CN107356741B - Application of reagent for detecting biomarkers in preparation of kit for evaluating sensitivity of patient to brigatinib and kit thereof - Google Patents

Abstract

The present disclosure discloses the use of a reagent for detecting a biomarker in the preparation of a kit for assessing the sensitivity of a patient to brigatinib and a kit thereof; wherein the biomarker is p-Met and/or p-Stat3, and the reagent for detecting the biomarker is a reagent capable of quantitatively detecting the biomarker; the present disclosure also provides kits for assessing a patient's sensitivity to brigatinib using quantitative detection of the biomarkers as described above using reagents. The method and the kit provided by the disclosure can quickly screen out patients suitable for treatment with buritinib, and provide effective guidance for treatment of the patients.

Description

Application of reagent for detecting biomarkers in preparation of kit for evaluating sensitivity of patient to brigatinib and kit thereof

Technical Field

The present disclosure relates to the field of biotechnology, specifically to the use of a reagent for detecting a biomarker in the preparation of a kit for assessing the sensitivity of a patient to brigatinib, and a kit thereof.

Background

c-Met is an important high affinity receptor with tyrosine kinase activity. Activation of c-Met can lead to the spread, proliferation, migration and invasion of tumor cells as well as promote tumor angiogenesis and tumor metastasis. Preclinical studies show that brigatinib is a high-efficiency and high-selectivity small-molecule c-Met tyrosine kinase inhibitor. Among 100 tyrosine kinases, brigatinib has a remarkable inhibitory effect only on c-Met kinase. However, different patients have different sensitivities to brigatinib, so that the treatment effect varies from person to person, and therefore, a patient with better sensitivity to brigatinib needs to be screened out to obtain a good treatment effect.

Disclosure of Invention

The purpose of the disclosure is to provide a screening method for biritinib high-sensitivity patients, so that the patients can be effectively treated.

In order to achieve the above object, the present disclosure provides a use of a reagent for detecting a biomarker in the preparation of a kit for evaluating a patient's sensitivity to brigatinib, wherein the biomarker is p-Met and/or p-Stat3, and the reagent for detecting a biomarker is a reagent capable of quantitatively detecting the biomarker.

The present disclosure also provides a kit for assessing a patient's sensitivity to brigatinib, wherein the kit comprises a reagent for detecting a biomarker which is p-Met and/or p-Stat3, wherein the reagent for detecting a biomarker is a reagent capable of quantitatively detecting the biomarker.

The patients in the present disclosure are brain glioma patients, and it was found in earlier studies on brain glioma patients that patients with elevated expression of p-Met and p-Stat3 are more malignant in brain glioma patients; this class of glioma patients is characterized by excessive activation of the p-Met protein due to multiple pathways, whereas activation of the Met pathway can lead to the spread, proliferation, migration and invasion of tumor cells and promote angiogenesis and metastasis of tumors; beritinib is a high-efficiency and high-selectivity small-molecule c-Met tyrosine kinase inhibitor, so that patients with p-Met over-activation have better sensitivity to the Beritinib, and patients suitable for using the Beritinib can be quickly screened through the biomarker. p-Met and p-Stat3 are c-Met protein and Stat3 protein phosphorylation forms, respectively, and various pathways can activate c-Met in tumor development, and the most common way is to play a role by binding HGF and c-Met, and the binding of HGF and c-Met leads to receptor autophosphorylation, namely p-Met increase, and leads to tumor malignant progression. The protein family of Transcription Activator (Signal Transducer and Activator of Transcription, Stat) can be activated by different cytokine receptors, and can be used as a carrier in the process of cytokine-receptor interaction to keep the inherent specificity of Signal transmission in cells; among the Stat proteins, Stat3 is most closely related to tumors, its activation is found in many tumors, and p-Stat3 is its activated form.

Additional features and advantages of the disclosure will be set forth in the detailed description which follows.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the description serve to explain the principles of the disclosure and not to limit the disclosure. In the drawings:

FIG. 1 is a graph showing the measurement of the expression amounts of p-Met and p-Stat3 in tumor tissues of patients with brain glioma by immunohistochemistry.

FIG. 2 is an electrophoresis chart of immunoblotting to determine the expression levels of p-Met and p-Stat3 in tumor tissues of patients with brain glioma.

FIG. 3 is an electrophoresis chart of the expression amounts of p-Met and p-Stat in tumor cells treated with different drugs.

Figure 4 is the nude mouse subcutaneous tumor volume at buritinib treatment 11 d.

FIG. 5 shows the subcutaneous tumor volumes of nude mice treated with different drugs for 19 d.

Detailed Description

The following describes in detail specific embodiments of the present disclosure. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.

The first aspect of the present disclosure provides a use of a reagent for detecting a biomarker in the preparation of a kit for evaluating a patient's sensitivity to brigatinib, wherein the biomarker is p-Met and/or p-Stat3, and the reagent for detecting a biomarker is a reagent capable of quantitatively detecting the biomarker.

According to a first aspect of the present disclosure, the patient is a glioma patient. Patients with elevated expression of p-Met and p-Stat3, including patients with the fusion gene PTPRZ1-MET (ZM fusion gene), are more malignant in patients with brain gliomas; this class of glioma patients is characterized by excessive activation of the p-Met protein due to multiple pathways, whereas activation of the Met pathway can lead to the spread, proliferation, migration and invasion of tumor cells and promote angiogenesis and metastasis of tumors; beritinib is a high-efficiency and high-selectivity small-molecule c-Met tyrosine kinase inhibitor, so that patients with p-Met over-activation have better sensitivity to the Beritinib, and the addition of the biomarker p-stat3 is helpful for accurately and quickly screening patients suitable for using the Beritinib.

