WO2016200120A1 - Gene associated with ibrutinib susceptibility in glioblastoma patients, and use therefor - Google Patents

Abstract

The present invention provides: a method of assessing susceptibility or prognosis of glioblastoma patients with respect to ibrutinib, by testing for the presence of EGFRvⅢ mutation; and a kit for predicting the susceptibility of glioblastoma patients to ibrutinib. Use of the present invention makes it possible to predict reactivity to ibrutinib, which is a BTK/BMX/BLK specific inhibitor, in glioblastoma patients. The present invention can advantageously be used as evidence for allowing the administration of ibrutinib to EGFRvⅢ mutation patients.

Description

Genes Associated with Ibrutinib Sensitivity in Glioblastoma Patients and Their Uses

The present invention was made by the task number HI14C3418 under the support of the Ministry of Health and Welfare of the Republic of Korea, the research and management institution of the task is Korea Health Industry Development Institute, the research project name is "health medical research and development project", the research title is "leading intractable cancer research Project Group ”, the lead institution is Samsung Seoul Hospital, and the research period is from December 1, 2014 to November 30, 2015.

This patent application claims priority to Korean Patent Application No. 10-2015-0083534, filed with the Korean Patent Office on June 12, 2015, the disclosure of which is hereby incorporated by reference.

The present invention relates to genes associated with ibrutinib sensitivity and their use in glioblastoma patients.

Glioblastoma is the most common and deadly brain cancer, accounting for 60% of primary brain tumors. To date, glioblastoma is a very poor tumor with an average survival rate of about 15 months in radiation therapy and temozolomide-based therapy (1-3). Thus, there is a need for innovative glioblastoma treatment strategies. Recently, biomarkers of glioblastoma have been discovered through large-scale tumor genome analysis such as TCGA. Among them, EGFRvIII mutations found in about 15% of all glioblastomas are known as representative tumor-causing gene mutations, and researches on therapies targeting these mutations are being actively conducted.

Ibrutinib is a target anticancer agent specific to Bruton's tyrosine kinase (BTK), which is licensed for chronic lymphocytic leukemia. And genetic biomarkers are diversifying.

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

Prior art literature

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The present inventors have made extensive research efforts to select a group of genes related to the sensitivity of ibrutinib in glioblastoma patients. As a result, the present invention was completed by elucidating that the selected EGFRvIII (EGFRvIII) mutation is a predictor of ibrutinib response in glioblastoma patients.

Accordingly, it is an object of the present invention to provide a method for examining the presence of EGFRvIII mutations to provide useful information in determining the sensitivity or prognosis of ibrutinib in patients with glioblastoma.

Another object of the present invention is to provide a kit for predicting susceptibility to ibrutinib in glioblastoma patients.

Other objects and advantages of the present invention will become apparent from the following detailed description and claims.

The present invention provides a method for testing for the presence of EGFRvIII mutations to provide useful information in determining susceptibility or prognosis for ibrutinib in a glioblastoma multiforme patient comprising the following steps:

(a) isolating a nucleic acid molecule or protein from a biological sample isolated from the subject; And

(b) (i) detecting the polynucleotide of SEQ ID NO: 1 from the isolated nucleic acid molecule or (ii) detecting the protein of SEQ ID NO: 2 from the separated protein Or if the amino acid of SEQ ID NO: 2 is detected, the subject has increased ibrutinib sensitivity.

The present inventors have made intensive studies to select a group of genes related to ibrutinib susceptibility in glioblastoma patients, and as a result, the selected EGFRvIII mutation is a predictor of ibrutinib response in glioblastoma patients. It was.

As shown in the following examples, the specific susceptibility of ibrutinib in glioblastoma EGFRvIII mutations was confirmed by screening for glioblastoma-targeted anticancer agent derived from the patient, thereby predicting the sensitivity to ibrutinib.

As used herein, the term “ibrutinib susceptibility” refers to responsiveness to ibrutinib, and glioblastoma patients with EGFRvIII mutations exhibit increased sensitivity to ibrutinib, compared to glioblastoma patients without EGFRvIII mutations. The treatment effect by ibrutinib is excellent.

As used herein, the term “biological sample” refers to a biological sample of a glioblastoma patient separated in vitro and refers to a blood, plasma, serum, urine, cell, hair, or tissue sample.

As used herein, the term “nucleic acid molecule” is meant to encompass DNA (gDNA and cDNA) and RNA molecules inclusively, and the nucleotides, which are the basic structural units in nucleic acid molecules, are modified from sugar or base sites, as well as natural nucleotides. Analogues (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).

