WO2020159181A1 - Method for predicting therapeutic response in acute myeloid leukemia by using exosome-derived immunophenotype - Google Patents

Abstract

The present invention relates to a method for predicting the relapse of acute myeloid leukemia, the method comprising the steps of: 1) isolating exosomes from a sample collected from a patient confirmed to be completely cured of acute myeloid leukemia; and 2) measuring the expression levels of CD53 and CD47 in the exosomes.

Description

Method for predicting treatment responsiveness of acute myeloid leukemia using exosome-derived immunophenotype

The present invention relates to a method for predicting the recurrence of acute myeloid leukemia using an exosome-derived immunophenotype, and more specifically, by using an immunophenotype overexpressed in exosomes isolated from a patient's sample as a marker, standard therapy It relates to a method for predicting the likelihood of recurrence of a patient who has been cured through application.

Acute myeloid leukemia is one of the blood cancers caused by genetic mutations related to proliferation, differentiation, and cell natural death of progenitor cells or stem cells in the early stages of differentiation of myeloid leukocytes. According to the data of the Central Cancer Cancer Register in Korea, as of 2015, the incidence of cancer was 1.0% and the incidence rate of 4.4 cases per 100,000 population, and the number of new diagnoses by year is the most frequent.

The basis for the treatment of acute myelogenous leukemia is combined cytotoxic chemotherapy with anthracyclin, which has been recognized as a standard therapy for about 40 years. Meanwhile, FLT3 inhibitors (fms-like tyrosine kinase 3 inhibitors) in 2017 were used in clinical trials with approval from the U.S. FDA, followed by hypomethylating agents in the mid-2000s, and various targeted treatments for acute myeloid leukemia in the last 10 years. (Targeted therapies) have been developed and are waiting for clinical application.

About 70% of patients who have undergone standard therapy will eventually experience recurrence, and currently there is no apparent remedy for relapse, leading to the death of most relapsed patients. Therefore, it is very important to determine the optimal treatment method by selectively checking patients who are expected or suspected of recurrence. On the other hand, there is an existing study for diagnosing acute myeloid leukemia using an exosome-based biomarker, but development of a biomarker predicting the likelihood of recurrence of a patient who has been completely cured is insufficient.

Under this background, the present inventors overexpressed CD53 and CD47 in exosomes of patients who relapsed after undergoing a determination of cure of acute myeloid leukemia through cytotoxic anticancer drug treatment through exosome protein analysis. The present invention was completed by discovering that the degree of change in the expression levels of CD53 and CD47 was little.

Accordingly, an object of the present invention is to provide a method for predicting the recurrence of acute myelogenous leukemia by measuring the expression level of CD53 and CD47 in exosomes of a patient who has been completely cured of acute myeloid leukemia.

Another object of the present invention is to provide a method for providing information for predicting the recurrence of acute myelogenous leukemia by measuring the expression level of CD53 and CD47 in exosomes of a patient who has been judged to cure acute myelogenous leukemia.

However, the problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those having ordinary knowledge in the art from the following description.

According to an embodiment of the present invention, 1) separating the exosomes from a sample taken from a patient who has been determined to cure acute myeloid leukemia; And 2) measuring CD53 and CD47 expression levels of the exosomes; A method for predicting the recurrence of acute myeloid leukemia is provided.

According to one side, the patient may be a patient who has been treated with a cytotoxic anticancer agent.

According to one side, the cytotoxic anticancer agent may be at least one selected from the group consisting of anthracycline-based compounds, cytarabine and all-trans-retinoic acid (ATRA).

According to one side, CD53 and CD47 expression levels of the exosomes may be measured by exosome-ELISA.

According to one side, when CD53 and CD47 are overexpressed in the exosome, it can be predicted that the therapeutic response to a cytotoxic anticancer agent is low.

According to one side, when CD53 and CD47 are overexpressed in the exosome, it can be predicted that the likelihood of recurrence after treatment with cytotoxic chemotherapy is high.

According to another embodiment of the present invention, 1) separating the exosome from a sample taken from a patient who has been determined to cure acute myeloid leukemia; And 2) measuring CD53 and CD47 expression levels of the exosomes; A method of providing information for predicting the recurrence of acute myeloid leukemia is provided.

The method for predicting the recurrence of a patient with acute myeloid leukemia of the present invention is characterized by confirming the level of CD53 and CD47 expression of exosomes in a sample taken from a patient who has been completely cured through treatment with a cytotoxic anticancer drug, which is a standard treatment, and the patient relapses later. You can simply check the possibilities.

