KR20230029140A - Composition for detecting EGFR targeted therapy resistance and screening method for inhibiting EGFR targeted therapy resistance - Google Patents

Abstract

The present invention relates to a composition for predicting resistance of an EGFR target treating agent and a method for screening a drug of inhibiting resistance of an EGFR target treating agent. Specifically, the present invention manufactures a cell model for screening EGFR target treating agent resistance involved genes induced by an EGFR T790M mutation by using a cancer cell line showing sensitivity of the EGFR target treating agent; deduces the EGFR target treating agent resistance involved genes induced by the EGFR T790M mutation by performing gene expression profiling using the cells; uses the genes or protein encoded by the genes as a biomarker for predicting or diagnosing EGFR target treating agent resistance induced by the EGFR mutation; manufactures an EGFR target treating agent resistance cell model by using the same; and screens a drug for inhibiting the EGFR target treating agent resistance or a cancer treating agent.

Description

Composition for detecting EGFR targeted therapy resistance and screening method for inhibiting EGFR targeted therapy resistance {Composition for detecting EGFR targeted therapy resistance and screening method for inhibiting EGFR targeted therapy resistance}

The present invention relates to a composition for predicting EGFR target therapy resistance and a method for screening a drug for inhibiting EGFR target therapy resistance, and more particularly, to a biomarker involved in inducing EGFR target therapy resistance by EGFR mutagenesis and drug candidates using the same. It relates to a screening method and a method for producing a cell model resistant to an EGFR target therapy.

Epithelial cancer, such as prostate cancer, breast cancer, rectal cancer, lung cancer, pancreatic cancer, ovarian cancer, spleen cancer, testicular cancer, and thyroid cancer, is a disease characterized by abnormal accelerated growth of epithelial cells. This accelerated growth initially causes tumor formation and, eventually, metastases to other organs may also occur. Although progress has been made in the diagnosis and treatment of various cancers, these diseases still represent significant mortality rates.

Epidermal growth factor receptor (EGFR) is a 170 kDa membrane-bound protein expressed on the surface of epithelial cells, and EGFR is a member of the growth factor receptor family of protein tyrosine kinases and a class of cell cycle regulatory molecules. EGFR is activated when its ligand (EGF or TFRα) binds to its extracellular domain, resulting in autophosphorylation of the intracellular tyrosine kinase domain of the receptor.

EGFR is a protein product of the growth-promoting oncogene erbB or ErbB1, a member of the ERBB family of proto-oncogenes, and is believed to play a pivotal role in the onset and progression of many human cancers. In particular, increased EGFR expression has been observed in breast, bladder, lung, head, neck and stomach cancers as well as glioblastma. The ERBB family of oncogenes encodes four structurally related transmembrane receptors: EGFR, HER2/neu (erbB2), HER-3 (erbB3) and HER-4 (erbB4). Clinically, ERBB oncogene amplification and/or receptor overexpression in tumors has been reported to correlate with disease relapse and poor patient prognosis, as well as responsiveness to treatment.

Accordingly, EGFR-targeting therapeutics targeting EGFR have been developed as anti-cancer agents and are being applied to clinical practice. For example, AstraZeneca's Iressa (ingredient name: gefitinib) as a first-generation EGFR-targeted therapy, Roche's Tarceva (ingredient name: erlotinib), and Pfizer's Vizimpro (ingredient name: dacomitinib) as a second-generation EGFR-targeted therapy. (Dacomitinib)), Boehringer Ingelheim's Geotrip (ingredient: Afatinib), and AstraZeneca's Tagrisso (ingredient: Osimertinib), a third-generation EGFR-targeted therapy, are prescribed as non-small cell lung cancer treatments. It is becoming. However, it is known that relapse eventually occurs in most patients, including patients who have shown a dramatic response to EGFR-targeted therapy.

Recently, it has been found that resistance to gefitinib or erlotinib is acquired due to a secondary mutation (T790M) in exon 20 of EGFR. It is speculated. The EGFR T790M secondary mutation is the most important resistance mechanism, accounting for 50% of all types of EGFR target therapy resistance mechanisms. Drug resistance due to secondary mutations is estimated to be much higher than known. Since the first report in 2005 that an irreversible EGFR-targeted therapy that is much more powerful than a reversible EGFR-targeted therapy such as Gefitinib that is currently being used can overcome the EGFR T790M secondary mutation, second-generation drugs such as Geotrip EGFR target therapy has been developed, but negative research results are gradually increasing as to whether resistance can be overcome. Therefore, there is an urgent need for the development of new therapies capable of suppressing resistance caused by EGFR mutation and overcoming resistance.

Therefore, the present inventors have made efforts to develop a new approach to overcome resistance due to EGFR mutation in the use of these EGFR-targeted anticancer drugs and a therapeutic agent for solving these problems, using a cancer cell line showing sensitivity to EGFR-targeted therapeutics A cell model for screening genes involved in EGFR target therapy resistance induced by EGFR T790M mutagenesis was prepared, and gene expression profiling was performed using these cells to identify genes involved in EGFR target therapy resistance induced by EGFR T790M mutagenesis. It was derived, and it was revealed that the derived gene or the protein encoded by the gene could be used as a biomarker for predicting or diagnosing EGFR target therapy resistance induced by EGFR mutation, leading to this application.

Gullick W, Marsden J, Whittle N, et al: Expression of epidermal growth factor receptors on human cervical, ovarian, and vulval carcinomas. Cancer Res 1986;46:285-292. S Cohen et al: Epidermal growth factor-receptor-protein kinase interactions. Co-purification of receptor and epidermal growth factor-enhanced phosphorylation activity. J Biol Chem 1980;255:4834-4842. A. B. Schreiber et al: Biological Role of Epidermal Growth Factor-Receptor Clusterin. J. Biol. Chem 1983 258;846-853. L. Harris et al: C-erbB-2 in serum of patients with breast cancer. Int J Biol Markers 1999;14:8-15. J. Mendelsohn and J. Baselga: The EGF receptor family as targets for cancer therapy. Oncogene 2000;19(56):6550-6565 J. Downward et al: Close similarity of epidermal growth factor receptor and v-erb-B oncogene protein sequences. Nature 1984;307(5951):521-527. C. R. Carlin et al: Biosynthesis and glycosylation of the epidermal growth factor receptor in human tumor-derived cell lines A431 and Hep 3B. Mol. Cell. Biol 1986; 6:257-264. F. L. V. Mayes and M. D. Waterfield: Biosynthesis of the epidermal growth factor receptor in A431 cells. The EMBO J 1984;3:531-537 Kye Young Lee: Targeted Therapy of Lung Cancer. J Korean Med Assoc 2008;51(5):483-491. Kobayashi S, et al. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med 2005;352:786-792.

Conventional anticancer drugs targeting EGFR show a therapeutic effect at first, but show limitations in that the therapeutic effect does not appear due to resistance when administered for a long period of time. In particular, as the mechanism of resistance induction by EGFR T790M secondary mutation is known as the most important resistance mechanism, a new approach is needed to solve the problem of acquired resistance due to EGFR mutation in the use of EGFR target anticancer drugs.

Accordingly, an object of the present invention is to provide a biomarker for predicting or diagnosing EGFR target therapy resistance induced by EGFR mutation.

Another object of the present invention is to provide a method for providing information for predicting or diagnosing EGFR target therapy resistance in cancer in which EGFR mutations occur using the biomarker.

Another object of the present invention is to provide a method for preparing an EGFR target therapy resistant cell model using the biomarker.

Another object of the present invention is to provide a method for screening EGFR-targeted therapy resistance inhibitors or EGFR-targeted therapy-resistant cancer therapeutics using the biomarker.