If the starting material in the process of the invention is gDNA, isolation of gDNA can be carried out according to conventional methods known in the art (Rogers & Bendich (1994)).

If the starting material is mRNA, total RNA is isolated and performed by conventional methods known in the art (see Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press ( Ausubel, FM et al., Current Protocols in Molecular Biology, John Willey & Sons (1987); and Chomczynski, P. et al., Anal. Biochem. 162: 156 (1987)). The isolated total RNA is synthesized into cDNA using reverse transcriptase. Since the total RNA is isolated from animal cells, the end of the mRNA has a poly-A tail, and in the method of the oligo dT prabon using the sequence characteristics, in the step (b), the first sequence listing The method for detecting the polynucleotide of may be carried out by applying various methods known in the art.

According to one embodiment of the invention, the detection of the polynucleotide of SEQ ID NO: 1 sequence is PCR (Polymerase Chain Reaction), hybridization (hybridization), sequencing, pyrosequencing or next generation sequencing (NGS) The method can be used.

In addition, in the present invention, a technique applicable to the detection of polynucleotide of SEQ ID NO: 1 sequence, fluorescence in situ hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern blot analysis, single-strand conformation analysis ( SSCA, Orita et al., PNAS, USA 86: 2776 (1989)), RNase protection assay (Finkelstein et al., Genomics, 7: 167 (1990)), dot blot analysis, denaturation gradient gel electrophoresis (DGGE, Wartell) et al., Nucl. Acids Res., 18: 2699 (1990)), using methods that recognize nucleotide mismatches (eg, mutS proteins of E. coli ) (Modrich, Ann. Rev. Genet., 25: 229-253 (1991)) and gene amplification methods.

In step (b), detecting the protein of the second sequence is a process of confirming the presence of the EGFRvIII mutant protein in a biological sample, preferably, an antigen using an antibody that specifically binds to the protein of the gene. Can be carried out in an antibody reaction mode. Analytical methods for this purpose include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket Immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, Fluorescence Activated Cell Sorter (FACS), protein chip, etc. The method of analysis of the invention is not limited.

According to another aspect of the present invention, the present invention provides an antibody comprising (i) an oligopeptide, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, that specifically binds to a protein of SEQ ID NO: 2 For ibrutinib in patients with glioblastoma multiforme comprising a PNA (Peptide nucleic acid) or aptamer, or (ii) a primer or probe that binds to the polynucleotide of SEQ ID NO: 1 Provide a kit for predicting sensitivity.

According to one embodiment of the present invention, the kit for predicting susceptibility to ibrutinib in glioblastoma patients of the present invention may be a kit for immunoassay.

The immunoassay kit may be performed in an immunoassay mode, that is, in an antigen-antibody reaction mode. In this case, the antibody or aptamer specifically binds to the EGFRvIII mutant protein of the present invention described above.

As used herein, "antibody" refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody means an antibody that specifically binds to the protein, and includes all polyclonal antibodies, monoclonal antibodies and recombinant antibodies.

The antibody used in the present invention is a polyclonal or monoclonal antibody, preferably a monoclonal antibody. Antibodies may be commonly used in the art, such as fusion methods (Kohler and Milstein, European Journal of Immunology, 6: 511-519 (1976)), recombinant DNA methods (US Pat. No. 4,816,56) Or phage antibody library methods (Clackson et al, Nature, 352: 624-628 (1991) and Marks et al, J. Mol. Biol., 222: 58, 1-597 (1991)). General procedures for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY, 1991, which are incorporated herein by reference. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing immortalized cell lines with antibody-producing lymphocytes, and the techniques required for this process are well known to those skilled in the art and can be readily implemented.

Polyclonal antibodies can be obtained by injecting a protein antigen into a suitable animal, collecting antisera from the animal, and then isolating the antibody from the antisera using known affinity techniques.

When the method of the present invention is carried out using antibodies or aptamers, the present invention can be used according to conventional immunoassay methods to predict sensitivity to ibrutinib in glioblastoma patients.

Such immunoassays can be performed according to various quantitative or qualitative immunoassay protocols developed in the prior art. The immunoassay format includes radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or hardwood analysis, sandwich analysis, flow cytometry, and immunoassay. Including but not limited to fluorescent staining and immunoaffinity purification. The immunoassay or method of immunostaining is described in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzymelinked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J.M. ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.