The method of quantifying the biomarker according to the first aspect of the present disclosure may be any of various methods of quantifying protein content conventionally used by those skilled in the art; preferably, the kit for evaluating the sensitivity of the patient to the brigatinib can be a kit for detecting the content of the biomarker by a colloidal gold strip method, a western blotting method or an immunohistochemistry method.

According to the first aspect of the present disclosure, the reagent for quantitatively detecting the biomarker may be various reagents for quantitatively detecting protein content, which are conventionally used by those skilled in the art; preferably, the kit using western immunoblotting or immunohistochemistry may include a first monoclonal antibody and a second monoclonal antibody having a label; the first monoclonal antibody is a first monoclonal antibody of p-Met and/or a first monoclonal antibody of p-Stat 3; the reagent capable of quantitatively detecting the biomarker is a first monoclonal antibody against p-Met and/or a first monoclonal antibody against p-Stat 3.

A second aspect of the present disclosure provides a kit for assessing a patient's sensitivity to brigatinib, wherein the kit comprises reagents for detecting a biomarker which is p-Met and/or p-Stat3, wherein the reagents for detecting a biomarker are reagents capable of quantitatively detecting the biomarker.

According to the second aspect of the present disclosure, the method capable of quantitatively detecting the biomarker may be various methods for quantifying protein content, which are conventionally used by those skilled in the art, and preferably, the kit for evaluating the sensitivity of a patient to brigatinib is a kit for detecting the content of the biomarker by a colloidal gold strip method, western blotting or immunohistochemistry method.

According to the second aspect of the present disclosure, the reagent for quantitatively detecting the biomarker may be various reagents for quantitatively detecting protein content, which are conventionally used by those skilled in the art; preferably, the kit using western immunoblotting or immunohistochemistry may include a first monoclonal antibody and a second monoclonal antibody having a label; the first monoclonal antibody is a first monoclonal antibody against p-Met and/or a first monoclonal antibody against p-Stat 3.

According to the second aspect of the present disclosure, the label may be various labels capable of labeling the monoclonal antibody, which are conventionally used by those skilled in the art; preferably, the label is at least one selected from horseradish peroxidase, biotin, alkaline phosphatase, a fluorescent dye and a fluorescent protein.

According to the second aspect of the present disclosure, the first monoclonal antibody against p-Met is a monoclonal antibody purchased from CST corporation under item No. 4033 or 3077, and the first monoclonal antibody against p-Stat3 is a monoclonal antibody purchased from CST corporation under item No. 9145; the monoclonal antibody as described above has a better use effect when used in the present kit.

By the technical scheme, the application and the kit of the biomarker can efficiently and quickly screen out a patient with high burretinib sensitivity glioma, provide reliable guidance for the treatment of the patient, save the time of the patient and improve the effective rate of the treatment.

The invention is further illustrated by the following examples, but is not to be construed as being limited thereto.

In the disclosure, the first monoclonal antibody against p-Met is a monoclonal antibody purchased from CST company with a product number of 4033, the first monoclonal antibody against p-Stat3 is a monoclonal antibody purchased from CST company with a product number of 9145, the second monoclonal antibody labeled by horseradish peroxidase is purchased from China fir gold bridge company, and the others are commercial reagents.

Example 1

This example is an immunohistochemical method for detecting the expression levels of p-Met and p-Stat3 in tumor tissues.

The tumor tissue is derived from a brain glioma paraffin section, firstly, the tumor tissue section is dewaxed into water, and washed with PBS for three times and 15 minutes each time; place the sections in freshly prepared 3% H₂O₂-in PBS, for 5 minutes at room temperature; PBS washes three times, 5 minutes each; preparing a first monoclonal antibody according to the instruction, and dropwise adding a proper amount of the first monoclonal antibodyThe monoclonal antibody is completely covered on the specimen and is placed in a wet box at 4 ℃ for overnight; the sections were rewarmed for 30 minutes at room temperature and washed 4 times for 5 minutes each with PBS; preparing a second monoclonal antibody according to the instruction, dropwise adding a proper amount of the second monoclonal antibody, completely covering the specimen, placing the specimen in a wet box for 1h, and washing with PBS for 5 minutes each time for 4 times; 1ml of distilled water is taken, and one drop of DAB staining solution A, B, C liquid is respectively added dropwise in sequence. Dripping color development liquid, developing for 3-5 minutes, observing under a microscope to control the dyeing degree, and stopping developing timely; the sections were washed with tap water, counterstained with hematoxylin for 3 minutes, and rinsed with hydrochloric acid alcohol for 2 seconds. Washing with tap water for 10-15 min; dehydrating, transparent, sealing, and performing microscopic examination. The specific results are shown in FIG. 1.

As can be seen from the comparison of the brain glioma sections of different sources in FIG. 1, the expression levels of p-Met and p-Stat3 in different brain glioma cells are different, and the immunohistochemical staining result of the tumor tissue in the first row is negative, and the tumor tissue normally expresses p-Met and p-Stat 3; the second row tumor tissue has positive immunohistochemical staining result and is tumor tissue with high expression of p-Met and p-Stat 3.

Example 2

This example screens biritinib hypersensitive tumors by immunoblotting to quantify p-Met, p-Stat 3.