In order to achieve the object of the present invention, the present invention is OTOA, LOC400706, PARD6B, SPTB, PCDH9, PRDM1, DNAJB5, CNKSR3, SAA1, SERPINB2, ZNF396, HNRNPA1L2, C8orf31, CCDC115, ZNF718, LSM4, FN1, HOPX and BANK1 It provides a biomarker for predicting or diagnosing EGFR target therapy resistance induced by EGFR mutation, including one or more genes selected from the group consisting of or proteins encoded by the genes.

In addition, the present invention is one selected from the group consisting of OTOA, LOC400706, PARD6B, SPTB, PCDH9, PRDM1, DNAJB5, CNKSR3, SAA1, SERPINB2, ZNF396, HNRNPA1L2, C8orf31, CCDC115, ZNF718, LSM4, FN1, HOPX and BANK1 It provides a composition for predicting or diagnosing EGFR target therapy resistance induced by EGFR mutation, including an agent for measuring the expression or activity level of the gene or the protein encoded by the gene.

In addition, the present invention is OTOA, LOC400706, PARD6B, SPTB, PCDH9, PRDM1, DNAJB5, CNKSR3, SAA1, SERPINB2, ZNF396, HNRNPA1L2, C8orf31, CCDC115, ZNF718, LSM4, FN1, HOPX and BANK1 in samples isolated from cancer patients To provide information for predicting or diagnosing EGFR target therapy resistance in cancer in which EGFR mutations occur, including measuring the expression or activity level of one or more genes selected from the group consisting of or proteins encoded by the genes provides a way

In addition, the present invention

1) Overexpression of one or more genes selected from the group consisting of OTOA, LOC400706 and ZNF718 in cancer cells showing sensitivity to EGFR target therapy, or PARD6B, SPTB, PCDH9, PRDM1, DNAJB5, CNKSR3, SAA1, SERPINB2, ZNF396, HNRNPA1L2, Inhibiting the expression of one or more genes selected from the group consisting of C8orf31, CCDC115, LSM4, FN1, HOPX and BANK1; and

2) It provides a method for producing an EGFR-targeted therapeutic agent-resistant cell model, including the step of treating the cells of step 1) with an EGFR-targeted therapeutic agent.

In addition, the present invention

1) Overexpression of one or more genes selected from the group consisting of OTOA, LOC400706 and ZNF718 in cancer cells showing sensitivity to EGFR target therapy, or PARD6B, SPTB, PCDH9, PRDM1, DNAJB5, CNKSR3, SAA1, SERPINB2, ZNF396, HNRNPA1L2, Inhibiting the expression of one or more genes selected from the group consisting of C8orf31, CCDC115, LSM4, FN1, HOPX and BANK1; and

2) Provides a method for screening EGFR targeted therapy resistance inhibitors, comprising the steps of treating the cells of step 1) with the EGFR targeted therapy and a test substance and measuring drug reactivity.

In addition, the present invention

1) Overexpression of one or more genes selected from the group consisting of OTOA, LOC400706 and ZNF718 in cancer cells showing sensitivity to EGFR target therapy, or PARD6B, SPTB, PCDH9, PRDM1, DNAJB5, CNKSR3, SAA1, SERPINB2, ZNF396, HNRNPA1L2, Inhibiting the expression of one or more genes selected from the group consisting of C8orf31, CCDC115, LSM4, FN1, HOPX and BANK1; and

2) It provides a method for screening EGFR target therapy drug-resistant cancer therapeutics, including the steps of treating the cells of step 1) with a test substance and measuring drug reactivity.

The present inventors constructed a cell model for screening genes involved in EGFR target therapy resistance induced by EGFR T790M mutagenesis using cancer cell lines exhibiting EGFR target therapy sensitivity, and performed gene expression profiling using these cells to EGFR. Genes involved in EGFR target therapy resistance induced by T790M mutagenesis were derived. Therefore, the derived gene or the protein encoded by the gene can be used as a biomarker for predicting or diagnosing EGFR-targeted drug resistance induced by EGFR mutation, and using this, an EGFR-targeted drug-resistant cell model can be prepared. A drug that inhibits resistance to a targeted therapy or a cancer treatment can be screened.

Through this, it is possible to select patients in advance for whom treatment by EGFR-targeted therapy is not effective, and to provide information necessary for predicting a patient group that is likely to develop resistance among patient groups receiving EGFR-targeted therapy. It can provide a base technology that can be widely used in related research, such as screening for resistance inhibitory drugs and EGFR-targeted cancer treatment candidates.

1 is a diagram schematically illustrating a cell model production process for screening genes involved in EGFR mutation-induced EGFR-targeted drug resistance.
Figure 2 is a diagram confirming whether EGFR T790M mutation occurs in monoclonal cells isolated from PC9 cells in the process of preparing a cell model for screening genes involved in EGFR mutation-induced EGFR target therapy resistance.
Figure 3 shows 12 clones derived from monoclonal cells (T790M-bearing resistant clones) in which erlotinib resistance was induced by newly occurring EGFR T790M mutation obtained through the cell model production process for screening genes involved in EGFR mutation-induced EGFR target therapy resistance. It is a diagram confirming the EGFR T790M mutation in .
Figure 4 is a diagram showing the time point of acquiring erlotinib resistance in the 12 clones.
Figure 5 is a diagram showing genes selected based on p-values in EGFR T790M mutation-induced EGFR target therapy drug-resistant monoclonal cells that initially acquired resistance (Early, 100 days ago).
Figure 6 is a diagram showing genes selected based on GESA in EGFR T790M mutation-induced EGFR target therapy drug-resistant monoclonal cells that initially acquired resistance (Early, 100 days ago).
7 is a diagram showing genes selected based on p-values in EGFR T790M mutation-induced EGFR target therapy drug-resistant monoclonal cells that acquired resistance at a late stage (after 100 days).
8 is a diagram showing genes selected based on GESA in EGFR T790M mutation-induced EGFR target therapy drug-resistant monoclonal cells that acquired resistance at a late stage (Late, after 100 days).
9 is a diagram confirming the induction of erlotinib resistance in resistance-inducible monoclonal cells in which the expression of each of genes involved in EGFR target therapy resistance HOPX, PRDM1, and DNAJB5 induced by EGFR mutation was suppressed.

Hereinafter, the present invention will be described in detail.

A feature of the present invention is to determine whether EGFR T790M mutation causes EGFR target therapy resistance by using a gene involved in the induction of EGFR target therapy resistance as a biomarker.

Accordingly, the present invention provides 1 selected from the group consisting of OTOA, LOC400706, PARD6B, SPTB, PCDH9, PRDM1, DNAJB5, CNKSR3, SAA1, SERPINB2, ZNF396, HNRNPA1L2, C8orf31, CCDC115, ZNF718, LSM4, FN1, HOPX and BANK1 Provided is a biomarker for predicting or diagnosing EGFR target therapy resistance induced by EGFR mutation, including a gene of more than one species or a protein encoded by the gene.

The biomarker of the present invention can be an indicator of resistance to anticancer drugs, which are EGFR-targeted therapeutic agents, and can be specifically an indicator of resistance to EGFR-targeted therapeutic agents induced by EGFR mutation, and more specifically, due to EGFR T790M mutation. It can be an indicator of resistance to induced EGFR target therapy. In addition, anticancer drug resistance predictive or diagnostic markers, specifically anticancer drug resistance predictive or diagnostic markers induced due to EGFR mutations, more specifically, anticancer drug resistance predictive or diagnostic markers induced due to EGFR T790M mutations, the occurrence, development and / or for the diagnosis and treatment of metastases.

As used herein, the term "tolerance" or "resistance" means that the efficacy of a drug does not work because it does not react sensitively to the corresponding drug.