It should be understood that the effects of the present invention are not limited to the above-described effects, and include all effects that can be deduced from the configuration of the invention described in the detailed description or claims of the present invention.

Figure 1 (A) is a step-by-step schematic of a method for separating exosomes from a sample taken from a patient and a culture medium of a cell, Figure 1 (B) is a schematic diagram of multiple columns used for exosome separation, 1(C) shows the comparison of the results of separation of exosomes using the existing single column and the multiple columns.

Figure 2 shows the separated exosomes by Western blot by performing size exclusion chromatography using multiple columns.

3 is a result of protein analysis using liquid chromatography-mass spectrometry (LC-MS) of exosomes derived from human acute myeloid leukemia cell lines HL60, KG-1, THP-1 and normal cell lines HDF, MSC in human bone marrow.

Figure 4 is a human acute myeloid leukemia cell line derived from the results of protein analysis of the exosomes, respectively, overexpression, confirmed the underexpressed immune phenotype.

Figure 5 is a human acute myeloid leukemia cell line lysate and CD53, CD47 expression in exosomes confirmed by Western blot.

Figure 6 shows the difference in survival rate according to the difference in the amount of expression of CD53 or CD47 before and after treatment in the two patient groups to which standard therapy was applied.

FIG. 7 shows the difference in survival rates when the differences in the expression levels of CD53 and CD47 were combined before and after treatment in the two patient groups to which standard therapy was applied.

Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. However, various changes may be made to the embodiments, and the scope of the patent application right is not limited or limited by these embodiments. It should be understood that all modifications, equivalents, or substitutes for the embodiments are included in the scope of rights.

The terms used in the examples are used for illustrative purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as “include” or “have” are intended to indicate that a feature, number, step, operation, component, part, or combination thereof described on the specification exists, and that one or more other features are present. It should be understood that the existence or addition possibilities of fields or numbers, steps, operations, components, parts or combinations thereof are not excluded in advance.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person skilled in the art to which the embodiment belongs. Terms, such as those defined in a commonly used dictionary, should be interpreted as having meanings consistent with meanings in the context of related technologies, and should not be interpreted as ideal or excessively formal meanings unless explicitly defined in the present application. Does not.

In addition, in the description with reference to the accompanying drawings, the same reference numerals are assigned to the same components regardless of reference numerals, and redundant descriptions thereof will be omitted. In describing the embodiments, when it is determined that detailed descriptions of related known technologies may unnecessarily obscure the subject matter of the embodiments, the detailed descriptions will be omitted.

The present inventors are divided into a large change group (Group 1) and a small change group (Group 2) according to the following examples according to the difference in the expression level of CD53 or CD47 in acute myeloid leukemia patients to which standard treatment is applied. When comparing the survival rate, it was confirmed that a difference in disease-free survival occurred according to the degree of change in each immunophenotype, and also when comparing the disease-free survival rate by combining the changes in the expression levels of CD53 and CD47, each immunophenotype was used alone. The present invention was completed by discovering that it is possible to accurately distinguish disease-free survival rates.

Thus, according to an embodiment of the present invention, 1) separating the exosome from a sample taken from a patient who has been determined to cure acute myeloid leukemia; And 2) measuring CD53 and CD47 expression levels of the exosomes; A method for predicting the recurrence of acute myeloid leukemia is provided.

The sample taken from the patient may be blood, lymphatic fluid, cerebrospinal fluid, urine, saliva, cerebrospinal fluid, etc., but serum or plasma is preferred.

The term "therapeutic responsiveness" in the present invention means that the patient responds preferentially or unfavorably to the standard therapy of acute myeloid leukemia.

In the present invention, the term "prediction" means that the patient is treated with standard treatment of acute myelogenous leukemia, and it is determined in advance whether the likelihood of recurrence, survival or survivability.

In the present invention, the term "exosome" is a small size (30-150nm) vesicle secreted from various types of cells, and is known to play a variety of roles, such as delivering membrane components, proteins, and RNA.

In the case of using the method of the present invention, among the patients who are judged to be completely cured by applying standard therapy, recommend a clinical trial for a new drug to a patient with a high probability of recurrence of acute myeloid leukemia or help to select the most appropriate treatment method Can.

In step 1), the separation of the exosomes is ultracentrifuge, density centrifuge, the use of a column, PEG precipitation, chromatography, immuno-magnetic separation (immuno-magnetic separation, IMS) and acoustic separation (acoustic separation) methods can be used, but the pore size is 20nm to 100nm porous beads a), the porous beads a) laminated on top, the pores It is preferable to perform size exclusion chromatography using multiple columns including a porous bead having a size of 20 nm or less and a separation membrane positioned between the porous bead a) and the porous bead b). As such, by using multiple columns for size exclusion chromatography, only exosomes in which proteins and lipids are not mixed from a patient's sample can be separated with high purity (see FIGS. 1B and 1C).