(1) Extracting tumor tissue protein: taking the brain glioma tissues preserved in liquid nitrogen, placing the brain glioma tissues in 1 multiplied by protein lysate, grinding, collecting the brain glioma tissues in an EP tube, placing the brain glioma tissues in ice for 30 minutes to fully crack the brain glioma tissues, and setting a centrifuge to be 4 ℃ in advance. After 30 min centrifugation at 12000rpm for 15min, the supernatant was aspirated into a new EP tube. And detecting the protein concentration, adding 300 mu L of Coomassie brilliant blue solution into each hole of a 96-hole plate, then adding 10 mu L of protein sample, taking 3 multiple holes of each protein, placing the protein on a shaking table for 10 minutes at room temperature, and detecting the protein concentration by using an enzyme labeling instrument. And adding a proper amount of deionized water according to the result to adjust the protein concentration to be at the same level, adding a loading buffer solution, uniformly mixing, carrying out water bath at 100 ℃ for 10 minutes, and immediately placing on ice for cooling to obtain the tumor tissue protein.

(2) Immunoblotting method for detecting p-Met and c-Met protein expression level

Adding tumor tissue protein liquid on the polyacrylamide precast gel for electrophoresis; 100v constant pressure conventional electrophoresis, 0.45 μm pore size PVDF membrane conventional membrane (constant pressure 100v, 100 min). 5% skim milk powder solution (in 1 Xblock-wash buffer) was shake-sealed for 1h at room temperature. The blocked membrane was cut at 70kD, incubated with c-Met or p-Met primary monoclonal antibody diluted 1:2000 (5% milk as diluent) above 70kD, and incubated overnight on a 4 ℃ tumbling shaker. And (4) eluting the primary antibody and the secondary antibody conventionally, eluting the secondary antibody, soaking the ECL electrochemiluminescence solution, and then exposing.

The specific results of the immunoblotting of the expression levels of p-Met and p-Stat3 are shown in FIG. 2, wherein the lanes Normalbrain tissue 1 and Normal brain tissue 2 are healthy brain tissues as controls, and the remaining lanes are brain tissues of 12 patients with brain gliomas collected. As shown in FIG. 2, it was found that the patients with brain gliomas with higher expression levels of p-Met and p-Stat3 are mostly positive for ZM fusion gene.

Example 3

This example was used to study the effect of brigatinib and other drugs on Met, p-Met, Stat3, p-Stat3, Akt, p-Akt, Erk and p-Erk protein expression in tumor cells.

Wherein the control group is untransfected U87 tumor cells, which are marked as Parental; negative control Vector-GFP tumor cells were prepared by the following steps: the GFP gene is constructed into an adenovirus Vector (pShuttle-CMV) to obtain a recombinant Vector, and the recombinant Vector is used for transfecting U87 tumor cells to obtain Vector-GFP tumor cells; the Met tumor cells are prepared by the following steps: constructing a nucleotide sequence shown as SEQ ID NO.1 into an adenovirus vector to obtain a recombinant vector, and transfecting U87 tumor cells by using the recombinant vector to obtain Met tumor cells; ZM1-2 tumor cells are prepared by the following steps: constructing a nucleotide sequence shown as SEQ ID NO.2 into an adenovirus vector to obtain a recombinant vector, and transfecting U87 tumor cells by using the recombinant vector to obtain ZM1-2 tumor cells; ZM2-2 tumor cells are prepared by the following steps: the nucleotide sequence shown as SEQ ID NO.3 is constructed into an adenovirus vector to obtain a recombinant vector, and the recombinant vector is used for transfecting U87 tumor cells to obtain ZM2-2 tumor cells.

Culturing the 5 tumor cells in a complete cell culture medium for 48h, then dividing the Met tumor cells, ZM1-2 tumor cells and ZM2-2 tumor cells into 3 groups, adding distilled water or 30 mu M Burretinib (marked as + PLB) or 3 mu M Crizotinib (marked as + Crizotinib) into each group, and then continuously culturing for 6 h; the protein content of Met, p-Met, Stat3, p-Stat3, Akt, p-Akt, Erk and p-Erk in the tumor cells is detected by an immunoblotting method, and the method for detecting the expression level of each protein by the immunoblotting method is shown in example 2. The immunoblotting assay results are shown in FIG. 3.

Figure 3 shows that both brigatinib and crizotinib can significantly inhibit the expression of p-Met and p-Stat 3:

compared with the Met group, the Met + PLB group has completely inhibited p-Met expression and p-Stat3 expression, and compared with the Met group, the Met + Crizotinib group has completely inhibited p-Met expression and p-Stat3 expression;

in the ZM1-2+ PLB group, compared with the ZM1-2 group, the expression of p-Met is completely inhibited and the expression of p-Stat3 is completely inhibited, while in the ZM1-2+ Crizotinib group, compared with the ZM1-2 group, the expression of p-Met is completely inhibited and the expression of p-Stat3 is completely inhibited;

in the ZM2-2+ PLB group, the expression of p-Met was completely suppressed and the expression of p-Stat3 was completely suppressed compared with the ZM2-2 group, whereas in the Met + Crizotinib group, the expression of p-Met was completely suppressed and the expression of p-Stat3 was completely suppressed compared with the ZM2-2 group.

It can be seen that Burretiinib can obviously inhibit the expression of p-met and p-stat3 in tumor cells. Meanwhile, the influence of Beritinib on Akt and Erk signal paths is smaller than that of crizotinib, and no larger side effect is generated during treatment.