In addition, the term "prediction" or "diagnosis" means to confirm the pathological state, and for the purpose of the present invention, the prediction or diagnosis is to confirm the expression of resistance prediction or diagnostic biomarkers for EGFR target therapy. It means to check whether there is, development and reduction of

In the present invention, at least one gene selected from the group consisting of OTOA, LOC400706, PARD6B, SPTB, PCDH9, PRDM1, DNAJB5, CNKSR3 and SAA1, or a protein encoded by the gene, EGFR mutation occurs and EGFR target therapy is administered Early resistance can be predicted or diagnosed. Here, the initial stage after administration of EGFR-targeted therapy is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, and 13 weeks after administration of EGFR-targeted therapy. weeks or less than 14 weeks.

In addition, at least one gene selected from the group consisting of SERPINB2, ZNF396, HNRNPA1L2, C8orf31, CCDC115, ZNF718, LSM4, FN1, HOPX and BANK1, or a protein encoded by the gene, EGFR mutation occurs and EGFR target therapy administration Later resistance can be predicted or diagnosed. Here, the late period after administration of the EGFR target therapy refers to a period after 14 weeks, 15 weeks, or 16 weeks.

In the present invention, the EGFR-targeted therapeutic agent refers to an anticancer agent, and any EGFR-targeted therapeutic agent that exhibits an anti-cancer effect may be applied, and may be used interchangeably with an EGFR-targeted drug. Specifically, the EGFR target therapy is erlotinib, gefitinib, afatinib, dacomitinib, osimertinib, lazertinib, felitinib (Pelitinib), Neratinib, Icotinib, brigatinib, lapatinib, canertinib, vandetanib, PKI-166 and AEE788 It may be one or more selected from, and more specifically, it may be erlotinib, but is not limited thereto.

In a specific embodiment of the present invention, the present inventors show a short-term response to erlotinib, a first-generation EGFR-targeted therapy, in order to screen genes involved in EGFR mutation-induced EGFR-targeted therapy resistance, but are known to show resistance during long-term treatment. Using PC9 cells, a lung cancer cell line, as shown in the schematic diagram shown in FIG. 1, monoclonal cells incapable of inducing resistance, monoclonal cells in which erlotinib resistance was induced by newly occurring EGFR T790M mutation (T790M-bearing resistant clones) and no EGFR T790M mutation Monoclonal cells (non-T790M-bearing resistant clones) in which resistance was induced were prepared. In addition, in the case of monoclonal cells in which the EGFR T790M mutation was newly generated and erlotinib resistance was induced, it was confirmed that there was a difference in resistance acquisition time (see FIGS. 3 and 4).

In addition, the present inventors profiled gene expression changes in monoclonal cells in which erlotinib resistance was induced due to the newly occurring EGFR T790M mutation, parental cells before the EGFR T790M mutation, and monoclonal cells in which resistance was induced without EGFR T790M mutation. , 9 genes [OTOA, LOC400706, PARD6B, SPTB, PCDH9, PRDM1, DNAJB5, CNKSR3, SAA1] and late (100 After), the expression of 10 genes [SERPINB2, ZNF396, HNRNPA1L2, C8orf31, CCDC115, ZNF718, LSM4, FN1, HOPX, BANK1] was significantly changed in EGFR T790M mutation-induced EGFR-targeted therapy-resistant monoclonal cells that acquired resistance. It was confirmed that (see FIGS. 5 to 8).

In addition, the present inventors found that genes whose expression was decreased among the 19 genes [PARD6B, SPTB, PCDH9, PRDM1, DNAJB5, CNKSR3, SAA1, SERPINB2, ZNF396, HNRNPA1L2, C8orf31, CCDC115, ZNF718, LSM4, FN1, HOPX, BANK1] Inhibition of gene expression in the monoclonal cells incapable of induction of tolerance using shRNA against, and cDNA for genes whose expression was increased among the 19 genes [OTOA, LOC400706, ZNF718] were used in the monoclonal cells incapable of induction of tolerance. After overexpressing the gene in , as a result of treatment with erlotinib, it was confirmed that resistance was induced.

Therefore, the inventors of the present invention used cancer cell lines showing sensitivity to EGFR target therapy to develop monoclonal cells incapable of inducing resistance, monoclonal cells in which erlotinib resistance was induced by newly occurring EGFR T790M mutation (T790M-bearing resistant clones), and EGFR T790M mutation. We produced monoclonal cells (non-T790M-bearing resistant clones) in which resistance was induced without, and gene expression profiling was performed using these cells to derive genes involved in EGFR target therapy resistance induced by EGFR T790M mutagenesis. , The gene or the protein encoded by the gene can be used as a biomarker for predicting or diagnosing EGFR target therapy resistance induced by EGFR mutation, and using it as a composition for predicting or diagnosing EGFR target therapy resistance induced by EGFR mutation. can be utilized

Accordingly, the present inventors found that one selected from the group consisting of OTOA, LOC400706, PARD6B, SPTB, PCDH9, PRDM1, DNAJB5, CNKSR3, SAA1, SERPINB2, ZNF396, HNRNPA1L2, C8orf31, CCDC115, ZNF718, LSM4, FN1, HOPX and BANK1 It provides a composition for predicting or diagnosing EGFR target therapy resistance induced by EGFR mutation, including an agent for measuring the expression or activity level of the gene or the protein encoded by the gene.

In the present invention, the agent for measuring the gene expression level may be an antisense oligonucleotide, a primer pair or a probe that specifically binds to mRNA of the gene, but is not limited thereto.

The primer refers to a single-stranded oligonucleotide capable of serving as the starting point of template-directed DNA synthesis under suitable conditions (i.e., four different nucleoside triphosphates and polymerization enzymes) at a suitable temperature and in a suitable buffer. . The suitable length of the primer may vary depending on various factors, such as temperature and use of the primer. In addition, the sequence of the primer does not have to have a sequence completely complementary to a part of the sequence of the template, and it is sufficient that it has sufficient complementarity within a range capable of hybridizing with the template and performing the intrinsic function of the primer. Therefore, the primer in the present invention does not have to have a sequence perfectly complementary to the nucleotide sequence of the gene as a template, and it is sufficient if it has sufficient complementarity within the range in which it can hybridize to this gene sequence and act as a primer. In addition, it is preferable that the primers according to the present invention can be used in a gene amplification reaction. The amplification reaction refers to a reaction to amplify a nucleic acid molecule, and amplification reactions of such genes are well known in the art, such as polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), ligase These may include lase chain reaction (LCR), electron mediated amplification (TMA), nucleotide sequence substrate amplification (NASBA), and the like.

The probe refers to a linear oligomer of natural or modified monomers or linkages, includes deoxyribonucleotides and ribonucleotides, can specifically hybridize to a target nucleotide sequence, and is naturally present or artificially synthesized. say that The probe according to the present invention may be a single chain, and specifically may be an oligodeoxyribonucleotide. Probes of the present invention may include natural dNMPs (i.e., dAMP, dGMP, dCMP and dTMP), nucleotide analogues or derivatives. In addition, the probe of the present invention may also contain ribonucleotides. For example, the probes of the present invention can be used with backbone modified nucleotides such as peptide nucleic acids (PNA), phosphorothioate DNA, phosphorodithioate DNA, phosphoroamidate DNA, amide-linked DNA, MMI-linked DNA, 2 '-O-methyl RNA, alpha-DNA and methylphosphonate DNA, sugar modified nucleotides such as 2'-O-methyl RNA, 2'-fluoro RNA, 2'-amino RNA, 2'-O-alkyl DNA, 2'-O-allyl DNA, 2'-O-alkynyl DNA, hexose DNA, pyranosyl RNA and anhydrohexitol DNA, and nucleotides with base modifications such as C-5 substituted pyrimidines ( Substituents are fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, ethyl-, propynyl-, alkynyl-, thiazoyl-, imidazoyl- , including pyridyl-), 7-deazapurines with C-7 substituents (substituents are fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, alky yl-, alkenyl-, thiazoyl-, imidazoyl-, pyridyl-), inosine and diaminopurines.

In the present invention, the agent for measuring the protein expression or activity level may be an antibody, peptide or nucleotide that specifically binds to the protein, but is not limited thereto.

