 1.54. The substrate may be any material that satisfies the refractive index range and may be, for example, from a material selected from the group consisting of glass (refractive index: about 1.52), quartz, and the like, but is not limited thereto. The substrate may be of any type conventionally used for biological specimen observation and may be, for example, a well type, a slide type, a channel type, an array type, a microfluidic chip, a microtubule (capillary) It is not. When observing a fluorescent microscope, a cover glass can be observed on a substrate to which the sample is applied. The material of the cover glass is as described above for the substrate, and the thickness may be the range described in the substrate or may be thinner (for example, a refractive index of 1.52, 0.0 > 0.17, < / RTI > but not limited thereto). Substance specifically binding to the first protein can be selected from any substance capable of binding to the first protein and specifically, for example, a specific antibody or anti thereof that binds to the first _protein: One-binding fragment (E.g., 1, () acid-! ⁷ , Fab, ¹ and (2) 2), platamer (protein or nucleic acid molecule), small molecule compound, and the like. At this time, the substance specifically binding to the first protein is a site that does not interfere with the interaction between the first protein and the second protein, that is, a site where the first protein and the second protein interact (bind) And may bind to the first protein at a non-specific site.

In one example, the substrate is suitably surface-modified to include (immobilize) a biological substance (for example, an antibody or the like) that specifically binds to the first protein on the surface, or may be surface- The binding material may be fixed. When the substrate is surface-modified, the substrate may be treated (e.g., coated) with all of the compounds having a functional group capable of purifying a biological substance (for example, an antibody or the like) that specifically binds to the first protein on one surface, For example, a compound containing a functional group selected from the group consisting of an aldehyde group, a carboxyl group and an amine group. In one embodiment, The compound containing a functional group selected from the group consisting of an aldehyde group, a carboxyl group and an amine group may be selected from the group consisting of biotin, bovine serum albumin (BSA), bovine serum albumin bound with biotin, polyethylene glycol (PEG) Biotin-conjugated PEG (polyethylene glycol-biotin), polysorbate (e.g., Tween 20), and the like, but the present invention is not limited thereto. The surface-treated substrate may be further treated (e.g., coated) with at least one selected from the group consisting of neutravidin, streptavidin, avidin, and the like.

In one example, the first protein may be PD-L1 or egg-1. Specifically, the first protein may be PD-L1. When protein-protein interactions are measured by immobilizing PD-L1 on a substrate using a substance (such as an antibody) that specifically binds to PD-L1 using PD-L1 as the first protein, PD- This clustered spot is formed, which has the advantageous effect of qualitative and / or quantitative analysis.

In another example, the first protein may be PD-I. When protein-protein interactions are measured by immobilizing PD-1 on a substrate using a substance (for example, an antibody) that specifically binds to egg-1 using PD-1 as a first protein, So that a favorable effect can be obtained in the analysis requiring a single spot detection.

The test sample is used to test the level of interaction between PD-L1 and egg-1, the activation of the signal transduction pathway involved in the first protein, and the reactivity to the drug targeting the first protein, among cells or tissues It may be all possible biological samples.

In addition, complexes formed by binding between PD-L1 and PD-1 (for example, on the surface of xenogeneic cells) are clusters, and the in vivo environment can be simulated more similarly. In order to form clusters more similar to the in vivo environment, it may be advantageous to select PD-L1 as the first protein to be immobilized on the substrate.

In one example, the case of using the PD-L1 as a first protein, ^a substance that binds to the first protein may be a specific antibody or an antigen-binding fragment that specifically binds to PD-L1. In order not to affect the mutual binding between PD-L1 and suppress-1, the antibody specifically binding to PD-L1 or The antigen-binding fragment may specifically bind to a non-binding site (for example, does not overlap with the binding site) of the PD-L1 binding site, for example, the C-terminal site of PD-L1. The site where PD-L1 binds to PD-1 is generally the N-terminal region (extracellular domain site) of PD-L1, and specifically, the 20th to 150th, 20th to 130th, To 125th,

25 to 150, 25 to 130, or 25 to 125 amino acid residues, and examples thereof include PD-L1 At least one amino acid residue selected from the group consisting of D26, Y56, E58, R113, A121, D122, Y123, and R125. NP_001300958.1,

Human PD-L1 reference such as NP_054862.1) or a corresponding site (see Fig. 32). The non-binding site except for the binding site of PD-L1 with PD-1 is a PD-L1 site in which the N-terminal PD-L1 of the PD-L1 described above does not overlap with the site binding to PD- And the antibody or antigen-binding fragment thereof that specifically binds to PD-L1 binds to the PD-

1 to 30, 1 to 25, 1 to 20, 1 to 15, or 1 to 20 of the non-binding sites (for example, the C-terminal sites of PD-L1) An antibody or an antigen-binding fragment which binds to 1 to 10 amino acids. In one example, the antibody or antigen-binding fragment thereof that specifically binds to PD-L1 binds to the PD-L1 protein in the N-terminal to C-terminal direction, after the 250th, after the 260th, The amino acid sequence may be one that specifically binds to one or more amino acids selected from the amino acid positions after the 280th position, for example, from the 250th position to the 300th position, or from the 280th position to the 290th position.

In another example, when PD-1 is used as the first protein, the substance specifically binding to the first protein may be an antibody or an antigen-binding fragment thereof that specifically binds to egg-1. In order to prevent mutual binding between PD-1 and PD-L1, the antibody or antigen-binding fragment that specifically binds to PD-1 binds to the binding site of PD- Binding site at the C-terminal site of the repressor-1, i.e., not overlapping the C-terminal site of the repressor-1. The site where PD-1 binds to PD-L1 is generally the N-terminal region (extracellular domain site) of egg-1, and specifically the 60th to 200th, 60th to 150th, 60th From the 135th to the 135th, 65 to 150, or 65 to 135 amino acid residues, and examples thereof include a region containing at least one amino acid residue selected from the group consisting of Y68, Q75, K78, T76, 1126, 1134, and the like. The non-binding site other than the binding site of PD-L with PD-L1, for example, the C-terminal site of PD-1, is a site in which the PD-1 at the N- And the antibody or antigen-binding fragment thereof that specifically binds to PD-1 may be a non-binding site other than the binding site of PD-1 with PD-L1 (for example, 1 to 30, 1 to 25, 1 to 20, 1 to 15, or 1 to 10 amino acids in the C-terminal region of PD-1) Can be.

In another example, the first protein may be a HER2-HER3 heterodimer with HER2 and HER3 dimerized. In this case, the sample containing the first protein may comprise HER2 and / or HER3, or a HER2-HER3 heterodimer,

A cell isolated from a patient to be tested comprising recombinant cells expressing HER2 and HER3, a cell lysate, or a cell lysate, a recombinant cell engineered to express HER2 and HER3 simultaneously (for example, a recombinant cell expressing HER2 A recombinant cell into which a recombinant vector containing a HER3 gene is introduced into a cell, a recombinant cell into which a recombinant vector containing a HER2 gene is introduced into a cell expressing HER3, etc.), or a lysate or a lysate of the cell.

When the first protein is a HER2-HER3 heterodimer, the heterodimer of HER2 and HER3 may be characterized by treating the dimerization agent with cells that simultaneously express or express HER2 and HER3. The dimerization agent may be a ligand of HER3 (e.g., Neuregulin pi (NRGbl);

NP_039250, NP_039250.2, etc.), cholesterol-like detergent (e.g., DGTN (digitonin; CAS Number: 11024-24-1), GDN

(Glycol-diosgenin; CAS Number: 1402423-29-3)), and the like. In one embodiment, when NRGbl is treated (injected) into HER2 and HER3 expressing cells to form a HER2-HER3 heterodimer, the NRGbl may be injected into the cell prior to lysis after HER2 and HER3 expression have. In another embodiment, a cell that simultaneously expresses HER2 and HER3 (including) a cholesterol-like detergent to treat HER2-HER3 In the case of forming a heterodimer, the cholesterol-like cleansing agent may be treated (added) after cesiolysis and prior to measurement of activation of HER2 and / or HER3 (in one example, the cell has an NRGbl of between 10 ng / mL, 50 ng / mL to 200 ng / mL, or 10 ng / mL to 100 ng / mL). In this case, the concentration of the cholesterol-like detergent to be used may be in excess of the concentration of the cleaning agent used, for example, the concentration of the cholesterol-like detergent may be, for example, for DGTN 0.05% (w / v) or more, 0.05% (w / v) in excess, or 0.06% (w / v) or more, e.g., 0.05 to 2% (w / v) or 0.06 to _{2¾> (w}八), 0.003% (w or more, 0.005% (w) or more, 0.007% (w or more), or 0.01% (w / v) or more, such as 0.003 to 2% v), 0.005 to 2% (w / v), 0.007 to 2% (w / v), or 0.01 to 2¾ _{> (} w 8 0) It is difficult to maintain the membrane permeability of the protein and maintain the tyrosine kinase activity, and the activity of the HER2-HER3 heterodimer (for example, phosphorylation degree) is lowered and is not restored. Thus, suggestion Digi tonin is highly unstable at dissolution and precipitates within a few hours, so it is recommended that 10 to 20% (w / v) or 8 to 12% (w / v) GDN is stable for several days even when dissolved GDN is stable for several days All solutions can be maintained at pH 7 to 8.7, 2 to 7.6, or 7.4 using HEPES buffer, and NaCl

150 mM should be added to maintain proper ion concentration. To inhibit protein degradation by intracellular proteases during cell lysis, protease inhibi tor cocktail can be added (eg, Sigma-Aldrich P8340, approximately 2% (w / v)).

When the first protein is a HER2-HER3 heterodimer, the substance specifically binding to the low U protein may be selected from the first protein, i.e., HER2, HER3, or any substance capable of specifically binding to all of them (ScFv) 2, scFv-Fc, Fab, Fab 'and F (ab') 2 (SEQ ID NO: 2) of an antibody that specifically binds to HER2, HER3, Etc.), platamer (protein or nucleic acid molecule), small molecule compound, and the like. , A substance that specifically binds to the first protein (rule: R2, HER3, or both of them) is a region that does not interfere with the interaction between the first protein and the second protein, that is, In the region where the second protein does not interact (bind), the first It can be associated with proteins.

Step (2): Step of reacting the first protein task 2 protein

In the step (2), the labeled second protein is added to the substrate on which the prepared first protein is immobilized and reacted.

The second protein is as described above.

The labeled second protein may be labeled with a labeling substance that generates a detectable signal of the second protein (for example, the labeling substance is chemically (e.g., covalent or noncovalent), recombinantly, or physically bound) , And a tag-attached form to which a labeling substance can be bound. The detectable signal may be selected from any signal (e.g., light, radiation, etc.) that can be measured through conventional enzymatic reactions, fluorescence, luminescence, and / or radiation detection. The labeling substance may be at least one selected from the group consisting of all small molecule compounds capable of generating the label signal, proteins, peptides, and nucleic acid molecules. Examples of the labeling substance include fluorescent dyes (small molecule compounds: Cyanine, Alex, Dylight, Fluoprobes Etc.), fluorescent proteins (e.g., green fluorescent protein (GFP), enhanced GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), blue fluorescent protein (BPF), red fluorescent protein Lt; / RTI > The tag may be at least one selected from all kinds commonly used such as Hi s-tag / Ni-NTA. The concentration of the labeling substance to be used may be appropriately determined in the range of about luM or less, for example, in order to allow accurate and easy detection without generating noise. For example, InM to 1000 nM, InM to 500 nM, InM to 100 nM, To about 100 nM, from about 10 nM to about 500 nM, or from about 10 nM to about 100 nM, but are not limited thereto. The signal generated from such a labeling substance can be measured by any signal detecting means (for example, a conventional fluorescence microscope, a fluorescence camera, a fluorescence intensity measuring apparatus, etc.) commonly used for detecting or measuring the signal.

Between the step of reacting (step (2)) and the step of measuring the subsequent protein-protein interaction (step (3)), the substrate May be further washed in a conventional manner.

In one example, the second protein may be PD-L1 or PD-1. In one embodiment, when the first protein is PD-L1, the second protein may be PD-1. In another embodiment, when the first protein is PD-1, the second protein may be PD-L1.

In another example, when the first protein is a HER2-HER3 heterodimer, (2) the step of reacting the first protein task 2 protein comprises (2-1) phosphorylating the HER2-HER3 heterodimer and contacting the phosphorylated tyrosine- And the like.

When the first protein is a HER2-HER3 heterodimer, the step (2) or (2-1) may be carried out by adding the labeled second protein to the substrate on which the prepared first protein is immobilized, And adding the labeled phosphorylated tyrosine specific binding substance to react. The labeled phosphorylated tyrosine specific binding material may be an antibody that specifically binds to phosphorylated tyrosine of HER2 or HER3.

When the first protein is a HER2-HER3 heterodimer, the second protein is a sub-protein of HER2 and / or HER3 (e.g., HER2-HER3 heterodimer) in various biological signaling pathways, And / or proteins capable of interacting (binding) with HER3 (e.g., HER2-HER3 heterodimer).

When the first protein is a HER2-HER3 heterodimer, in the step (2-1), the phosphorylation step may comprise treating the substrate or the HER2-HER3 heterodimer immobilized on the substrate with a phosphorylating agent . The phosphate reagent may be a HER2 and / or can be at least one selected from every substances capable of HER3 phosphorylation, e.g., ATP and magnesium (e.g., MgCl _2, etc.), without being limited thereto. HER2-HER3 heterodimer immobilized on the substrate or substrate before, after, or simultaneously with the phosphorylation, and prior to the activation measurement of step (3-1), as described in step (1) or , HER2-HER3 heterodimers at a concentration exceeding one or more detergents selected from cholesterol-like detergents including DGTN, GDN, etc., in excess of the CMC crest micellar concentrate, Or functional activity thereof.

Step (3) or (3-1): Measurement of protein-protein interaction step

The step (3) is a step of measuring a signal from the reactant obtained in the step (2). The signal measurement may be carried out using a conventional signal (e. G., An enzyme reaction, fluorescence, luminescence, or radiation detection) 2019/132517 1 »(: 1 ^ {2018/016675

(Or measuring or verifying) a signal (e.g., a signal measurable via a method).

In one example, the measurement of protein-protein interaction in step (3) may be by real-time analysis.

In one example, when the label signal is a fluorescence signal, the signal detection is performed by supplying a fluorescence signal generated by supplying a light source absorbed by a labeling substance for generating the fluorescence signal to a fluorescence microscope, a fluorescence camera, and / And may be imaged, imaged, and / or quantified using, for example, an intensity measuring device.

In one embodiment, when the signal is a fluorescence signal, the fluorescence signal can be imaged and / or quantified using a fluorescence camera.

If the signal is a fluorescent signal, step (3) (protein-protein interaction measurement step)

(0) supplying a light source to the reactant of the step (2); and

(11) detecting a fluorescence signal generated by the supplied light source.

The step of supplying the light source of the step 0) is a step of supplying a light source to the reactant of the first protein task 2 protein obtained in the step (2). If the above purpose can be achieved, There is no. For example, the light source may be continuously supplied before step (1), simultaneously or after step (2), or may be supplied for a predetermined time immediately before, simultaneously with, or immediately after step (2) no.

The light source may be any light source having a wavelength corresponding to a fluorescent signal, for example, a laser, a halogen lamp, or the like.

Wavelength of the light source can be adjusted depending on the fluorescence signal using, for example, it can be selected from about 300 to about 600 ^ ₁ ^, or about 350 to about 560 ™ ™ range. More specifically, the green fluorescent protein absorbs about 480 ₁₁₁₁₁ , the yellow fluorescent protein absorbs about 540, the blue fluorescent protein absorbs about 375 < ₁ _> , and the blue fluorescent protein absorbs about 425, When the green fluorescence is used, the wavelength of the light source is about 460 to about 500, the wavelength of the light source is about 520 to about 560 when the yellow fluorescence is used as the fluorescence signal, and when the blue fluorescence is used as the fluorescence signal, The wavelength of the light source is ₁ to about 350 to about 400, and when the cyan fluorescence is used as the fluorescence signal, The wavelength can be selected in the range of about 400 to about 450 nm.

In one embodiment, the protein of step (3) - protein interaction measuring step, by using the total internal reflection fluorescence microscopy (Total Internal Ref lect ion Fluorescence (TIRF) mi croscope) or confocal microscopy, and supplies the light source, the typical ₅ Lt; RTI ID = 0.0 > fluorescence < / RTI > signal. In another example, a fluorescence camera for signal imaging, such as an EMC suppressor

(Electron-mul t iplying charge- coupled devi ce) The use equipped with a camera or a CMOS (Complementary metal oxide semi conductor) camera can perform imaging and / or quantification of the light source and fluorescence signals are fed _0.

In the following, the steps of measuring the protein-protein interaction in step (3) are carried out using a total internal reflection microscope and a fluorescent camera, for example, in more detail:

A) The substrate of step (1) or step (2) is mounted on the total reflection microscope ₅ . The supply position of the light source in the total reflection microscope is normally downward and the fluorescence signal is emitted from above the substrate (in this case, from the lower side to the upper side, the light source supply portion, the substrate, the lens or the substrate, under that it can position in the sequence) or (observed in this case, the upward direction from below, may be located in the lens, the light source supply, substrate, or the light source supply unit, lenses, substrate, and _0, or the lens, substrate, order of the light source supply unit) can do.

B) the light source may be a laser and the intensity of the light source may be from about 0.5 mW to about 5 mW, from about 0.5 mW to about 4.5 mW, from about 0.5 mW to about 4 mW, from about 0.5 mW to about 3.5 mW, mW, about 0.5 mW to about 2.5 mW, about 0.5 mW to about 2 mff, about 1 mff to about 5 mW, about 1 mW to about 4.5 mW, about 1 mW ₅ to about 4 mW, about 1 mW to about 3.5 mW From about 1 mW to about 4 mW, from about 1.5 mW to about 4 mW, from about 1 mW to about 3 mW, from about 1 mW to about 2.5 mW, from about 1 mW to about 2 mW, from about 1.5 mW to about 5 mW, 1.5 mff to about 3.5 mW,

And a strong light source can be used when an attenuation filter is used).

C) The fluorescence signal generated by the light source supply is transmitted to the fluorescent camera Imaging and imaging and / or quantification.

The imaging (or imaging) of the fluorescence signal can be performed simultaneously with the light source supply or within the signal generation holding time, taking into account the fluorescence signal generation holding time (luminescence time) of the labeling material.

In taking a fluorescent signal (or imaging) with a fluorescent camera (for example, an EMCCD camera), the exposure time per one frame, the laser power, the camera gain value, and the total shooting frame can be appropriately adjusted. For example, the shorter the dwell time per frame is, the less the accumulated signal in one frame. In order to compensate for this, the laser power can be increased or the sensitivity of the fluorescent camera can be increased. In one example, the exposure time per frame is from about 0.0 () 1 second to about 5 seconds, from about 0.001 second to about 3 seconds, from about 0.0 second to about 2 seconds, from about 0.0 () second to about 1 second, from about 0.001 From about 0.01 second to about 5 seconds, from about 0. 1 second to about 3 seconds, from about 0.01 second to about 2 seconds, from about 0.01 second to about 0.5 second, from about 0.001 second to about 0.3 second, from about 0.001 second to about 0.1 second, From about 0.01 second to about 1 second, from about 0.01 second to about 0.5 second, from about 0.01 second to about 0.3 second, from about 1 second to about 0.1 second, from about 0.05 second to about 5 seconds, From about 0.05 second to about 2 seconds, from about 0.05 second to about 1 second, from about 0.05 second to about 0.5 second, from about 0.05 second to about 0.3 second, from about 0.05 second to about 0.1 second, from about 0.07 second to about 5 seconds About 0. 07 second to about 3 seconds, about 0. 07 second to about 2 seconds, about 0. 07 second to about 1 second, about 0. 07 second to about 0.5 second, about 0. 07 second to about 0.3 second From about 0.07 seconds to about 0.1 seconds, from about 0.1 seconds to about 5 seconds, from about 0.1 seconds to about 3 seconds, from about 0.1 seconds To about 2 seconds, from about 0.1 second to about 1 second, from about 0.1 second to about 0.5 second, or from about 0.1 second to about 0.3 second, such as about 0.1 second.

For example, when an EMCCD camera is used, photons generated from a labeling substance (eg, eGFP) are converted to electrons through the EMCXD element (photoelectric effect). At this time, the number of electrons generated per photon can be changed through the gain value. The higher the set gain value, the greater the number of electrons generated per photon, and the higher the sensitivity of the EMCCD and the higher the background noise, the higher the signal-to-noise ratio. In one embodiment, to obtain an excellent signal-to-noise ratio, the gain value can be adjusted to a value in the range of from about 10 to about 100, from about 10 to about 80, from about 10 to about 60, from about 10 to about 50, from about 20 to about 100, From about 20 to about 80, from about 20 to about 60, from about 20 to about 50, from about 30 to about 100, from about 30 to about 80, 2019/132517 1 »(: 1 ^ {2018/016675

About 60 to about 60, or about 30 to about 50, such as about 40, but the present invention is not limited thereto, and can be appropriately selected in consideration of sensitivity, life span, equipment construction status, noise,

The total shooting time is obtained by multiplying the total number of shooting frames by the exposure time (exposure time _* total shooting frame number = shooting time). Since the fluorescence signal disappears after the emission time of the fluorescent material ( ₁₆₎ , the total number of photographed frames and / or the exposure time can be adjusted so that the photographed time progresses within the light emission time. In order to increase the accuracy of the protein-protein interaction measurement results, it is preferable that the imaging is performed by using one or more, for example, two or more, three or more, four or more, five or more, seven or more, performed on a substrate including one or more channels (each channel is at least two substrates comprising the same according to the determined size and the imaging area as possible)), and the signal appears ₃₁ be

: &Lt; / RTI > EIA), and quantify the fluorescence signal obtained, which can be seen as quantifying protein-protein interactions _.

In another example, the fluorescence intensity measured in step (3) may be quantified using conventional equipment to quantify protein-protein interactions.

When two or more of the first protein and / or the second protein are used, steps (1) to (3) can be performed for each first protein task 2 protein combination (that is, steps (1) to 3) can be repeated as many times as the number of combinations of the first protein and the second protein.

In the case where the first protein is quadrature 2-y3 heterodimer, the step (3) of measuring the protein-protein interaction may be applied to the step (3-1) of measuring the phosphorylation of the tyrosine residue.

The step (3) or (3-1) is a step of measuring a signal from the reactant obtained in the step (2) or (2-1). The signal measurement can be used to detect (or measure or verify) the label signal used in step (2) or (2-1) (e.g., a signal measurable via conventional means such as enzyme reaction, fluorescence, luminescence, ), Which can be performed using any means available.

In the case where the first protein is a first-order? 2-quaternary 3-heterodimer, the step of measuring the phosphorylation of the tyrosine residue in step (3-1) comprises the steps of: And detecting and / or quantifying a signal generated in a reaction between a substance (e. G., An antibody) that specifically binds to a phosphorylated tyrosine residue of 1 to 3. In one example, a fluorescent material (e.g., fluorescent Protein) is used, phosphorylation of the tyrosine residue can be measured by measuring and quantifying the fluorescence intensity detected in the reactant as described above.

Step (4): measuring the activation level of the first protein

The step of measuring the activation level of the first protein of the step (4) may include the step of measuring the activation level of the first protein in the test sample added in step (1) using the signal measured in step (3) ).

As used herein, "activation of a first protein " means interaction (binding) of a first protein with a second protein, and" activation level of a first protein " (Binding), and the "activated first protein " may mean a first protein that interacts with (binds to) the second protein.

The signal value for the unit amount of the first protein refers to the unit weight or concentration (for example, lug / ml) of the first protein or the signal value measured in the step (3) Value)

(a) dividing the signal value measured in step (3) by the weight, concentration, or fluorescence signal value of the first protein in the test sample,

(b) the increment of the signal value measured in step (3) as the first protein weight, concentration, or fluorescence signal value in the test sample increases (i.e., the first protein weight, concentration, or fluorescence signal value in the test sample is x Axis, and the slope of the graph obtained by taking the signal value measured in step (3) as the y value).

The present specification suggests that the ratio of the activated first protein to the amount of the first protein in the sample shows a more significant correlation with the drug reactivity targeting the first protein in the sample from which the sample is derived. That is, when the level of the first protein in the sample is low (that is, the amount is at least) and the ratio of the activated U protein is high (activation level: act iatv ion score), the amount of the first protein The drug reactivity is remarkably superior as compared with the case where the ratio is low (see FIGS. 19 and 20). Therefore, the present invention is characterized in that the protein-protein interaction is measured and calculated as a value for the first protein unit amount (divided by the amount of the first protein), thereby providing more accurate information on drug reactivity determination.

The step of measuring the activation level of the first protein of step (4) (i) directly measuring the signal value measured in step (3) by dividing the signal value by the weight _, concentration _, or fluorescence signal value of the first protein in the test sample added in step (1)

(ii) obtaining a signal value for the unit amount of the test sample by dividing the signal value measured in step (3) by the weight, concentration, or fluorescence signal value of the test sample added in step (1) The signal level for the unit amount of the test sample obtained in step (u-1)), and the step (4-1), the weight or concentration of the first protein in the test sample added in step (4-2) of obtaining the signal value of the unit amount of the first protein contained in the test sample (activation level immediate) by dividing the fluorescence signal value by the fluorescence signal value.

Protein-Protein Interaction Level Measurement Step (4-1)

The step of measuring the protein-protein interaction level in the step (4-1) is a step of obtaining a signal value of the unit amount of the test sample added in the step (1) using the signal measured in the step (3).

The protein-protein interaction level is determined by the protein-protein interaction intensity

(PPI strength), and the signal value of the unit amount of the test sample added in the step (1) by using the signal measured in the step (3) is determined by the test sample condition such as the amount of the test sample Error can be reduced.