Example 4

The MET tumor cells in this example were prepared as follows: the nucleotide sequence shown as SEQ ID NO.1 is constructed into a lentiviral vector (pLent-RFP-Puro-CMV) to obtain a recombinant vector, and the recombinant vector is used for transfecting U87 tumor cells to obtain MET tumor cells. The MET tumor cells are used for simulating brain glioma cells with high expression of p-MET and p-stat3 caused by various pathways.

The subcutaneous tumor-forming mice of MET in this example were prepared by the following steps: recovering MET tumor cells, and culturing with a whole cell culture medium; meanwhile, U87 tumor cells transfected with U87-lentivirus empty vectors are set as a control group; tumor cells in logarithmic growth phase were collected, centrifuged and counted. F0 generation tumor-bearing mice were prepared by inoculating the mice with the cell concentration adjusted based on the actual cell number under the right axilla of the nude mice (the mice were further passaged in vivo and recorded as F1, and so on). Passaging for 1-2 times in vivo according to the condition, selecting tumor-bearing animals with vigorous tumor growth, no rupture and good health condition, taking out the tumor under aseptic condition, and preparing into 2-3 mm³Small tumor mass, inoculated under the right axilla of nude mice; observing the growth condition of the tumor after inoculation, and selecting the tumor with the volume of 85.17-199.75 mm³Subcutaneous tumorigenic nude mice within the range were subjected to the experiment, and MET group and control group (U87 no-load group) were selected 8 nude mice with subcutaneous tumorigenic tumor to be administered with 10mg/kg brigatinib (PLB-1001), and 8 nude mice with subcutaneous tumorigenic tumor were selected to be treated with physiological saline (untreated group), respectively. After 11d treatment, the mice were sacrificed by cervical dislocation and subcutaneous nodules were dissected, and the comparison of the volumes of subcutaneous nodules of nude mice at 11d is shown in fig. 4; subcutaneous tumorigenic volume (V) 1/2ab²Wherein a is the major diameter of the tumor and b is the minor diameter of the tumor.

The MET group simulates brain gliomas with increased MET protein expression and finally increased P-MET and P-stat3 expression, and fig. 4 shows that the tumor growth speed of the MET group after treatment with brigatinib is obviously lower than that of the MET untreated group, the tumor inhibition rate of the brigatinib on the MET group reaches 89.13%, and the tumor inhibition rate is significant compared with the tumor inhibition rate of 58.42% of a brigatinib-treated control group (U87 no-load-PLB treated group) (P < 0.05). The results show that the brain glioma with the p-met and p-stat3 expression remarkably increased is superior to other tumors in sensitivity to Burretiinib, can obviously inhibit the growth of the brain glioma with the p-met and p-stat3 expression remarkably increased, and has a very good treatment effect.

Example 5

ZM2-2 tumor cells in this example were prepared as follows: a ZM2-2 tumor cell obtained by constructing a nucleotide sequence shown as SEQ ID NO.3 into a lentiviral vector (pLent-RFP-Puro-CMV) to obtain a recombinant vector and transfecting U87 tumor cells with the recombinant vector. The ZM2-2 tumor cells are used for simulating ZM fusion gene positive brain glioma cells with higher expression of p-met and p-stat3 obtained by screening in example 2.

In this example, ZM2-2 subcutaneous tumorigenic mice were prepared by the following steps: recovering ZM2-2 tumor cells, and culturing with a whole cell culture medium; tumor cells in logarithmic growth phase were collected, centrifuged and counted. F0 generation tumor-bearing mice were prepared by inoculating the mice with the cell concentration adjusted based on the actual cell number under the right axilla of the nude mice (the mice were further passaged in vivo and recorded as F1, and so on). Passaging for 1-2 times in vivo according to the condition, selecting tumor-bearing animals with vigorous tumor growth, no rupture and good health condition, taking out the tumor under aseptic condition, and preparing into 2-3 mm³Small tumor mass, inoculated under the right axilla of nude mice; observing the growth condition of the tumor after inoculation, and selecting the tumor with the volume of 85.17-199.75 mm³The range of ZM2-2 subcutaneous tumorigenic nude mice were tested with 8 mice each.

Using ZM2-2 subcutaneous tumorigenic nude mice injected with sterilized water as a control group, and marking as a group 1; nude mice subcutaneously tumorigenic of ZM2-2 administered Beritinib (PLB-1001) at 3mg/kg were assigned as group 2; nude mice subcutaneously tumorigenic of ZM2-2 administered Beritinib (PLB-1001) at 10mg/kg were assigned as group 3; nude mice subcutaneously tumorigenic ZM2-2 administered Beritinib (PLB-1001) at 30mg/kg were assigned as group 4; nude mice subcutaneously tumorigenic ZM2-2 administered temozolomide at 60mg/kg were assigned group 5; nude mice subcutaneously tumorigenic ZM2-2 administered Crizotinib (Crizotinib) at 50mg/kg were assigned as group 6. The tumor volumes of groups administered with 1d, 5d, 8d, 12d, 15d and 19d were recorded, and the results are shown in table 1; in Table 1, P is 0.05 or less as compared with group 1; in comparison with group 2, # means P.ltoreq.0.05, in comparison with group 3,. DELTA.means P.ltoreq.0.05, and in comparison with group 4,. DELTA.means P.ltoreq.0.05.