As the antibody, polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, or fragments thereof may be used. Examples of the antibody fragments include Fab, Fab', F(ab')2, Fv fragments, diabodies, and linear antibodies (Zapata et al., Protein Eng.8(10): 1057-1062 (1995)). , multispecific antibodies formed from single chain antibody molecules or antibody fragments, and the like.

Methods for preparing the polyclonal antibodies are known to those skilled in the art. A polyclonal antibody can be prepared by injecting an immunizing agent into a mammal one or more times, and injecting it together with an adjuvant, if necessary. Typically, the immunizing agent and/or adjuvant is injected into the mammal multiple times by subcutaneous or intraperitoneal injection. It may be effective to inject the immunizing agent together with a protein known to be immunogenic to the mammal being immunized.

Such monoclonal antibodies can be prepared by the hybridoma method described by Kohler et al., Nature, 256:495 (1975), or by recombinant DNA methods (see, eg, US Pat. No. 4,816,576). can be manufactured with Monoclonal antibodies are also described, for example, in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991). It can be isolated from phage antibody libraries using the techniques described. The monoclonal antibody is specifically such that, if it exerts the desired activity, portions of its heavy and/or light chains are identical or homologous to the corresponding sequences of antibodies derived from a particular species or of an antibody belonging to a particular antibody class or subclass. but the remainder of the chain(s) includes "chimeric" antibodies that are identical or homologous to antibodies or fragments of such antibodies, either from antibodies derived from other species or belonging to other antibody classes or subclasses (Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

"Humanized" forms of non-human (eg murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (eg Fv, Fab, Fab', F(ab')2 or other antigen binding sequence of an antibody). In most cases, humanized antibodies are human immunoglobulins in which residues from a complementarity determining region (CDR) of the recipient are substituted with CDR residues from a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. (recipient antibody). In some cases, Fv framework residues of a human immunoglobulin are replaced by corresponding non-human residues. In addition, a humanized antibody may contain residues that are not found in the recipient antibody or in the imported CDR or framework sequences. Generally, a humanized antibody comprises substantially all of one or more, and usually two or more, variable domains, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are Corresponds to a region of the human immunoglobulin sequence. A humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin region (Presta, Curr.Op.Struct.Biol. 2:593-596 (1992)).

In addition, the antibody is preferably used in an immunoassay method, for example, radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), and flow cytometry. (flow cytometry) or immunofluorescence staining, but is not limited thereto.

In the present invention, the composition is used for a biological sample of a cancer patient suspected of having EGFR target therapy resistance. In addition, the biological sample is a biological sample isolated from a cancer patient suspected of having EGFR target therapy resistance, and may be, for example, tissue, cell, blood, serum, plasma, saliva or urine, but is not limited thereto.

The composition for predicting or diagnosing EGFR target therapy resistance induced by the EGFR mutation of the present invention may be included in the form of a kit.

The kit may include primers, probes, or antibodies capable of measuring the expression level of a gene or the amount of a protein, the definitions of which are as described above.

Optionally, when the kit is applied to the PCR amplification process, reagents necessary for PCR amplification, such as buffers, DNA polymerases (eg, Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis or heat stable DNA polymerase obtained from Pyrococcus furiosus (Pfu)), DNA polymerase cofactors and dNTPs, and when the kit is applied to an immunoassay, the kit of the present invention optionally comprises a secondary antibody and a label may include the substrate of Furthermore, the kit according to the present invention may be manufactured in a plurality of separate packaging or compartments containing the reagent components described above.

The composition for predicting or diagnosing EGFR target therapy resistance induced by EGFR mutations of the present invention may be included in the form of a microarray.

In the microarray of the present invention, primers, probes, or antibodies capable of measuring the expression level of the protein or the gene encoding it are used as hybridizable array elements and immobilized on a substrate. Preferred substrates may include suitable rigid or semi-rigid supports such as membranes, filters, chips, slides, wafers, fibers, magnetic or non-magnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. there is. The hybridization array elements are arranged and immobilized on the substrate, and such immobilization may be performed by a chemical bonding method or a covalent bonding method such as UV. For example, the hybridization array element may be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and may also be bonded by UV to a polylysine coated surface. In addition, the hybridization array element may be coupled to a substrate through a linker (eg, ethylene glycol oligomer and diamine).

Meanwhile, when a sample applied to the microarray of the present invention is a nucleic acid, it can be labeled and hybridized with an array element on the microarray. Hybridization conditions may vary, and the degree of hybridization may be detected and analyzed in various ways depending on the labeling material.

In addition, the present invention is OTOA, LOC400706, PARD6B, SPTB, PCDH9, PRDM1, DNAJB5, CNKSR3, SAA1, SERPINB2, ZNF396, HNRNPA1L2, C8orf31, CCDC115, ZNF718, LSM4, FN1, HOPX and BANK1 in samples isolated from cancer patients To provide information for predicting or diagnosing EGFR target therapy resistance in cancer in which EGFR mutations occur, including measuring the expression or activity level of one or more genes selected from the group consisting of or proteins encoded by the genes provides a way

In the method according to the present invention, the cancer is a cancer to which an EGFR target therapy is applied, and may be, for example, breast cancer, colorectal cancer, head and neck cancer, non-small cell lung cancer, pancreatic cancer, squamous cell cancer or thyroid cancer, but is not limited thereto. In addition, the cancer is a cancer having EGFR mutation, more specifically, a cancer in which EGFR T790M mutation occurs.

In the method according to the present invention, the contents of the EGFR target therapeutic agent are as described above.

In the method according to the present invention, the method for measuring the expression or activity level of the gene or the protein encoded by the gene may be performed including a known process of isolating mRNA or protein from a biological sample using a known technique. can

The biological sample refers to a sample collected from a living body in which the expression or activity level of the gene or protein is different from that of a normal control according to the development or progression of resistance to an EGFR target therapeutic agent, and the sample is, for example, not limited thereto. but may include tissues, cells, blood, serum, plasma, saliva and urine.

The measurement of the expression level of the gene is specifically to measure the level of mRNA, and methods for measuring the level of mRNA include reverse transcription polymerase chain reaction (RT-PCR), real-time reverse transcription polymerase chain reaction, RNase protection assay, Northern blot and DNA chips, but are not limited thereto.

The protein level may be measured using an antibody. In this case, the protein in the biological sample and an antibody specific thereto form a binding product, that is, an antigen-antibody complex, and the amount of the antigen-antibody complex formed is a detection label ( It can be measured quantitatively through the size of the signal of the detection label). These detection labels may be selected from the group consisting of enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules, and radioactive isotopes, but are not limited thereto. Assay methods for measuring protein levels include, but are not limited to, Western blot, ELISA, radioimmunoassay, radioimmunoassay, Oukteroni immunodiffusion assay, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, There are complement fixation assay, FACS, and protein chip.

Therefore, the present invention can confirm the mRNA or protein level of the control group and the mRNA or protein level of a cancer patient resistant to an EGFR target therapy or a suspected cancer patient resistant to an EGFR target therapy through the detection methods described above. By comparing the amount or the degree of activity with the control group, it is possible to diagnose the presence or absence of resistance to EGFR-targeted therapeutics due to EGFR mutation and the stage of progression.

Specifically, at least one gene selected from the group consisting of OTOA, LOC400706, PARD6B, SPTB, PCDH9, PRDM1, DNAJB5, CNKSR3, and SAA1, or a protein encoded by the gene Expression or activity level measurement results,

Compared to normal control samples, the expression or activity level of at least one gene selected from the group consisting of OTOA and LOC400706 or a protein encoded by the gene is increased, or the group consisting of PARD6B, SPTB, PCDH9, PRDM1, DNAJB5, CNKSR3 and SAA1 If the expression or activity level of at least one gene selected from or the gene encoded by the gene is reduced, the cancer patient may have an EGFR mutation, which may be determined to be initially resistant after administration of an EGFR target therapy.