The step of obtaining the signal value for the unit amount of the test sample added in the step (1) using the signal measured in the step (3) is the step of measuring the signal measured in the step (3) by the amount , Or by calculating the slope of the graph obtained by taking the signal measured in step (3) as the y-axis and the amount (concentration or weight) of the test sample added in step (1) as the x-axis.

When two or more of the first protein and / or the second protein are used, the step of measuring the protein-protein interaction level may be performed for each protein combination.

When two or more downstream proteins are present, the sum of the PPI strengths obtained for each second protein can be determined as the protein-protein interaction level (PPI score), as expressed in the following equation :

c _{DD T} First protein

Sum of PPI test sample = 2V_ _, ^ DDT _f protein 1 protein

( ^PPI strength) _k

k = second protein When two or more first proteins are present, the sum of the PPI scores obtained for each first protein can be determined as the protein-protein interaction level (PPI score).

In another example, the obtained protein-protein interaction level (PPI strength or PPI score) is normal izat ion such that the protein-protein interaction level of the reference sample described below is 1, and the PPI strength of the test sample is set as a reference sample Of the PPI strength.

The activation level measurement step (step 4 or 4-2)

(4) (the protein-protein interaction level of step (4-1) described above) is directly measured without the measurement step) or step (4-2) Value or the signal value of the unit amount of the test sample obtained in the step (4-1) to obtain the value for the unit amount of the first protein contained in the test sample. In this specification, the result obtained in the step (4) or the step (4-2) is referred to as an activation level (or an act ivat ion score).

The activation level is determined by dividing the signal value obtained in step (3) or the protein-protein interaction level obtained in step (4-1) by the amount of the first protein contained in the test sample, It is possible to more accurately measure the degree of activation of the first protein by reducing the error due to the amount and / or distribution.

The method provided herein may further comprise the step of measuring the amount of the first protein in the test sample. The step of measuring the amount of the first protein in the test sample may be carried out before or simultaneously with the step (4) or the step (4-2).

The amount of the first protein can be measured by any conventional method. For example, the amount of the first protein can be measured by a conventional immunoblotting method (for example, quantitative assay, western blotting), ELISA (enzyme-linked immunosorbent assay; sandwich assay, etc.), but the present invention is not limited thereto. In one example, the first protein may be quantified by adding a detection antibody to the substrate to which the first protein is immobilized, and measuring a signal generated from the label, but the present invention is not limited thereto.

When two or more of the first protein and / or the second protein are used, the step of measuring the activation level may be performed for each combination of the first protein task 2 protein Respectively.

When there are two or more downstream proteins, the protein-protein interaction level score obtained from the sum of the PPI strengths obtained for each second protein or the sum of the PPI strengths as described above) (act ivat ion score), or the sum of the act iat ion scores obtained for each second protein, and determine the act iat ion score for the two or more second proteins.

When the first protein is in the form of two proteins, as described above, the protein level (PPI score) obtained from the sum of the PPI strengths obtained for each first protein or from the sum of the PPI strengths is used to determine the activation level act ivat ion score) or the sum of the act iat ion scores obtained for each first protein, and determine the act iat ion score for the two or more first proteins.

In another example, the activity level obtained can be normalized to an activation level of the reference sample described below, such that the activation level of the test sample is relative to the activation level of the reference sample.

In one example, when the first protein is a HER2-HER3 heterodimer, step (4) may comprise comparing the results (protein-protein interaction level or degree of tyrosine kinase activation) obtained in step (3) To the result obtained in the sample (protein-protein interaction level or degree of tyrosine kinase activation).

When the first protein is a HER2-HER3 heterodimer, the reference sample can be appropriately selected according to the purpose of the invention. For example, in the case of a method for measuring the activation (or confirmation or determination or analysis) of a signal transduction pathway in a cell or tissue and / or between cells or tissues, or a method for providing information on the activity measurement, the reference sample may be (1) And / or (2) cells whose known degree of activation of the signal transduction pathway involved in the first protein (HER2, HER3, or both (e.g., HER2-HER3 heterodimer forms) Cancer cells), and / or (3) the degree of activation of the signal transduction pathway involving the first protein (HER2, HER3, or both (e.g., HER2-HER3 heterodimer forms) Cells (e. G., Normal cells or cancer cells). In one embodiment, the test sample is from a subject In the case of including isolated cancer cells, it may include normal cells of the same tissue or the same organ as the cancer cells isolated from the individual.

In order to carry out the step (4), the methods further comprise, before step (4), measuring the degree of protein-protein interaction or tyrosine kinase activation between the first protein and the second protein with respect to the reference sample These steps can be performed with reference to the steps (1), (2), and (3) described above, or (1-1), (2-1), and (3-1).

In the case where the first protein is a HER2-HER3 heterodimer, after the step (4), a step of confirming (determining) a desired item from the comparison result obtained in step (4) may be further included. More specifically, it is as follows:

If the first protein is a HER2-HER3 heterodimer, the step of determining may comprise the following steps:

When the protein-protein interaction level of the test sample measured in step (3) is higher than the protein-protein interaction level measured in the reference sample, the signal that the first protein participates in the test sample or the individual from which the test sample originates Confirming (determining) the degree of activation of the delivery path is higher than the degree of activation of normal cells or the level of activation (known) recognized in the reference sample;

When the protein-protein interaction level of the test sample measured in step (3) is equal to the protein-protein interaction level measured in the reference sample, the signal that the first protein participates in the test sample or the individual from which the test sample originates Confirming (determining) the degree of activation of the delivery path is equivalent to the degree of activation of normal cells or the degree of activation (known) recognized in the reference sample; And / or the protein-protein interaction level of the test sample measured in step (3) is lower than the protein-protein interaction level measured in the reference sample, the test sample or the first protein from the test sample- (Determination) that the degree of activation of the involved signaling pathway is lower than the degree of activation of normal cells or the level of activation (known) known in the reference sample.

Step (5): Step of comparing with reference sample

Step 5 is a step of comparing the results obtained in step (4) or step (4-1) or step (4-2) (protein-protein interaction level (PPI score or Activat ion score) (Protein-protein interaction level (PPI score) or activation level (Act iat ion score)) obtained from the sample 2019/132517 1 »(: 1 ^ {2018/016675

.

The reference sample can be appropriately selected according to the purpose of the invention. For example, in the case of a method for measuring the activation (or confirmation or determination or analysis) of a signal transduction pathway in a cell or tissue and / or between a cell or tissue, or a method for providing information on a 5 activation measurement, And / or (2) a cell in which the degree of activation of the signal transduction pathway involved in the first protein is known (confirmed) (e.g., normal or cancer cells), and / or (3) the degree of activation can ₁₀ may comprise a known (identified), the cells (e.g., normal cells or cancer cells) isolated from the object. In one embodiment, when the test sample comprises cancer cells isolated from an individual, it may include normal cells of the same tissue or the same organ as cancer cells isolated from the test specimen.

As used herein, the term "normal cell" may refer to any cell in a non-pathological state, wherein the "non-pathological condition" refers to a condition in which the disease state is not ₁₅ , Functional, and / or morphological abnormality, or the like. For example, a normal cell may be a disease associated with the first protein or a cell that does not have a disease to be treated of the drug to be tested, and may be derived from the same individual or tissue as the individual or tissue from which the test sample is derived.

₂₀ Also, in the case of a method for predicting the reactivity to a drug targeting the first protein or a method for providing information on the prediction, the reference sample may be a normal cell or a cell having known (confirmed) , Cancer cells), or when the test sample contains cancer cells isolated from an individual, ₂₅ normal cells of the same tissue or the same organ as the cancer cells may be included.

In addition, in the case of selecting the individual suitable for treatment targeting the first protein or providing the information to the selection, the reference sample may be a normal cell or a cell which has known the effect of treatment targeting the first protein (Eg, cancer cells), or when the test sample contains 30 cancer cells isolated from an individual, normal cells of the same tissue or the same organ as the cancer cells may be included.

To perform step (5), the methods comprise the following steps (1 '), (2), (3) and (4) ), 2019/132517 1 »(: 1 ^ {2018/016675

(2 '), (3'), (4-1), and (4-2)

(1 ') preparing a substrate to which a first protein is immobilized by applying a reference sample containing a first protein to a fixed substrate comprising a substance that specifically binds to the first protein on a surface thereof;

₅ (2) adding the labeled second protein to the substrate on which the prepared first protein is immobilized and reacting;

(3 ') measuring a signal from the reaction product obtained in the step (2') (measurement of protein-protein interaction); And

(Measurement of the level of activation) by the amount of the first protein contained in the reference ₁₀ sample added in the step (1 ') using the measured signal,

(4-1 ') dividing the measured signal by the weight or concentration of the reference sample added in the step (1') (measuring the protein-protein interaction level) and (4-2 ' Dividing the result obtained in the step (4-1 ') by the amount of the first protein contained in the reference sample (measurement of the activation level)). ₁₅ Details of each step are as described in the previous sections for the test sample.

Step (6)

Methods provided herein, may be included the step of: after the step (5), step (5) determine (determine) the locations of interest from the comparison results obtained in a ₂₀ added.

More specifically, it is as follows:

3 ₀ The degree of activation of the signal transduction pathway involved in the first protein in the test sample or the individual from which the test sample is derived when the interaction level or activation level is higher than the protein-protein interaction level or activation level measured in the reference sample The degree of activation of the home specimen or the known (confirmed) activation in the reference sample 2019/132517 1 »(: 1 ^ {2018/016675

(Determination) that the current time is higher than the predetermined time;

When the protein-protein interaction level or activation level of the test sample measured in step (4) or (4-2) is equivalent to the protein-protein interaction level or activation level measured in the reference sample, the test sample or the test sample (Determining) the degree of activation of the signal transduction pathway involved in the first protein in the individual from which the first protein is derived is equal to the degree of activation of the normal cell or the degree of activation (known) known in the reference sample; And / or

When the protein-protein interaction level or activation level of the test sample measured in step (4) or (4-2) is lower than the protein-protein interaction level or activation level measured in the reference sample, the test sample or the test sample (Determining) the degree of activation of the signal transduction pathway involved in the first protein in the individual from which the first protein is derived is lower than the degree of activation of the normal cell or the known (confirmed) activation level in the reference sample.

({&Lt; - >) method for predicting the response to a drug targeting a first protein or providing information on the prediction

Step (6) may include the following steps:

When the protein-protein interaction level or activation level of the test sample measured in step (4) or (4-2) is higher than the protein-protein interaction level or activation level measured in the reference sample, the test sample or the test sample (Determining) the reactivity of the first protein of the individual derived from the first sample to the drug that targets the first protein is higher than the drug reactivity of the reference sample;

When the protein-protein interaction level or activation level of the test sample measured in step (4) or (4-2) is equivalent to the protein-protein interaction level or activation level measured in the reference sample, the test sample or the test sample (Determining) the reactivity of the first protein of the individual derived from the target sample to the drug that targets the drug is equivalent to the drug response of the reference sample; And / or

When the protein-protein interaction level or activation level of the test sample measured in step (4) or (4-2) is lower than the protein-protein interaction level or activation level measured in the reference sample, the test sample or the test sample (Determination) that the reactivity of the first protein of the individual derived from the target is lower than the drug reactivity of the reference sample.

The reference sample is selected so as to include cells having reactivity to the drug targeting the first protein to a degree required for the subject to be tested, 2019/132517 1 »(: 1 ^ {2018/016675

It is possible to examine whether the drug has a desired effect on the test sample or the test subject from which the test sample is derived, or to select the reference sample as a normal cell, Specific illnesses < RTI ID = 0.0 > associated with < / RTI > For example, when a cell having a reactivity to a drug targeting a first protein to a degree required for a subject to be tested is selected as the reference sample, step (6) is performed in step (4) or For example, when the protein-protein interaction level or the activation level of the test sample is equal to or higher than the protein-protein interaction level or the activation level measured in the reference sample, the test sample or the sample Determining whether the drug is more reactive to the drug targeting the first protein and / or that the drug is effective on the test sample or on the individual from which the test sample is derived.

A method for predicting reactivity to a drug or a method for predicting reactivity to a drug that targets the first protein, comprising the steps of: (a) providing a test sample or a first protein of the individual from which the test sample is derived Administering a drug targeting the first protein to the subject, if it is determined that the drug has an excellent response to the drug, and / or that the drug has an effect on the test sample or an individual from which the test sample is derived You can include it.

On the other side, individual customized treatment means suitable for each individual are provided based on the above decision content. One example is that the drug is excellent in reactivity to a drug targeting the first protein in step (6), which comprises a drug targeting the first protein as an active ingredient, and / or the drug has an effect A pharmaceutical composition for the treatment of a disease associated with a first protein in a determined individual. Another example is the use of a drug targeting the first protein, an agent having an excellent reactivity to the drug targeting the first protein in step (6), and / or an agent in the subject determined to be effective 1 < / RTI > protein-related diseases _. Another example is the step of administering a drug targeting the first protein to an individual having an excellent response to the drug targeting the first protein in step (6) and / or determining that the drug is effective and it provides a method of treating diseases which are associated with the low-i ₁ protein in the object containing _the. 2019/132517 1 »(: 1 ^ {2018/016675

0) a method for screening an object suitable for treatment targeting the first protein or a method for providing information to screening

The reference sample includes normal cells or cells (for example, cancer cells) whose effects of treatment targeting the first protein are known (confirmed), or when the fifth test sample contains cancer cells isolated from the subject, And may include normal cells of the same tissue or the same organ as the cancer cells.

Step (6) is a step (6) wherein the protein-protein interaction level or activation level of the test sample measured in step (4) or (4-2) is compared with the protein- For example, 0, the test sample or the individual from which the test sample is derived may be identified (determined) as a patient suitable for treatment targeting the first protein.

Treatment targeting the first protein may mean prescribing and / or administering a drug targeting the first protein.

5 The reference sample may include a cell having a therapeutic effect that targets the first protein.

A method for screening a subject suitable for treatment targeting the first protein or a method for providing information to screening, wherein in step (6), the test sample or the individual from which the test sample is derived is selected from the group consisting of 0 < / RTI > identified as a patient suitable for treatment, the subject is treated with a first protein

^_ Perform the treatment of the target can contain the further step of (e. G., A drug for the first protein with the target prescription and / or dosage).

On the other side, individual personalized treatment means suitable for each individual are provided based on the above-mentioned confirmation (decision) contents. One example is a method for treating a disease associated with a first protein in an individual identified as being suitable for treatment targeting the first protein in step (6), comprising the step of administering a drug that targets the first protein as an active ingredient To provide a pharmacological ancestor for. Another example provides the use of a drug targeting said first protein for the treatment of a disease associated with a first protein in said individual identified as being suitable for treatment targeting said s1 protein in said step (6) do. Another example is to perform treatment targeting the first protein to a subject identified as suitable for treatment targeting the first protein in step (6) (e.g., administering a drug targeting the first protein and / Or administration) of the first protein in the subject, 2019/132517 1 »(: 1 ^ {2018/016675

And provides a method of treating a related disease.

(^) A method for monitoring the effect of a treatment targeting a first protein (reactivity with a drug targeting the first protein) or a method for providing information to monitoring

The reference sample includes normal cells or cells (for example, cancer cells) for which the effect of the treatment targeting the first protein is known (confirmed), or when the test sample contains cancer cells isolated from an individual, And normal cells of the same organ or the same organ.

Step (6) is a step (6) wherein the protein-protein interaction level or activation level of the test sample measured in step (4) or (4-2) is compared with the protein- , For example, when the test sample or an object to which the test sample is derived is subjected to treatment with a low protein targeting effect (for example, after administration of a drug targeting the first protein, The response to the drug is maintained or the drug does not develop resistance to the drug). Treatment targeting the first protein may mean prescribing and / or administering a drug targeting the first protein.

The reference sample may include a cell having a therapeutic effect that targets the first protein.

A method for monitoring the effect of treatment targeting the first protein or a method for providing information to monitoring, comprising the steps of: (a) administering a test sample or a first protein to a subject in a step (6) (For example, prescribing and / or administering a drug targeting a low protein) to the subject when it is confirmed that the effect of the low protein is exerted Or in the step (6), the test sample or an object to which the test sample is derived does not exhibit the effect of treatment targeting the first protein (for example, reduction in therapeutic effect (drug reactivity) or acquisition of resistance, etc.) The method comprising: stopping treatment to target the first protein to the subject, and / or stopping the first protein Administering another drug to be targeted, or performing a treatment targeting a different first protein.

Another example is protein-protein interaction between a first protein and a second protein 2019/132517 1 »(: 1 ^ {2018/016675

Wherein the first protein is a protein involved in a signal transduction pathway in a cell or tissue and / or between a cell or tissue, and the second protein is a protein in the cell or tissue and / Wherein the step of measuring the protein-protein interaction is performed on two or more first proteins, wherein the cells or tissues or the individual from which the cells or tissues are derived A method for screening a _first protein or a drug targeting the _first protein as a therapeutic target suitable for application to a subject, or providing information on screening.

The method comprises:

(2) adding a labeled second protein to the substrate on which the prepared first protein is immobilized and reacting;

(3) measuring the signal from the reactant obtained in step (2)

(Protein-protein interaction measurement); And

(4) obtaining a value for the unit amount of the first protein included in the sample added in step (1) (measuring the activation level) using the signal measured in step (3); or

(4-1) obtaining a signal value (measurement of protein-protein interaction level) for the unit amount of the test sample added in step (1) using the signal measured in step (3), and (4-2 Obtaining a value for a unit amount of the first protein included in the test sample (measuring the activation level) using the signal value of the unit amount of the test sample obtained in the step (4-1); And

(5) comparing the results obtained in step (4) or step (4- ₂₎ with respect to two or more first proteins

.

At this time, as the first protein, two or more proteins selected from proteins involved in signal transduction pathways of cells or tissues are used,

(1), (2), (3) and (4) or steps (1), (2), (3), (4-1) Respectively,

The comparing step of step (5) is performed on two or more species of the first protein 2019/132517 1 »(: 1 ^ {2018/016675

The results can be compared to each other.

As a result of the comparison in the step (5), a first protein having a high protein-protein interaction level or activation level is selected as a therapeutic target of the test sample or a test sample derived from the test sample, (Step (6)) of selecting the drug as a candidate drug for the treatment of a test sample or an individual from which the test sample is derived.

The description of the steps (1) to (6) and the test sample, the first protein, the second protein and the like are as described above.

Another example includes measuring a protein-protein interaction between a first protein and a second protein, wherein the first protein is a protein involved in a signal transduction pathway in a cell or tissue and / or between cells or tissue, 2 protein is a downstream protein of the first protein in the cell or tissue and / or in a signal transduction pathway between cells or tissues, and the step of measuring the protein-protein interaction is performed before and after the candidate substance treatment. 1 < / RTI > protein, or a method for confirming the efficacy of a candidate drug targeting the first protein.

The method comprises:

(1), (2), (3), and (3) for each of the test sample that has not been treated with the candidate compound and the treated test sample, (3), (4), and (5) or (1), (2) (1), (2), (3), (4-1), (4-2), and (5)

(3) measuring a signal from the reactant obtained in step (2) (protein-protein interaction measurement); And

(4) obtaining a value for a unit amount of the first protein included in the test sample added in the step (1) (measuring the activation level) using the signal measured in the step (3); or

(4-1) Using the signals measured in step (3), the signals added in step (1) 2019/132517 1 »(: 1 ^ {2018/016675

Using the signal values for the unit amount of the test sample obtained in step (4-1) and the step of obtaining the signal value for the unit amount of the test sample (measurement of protein-protein interaction level) and (4-2) Obtaining a value for a unit amount of the first protein contained in the sample (measurement of the activation level); And

(5) The respective results obtained in step (3), step (4), step (4-1), or step (4- 2) for the test sample not treated with the candidate compound and the treated test sample Comparing them to each other.

The test sample not treated with the candidate compound and the treated test sample means the test sample before treatment and the test sample after treatment of the candidate compound, respectively, or a part of the test sample treated with the candidate compound and the candidate May refer to some other test sample that has not been treated with the compound.

If the result of the comparison in the step (5) is that the protein-protein interaction level or activation level of the test sample treated with the candidate compound is lower than that of the test sample in which the candidate compound is not treated, The candidate compound may be selected as a candidate candidate drug, or the candidate compound may be effective as a drug targeting the first protein.

Therefore, the method for selecting a candidate drug targeting the first protein or the method for confirming the efficacy of a candidate drug targeting the first protein can be performed after the step (5), as a result of the comparison in the step (5) When the compound-protein interaction level or activation level of the test sample treated with the compound is lower than that of the test sample in which the candidate compound is not treated, the candidate compound is selected as a candidate drug targeting the first protein, (Step (6)) of confirming that the compound is effective as a drug targeting the first protein.

One example includes measuring protein-protein interactions between a first protein and a second protein, wherein the first protein comprises

And? 0-1, the second protein is another one, and the step of measuring the protein-protein interaction is carried out before and after the candidate substance treatment, wherein the first protein and / or the second protein It provides the first protein and / or the second method confirmed the efficacy of candidate drugs which target _proteins: the candidate drug to target screening methods, or. Another example comprises measuring a protein-protein interaction between a first protein and a second protein, 2019/132517 1 »(: 1 ^ {2018/016675

0-1, and the second protein is another one, and the step of measuring the protein-protein interaction is performed before and after the candidate substance treatment, wherein the first protein and / or the second protein The present invention provides a method for screening candidate drugs for the prevention or treatment of diseases (e.g., diseases associated with the expression and / or activation of the first protein and / or the second protein; cancer, immune diseases, etc.).

The method comprises:

(1), (2), (3), and (3) for each of the test sample that has not been treated with the candidate compound and the treated test sample, (1), (2), (3), (4), and (5) ), Or step

(1), (2), (3), (4-1), (4-2), and (5)

(4) obtaining a value for the unit amount of the first protein included in the test sample added in step (1) (measuring the activation level) using the signal measured in step (3); or

The concrete description of each step is as described above.

As a result of the comparison in the above step (5), the protein-protein interaction level or the activation level of the test sample to which the candidate compound has been treated, , The candidate compound may be selected as a candidate drug targeting the first protein or the candidate compound may be found to be effective as a drug targeting the first protein. Therefore, the method for screening a candidate drug targeting the first protein or the method for confirming the efficacy of a candidate drug targeting the first protein is characterized in that after the step (5), the result of the comparison in the step (5) When the protein-protein interaction level or the activation level of the treated test sample is lower than that of the test sample in which the candidate compound is not treated, the candidate compound is selected as a candidate drug targeting the first protein or the second protein, (Step (6)) of confirming that the candidate compound is effective as a drug targeting the first protein or the second protein.

The candidate compound may be selected from among all biocompatible materials that can be used as a target drug of the first protein, for example, small molecule chemicals, proteins (e.g., antibodies, antibody fragments, ), Peptides, nucleic acid molecules (such as DNA, RNA (e.g., siRNA, miroRNA, shRNA, etc.), peptid nucleic acid (PNA), aptamer, And the like. However, the present invention is not limited thereto.

The test sample can be used for a disease associated with a first protein, or a disease associated with a first protein to be screened or a first protein to be screened, such as a cell (for example, a cell lysate or the like) (E.g., cell lysate, etc.) or tissue (e. G., Tissue lysate, etc.) associated with the application (treatment) target disease of the protein-targeting drug, and in one example, (E.g., cancer). For example, the test sample may be a cell or tissue isolated from an established cancer cell line or cancer patient (the cancer may be associated with overexpression and / or activation of the first protein).

When the first protein is any one selected from PD-L1 and PD-1 and the low-12 protein is the other, the test sample used for the drug screening is a cell in which the first protein is overexpressed and / (Treatment) of a drug targeting a disease associated with the first protein or a first protein to be screened (for example, a cell lysis solution or the like) or a tissue (for example, a tissue lysis solution) Cell lysate, etc.) or tissue (e.g., Tissue lysate, etc.), or a purified first protein, and in one example, a cell or tissue isolated from an established cell line or a patient suffering from the disease (e.g., cancer, immune disorders, etc.) Lt; RTI ID = 0.0 > 1 < / RTI > The second protein may be in a purified (or isolated form) cell.

The above steps (1) to (6) may be performed in in / iro. The description of the above steps (1) to (6) and the first protein, the second protein and the like are as described above.

The method for screening a candidate drug targeting the above-mentioned first protein can be usefully applied to the efficacy (or confirmation or testing) of a drug developed as a candidate drug in the development of a new drug targeted by the first protein.

Selecting the candidate compound as a candidate drug targeting the first protein when the protein-protein interaction level or activation level of the test sample treated with the candidate compound is higher than that of the test sample not treated with the candidate compound Step (6)).

The candidate compound may be selected from among all biocompatible materials that can be used as a target drug of the first protein, for example, small molecule chemicals, proteins (e.g., antibodies, antibody fragments, ), Peptides, nucleic acid molecules (e.g., DNA, RNA (e.g., siRNA, microRNA, shRNA), peptid nucleic acid (PNA), aptamer, etc.), plant extracts, animal extracts, But it is not limited thereto.

The test sample can be used for a disease associated with a first protein, or a disease associated with a first protein to be screened or a first protein to be screened, such as a cell (for example, a cell lysate or the like) (E.g., cell lysate, etc.) or tissue (e. G., Tissue lysate, etc.) associated with the application (treatment) target disease of the protein-targeting drug, and in one example, (E.g., cancer). For example, the test sample may be a cell or tissue isolated from an established cancer cell line or a cancer patient _. Or may be related to overexpression and / or activation of the protein).

The description of the terms (1) to (6) and the terms of the first protein, the second protein and the like are as described above. 2019/132517 1 »(: 1 ^ {2018/016675

The screening method of the candidate drug targeting the first protein can be usefully applied to drugs and efficacy testing (or confirmation or testing) developed as a candidate drug in the development of a new drug targeted by the first protein.