The neck was cut off to kill mice and the subcutaneous nodules were dissected, and the volume comparison of subcutaneous nodules in nude mice at 19d is shown in FIG. 5.

TABLE 1

Group of

1d

5d

8d

12d

15d

19d

1

129.90±17.45

317.06±70.22

612.87±217.02

952.39±398.67

1100.51±670.93

1462.78±733.08

2

130.48±26.59

171.94±34.47*

205.48±65.13*

276.87±147.78*

404.34±234.34*

499.83±259.44*

3

129.57±30.03

136.80±31.18*

111.51±32.51*

92.01±26.99*

99.26±30.39*

113.31±49.97*#

4

130.28±41.24

88.76±39.65*#

65.60±38.13*#

56.24±55.07*#

58.13±66.34*#

55.62±65.72*#

5

131.01±29.46

211.32±86.57*Δ☆

230.93±97.81*☆

205.00±78.50*

245.09±124.75*

242.78±128.37*

6

129.28±29.91

219.27±68.79*Δ☆

301.66±155.04*Δ☆

361.59±273.98*Δ☆

466.57±369.03*Δ☆

572.88±456.60*Δ☆

Table 1 and fig. 5 show that the tumor growth rate was significantly lower in groups 2, 3, and 4 treated with different concentrations of brigatinib (PLB-1001) than in groups administered with temozolomide and crizotinib; compared with a control group 1, the tumor inhibition rate of the group 2 reaches 64.08%, the tumor inhibition rate of the group 3 reaches 91.77%, the tumor inhibition rate of the group 4 reaches 96.44%, the tumor inhibition rate of the group 5 reaches 84.02%, the tumor inhibition rate of the group 6 reaches 61.12%, and the comparative difference between each group and the control group is significant (P < 0.05).

ZM2-2 tumor cells simulate brain glioma cells with significantly increased p-met and p-stat3 expression, and the results show that patients screened for brain gliomas with significantly increased p-met and p-stat3 expression can effectively inhibit the growth of brain gliomas by targeted administration of Beritinib drug therapy.

When the biomarker p-met and p-stat3 provided by the disclosure are combined for screening Buretinib high-sensitivity patients, brain glioma patients suitable for effective treatment of Buretinib can be efficiently, accurately and quickly screened, reliable guidance is provided for treatment of patients, time of the patients can be saved, and treatment efficiency can be improved.

The preferred embodiments of the present disclosure have been described in detail above, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

It should be noted that the various features described in the above embodiments may be combined in any suitable manner without departing from the scope of the invention. In order to avoid unnecessary repetition, various possible combinations will not be separately described in this disclosure.

In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.

SEQUENCE LISTING

<110> Neuko department of neurosurgery research in Beijing

<120> use of reagent for detecting biomarker for preparing kit for evaluating sensitivity of patient to brigatinib

<130>5721BJNI

<160>3

<170>PatentIn version 3.5

<210>1

<211>4173

<212>DNA

<213>Artificial Sequence

<220>

<223>This sequence is synthesized in lab.

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gatatatcca gtccattact gcaaaatact gtccacattg acctcagtgc tctaaatcca 3180

gagctggtcc aggcagtgca gcatgtagtg attgggccca gtagcctgat tgtgcatttc 3240

aatgaagtca taggaagagg gcattttggt tgtgtatatc atgggacttt gttggacaat 3300

gatggcaaga aaattcactg tgctgtgaaa tccttgaaca gaatcactga cataggagaa 3360

gtttcccaat ttctgaccga gggaatcatc atgaaagatt ttagtcatcc caatgtcctc 3420

tcgctcctgg gaatctgcct gcgaagtgaa gggtctccgc tggtggtcct accatacatg 3480

aaacatggag atcttcgaaa tttcattcga aatgagactc ataatccaac tgtaaaagat 3540

cttattggct ttggtcttca agtagccaaa ggcatgaaat atcttgcaag caaaaagttt 3600

gtccacagag acttggctgc aagaaactgt atgctggatg aaaaattcac agtcaaggtt 3660

gctgattttg gtcttgccag agacatgtat gataaagaat actatagtgt acacaacaaa 3720

acaggtgcaa agctgccagt gaagtggatg gctttggaaa gtctgcaaac tcaaaagttt 3780

accaccaagt cagatgtgtg gtcctttggc gtgctcctct gggagctgat gacaagagga 3840

gccccacctt atcctgacgt aaacaccttt gatataactg tttacttgtt gcaagggaga 3900

agactcctac aacccgaata ctgcccagac cccttatatg aagtaatgct aaaatgctgg 3960

caccctaaag ccgaaatgcg cccatccttt tctgaactgg tgtcccggat atcagcgatc 4020

ttctctactt tcattgggga gcactatgtc catgtgaacg ctacttatgt gaacgtaaaa 4080

tgtgtcgctc cgtatccttc tctgttgtca tcagaagata acgctgatga tgaggtggac 4140

acacgaccag cctccttctg ggagacatca tag 4173

<210>2

<211>4245

<212>DNA

<213>Artificial Sequence

<220>

<223>This sequence is synthesized in lab.