In addition, at least one gene selected from the group consisting of SERPINB2, ZNF396, HNRNPA1L2, C8orf31, CCDC115, ZNF718, LSM4, FN1, HOPX and BANK1 or the expression or activity level of a protein encoded by the gene is measured,

One or more genes selected from the group consisting of SERPINB2, ZNF396, HNRNPA1L2, C8orf31, CCDC115, LSM4, FN1, HOPX, and BANK1, wherein the expression or activity level of the ZNF718 gene or a protein encoded by the gene is increased compared to a normal control sample Alternatively, if the expression or activity level of the protein encoded by the gene decreases, the cancer patient may have EGFR mutation, and it may be determined that the drug is resistant to the late stage after administration of an EGFR target therapy.

In addition, the present invention

1) Overexpression of one or more genes selected from the group consisting of OTOA, LOC400706 and ZNF718 in cancer cells showing sensitivity to EGFR target therapy, or PARD6B, SPTB, PCDH9, PRDM1, DNAJB5, CNKSR3, SAA1, SERPINB2, ZNF396, HNRNPA1L2, Inhibiting the expression of one or more genes selected from the group consisting of C8orf31, CCDC115, LSM4, FN1, HOPX and BANK1; and

2) It provides a method for producing an EGFR-targeted therapeutic agent-resistant cell model, including the step of treating the cells of step 1) with an EGFR-targeted therapeutic agent.

In the method according to the present invention, the cancer cells are cancer cells in which EGFR is abnormally activated, and may be, for example, breast cancer, colon cancer, head and neck cancer, non-small cell lung cancer, pancreatic cancer, squamous cell cancer or thyroid cancer cells, but are not limited thereto. don't

In addition, the description of the EGFR target therapeutic agent is as described above.

The present inventors used cancer cell lines showing sensitivity to EGFR target therapy to treat resistance refractory clones, monoclonal cells in which erlotinib resistance was induced by newly occurring EGFR T790M mutation (T790M-bearing resistant clones), and Monoclonal cells (non-T790M-bearing resistant clones) in which resistance was induced without EGFR T790M mutation were prepared, and gene expression profiling was performed using these cells, and genes involved in EGFR target therapy resistance induced by EGFR T790M mutation were induced. was derived, and since it was confirmed that erlotinib resistance was induced in cells suppressing or overexpressing the derived gene expression, cells suppressing or overexpressing the gene expression could be used for the preparation of EGFR target therapy resistant cell models.

In addition, the present invention

1) Overexpression of one or more genes selected from the group consisting of OTOA, LOC400706 and ZNF718 in cancer cells showing sensitivity to EGFR target therapy, or PARD6B, SPTB, PCDH9, PRDM1, DNAJB5, CNKSR3, SAA1, SERPINB2, ZNF396, HNRNPA1L2, Inhibiting the expression of one or more genes selected from the group consisting of C8orf31, CCDC115, LSM4, FN1, HOPX and BANK1; and

2) Provides a method for screening EGFR targeted therapy resistance inhibitors, comprising the steps of treating the cells of step 1) with the EGFR targeted therapy and a test substance and measuring drug reactivity.

In addition, the present invention

1) Overexpression of one or more genes selected from the group consisting of OTOA, LOC400706 and ZNF718 in cancer cells showing sensitivity to EGFR target therapy, or PARD6B, SPTB, PCDH9, PRDM1, DNAJB5, CNKSR3, SAA1, SERPINB2, ZNF396, HNRNPA1L2, Inhibiting the expression of one or more genes selected from the group consisting of C8orf31, CCDC115, LSM4, FN1, HOPX and BANK1; and

2) It provides a method for screening EGFR target therapy drug-resistant cancer therapeutics, including the steps of treating the cells of step 1) with a test substance and measuring drug reactivity.

In the method according to the present invention, descriptions of the cancer cells and the EGFR targeting agent are as described above.

In the method according to the present invention, the test substance in step 2) may be an oligonucleotide, peptide, protein, non-peptidic compound, active compound, fermentation product, metabolite, enzyme, antibody or bioactive molecule, but is limited thereto. It doesn't work.

The oligonucleotide may be an antisense-oligonucleotide for the gene, siRNA, shRNA, miRNA, or a vector containing them. These antisense-oligonucleotides, siRNA, shRNA, miRNA or vectors containing them can be prepared using methods known in the art.

The "antisense nucleotide", as defined in Watson-Crick base pairing, interferes with the flow of genetic information from DNA to protein by binding (hybridizing) to complementary nucleotide sequences of DNA, immature-mRNA or mature mRNA. The specific nature of antisense nucleotides for their target sequences makes them exceptionally multifunctional. Since antisense nucleotides are long chains of monomeric units, they can be easily synthesized against the target RNA sequence. A number of recent studies have demonstrated the usefulness of antisense nucleotides as a biochemical means to study target proteins (Rothenberg et al., J. Natl. Cancer Inst., 81:1539-1544, 1999). Since many recent advances have been made in the field of oligonucleotide chemistry and the synthesis of nucleotides that exhibit improved cell adhesion, target binding affinity and nuclease resistance, the use of antisense nucleotides can be considered as a new type of inhibitor.

The "siRNA" refers to a double-stranded RNA that induces RNA interference through cleavage of mRNA of a target gene, and is composed of an RNA strand of a sense sequence having the same sequence as the mRNA of a target gene and an antisense sequence having a complementary sequence thereto. It is composed of RNA strands. The siRNA may include siRNA itself synthesized in vitro or a form expressed by inserting a nucleotide sequence encoding the siRNA into an expression vector.

The "shRNA" means a single strand composed of 50-60 nucleotides, and forms a stem-loop structure in vivo . That is, shRNA is an RNA sequence that creates a tight hairpin structure to inhibit gene expression through RNA interference (RNAi). Long RNAs of 15-30 nucleotides complementary to both sides of the loop of 5-10 nucleotides are base-paired to form a double-stranded stem. shRNAs are generally transduced into cells via a vector containing a U6 promoter for expression and are usually passed on to daughter cells to allow inheritance of gene repression. The shRNA hairpin structure is cleaved by intracellular mechanisms to become siRNA, which then binds to RNA-induced silencing complex (RISC). These RISCs bind to and cleave mRNA. shRNA is transcribed by RNA polymerase.

The vector refers to a genetic construct comprising foreign DNA inserted into a genome encoding a polypeptide. The vector related to the present invention is a vector in which a nucleic acid sequence that inhibits the gene is inserted into the genome, and examples of these vectors include DNA vectors, plasmid vectors, cosmid vectors, bacteriophage vectors, yeast vectors, or viral vectors.

The "miRNA" refers to a short non-coding RNA consisting of about 22 nucleotide sequences. It is known to function as a post-transcriptional regulator in the process of gene expression. By complementarily binding to target mRNAs having complementary nucleotide sequences, target mRNAs are degraded or translation into proteins is inhibited.

In addition, the compound may have a low molecular weight therapeutic effect, for example, a compound with a weight of about 1000 Da, such as 400 Da, 600 Da or 800 Da. Depending on the purpose, these compounds may constitute part of a compound library, and the number of compounds constituting the library may vary from tens to millions. Such compound libraries include peptides and other cyclic or linear oligomeric compounds, and template-based small molecule compounds, such as benzodiazepines, hydantoins, biaryl, carbocyclic and polycyclic compounds (such as naphthalene, phenothiazine, acri deine, steroids, etc.), carbohydrates and amino acid derivatives, dihydropyridine, benzhydryl and heterocycles (such as triazine, indole, thiazolidine, etc.), but this is only illustrative and not limited thereto. .

Therefore, the present invention treats the test substance as described above with an EGFR-targeted therapy in combination, and/or treats the test substance and measures the drug reactivity, thereby converting the test substance showing drug reactivity into an EGFR-targeted therapy resistance inhibitor or an EGFR-targeted therapy-resistant cancer treatment. can be selected

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are only to illustrate the present invention, and the content of the present invention is not limited to the following examples.