Another example includes measuring a protein-protein interaction between a first protein and a second protein, wherein the first protein is a protein involved in a signal transduction pathway in a cell or tissue and / or between cells or tissue, 2 protein is at least one selected from among proteins downstream of the first protein in the cell or tissue and / or signal transduction pathway between cells or tissues, a method for selecting a concurrent therapeutic target for concurrent with the target treatment of the first protein, or A method of providing information to the selection, or a method of selecting a concomitant drug to be co-administered with a target drug of the first protein, or a method of providing information to the selection.

The method comprises:

(3) determining the signal from the reactant obtained in step (2) (protein-protein interaction measurement); And

(5) comparing the results obtained in step (3), step (4), step (4-1), or step (4-2)

.

Wherein the step of comparing (5)

(a) the step (4) or (4-2) obtained for the test sample 2019/132517 1 »(: 1 ^ {2018/016675

(1), (2 '), (3'), and (4) described above with reference to the reference sample, and the results are compared with the results obtained for the reference sample , Or may further comprise (I), (2 '), (3), (4-1'), and (4-2 '))

(Comparing the results of step (4) or step (4-2) obtained for two or more second proteins with each other).

As a result of the comparison in the step (5)

( _{3) When} the protein-protein interaction level or the activation level of the step (4) or step (4-2) obtained for the test sample is higher than the reference sample, the second protein is combined with the target treatment of the first protein Or a drug targeting the second protein is selected as a drug for use in combination with a target drug of the first protein;

(The second protein having the highest protein-protein interaction level or activation level obtained in the second or more second protein heavy chain stage (4) or step (4-2) is used in combination with the target treatment of the first protein, Or a drug targeting the second protein may be selected as a drug for administration in combination with a target drug of the first protein.

Therefore, a method for selecting a concurrent treatment target (or a method for providing information on the selection) for concurrently with the target treatment of the first protein, or a method for selecting a concomitant drug for co-administration with the target protein of the first protein (Or a method for providing information to the selection), after the step (5)

(6-1) When the protein-protein interaction level or the activation level of the step (4) or the step (4-2) obtained on the test sample is higher than the reference sample, And selecting a drug targeting the second protein as a drug to be used in combination with a target drug of low 11 protein; or

(6-2) The second protein having the highest protein-protein interaction level or activation level obtained in step (4) or step (4-2) among the second protein on the two or more species is administered in parallel with the target treatment of the first protein Or a drug for targeting the second protein to a drug for use in combination with a target drug of the first protein,

May be further included.

In another aspect, a combination therapy means based on the above decision content / RTI > One example includes a drug targeting the first protein and a drug targeting (e.g., inhibiting) a second protein selected as a concurrent therapeutic target in step (6-1) or (6-2) , &Lt; / RTI > a pharmaceutical composition for coadministration for the treatment of diseases associated with the first protein. The pharmaceutical composition may be used for the combined administration for the treatment of diseases related to the first protein in the test sample or the individual from which the test sample is derived. Another example is a pharmaceutical composition comprising a drug targeting the first protein and a drug targeting (e.g., inhibiting) a second protein selected as a concurrent therapeutic target in step (6-1) or (6-2) 1 < / RTI > protein. Another example is a method for treating a disease associated with a first protein, comprising administering to a patient in need of such treatment a therapeutically effective amount of a first protein (e. G., Administering a drug targeting the first protein) (E.g., administration of a drug that targets (e. G., Inhibits) the second protein) targeted to a second protein selected as a concurrent therapeutic target in step A method of treating a disease associated with a first protein. The patient may be the test sample or an individual from which the test sample is derived.

In another example, there is provided a method of predicting or predicting responsiveness to a drug that targets the provided first protein, a method of screening an entity suitable for treatment targeting the first protein, , And methods of screening or screening for a first protein or a target thereof as a therapeutic target suitable for application to a cell or tissue or an individual (individual patient) from which the cell or tissue is derived (Step 7) of confirming whether the first protein is post-translationally modified (PTM) in addition to the protein-protein interaction measurement and activation level measurement step described above, ). PTM is a common marker showing the activation status of the first protein. It is therefore used as a control against existing methods.

The post-translational modification of the first protein refers to a modification (a post-translational modification related to the first protein of the first protein) that occurs more frequently in a cancer or tumor patient than a normal individual (a cancer or a tumor-free individual) A gene encoding a first protein or a first protein may comprise at least one nucleotide or at least one amino acid mutation (or at least one amino acid mutation A nucleotide variation), phosphorylation (e.g., phosphorylation of a Tyr residue), and the like. For example, if the first protein is EGFR, the mutation may be intensively present in exon 19 and / or exon 21, and in one embodiment, the mutation is a deletion of one or more genes in exon 19 and exon 21 of EGFR , Deletion of exon 19 and / or exon 21), gene mutation causing L858R substitution, phosphorylation at Tyr 1068 and / or Tyr 1086, but not limited to, normal individuals (Not present) and can be selected from all known EGFR (gene / protein) variants with high incidence in cancer or tumor patients. When the first protein is HER2, the mutation may be phosphorylation at Tyr 1221, but is not limited thereto, and may occur in a cancerous or tumor patient with a high incidence in comparison with a normal individual (a cancer or a tumor-free individual) It can be selected from all known HER2 (gene / protein) variants. When the first protein is HER3, the mutation may be phosphorylation at Tyr 1289, but is not limited thereto, and is more frequent in cancer or tumor patients than in normal individuals (individuals without cancer or tumor) It can be selected from all known HER3 (gene / protein) variants. When the first protein is MET, the mutation may be phosphorylation at Tyr 1349, but is not limited thereto, and is more frequent in cancer or tumor patients than in a normal individual (a cancer or a tumor-free individual) It can be selected from all known MET (gene / protein) variants.

As used herein, the term "targeting a first protein " may mean promoting or inhibiting the activity of a first protein, e. G., Inhibiting the activity of a first protein The inhibition of the activity of the first protein may be achieved by binding to the first protein and / or degrading and / or structurally modifying the first protein to produce a unique function, e. G., A unique bio signaling function in cells and / Lt; RTI ID = 0.0 > and / or < / RTI >

As used herein, the term "drug" refers to any substance that exhibits a pharmacological effect, such as a small molecule compound, a protein (such as an antibody, an antibody fragment, or an analogue thereof), a peptide, a nucleic acid molecule And at least one compound selected from the group consisting of RNA (for example, siRNA, miroRNA, shRNA, etc.), peptidase nucleic acid (PNA), aptamer, etc., plant extracts, animal extracts, It can be selected.

In another example,

(a) an expression level of the first protein and / or post-translational modification (PTM)

(b) a first protein task 2 protein-protein interaction measurement unit,

Wherein the first protein is a receptor tyrosine kinase (RTK), an RTK profiling kit, or a kit for predicting the efficacy of a drug targeting the first protein. In one example, the drug may be, but is not limited to, a therapeutic agent for lung cancer (such as Afatinib).

As used herein, the term "drug targeting a first protein" refers to any substance that inhibits the activity of a first protein, such as a small molecule, a protein, (E.g., small interfering RNA (siRNA), small hairpin RNA, miRNA (microRNA), etc.), PNAs (such as antibodies, antibody fragments or analogs thereof), peptides, nucleic acid molecules a peptide nucleic acid, an aptamer, etc.), a plant stem, an animal stem, a cell stem, and the like. More specifically, "a drug targeting a first protein " is a substance that binds to a first protein and / or degrades and / or structurally modifies a first protein to produce a unique function, (E. G., Antibodies, antibody fragments, or analogs thereof), peptides, nucleic acid molecules (e. G., DNA < / RTI > , RNA (such as siRNA, microRNA, shRNA), PNA (peptide nucleic acid), aptamer, etc.), plant extracts, animal extracts, cell extracts and the like.

In one embodiment, the "drug targeting a first protein " may refer to a therapeutic agent targeting a first protein, such as a target inhibitor of a first protein. In one example, Targeted drugs include antiretroviral drugs such as cetuximab, gefitinib, erlotinib, afatinib, osimertinib (AZD9291), various anti-EGFR antibodies, MET target therapeutics (various anti-MET antibodies, Crizotinib, Cabozant inib etc.) Therapeutic agents (trastuzumab, Trastuzumab, Pertuzumab, Lapatinib, etc.), HER3 target therapy (Eg, various anti-HER3 antibodies), FGFR1, 2) Target therapeutic agents (Lenvatinib, Nintedanib, Regorafenib etc.), VEGFR (1,2,3) Target therapeutic agents (Bevacizumab, Axitinib, Lenvat inib etc.), PDGFR target treatment agent , Vetetinib, etc.), BRAF target treatment (Vemurafenib, Dabrafenib, etc.), MEK (Gefitinib, Imatinib etc.), IGF1R target therapy (Ceritinib etc.), c-KIT target treatment (Axitinib, Cabozant inib, Dasatinib etc.) (Czototinib, Cabozant inib, etc.), CDK (4, 6) Target therapy (Palbociclib, Sorafenib, etc.), R0S1 Target Therapeutics (Ceritinib, Crizotinib, etc.), ALK Target Therapeutics (Ceritinib, Crizotinib, etc.), BCR-Abll Target Therapeutic Agents

(Ivilimumab, Tremel imumab, etc.), PD-1 target therapeutic agent (Nivolumab etc.), and the like, which are selected from the group consisting of anti-inflammatory drugs (Bosutinib, Dasatinib, Imatinib, Ni lotinib etc.), AR target therapeutic agents (Abiraterone, Enzalut amide etc.) Or more, but is not limited thereto.

In another embodiment, the "drug targeting the first protein" (drug response measure or screening drug) competes with the second protein in binding to the first protein such that the first protein task 2 protein (E.g., a small molecule, a protein (e.g., an antibody, an antibody fragment, or an analogue thereof), a peptide, a nucleic acid molecule ), shRNA (small hairpin RNA), miRNA (microRNA), etc.), PNA (peptide nucleic acid), aptamer, etc.), plant trunk, animal trunk, Or more).

As used herein, the term " treatment targeting a first protein "may refer to any medical and / or pharmaceutical activity that inhibits the activity of a first protein, for example, Quot; first protein-targeting treatment " may include administering a first protein and / or a second protein to a subject in need of inhibiting the activity of the first protein, To a subject in need thereof a drug that reduces or eliminates the inherent functions of, for example, inherent bio-signal transduction functions in cells and / or tissues, by binding and / or degrading and / or structurally modifying the first protein Prescription and / or administration.

Wherein the first protein is a first class 2 and the third class 3, the first class 2 and the third class 3 form a heterodimer and the second protein is a cell or a tissue and / or a cell or a cell, Wherein the protein-protein interaction is a sub-protein of the other of the inter-tissue signal transduction pathways, and wherein the step of measuring the protein-protein interaction is performed in the sample treated with the candidate substance and the untreated sample, A method for confirming the efficacy of a candidate drug targeting a protein or a method for confirming the efficacy of a candidate drug for the prevention or treatment of the disease. Another example involves measuring the activation level of the inhibitor 2 and / or 1-3 of the quadrivalent 2-thymine 3 heterodimer (e.g., phosphorylation of the tyrosine residue), wherein the step of measuring activation is referred to as the candidate substance A sample containing a heterodimer or a heterodimer of the 2-tetradecane heterodimer and an untreated heterodimer or an untreated heterodimer of 2- A method for confirming the efficacy of a candidate drug targeting the first protein or a method for confirming the efficacy of a candidate drug for prevention or treatment of the disease.

The method comprises:

(For example, contacting) the candidate material with the test sample, and performing the following steps ( ₃ ), (0), ((), and

( ₃ ) Prepare a test sample containing a 1-thymine 3 heterodimer or a test sample containing a tetra-quadrone-3-hetero dimer on the surface

And / or to a substrate comprising a substance that specifically binds to the other epitope 3, thereby preparing a substrate on which quaternary 2,4-heterodimer is immobilized;

Phosphorylating the 1,2-to 4-membered heterodimer and adding and reacting the labeled phosphorylated tyrosine specific binding substance;

( ₀ ) step of measuring the signal from the reactant obtained in step

(Protein-protein interaction measurement or

And / or the degree of activation of the tyrosine kinase activity of inhibitor 3); And

(1) comparing the respective results obtained in the above step with respect to the test sample not treated with the candidate substance and the treated test sample with each other.

As described above, the phosphorylated tyrosine specific binding substance may be an antibody specifically binding to the phosphorylated tyrosine of the first class II and / or the third class. 2019/132517 1 »(: 1 ^ {2018/016675

The candidate material may be treated in the step (e. G., Before, after, or simultaneously with the treatment of the phosphorylated or phosphorylated tyrosine specific binding material of step (a). Phosphorylation of this step can be carried out by treating the phosphorylating agent. The phosphorus-

And / or all substances capable of phosphorylating urine 3, for example, may be selected from the group consisting of: ^3, and magnesium (e.g., 1, ₂ , 1, _2, etc.).

In order to compare the results of the untreated test sample with the treated test sample in step ( ₃ ) - (0), the candidate substance is subjected to the test sample not treated with the candidate substance and the treated test sample, respectively .

The test sample not treated with the candidate substance and the treated test sample mean a test sample before treatment and a test sample after treatment of the candidate substance, respectively, or a part of the test sample treated with the candidate substance and the candidate May refer to some other test sample that has not been treated with the substance.

If the protein-protein interaction level of the test sample treated with the candidate substance or the tyrosine kinase activation degree of quadrature 2 and / or quadruple 3 is lower than that of the test sample in which the candidate substance is not treated, (I. E., When the candidate substance inhibits tyrosine kinase activation of < RTI ID = 0.0 > 41 < / RTI &

(I. E., Inhibiting the activation of tyrosine kinase 2, and / or < RTI ID = 0.0 > 1-3). &Lt; / RTI >

Therefore, the method for confirming the efficacy of the candidate drug is a method for determining the efficacy of the candidate drug by comparing the protein-protein interaction level of the test sample treated with the candidate substance or the tyrosine 4 of tetrahedral 2 and / When the degree of kinase activation is lower than that of the test sample in which this candidate substance is not treated (i.e., the candidate substance inhibits the interaction between the first protein and the second protein or the activation of tyrosine kinase of quadruplex 2 and / or 1-3) ), The candidate material is targeted to four quadrants 2, 4 quadrature 3, or both (e. G., In the form of a 4 1 2-quadrature 3 heterodimer) Or inhibiting tyrosine kinase activation of 1? 2 and / or 1? 3) (step (i) . The level of protein-protein interaction level or tyrosine kinase activation level of HER2 and / or HER3 of the test sample treated with the candidate drug is lower than that of the test sample not treated with the candidate substance. This means that the protein- Mutual levels or degree of tyrosine kinase activation of HER2 and / or HER3 is less than or equal to 75% (i.e., greater than or equal to 25% inhibition) of the protein-protein interfering level of the test sample to which the candidate agent has not been treated or the degree of tyrosine kinase activation of HER2 and / ), 50% or less (i.e., 50% or more inhibition), 45% or less, 40% or less, 35% or less, 30% or less, 25% or less Inhibition), 15% or less, or 10% or less (i.e., 90% or more inhibition).

The candidate agent may be selected from any biocompatible material available as a target drug for the first protein (HER2, HER3, or both (e.g., HER2-HER3 heterodimer form)), such as small molecular (eg, DNA, RNA (eg, siRNA, microRNA, shRNA, etc.), PNA (peptide nucleic acid), aptamer, etc.), proteins (eg, antibodies, antibody fragments or analogs thereof) ), Plant extracts, animal extracts, cell extracts, and the like, but the present invention is not limited thereto.

The test sample may be a cell (e.g., a cell lysate, etc.) or a tissue (e.g., a cell lysate, etc.) overexpressing and / or activating a first protein (HER2, HER3, or both (in particular, a HER2-HER3 heterodimer form) (E.g., cell lysate) or tissue (e.g., cell lysate) associated with the disease (s) involved in the application (treatment) of a drug targeting the first protein for which the disease or efficacy associated with the first protein is to be identified Tissue lysate, etc.), and in one example, may be a cell or tissue isolated from an established cell line or a patient suffering from the disease (e.g., cancer). For example, the test sample may be a cell or tissue isolated from an established cancer cell line or cancer patient (the cancer may be associated with overexpression and / or activation of the first protein).

The steps (a) to () may be performed in F / ZTO. The terminology of each step is as described above.

As used herein, the term "targeting a first protein" refers to promoting or inhibiting the activity of a first protein (HER2, HER3, or both (eg, a HER2-HER3 heterodimer form) You can, 2019/132517 1 »(: 1 ^ {2018/016675

May mean inhibiting the activity (tyrosine kinase activity) of the first protein, e.g., the first protein, the second protein, the second protein, the third protein, or both (e.g., other 1? . Inhibition of the activity of the first protein may be associated with the first protein and / or by degradation and / or structural modification of the first protein to provide a unique function, e. G., A unique bio signaling function in 5 cells and / It can be reduced or eliminated.

The administration can be by oral or parenteral routes. In the case of parenteral administration, it can be carried out by ₁₀ routes such as intravenous infusion, subcutaneous infusion, muscle infusion, intraperitoneal injection, endothelial administration, local administration of lesion site, intranasal administration, intrapulmonary administration or rectal administration.

The subject, patient or subject may be any mammal such as a primate such as a human, a monkey, a rodent such as a mouse, a rat, or the like, and may be, for example, a patient having a disease associated with the first protein _. The disease associated with the said protein may be a disease associated with overexpression of the first protein or activation of a signal transduction pathway in a cell or tissue ₁₅ and / or between cells or tissues in which the first protein participates, such as cancer, inflammation, Immune diseases, and the like. In an embodiment, the subject, patient or subject may be a cancer patient.

The cancer may be selected from all solid tumors and blood cancers and may be, for example, a cancer associated with overexpression of a first protein or activation of a signal transduction pathway between a cell or tissue ₂₀ and / or a cell or tissue involved in the first protein. For example, the cancer is selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, skin cancer, skin or intraocular melanoma, rectal cancer, adenocarcinoma, part thyroid cancer, adrenal cancer, soft tissue sarcoma, uterine cancer, chronic or acute leukemia, lymphocytic lymphomas, gastric cancer, ₂₅ pancreatic cancer, schools subspecies, cervical cancer, ovarian cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland Cancer, kidney cancer, prostate cancer, mucin cancer, thyroid cancer, head and neck cancer, brain cancer, and the like, but the present invention is not limited thereto. The cancer may include metastatic cancer as well as primary cancer. In addition, the cancer may be resistant to conventional chemotherapy or obtained _{30 times} .

As used herein, the term "responsiveness to a drug " may refer to the degree to which the drug exerts an effect on an individual receiving the drug. As used herein, the term & Medication or treatment cured Means a medical and / or pharmaceutical effect intended to be achieved by the subject, which may mean prevention and / or treatment of the disease to be treated, and / or alleviation and / or amelioration of symptoms. For example, when the drug is an anticancer agent and the treatment is chemotherapy and the subject is a cancer patient, the effect may be an anticancer effect (prevention and / or therapeutic effect of cancer), and the anticancer effect may inhibit the growth of cancer cells As well as the effects of inhibiting migration, invasion, and / or metastasis, and / or inhibiting the deterioration of cancer and / or reducing or eliminating resistance, and the like. have.

In another example, a method for measuring activation of a signal transduction pathway in a cell or tissue and / or between cells or tissues as described above, a method for predicting responsiveness to a drug targeting the first protein, a method for monitoring reactivity for a drug targeting the first protein There is provided a method for measuring protein-protein interactions for use in a method for screening a first protein, a method for selecting an individual suitable for treatment targeting the first protein, and / or a method for screening a drug. The apparatus for measuring protein-protein interactions may be a device for measuring activation of a signal transduction pathway in a tissue, an apparatus for predicting and / or monitoring reactivity to a drug targeting the first protein, a device suitable for treatment targeting the first protein An apparatus for screening an individual, and / or an apparatus for confirming the efficacy of a drug targeting the first protein.

In one example, the first protein in the device may be PD-L1 or PD-1.

In another example, the first protein in the device may be HER2, HER3, or both (e. G., A HER2-HER3 heterodimer form).

The device for measuring protein-protein interactions can be used for measuring the activation of a signal transduction pathway in cells or tissues and / or between cells or tissues, a method for predicting the reactivity of a drug targeting a first protein, The present invention can be applied to a method for monitoring the reactivity of a drug to a drug, a method for screening an individual suitable for treatment targeting the first protein, and / or a screening method for a drug, so that the interaction between biomolecules in a small amount of sample can be accurately and efficiently Can be observed, analyzed, detected, and / or measured. An apparatus for measuring protein-protein interactions or an analytical method using the same may be usefully and effectively applied to very small amounts of samples such as biopsy (e.g., needle biopsy) samples. In addition, the protein-protein 2019/132517 1 »(: 1 ^ {2018/016675

The interaction measurement device or analysis method using the same can accurately and efficiently observe, analyze, detect, and / or measure interaction between various biomolecules (e.g., protein, nucleic acid, etc.) using a small amount of sample.

The apparatus for measuring protein-protein interactions includes:

A multiwell containing a substance for capturing a first protein that specifically binds to the first protein,

In addition, it is possible to additionally include a signal detecting means.

The apparatus for measuring protein-protein interaction includes a protein (second protein) that interacts with the first protein (e.g., participates in a lower pathway of a biological signal transduction pathway of a cell or tissue involved in the first protein) In one example, the second protein is labeled with a labeling substance that generates a detectable signal (the labeling substance may be chemically (e.g., covalent or noncovalent), recombinant, or physical , Or it may be in the form in which ₁₃ sugars to which the labeling substance can be attached are attached. Also, the protein-protein interaction measuring devices for the order to ₍₁₁ 1 1 ₀₁₁ ₁₁₎ level with the normalization of the first protein, the signal measured by the signal detecting means, detecting substances and the for binding to the first protein, A labeling substance that binds to the detection substance may be further included.

The detection substance binding to the first protein may be a biomolecule (for example, an antibody) binding to the first protein at a site different from the capturing substance for the first protein contained in the multiwell described above, (The labeling substance is chemically (e _. G., Covalently or non-covalently bound)) to generate a detectable signal _. (E. G., An antibody) capable of binding to a detection substance that binds to the first protein (see Fig. 6).

If the first protein is? - 11 or 1) -1, the apparatus for measuring protein-protein interactions includes a reaction unit comprising a substance capable of capturing a first protein that specifically binds to a first protein And may additionally include signal detection means.

The apparatus for measuring protein-protein interactions can be used to measure the presence and / or the degree of interaction (binding) between 1) - [the and 1) -1.

When the first protein is P? -Ll or (1) -1, the reaction unit may include a substrate on which a substance for capturing a first protein is immobilized, 2019/132517 1 »(: 1 ^ {2018/016675

The capturing material may be a substance that specifically binds to the first protein described above in the substrate preparation step in which the first protein is immobilized, for example, an antibody or an antigen-binding fragment thereof. For example, the antibody or antigen-binding fragment thereof, as described above, binds to a non-binding site (for example, (: -terminal site) other than the binding site between 1) -1 term

Antibody or antigen-binding fragment thereof,

Linkage

1 antibody or an antigen-binding fragment thereof which binds to a non-binding site (for example, the 0-terminal site) except for the terminal region of the antibody. The reaction unit may be, but is not limited to, a well (e.g., multiwell) type, a slide type, a channel type, an array type, a microfluidic chip, a microtubule (capillary), and the like.

If the first protein is p -ll or suppress-1, the apparatus for measuring protein-protein interaction may further comprise a second protein interacting with the first protein. In one example, the second protein is labeled with a labeling substance that generates a detectable signal (the labeling substance is chemically (e. G., Covalent or noncovalent), recombinantly or physically coupled) It may be in the form of a 3- _sided attachment to which a labeling substance can be attached.

If the first protein is? - 11 or suppress-1, the first protein is any one selected from the group consisting of suppression-1 and suppression-1, and the second protein is the other. For example, when the first protein is 1) -1, the second protein is 1) -1, and when the first protein is suppressed 1, the second protein is 1) -1.

In the case where the first protein is a 4-1,2 and 4-1 < - >, in particular a 1-thymine 2- 4-tetrahedral heterodimer, the analyzing apparatus is capable of capturing a first protein specifically binding to the first protein A substance or a first protein, or may additionally include signal detection means.

In one example, the first protein comprises a cell that comprises (expresses) a 2-quaternary 3-heterodimer or a 1-12 (3) heterodimer on a cell surface or a lysate of the cell And the reaction part can be used in the form of (1) a cell that contains (expresses) a 2-quaternary 3-heterodimer or a 2-quaternary heterodimer on the cell surface or a cell Seaweed, or (2) a substrate on which they are immobilized. Cells expressing (expressing) the quadrature 2-quadrant 3 heterodimer on the cell surface express the quadruplex and inhibitor 3 and are capable of expressing a dimerizing agent (eg, a ligand of other moieties (eg, 31 (NRGbl), etc.), cholesterol-like cleanser (Cholesterol-1 ike detergent; For example, DGTN (digi tonin; CAS Number: 11024-24-1), GDN (glyco-diosgenin; CAS Number: 1402423-29-3), and may be, for example, one expressing HER2 and HER3 and expressing DGTN and GDN Or a cell lysate treated with at least one cholesterol-like detergent selected from the group consisting of < RTI ID = 0.0 >

The analyzer is capable of detecting the presence and / or degree of interaction (binding) of HER2 and HER3, particularly HER2-HER3 heterodimers and their downstream proteins in the signal transduction pathway, or the degree of activation of HER2 and / or HER3 &Lt; / RTI >

The reaction unit may include a substrate on which a substance for capturing a first protein is immobilized on a surface, or a substrate on which the first protein is immobilized (directly) without passing through a capturing substance. The first protein is HER2 and HER3, in particular HER2-HER3 heterodimer, and the first protein capturing substance is a protein having the first protein (HER2 and / or HER3) described in the substrate preparation step in which the first protein is immobilized Specific binding material, such as an antibody or an antigen-binding fragment thereof. The reaction unit may be, but is not limited to, a well (e.g., multi-well) type, a slide type, a channel type, an array type, a microfluid chip, and a microtubule (capillary).