<400>2

atgcgaatcc taaagcgttt cctcgcttgc attcagctcc tctgtgtttg ccgcctggat 60

aaacctctca taatgaaggc ccccgctgtg cttgcacctg gcatcctcgt gctcctgttt 120

accttggtgc agaggagcaa tggggagtgt aaagaggcac tagcaaagtc cgagatgaat 180

gtgaatatga agtatcagct tcccaacttc accgcggaaa cacccatcca gaatgtcatt 240

ctacatgagc atcacatttt ccttggtgcc actaactaca tttatgtttt aaatgaggaa 300

gaccttcaga aggttgctga gtacaagact gggcctgtgc tggaacaccc agattgtttc 360

ccatgtcagg actgcagcag caaagccaat ttatcaggag gtgtttggaa agataacatc 420

aacatggctc tagttgtcga cacctactat gatgatcaac tcattagctg tggcagcgtc 480

aacagaggga cctgccagcg acatgtcttt ccccacaatc atactgctga catacagtcg 540

gaggttcact gcatattctc cccacagata gaagagccca gccagtgtcc tgactgtgtg 600

gtgagcgccc tgggagccaa agtcctttca tctgtaaagg accggttcat caacttcttt 660

gtaggcaata ccataaattc ttcttatttc ccagatcatc cattgcattc gatatcagtg 720

agaaggctaa aggaaacgaa agatggtttt atgtttttga cggaccagtc ctacattgat 780

gttttacctg agttcagaga ttcttacccc attaagtatg tccatgcctt tgaaagcaac 840

aattttattt acttcttgac ggtccaaagg gaaactctag atgctcagac ttttcacaca 900

agaataatca ggttctgttc cataaactct ggattgcatt cctacatgga aatgcctctg 960

gagtgtattc tcacagaaaa gagaaaaaag agatccacaa agaaggaagt gtttaatata 1020

cttcaggctg cgtatgtcag caagcctggg gcccagcttg ctagacaaat aggagccagc 1080

ctgaatgatg acattctttt cggggtgttc gcacaaagca agccagattc tgccgaacca 1140

atggatcgat ctgccatgtg tgcattccct atcaaatatg tcaacgactt cttcaacaag 1200

atcgtcaaca aaaacaatgt gagatgtctc cagcattttt acggacccaa tcatgagcac 1260

tgctttaata ggacacttct gagaaattca tcaggctgtg aagcgcgccg tgatgaatat 1320

cgaacagagt ttaccacagc tttgcagcgc gttgacttat tcatgggtca attcagcgaa 1380

gtcctcttaa catctatatc caccttcatt aaaggagacc tcaccatagc taatcttggg 1440

acatcagagg gtcgcttcat gcaggttgtg gtttctcgat caggaccatc aacccctcat 1500

gtgaattttc tcctggactc ccatccagtg tctccagaag tgattgtgga gcatacatta 1560

aaccaaaatg gctacacact ggttatcact gggaagaaga tcacgaagat cccattgaat 1620

ggcttgggct gcagacattt ccagtcctgc agtcaatgcc tctctgcccc accctttgtt 1680

cagtgtggct ggtgccacga caaatgtgtg cgatcggagg aatgcctgag cgggacatgg 1740

actcaacaga tctgtctgcc tgcaatctac aaggttttcc caaatagtgc accccttgaa 1800

ggagggacaa ggctgaccat atgtggctgg gactttggat ttcggaggaa taataaattt 1860

gatttaaaga aaactagagt tctccttgga aatgagagct gcaccttgac tttaagtgag 1920

agcacgatga atacattgaa atgcacagtt ggtcctgcca tgaataagca tttcaatatg 1980

tccataatta tttcaaatgg ccacgggaca acacaataca gtacattctc ctatgtggat 2040

cctgtaataa caagtatttc gccgaaatac ggtcctatgg ctggtggcac tttacttact 2100

ttaactggaa attacctaaa cagtgggaat tctagacaca tttcaattgg tggaaaaaca 2160

tgtactttaa aaagtgtgtc aaacagtatt cttgaatgtt ataccccagc ccaaaccatt 2220

tcaactgagt ttgctgttaa attgaaaatt gacttagcca accgagagac aagcatcttc 2280

agttaccgtg aagatcccat tgtctatgaa attcatccaa ccaaatcttt tattagtggt 2340

gggagcacaa taacaggtgt tgggaaaaac ctgaattcag ttagtgtccc gagaatggtc 2400

ataaatgtgc atgaagcagg aaggaacttt acagtggcat gtcaacatcg ctctaattca 2460

gagataatct gttgtaccac tccttccctg caacagctga atctgcaact ccccctgaaa 2520

accaaagcct ttttcatgtt agatgggatc ctttccaaat actttgatct catttatgta 2580

cataatcctg tgtttaagcc ttttgaaaag ccagtgatga tctcaatggg caatgaaaat 2640

gtactggaaa ttaagggaaa tgatattgac cctgaagcag ttaaaggtga agtgttaaaa 2700

gttggaaata agagctgtga gaatatacac ttacattctg aagccgtttt atgcacggtc 2760

cccaatgacc tgctgaaatt gaacagcgag ctaaatatag agtggaagca agcaatttct 2820

tcaaccgtcc ttggaaaagt aatagttcaa ccagatcaga atttcacagg attgattgct 2880

ggtgttgtct caatatcaac agcactgtta ttactacttg ggtttttcct gtggctgaaa 2940

aagagaaagc aaattaaaga tctgggcagt gaattagttc gctacgatgc aagagtacac 3000

actcctcatt tggataggct tgtaagtgcc cgaagtgtaa gcccaactac agaaatggtt 3060

tcaaatgaat ctgtagacta ccgagctact tttccagaag atcagtttcc taattcatct 3120

cagaacggtt catgccgaca agtgcagtat cctctgacag acatgtcccc catcctaact 3180

agtggggact ctgatatatc cagtccatta ctgcaaaata ctgtccacat tgacctcagt 3240

gctctaaatc cagagctggt ccaggcagtg cagcatgtag tgattgggcc cagtagcctg 3300

attgtgcatt tcaatgaagt cataggaaga gggcattttg gttgtgtata tcatgggact 3360

ttgttggaca atgatggcaa gaaaattcac tgtgctgtga aatccttgaa cagaatcact 3420

gacataggag aagtttccca atttctgacc gagggaatca tcatgaaaga ttttagtcat 3480

cccaatgtcc tctcgctcct gggaatctgc ctgcgaagtg aagggtctcc gctggtggtc 3540

ctaccataca tgaaacatgg agatcttcga aatttcattc gaaatgagac tcataatcca 3600

actgtaaaag atcttattgg ctttggtctt caagtagcca aaggcatgaa atatcttgca 3660

agcaaaaagt ttgtccacag agacttggct gcaagaaact gtatgctgga tgaaaaattc 3720

acagtcaagg ttgctgattt tggtcttgcc agagacatgt atgataaaga atactatagt 3780

gtacacaaca aaacaggtgc aaagctgcca gtgaagtgga tggctttgga aagtctgcaa 3840

actcaaaagt ttaccaccaa gtcagatgtg tggtcctttg gcgtgctcct ctgggagctg 3900

atgacaagag gagccccacc ttatcctgac gtaaacacct ttgatataac tgtttacttg 3960

ttgcaaggga gaagactcct acaacccgaa tactgcccag accccttata tgaagtaatg 4020

ctaaaatgct ggcaccctaa agccgaaatg cgcccatcct tttctgaact ggtgtcccgg 4080

atatcagcga tcttctctac tttcattggg gagcactatg tccatgtgaa cgctacttat 4140

gtgaacgtaa aatgtgtcgc tccgtatcct tctctgttgt catcagaaga taacgctgat 4200

gatgaggtgg acacacgacc agcctccttc tgggagacat catag 4245

<210>3

<211>4311

<212>DNA

<213>Artificial Sequence

<220>

<223>This sequence is synthesized in lab.