<Example 1> Cell model production for screening genes involved in the induction of EGFR target therapy resistance

As shown in the schematic diagram of FIG. 1, monoclonal cells without the EGFR T790M mutation were isolated from cancer cell lines showing sensitivity to EGFR-targeted therapeutics, and drug resistance was induced by treating the isolated monoclonal cells with EGFR-targeted therapeutics for a long period of time. As a result, 3 Types of monoclonal cells, specifically, monoclonal cells incapable of inducing resistance, monoclonal cells in which erlotinib resistance was induced by newly occurring EGFR T790M mutation (T790M-bearing resistant clones), and monoclonal cells in which resistance was induced without EGFR T790M mutation (non-T790M-bearing resistant clones) were obtained.

<1-1> Isolation of monoclonal cells from cancer cell lines showing sensitivity to EGFR targeted therapy

PC9 cells, a non-small cell lung cancer cell line, have an EGFR del19 mutation, and show a short-term response to Erlotinib, a first-generation EGFR-targeted therapy, but a long-term response of 6 months to 1 year. It is known that tolerance develops upon treatment. Accordingly, as a cancer cell line exhibiting sensitivity to EGFR target therapeutics, single claw cells were isolated from PC9 cells.

Specifically, PC9 cells were cultured in RPMI-1640 medium (Corning, USA) supplemented with 10% fetal bovine serum (FBS, Merck, USA) and 1% antibiotics (Invitrogen, USA) under 5% CO ₂ , 37°C conditions. cultured in a cell culture medium. For single cell isolation, 10 ml of a mixture of RPMI-1640 medium, 10% FBS, 1% antibiotic (penicillin-streptomycin, RPMI 10%) and 0.5% Select agar was added to a 100 mm cell culture dish. After adding, it was hardened at room temperature for 30 minutes. Thereafter, 5 ml of a mixture of 1×10 ³ PC9 cells and RPMI-1640 medium 10% and 0.4% select agar was dispensed onto the dish, and then solidified at room temperature for 15 minutes. Then, after confirming that the cells were separated into single cells in the dish, they were cultured in a cell culture medium under 5% CO ₂ and 37°C conditions.

After 2-3 weeks, a single cell line with a colony was taken using a 200 μl tip, and then dispensed into a 96-well plate containing 100 μl of culture solution (RPMI-1640 medium, 10% FBS, 1% PS mixture). . After that. When the cell density of each well plate reached 80% at intervals of 5 to 7 days, monoclonal cells were obtained by sequentially transferring to large plates such as a 24-well plate, a 12-well plate, and a 6-well plate.

In addition, when cells with a density of 80% in a 6-well plate were transferred to a 100 mm cell culture dish, 40% were subjected to DNA extraction using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer's procedure. Using the extracted DNA, to amplify the EGFR T790 site, forward primer: 5'-TGA AAC TCA AGA TCG CAT TC-3' and reverse primer: 5'-ATA TCC CCA TGG CAA PCR was performed using ACT CTT-3' and ExPrime TaqTM DNA Polymerase (Genet bio). Thereafter, Sanger sequencing was performed to confirm the presence of the EGFR T790M mutation.

As a result, as shown in FIG. 2, it was confirmed that the EGFR T790M mutation, which is well known as an existing resistance induction mechanism, does not exist in all 30 monoclonal cells.

<1-2> Selection of monoclonal cells incapable of inducing EGFR-targeted therapy resistance in monoclonal cells

Each of the 30 monoclonal cells obtained in Example <1-1> was dispensed in a 96-well plate at 5×10 ³ cells/well and treated with 1 μM of erlotinib. It was confirmed whether resistance was induced by changing to a new drug every 3-4 days and observing cell proliferation. When the reactivity to erlotinib is no longer seen and the proliferation of the cells is confirmed, they are sequentially transferred to a large plate and Sanger sequencing is performed in the same manner as described in Example <1-1> to determine the presence of the EGFR T790M mutation. Confirmed.

Through this, as shown in FIG. 1, resistance-inducible monoclonal cells in which resistance is not induced at all, monoclonal cells in which resistance is induced by EGFR T790M mutation, and monoclonal cells in which resistance is induced by causes other than EGFR T790M mutation Cells were identified and isolated. Specifically, the time point at which cell proliferation was confirmed without showing reactivity to erlotinib was about 6 months after treatment with erlotinib. At this time, 16 monoclonal cells with erlotinib resistance were identified. Seven of these (non-T790M-bearing resistant clones) were confirmed to induce resistance without EGFR T790M mutation. That is, it was confirmed that resistance was induced by a cause other than the EGFR T790M mutation. In addition, it was confirmed that the remaining 9 species (T790M-bearing resistant clones) were newly generated with the previously well-known EGFR T790M mutation, resulting in resistance.

In addition, it was confirmed that the 14 types of monoclonal cells (resistance refractory clones) were monoclonal cells incapable of inducing resistance to erlotinib.

In addition, as shown in Figures 3 and 4, 12 clones derived from the 9 types of monoclonal cells (T790M-bearing resistant clones) were secured, and these EGFR T790M mutation-induced EGFR target therapy resistance resistance of the monoclonal cells It was confirmed that the acquisition time point was different for each clone.

<Example 2> EGFR mutation-induced EGFR target therapy resistance-related gene screening

If there is a difference in the expression of genes between cells before EGFR mutation occurs and after EGFR mutation occurs and resistance is acquired by treatment with EGFR-targeted therapy, it can be seen that these genes are involved in EGFR mutation, and this induces resistance to EGFR-targeted therapy. there is. Accordingly, in order to confirm the level of mRNA expression between the EGFR T790M mutation-induced EGFR-targeted therapy-resistant monoclonal cells of Example <1-2> and parental cells before the EGFR T790M mutation of the cells newly occurred, expression profiling was performed through the NGS method. (expression profiling) was performed.

Specifically, the parental cells of the EGFR T790M mutation-induced EGFR-targeted therapy-resistant monoclonal cells of Example <1-2> before erlotinib treatment were seeded in a 100 mm cell culture dish at a cell number of 5×10 ⁶ and then transferred to a serum-free medium. The experiment was conducted by dividing the cells into a condition not treated with 1 μM erlotinib and a condition treated with 1 μM erlotinib for 24 hours. In addition, EGFR T790M mutation-induced EGFR-targeted therapy-resistant monoclonal cells of Example <1-2> were aliquoted in the same amount as the parental cells, and treated with 1 μM of erlotinib for 24 hours. In addition, monoclonal cells in which resistance was induced without the EGFR T790M mutation of Example <1-2> were divided in the same manner as above and treated with erlotinib. Then, mRNA was extracted from the cells using RNeasy Mini Kit (Qiagen), expression profiling data was obtained using NovaSeq 6000, and profile analysis (p-value and GSEA) was performed.

The P-value based gene selection method was as follows. Among EGFR T790M mutation-induced EGFR-targeted therapy-resistant monoclonal cells, 6 clones that acquired resistance early (early, before 100 days) and 6 clones that acquired resistance later (after 100 days) were each erlotinib. DEG analysis was performed with parental cells that were not treated. After that, based on the calculated p-value, 600 genes of the upper/lower rank were selected (Early = 600, Late = 600). After that, in order to select genes showing a fold change of 1.5 fold or more in 5 or more of each 6 samples, and then selecting genes that show changes due to EGFR T790M mutation, not changes caused by erlotinib treatment, Compared to the group treated with erlotinib, genes whose fold change was up-regulated by 1.2 times or more or down-regulated by 1.2 times or more were removed. Lastly, in order to confirm changes that appear specifically only in the EGFR T790M mutation, compared to the monoclonal cells in which resistance was induced without the EGFR T790M mutation, the fold change was more than 1.5-fold in the EGFR T790M mutation-induced EGFR target therapy-resistant monoclonal cells. And fold change is -1.2 fold or less in monoclonal cells in which resistance is induced without EGFR T790M mutation, or fold change is -1.5 fold or less in monoclonal cells resistant to EGFR T790M mutation-induced EGFR target therapy and resistance is induced without EGFR T790M mutation Genes with a fold change of 1.2 fold or more in monoclonal cells were selected.