The analyzer may be a device for measuring protein-protein interaction between a first protein and a second protein, and may further comprise a second protein that interacts with the first protein. In another example, the analyzer may be a device for measuring the degree of activation of HER2 and / or HER3 (e.g., phosphorylation of a Tyr residue), and for this purpose, a substance (e.g., an antibody) that specifically binds phosphorylated Tyr May be further included. In one example, the substance that specifically binds to the second protein or phosphorylated Tyr is labeled with a labeling substance that generates a detectable signal (the labeling substance may be labeled, for example, chemically (e.g., covalently or noncovalently) Or physically), or may be attached to a tag to which a labeling substance can be attached.

The multiwells included in the apparatus for measuring protein-protein interactions according to an embodiment may include a plurality of tubes whose one surface is open or a plurality of non-through holes (for example, grooves formed in the support plate )of Wherein the one or more non-through holes are defined as wells, and wherein the wells are arranged in a first direction in a second direction in which two or more arrangement tube array structures intersect the first direction, Or a structure having two or more layouts (grid structure) (see FIG. 30). In this case, the multi-well may include a sample injecting part located on one opened surface of the tube or the non-through hole, a protein-protein interaction inside the tube or the non-penetrating hole (for example, (For example, a substance that specifically binds to a first protein (for example, a substance that binds to an antibody ) May comprise a capture portion (or substrate) of the immobilized or immobilizable first protein. In one embodiment, the multi-well comprises one or more wells comprising a reaction portion (a reaction portion of the first protein and the second protein: the first reaction portion) where interaction between the first protein and the second protein takes place, And a reaction unit (first protein detection unit: second reaction unit) to which a detection substance binding to the first protein binds. The detection substance binding to the first protein is as described above. The first protein detection unit may be used to measure the first protein level in the test sample and to normalize the signal value measured by the signal detection unit included in the device to the level of the first protein.

The signal detecting means may be any signal detecting means normally used according to a signal generated from the used labeling material. For example, the signal detecting means may include a signal stimulating portion and a signal detecting portion, and may further include a signal analyzing portion for analyzing (e.g., quantifying or imaging) the measured signal. In one example, signal stimulation, signal detection, and signal analysis may be performed at different sites, respectively, or at least two of them may be performed simultaneously or sequentially in one site. In one example, when the label is a fluorescent material, the signal detecting means may be selected from among all means capable of generating and detecting a fluorescent signal, and may include, for example, a fluorescent signal stimulating portion (e.g., a light source) , And / or a fluorescence signal analyzer. In one embodiment, the signal detection means comprises a Total Internal Reflection Fluorescence (TIRF) microscope or a confocal microscope (for detecting a light source and a fluorescence signal), or additionally a fluorescence camera, such as an EMCCD (Electron-multiplying charge-coupled device) A camera or a CMOS (complementary metal oxide semi conductor) camera may be further included to perform illumination and / or quantification of the light source and the fluorescent signal. The wavelength, intensity, and fluorescent camera measurement conditions (e.g., exposure time per frame, laser power, camera gain value, total shooting frame, etc.) of the light source are as described above.

The signal detecting means according to the first protein, the substrate, the substance specifically binding to the first protein, the second protein, the labeling substance, and the labeling substance are as described above.

The apparatus for measuring protein-protein interactions provided herein is capable of observing, analyzing, detecting _, and / or measuring interactions (e.g., protein-protein interactions) between various biomolecules Can be usefully applied.

Accordingly, another example is a method for measuring a protein-protein interaction, comprising contacting a sample containing a biomolecule (for example, a first protein) to be analyzed with a device for measuring the protein-protein interaction or a multi- (E. G., Protein-protein interaction). The analysis method may further include a step of measuring a signal generated in the sample after the contacting step. At this time, the signal can be appropriately selected from among all signals (for example, fluorescence, luminescence, etc.) conventionally used for biomolecule analysis, and the measurement of the signal is typically used according to the kind of the used signal All of these methods can be selected appropriately. The biomolecule may be at least one selected from the group consisting of proteins, nucleic acids, and cells isolated from a living body, and may be, for example, a protein. When the biomolecule is a protein, a multi-well contained in a device for measuring protein-protein interaction used for protein-protein interaction analysis is a molecule that specifically binds to one of the proteins to be analyzed on the surface (for example, ) May be fixed.

In another example, there is provided a kit for measuring protein-protein interactions comprising a multi-well comprising a substance capable of capturing a first protein that specifically binds to a first protein as described above. The kits for measuring protein-protein interactions include kits for measuring the activation of signal transduction pathways in tissues, kits for predicting and / or monitoring the response to a drug targeting the first protein, kits suitable for treatment targeting the first protein For screening individuals Kit, and / or kit for confirming the efficacy of a drug targeting the first protein.

The apparatus for measuring protein-protein interactions provided herein has the advantage of being able to accurately and efficiently observe, analyze, detect, and / or measure biomolecular interactions in small amounts of sample. Thus, the multiwells or analytical methods using them can be usefully and effectively applied to very small samples, such as biopsy (e.g., needle biopsy) samples. Another example provides a method for preparing heterodimers of HER2 and HER3. The method may comprise treating the dimerizing agent to cells that simultaneously express or express HER2 and HER3. The dimerization agent may be a ligand of HER3 (for example, Neuregul in pi (NRGbl), NP_039250, NP_039250.2, etc.), a cholesterol-like detergent (e.g., DGTNCdigi tonin; CAS Number : 11024-24-1), GDN (glycol-diosgenin; CAS Number: 1402423-29-3, etc.), and the like. In one embodiment, the method comprises contacting HER2 with

When the step of treating (injecting) NRGbl into cells simultaneously expressing HER3, the NRGbl may be injected into cells before expression after HER2 and HER3 lysis. In another embodiment, the cholesterol-like cleansing agent can be treated (added) to the washing step after cell lysis if it comprises the step of treating the cholesterol-like cleansing agent to cells simultaneously expressing HER2 and HER3 , The cell may be NRGbl injected in an amount of 10 ng / mL to 1000 ng / mL or 50 ng / mL to 200 ng / mL). In this case, the concentration of the cholesterol-like detergent to be used may be a concentration exceeding that of the cleaning agent used. For example, the concentration of the cholesterol-like detergent may be DGTN (W / v), 0.05% (w / v), or 0.06% (w / v) or more, such as 0.05 to 5% ), 0.05 to 3% (w / v), 0.05 to 2% (w / v) or 0.06 to 2% (W / v), 0.005 to 2% (w / v), 0.007 to 2% (w / v) ), Or 0.01 to 2% (w8).

【Effects of the Invention】

The techniques provided herein measure protein-protein interactions intracellularly and / or intercellularly, thereby preventing the disease that the protein is involved in And / or screening or selecting effective drugs for treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

Brief Description of the Drawings Fig. 1 is a schematic diagram of a method for measuring short-term molecular protein interactions.

FIG. 2 is a graph showing the result of identification of a target protein (first protein) immobilized on a substrate _.

Figure 3 is a fluorescence image showing protein interactions after infusion of the fluorescently labeled interacting protein (second protein).

FIG. 4 is a graph quantifying the number of PPI compexes observed in FIG. FIG. 5 is a graph showing changes in the number of PPI complexes according to the amount of injected cell lysate.

6 is a schematic diagram showing a process of quantifying a first protein through a monoclonal sandwich ELISA.

FIG. 7 is a graph showing the specificity obtained by the single-molecule sandwich ELISA method.

FIG. 8 is a graph showing changes in the number of PPI complexes according to the cell line type (red ① vs light blue ③) and the state (red ① vs black ②).

FIG. 9 is a graph showing changes in the number of PPI complexes according to various target RTKs (first proteins) according to cell conditions.

FIG. 10 is a graph showing the change in the PPI complex according to the EGFR mutation state and the calculated ratio of EGFR activated per cell based on the change. FIG.

FIG. 11 is a graph showing the results of performing the same method for HER2 and HER3 performed on EGFR in FIG.

Fig. 12 shows the result of measuring the interactions between EGFR, MET, HER2, HER3 (first protein) and the lower signal transduction protein (second protein) for each cell line and displaying them in a heatmap format.

FIG. 13 is a graph (left side and middle) graphically showing the results of FIG. 12 (left side) and a graph showing the reactivity results of AZD9291 (right side) as an EGFR target anticancer drug.

FIG. 14 is a graph showing the correlation between the reactivity (left, y axis) of the EGFR target anticancer agent (AZD9291) and Act iat ion score (left, x axis) and the diversity of the target anticancer response (right side) according to the gene type.

15 shows the intensity of HER2 and HER3 signals in breast cancer cell lines It is a heatmap.

FIG. 16 shows the results of measurement of biomarker HER2 (upper) and HER3 (intermediate) expression levels used for predicting the reactivity of trastuzumab anticancer drugs in breast cancer cell lines and the degree of suppression of cell growth by trastuzumab (lower) FIG.

17 is a graph showing the correlation between the result of measuring the PPI score using the HER2 or HER3 signal and the reactivity of trastuzumab (logGI50). Figure 18 is a heatmap showing the results of the PPI complex signal with the three sub-signal proteins of EGFR, MET, HER2, and HER3 measured in the PDTX mouse model, respectively.

FIG. 19 is a graph showing the result of calculating the act iat ion score (bottom) using the expression level (top) of EGFR and the expression level of EGFR in the PDTX mouse model.

20 is a graph showing the results of measurement of changes in tumor size by administering gefitinib to a PDTX mouse model.

Figure 21 shows tumor suppression by gef i t inib in a PDTX mouse model

(Tumor growth inhibi- tion) and EGFR act iat ion score.

FIG. 22 is a graph showing the results of measurements of EGFR PPI complexes measured in tissues before and after the administration of gef i t inib in a PDTX mouse model, respectively.

Figures 23A-23I show EGFR target inhibitor reactivity in a lung cancer PDTX model,

23a and 23b are graphs showing tumor volume changes in PDTX during the treatment of the vehicle or the indicated EGFR-specific inhibitor, and PDTXs (PDTX-A1 A3) Osimert inib. (PDTX-S1 S5) was treated with 5 mg per kg of weight of dairy (PDTX). 50 mg per kg of weight dairy), showing the size of the tumors obtained (test population per 3 PDTX)

23c is the labeled receptor tyrosine kinase (RTK; EGFR, HER2, HER3, and MET), wherein the PPI complex counts (number of PPI complexes) 23d is a graph showing the results of normalization of EGFR expression levels in 8 PDTX (A1-A3 and S1-S5) individuals to EGFR expression levels in A549 cells (control group)

23e and 23f are graphs showing the tumor growth inhibition rate (%) as the y-axis and the value obtained by dividing the EGFR PPI sum obtained from the PDTX model (E) and the SQCC PDTX model (0) by the EGFR level as the x axis.

23g is a graph showing changes in PPI complex counts (the number of PPI complexes between the second protein expressed on the EGFR and the x-axis) when the gefitinib was treated daily for 15 days,

23h is a graph showing the degree of tumor growth when PDTX-Sl (n = 2) was treated with gefitinib and BKM120 for 15 days in comparison with that in single treatment,

23i is a graph showing the value obtained by dividing the PPI sum by the EGFR level obtained from all 8 PDTX (A1-A3 and S1-S5) individuals as the x axis and the tumor growth inhibition rate as the y axis (Error bars: sd).

Figures 24a to 24d show an example of applying single-molecule co-IP and singlenolecule immunolabeling to human tumor samples,

24a shows human tumor tissues obtained by tumor resection of two tumor patients (P1 and P2)

24b is the amount of 1o protein produced by monoclonal immuno-labeling

A graph showing the PPI levels of 10 protein-protein pairs obtained by monomolecular co-IP using PTM (Immunolabel 1 ing level) and a high-efficiency monomolecular imaging system. As a positive control, PC9 cells (for EGFR ), HCC827 cells (for MET), and SKBR3 cells (for HER2 and HER3)

24c is a graph showing PPI complex counts for the indicated RTKs at P1 and P2,

24d is a graph showing the change in count PPI complex PLC _ga s _H2 and Grb2 in the case of the EGFR on the surface treatment hanhu pul l ing down PTPN1 (Error bars :. S d.).

Figs. 25A to 25H show the characteristics of the PDTX-model (n = 3)

25a to 25c show MET, HER2, and HER3 levels in HCC827 cells (for MET) and SKBR3 cells (for HER2 and HER3) for MET levels (a), HER2 levels And HER3 levels (Error bars: sd; JT = 5), indicating that these RTKs were not overexpressed,

25d is an image showing the immunohistochemical staining (IHC) of EGFR measured in five SQCC PDTX. EGFR expression was calculated by calculating the EGFR H-score as a magnification rule,

25e is a scatter plot showing the correlation between EGFR level and EGFR H-score determined by monomolecular immuno-labeling, and IHC H-score has a complete linear correlation with total EGFR expression level determined by monomolecular immunodiffusion ,

25f and 25g are scatter diagrams showing the correlation between EGFR level (g) and PPI sum (h) of SQCC egg TX and tumor growth inhibition,

25h shows Immunoblot analysis of vehicle or gefitinib-treated PDTX-S2. After 15 days of treatment with gefitinib, phosphorylation (pEGFR) of 1068th residue of EGFR is completely disappeared and phosphorylation of AKT and S6K by gefitinib treatment pAkt and pS6K), and these results show that the inhibition of tumor growth in PDTX-S2 is obtained by inhibiting the EGFR / AKT / mT0R / S6K signaling pathway by treatment with gefitinib.

26A and 26B show the effects of gefitinib treatment on PDTX-S1 and PDTX-S2,

26a is a graph showing the change in EGFR level when treated with gefitinib for 15 days (Error bars: s.d .; TF5)

26b is a graph showing the degree of inhibition of EGFR PPI by gefitinib treatment and EGFR PPI complex count of A549 cells as a negative control group (Error bars: s.d .; n = 5).

FIG. 27A is a schematic diagram showing a process of acquiring a protein, analyzing protein-protein interaction, and quantifying using a cell lysate expressing GFP-PD-1 and mcherry PD-L1, respectively.

27B is a fluorescence image of the protein-protein interaction obtained by using 10 nM of GFP-egg-1 and mcherry PD-L1. FIG. 27C shows the result of quantitative analysis (number of PPI complexes: fluorescence- counts).

28A schematically shows a process of screening for an inhibitor candidate drug. FIG. 28B is a graph showing test results for optimizing the reaction conditions (reaction time) of one-step reaction in a tube for drug screening. FIG. Figure 28c shows the number of PPI complexes (F-counts) between PD-1 / PD-L1 obtained using two commercially available egg-1 / PD-L1 inhibitors BMS202 and S7911 (purchased from Sel leck Chem) It is a graph showing.

FIG. 28d shows the PPI librarians screening (http: / /. (F-counts) between egg-1 / PD-L1 obtained using a non-macrocyclic ic PPI drug libbrary compound provided by Asinex.com/ppi/.

FIG. 28E is a graph showing the results obtained by conducting t i tratation on four drugs (Bl, B2, B3, and E2) selected to exhibit excellent inhibitory effect in FIG. 28D.

29 is a graph showing the number of PPI complexes (F-counts) between inhib-1 / PD-L1 obtained by immobilizing various antibodies on a substrate and using GFP-PD-1 and mcherry PD-L1.

FIG. 30A shows Total GFP-1 abe 1 ed count (left) and fluorescent image (right) when GFP PD-L1 is immobilized on a substrate and when GFP-egg-1 is immobilized.

FIG. 30B is a graph showing the relationship between the cluster rat io (%) (left) and fluorescence image (right) of GFP PD-L1 and GFP suppression-1 when GFP PD-L1 was immobilized on a substrate, Lt; / RTI >

31A schematically shows a design process of a PD-L1 mutant construct for epitope mapping.

FIG. 31B shows the degree of binding of PD-L1 antibody (9A11) to PD-L1 WT (wild-type), d280, or d270 variant.

Figure 31c shows the degree of protein-protein interaction between egg-1 and PD-L1 WT, d280, or d270 variants.

32 schematically shows the interaction (binding) site between egg-1 and PD-L1.

Figure 33 shows the structures of drugs Bl, B2, B3, and E2.

Figure 34A depicts expression of HER3-eGFP in SKBR3, followed by treatment with NRGbl to generate HER2-

Lt; RTI ID = 0.0 > HER3-eGFP heterodimer. &Lt; / RTI >

FIG. 34B shows the HER2-eGFP concentration in the cell extract according to the amount of HER3- HER3-eGFP heterodimer. &Lt; / RTI >

FIG. 34C is a graph showing the amount of HER2-HER3 heterodimer detected when NRGbl was treated before and after cell lysis. FIG. FIG. 34D is a schematic diagram illustrating a process of inducing, extracting, and detecting heterodimers of HER2 and HER3 in cells by treating NRBbl with SKBR3 cells. FIG.

Figure 34E is a graph showing the amount of HER2-HER3 heterodimer detected according to the amount of cell extract added to the substrate.

FIG. 34f is a graph showing that HER2-HER3 heterodimer is mainly present when a cell extract extracted according to the procedure of 34d is attached to a substrate using another kind of antibody (when the ligand is not treated, EGFR-HER3, When EGFR-HER2, HER2-HER3 heterodimer, and ligand were treated, the EGFR-HER3 heterodimer was much less than the amount of HER2-HER3 heterodimer when treated with ligand)

Figure 34g illustrates the process of overexpressing HER2-eGFP in four K293T cells to form a HER2-eGFP-HER2-eGFP homodimer and attaching it to a substrate (PEG, neutravidin, biot in, and ant i -HER2 ant ibody surface treated) It is a schematic diagram showing the enemy.

Figure 34h shows the results of measuring the fluorescence signal from the HER2_eGFP-HER2-eGFP homodimer or HER2-eGFP monomer attached to the substrate at 34g. FIG. 34I is a graph showing the ratio of the dimer to the fluorescence signal obtained from the homodimer or monomer at 34h, and the ratio of the fluorescence signal to the fluorescence signal at the second step and the olimeric state. FIG. It can be seen that the ratio of HER2-eGFP to that of step 2 and step 2 is higher than that of eGFP or HER3_eGFP.

FIG. 35A is a schematic diagram showing an example of a process for measuring the Tyr phosphorylation level of the HER2-HER3 heterodimer. FIG.

FIG. 35B is a graph showing the results of measuring the degree of phosphorylation (HER3 pTyr counts) of HER3 Tyr residues after treating ATP and ¾ ²⁺ on a substrate immobilized with HER2-HER3 heterodimer.

Figures 35c and 35d show the degree of activation of HER3 obtained by washing the HER2-HER3 heterodimer-forming cell lysate with various concentrations of digi tonin as the degree of phosphorylation of HER3 Y1289 according to digi tonin concentration Graph.

FIG. 35E is a graph showing the degree of HER3 activation according to the kind of detergent used for washing the HER2-HER3 heterodimer-forming cell lysate through the degree of phosphorylation of HER3 Y1289.

Figure 35f shows that the degree of activation of the HER2-HER3 heterodimer is decreased when HER2-HER3 heterodimers are subjected to HER2 and HER3, respectively, corresponding to EGFR, in order to show that they follow the same activation mechanism as previously known by the EGFR homodimer beam ^"gourd is a graph that.

FIG. 35G is a schematic diagram showing an example of a process for measuring the Tyr phosphorylation level of the HER2-HER2 homodimer.

FIG. 35h is a graph showing the results of measuring the degree of phosphorylation (HER2 pTyr counts) of HER2 Tyr residues after treating ATP and sugar ²⁺ on a substrate immobilized with a suppressor R2-HER2 homodimer.

FIG. 35I is a graph showing the degree of HER2 activation according to the type of detergent used for washing HER2-HER2 homodimer-forming cell lysates through the degree of phosphorylation of HER2 Y1196.

36A is a schematic diagram showing that a HER2-HER3 heterodimer attached to a substrate induces an interaction with an eGFP-labeled lower signaling protein when subjected to a phosphorylation process.

FIG. 36B is a graph showing the magnitude of the fluorescence signal as to how much amount of the lower signaling protein (PLC gamma 1) interacts with one HER2-HER3 heterodimer.

Figure 36c is a graph showing the number of single molecular off events from a fluorescence signal to see how much of the HER2-HER3, heterodimer, interacts with the lower signal transduction protein (PLC gamma 1).

Figure 36d is a graph showing the magnitude of the fluorescence signal as to how much of the HER2-HER3 heterodimer interacts with the amount of the lower signal transduction protein (p85 alpha).

Figure 36E is a graph showing how much of the HER2-HER3 heterodimer interacts with the amount of the lower signaling protein (p85 alpha) as the number of single molecule off events from the fluorescence signal.

Figure 36f shows how much amount of the lower signaling protein (PI3K: p85 alpha-pi 10 alpha complex) is associated with one HER2-HER3 heterodimer And the intensity of the fluorescence signal.

FIG. 36g is a schematic diagram showing that the HER2 homodimer attached to the substrate induces an interaction with the eGFP-labeled lower signal transduction protein upon oxidation.

Figure 36h is a graph showing the magnitude of the fluorescence signal as to how much of the HER2 homodimer interacts with the amount of the lower signaling protein (PLC gamma 1).

Figure 36i is a graph showing how much of the HER2 homodimer interacts with PLC signaling protein (PLC gamma 1) as the number of single molecular off events from the fluorescence signal.

Figure 36j is a graph showing the magnitude of the fluorescence signal as to how much of the HER2 homodimer interacts with the amount of the lower signaling protein (p85 alpha).

Figure 36k is a graph showing how much of the HER2 homodimer interacts with the amount of the lower signaling protein (p85 alpha) as the number of single molecular off events from the fluorescence signal.

Figure 37A is a graph showing the phosphorylation rate of the HER3 Tyr residues during ATP and Mg2 + treatment of the HER2-HER3 heterodimer (x-axis is the time taken to phosphorylate and y-axis is the amount of phosphorylated Tyr during that time).

Figure 37b shows the results of increasing the ATP treatment concentration in the HER2-HER3 heterodimer

Lt; RTI ID = 0.0 > Tyr < / RTI >

FIG. 37C is a graph showing the rate of phosphorylation of HER2 Tyr residues measured while increasing the ATP treatment concentration using a HER2-HER2 homodimer. Figure 37d shows the results of pretreatment of HER2-HER3 heterodimer and HER2 homodimer at various concentrations of Lapat inib followed by treatment with ATP and magnesium chloride ide

The activity of HER2 is shown by the concentration of Lapat inib.

FIG. 37E is a graph showing the degree of activation of HER2 obtained by treating LER2-HER3 heterodimer and HER2 homodimer with Lapat inib, ATP, and magnesium chlorid ide according to Lapat inib concentration.

Figure 37f is a graph showing the HER3 Y1289 phosphorylation level obtained by treating Lapat inib, ATP, and magnesium chloride ide with various concentrations of PTPN1 (Protein Tyrosine Phosphatase, Nonreceptor type 1) in the HER2-HER3 heterodimer according to Lapat inib concentration . Figure 38 is a graph showing the results of application of single-molecule co-IP and single-molecule immunolabeling on human tumor samples. Figures 39a-39g show SH3 domain-mediated interaction (pTyr-independent manner) between Grb2 and mutant EGFR ((a) Suppression of EGFR_Grb2 interaction by gef itinib (100 nM for 24 h) treatment during cell culture. Osimertinib dose as gefitinib) was treated to H1975 for further suppression of EGFR_Grb2 interaction due to the presence of the gatekeeper mutation, T790M, in H1975 cell (bg) single-molecule immunolabeling assay (b), MIG6 (c), GAPDH (d), Shcl (e), EGFR (f) or c-Cbl The amounts of cell extracts used are shown in the following table, and the amount of cell extracts used are as follows: shown at bottom. Error bars in Fig. 40 show sd)

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the following examples.

Example 1: Preparation of first protein (EGFR, MET, HER2, and HER3)

EGFR, MET, HER2, and HER3 were selected as the first protein, and the first protein was obtained from a lysate obtained by dissolving a cell line (for example, cancer cell line) or cancer cell tissue containing the same. This process is described in more detail as follows:

1.1 Preparation of cell lysate

1.1.1. Cell line preparation

Cell lines were cultured in medium (RPMI 1640, high glucose (Thermo 11965-092)) at 2 x

10 ⁶ cel Is. When the cell line was over 90% confluency in a 100-pi culture dish, the cell line was collected and divided into two 1.5 ml tubes. The tube centrifuged (5 min x 15,000g) to leave only the cell after removing the culture medium were kept eolryeoseo to 80 ^° C.

The prepared cell lines are listed in Table 1 below: [Table 1]

1.1.2 Preparation of cell lysate

100 mM Tris-HCl (pH 7.4), 1% (v / v) Triton X-100, 150 mM NaCl, 1 mM EDTA, 10% (v / v) glycerol, protease inhibitor cocktail tyrosine phosphatase inhibitor cocktail (Sigma, P5726) 100).

The cell line sample prepared in Example 1.1.1 was pipetted and the cells were collected by pipetting. The prepared cell lysis buffer was added to the pipetted cell lysate in an amount of 200 ul per tube, and the cells were reacted for 30 minutes in a cold block (0-4 ^° C) placed on ice. At this time, every 10 minutes The physically mixing process was repeated by cyclic pipetting so that the cell lysis reaction by the surfactant was actively performed.

After 30 min of reaction as described above, centrifugation was performed (10 min, 15,000 g, 4 ° C). Thereafter, the pellet is discarded, the supernatant is removed, the membrane is filtered using a membrane having a pore size of 0.2 mu, and the portion of the membrane passed through the membrane is transferred to a new tube and stored Respectively.