For example, when the method of the present invention is carried out in accordance with radioimmunoassay methods, antibodies labeled with radioisotopes (eg, C ¹⁴ , I ¹²⁵ , P ³² and S ³⁵ ) are EGFRvIII variant proteins as markers of the present invention. Can be used to detect.

When the method of the invention is carried out in an ELISA manner, certain embodiments of the invention comprise the steps of: (i) coating an unknown cell sample lysate to be analyzed on the surface of a solid substrate; (ii) reacting said cell lysate with an antibody against a marker as a primary antibody; (iii) reacting the resultant of step (ii) with the secondary antibody to which the enzyme is bound; And (iv) measuring the activity of the enzyme.

Suitable as the solid substrate are hydrocarbon polymers (eg polystyrene and polypropylene), glass, metal or gel, most preferably microtiter plates.

Enzymes bound to the secondary antibody include, but are not limited to, enzymes catalyzing color reaction, fluorescence, luminescence or infrared reaction, for example, alkaline phosphatase, β-galactosidase, hose Radish peroxidase, luciferase and cytochrome P450. When alkaline phosphatase is used as the enzyme binding to the secondary antibody, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-ASB1-phosphate (naphthol-AS-B1) as a substrate chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N) when colorimetric substrates such as -phosphate) and enhanced chemifluorescence (ECF) are used, and horse radish peroxidase is used. -Methylacridinium nitrate), resorupin benzyl ether, luminol, amplex red reagent (10-acetyl-3,7-dihydroxyphenoxazine), p-phenylenediamine-HCl and pyrocatechol (HYR), tetramethylbenzidine ), ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronine, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS (phenzaine) substrates such as methosulfat e) can be used The.

When the method of the invention is carried out in a capture-ELISA mode, certain embodiments of the invention comprise the steps of: (i) coating the surface of a solid substrate with an antibody against the marker of the invention as a capturing antibody; (ii) reacting the capture antibody with the sample; (iii) reacting the result of step (ii) with a detecting antibody having a label that generates a signal and which specifically reacts with a marker protein; And (iv) measuring the signal resulting from the label.

The detection antibody carries a label which generates a detectable signal. The label may include chemicals (eg biotin), enzymes (alkaline phosphatase, β-galactosidase, horse radish peroxidase and cytochrome P450), radioactive substances (eg C ¹⁴ , I ¹²⁵ , P ³² and S ³⁵ ), fluorescent materials (eg, fluorescein), luminescent materials, chemiluminescent and fluorescence resonance energy transfer (FRET), including but not limited to various labels and labeling methods. Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999.

Measurement of the final enzyme activity or signal in the ELISA method and the capture-ELISA method can be carried out according to various methods known in the art. If biotin is used as a label, the signal can be easily detected with streptavidin and luciferin if luciferase is used.

According to another variant of the present invention, an aptamer that specifically binds to the marker of the present invention may be used instead of the antibody. Aptamers are oligonucleic acid or peptide molecules, the general contents of which are described in Bock LC et al., Nature 355 (6360): 5646 (1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78 (8): 42630 (2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95 (24): 142727 (1998).

By analyzing the final signal by the above-described immunoassay, sensitivity to ibrutinib in patients with glioblastoma can be predicted. In other words, when a signal for the marker of the present invention emerges from the sample, it is determined to have an increased sensitivity to ibrutinib.

Examples of the types of kits in the present invention include immunochromatography strip kits, luminex assay kits, protein microarray kits, eliza kits, or immunological dot kits. Kind is not limited.

According to another embodiment of the present invention, the kit for predicting susceptibility to ibrutinib in glioblastoma patients of the present invention may be a kit for microarray.

The probe or primer used in the kit for predicting susceptibility of the present invention has a sequence complementary to the nucleotide sequence of SEQ ID NO: 1. As used herein, the term “complementary” means that the composition has sufficient complementarity to selectively hybridize to the nucleotide sequence of the first sequence in the specific sequence under certain hybridization or annealing conditions. Thus, the term "complementary" has a meaning different from that of the term perfectly complementary, and the primer or probe of the present invention may be selectively hybridized to the nucleotide sequence of the first sequence of the above-mentioned sequence, or It can have more mismatch sequences.

As used herein, the term “primer” can serve as an initiation point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) in suitable buffers at suitable temperatures. By single-stranded oligonucleotide. Suitable lengths of primers are typically 15-30 nucleotides, although varying with various factors, such as temperature and the use of the primer. Short primer molecules generally require lower temperatures to form hybrid complexes that are sufficiently stable with the template.