The GSEA-based gene selection method was as follows. Among EGFR T790M mutation-induced EGFR-targeted therapy-resistant monoclonal cells, 6 clones that acquired resistance early (before 100 days) and their fold change values of parental cells not treated with erlotinib or EGFR T790M mutation-induced EGFR-targeted therapy resistance After ranking all genes based on the fold change value of 6 clones that acquired resistance at late stages (after 100 days) among monoclonal cells and their parental cells not treated with erlotinib, Hallmark gene set and C6 gene Using the set, a signal transduction system that is statistically significantly regulated was secured, and all genes (Leading edge genes) that played an important role in securing such a signal transduction system were secured. From this, Early selected 1725 leading edge genes and Late selected 1694 leading edge genes. After that, gene selection was performed in the same way as the p-value-based gene selection method.

As a result, as shown in FIGS. 5 to 8, nine genes [OTOA, LOC400706, PARD6B, SPTB, PCDH9, PRDM1, DNAJB5, CNKSR3, SAA1] expression showed a statistically significant change when compared to the parental control group. In addition, expression of 10 genes [SERPINB2, ZNF396, HNRNPA1L2, C8orf31, CCDC115, ZNF718, LSM4, FN1, HOPX, BANK1] in monoclonal cells resistant to EGFR T790M mutation-induced EGFR target therapy that acquired resistance at the late stage (after 100 days) It was confirmed that a statistically significant change was observed when compared to the parental control group.

Through the above results, it can be seen that the 19 genes or proteins encoded therefrom are involved in the generation of EGFR T790M mutation and the induction of resistance to EGFR target therapy agents, and these are EGFR targets induced by EGFR mutations such as EGFR T790M mutation. It can be seen that it can be used as a biomarker for predicting drug resistance.

<Example 3> EGFR mutation induction Confirmation of induction of EGFR target therapy resistance through suppression and overexpression of genes involved in EGFR target therapy resistance

In order to determine whether the gene involved in EGFR target therapy resistance induced by the EGFR mutation identified in <Example 2> is involved in the induction of EGFR target therapy resistance, involvement in EGFR target therapy resistance induced by the EGFR mutation identified in <Example 2> Induction of resistance to EGFR target therapy was confirmed using cells that suppress or overexpress gene expression.

Specifically, EGFR induced by the EGFR mutation identified in <Example 2> was obtained by randomly selecting two types of inducible resistant monoclonal cells obtained in Example <1-2> (PC9-1 and PC9-6). Among the genes involved in resistance to targeted therapy, genes with decreased expression compared to the control group [PARD6B, SPTB, PCDH9, PRDM1, DNAJB5, CNKSR3, SAA1, SERPINB2, ZNF396, HNRNPA1L2, C8orf31, CCDC115, ZNF718, LSM4, FN1, HOPX, BANK1] shRNA was produced by requesting Sigma. Then, lentiviruses were prepared using each of the prepared shRNAs. 293T cells (culture medium conditions = DMEM medium, 10% FBS, 1% PS) were dispensed with the virus in a 6-well cell culture dish (Corning, USA), and after confirming that the cell density was 30% after 24 hours, pMD2. G (addgene, 12259) 0.5 μg, psPAX2 (addgene, 12260) 0.5 μg, ORF DNA (each of 589 ORF library introduction vectors) 1 μg, Fugene (Promega, USA) 6 μl and optimem (Gibco, UK) 100 μl A total of 51 viruses were prepared by mixing. Then, two kinds of monoclonal cells incapable of induction of resistance were randomly selected (PC9-1 and PC9-6), cultured in a 6-well plate at 3×10 ⁵ cells/well, and the culture medium was removed after 24 hours, 1 ml of a mixture of the culture medium and the virus in a ratio of 1:1 was treated. After 16 hours, the mixture of the culture medium and the virus was removed and a new culture medium was added. After 48 hours, 2 μg/ml of puromycin was treated for 72 hours to select only virus-infected cells, thereby obtaining 51 cell lines in which the gene expression was suppressed. Then, 5×10 ³ cell/well was dispensed into a 96-well plate, and erlotinib 1 μM was treated after 24 hours. Then, 48 hours later, the culture medium was replaced with a new one and erlotinib 1 μM was treated. Thereafter, the culture medium and erlotinib were replaced with new ones every 7 days. In addition, cell growth before replacement was observed under a microscope to confirm drug reactivity. In addition, to ensure the validity of the experiment, the experiment was repeated twice.

In addition, cDNA for genes [OTOA, LOC400706, ZNF718] whose expression is increased compared to the control group among genes involved in EGFR target therapy resistance induced by the EGFR mutation identified in <Example 2> were ordered and secured from origene. Two types of inducible resistant monoclonal cells (PC9-1 and PC9-6) were randomly selected and dispensed into 6-well cell culture dishes (Corning, USA), and after 24 hours, it was confirmed that the cell density reached 30%, Viruses were prepared by mixing 2 μg of the obtained cDNA (each cDNA introduction vector), 6 μl of Fugene (Promega), and 100 μl of optimem (Gibco). To transfect the virus, the monoclonal cells incapable of inducing resistance were seeded in a 96-well plate at 5×10 ³ cell/well, and the culture medium was removed after 24 hours. Then, after adding 70 μl of the virus and 10 μg/ml of polybrene, the cells were cultured for 12 hours in a cell incubator under 5% CO ₂ and 37°C conditions. After removing the virus after 12 hours, 100 μl of RPMI-1640 medium supplemented with 10% FBS and 1% antibiotics was added. After 48 hours, G418 was treated for 72 hours to select only virus-infected cells. The sorted cells were dispensed in a 96-well plate at 5×10 ³ cell/well and treated with 1 μM of erlotinib. After 48 hours, the culture medium was replaced with a new one, and erlotinib 1 μM was treated. Thereafter, the culture medium and erlotinib were replaced with new ones every 7 days. In addition, cell growth before replacement was observed under a microscope to confirm drug reactivity. In addition, to ensure the validity of the experiment, the experiment was repeated twice.

On the other hand, the experimental results are shown for cells in which HOPX, PRDM1, and DNAJB5 gene expression was suppressed.

As a result, as shown in FIG. 9 , it was confirmed that erlotinib resistance was induced in all cells in which the expression of the genes was suppressed by treatment with shRNAs against HOPX, PRDM1, and DNAJB5.