Total protein concentration in the reaction product was measured using a total protein measurement method (Bradford, BCA, DC protein assay, etc.), and the protein concentration was measured to be about 5-10 mg / ml.

1.2 Preparation of tissue solution

1.2.1. Patient-derived tumor xenograft model preparation

Tumor xenografts from patients with Lung squamous cell carcinoma (SQCC) were obtained from Yonsei University research team. A brief review of the production process of Patient's tumor xenograft (PDTXs) is as follows: 6 to 8 weeks old female severe combined immunodeficient mice (NOG) and nude mice (nu / nu mi ce; Or ientBio). All animal studies were carried out in accordance with guidelines approved by the Insti- tional Animal Care and Use Committee (IACUC). A patient-derived clinical tumor sample was cut into sections of 3 mm or less in size and subcutaneously transplanted into the side of the prepared NOG mouse. Tumor size was measured twice weekly with calipers to determine the growth rate in subcutaneous tissue. When the size of the tumor reached about 1.5 cm in diameter, the tumor tissue was excised and cut into small sections (hexagon with one side length of about 5 mm). Thereafter, the excised tissue was re-implanted in another mouse to obtain consecutive individuals containing subsequent tumors. Mice with the patient-derived tumors thus obtained were named as mice, and mice having subsequent tumors continuously derived therefrom were referred to as FI, F2, F3, F4 and the like with serial numbers attached thereto. Mice (F3) with third generation subsequent tumor were injected with vehicle (PBS) or gefitinib Xgef it inib). The prepared PDTXs were intraperitoneally administered 50 mg / kg of gefitinib or vehicle once a day. Tumor tissues were collected from PDTX 15 days after the administration of gefitinib and used to monitor PPI and expression level changes described below. 1.2.2 Preparation of tissue solution

The tumor tissue obtained in Example 1.2.1 was prepared in an amount of about 20 mm ³ , but it may be larger.

Approximately 300 μL of the lysis buffer prepared in Example 1.1.2 was added to 20 mm ^{3 of} the prepared tumor tissue, and the mixture was reacted while continuing to rotate in a 4 ° C refrigerator for 1 hour. At this time, the size of the tissue was made as small as possible using surgical scissors, and the surface area per unit volume was widened so that the chemical reaction by the surfactant in the dissolution buffer occurred as efficiently as possible. After 1 hour of reaction as described above, centrifugation was performed (10 min, 15,000 g, 4 ° C). Thereafter, the pellet is discarded, the supernatant is removed, the membrane is filtered using a membrane having a pore size of 0.2 mu, and the portion of the membrane passed through the membrane is transferred to a new tube and stored Respectively.

Example 2: Preparation of second protein

In this example, preparation of a _second protein in which a fluorescent protein is attached to a lower signal transduction protein of the _first protein prepared in Example ₁ is exemplified.

The second protein exemplified in this example is summarized in Table 2 below:

HEK293 cells (ATCC) and HeLa cells (ATCC), which are low cell expressing proteins, were prepared.

An expression vector for each of the second proteins listed in Table 2 was injected into the prepared HEK293 cells or HeLa cells and cultured to express the second protein. After 24 h incubation, the cells were harvested, dispensed in the appropriate volume and stored at -80 ^° C.

The cells were lysed in a solution (50 mM Tris-HCl (pH 7.4), 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 10% glycerol, protease inhibitor cocktail (Sigma, P8340) 100X, tyrosine phosphatase inhibitor cocktail (Sigma, P5726) 100X). High concentrations of surfactant (Triton X-100) was added 60 uL of the solution with respect to the first 5x10 ^s cel Is, since it can interfere with the protein interactions.

The cells in the obtained reaction mixture were released by pipetting and reacted for 30 minutes in a cold block (0-4 ° C) placed on ice. At this time, physical mixing was performed by periodic pipetting at intervals of 10 minutes so that dissolution reaction by the surfactant was actively performed.

After 30 minutes of reaction, centrifugation was performed (10 min, 15,000 g, 4 ° C). The sinking was discarded (pellet) and transferred to a new tube for supernatant experiments.

The obtained aqueous solution 60 was added to 140 uL of PBS. Finally, a solution of the second protein containing 0.3% Triton X-100 can be obtained. Fluoremeter was used to measure the concentration of fluorescent protein attached to the second protein. As a result, the concentrations of the three second proteins used were measured within the range of about 400 to 1000 nM.

Example 3: Preparation of substrate

Coverslip was immersed in R0H 1M solution and washed with a sonicator (20-30 min). After that, it was washed well with tertiary distilled water and then washed with piranha solution (sulfuric acid: hydrogen peroxide = 2: 1 - 3: 1 (v 八 0). The washed coverslip surface was treated with aminopropyl lane and PEG in order.

After 2 hours of reaction, the cells were washed with 3 rd distilled water and stored ^at -20 ^° C until used without contact with the PEG-coated surface.

At the same time, a quartz channel type substrate or an acrylic type well type substrate was prepared. In the case of a quartz substrate, cleaning and PEG coating processes were performed with reference to the cover si ip process described above.

In the case of an acrylic substrate, the substrate was immersed in the third distilled water and sonicated for washing. The washed acrylic substrate was immersed in a 5% BSA solution and reacted for 2 hours to prevent nonspecific protein adsorption and stored at -20 C until use.

In the following test, the prepared cover lip and acrylic substrate were used. Before the test, the coverslip and the acrylic substrate were thawed or assembled, or they were pre-assembled after the PEG coating and stored in -20C And thawed before use.

Example 4: Protein-protein interaction imaging between a first protein and a second protein

Neutravidin (Thermo, A2666), an Avidin family protein, was added to the prepared substrate at a concentration of 0.1 mg / ml. After 5 minutes of reaction at room temperature, the substrate was washed twice with 30 μl of PBS buffer.

An antibody against the first protein to be a target was added to the prepared substrate. At this time, the antibody to be used was prepared as a biotin-conjugated form. The concentration of the antibody can be appropriately adjusted according to the affinity (dissociation constant, KD) of the antibody-antigen. In this experiment, the antibody concentration was about 2 ug / ml and the reaction time was about 5 minutes. If a biotin-conjugated antibody is used, the antibody of the first protein can be attached using a secondary antibody.

Antibodies to the first protein used at this time are summarized in the following Table 3:

[Table 3]

The substrate treated with the antibody was washed twice with 30 ul of PBS buf fer. A cell lysis solution or a tissue lysis solution containing the first protein well prepared in Example 1 was added to the prepared substrate. In the case of antigen-antibody reaction, the antigen-antibody reaction efficiency could be lowered to 15 minutes and the reaction time could be lowered to 15 minutes or more.

After the reaction, the substrate was washed with PBS containing 0.05% (v / v) of tween 20. Tween 20 0.05% reduces nonspecic binding and helps prevent the hydrophobic c region of membrane proteins from breaking down.

Thereafter, the second protein solution obtained in Example 2 was added to the above-mentioned substrate. The concentration of the second protein in the first protein lysis solution used was between 1 and 50 nM (about 30 nM) based on the fluorescent protein. If the concentration of the second protein is more than 100 nM, the background noise increases in the fluorescence microscope, which hinders accurate fluorescence signal measurement.

The substrate was immobilized on a fluorescence microscope and imaged to obtain data for each first protein / second protein.

Example 5: Analysis of protein complex (PPI complex)

Protein complexes were analyzed based on the toolkits provided in the Mat 1 ab program (provided by MathWorks).

The fluorescence image obtained in Example 4 was stored in a 16-bit unsigned integer format. The fluorescence signal was obtained from eGFP (enhanced green fluorescence protein). To observe the signal, the wavelength of the laser was set to 488 nm and the laser power was adjusted to 2 mW to maintain the emission time of eGFP for about 11 seconds. In the whole frame (30 frames), the first frame is discarded, and the image of the frame is averaged to generate one image. This process is repeatedly carried out while shifting the position in the wel l, Respectively. The reason for discarding the early frame is to selectively use the section where the autofluorescence on the surface of the substrate disappears and the eGFP signal is maintained. The selection interval may vary depending on the imaging condition / equipment construction status. In this embodiment, the exposure time per frame is set to 0.1 second by using an EMCCD (Electron-Multi loading charging-coupled device; Andor iXon Ul tra 897 EX2 (DU-897U-CS0-EXF) A fluorescence image was obtained with an EMCCD gain value of 40.

To eliminate noise, the following procedure was performed for each frame:

(a) The initial start begins at the top left corner. One frame consists of 512x512 pixels. 11 pixels to the right based on the base pixel, down

The median value is obtained from 11 11x11 individual pixels, and this median value is subtracted from the reference pixel value [(Intensi_pixel) _

(median anintensi ty_llxllneighborhood)]. The above procedure was performed for all pixels of 512x512. Median filtering was used to remove pepper & salt noi se.

(b) Gaussian smoothing of s i gma = 0.7 and si ze = 5x5 was performed on the processed image to smooth the image.

(c) Threshold was set. Threshold is the threshold value that makes the pixel intensities below the threshold in the whole image (using the algorithm to find the local maximum in the Matlab toolkit). This eliminates the local maximum that was not produced by the fluorescence signal in the image. The threshold value used in the imaging conditions of this embodiment is 70.

The signal from the fluorescent protein occurs as a localized spreading func- tion (PSF). The PPI complex between the first and second proteins to measure the PSF count (physical value) It is a number (biological value). The PSF value was converted to the biological value PPI complex value through the following procedure:

(a) Locate the local maximum (for example, i-th row, j-th column pixel). As described above, the single-molecule fluorescence signal is formed by collecting at 512x512 pixels at a specific position (approximately 5x5 pixel size under the current observation equipment, 1 pixel = 0.167 micrometer). This can be obtained using the toolkit provided in the mat lab.

(b) It is judged whether the local maximum obtained in the above (a) is generated from the actual PSF. First, the minimum value of the local maximum is defined, and only the case where the local maximum is greater than the minimum value is used for the analysis. The minimum value used in this embodiment is 2019/132517 1 »(: 1/10/06 018/016675

75, and this value may vary depending on the laser power / exposure time / equipment construction situation. The 5x5 pixel information centered on the finally obtained local maximum coordinate was obtained, and the center of brightness was obtained from the 5x5 pixel (centroid of intensities). At this time, if the obtained center of brightness deviates by more than 0.5 pixels relative to the existing local maximum coordinates (when the 2D symmetry about the PSF shape disappears), it is judged to be a non-normal fluorescence signal and excluded from the analysis.

(c) Only the PSF that passed all the conditions were finally selected to obtain the coordinates and the total number. The total number of PSFs obtained at this time is the number of PPI comp lex. I

The above procedure is performed for all files photographed under the same conditions

The number of PSFs was calculated, and the mean and standard deviation were calculated. This value finally represents the number of PPI complexes under certain conditions (indicated as "Number of single PPI complexes" in the figure).

Example 6 Determination of PPI Strength, PPI Score, and Activative Score

The concentration of the cell lysate (see Example 1.1.2) is plotted on the x-axis and the slope of the graph obtained by plotting the PPI complex value measured in each cell lysate (see Example 5), that is, [(PPI complex ) / (Number of PPI complexes per unit concentration of cell lysate (Wml))] was defined as the PPI intensity (or PPI slope) between the first protein and the second protein in the cells. All PPI intensities obtained for each cell line were combined to obtain the total PPI intensity, which was defined as the PPI score for the cell line. The PPI intensity sum (or PPI score) represents the total PPI level of the tested first and low 12 proteins at the cell lysate unit concentration of each cell line.

The method of obtaining the PPI intensity sum is expressed as follows:

(First protein: RTK (EGFR, MET, HER2, or HER3 for lung cancer; HER2 and HER3 for breast cancer);

Second protein: downstream protein (PLC-ga_a_SH2, Grb2, p85_alpha)). In addition, in order to show the relative PPI scores among the cell lines, (Except for the reference cells), so that the PPI score of the cell line (hereinafter referred to as reference cell, in this test example, PC9 cells among lung cancer cell lines and SKBR3 cells among breast cancer cell lines, respectively) Hereinafter referred to as "test cells") were normalized, and the values obtained for each cell line were defined as normalized PPI scores for the cell lines.

In addition, the total amount of the first protein (e.g., RTK (EGFR, MET, HER2, or HER3 for lung cancer; HER2 and R4 for breast cancer) in each cell lysate was measured. The total amount of the first protein was determined by sandwich ELISA or quantitative western blot using each of the antibodies described above (see Table 3), and then divided by the weight of the cell lysate (total protein weight in the cell lysate) Respectively.

A value obtained by dividing the obtained PPI score or normalized PPI score by the total amount of the first protein was defined as an activation score. At this time, activation scores obtained from the test cells were normalized so that the activation score of the reference cells (lung cancer cell line: PC9 cell, breast cancer cell line: SKBR3 cell) was 1, The value obtained for the cell line was defined as the normalized activation score for that cell line.

When a PPI score is obtained in a PDTX (patient-dependent tumor xenograft) mouse model, background noise can be reduced by measuring a negative background value from the value obtained by the above method. In this case, the negative backgromid can use normal tissue of the same patient or cancer cell lysate whose test protein (for example, EGFR) is normal. For example, in the A549 cell, in which the EGFR gene is in a normal state, the degree of interaction between the EGFR and the sub-protein is measured to obtain the PPI score. We set this as a negative background and subtract the negative background from the PPI score obtained from each PDTX mouse model to yield the final PPI score.

Example 7: Heatmap creation

In addition to quantifying the data in Example 5, a Heatmap was created to add supplemental judgment to its analysis. Heatmap is only one way to show data and is not intended to limit data interpretation.

Among the X axis and the Y axis, one axis is a second protein (lower signal protein) The other axis is the number of cell lines (15 kinds (lung cancer cell line) or 11 (breast cancer cell line): 3x16 (number of the second protein (total 3: see Table 2 (p85_alpha, Grb2, PLC-ga_a_SH2) (EGFR, MET, HER2, and HER3; lung cancer), or a total of two (HER2 and HER3; breast cancer) by the first protein (see Table 1) However, in the case of HER3, only p85-alpha is used as the second protein). Then, a heatmap was prepared by varying the color and brightness according to the PPI intensity between the first protein and the second protein obtained from the corresponding cells (for example, as the PPI intensity is increased, dark, black, , Which is not the value determined by the tester for each test). Regardless of which cell is the reference, the relative differences between the cell lines remain unchanged.

Example 8 Correlation Between Drug Reactivity, PPI Score and Activation Score

The results of tests conducted by the methods described in Examples 1 to 7 are described below with reference to the drawings:

1 is a schematic diagram of a method for measuring monomolecular protein interactions. On the left side, the Neutravidin, RTK antibody, and the cell lysis solution or tissue lysis solution to be analyzed are sequentially injected into the substrate coated with PolyeUlyene glycol, and then the sample is washed. In the middle, the target RTK protein It shows how it is fixed to the substrate. On the right side, the fluorescence signal is observed and quantified by injecting fluorescently labeled interacting protein to the substrate, and the degree of monomolecular protein interaction is measured.

FIG. 2 is a graph showing the result of identification of a target protein (first protein) immobilized on a substrate. With reference to the methods described in Examples 4 and 5. EGF was treated for 3 minutes in an amount of 100 ng 8 il, and the graph on the left side of Fig. 2

H antigens were used to attach to the substrate via an ant ibody (MAS-13266, Themof i sher) that binds to the extracelular domain of EGFR using H1666 and an ant ibody for the intracelular domain of EGFR (# 4267, Cel l signaling technology) 2 shows the result of confirming whether or not EGFR forms a dimer with HER2. The graph on the right side of FIG. 2 shows whether or not EGFR forms a dimer with Shcl. Shcl The results are shown by inserting the antibody.

Depending on whether the substrate is injected with the antibody, the target RTK protein (first protein) It is fixed (+), not fixed (-). Through this, various target proteins can be attached to the substrate through appropriate antibody screening. In addition, as in the case of EGFR-HER2 or EGFR-Shcl, it can be confirmed that not only a single target protein but also a protein conjugate existing in vivo can be immobilized on a substrate.

FIG. 3 is an image showing protein interactions after the injection of fluorescently labeled interacting protein (second protein) onto a substrate to which the first protein is immobilized. The PPI complexes identified in the method described in Example 5 appear in the form of a point spread function (PSF), and the PPI complexes were selected through computer algorithms. It can be seen that the fluorescence signal occurs only when the lower signaling protein is injected. Green circles represent the observed PPI complexes.

FIG. 4 is a graph quantifying the number of PPI compexes observed in FIG. High PPI complexes were selectively observed only when the lower signaling protein (the second protein) was injected (designated PLCgammaSH2, Grb2, and p85-alpha in the x-axis). On the other hand, when the target RTK protein (the first protein (EGFR) antibody is absent (black bar) and the injected lower signaling protein is absent (buf fer of the x axis), the observed signal is very small. In both cases, it can be interpreted as background noise.

5 is a graph showing the increase in the number of PPI complexes according to the amount of injected cell lysate. It can be seen that the amount of observed PPI complex (y axis) increases linearly as the amount of cell lysate containing the target RTK protein (first protein: EGFR) increases. This allows a quantitative comparison of the PPI compex between samples in the amount of specific cell lysate.

6 is a schematic diagram showing a process of quantifying a first protein through a monomolecular sandwich ELISA. The process of attaching the target RTK protein (first protein) to the surface of the substrate is the same as in Fig. Instead of the fluorescently labeled lower signal protein (second protein), a second antibody recognizing the target RTK protein can be injected to measure the amount of target RTK protein. The second antibody used here is a first antibody (pull down ant ibody) used to immobilize the target RTK protein on the surface of the substrate and a second antibody (epi toe) on the target RTK protein ). The amount of RTK protein immobilized on the surface of the substrate through the labeled ant i body recognizing the second antibody can be measured via a single molecule technique (see Example 5). FIG. 7 is a graph showing the specificity obtained by the single-molecule sandwich ELISA method. It can be seen that the single molecule sandwich ELISA signal result is suppressed even if only one component out of the components shown in the schematic diagram of FIG. 6 is omitted.

FIG. 8 is a graph showing changes in the number of PPI complexes according to the cell line type (red ① vs light blue ③) and the state (red ① vs black ②). The target RTK protein (first protein; EGFR) was activated by the corresponding ligand (EGF +), a higher PPI complex was observed at the same dose compared to the case without EGFR. In addition, the presence of active mutations in the target RTK (PC9, light blue) indicates that the number of PPI complexes in the observed target RTK increases.

FIG. 9 is a graph showing the PPI complex number change (PPI slope) per unit concentration of a sample for various target RTKs (first protein) according to cell conditions. Using the monomolecular co-IP technique described in Example 6, when the target protein (first protein) of each of EGFR, MET, HER2 and HER3 was subjected to 1-stigm imitation (gray) and not (black) The number of PPI complexes was measured quantitatively. Based on this, the activity of the target RTK can be measured through PPI complex quantification.

FIG. 10 is a graph showing the change in the PPI complex according to the EGFR mutation state and the calculated ratio of EGFR activated per cell based on the change. FIG. The upper part shows the result of measuring the interaction between the EGFR and the lower signal transduction protein for each individual cell by the PPI complex measurement method and the lower part shows the EGFR expression per cell through the single molecule sandwicz ELISA (see FIG. 6) After the two values were divided, the amount of activated EGFR per cell was measured (Absolute occupancy (%)).

FIG. 11 is a graph showing absolute occupancy (%) results obtained by performing the same method for HER2 and HER3 performed on EGFR in FIG. In the case of HER2, the activity is very low, while HER3 shows a very high activity ratio.

FIG. 12 is a graph showing the interactions (signal intensity) between EGFR, MET, HER2, HER3 (first protein) and the lower signal transduction protein (second protein) Show one result. The color indicators for each signal strength are shown below. FIG. 13 is a graph (left and middle) showing values obtained by adding numerical values obtained by quantifying the signal intensities between EGFR (first protein) and 3 kinds of second proteins among the results of FIG. 12, and AZD9291 0.0 >(IC50;< / RTI > And the cell survival rate is 50% as compared with that before treatment) (right side).

The color of each bar is distinguished by the group shown on the right depending on the EGFR gene mutation status. The Act iat ion score showed a more significant correlation with the drug response (IC50) compared to the PPI score. (The higher the Act iat ion score, the lower the IC50 value (the higher the drug reactivity)). 14 is a graph showing the correlation between the reactivity (y axis) and the Act iat ion score (x axis) of the EGFR target anticancer agent (AZD9291) (left) and the diversity of the target anticancer response (right) depending on the gene type. The Act iat ion score showed a high correlation with the reactivity of AZD9291 (r = 0.85), and the EGFR gene test can show different drug reactivity even if it has the same genotype.

FIG. 15 is a diagram showing the intensity (interaction) of signals between HER2 and HER3 (first protein) and a lower signal transduction protein (second protein) in a breast cancer cell line in the heatmap format (Example 7).

17 is a graph showing the correlation between the result of measuring the PPI score using the HER2 or HER3 signal and the reactivity of trastuzumab (logGI50). The PPI score (r = 0.91) showed a higher correlation with trastuzumab reactivity than the conventional biomarker HER2 (r = 0.54) or pHER2 (r = 0.44)

Fig. 18 shows the results of the tissue lysis (Example 1.2) (n = 5; And METH, HER2, and HER3 as measured by the PDTX-1, -2, -3, -4, and -5, respectively.

19 shows the expression levels (top) of EGFR and the expression levels of EGFR (results of FIG. 18) in the tissue lysate (Example 1.2) obtained in the PDTX mouse model This is a graph showing the result of calculating the activation score (bottom).

FIG. 20 is a graph showing the result of measuring the change in tumor size by administering gefitinib (50 mg / kg) to a PDTX mouse model (Example 1.2.1) in comparison with the results in Vehicle (PBS) administration group. In the case of PDTX-2, the expression level of EGFR was not high, but the activation score was high (see FIG. 19) and the antitumor effect was also excellent (see FIG. 20). These results confirm that the activated activation score (ie, the activated EGFR ratio) is more closely correlated with the drug response than the EGFR expression level. 21 is a graph showing the correlation between the degree of tumor growth inhibition by gefitinib and the EGFR activation score in the PDTX mouse model (Example 1.2.1). As described above, there is a significant correlation (r = 0.96) between the extent of tumor growth inhibition by gefitinib and the EGFR activation score.

FIG. 22 is a graph showing the results of measuring the number of EGFR PPI complexes per unit concentration of the samples measured in the tissue lysate samples before and after the administration of gefit inib (50 mg / kg) in the PDTX mouse model (Example 1.2.1) to be. In the tissues obtained after treatment with Gefitinib, the EGFR PPI complexes were significantly reduced. This may be evidence that the EGFR signal is suppressed by gefitinib. Example 9

9.1. Antibody and reagent preparation

The following antibodies were used for the pulling down of respective proteins: anti-EGFR antibody (MS-378-B0 ThermoFisher), anti-MET antibody (ab89297 Abeam), anti- HER2 antibody (BMS120BT ThermoFisher) HER3 antibody (BAM348 R & D systems) mCherry (ab34771 Abeam), and anti-KRas antibody (sc-521

Santa Cruz).

EGFR antibody (4267 Cell signaling), anti-EGFR (pTyr 1068) antibody (ab32430 Abeam) was used as the detection antibody for each of the corresponding proteins and PTMs (post-translational modi fi cat ions) , Anti-EGFR (pTyr 1086) antibody (ab32086 Abeam), anti-EGFR (pTyr 1173) antibody (4407 cell signaling), anti-MET antibody (8494 Cell signaling)

HER2 (pTyr 1221/1222) antibody (2243 Cell signaling), anti-HER3 antibody (ab32121 Abeam), anti-HER3 (pTyr 1289) technology, cat. No. (Ab32037 Abeam), anti-Shcl antibody (ab33770 Abeam), anti-She pTyr 239/240) antibody (abl09455 Abeam), anti-HSP90 antibody (PA3-013 ThermoF i sher), anti- MIG6 antibody (11630-1-AP Proteintech), anti-GAPDH antibody (3906 cell signaling), and anti-c-Cbl antibody (2179 Cell signaling).

The biotin conjugated anti-mouse immunoglobulin G (IgG) (405303 BioLegend) and Cy3 conjugated anti-rabbit IgG (111-165-046 Jackson

ImmunoResearch antibodies were used as secondary antibodies.

Western blotting was performed using the following antibodies: anti-EGFR (pTyr 1068) antibody (2234 Cell signaling), anti-EGFR antibody (2232 Cell signaling), anti-Erk (pThr202 / Tyr204) ), Anti-Erk antibody (4696 cell signaling), anti-Akt (pSer473) antibody (4060 cell signaling), anti-Akt antibody (4691 cell signaling), anti- S6K (pSer235 / 236) Anti-S6K antibody (2217 Cell signaling), and anti-actin antibody (ab8227 Abeam) were used.

EGFR was stimulated using lOOng / ml EGF (PHG0311L Life technologies) (3 min).

The changes in PPI and lung cancer cells in lung adenocarcinoma cells were compared with that of Gefitinib (S1025 Sel leckchem), Osimert inib (S7297 Sel leckchem), BKM120 (S2247 Sel leckchem), Dabrafenib (S2807 Sel leckchem), and Trastuzumab HER2- / HER3-PPI assay, MTT assay, and tumor growth assay in the PDTX model.

9.2. Cell culture

All cell lines were incubated with 10% (w / v) fetal bovine serum (26140-079 Life technologies), 10 // g / ml gentamicin (15710-063 Life technologies), 100 units / ml penicillin, 15140-122 Life technologies) were supplemented in RPMI1640 medium (22400-105 Life technologies). Cell lines of PC9-GR (gefitinib resistant cell line: Accession No. CVCL_S706), HCC827-GR5 (gefitinib resistant cell line; Accession No. CVCL_V622), and HCC4006-ER (erlotinib resistant cell line; Accession No. CVCL_S746) . &Lt; / RTI > All cell lines were cultured in a humidified incubator at 37 ^° C and 5% CO ₂ . Cultured cells were washed with cold phosphate buffered saline (PBS). With 1 ml of cold PBS Cells were rapidly harvested using a scraper (90020 SPL Life Science). The cell suspension from one petri dish (100 mm diameter) was divided into 3-4 aliquots. This fraction was centrifuged for 5 minutes at 3,000xg at 4 ^° C. The supernatant was discarded and the resulting pellet was stored at -80 ^° C and was then used for analysis.