The sequence of the primer does not need to have a sequence that is completely complementary to some sequences of the template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing with the template to perform the primer-specific function. Therefore, the primer in the present invention does not need to have a sequence that is perfectly complementary to the nucleotide sequence of the first sequence of the sequence listing as a template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing to the gene sequence and acting as a primer. Do. The design of such primers can be easily carried out by those skilled in the art with reference to the nucleotide sequence of SEQ ID NO: 1, for example, by using a primer design program (eg, PRIMER 3 program).

As used herein, the term “probe” refers to a linear oligomer of natural or modified monomers or linkages, including deoxyribonucleotides and ribonucleotides, and capable of specific hybridization to a target nucleotide sequence, and naturally Present or artificially synthesized. Probes of the invention are preferably single chain and oligodioxyribonucleotides. Probes of the invention can include naturally occurring dNMPs (ie, dAMP, dGMP, dCMP and dTMP), nucleotide analogues or derivatives. In addition, the probes of the present invention may also comprise ribonucleotides. For example, probes of the present invention may be selected from the group consisting of backbone modified nucleotides such as peptide nucleic acids (PNA) (M. Egholm et al., Nature, 365: 566-568 (1993)), phosphorothioate DNA, phosphorodithioate DNA , Phosphoramidate DNA, amide-linked DNA, MMI-linked DNA, 2'-0-methyl RNA, alpha-DNA and methylphosphonate DNA, sugar modified nucleotides such as 2'-0-methyl RNA, 2 '-Fluoro RNA, 2'-amino RNA, 2'-0-alkyl DNA, 2'-0-allyl DNA, 2'-0-alkynylDNA, hexose DNA, pyranosyl RNA and anhydrohexitol DNAs, and nucleotides with base modifications such as C-5 substituted pyrimidines (substituents are fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, ethyl -, Propynyl-, alkynyl-, thiazolyl-, imidazoryl-, pyridyl-, 7-deazapurine with C-7 substituents (substituents are fluoro-, bromo-, chloro-, Iodo-, methyl- on Methyl-, vinyl-, formyl-, alkynyl-, alkenyl-, thiazolyl-, imidazoryl-, pyridyl-), inosine and diaminopurine.

In the microarray of the present invention, the probe is used as a hybridizable array element and is immobilized on a substrate.

Preferred gases include suitable rigid or semi-rigid supports such as membranes, filters, chips, slides, wafers, fibers, magnetic beads or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. Said hybridization array element is arranged and immobilized on said gas. This immobilization is carried out by chemical bonding methods or by covalent binding methods such as UV. For example, the hybridization array element can be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and can also be bonded by UV at the polylysine coating surface. In addition, the hybridization array element can be coupled to the gas through a linker (eg, ethylene glycol oligomer and diamine).

Meanwhile, the sample DNA applied to the microarray of the present invention can be labeled and hybridized with the array elements on the microarray. Hybridization conditions can vary. Detection and analysis of the degree of hybridization can be carried out in various ways depending on the labeling substance.

The kit for predicting susceptibility to ibrutinib of the present invention can be carried out based on hybridization. In this case, a probe having a sequence complementary to the nucleotide sequence of the sequence listing first sequence is used. Hybridization-based analysis may be performed using a probe hybridized to the nucleotide sequence of the first sequence listing sequence to determine whether it is sensitive to ibrutinib. The label of the probe can provide a signal that allows detection of hybridization, which can be linked to oligonucleotides. Suitable labels include fluorophores (eg fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5 (Pharmacia), chromophores, chemilumines, magnetic particles, radioisotopes Elements (P32 and S35), mass labels, electron dense particles, enzymes (alkaline phosphatase or horseradish peroxidase), cofactors, substrates for enzymes, heavy metals (eg gold) and antibodies, streptavidin, biotin And hapten with specific binding partners such as digoxigenin and chelating groups. Labeling is performed in a variety of ways conventionally practiced in the art, such as nick translation methods, random priming methods (Multiprime DNA labelling systems booklet, "Amersham" (1989)), and chination methods (Maxam & Gilbert, Methods in Enzymology, 65: 499 (1986)). Labels provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetric, X-ray diffraction or absorption, magnetism, enzymatic activity, mass analysis, binding affinity, hybridization high frequency, nanocrystals.

The nucleic acid sample to be analyzed can be prepared using mRNA obtained from various biological samples.

The hybridization reaction-based assay may be performed by labeling the cDNA to be analyzed instead of the probe.

If a probe is used, the probe is hybridized with the cDNA molecule. In the present invention, suitable hybridization conditions can be determined in a series of procedures by an optimization procedure.