Claims (22)

One or more genes selected from the group consisting of OTOA, LOC400706, PARD6B, SPTB, PCDH9, PRDM1, DNAJB5, CNKSR3, SAA1, SERPINB2, ZNF396, HNRNPA1L2, C8orf31, CCDC115, ZNF718, LSM4, FN1, HOPX and BANK1, or the gene A biomarker for predicting or diagnosing EGFR target therapy resistance induced by EGFR mutation, including a protein encoded by.
The method of claim 1, wherein at least one gene selected from the group consisting of OTOA, LOC400706, PARD6B, SPTB, PCDH9, PRDM1, DNAJB5, CNKSR3 and SAA1 or a protein encoded by the gene is EGFR mutated to target EGFR Characterized in that for predicting or diagnosing initial resistance after administration of a therapeutic agent, a biomarker for predicting or diagnosing EGFR target therapy resistance induced by EGFR mutation.
The method of claim 1, wherein at least one gene selected from the group consisting of SERPINB2, ZNF396, HNRNPA1L2, C8orf31, CCDC115, ZNF718, LSM4, FN1, HOPX and BANK1 or a protein encoded by the gene is EGFR mutation occurs A biomarker for predicting or diagnosing EGFR target therapy resistance induced by EGFR mutation, characterized in that for predicting or diagnosing late resistance after administration of EGFR targeted therapy.
The method of claim 1, wherein the EGFR target therapy is erlotinib, gefitinib, afatinib, dacomitinib, osimertinib, lazertinib , Pelitinib, Neratinib, Icotinib, brigatinib, lapatinib, canertinib, vandetanib, PKI-166 and AEE788 A biomarker for predicting or diagnosing EGFR target therapy resistance induced by EGFR mutation, characterized in that at least one selected from the group consisting of.
The biomarker for predicting or diagnosing resistance to EGFR-targeted therapeutics induced by EGFR mutation according to claim 4, wherein the EGFR-targeted therapeutics is erlotinib.
According to claim 1, wherein the EGFR mutation is characterized in that the EGFR T790M mutation, a biomarker for predicting or diagnosing EGFR target therapy resistance induced by EGFR mutation.
One or more genes selected from the group consisting of OTOA, LOC400706, PARD6B, SPTB, PCDH9, PRDM1, DNAJB5, CNKSR3, SAA1, SERPINB2, ZNF396, HNRNPA1L2, C8orf31, CCDC115, ZNF718, LSM4, FN1, HOPX and BANK1, or the gene A composition for predicting or diagnosing EGFR target therapy resistance induced by EGFR mutation, including an agent for measuring the expression or activity level of a protein encoded by.
The method of claim 7, wherein the agent for measuring the gene expression level is characterized in that at least one of antisense oligonucleotides, primer pairs and probes that specifically bind to mRNA of the gene, EGFR target induced by EGFR mutation A composition for predicting or diagnosing drug resistance.
The method of claim 7, wherein the agent for measuring the protein expression or activity level is characterized in that one or more of antibodies, peptides and nucleotides that specifically bind to the protein, EGFR target drug resistance prediction induced by EGFR mutation or a diagnostic composition.
Selected from the group consisting of OTOA, LOC400706, PARD6B, SPTB, PCDH9, PRDM1, DNAJB5, CNKSR3, SAA1, SERPINB2, ZNF396, HNRNPA1L2, C8orf31, CCDC115, ZNF718, LSM4, FN1, HOPX and BANK1 in samples isolated from cancer patients A method for providing information for predicting or diagnosing EGFR target therapy resistance in cancer in which EGFR mutations occur, comprising measuring the expression or activity level of at least one gene or a protein encoded by the gene.
The method of claim 10, wherein the cancer is characterized by one or more of breast cancer, colorectal cancer, head and neck cancer, non-small cell lung cancer, pancreatic cancer, squamous cell cancer and thyroid cancer, EGFR target drug resistance prediction in cancer in which EGFR mutation occurs or How to provide information for diagnosis.
The method of claim 10, wherein the sample is at least one of tissue, cell, blood, serum, plasma, saliva and urine, characterized in that, information for predicting or diagnosing EGFR target therapy resistance in cancer in which EGFR mutations occur How to provide.
11. The method of claim 10, wherein at least one gene selected from the group consisting of OTOA, LOC400706, PARD6B, SPTB, PCDH9, PRDM1, DNAJB5, CNKSR3 and SAA1 or a protein encoded by the gene Expression or activity level measurement result,
Compared to normal control samples, the expression or activity level of at least one gene selected from the group consisting of OTOA and LOC400706 or a protein encoded by the gene is increased, or the group consisting of PARD6B, SPTB, PCDH9, PRDM1, DNAJB5, CNKSR3 and SAA1 When the expression or activity level of one or more genes selected from or the gene encoded by the gene is reduced, the cancer patient has EGFR mutation, characterized in that it is judged to be initially resistant after administration of EGFR target therapy, EGFR A method for providing information for predicting or diagnosing EGFR target therapy resistance in cancers in which mutations occur.
The method of claim 10, wherein at least one gene selected from the group consisting of SERPINB2, ZNF396, HNRNPA1L2, C8orf31, CCDC115, ZNF718, LSM4, FN1, HOPX and BANK1 or a protein encoded by the gene is measured for expression or activity level ,
One or more genes selected from the group consisting of SERPINB2, ZNF396, HNRNPA1L2, C8orf31, CCDC115, LSM4, FN1, HOPX, and BANK1, wherein the expression or activity level of the ZNF718 gene or a protein encoded by the gene is increased compared to a normal control sample Or, when the expression or activity level of the protein encoded by the gene decreases, the cancer patient has an EGFR mutation and is judged to be resistant to the late stage after administration of an EGFR target therapy. A method for providing information for predicting or diagnosing EGFR target therapy resistance.
11. The method of claim 10, wherein the EGFR target therapy is erlotinib, gefitinib, afatinib, dacomitinib, osimertinib, lazertinib, felitinib, neratinib, icotinib, brigatinib, lapatinib, ca A method for providing information for predicting or diagnosing EGFR target therapy resistance in cancer in which EGFR mutations occur, characterized in that at least one selected from the group consisting of nertinib, vandetanib, PKI-166 and AEE788.
16. The method of claim 15, wherein the EGFR-targeted therapy is erlotinib.
11. The method of claim 10, wherein the mutation is an EGFR T790M mutation, a method for providing information for predicting or diagnosing resistance to EGFR target therapy in cancers in which EGFR mutations occur.
1) Overexpression of one or more genes selected from the group consisting of OTOA, LOC400706 and ZNF718 in cancer cells showing sensitivity to EGFR target therapy, or PARD6B, SPTB, PCDH9, PRDM1, DNAJB5, CNKSR3, SAA1, SERPINB2, ZNF396, HNRNPA1L2, Inhibiting the expression of one or more genes selected from the group consisting of C8orf31, CCDC115, LSM4, FN1, HOPX and BANK1; and
2) A method for producing an EGFR-targeted therapeutic agent-resistant cell model comprising the step of treating the cells of step 1) with an EGFR-targeted therapeutic agent.
1) Overexpression of one or more genes selected from the group consisting of OTOA, LOC400706 and ZNF718 in cancer cells showing sensitivity to EGFR target therapy, or PARD6B, SPTB, PCDH9, PRDM1, DNAJB5, CNKSR3, SAA1, SERPINB2, ZNF396, HNRNPA1L2, Inhibiting the expression of one or more genes selected from the group consisting of C8orf31, CCDC115, LSM4, FN1, HOPX and BANK1; and
2) A method for screening EGFR-targeted therapeutics resistance inhibitors comprising the steps of treating the cells of step 1) with the EGFR-targeted therapeutics and the test substance and measuring drug reactivity.
The method of claim 19, wherein the test substance in step 2) is one selected from the group consisting of oligonucleotides, peptides, proteins, non-peptide compounds, active compounds, fermentation products, metabolites, enzymes, antibodies, and bioactive molecules. Characterized in that the above, the screening method of the EGFR target therapy drug resistance inhibitor.
1) Overexpression of one or more genes selected from the group consisting of OTOA, LOC400706 and ZNF718 in cancer cells showing sensitivity to EGFR target therapy, or PARD6B, SPTB, PCDH9, PRDM1, DNAJB5, CNKSR3, SAA1, SERPINB2, ZNF396, HNRNPA1L2, Inhibiting the expression of one or more genes selected from the group consisting of C8orf31, CCDC115, LSM4, FN1, HOPX and BANK1; and
2) A method of screening for a cancer treatment resistant to EGFR target therapy, comprising the step of treating the cells of step 1) with a test substance and measuring drug reactivity.
The method of claim 21, wherein the test substance in step 2) is one selected from the group consisting of oligonucleotides, peptides, proteins, non-peptide compounds, active compounds, fermentation products, metabolites, enzymes, antibodies, and bioactive molecules. Characterized in that the above, the screening method for EGFR target therapy resistant cancer therapeutics.

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