9.3. Preparation and transfections of eGFP-labeled prey proteins

Using the EcoRI and Bglll were isolated directly Rat PLC Y _S H2 cDNA containing the tandem SH2 domain (542 to 765 amino acids of NM_013187.1 ) from Rat cDNA library. Shcl (human Shcl, Addgene 73255), Eat2 (human Eat 2, Addgene 46423), APCS (human APCS, Addgene 46477), Nckl (human), Grb2 (human Grb2; Addgene 46442), p85a (mouse p85a Addgene 1399) Nckl, Addgene 45903) and S0S1 (human S0S1, Addgene 32920) were excised using restriction enzymes corresponding to the restriction sites contained in each original plasmid. The eGFP-tagged CARM1 (human CARM1) and EGFR genes were obtained from Seoul National University (Korea) and KAIST (Korea), respectively. All cDNAs were cloned into pEGFP-Cl (Clontech Laboratories) to produce corresponding eGFP-1 abe 1 ed prey proteins. Mutations of W36K, R86M and W193K were introduced into the Grb2 gene, respectively, to produce N * -, SH2 * -, and C * - constructs of Grb2 variants. EGFR mutants were prepared by either deleting E746-A750 from the EGFR gene or replacing the 858th residue, lysine, with arginine.

The obtained plasmid was introduced into HEK293 cells by electroporation using a Neon transfection system (MPK5000 Life technologies) according to the manufacturer's instructions. Plasmid DNA 30! Were mixed with 100 M of HEK293 cell suspension containing 2x10 ⁶ cells. Two 950V electric pulses (with a duration of 35 ms for each pulse) were applied to HEK293 cells. Transfected cells were harvested 24 hours after transfection and stored at -80 ^° C.

9.4. Tumor xenograft model from lung cancer patient

All animal studies were conducted by the Institutional Animal Care and Use Committee

(IA, C). (NOG) and nude (nu / nu) mice (OrientBio) at 6 to 8 weeks of age were used. Clinical tumor samples (lung adenocarcinoma) Patient-derived or lung squamous cell carcinoma (SQCC) patients) were cut into ~ 3 mm ³ pieces and subcutaneously implanted into the lateral side of the N0G mice. After 1 to 4 months after transplantation, a tumor was observed at the transplanted site. The volume of the tumor was measured twice weekly with caliper to determine the subcutaneous growth rate of the tumor. Tumor size is diameter

Upon reaching 1.5 cm, the tumor tissue was excised and cut into small sections (approximately 5 mm ³ size). The severed tissue was re-implanted in another group of mice to obtain subsequent tumors. Mouse generations with patient-derived tumors were named and subsequent generations were numbered sequentially (FI, F2, F3, etc.) (see FIG. 23A). A third generation (F3)

(Phosphata buffered saline, PBS), osmate inib, or gefitinib.

In the obtained patient-derived tumor xenograft (PDTX), a mouse (F3; n = 3) transplanted with a malignant tumor-derived tumor was transplanted into an egg TX-A1, an egg TX- A3, and mice with FT (n = 5) implanted with lung SQCC patient-derived tumors were named as PDTX-S1, egg TX-S2, egg TX-S3, PDTX-S4 and PDTX-S5.

5 mg of osimert inib or 50 mg of gefitinib or vehicle per kg body weight was injected into the obtained patient-derived tumor xenograft (PDTX) by intraperitoneal injection once a day. Tumor tissues were excised from PDTX 15 days after the drug treatment to monitor changes in PPI and expression levels.

9.5. Single molecule co-IP (single-molecule co-IP) and immuno-labeling imaging

Detailed protocols for monomolecular co-IP and immunolabel imaging

Lee, H. W. et al., Real-time single-molecule coimmunoprecipitate ion of weak protein-protein interactions., Nat. Pro. Toe. 8, 2045-2060,

NeutrAvidin (10 jd of 0.1 mg / ml: A2666 Life technologies) was placed in each individual reaction chamber. After 10 minutes of incubation, unbound NeutrAvidin was removed. A miniaturized imaging chamber was filled with PBS After PBS was completely removed, biotinylated pul 1 down antibodies were incubated on a NeutrAvidin coated surface for 10 min, made. For MET antibodies, the primary antibody was conjugated using a biot inylated secondary antibody (alpha- mouse IgG). After washing with PBS, cancer cells or tumor tissue extracts were applied to the antibody coated surface. After 15 minutes, the unbound extract was removed and the chamber was soaked in a reservoir filled with PBS supplemented with 0.05% (v / v) Tween 20.

For single-molecule co-IP imaging, transformed HEK293 cell extracts were diluted with 30 nM (eGFP-tagged probe protein) and loaded into the imaging chamber. Implants were placed on a TIRF microscope and eGFP fluorescence was recorded with EMC suppression (20 frames; 100-ms exposure).

For single-molecule immunolabel imaging, dye-labeled detection antibody was used instead of eGFP-labeled probe protein for 5 frames. To avoid duplication between detection and pulldown antibodies, the detection antibody was chosen to have an epitope within the tyrosine residue on the cytoplasmic kinase site or end (tai l). Detection antibodies were directly labeled with Alexa488 (MET antibody) or indirectly with Cy3-labeled secondary antibodies (EGFR, HER2, HER3 and pTyr antibodies). After recording 5 or 20 frames of fluorescence (0.1 second exposure) on a TIFF stack, the number of fluorescent spots was counted and the number of unimolecular PPI complexes or immunolabeled proteins was immediate. The mean and standard deviation of single-molecule counts were determined from ten different positions in the same reaction chamber.

Example 9.6. The TIFF file obtained by counting fluorescence imaging of the counting PPI complex and immuno-labeled proteins was analyzed with a custom GUI (Matlab 2016a, MathWorks). Three frames (17-19 for eGFP, 3_5 for Cy3 and Alexa488) were used to confirm the local maxima of intensities representing a single PPI complex or immunoreactive protein. For the background correction, the images obtained by spatial aliasing (11 × 11 pixels) were subtracted frame by frame from the original image. The obtained images were averaged and used to detect thresholding to local maxima (using custom Mat 1 ab GUI).

Example 10: Prediction of responsiveness of PDTX to EGFR target inhibitor

10.1. Reactivity to Osimert inib in PDTX transplanted with lung carcinoma

The PPI measurement index of HER receptor is closely related to the drug reactivity of cancer And whether single-molecule immuno-labeling or co-IP analyses can be applied to screening for specific cancers that are reactive to HER receptor target therapy (HER receptor-targeted therapies have anticancer effects) Respectively. For this, three types of lung cancer xenograft-derived tumor xenografted mice (PDTXs; PDTX-

A1 A3).

These abundant carcinosarcoma TX were found to have an activating mutation (exon 19 or L858R mutation) in the EGFR gene.

Treatment of ovarian carcinoma PDTXs (PDTX-A1-A3; each population of 3 or more, the results of which are shown as the mean value) for 30 days with osmate inib (5 mg per kg of weight dairy) And compared with the control group (vehicle-treated group), the results are shown on the left side of FIG. 23B. As can be seen from the left-hand side of FIG. 23B, PDTX-A1 A3 showed a significant decrease in tumor size by osimert inib treatment (A1> A2> A3).

The PPI complex (represented by PPI count in Figure 23c) between each of the EGFR, HER2, HER3 and MET receptors and the lower signal proteins PLCgammaSH2, Grb2 and p85-alpha was measured in each egg TX (PDTX-A1 to A3) , And the left side of Fig. 23C. As shown in FIG. 23C, the PPI complex counts between EGFR and the three sub-signal proteins were in the order of A1> A2> A3. This is due to the effect of the EGFR inhibitor osimert inib on tumor size reduction The same can be confirmed. These results show that the PPI complex count between the target protein and the lower signal protein in the PDTX model of lung cancer carcinoma shows a significant correlation with the anticancer effect of the target therapeutic agent targeting the target protein.

Expression levels of EGFR and other receptors (MET, HER2 and HER3) in 8 PDTX (A1-A3 and S1-S5) individuals were also measured and the expression levels of the respective receptors were measured in a control (EGFR: A549 cells , MET: HCC827-GR5, HER2 and HER3: SKBR3), and the results are shown in FIGS. 23d and 25a_c.

As shown in FIG. 23C, all of the tested egg TX models (A1 to A3) were MET,

Did not show significant PPI complex levels for HER2 and HER3 receptors, but showed a somewhat significant PPI complex level for EGFR. These results suggest that PDTXs inhibit EGFR This means that the oncogene addiction is expressed on the signaling.

Next, we investigated whether the reactivity of egg TX to osimertinib treatment can be predicted, as evidenced by the lung cancer cell line, by normalized PPI count (PPI complex number per unit concentration of the first protein; corresponding to the activation score). The tumor growth inhibition (%) in each model obtained by treating the PDTX model (A1 A3) for 30 days with osmate inib (5 mg per kg of weight daily) EGFR level (PPI sum: PPI) was calculated by subtracting the tumor volume before and after the vehicle treatment and the tumor volume before and after treatment with Mgefitinib and gefit inib. score, and PPI sum / EGFR level: Activation score) is shown on the x-axis in FIG. 23E. As shown in FIG. 23E, it can be confirmed that the normalized PPI count (PPI sum / EGFR level: Activation score) actually shows a high correlation (r = 1) with tumor growth inhibition by osimert inib.

10.2. lung SQCCClung squamous cell carcinoma) Reactivity prediction for Osimert inib in xenotransplantation PDTX

While 29% of lung cancer is associated with sensitization mutations of EGFR, only 0.5% of lung SQCC have EGFR sensitization mutations. Therefore, at present, there is no appropriate biomarker for EGFR target treatment in lung SQCC.

In this example, five suppressed TX (PDTX_S1 S5) were constructed from lung SQCC patient tissues and monolayer immuno-labeling and co-IP profiling (Example 9.5) were performed. The five PDTX (PDTX-S1 to S5) mammary gland MET, HER2 and HER3 receptor proteins and their associated PPI complexes were found to exhibit minimal levels (Figure 23c, right and Figures 25a-c).

On the other hand, it was confirmed that the total EGFR count (EGFR level) and the EGFR PPI complex count were detected at a considerable level (FIGS. 23C and 23D, and FIGS. 25D and 25E). These results are expected to be due to the fact that lung SQCC is often dependent on EGFR in the proliferative signal.

10.3. lung SQCC (lung squamous cell cm "cinoma) reactivity prediction for gefitinib in xenotransplantation PDTX

Five suppressive TXs (PDTX-S1-S5) were treated with gefitinib for 15 days, The degree of tumor growth was measured and shown on the right side of FIG. 23B. PDTX-S1-S5 showed significant tumor suppression effects in S1 and S2. Of the various PDTX individuals tested, it was once again confirmed that the PPI complex count (activation score) normalized to EGFR level had a very high correlation (Spearman correlation of 0.9) with tumor growth inhibition (Figure 23 f and Figure 25f- h).

The results obtained show that the normalized PPI count is closely related to the response of non-small cell lung cancer to EGFR target inhibitors. Two lung SQCC PDTX (PDTX-S1 and S2) with intrinsic signaling phenotype and gefitinib reactivity were studied intensively to understand whether normalized PPI values, rather than the sum of PPI values, were more relevant to response to anti-tumor drugs Respectively.

The PPI complex counts in PDTX-S1 and PDTX-S2 were measured before and 15 days after gefitinib treatment and are shown in Figures 23g and 26a_b.

As shown in the above results, PDTX-S1 maintained a detectable level of EGFR PPI complex counts, especially in the regulatory p85 a subunit. PDTX-S2, on the other hand, exhibited reduced or indistinguishable levels of EGFR PPI complex counts compared to negative control with A549 cells (Figure 23g and Figure 26). Thus, the gefitinib dose (50 mg / kg) used in the study

In PDTX-S2, the hyperactive but smaller pool of EGFRs is completely inhibited, leading to the shrinkage of the cancer. On the other hand, the same gefitinib dose did not inhibit EGFR activity in PDTX-S1 and showed significant EGFR overexpression. These results suggest that TX-S2 with high normalized PPI cmint exhibits better reactivity to gefitinib than suppress TX-S1 with high EGFR level and total PPI count.

Based on the above results that PDTX-S1 exhibits increased EGFR-p85a binding, PDTX-S1 was treated with PI3K inhibitor BKM120 (50 mg / kg) (Figure 23h).

As shown in the above results, BKM120 showed strong tumor growth inhibitory effect at the same dose (50 mg / kg) than gefitinib. In addition, combination therapy of gefitinib and BKM120 (gefitinib (50 mpk (mg per lkg weight (mpk)) / BKM120 (50 mpk) atrophied the tumor. These results suggest that single-molecule co-IP profiling of PPI using different downstream proteins can be useful for selecting target signaling pathways and designing a combined treatment strategy for multiple drugs.

10.4. Prediction of reactivity in non-small cell lung cancer xenotransplantation PDTX

Normalized PPI complex counts from PDTX-A1-A3 and PDTX-S1 S5, two non-small cell lung cancer transplantation models, were pooled and single plots compared (FIG. 23I).

Despite all differences in the genetic and cancer subtypes, the data points displayed a consistent pattern and formed a shape that closely matched the tumor growth inhibition with a Spearman correlation of 0.95.

These results suggest that the normal ized PPI complex count is a measure of the EGFR signal intensity that can serve as an efficacy predictor for EGFR-targeted therapies.

Example 11. Single-molecule immunolabeling and co-IP profiling for human patient samples (co-IP profiling)

Tumor tissues of human patients were characterized using the micro chamber and high-throughput single-molecule imaging system of the present invention (Fig. 24).

The protocol (cryogenic lysi s protocol) was applied to two lung cancer patient tissues (Yonsei University Severance Hospital) obtained by surgical resection from a lung cancer patient (Fig. 24A, P1 and P2).

Briefly, the prepared patient tissue is pulverized (~0.6 cm) and immersed in liquid nitrogen. After further pulverization, PBS was added to dissolve completely, centrifuged, and pellets were taken. PBS was then added and incubated at 4 ° C with continued mixing. Then, the supernatant was taken by centrifugation. Using tissues of 15 mm ³ (P1) and 18 mm ³ (P2) in size, we obtained 10 different PPI levels for each tissue lysate obtained by the method described above and 1.0 for the PPI complex When P1 and

(Relative value of the PPI complex at P2) and the level of 1 < 0 > different proteins and PTM (expression levels) (see immunolabeling of Example 9.5) (Fig. As a positive control, PC9 cells (for EGFR), HCC827 cells (for MET), and SKBR3 cells (for HER2 and HER3).

Both tumor tissues and P2) had an exonl9 mutation (exonl9 deletion mutation) in the EGFR gene, but only the sample showed significant EGFR PPI complex counts (Figure 24b, c and Table 4).

[Table 4]

Biopsy type Deposite date EGFR genotype Treatment Response

(PR = partial response, PD = progressive disease)

For the other receptors HER and MET receptors, meaningful PPI counts were not observed in both samples (Fig. 24b, c).

Patient P1 had a partial response to gefitinib treatment for approximately one and a half years prior to the diagnosis of progressive disease (PD), a partial response (PR) to the response evaluation criteria in solid tumors (RECIST) While Patient P2 maintained a stable disease (SD) diagnosis for one year before PD designation.

Finally, in vitro dephosphorylation was performed by treating PTPNl against PDTX-A1 showing the highest active EGFR signal and mutant EGFR (exon 19 deletion) derived from human patient sample Pl (Fig. 24d).

After dephosphorylation, the binding of eGFP-labeled PLCgamma and Grb2 to the mutant EGFR complex was measured and is shown in Figure 24d. As shown in Figure 24d, the binding of PLCga_as _H 2, which is entirely dependent on the pTyr-SH2 domain interaction, is almost completely stopped by dephosphorylation (+ PTPN1), while in the case of Grb2 binding ccxints, % Were retained after dephosphorylation. These results suggest that the pTyr-independent signaling median ism of the mutant EGFR actually works in extracorporeal tumor tissue of the PDTX model.

Example 12 Preparation of Immune Proteins PD-1 and PD-L1

Sup-1 is labeled with GFP and labeled with GFP labeled egg-1, while PD-L1 is labeled with mcherry Labeled and prepared in the form of mcherry labeled PD-L1.

Egg-1-GFP has a protein sequence from 1 to 288 including the unique signal sequence of human egg-1 (NP_005009.2) and GFP linked to the carboxy terminal (C-teminal) of the amino acid sequence (Amino acid sequence: MVSKGEELFTGWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQ

CFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGH KLEYNYNSHNVYIMADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSK consists DPNEKRDHMVLLEFVTMGITLGMDELYK), the two proteins were prepared to be connected by a peptide linker consisting of 19 amino acid (N terminal side: PD-1, C terminal side: GFP).

The amino acid sequence of the linker used between PD-1-GFP and the nucleic acid sequence encoding it is as follows:

Nucleic acid sequence:

5 ¹ -AGAATTCTGCAGTCGACGGTACCGCGGGCCCGGGATCCACCGGTCGGATCTGGTAGT-3 '; Amino acid sequence:

N'-RILQSTVPRARDPPVGSGS-C.

However, the length and sequence of the linker are merely illustrative.

If the length of the linker is shortened to a certain number, for example, less than 8 (based on the number of amino acids), or consists of a rigid and / or bulky amino acid, the egg- Clashes between the GFPs at the back (C-terminal) and the backside (C-terminal) may cause misfolding of the protein or may easily result in degradation, which may affect the PPI immediate effect. The linker may include, but is not limited to, one or more G (glycine) and / or one or more S (serine). Glycine and serine are relatively flexible compared to other amino acids, which can be advantageous for protein folding and PPI measurement.

PD-Ll-mcherry has a protein sequence from 1 to 290 including a signal sequence unique to human PD-L1 (NP_054862.1) and a mcherry terminal linked to the carboxy terminal (C-terminal) of the protein sequence (Amino acid sequence: MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAffDILSPQF

Lt; / RTI & (N-terminal side: PD-L1, C-terminal side: mcherry).

The amino acid sequence of the linker used between PD-L1-mcherry and the nucleic acid sequence encoding it is as follows:

Nucleic acid sequence:

5 '-TACCCAACTTTCTTGTACAAAGTGGTTGATCCGGTACCGGTCGCCACC-3';

Amino acid sequence:

N'-YPTFLYKWDPVPVAT-C.

However, the length and sequence of the linker are merely illustrative.

The linker used is a structure consisting of 16 amino acids and connects the N-terminal PD-L1 and the C terminal mcherry. If the length of the linker is shortened to a certain number, e. G., Less than 8 amino acids by amino acid number, or if it consists of rigid and / or bulky amino acids, Clashes occur between GFP1 and the GFP at the back (C-terminal portion), which can result in mi-sfolding of proteins or degradation, which can affect PPI measurement. The linker may include, but is not limited to, one or more G (glycine) and / or one or more S (serine). Glycine and serine are relatively lexible compared to other amino acids, which can be advantageous for protein folding and PPI measurement. In addition, when the linker becomes too short, the binding with the PD_L1 antibody using the C-terminus as epi tope (for example, 9A11 (Cell Signaling Technology, # 29122S)) is seriously impaired and pul 1-down The efficiency may drop significantly, which may affect the PPI measurement.

However, the label (mcherry in this embodiment) bound to the PD-L1 immobilized on the substrate (i.e., used as the first protein) is not indispensable for the PPI measurement and can be suppressed without attaching mcherry to the PD- -L1 interactions can be measured.

On the other hand, when a label (for example, mcherry) is attached to PD-L1 used as the first protein, PD-L1 attached to the substrate can be quantified. In this case, fluorescence quantification using a spectrophotometer is possible, so that the desired concentration of PD-L1 can be coated on the substrate, and conversely, the number of PD-L1 coated on the substrate under the corresponding conditions can be quantitatively tracked.

The advantage of quantitative tracking of the number of PD-L1 is as follows have:

Tracking the number of PD-L1 coatings on the substrate can aid in the negativity of the inhibi tor when performing drug screening. 1) -1 _{* (*} indicates fluorescence labeling) / PD-L1 / inhibi tor mixture is incubated with Tube reactant and the PD-1 _* counts to inhibit the interaction of PEKL and PD-L1 , But drugs that sometimes interfere with the binding of PD-L1 and PD-L1 antibodies may also be misdiagnosed. At this time, the number of PD-L1 bound to the substrate can be observed to select the wrongly selected drug.

On the other hand, negated ive select ion is not impossible even when using unlabeled PD-L1. PD-L1 ELISA. However, if you use a labeled PD-L1 (labeled mcherry, for example), you can proceed with the process one-step.

Egg-1-GFP and soybean oil encoding egg Ll-mcherry were inserted into pCDNA 3.1 backbone, respectively, and introduced into HEK293T cells, which were expressed as proteins.

For introduction of a gene into a host cell, any means generally used can be used such as transfection using PEI (polyethyleneimine), 1 ipofect ion using 1 ipofectamine, electroporat ion, and the like.

In the present embodiment, transfect ion was exemplarily performed using PEI. PEI is a highly positively charged substance that penetrates DNA into the cell and expresses it as a protein. The specific procedure of transfect ion using PEI is as follows:

500,000 HEK293T cells (ATCC [293T (ATCC® CRL-3216 ™)] were prepared on the basis of 100 mm di sh. 2 ml of Opti -MEM buf fer (Thermo Fi sher Scientif ic) was prepared, 1 ml of DNA (vector containing the previously prepared egg-1-GFP and PDLl-mcherry gene) and 1 ml of the remaining 50 ml of PEI . After 5 minutes, the DNA and PEI were mixed together and incubated for an additional 20 minutes. The DNA and PEI mixture was treated with the prepared HEK293T cells, cultured for 48 hours, and then the cells were collected.

The composition of the lysis buffer (Lysi s buf fer) used in the cell lysis procedure is as follows.

[Table 5]

Re-suspension was added to dissolve buffer to the amount of cells collected, incubated in ice for 30 min, and then centrifuged at 14000 g for 15 min. At this time, protease inhibitor (Sigma-Aldrich (11836170001)) and phosphatase inhibitor (Sigma-Aldrich (P5726)) were added. The supernatant was taken and the fluorescence intensity was measured using a Fluoremeter and stored.

Example 13. Preparation of substrate

Covers lip was immersed in KOH 1M solution and washed with a sonicator (20-30 min). Then, after washing well with deionized water, piranha solution (sulfuric acid: aqueous hydrogen peroxide = 2: 1 3: 1 ( \ "v)). Was washed with a aminopropylsi against the covers 1 ip surface was washed lane and PEG (polyethylene glycol) were sequentially treated to perform coating.

After 2 hours of reaction, the cells were washed with 3 rd distilled water and stored at -20 ° C until used without contact with the PEG-coated surface.

In the following test, the prepared cover lip and acrylic substrate were used. Before the test, the coverslip and the acrylic substrate were thawed or assembled. After the PEG coating was completed, the assembly was pre-assembled and stored at -20 ° C And thawed before use _.

Example 14. Protein-protein interaction measurement between PD-L1 and PD-1

Induction of protein-protein interaction between PD-L1 and PD-1

The substrate prepared in Example 13 was added with the Avidin series protein Neutravidin (Thermo, A2666) was added at a concentration of 0.1 mg / ml. After 5 minutes of reaction at room temperature, the substrate was washed twice with 30 μl of PBS buf fer. To illustrate the use of PD-L1 or egg-1 as the first protein, the prepared substrate was treated with the antibody against the first protein. At this time, the antibody used was prepared as a biotin-conjugated form. The concentration of the antibody can be appropriately adjusted according to the affinity of the antibody-antigen (KD). In this experiment, the antibody concentration was about 2 μg / ml, and the reaction time was about 5 minutes. If a biotin-conjugated antibody is used, the antibody of the first protein can be attached using a secondary antibody.

Antibodies to the first protein used in this example are summarized in Table 6 below:

[Table 6]

The substrate treated with the anti-PD-L1 antibody (9A11 or 8T4X) shown in Table 6 was washed twice with PBS buf fer 30. PD-L1 protein solubilization solution (supernatant after centrifugation of mCherry-PD-L1 expressing cell lysis solution; used as the first protein) prepared in Example 12 was added to the prepared substrate, and PD-L1 was immobilized on the substrate . In the case of antigen-antibody reaction, it is possible to continue to increase until 15 minutes, and the efficiency of antigen-antibody reaction may be lowered over 15 minutes and the reaction time is set to about 15 minutes. After the reaction, the substrate was washed with PBS containing 0.05% (v / v) of tween 20. Tween 20 0.05% reduces nonspecic binding and helps prevent the hydrophobic region of the membrane protein from breaking down.

Thereafter, on the substrate on which the PD-L1 protein was immobilized on the obtained surface,

PD-1 protein solution (supernatant after centrifugation of GFP-PD-1 expressing cell lysate; used as the second protein) obtained in Example 12 was added. The PD-L1 protein contained 10 nM And egg-1 protein was used at a concentration of 10 nM, 30 nM, or 50 nM. The test procedure is schematically shown in Fig. 27A.

Imaging of the complex (PPI complex) by protein protein interaction

The PPI complex imaging process is as follows:

The prepared substrate (either a well type or a quartz slide can be used) was fixed on a total internal reflection fluorescence (TIRF) microscope. In order to observe the fluorescence signal of eGFP, a wavelength of 488 nm was used. However, the present invention is not limited thereto, and any wavelength laser capable of emitting eGFP can be used (laser wavelength range: 460 to 500 nm). Fluorescence images were obtained using an EMCCD (Electron-multiplying charge-coupled device; Andor iXon Ultra 897 EX2 (DU-897U-CS0-EXF)) camera.