<400>3

atgcgaatcc taaagcgttt cctcgcttgc attcagctcc tctgtgtttg ccgcctggat 60

tgggctaatg gatactacag acaacagaga aaacttgttg aagagattgg ctggtcctat 120

acagataaac ctctcataat gaaggccccc gctgtgcttg cacctggcat cctcgtgctc 180

ctgtttacct tggtgcagag gagcaatggg gagtgtaaag aggcactagc aaagtccgag 240

atgaatgtga atatgaagta tcagcttccc aacttcaccg cggaaacacc catccagaat 300

gtcattctac atgagcatca cattttcctt ggtgccacta actacattta tgttttaaat 360

gaggaagacc ttcagaaggt tgctgagtac aagactgggc ctgtgctgga acacccagat 420

tgtttcccat gtcaggactg cagcagcaaa gccaatttat caggaggtgt ttggaaagat 480

aacatcaaca tggctctagt tgtcgacacc tactatgatg atcaactcat tagctgtggc 540

agcgtcaaca gagggacctg ccagcgacat gtctttcccc acaatcatac tgctgacata 600

cagtcggagg ttcactgcat attctcccca cagatagaag agcccagcca gtgtcctgac 660

tgtgtggtga gcgccctggg agccaaagtc ctttcatctg taaaggaccg gttcatcaac 720

ttctttgtag gcaataccat aaattcttct tatttcccag atcatccatt gcattcgata 780

tcagtgagaa ggctaaagga aacgaaagat ggttttatgt ttttgacgga ccagtcctac 840

attgatgttt tacctgagtt cagagattct taccccatta agtatgtcca tgcctttgaa 900

agcaacaatt ttatttactt cttgacggtc caaagggaaa ctctagatgc tcagactttt 960

cacacaagaa taatcaggtt ctgttccata aactctggat tgcattccta catggaaatg 1020

cctctggagt gtattctcac agaaaagaga aaaaagagat ccacaaagaa ggaagtgttt 1080

aatatacttc aggctgcgta tgtcagcaag cctggggccc agcttgctag acaaatagga 1140

gccagcctga atgatgacat tcttttcggg gtgttcgcac aaagcaagcc agattctgcc 1200

gaaccaatgg atcgatctgc catgtgtgca ttccctatca aatatgtcaa cgacttcttc 1260

aacaagatcg tcaacaaaaa caatgtgaga tgtctccagc atttttacgg acccaatcat 1320

gagcactgct ttaataggac acttctgaga aattcatcag gctgtgaagc gcgccgtgat 1380

gaatatcgaa cagagtttac cacagctttg cagcgcgttg acttattcat gggtcaattc 1440

agcgaagtcc tcttaacatc tatatccacc ttcattaaag gagacctcac catagctaat 1500

cttgggacat cagagggtcg cttcatgcag gttgtggttt ctcgatcagg accatcaacc 1560

cctcatgtga attttctcct ggactcccat ccagtgtctc cagaagtgat tgtggagcat 1620

acattaaacc aaaatggcta cacactggtt atcactggga agaagatcac gaagatccca 1680

ttgaatggct tgggctgcag acatttccag tcctgcagtc aatgcctctc tgccccaccc 1740

tttgttcagt gtggctggtg ccacgacaaa tgtgtgcgat cggaggaatg cctgagcggg 1800

acatggactc aacagatctg tctgcctgca atctacaagg ttttcccaaa tagtgcaccc 1860

cttgaaggag ggacaaggct gaccatatgt ggctgggact ttggatttcg gaggaataat 1920

aaatttgatt taaagaaaac tagagttctc cttggaaatg agagctgcac cttgacttta 1980

agtgagagca cgatgaatac attgaaatgc acagttggtc ctgccatgaa taagcatttc 2040

aatatgtcca taattatttc aaatggccac gggacaacac aatacagtac attctcctat 2100

gtggatcctg taataacaag tatttcgccg aaatacggtc ctatggctgg tggcacttta 2160

cttactttaa ctggaaatta cctaaacagt gggaattcta gacacatttc aattggtgga 2220

aaaacatgta ctttaaaaag tgtgtcaaac agtattcttg aatgttatac cccagcccaa 2280

accatttcaa ctgagtttgc tgttaaattg aaaattgact tagccaaccg agagacaagc 2340

atcttcagtt accgtgaaga tcccattgtc tatgaaattc atccaaccaa atcttttatt 2400

agtggtggga gcacaataac aggtgttggg aaaaacctga attcagttag tgtcccgaga 2460

atggtcataa atgtgcatga agcaggaagg aactttacag tggcatgtca acatcgctct 2520