This procedure is carried out by a person skilled in the art in order to establish a protocol for use in the laboratory. For example, conditions such as temperature, concentration of components, hybridization and wash times, buffer components and their pH and ionic strength depend on various factors such as probe length and GC amount and target nucleotide sequence. Detailed conditions for hybridization can be found in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); And M.L.M. Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. N.Y. (1999). For example, among the stringent conditions, the high stringency conditions were hybridized to 65 ° C. in 0.5 M NaHPO 4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA, and 68 at 0.1 × standard saline citrate / 0.1% SDS. It means washing under the condition of ℃. Alternatively, high stringency conditions mean washing at 48 ° C. in 6 × SSC / 0.05% sodium pyrophosphate. Low stringency means washing at 42 ° C. conditions, for example, at 0.2 × SSC / 0.1% SDS.

After the hybridization reaction, the hybridization signal coming out of the hybridization reaction is detected. The hybridization signal can be performed by various methods, for example, depending on the type of label bound to the probe. For example, if the probe is labeled by an enzyme, the substrate of the enzyme can be reacted with the hybridization product to confirm hybridization. Combinations of enzymes / substrates that can be used include peroxidase (eg horseradish peroxidase) and chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium). Nitrate), lesorupine benzyl ether, luminol, Amplex red reagent (10-acetyl-3,7-dihydroxyphenoxazine), p-phenylenediamine-HCl and pyrocatechol (HYR), tetramethylbenzidine (TMB), ABTS (2, 2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronine; Alkaline phosphatase with bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate and ECF substrates; Glucose oxidase, t-NBT (nitroblue tetrazolium) and m-PMS (phenzaine methosulfate). When the probe is labeled with gold particles, it can be detected by silver dyeing using silver nitrate. Therefore, when the method of predicting the sensitivity to ibrutinib of a glioblastoma patient of the present invention is performed based on hybridization, specifically, (i) a probe having a sequence complementary to the nucleotide sequence of SEQ ID NO: 1 Hybridizing to a nucleic acid sample; (ii) detecting whether the hybridization reaction occurs. By analyzing the intensity of the hybridization signal by the hybridization process, sensitivity to ibrutinib in patients with glioblastoma can be determined. In other words, when a hybridization signal for the first sequence of SEQ ID NO is given, it is determined to exhibit increased sensitivity to ibrutinib.

According to one embodiment of the present invention, the kit for predicting susceptibility to ibrutinib in glioblastoma patients of the present invention may be a gene amplification kit.

As used herein, the term "amplification" refers to a reaction that amplifies a nucleic acid molecule. Various amplification reactions have been reported in the art, which include polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcriptase-polymerase chain reaction (RT-PCR) (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001)), Miller, HI (WO 89/06700) and Davey, C. et al. (EP 329,822), ligase chain reaction (LCR) ( 17, 18), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA) (19) (WO 88/10315), autologous Self sustained sequence replication (20) (WO 90/06995), selective amplification of target polynucleotide sequences (US Pat. No. 6,410,276), consensus sequence priming polymerase chain Reaction (consensus sequence primed polymerase chain reaction (CPPCR)) (US Pat. No. 4,437,975), optionally Arbitrarily primed polymerase chain reaction (AP-PCR) (US Pat. Nos. 5,413,909 and 5,861,245), nucleic acid sequence based amplification (NASBA) (US Pat. No. 5,130,238, No. 5,409,818, 5,554,517 and 6,063,603), strand displacement amplification (21, 22) and loop-mediated isothermal amplification; LAMP) 23, but is not limited thereto. Other amplification methods that can be used are described in US Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and US Pat. No. 09 / 854,317. PCR is the best known nucleic acid amplification method, and many modifications and applications thereof have been developed. For example, touchdown PCR, hot start PCR, nested PCR, and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR. In addition, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction : IPCR), vectorette PCR and thermal asymmetric interlaced PCR (TAIL-PCR) have been developed for specific applications. For more information on PCR see McPherson, M.J., and Moller, S.G. PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, N.Y. (2000), the teachings of which are incorporated herein by reference. When the diagnostic kit of the present invention is carried out using a primer, a gene amplification reaction is performed to investigate the presence or absence of a nucleotide of SEQ ID NO: 1. In principle, the present invention performs a gene amplification reaction using primers that bind to mRNA or cDNA as a template of mRNA in a sample.