More specifically, the porous beads a) separate exosomes and lipid proteins from the biological sample, and the porous beads b) separate exosomes and water-soluble proteins from the biological sample, and the porous beads a) and the porous The volume ratio of beads b) may be 95:5 to 5:95. Beads used in multiple columns include agarose, allyl dextran and N, N'-methylene bisacrylamide compounds, methacryl, and dex. It may be to include a polymer consisting of one or more selected from tran (dextran) and polyacrylamide (polyacrylamide), but is not limited thereto.

The cytotoxic anti-cancer agent may be one or more selected from the group consisting of anthracycline-based compound compounds, cytarabine and all-trans-retinoic acid (ATRA), which are known to be used in standard therapy for acute myeloid leukemia.

The CD53 or CD47 expression level of exosomes in step 2) may be measured by exosome-ELISA. "Exosomal-ELISA", unlike conventional ELISA, can use the exosome itself as a sample to identify an immunophenotype that is specifically expressed only in exosomes, and is an exosome-ELISA marker that uses the separated exosome itself as a sample Since the expression level is confirmed, the results can be quickly confirmed.

According to the result confirmed in step 2), when CD53 or CD47 is overexpressed in the exosome, it can be predicted that the therapeutic reactivity to the cytotoxic anticancer agent is low, and the likelihood of recurrence after the cytotoxic anticancer therapy is high.

According to another embodiment of the present invention, 1) separating the exosome from a sample taken from a patient who has been determined to cure acute myeloid leukemia; And 2) measuring CD53 and CD47 expression levels of the exosomes; A method of providing information for predicting the recurrence of acute myeloid leukemia is provided.

Hereinafter, the present invention will be described in more detail through examples. The following examples are described for the purpose of illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1: Selection and culture of cell lines

Human acute myeloid leukemia cell lines HL60, KG-1, THP-1, HDF was purchased from the American Type Culture Collection (Manassasm VA, USA) as a normal cell line in human bone marrow, and human bone marrow derived mesenchymal stem cell as a normal cell line in human bone marrow. (hMSC) was obtained by primary culture in normal human bone marrow. HL60, KG-1 for IMDM, THP-1 for RPMI-1640, HDF for 10% exosome removal in DMEM cultures Fetal Bovine Serum (Gibco, CA, USA), 1% Penicillin (Gibco), 1% Streptomycin (Gibco ) And cultured. hMSC was cultured using Mesenchymal Stem Cell Growth Medium (Lonza, Walkersville, USA). Removal of exosomes Fetal bovine serum was removed by centrifuging normal fetal bovine serum in an ultracentrifuge 100,000 xg for 3 hours to remove exosomes, which were then filtered through a 0.2 micrometer filter.

Example 2: Isolation of exosomes

2-1. Production of multiple columns

After filling the chromatography column (diameter: 10 mm, height: 50 mm) with Sepharose® CL-6B at 70% (v/v), Sephacryl 200-HR at the top is filled with 30% (v/v) The multi-column was manufactured so that the height ratio between the lower part and the upper part was 7:3.

2-2. Size exclusion chromatography

After culturing the HL60, KG-1, THP-1, HDF, and MSC cell lines of Example 1 for 48-72 hours, the culture solution was collected, and 10 minutes at 300 xg, 30 minutes at 5,000 xg, 30 minutes at 10,000 xg Sequential centrifugation was performed for a minute to obtain a supernatant from which cells, cell death, and cell debris were removed (FIG. 1). After concentration using Amicon® ultrafilter 100 kDa (Merch Millipore, Temecula, CA, USA), size exclusion chromatography was performed using the multiple columns prepared in Example 2-1.

When using a patient plasma or serum sample, size exclusion chromatography was performed using the multiple columns prepared in Example 2-1 without concentration.

2-3. Exosome separation result confirmation

The exosomes isolated through Example 2-2 were dissolved using PRO-PREP Protein Extraction Solution (Intron Biotech), and then quantified using the BCA Protein Assay method. 30 micrograms of protein were electrophoresed and 5% Sodium dodecyl sulfate (SDS)-polyacrylamide stocjing gel and 8-12% SDS-polyacrylamide separating gel were used. Mouse monoclonal anti-CD4 antibody (1:1000), Mouse monoclonal anti-CD9 antibody (1:1000), Mouse monoclonal anti-CD33 antibody (1:1000), Mouse monoclonal anti-CD47 antibody (1:1000), Mouse monoclonal Anti-CD53 antibody (1:1000) (all Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit monoclonal anti-calnexin antibody (1:1000) (Cell Signaling Technology, Beverly, MA, USA) as primary antibody Peroxidase-conjugated anti-mouse (1:2000) and anti-rabbit (1:2000) were used as secondary antibodies.