aattcagaga taatctgttg taccactcct tccctgcaac agctgaatct gcaactcccc 2580

ctgaaaacca aagccttttt catgttagat gggatccttt ccaaatactt tgatctcatt 2640

tatgtacata atcctgtgtt taagcctttt gaaaagccag tgatgatctc aatgggcaat 2700

gaaaatgtac tggaaattaa gggaaatgat attgaccctg aagcagttaa aggtgaagtg 2760

ttaaaagttg gaaataagag ctgtgagaat atacacttac attctgaagc cgttttatgc 2820

acggtcccca atgacctgct gaaattgaac agcgagctaa atatagagtg gaagcaagca 2880

atttcttcaa ccgtccttgg aaaagtaata gttcaaccag atcagaattt cacaggattg 2940

attgctggtg ttgtctcaat atcaacagca ctgttattac tacttgggtt tttcctgtgg 3000

ctgaaaaaga gaaagcaaat taaagatctg ggcagtgaat tagttcgcta cgatgcaaga 3060

gtacacactc ctcatttgga taggcttgta agtgcccgaa gtgtaagccc aactacagaa 3120

atggtttcaa atgaatctgt agactaccga gctacttttc cagaagatca gtttcctaat 3180

tcatctcaga acggttcatg ccgacaagtg cagtatcctc tgacagacat gtcccccatc 3240

ctaactagtg gggactctga tatatccagt ccattactgc aaaatactgt ccacattgac 3300

ctcagtgctc taaatccaga gctggtccag gcagtgcagc atgtagtgat tgggcccagt 3360

agcctgattg tgcatttcaa tgaagtcata ggaagagggc attttggttg tgtatatcat 3420

gggactttgt tggacaatga tggcaagaaa attcactgtg ctgtgaaatc cttgaacaga 3480

atcactgaca taggagaagt ttcccaattt ctgaccgagg gaatcatcat gaaagatttt 3540

agtcatccca atgtcctctc gctcctggga atctgcctgc gaagtgaagg gtctccgctg 3600

gtggtcctac catacatgaa acatggagat cttcgaaatt tcattcgaaa tgagactcat 3660

aatccaactg taaaagatct tattggcttt ggtcttcaag tagccaaagg catgaaatat 3720

cttgcaagca aaaagtttgt ccacagagac ttggctgcaa gaaactgtat gctggatgaa 3780

aaattcacag tcaaggttgc tgattttggt cttgccagag acatgtatga taaagaatac 3840

tatagtgtac acaacaaaac aggtgcaaag ctgccagtga agtggatggc tttggaaagt 3900

ctgcaaactc aaaagtttac caccaagtca gatgtgtggt cctttggcgt gctcctctgg 3960

gagctgatga caagaggagc cccaccttat cctgacgtaa acacctttga tataactgtt 4020

tacttgttgc aagggagaag actcctacaa cccgaatact gcccagaccc cttatatgaa 4080

gtaatgctaa aatgctggca ccctaaagcc gaaatgcgcc catccttttc tgaactggtg 4140

tcccggatat cagcgatctt ctctactttc attggggagc actatgtcca tgtgaacgct 4200

acttatgtga acgtaaaatg tgtcgctccg tatccttctc tgttgtcatc agaagataac 4260

gctgatgatg aggtggacac acgaccagcc tccttctggg agacatcata g 4311

Claims (3)

1. Use of a reagent for detecting a biomarker in the manufacture of a kit for assessing a patient's sensitivity to brigatinib, wherein the biomarker is p-Met and/or p-Stat3 of a tumor tissue and the reagent for detecting a biomarker is a reagent capable of quantitatively detecting the biomarker; wherein the patient is a glioma patient.

2. The use according to claim 1, wherein the kit for assessing the sensitivity of a patient to brigatinib is a kit for detecting the biomarker content by colloidal gold dipstick, western blotting or immunohistochemistry.

3. Use according to claim 1, wherein the reagent capable of quantitatively detecting the biomarker is a first monoclonal antibody against p-Met and/or a first monoclonal antibody against p-Stat 3.

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