To obtain mRNA, total RNA is isolated from the sample. Isolation of total RNA can be carried out according to conventional methods known in the art. See Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001); Tesniere. , C. et al., Plant Mol. Biol. Rep., 9: 242 (1991); Ausubel, FM et al., Current Protocols in Molecular Biology, John Willey & Sons (1987); and Chomczynski, P. et al. , Anal.Biochem. 162: 156 (1987)). For example, Trizol can be used to easily isolate total RNA in cells. Subsequently, cDNA is synthesized from the isolated mRNA, and the cDNA is amplified. Since the total RNA of the present invention is isolated from human samples, the end of the mRNA has a poly-A tail, and cDNA can be easily synthesized using oligo dT primers and reverse transcriptases using these sequence characteristics. PNAS USA, 85: 8998 (1988); Libert F, et al. Science, 244: 569 (1989); and Sambrook, J. et al., Molecular Cloning.A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001)). Then, the synthesized cDNA is amplified by a gene amplification reaction.

Primers used in the present invention are hybridized or annealed to one site of the template to form a double chain structure. Suitable nucleic acid hybridization conditions for forming such a double-chain structure include Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization , A Practical Approach, IRL Press, Washington, DC (1985).

Various DNA polymerases can be used for amplification of the present invention, E. coli “Clenow” fragments of DNA polymerase I, thermostable DNA polymerase and bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtainable from various bacterial species, which include Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu). Include.

When carrying out the polymerization reaction, it is preferable to provide an excess amount of components necessary for the reaction to the reaction vessel. Excess of components necessary for the amplification reaction means an amount such that the amplification reaction is not substantially limited to the concentration of the components. To provide joinja, dATP, dCTP, dGTP and dTTP, such as Mg ^{+ 2} to the reaction mixtures to have a desired degree of amplification can be achieved is required. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, the buffer ensures that all enzymes are close to optimal reaction conditions. Thus, the amplification process of the present invention can be carried out in a single reactant without changing conditions such as addition of reactants.

Annealing in the present invention is carried out under stringent conditions allowing specific binding between the target nucleotide sequence and the primer. Stringent conditions for annealing are sequence-dependent and vary with ambient environmental variables.

The result of the amplification reaction described above is subjected to gel electrophoresis, and the presence or absence of the nucleotide of the first sequence of the sequence listing is examined by observing and analyzing the resulting band. When the amplification reaction indicates that the nucleotide of SEQ ID NO: 1 is present in the biological sample, it is determined to exhibit increased sensitivity to ibrutinib. Therefore, when the method of detecting a susceptible marker for ibrutinib is performed based on an amplification reaction using cDNA, specifically, (i) an amplification reaction using a primer annealed to the nucleotide sequence of SEQ ID NO: 1 Performing; And (ii) analyzing the product of the amplification reaction to determine the presence or absence of a nucleotide of SEQ ID NO: 1.

The kit of the present invention may further include other components in addition to the above components. For example, when the kit of the present invention is subjected to a PCR amplification process, the kit of the present invention may optionally contain reagents necessary for PCR amplification, such as buffers, DNA polymerases (eg, Thermus aquaticus (Taq), Thermus thermophilus). (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis or thermally stable DNA polymerase obtained from Pyrococcus furiosus (Pfu)), DNA polymerase cofactors and dNTPs. Kits of the invention can be prepared in a number of separate packaging or compartments containing the reagent components described above.

The features and advantages of the present invention are summarized as follows:

(i) The present invention provides a method for determining the sensitivity or prognosis of ibrutinib in glioblastoma patients by examining the presence of EGFRvIII mutations, and a kit for predicting susceptibility to ibrutinib in glioblastoma patients.

(ii) The present invention can predict the reactivity of ibrutinib, a BTK / BMX / BLK specific inhibitor, in glioblastoma patients.

(iii) The present invention can be usefully used as a basis for administering ibrutinib to patients with EGFRvIII mutations.

1 is a result of measuring sensitivity to ibrutinib in patient-derived cells and mutant cells with EGFRvIII mutation (p = 0.0001).

2 is a result confirming that tumor sphere formation ability of EGFRvIII mutant cells is reduced by ibrutinib treatment.

3 is a result confirming that the activity of EGFR-related signaling system (pEGFR, pAKT, pS6K) is reduced by ibrutinib treatment.