As a result, it was confirmed that the exosomes were purely separated as shown in FIG. 2.

Example 3: Protein analysis of exosomes

3-1. Tryptic digestion using FASP method

30 μg of human acute myeloid leukemia cell lines HL60, KG-1, THP-1, normal cell lines HDF in human bone marrow and MSC-derived exosome proteins cultured in Example 1 were digested with peptides using the FASP method. The FASP method is briefly summarized as follows. 30 μg of exosome protein was reduced by decomposition with 0.1 M Tris-HCl, pH 7.6 to 4% SDS and 0.1 M DTT, incubated at 37 °C for 45 min, and boiled for 7 min under mild vibration. Then, the microcon Ultracel 30kDa (Millipore) was centrifuged for 40 minutes at 14,000 xg, 16 °C, and the prepared sample was FASP-urea buffer (0.2 mL of 8 M urea in 0.1 M Tris-HCl, pH 8.5). Washed and centrifuged 3 times under the same conditions. Next, alkylated in a UA solution containing 55 mM IAA, 0.1 mL, incubated for 20 minutes in a state of blocking light, and then centrifuged for 14,000 xg, 16 °C, 40 minutes, and the prepared sample was prepared in FASP-urea buffer ( 0.2 mL of 8 M urea in 0.1 M Tris-HCl, pH 8.5) and centrifuged 3 times under the same conditions. To prepare buffer conditions for trypsin, 0.2 mL of 100 mM TEAB was added to the sample. Protein digestion was performed using a sequencing grade trypsin filter under 100 mM TEAB, 37 °C, overnight. Subsequently, the peptide was reduced by centrifugation for 14,000 xg, 16°C, and 20 minutes, and further reduced by adding 100 mM TEAB and 75 μL twice more. The reduced peptide was dried using a SpeedVac concentrator. The dried sample was dissolved in 5% ACN, water containing 180 μL, 0.1% Formic acid, desalted using a C-18 Spin Column (Thermo Scientific, USA), and then dried again with a SpeedVac concentrator.

3-2. TMT 5 plex labeling

Tandem Mass Tag (TMT) Labeling Reagents TMT-126, TMT-127, TMT-128, TMT-129, TMT-130 0.8mg vials of Anhydrous ACN were added to 41 μL and the prepared TMT labeling reagent 41 μL was prepared in the above example After adding to the peptide sample prepared in 3-1 and reacting for 1 hour, 8 μL of 5% hydroxylamine was added to each sample and reacted for 15 minutes.

3-3. High pH fractionation

High pH fractionation was performed to increase the number of peptides identified in the sample and to separate each peptide using hydrophobicity during liquid chromatography-mass spectrometry (LC-MS). Each peptide sample was separated into 12 fractions using an Agilent 1100 series HPLC system (Agilent Technologies, Santa Clara, CA, USA) using Waters, XBridge BEH C18 column, 3.5 μm, 1.0×100 mm. Specifically, equilibrium was maintained in buffer A (10 mM Ammonium formate in water (pH 10.0)) before sample injection, and separated using 0-70% of buffer B (10 mM Ammonium formate in 90% ACN (pH 10)). (The gradient followed as: 0-10 min, 5% B; 10-20 min, 5% B; 20-80 min, 35% B; 80-95 min, 70% B; 95-105 min, 70% B; 105-115 min, 0% B).

3-4. Identification of proteins using mass spectrometry

The peptide sample fractionated in Example 3-3 was resuspended in water containing 0.1% formic acid, and then analyzed using a Q-Exactive Orbitrap Hybrid Mass Spectrometry connected with EASY-nLC 1000 system (ThermoScientific, Bremen, Germany). The gradient was as follow: from 5% to 40% of solvent B for 120 min, from 40% to 80% of solvent B for 5 min, maintaining at 80% of solvent B for 10 min, and equilibrating the column at 1% of B for 30% (Sol A: 0.1% formic acid in water, Sol B: 0.1% formic acid in acetonitrile). The peptide was eluted with a trap column, and ionization was performed with an EASY-spray column (50 cm×75 μm ID) filled with 2 μm C18 particles and an electric spray voltage of 1.8 kV. Mass spectrometry data were collected at an automatic gain control target value of 1.0 x 10 ⁶ , a maximum ion implantation of 120 ms, a resolution of 200-70,000 m/z, and a scan range of 400-2,000 m/z. The maximum ion implantation time of MS/MS was set at a resolution of 17,500 to 60 ms. The dynamic exclusion time was set to 30 seconds.