Illustratively, various values of the EMCCD camera for imaging were adjusted as follows:

a. Laser power: 2 mW

If the laser power is too high, eGFP will lose the fluorescence signal quickly (photob leaching), and if it is too low, it will be difficult to observe the signal by the EMCCD. The laser power range is a range that maintains the light-emitting time (lifetime) of _e GFP about 11 seconds.

b. Exposure time per frame of EMCCD camera: 0.1 second

The shorter the exposure time per frame is, the fewer signals accumulated in one frame. To overcome this, the laser power must be increased or the sensitivity of EMCCD must be increased.

c. The gain of the EMCCD (the larger the gain, the more the measured signal is amplified and the higher the noise): 40

The photons generated in eGFP are converted to electrons through the EMCCD element (photoelectric effect, photoelectric effect). At this time, the number of electrons generated per photon can be changed through the gain value. The higher the set gain, the greater the number of electrons generated per photon, and the higher the sensitivity of the EMCCD. At the same time, background noise also increases together, so the signal-to-noise ratio is important. Therefore, although the gain of the EMC suppression is adjusted to 40 in the present embodiment, the present invention is not limited to this, and can be appropriately changed based on the foregoing description. d. Total shooting frame: 10 frames

If you take a 10 frame shot, you can observe the PPI change for 1 second based on the current exposure time. In this embodiment, the intensity of the signal observed in the imaging is extremely stable since the imaging is performed in succession of the incubation for 30 minutes, the incubation in the substrate for 15 minutes, and the washing step. Therefore, to shorten the experiment time, it is fixed to 10 frames, and the analysis mainly proceeds with the frame (5-7). However, a similar value can be obtained even if the initial frame 1-3 and the second half frame 8-10 of 10 frames are measured.

A minimum of 10 images were obtained at different positions, each position was shifted for one condition (one wel l or one channel), and the 10 images obtained were analyzed. The mean and the standard deviation were compared with the representative values of each condition Respectively. Chapter 10 is the minimum standard, and the higher the number, the higher the statistical accuracy. Once all the images have been acquired under one condition, move to the next one (next wel or next channel) and repeat the same operation.

As described above, when the PD-L1 is fixed to the substrate, the PPI signal can form a cluster. In this case, if the laser power is reduced or the gain number is reduced, the fluorescence intensity of the spot is reduced, the single GFP spot is almost invisible, only the cluster signal can be left, only the cluster is analyzed, , Or if you want to analyze the correlation, you can proceed with the step further.

PPI complex analysis

The imaged protein complex (PPI complex) was analyzed by the following procedure:

In this example, the protein complexes were analyzed based on the toolkits provided in the Matlab program (provided by MathWorks).

1. The fluorescence images obtained were stored in a 16-bit unsigned integer format. In PD-1-PDL1 analysis, the following procedure was performed using a total of three frames of 5-7 frames. As described above, since the washing process is included, the intensity and stability of the signal seen in the imaging are excellent. As a result of analyzing 1-3 frames of 10 frames and 8-10 frames, There was no significant difference in the results of the analysis of all the frames. this The interval may vary depending on the imaging condition / equipment construction status.

2. To eliminate noise, the following procedure was performed for each frame:

a. The initial start begins at the top left corner. One frame consists of 512x512 pixels. 11 pixels to the right based on the base pixel, down

The median value was obtained from 11 11x11 individual pixels, and this median value was subtracted from the reference pixel value [(Intensi_pixel) -

(median anintensi ty_llxllneighborhood)]. The above procedure was performed for all pixels of 512x512. The mediating effect of pepper & sal noi was eliminated.

b. In the above processed image, Gaussian smoothing of sigma ^ O.?, Si ze = 5 x 5 was performed. This helped to soften the image. c. Threshold was set. Threshold is the threshold value of the pixel that the pixel intensities become below the threshold in the whole image. This can remove local maximums not created by the fluorescence signal in the image. In this embodiment, the threshold value used in the imaging condition is 70.

3. The signal from the fluorescent protein occurs in a specific location (localized spread function (PSF)). That is, this PSF (physical value) is the PPI complex (biological value) to be measured. The following steps are for determining this PSF:

a. The local maximum is obtained (for example, i-th row, j-th column pixel). As described above, since the monomolecular fluorescence signal is formed at a specific position among 512x512 pixels (approximately 5x5 pixel size, 1 pixel = 0.167 micrometer under the present observation equipment), when the local maximum is found,

PSF can be selected. This can be obtained using the toolkit provided in the mat lab.

b. The process of determining whether the local maximum obtained from the actual PSF is performed is performed. First, the minimum value of the local maximum is defined (minimum intensities), and only the maximum of the obtained local maximums is used for the analysis. In this embodiment, the minimum value is defined as 75, which may vary depending on the laser power / exposure time / equipment construction situation. 5x5 pixel centered on the finally obtained local maximum coordinate The information was retrieved, and the centroid of intensities was obtained for this 5x5 pixel. At this time, if the brightness center deviates by more than 0.5 pixels from the existing local maximum coordinates (when the 2D symmetry about the PSF shape disappears), it is judged to be a non-normal fluorescence signal and removed from the analysis.

c. Only the PSF that passed all the conditions were finally classified and the coordinates and the total number were obtained.

The number of PPI complexes was determined for all the files by performing steps 1-3 above. The information of each file taken under the same conditions was collected and the mean and standard deviation were obtained. This value finally represents the number of PPI complexes under certain conditions.

The number of the obtained PPI complexes can be obtained by obtaining the count of the PPI signal or by calculating the sum of the intensities of the PPI signals or by measuring the rat io of the spot in the measurement region.

In one example, when only the signal of the cluster is measured, the laser power and the gain number are adjusted to reduce the size of the cluster signal, that is, the spot where the intensity of the fluorescence is too low, (The local maximum may be reduced or overlapped spots may be distinguished). By measuring cluster intensities, we will be able to more closely characterize the drug. In some drugs, there are drugs that inhibit PD-1 and PD-1 clustering. In the case of the drug, the number of clusters will be reduced, although the number of single spot counts may be rather increased or similar, and effectively inhibiting the PD-1-PDL1 interaction in the body with cluster ing. Therefore, it would be meaningful to analyze the distribution and number of c luster.

Also, by subtracting the cluster intensities from the total intensities, intensities of only single molecules could be derived.

When accessing a specific cluster, it can be analyzed as count or sum of signals with cluster intensities above a certain threshold, or cluster rat io can be analyzed. The same is true for single molecule identification.

Analysis of fluorescence signal value of PD-1 _* versus fluorescence signal of PD-L1 _*

1) After supplying m -erry-attached PD-L1 to the substrate, the fluorescence signal a formed at a certain threshold or higher among the signals formed by the m- .

2) Detection of a fluorescent signal b formed at a certain threshold or higher among the signals formed by GFP after supplying suppression 1 with GFP attached thereto.

3) Analyze the fluorescence signal b value for the fluorescence signal a value. The fluorescence signal value may be PPI count, sum, rat io, and the like.

By analyzing the fluorescence signal b value for the fluorescence signal a value, more qualitative data can be confirmed than when analyzing only the fluorescence signal b value. The measurement of the fluorescence signal a value can also be taken into account for the influence of PDL1 *, which is injected onto the substrate but not attached to the substrate, at a specific concentration of mcherry-PDL1. In addition, the accuracy can be improved by quantifying the interaction of PD-L1 and PD-1 *.

Comparative Example

For comparison, the PD-1 mutant containing the PD-1 mutation K78A, which inhibits the binding of the suppression-1 and PD-L1, or the PD-L1 mutant D122A + The same test as above was carried out using an egg-1 mutant containing the PD-L1 mutant containing K124A, or A132L, a PD-1 mutation that enhances the binding of egg-1 to PD-L1.

result

A fluorescence image obtained by using the anti-PD-L1 antibody 9A11 (# CST-29122; Cell Signaling Technology) is shown in FIG. 27B, and the result of quantitation (PPI complex number: F-counts) Respectively. As shown in FIGS. 27B and 27C, when PD-1 and PD-L1 are both wild type (WT), PD-1 binds normally to PD-L1 and GFP signal is observed in a concentration- On the other hand, when the mutant egg-1 K78A mutant or PD-L1 D122A + K124A mutant inhibiting the binding of PD-1 and PD-L1 was used, no GFP signal could be observed regardless of the concentration of PD-1 , And the PD-1 A132L mutant, which enhances the binding strength between Sup-1 and egg-Ll, was able to observe more signals than WT. The above results confirm that the protein-protein interaction (PD-L1 / PD-1 interaction) between egg-1 and PD-L1 can be accurately measured by the method performed in this example _.

In the PD-L1 / PD-1 interaction measurement, when the anti-PD-L1 antibody was immobilized on the substrate, spot visible as a duster in the fluorescence signal was observed. On the contrary, PD-L1 was used as the second protein, the anti-PD? 1 antibody was immobilized on the substrate, As a result of the test, no images of cluster electrons were observed, and a single GFP spot image was observed.

Therefore, PD-L1 antibody was immobilized on a substrate using PD-L1 antibody in the physiologic conformation of PD-1 / PD-L1. It was predicted that it would be more advantageous to approach the analysis.

It is not excluded to measure the PPI by immobilizing PD-1 by treating the substrate with the anti-PD-1 antibody. In such a case, there is no difference in the above-mentioned PPI measurement method except that the protein is recombined with egg-1-mcherry and suppressed L1-GFP. However, since there is a difference in signal generation due to cluster formation, the analysis method can be appropriately changed accordingly.

When the PD-L1 is immobilized on the substrate, the antibody used for immobilizing the PD-L1 on the substrate may be appropriately selected so as to expose a specific domain of PD-L1 (binding site with egg-1) have.

Of the anti-PD-L1 antibodies exemplified in Table 6, 9A11 (# CST-29122; Cel l

Signaling technology is an antibody that binds to the C_ terminus of PD-L1, # D8T4X (Cell Signaling Technology) is an antibody that binds to the N_ terminus of PD-L1, and D4W2J (Cell Signaling Technology) is an antibody that binds to the C-terminal of egg-1, and NAT105 (Abeam) is an antibody that binds to the N-terminal of egg-1. FIG. 29 shows the number of PPI complexes (F-counts) obtained by fixing the anti-PD-L1 antibody on the substrate and performing the above test. In FIG. 29, when tested with the anti-PD-L1 antibody, the concentration of PD-L1 was fixed at 5 nM and the PD-1 concentration was tested with 0 nM, 5 nM, -1 antibody, the concentration of PD-1 was fixed at 5 nM and the PD-L1 concentration was tested while increasing to 0 nM, 5 nM, and 15 nM.

29, when # D8T4X (Cell Signaling Technology), which is an antibody that binds to the N-terminal of PD-L1, is used, 9A11 (CST # 29122; 1 / PD-L1 interaction is less counted under the condition, compared to the case of using an in-vitro technology.

The above results indicate that fixing the C-terminal region of PD-L1 toward the substrate is difficult because the interface between the egg-1 and the PD-L1 interacts with the N-terminal region, It is advantageous to induce the action. Example 15. Application to PD-1 / PD-L1 interaction inhibition drug screening

Neutravidin (Thermo, A2666), an Avidin series protein, was added to the substrate prepared in Example 13 at a concentration of 0.1 mg / ml. After 5 minutes of reaction at room temperature, the substrate was washed twice with PBS buffer 30. The prepared substrate was treated with the anti-PD-L1 antibody 9A11 (CST # 29122) confirmed to have excellent effect in Example 14 and FIG. 29 in the amount of about 2 ug / ml for about 5 minutes, A substrate coated with anti-PD-L1 antibody (9A11) was prepared. The prepared substrate was washed twice with 30 ul of PBS buf fer and used for further testing.

The GFP-PD-1 (ONM) and mcherry-egg-L1 (30 nM) (PD-

L1 in the absence of mcherry) was reacted with PD-1 / PD-L1 interaction inhibitor candidate material in a tube for 30 minutes, and the reaction was applied to the prepared substrate in an amount of 20 ul, followed by reaction for 15 minutes .

PD-L1, and PD-MGFP labeled PD-1), and the reason why the candidate drug was reacted in the tube (one-step reacted) same:

Inhibitors inhibit the interaction of PD-L1 / PD-1 _* in three ways: 1) when the inhibitor binds to the binding interface of PD-L1 and PD-1 with strong intensity; 2) inhibitors (PD-1 independent ly) bind to specific sites of PD-L1 to induce structural changes of PD-L1; And 3) inhibitors (PD-

L1 independent ly) when it binds to a specific site of PD-1 and induces a structural change of PD-1.

If this test is carried out by a stepwise reaction ion (PD-L1, inhibi tor, PD_1) which is not one-step react ion [ ⁰ ], the above step 1) can be effectively screened On the other hand, in cases 2) and 3), since the coli lion itself of the drug and the target may not be induced, a missed populat ion is generated, so that there is a limitation in testing the correct inhibitory effect. Also, since one-step reaction is efficient in terms of time, the one-step reaction method in the tube (Standard 1.5 ml ep-tube) is adopted in this embodiment.

The subsequent test procedure was the same as the imaging of the PPI complex of Example 16 (488 nm) and the PPI complex analysis.

28A schematically shows the inhibitor candidate drug screening process of the present embodiment Show.

FIG. 28B shows the results of a test for optimizing the reaction conditions of one-step reaction in a tube. The GFP-egg-1 and mcherry-PD-Ll prepared in Example 12 were mixed in a tube for 30 minutes or 2 Incubation time, and fluorescence counts (F-counts). In FIG. 28B, 'Negat ive' indicates a reaction product in which mch-PDLl was not added to the tube,

'Negat ived means reactants without GFP-PD1 added to the tube, respectively. As can be seen in FIG. 28 (b), the number of PPI complexes did not show a significant difference between the incubation time of 30 minutes and the incubation time of 2 hours.

The number of PPI complexes obtained using two commercially available PD-1 / PD-L1 inhibitors BMS202 and S7911 (purchased from Sel leck Chem) as candidate agents for egg-1 / PD-L1 interaction inhibition ) Is shown in Fig. In Fig. 28C, it can be seen that the PD-1 / PD-L1 complex is reduced in dependence on the inhibitor treatment concentration. Also, in the case of BMS202, it was confirmed that the IC50 value (18 nM) similar to the IC50 value calculated using the purified protein was shown. The results show that the PD-1 / PD-L1 inhibitor can be accurately determined by the PD-1 / PD-L1 interaction assay performed in this example.

Eighty compounds sampled in the non-macrocyclic PPI drug librium provided by PPI libris screening (http: / / vw.asinex.com/ppi/) as candidates for PD-1 / PD- The number of PPI complexes (F-counts) obtained by the use of the amount of each of the PPI complexes is shown in FIG. In the results of FIG. 28 (d), four drugs with PD-1 / PD-L1 complex number less than 150 (for example, less than 100) were selected (plate numbers: B1, B2, B3, and E2; 33). DMSO was used instead of the drug and used as a negative control. BMS202 was used as a positive control. The results are shown in FIG. 28E (the four scales displayed on each drug are lmM, 10 uM (in order from left), 10 uM , 100 nM, and InM. P indicates the result of confirming the normal PPI level by treating DMSO instead of the drug, and shows the result when the mch-PDLl is not coated. As shown in FIG. 28 (e), the drug A1 used as the reference drug showed no effect at all, while the four selected drugs (Bl, B2, B3, and E2) Dependent inhibition of PD-1 / PD-L1 interaction. In particular, B2 showed the best efficacy, which was found to be equal to or better than the efficacy of BMS202, a commercially available drug.

Example 16 Comparison between PD-1 and PD-1 immobilized on a substrate

Protein-protein interaction between PD-L1 and PD-1 when GFP PD-L1 was immobilized on a substrate and GFP-PD-1 was immobilized on the substrate (fluorescence complex number : F counts) were measured.

Total GFP-labeled counts and fluorescence images are shown in FIG. 30A (PSF cut of f> 150). As shown in Fig. 30 (a), it can be confirmed that GFP PD-L1 is fixed on the substrate and GFP-suppress-1 is fixed.

The cluster rat io (%) of GFP PD-L1 and GFP PD-1 (calculated by [Count @ PSF cut of f> 350 / Count @ PSF cut of f> 150]) is shown in FIG. The cluster rat io% is a value obtained by dividing a cluster population defined as a point spread function (PSF) cutoff of 350 or more by a total count of a cutoff of 150 or more as a percentage. As shown in Fig. 30B, it can be seen that the cluster rat io increased by 40% or more as compared with the condition in which GFP-O-1 was fixed under the condition that GFP PD-L1 was immobilized on the substrate.

Example 17. Epitope mapping of anti-PD-L1 antibodies

For epitope mapping of anti-PD-L1 antibodies suitable for immobilizing PD-L1 on a substrate, a PD-L1 mutant construct was designed (see FIG. 31A). Specifically, the PD-L1 cytosol ic domain deletion mutant construct was designed to identify the epi tope of the PD-L1 cytosol ic domain targeting antibody. The mutant was designed as a PD-L1 amino acid sequence (NP_001300958.1) The cytosol ic part corresponding to amino acids 260-290 was divided into 10 units to produce a protein containing a mutation of d280 (amino acid deletion after 281) or d270 (deletion of amino acid deletion after the 2nd) .

PD of the PD-L1 ant ibody (9A11) shown to be effective for measuring the PD-L1 / PD-1 interaction (see FIG. 29) previously, through the PD-L1 pul 1-down assay, The degree of binding (number of fluorescent complexes due to binding between antibody and PD-L1: F-counts) to the L1-WT (wild type), d280, or d270 mutant was measured and shown in Fig. 31B, In the case of the d280 or d270 variant of PD-L1, it does not bind to the antibody at all, while in the case of the antibody it binds sensitively. It is predicted that the sequences corresponding to amino acids 280-290 of PD-L1 will play an important role in the reaction with antibodies used to immobilize PD-L1 on the substrate in the suppression-L1 / egg-1 interaction assay have.

Furthermore, in order to test the effect of the d280 or d270 mutation of PD-L1 on the interaction with PD-1, an immunoprecipitation experiment based on the PD-1 / PD-L1 interaction (see Example 14) The degree of protein-protein interaction between PD-1 (immobilized on substrate with anti-PD-1 antibody) and PD-Ll WT, d280, or d270 mutant was measured and is shown in FIG. As shown in FIG. 31C, the d270 mutant and the d280 mutant of PD-L1 showed a binding strength to PD-1 at a level of about 70% of 1T.

Example 18: Formation of HER2-HER3 heterodimer

HER3_eGFP recombinant gene in which eGFP is connected to the intracellular domain termini of HER3 (R4 R3: NM_001982.3 BC082992.1, eGFP: MH087225 linker:

PEGFP-Nl-FLAG) is injected into the HER2-ove rexpressing SKBR3 breast cancer cell line (Korean Cell Line Bank or ATCC) through electroporation to induce overexpression of HER3. 30 ug of HER3_eGFP pi asm ids were transfected in 6X1CT6 SKBR3 cells using electroporat ion (electroporation condition:

950V, 35ms, 2 pulses). (RPMI1640, FBS 10%) in 90 mm cul ture di sh to allow SKBR3 to express HER3_eGFP. Existing medium was removed and cultured for 12-18 hours with FBS-free medium (RPMI1640) to remove the influence of unspecified growth factors.

NRGbl (lOOng / ml) was added to the prepared SKBR3 and incubated for 10 minutes to induce formation of a heterodimer of HER2 protein and HER3_eGFP protein on the cell surface (see Fig. 34A).

The level of HER2-HER3_eGFP heterodimer production according to the expression level of HER3-eGFP was determined by a single-molecule Total Internal Reflection Fluorescence Microscope, and the result is shown in FIG. 34B. HER2-HER3 heterodimer was not produced when NRGbl was not treated after introduction of HER3-eGFP plasmid into SKBR3 as shown in FIG. 34B, whereas HER2-HER3 heterodimer was not produced when HER2-HER3 heterodimer was treated with NRGbl And increased in proportion to the expression level. The results show that NRGbl should be injected to generate the HER2-HER3 heterodimer.

In addition, the level of HER2-HER3 heterodimer production when NRGbl was treated before and after cell lysis was analyzed by total internal fluorescence microscopy

Mi croscope). The results are shown in Fig. 34C. Cell lysis was carried out in lysis buffer (1% Digi tonin or 1% GDNO, 10% glycerol, 50 mM HEPES-NaOH [pH 7.4], 150 mM NaCl, 2% protease inhibitor cocktail

P8340)% concentrations were all performed using w0. As shown in FIG. 34C, when NRGbl was treated after cell lysis (i.e., treatment of NRGbl with cell lysate; Extract + NRGbl), the HER2-HER3 heterodimer was produced to a similar extent as that in the case where NRGbl was not treated On the contrary, when Gbl was treated before cell lysis, the level of HER2-HER3 heterodimer production was remarkably high. These results suggest that administration of rnGbl prior to cell lysis can produce a HER2-HER3 heterodimer, and that administration of NRGbl concurrently with or after cell lysis does not produce a significant level of HER2-HER3 heterodimer.

After the cell lysis, the cell lysate was treated on the substrate to attach the HER2-HER3 heterodimer to the substrate and washed with detergent. DGTN (digi tonin) 0.1% (w / v), GDN 0.01% (w / v), CHAPS0 (3- (3-Cho 1am (w / v), OG (n-octyl P-D-glucoside) 1.00% (w / v) , DDM (n-Dodecyl P-D-maltose ide) 0.01% (w / v), and TX100 0.10% Fluorescence microscope (s single-mo lecule

Total Internal Reflectance Fluorescence Microcope), and the result is shown in Fig. 35E.

35E, when using digi tonin or detergent as a detergent when washing a HER2-HER3 heterodimer attached to a substrate, the degree of phosphorylation of Y1289 of HER3 was high, whereas when CHAPS0, 0G, DDM, and TX100 were used In the first place.

In order to test the degree of HER3 activation according to the detergent concentration, digi tonin was added in an amount of 0.1% (w / v), 0.05% (w / v), 0.01% The degree of phosphorylation of Y1289 of HER3 when the cell lysate was washed using various concentrations such as 0.0% (W8) (no treatment) was measured with a single molecule Total Internal Reflection Fluorescence Microscope, The results are shown in Figs. 35C and 35D. As shown in FIGS. 35C and 35D, in the case of digitonin, the degree of HER3 activation is excellent when the concentration used for cell washing is about 0.05% (w / v) or more, You can check.

The HER2-HER3 heterodimer thus obtained was attached to the substrate. To this end, the surface of the substrate was treated with PEG, neutravidin, biotin, and anti-HER3 antibody (see below) and the cell lysate was injected to the HER2-

After attaching the HER3 heterodimer to the substrate surface, rinse with digitonin (0.1% (w / v)).

(1) PEG surface treatment

a. Coverslip and quartz slide are reacted with sulfuric acid and hydrogen peroxide 2: 1 solution (piranha solution) for 30 minutes. When the reaction is over

In distilled water

Neutralized for 10 minutes.

b. Neutralized coverslips and slides were rinsed in distilled water 18 times more than 5 times.

c. Coverslip and slide were incubated for 20 minutes in ami nos i lane solution (3 ml N_ [3_ (Tr imethoxysilyl) propyl] ethyl enediamine, 5 ml acetic acid, 92 ml methanol) .

d. The prepared coverslip and slide were dried with compressed nitrogen gas. PEG buffer (42 mg sodium bicarbonate, 347.5 mg potassium sulfate dissolved in 5 m 1 18 MQ distilled water) was prepared.

e. The slides were coated with a solution of PEG (3 mg biotin-PEG, lOOmg mPEG, dissolved in at lml of PEG buffer), covered with a coverslip and allowed to react with the PEG solution evenly between the slide and coverslip for 2 hours.

f. The coverslip and slide 18

Washed with distilled water and dried with compressed nitrogen gas. The completed coverslip and slide were vacuum packed in a 50 ml tube (-20 ^° C) to prevent contact with the treated surface of the PEG solution. The Coverslip and the slide PEG solution were kept free of any contact with the treated surface. (2) Assembly and use

a. The frozen coverslips and slides were dissolved in a 37 ^° C incubator to prevent water droplets from forming.

b. A reaction chamber was made between the coverslip and the slide using a double-sided tape and epoxy, with the PEG-treated coverslip and the sides of the slide facing each other.

c. Once the epoxy is sufficiently cured, the neutravidin solution (lOOug / ml neutravidin,

PBS or 0.1% Triton-X-100, 50 mM HEPES [pH 7.4], 150 mM NaCl) was added to the chamber and reacted for 5 minutes.

d. The neutravidin solution remaining in the chamber was washed with PBS and incubated with biotin conjugated anti-HER2 antibody (eBioscience, Cat. No. BMS120BT, clone 2G11) or anti-HER3 antibody (R & D systems, Cat. No. BAM348, clone: # 66201 5 μg / ml, PBS or 0.1% Tr iton-X-100, 50 mM HEPES [pH 7.4], 150 mM NaCl) was added and reacted for 5 minutes.

e. The antibody remaining in the chamber was washed with PBS, and the above cell lysate was injected, followed by reaction for 15 minutes.

f. The lysates remaining in the chamber were washed with washing buffer (0.1% digitonin, or 0.01% GDN, 50 mM HEPES [pH 7.4], 150 mM NaCl, 1% glycerol).