4 is a result confirming that the sensitivity of Ibrutinib is increased in the EGFRvIII gene injection cells compared to the control.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Experiment method

Glioblastoma Cells

Glioblastoma samples and clinical records were obtained from glioblastoma patients undergoing surgery at Samsung Medical Center according to Institutional Review Boards (IRB). About 5 × 5 × 5 mm ³ surgical samples were frozen with liquid nitrogen for genetic analysis. Some of the surgical samples were separated into single cells using enzymes according to previously known immune cell removal methods (7). Tumor cells were cultured in neuronal medium further comprising N2 and B27 (0.5X each; Invitrogen), human recombinant basic fibroblast growth factor (bFGF) and epidermal growth factor (20 ng / ml each; R & D systems). Standard assays and immunoblots comprising tumorigenic in vitro restriction dilution assays were performed according to known methods (7).

2. Full Exome Sequencing

Alignment

The FASTQ file was aligned with the human genome assembly (hg19) using Burrows-Wheeler Aligner (version 0.6.2) (22). Use SAMtools, Picard (version 1.73, http://picard.sourceforge.net), and Genome Analysis ToolKit (GATK, version 2.5.2) to sort, remove duplicate reads, and insert / read initial aligned BAM files prior to analysis Partial realignment of the sequences around the INDELs and recalibration of the base quality scores were performed (23).

Mutation calling

Somatic mutation calling from tumors and normal tissues was performed using MuTect (version 1.1.4) and Somatic Indel Detector (GATK version 2.2) (24, 25). Variant Effect Predictor (VEP, version 73) was used to extract biological information about the somatic mutations called. Nonsynonymous somatic mutations in the present invention were called for later analysis when the MAF (minor allele frequency)> 0.20 and the read number for the mutant allele were> 2.

Copy number change

The ngCGh python package was used to measure copy number changes in tumor WES data compared to matched normal WES data. The measured DNA copy number split was performed using the R package “DNAcopy” (version 1.30.0), and the copy number of each gene was calculated as the average copy number of all exon segments of the gene. If the log2 ratio of the tumor group to the normal group was greater than 0.5 or less than -0.5, the copy number was assumed to be "amplified" or "deleted", respectively.

Exon Skipping & Gene Fusion Analysis

In single-ended mapping mode, GSNAP was used to align the readings for the uncleaved sequence at hg19 allowing splicing. “Split” readings spanning non-canonical splicing splices were separated and exon-skipping events were called when the split read count was 2 or more. For gene fusion analysis, span fusion junctions were isolated using the same method as exon-skipping analysis and fusion events were called.

3. RNA Sequencing

Without allowing any mismatches, insertions / deletions or splicings, 30 nt-cleaved sequence reads were mapped on hg19 using GSNAP (version 2012-12-20). SAMtools categorized as alignment SAM files and bedTools (bamToBed, version 2.16.2) were used to organize them into BED files. RPKM values were measured using the R package DEGseq.

4. Cell Based HTS Drug Screening

Primary glioblastoma cells incubated in serum free medium were seeded in 384-well plates at 500 cells per well. After 2 hours of plating, the drug was treated with cells using 4-fold and 7-point step dilution using Janus Automated Workstation (PerkinElmer). After 6 days incubation in a 37 ° C., 5% CO ₂ incubator, cell viability was analyzed using an adenosine triphosphate monitoring system based on Firefly Luciferase (ATPLite ™ 1step, PerkinElmer, Waltham, Mass., USA). . Viable cells were measured using EnVision Multilabel Reader (PerkinElmer). In vitro drug screening allowed control wells containing only cells and DMSO (vehicle) to be included in each assay plate. These controls were used to calculate relative cell viability in plates and to normalize data per plate. The data was curved using PRISM (Graphpad USA) and the area under the curve was calculated.

5. Analysis of drug response and genetic changes

Normalized to analyze the association between drug responsiveness and genetic changes to discrete events such as point mutations, insertion / deletion, exon skipping, gene fusion and copy number changes (“amplification”, “deletions” and “wild type”) The Kolmogorov-Smirnov test (null hypothesis: uniform distribution) and the Wilcoxon rank sum test were performed. Pearson's correlation assay was used to analyze the relationship between drug reactivity and gene expression.

6. Western Blot

Patient-derived glioblastoma cells were washed with cold PBS and lysis buffer (150 mM sodium chloride, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing protease and phosphatase inhibitor cocktail (Thermo scientific) , 50 mM Tris-HCL and 2 mM EDTA) were obtained after treatment. Insoluble materials were removed by centrifugation at 12,000 rpm for 15 minutes at 4 ° C. Proteins were separated by SDS-PAGE. Immunoblotting was performed using antibodies specific for pEGFR (Y1068), EGFR, pAKT (S473), AKT, pERK (T202 / Y204), ERK, pSTAT3 (Y705) and all from Cell Signaling Technology (STAT3).