Mass spectrometry data was retrieved against the Uniprot human database (released in June, 2017) using SEQUEST® from Proteome Discoverer 2.1 (Thermo Fisher Scientific Inc., Germany). Carbamidomethylation of cysteine, TMT tag and N-terminal of Lysin, N-acetylation or oxidation of Methionine were used for each search. Peptide spectrum match and 1% False discovery rate standard at the protein level are applied. The tolerance was set to 10 ppm for the precursor mass and 0.8 Da for the fragment mass.

As a result, as shown in Figures 3 and 4, human acute myeloid leukemia cell lines HL60, KG-1, THP-1 and marker candidates showing significant differences in expression levels in exosome proteins derived from normal cell lines HDF and MSC in human bone marrow Could be selected.

Example 4: Analysis of survival rate according to the difference in the expression level of CD53 or CD47 in a patient to which standard therapy is applied

By selecting an overexpressed immunophenotype among acute myeloid leukemia-derived exosome proteins, ELISA conducted an experiment for predicting treatment response by this method, including Human leukocyte surface antigen CD47 ELISA (Cat.MBS926478, MyBiosource, Inc., CA, USA) , Exo-TESTTM CD53-exosome ELISA (Cat. EKC34422, Lonza) was used. The experiment proceeded according to the protocol provided by the company.

For patients with acute myeloid leukemia to which standard therapy was applied, disease-free survival was compared by dividing the patients into two groups according to the difference in the expression levels of CD53 or CD47. The independent variable is based on the median value for the difference in the expression level of CD53 or CD47 in the entire patient group, and the difference in the expression level is greater than the median value in the entire patient group (Group 1) and the expression level is less than the median value in the entire patient group. Divided into groups (Group 2) showing differences. The dependent variable was disease-free survival according to each group, and was set as the period from diagnosis of acute myeloid leukemia to relapse or death.

As a result, as shown in FIG. 6, it was confirmed that a difference in disease-free survival occurred after being judged as a cure by standard treatment according to a difference in the expression level of CD53 or CD47.

Next, the results of analyzing the disease-free survival rate using CD53 and CD47 together are shown in FIG. 7. Referring to FIG. 7, CD53 and CD47 expression levels are greater than the median value of the entire patient group, or the difference between CD53 and CD47 expression levels is small and the difference is greater (Group 1), CD53 than the median value of the entire patient group When comparing the CD47 and CD47 expressions, the patients (Group 2) had little difference in expression level, and it was confirmed that disease-free survival can be predicted more accurately when both CD53 and CD47 were used.

These results suggest that CD53 and CD47 are overexpressed even after standard therapy is applied to patients whose CD53 and CD47 expression levels are less than the median value of the entire patient group.

As described above, although the embodiments have been described by the limited drawings, those skilled in the art can apply various technical modifications and variations based on the above. For example, even if the described techniques are performed in a different order than the described method, and/or the described components are combined or combined in a different form from the described method, or replaced or replaced by another component or equivalent Appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents to the claims are also within the scope of the following claims.

Claims (8)

1) separating the exosomes from a sample taken from a patient who has been determined to have completely cured acute myeloid leukemia; And

2) measuring CD53 and CD47 expression levels of the exosomes; A method of predicting the recurrence of acute myeloid leukemia, which includes.

According to claim 1,

The patient is a patient who has been treated with a cytotoxic anticancer agent.

According to claim 2,

The cytotoxic anticancer agent is at least one member selected from the group consisting of anthracycline-based compounds, cytarabine and all-trans-retinoic acid (ATRA).

According to claim 1,

The level of CD53 and CD47 expression of the exosomes is measured by exosome-ELISA, method.

According to claim 1,

If CD53 and CD47 are overexpressed in the exosome, the method predicts that the therapeutic response to a cytotoxic anticancer agent is low.

According to claim 1,

If CD53 and CD47 are overexpressed in the exosomes, the method is predicted to have a high probability of recurrence after treatment with cytotoxic chemotherapy.

1) separating the exosomes from a sample taken from a patient who has been determined to have completely cured acute myeloid leukemia; And

2) measuring CD53 and CD47 expression levels of the exosomes; A method of providing information for predicting the recurrence of a patient with acute myeloid leukemia comprising a.

The method of claim 7,

The patient is a patient who has been treated with a cytotoxic anticancer agent.

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