Example 19. Formation of HER2-HER2 homodimer

HER2-eGFP recombinant gene (HER2: NMJ304448, linker PTFLYKWDPVPVAT or GGGSGGGT, eGFP: MH087225, vector: pEGFP-Nl addgene pEGFP-Nl-FLAG) in which eGFP is connected to the intracellular domain termini of HER2, Was injected into HEK293T cell line (Korean Cell Line Bank, or ATCC) through electroporation to induce HER2 overexpression and induce the formation of HER2-HER2 homodimer (also referred to as HER2 homodimer) on the cell surface ). The prepared HER2 homodimer-forming cells are lysed and then treated with a HER2 homodimer. The attached HER2 homodimer is washed with detergent. Referring to the test procedure of Example 18, the phosphorylation level (degree of activation) of HER2 Y1196 obtained as a result of washing the attached HER2 homodimer using various detergents at various concentrations is shown in Fig. 35i. As shown in FIG. 35i, the amount of HER2 adhered using more than 0.05% (w / v) or 0.10% (w / v) of DGTN (digitonin) or 0.01% When the homodimer was washed, the degree of HER2 activation was high.

After the surface of the substrate was treated with PEG, neutravidin, biotin, and ant i-HER2 antibody, the cell lysate was injected to adhere the HER2 homodimer to the substrate surface, and the DGTN (digitonin 0.1%).

The fluorescence signal thus obtained is shown in Fig. 34H. In FIG. 34H, a decrease in fluorescence intensity in one step corresponds to a monomer, and a decrease in fluorescence signal in a two-step corresponds to a dimer. In Figure 34h, HER2_eGFP has a high 2-step ratio due to a high ratio of eGFP and HER3_eGFP, which are likely to be present as relatively monomers, thus demonstrating the production of the HER2-eGFP homodimer.

Based on the Hidden Markov Model, the most probable step (recursive calculation convergence step) from raw data was determined and analyzed.

Example 20. Measurement of activity of tyrosine kinase of HER2-HER3 heterodimer

ATP and ¾ ²⁺ were added to the reaction chamber of the immunoprecipitated HER2-HER3 heterodimer (HER2-HER3 heterodimer bound to the substrate treated antibody in Example 18) and incubated for 5 minutes to induce Tyr phosphorylation. Afterwards, changes in HER3 pTyr levels were observed by single-molecule immunolabeling with pTyr-specific antibodies. The test procedure is schematically shown in FIG. 35A (1. cell lysis, 2. dimer attachment to the substrate, 3. dimer washing attached, 4. phosphorylation by treating ATP and Mg2 +, 5. phosphorylation of HER3 or HER2 Antibody treatment, 6. fluorescence signal detection), the specific test method is as follows:

HER2-HER3 heterodimer was pulled down from the cell lysate (1 to 4 mg / ml) onto the surface of the sample using the biotin-conjugated HER3 antibody in Example 2 (2) The remaining cell lysates were washed with DGTN or 0.01% GDN, 50 mM HEPES [pH 7.4], 150 mM NaCl, 1% glycerol and the kinase assay solution (0.1% DGTN or 0.01 Â »GDN, 200 ÂM ATP, 10 mM magnesium chloride, 50 mM HEPES [pH 7.4], 150 mM NaCl, 1% glycerol) and reacted for 5 minutes.

b. The remaining kinase assay solution was washed with PBS (or 0.1% Triton-X-100, 50 mM HEPES [pH 7.4], 150 mM NaCl), and the HER3 pTyr antibody solution (HER3 pTyr antibody (monoclonal rabbit host) Cat. No. # 479 IS, clone: 21D3) to 2 ug / ml, PBS or 0.1% Triton-X-100, 50 mM HEPES [pH 7.4], 150 mM NaCl) and reacted for 5 minutes.

c. The remaining HER3 pTyr antibody solution was washed with PBS (or 0.1% Triton-X-100, 50 mM HEPES [pH 7.4], 150 mM NaCl) and incubated with cy3 conjugated anti-rabbit FC antibody

(Jackson ImmunoResearch Labs, Cat. No. 11-165-046) at 5 nM, PBS or 0.1% Triton-X-100, 50 mM HEPES [pH 7.4], 150 mM NaCl) and reacted for 5 minutes.

d. The remaining cy3 conjugated anti-rabbit FC antibody solution was washed with PBS (or 0.1% Triton-X-100, 50 mM HEPES [pH 7.4], 150 mM NaCl)

Imaging and quantification were performed using an Internal Reflection Microscope.

The HER3 pTyr levels thus obtained are shown in FIG. 35B. The Tyr residues of the HER3 tail are nine, and in this example a total of five Tyr residues of Y1197, Y1222, Y1276, Y1289, and Y1328 were tested. As shown in FIG. 35B, the degree of phosphorylation was increased by culturing both ATP and Mg2 ^{+ in} pTyr residues.

Example 21. Measurement of activity of tyrosine kinase of HER2-HER2 homodimer

ATP and Mf ⁺ were added to the reaction chamber of the immunoprecipitated HER2-HER2 heterodimer (HER2-HER2 homodimer bound to the substrate treated antibody in Example 19) and incubated for 5 minutes to induce Tyr phosphorylation. Subsequently, changes in HER2 pTyr levels were observed by single-molecule immunolabeling with pTyr-specif ic antibodies. The above test procedure is schematically shown in Figure 35G, and its specific test method is as follows:

a. HER2 homodimer was pulled down from the cell lysate to the surface using a biotin-conjugated HER2 antibody in the preparation and use of Example 18 (2), and then 0.1% DGTN or 0.01% GDN 50 mM HEPES [pH (0.1% DGTN or 0.01% GDN, 200 uM ATP, lOmM magnesium chloride, 50 mM HEPES [pH 7.4], 150 mM NaCl, 1% glycerol) was used to wash out the remaining cell lysate using a buffer solution glycerol) and reacted for 5 minutes.

b. The remaining kinase assay solution was washed with PBS (or 0.1% Triton-X-100, 50 mM HEPES [pH 7.4], 150 mM NaCl), and the HER2 pTyr antibody solution (HER2 (PBS) or 0.1% Tr iton-X-100, 50 mM HEPES [pH 7.4], 150 mM NaCl) in the presence or absence of pTyr antibody (monoclonal rabbit host (Cell Signaling Technology, Cat. No. 6942S, clone: D66B7) Treated and reacted for 5 minutes.

c. The remaining HER2 pTyr antibody solution was washed with PBS (or 0.1% Triton-X-100, 50 mM HEPES [pH 7.4], 150 mM NaCl) and incubated with a cy3 conjugated anti-rabbit FC antibody solution Treated with 0.1% Triton-X-100, 50 mM HEPEStpH 7.4, 150 mM NaCl) and reacted for 5 minutes.

d. The remaining cy3 conjugated anti-rabbit FC antibody solution was diluted with PBS (or

Washed with 0.1% Triton-X-100, 50 mM HEPES [pH 7.4], 150 mM NaCl) and imaged and quantified using a Total Internal Reflection Microscope.

The thus obtained HER2 pTyr level level is shown in Fig. 35H. As shown in FIG. 35h, phosphorylation was increased by ATP and Mg2 ⁺ cultivation in almost all of the Tyr residues Y1139, Y1196, Y1121 / 1222 and Y1248 of HER2 tail (degree of phosphorylation increase: Y1139, control (ATP + EDTA ), About 13 times in the case of Y1196, about 10 times in the case of Y1121 / 1222 and about 13 times in the case of Y1248 in the case of Y1121 / 1222).

Example 22. Signal transduction test of HER2-HER3 heterodimer

FIG. 36A schematically shows that the HER2-HER3 heterodimer attached to the substrate induces an interaction with the eGFP-labeled lower signal transduction protein when it undergoes a phosphorylation process.

With reference to the method of Example 20 or 21, the degree of signal transduction of the HER2-HER3 heterodimer or HER2 homodimer was measured.

Figure 36b shows how much of the HER2-HER3 heterodimer interacts with PLC signaling protein (PLC gamma 1) in terms of the size of the fluorescence signal.

Figure 36c shows the number of single molecular off events obtained from the fluorescence signal to determine how much of the HER2-HER3 heterodimer interacts with PLC signaling protein (PLC gamma 1) (black: ATP + EDTA, white: ATP + MgCh).

By measuring the magnitude of the fluorescence signal how much of the HER2-HER3 heterodimer interacts with the amount of the lower signaling protein (p85 alpha) 36d.

Figure 36e shows the number of single molecular off events from the fluorescence signal as to how much of the HER2-HER3 heterodimer interacts with the amount of the lower signaling protein (p85 alpha) (black: ATP + EDTA, whi te : ATP + MgCh).

FIG. 36f shows how much of the HER2-HER3 heterodimer interacts with the amount of the lower signaling protein (PI3K: p85 alpha -pllO alpha complex) in terms of the magnitude of the fluorescence signal.

FIG. 36g schematically shows that the HER2 homodimer attached to the substrate induces an interaction with the eGFP-labeled lower signal transduction protein when oxidized.

How much amount of the lower signaling protein in one HER2 homodimer

(PLC gamma 1) was measured by the magnitude of the fluorescence signal, and FIG. 36h shows the result.

How much amount of the lower signaling protein in one HER2 homodimer

(ATP + EDTA, whi te: ATP + MgCl ₂₎ as measured by the number of single molecular off events obtained from the fluorescence signal _.

Figure 36j shows how much of the HER2 homodimer interacts with the amount of the lower signaling protein (p85 alpha) in terms of the size of the fluorescence signal.

How much amount of the lower signaling protein in one HER2 homodimer

(p85 alpha), as shown in Figure 36k (black: ATP + EDTA, whi te: ATP + MgCl ₂₎ as measured by the number of single molecular off events from the fluorescence signal.

Example 23. Comparison of phosphorylation rate between HER2-HER3 heterodimer and HER2-HER2 homodimer

Referring to the method of Example 20 using the HER2-HER3 heterodimer, the degree of phosphorylation of HER3 Y1289 over time was measured and shown in Figure 37A. The HER2-HER3 heterodimer was known to strongly induce the prolipid ion of the cell, but the enzymatic character was unknown. Figure 37A is a measure of the enzymatic performance of the HER2-HER3 heterodimer (the HER2-HER3 heterodimer at a specific concentration of phosphorus used for phosphorylation 2019/132517 1 »(: 1 ^ {2018/016675

Indicating rapid phosphorylation). FIG time from ₃ to 37 X-axis phosphorylation, V-axis represents the amount of phosphorylated during that time.

Also, with reference to the method of Example 20 using the constraint 2 - 4 31 ₁ ₀ (1 _{61 '} ,? And the phosphorylation rate of the first and third residues was measured while increasing the concentration. this is ! ³ , the rate of oxidation of _{1 6110} (1 ^ _{61 ·} g) is changed. When the rate of phosphorylation was measured at various concentrations, the ¾ (Mycalis constant) and

(You can measure up to. And the phosphorylation rate of ¾ is the maximum phosphorylation rate

Concentration, and measuring the phosphorylation rate at the ^ TP concentration corresponding to < RTI ID = 0.0 > (I) < / RTI >

It is the maximum phosphorylation rate that can _{reach 11336 at the} maximum phosphorylation rate.

The obtained results are shown in Figure 371 (the dotted line indicates the convergence value of the red line). 371) is a measurement of 1289 of 1 to 3, and a similar level of phosphorylation rate is measured when the other (1197, 1222, 1276, 1328) is measured.

It can be seen that this does not apply. 371)

As the concentration increases,

And the phosphorylation rate of the residue is also increased. For comparison, two seedlings四-四to refer to the process of John 21 _{101 110} (1 in Example 21 with _161, while increasing the concentration required四frozen oxidation rate for the second打residue (凡196), ¾,

The measurement is shown in FIG. 37 (:).

As can be seen from the comparison of Figures 3 and 37 (:),

,

It means that it is 100 times faster. On the other hand,四two graves? - by ¾ ₁ phosphorylation in the grave three四slow modernity "is compared with the first 2] 11 161 101 110, more than twice

The substrate is attached to the substrate at the inhibitor 18

1 _{1 61'0 11161 ·} Example

Attaching a}世seedlings 21 _{101 110,} each processing a ₁₁₁ Ah after Example 20 and with reference to the 21 courses, (100 ₁虛) and _{_{_{1卵63 1111 (± 1 0}}} : ^ (1 6 (10 1) (At this time, 0.1%, 0% and 0.01% And the degree of activation of each HER2 dimer was measured. The obtained results are shown in Fig. 37D. In Figure 37d, the x-axis represents the concentration of used Lapat inib and the y-axis represents the normalized degree of phosphorylation. Activation of the HER2-HER3 heterodimer was measured using HER3 pY1289 antibody, which was normalized to 100% in the absence of Lapat inib. Activation of the HER2 homodimer was measured using HER2 pY1196 antibody. 100% And the standardized value. 37D,

Lapat inib has been shown to exhibit similar inhibition curves to HER2-HOM3 and HER2-HER3 heterodimers that are not resistant to Lapat inib.

The HER2-HER3 heterodimer attached to the substrate in Example 18 and the Example

19, the HER2 homodimer attached to the substrate was treated together with Lapat inib, ATP (lOOuM), and magnesium chloride ide (10 mM) (at this time DGTN should be kept at 0.1% and GDN at 0.0B) The degree of activation was measured. The result is shown in Fig. 37E. As shown in Figure 37 (e), the HER2-HER3 heterodimer had a lower inhibitory effect of Lapat inib compared to the HER2 homodimer, as compared to the HER2 homodimer. These results suggest that HER2-HER3 heterodimer-expressing cells can be activated by ATP and magnesium (when Tyr is phosphorylated)

Lt; RTI ID = 0.0 > Lapat inib. &Lt; / RTI > It has been previously shown that the HER2-HER3 heterodimer is significantly faster than the HER2-homodimer in phosphorylation rate, so this phosphorylation rate may be related to the resistance to the inhibitor (Lapat inib) (ie, Resistance can be seen as well).

HER2-HER3 heterodimer expressing cell lysates were incubated with Lapat inib, ATP (lOOuM), and magnesium chlor ide (10 mM) (again, DGTN was added to the HER2-HER3 heterodimer expressing cell lysate to confirm the correlation between phosphorylation rate and inhibitor resistance

0.1%, GDN should be maintained at 0.01%), PTPN1 (Protein Tyrosine Phosphatase, Non-receptor type 1; ProSpecbio, Cat. PKA-219) was treated and the phosphorylation level of HER3 Y1289 was measured and is shown in Figure 37f. PTPN1 is a dephosphorylation enzyme that forms an equilibrium between Tyr phosphorylation and dephosphorylation and effectively slows down the rate of substantial phosphorylation without changing the HER2-HER3 heterodimer into its structure. As shown in FIG. 37F, as the treatment concentration of PTPN1 increases (from the red graph (rightmost) to the purple graph (leftmost) in FIG. 37F) Phosphorylation is slowed down and the inhibition of phosphorylation by lapatinib is increased (ie, phosphorylation is inhibited at lower concentrations of lapatinib).

Example 25. Single-molecule immunolabelin and co-IP profiling for xenograft models and human patient samples.

All animal studies were conducted in accordance with guidelines approved by the Institutional Animal Care and Use Committee (IA, C). (NOG) and nude (nu / nu) mice (OrientBio) at 6 to 8 weeks of age were used. Clinical tumor samples (derived from lung adenocarcinoma patients) were cut into ~ 3 nun ³ size pieces and subcutaneously implanted into the lateral side of the N0G mice. After 1 to 4 months of transplantation, the tumor was observed at the transplanted site. The volume of the tumor was measured twice weekly with caliper to determine the subcutaneous growth rate of the tumor. When the tumor size reached 1.5 cm in diameter, tumor tissue was excised and cut into small sections (approximately 5 mm ³ size). The severed tissue was re-implanted in another group of mice to obtain subsequent tumors. Mouse generations with patient-derived tumors were named F0, and subsequent generations were numbered sequentially (FI, F2, F3, etc.). A third generation (F3) mouse was used in the vehicle (Phosphata buffered saline, PBS), osimert inib, or gefitinib treatment test (PDTX-A1).

Patient PI was assessed for partial response (PR) to gefitinib therapy for about a year and a half before diagnosis of progressive disease (PD). Partial response to the response evaluation criteria in solid tumors (RECIST) While Patient P2 maintained a stable disease (SD) diagnosis for one year before PD designation.

The PDTX-A1 and the human patient sample P1 (partial response: PR; response evaluation criteria in sol (PD) for the treatment of gefitinib for about a year and a half before the diagnosis of progressive disease In vitro dephosphorylation was performed on mutant EGFR complexes derived from EGFR genotype: exon 19) (Fig. 38). After dephosphorylation, the binding of eGFP-labeled PLC-gammasH2 and Grb2 to the mutant EGFR complex was measured _. By dephosphorylation, binding of PLC-gamma _SH2 entirely dependent on pTyr-SH2 domain interaction was almost completely stopped, while over 50% and over 80% of Grb2 binding counts were maintained after dephosphorylation. These results suggest that the pTyr-independent signaling mechanism of the mutant EGFR actually works in extracorporeal tumor tissue of the PDTX model.

Example 26. Effect of EGFR mutation on interaction with Grb2 To test whether mutated EGFRs require tyrosine kinase activity in order to interact with Grb2, gefitinib, a TKI specific for EGFR, was incubated for 24 hours Were treated with four cells (PC9, HCC827, HCC1006, H1975) with EGFR mutations (pTyr 1068 and 1086). Then, it was dissolved in a nip Pitti treated cells, EGFR it was performed _{11111111110-1)} (^ ₁₎ 11 ₃₁ ^ _011. EGFR tyrosine kinase inhibition by gefitinib almost completely inhibited Grb2 binding to three cells with exon 19 deletion (no gatekeeper mutations) (Fig. 39A). Some residual EGFR-Grb2 PPI counts were observed in H1975 cells with L858R and T790M gatekeeper mutations. It was also confirmed to completely inhibit Grb2 binding in H1975 cells by treating osimert inib (third generation TKI that equally well binds EGFR with gatekeeper mutations) (Fig. 39a, blue dot).

As previously noted, the binding between Grb2 and mutant EGFR was completely inhibited by TKI treatment. To further observe this phenomenon, we used a single-molecule immunolabel 1 ing scheme to identify other known interacting partners of EGFR Co-IP was tested and the results are shown in Figure 39b_g.

First, the co-IP level of endogenous HSP90 (alpha isoform) was quantitated. The association of HSP90 was selectively observed with mutant EGFR (Figure 39b; blue-black bars), and this HSP90 association was shown to be resistant to in vitro dephosphorylation (Figure 39b, black versus red bars) (Fig. 39b; black versus yellow bars) by treating TKI for a period of time. A similar pattern was observed for MIG6 (natural EGFR inhibitor) and GAPDH35 (Figure 39c, d), although GAPDH associated with mutant EGFR was more abundant after 24 hours of gefitinib treatment. In contrast, Shbl (a well known support for Grb2 binding 20) exhibited similar levels of binding across WT and mutant EGFR (Figure 39e).

Although the interaction of Shcl and WT EGFR was reduced after in vitro dephosphorylation, the binding to the mutant EGFR showed resistance to in vitro dephosphorylation similar to that observed in HSP90, MIG6 and GAPDH. In addition, the associat ion of Shcl, WT and mutant EGFR was stabilized or even increased due to 24 hours of gefitinib treatment. These results indicate that Shcl behaves differently from other proteins selectively associated with the mutant EGFR. Finally, using the single molecule immunolabel 1 ing technique, it was found that TKI treatment did not affect the total EGFR level (Figure 39f).

The results suggest that the binding of Grb2 to WT EGFR is primarily mediated by the canonical pathway, i.e. through the interaction between pTyr 1068 and 1086 and the Grb2 SH2 domain.

In contrast, mutant EGFR utilizes fundamentally different mechanisms that are least dependent on surface exposed pTyr residues. We observed that mutant EGFRs form a larger signaling complex than WT EGFR. Some components of these signaling complexes engage GrB2 in a pTyr-independent manner. This non-canonical pathway is highly resistant to dephosphorylation of phosphatase

It is possible to explain how cancer with an EGFR mutation maintains EGFR signaling in the absence of a stimulatory ligand. In addition, we found that co-IP levels of mutant EGFR and c-Cbl (and Cbl-b) were significantly lower than WT EGFR (Figure 39g). These results suggest that a signaling complex unique to the mutant EGFR hinders the degradation of EGFR.

The results in Figures 39a-g show that when sedimented with EGFR,

In the case of PC9 / HCC827 / HCC4006, it can be seen that HSP90a is remarkably abundant with EGFR. This suggests that the cells depend on EGFR signaling as well as accessory proteins called HSP90a. Therefore, it is expected that ESPR inhibitor and HSP90a may be used at the same time, or the desired effect may be obtained to some extent even when the HSP90a inhibitor alone is used. On the other hand, it can be confirmed that EGFR does not work at all in the normal H1666 + cell line. 2019/132517 1 »(: 1/10/06 018/016675

Claims

(I) treating a candidate compound with a test sample, and (II) contacting the candidate compound with the treated test sample and the treated sample. (1), (2), (3) and (5), or (1), (2), (3), (4) and (1), (2), (3), (4-1), and (5) 5). &Lt; / RTI > A method of screening candidate drugs targeting a first protein comprising:

(3) measuring a signal from the reactant obtained in step (2) to measure protein-protein interaction;

(4) measuring an activation level by obtaining a value for a unit amount of the first protein included in the test sample added in step (1) using the signal measured in step (3);

(4-1) measuring a protein-protein interaction level by obtaining a signal value for a unit amount of the test sample added in step (1) using the signal measured in step (3);

(4-2) measuring the activation level by obtaining a value for the unit amount of the protein contained in the test sample using the signal value of the unit amount of the test sample obtained in the step (4-1);

[Claim 2]

The method of claim 1, wherein after step (5)

(6) As a result of the comparison in the above step (5), when the protein-protein interaction, the protein-protein interaction level, or the activation level of the test sample treated with the candidate compound is lower than that of the test sample in which the candidate compound is not treated , The candidate compound is selected as a candidate drug targeting the first protein 2019/132517 1 »(: 1 ^ {2018/016675

The method further comprising the step of selecting.

[Claim 3]

3. The method of claim 1 or 2, wherein the steps (1), (2), (3) and (5), or (1), (2), (3), (4) (1), (2), (3), (4-1), and (5) , And (5)

( ₃₎ the interaction between the receptor (first protein) and the ligand (second protein)

(Interaction between a first protein and a second protein when at least one of the first protein and the second protein has a phosphorylated tyrosine residue, and

₍₀₎ the first protein and the interaction between the lower protein ingest 2 protein of the ₁₀ proteins in the first transmission path to engage the first protein

&Lt; / RTI >

[4]

3. The method of claim 1 or 2, wherein the steps (1), (2), (3) and (5), or (1), (2), (3), (4) , or (1), (2), (3), (4-1), and (5), or ₁₅ (1), (2), (3), (4-1), (4-2) , And (5)

₍₀₎ the first protein and the interaction between the lower protein ingest 2 protein of the ₂₀ proteins in the first transmission path to engage the first protein

&Lt; / RTI >

[Claim 5]

4. The method according to claim 2, wherein the candidate drug targeting the first protein is selected from the group consisting of an activity inhibitor of the first protein, a therapeutic agent for a disease associated with the first protein, and an inhibitor of interaction between the first protein and the second protein Wherein one selected from the group consisting of.

[Claim 6]

1) -1: supplying egg-1 bound with a labeling substance to the substrate to which the labeling substance is bound and reacting; And

₃₀ Measuring the signal (show) generated in the reacting step

Lt; / RTI >

Wherein the reactants of 1) -1 and egg-1 form clusters, and 1) a method of measuring the interaction of 1) and 1) -1.

[7]

7. The method of claim 6, wherein, prior to the step of reacting,

A step of supplying PD-L1 to a substrate to which a substance bound to the substrate is attached and attaching the PD-L1 to the substrate

Wherein the PD-L1 and the PD-1 interact with each other.

[8]

The method according to claim 6,

(PD-L1) to which a labeling substance binds to a substrate to which a substance binding to the PD-L1 has been attached, and a step of measuring a signal

Wherein the PD-L1 and the PD-1 interact with each other.

9]

The method according to claim 8, further comprising the step of obtaining and quantifying the value of the signal (A) for the signal (PD-L1 and PD-1).

[Claim 10]

7. The method according to claim 6, wherein in the step of reacting, a substance which competes with PD-L1 or PD-1 for the binding between PD-L1 and PD-1 is added and reacted, (PD-1) and PD-1 (PD-1).

Claim 11

11. A method according to any one of claims 6 to 10, wherein the signal (A) or () is measured in a near-field region.

Claim 12

11. A method according to any one of claims 6 to 10, wherein the PD-

The method for measuring the interaction between PD-L1 and PD-1, wherein the substance binding to L1 is a substance binding to the terminal region of PD-L1.

Claim 13

Supplying a mixture of a candidate substance, PD-L1, and a labeled substance-bound mixture of PD-1 with a substance that binds to the PD-L1 to a surface-attached substrate; And

The step of measuring the signal (obtained in the step of reacting)

Lt; RTI ID = 0.0 > PD-L1 < / RTI > and PD-1. 2019/132517 1 »(: 1 ^ {2018/016675

14.

14. The method of claim 13,

(1) measuring the signal generated by the reaction between the substance on the surface of the substrate and the substrate,

Wherein the method further comprises the step of:

15.

15. The method of claim 14, further comprising: obtaining a signal (a value for the signal,

Selection method of interaction inhibitor.

Claim 16

Lt; / RTI >

Lt; RTI ID = 0.0 > in vitro, < / RTI >

Wherein the activated receptor tyrosine kinase dimer is produced by the method.

17.

17. The method of claim 16, wherein the first receptor tyrosine kinase is iodine 3 and the second receptor tyrosine kinase is Seed 2.

Claim 18

18. The method according to claim 16 or 17,

The cholesterol-

And 0.003 to 2% (八0 of _{_{(1 ¥ 1- 03 6 11)}} 1 ahsang one selected from the method for the preparation of the activated receptor tyrosine kinases for dimer.