7. Tumorsphere Formation Assay

Glioblastoma cells and xenograft tumors from glioblastoma patient samples were isolated by single cell suspension and then plated in 96-well plates at various seeding concentrations (1-500 cells per well). Cells were incubated for 1-2 weeks at 37 ° C. Neurosphere-like cell population formation was measured for each well. Statistical significance was calculated using Extreme Limiting Dilution Analysis (ELDA; Walter + Eliza Hall Bioinformatics).

Experiment result

1. EGFRvIII group with high sensitivity to ibrutinib

After observing the drug sensitivity of Ibrutinib at 7 doses using 50 glioblastoma-derived cells (20 μM-5 nM), the DRC (Dose response) was calculated using the% cell viability compared to the DMSO control on day 6 of the drug treatment. curve) was measured based on the area under curve. When comparing ibrutinib responses after sorting cells with EGFRvIII mutations and non-mutant cell groups, the group showed significantly high sensitivity to ibrutinib in the EGFRvIII mutation group (Wilcox P = 0.0001) (FIG. 1).

2. Changes in Tumor Formation Capacity by EGFRvIII Mutation

EGFR WT (GBM591) and EGFRvIII variant cell lines (NS626 and GBM352R) were treated with DMSO or ibrutinib (100 or 500 nM) and tumor cell formation was observed using Limiting Dilutiion Assay. Cells were plated at various concentrations (1-250 cells per well) in 24 well plates and incubated for 2 weeks, and then the presence of tumor cells was observed. Representative images of cells are shown in FIG. 2. In EGFR WT cells (GBM591), there was no difference in tumor cell formation ability according to ibrutinib treatment, but EGFRvIII cells (NS626, GBM352R) showed significant tumor cell formation inhibitory effect depending on ibrutinib concentration.

3. Changes in EGFR Signaling Activity According to EGFRvIII Variation

GBM591 and NS626 Cells were treated with ibrutinib (0, 100 and 500 nM) and cells were harvested and lysed 24 hours later, followed by Western blot using antibodies specific for pEGFR (Y1068), EGFR, pAKT, AKT, pS6K and S6K. Was carried out. Beta-actin was used as loading control (FIG. 3). Downstream signals such as EGFR and pAKT and pSTAT3 were significantly reduced in a concentration dependent manner in EGFRvIII cells (NS626) by ibrutinib treatment.

4. Susceptibility of Ibrutinib in EGFRvIII Gene-Injected Cells

The pLenti control vector or EGFRvIII (nucleotides of SEQ ID NO: 1) was injected into GBM352L cells using a lentiviral system. GBM352L-vector or -EGFRvIII injected cells were treated with ibrutinib (7 doses at 20 μM-5 nM) for 6 days. Cell viability was measured using ATPlite cell viability kit, and survival was evaluated in comparison with DMSO control viability (FIG. 4). The sensitivity to ibrutinib was markedly increased when EGFRvIII was injected into EGFR WT cells (GBM352L).

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (8)

Methods to test for the presence of EGFRvIII mutations to provide useful information in determining the susceptibility or prognosis for ibrutinib in patients with glioblastoma multiforme, comprising the following steps: (a) isolating a nucleic acid molecule or protein from a biological sample isolated from the subject; And (b) (i) detecting the polynucleotide of SEQ ID NO: 1 from the isolated nucleic acid molecule or (ii) detecting the protein of SEQ ID NO: 2 from the separated protein Or if the amino acid of SEQ ID NO: 2 is detected, the subject has increased ibrutinib sensitivity. The method of claim 1, wherein the detection of the polynucleotide of the first sequence is performed by PCR (Polymerase Chain Reaction), hybridization, sequencing, pyrosequencing, or next generation sequencing (NGS). Method of carrying out. The method of claim 1, wherein the detection of the protein of SEQ ID NO: 2 is performed by an antigen-antibody reaction method. The method of claim 1, wherein the biological sample is a blood, plasma, serum, urine, cell, hair or tissue sample.  (i) oligopeptides, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, ligands, peptide nucleic acids or aptamers that specifically bind to proteins of SEQ ID NO: 2 Or (ii) a kit for predicting susceptibility to ibrutinib in a glioblastoma multiforme patient comprising a primer or probe that binds to the polynucleotide of SEQ ID NO: 1. The kit according to claim 5, wherein the kit is a kit for immunoassay. 6. The kit according to claim 5, wherein the kit is a kit for microarray. 6. The kit of claim 5, wherein said kit is a gene amplification kit.

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