WO2014030683A1 - Biomarqueur pour la détection d'un facteur pour l'anémie chez un patient anémique - Google Patents

Biomarqueur pour la détection d'un facteur pour l'anémie chez un patient anémique Download PDF

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WO2014030683A1
WO2014030683A1 PCT/JP2013/072335 JP2013072335W WO2014030683A1 WO 2014030683 A1 WO2014030683 A1 WO 2014030683A1 JP 2013072335 W JP2013072335 W JP 2013072335W WO 2014030683 A1 WO2014030683 A1 WO 2014030683A1
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acvr2b
anemia
patient
antibody
peripheral blood
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PCT/JP2013/072335
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Japanese (ja)
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杉山　大介
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国立大学法人九州大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)

Definitions

  • the present invention relates to a biomarker for determining that an anemic patient is MDS, a method for detecting a factor of anemia in an anemic patient, a kit therefor, and a diagnostic agent.
  • the present invention also relates to a biomarker for determining the risk of developing leukemia in an anemic patient, a method for determining the risk of developing leukemia in an anemic patient, and a kit and diagnostic agent therefor.
  • the present invention relates to a biomarker for predicting the necessity and frequency of blood transfusion in an anemic patient, a method for predicting the necessity and frequency of blood transfusion in an anemic patient, and a kit and a diagnostic agent used therefor.
  • MDS myelodysplastic syndrome
  • MDS is a type of blood tumor and is a disease in which the quantity and quality of blood cells are abnormal. And it is known that MDS causes a pre-leukemia state to cause hematopoietic disorder, has a poor prognosis, and shifts to acute myeloid leukemia. It is also a disease in which disorders such as blood cell differentiation and maturation are observed. Specifically, symptoms such as a decrease in the number of red blood cells (anemia state), a decrease in the number of platelets, an abnormality (decrease or increase) in the number of white blood cells are observed.
  • the number of patients with MDS is 300,000 worldwide, and 7,100 in Japan, and the average age is 70s and the elderly. For this reason, there is concern about an increase in the number of MDS patients because it will enter an aging society in the future.
  • MDS includes refractory anemia (RA), refractory cytopenia with multicytic dysplasia (RCMD), ironblastic refractory anemia with multicytic dysplasia (RCMD-RS), and increased blasts Refractory anemia with (RAEB) and the like are included.
  • the diagnostic criteria for RA is based on the presence of blasts less than 1% in peripheral blood, less than 5% in bone marrow, and no cyclic iron blasts, with a probability of 10 becoming a leukemia from RA. %.
  • As a method for treating RA no treatment, follow-up, or blood transfusion treatment is mainly used, and 60 to 80% of RA patients have worsened symptoms.
  • RCMD in addition to the diagnostic criteria for RA, clear morphological dysplasia of multiblood cells is observed at 10% or more, and the probability of transition from RCMD to leukemia is 15 to 20%.
  • RA and RARS are mild, RCMD and RCMD-RS are moderate, and RAEB is severe.
  • kits using mRNA encoding WT1 protein have been put on the market for diagnosis of RAEB, which is a severe myelodysplastic syndrome.
  • RAEB a severe myelodysplastic syndrome.
  • a kit requires a step of collecting total RNA from peripheral blood and the like, and further improvement is required for easy use in medical practice.
  • kits are not suitable for diagnosis of mild cases of MDS.
  • Patent Document 1 discloses a method for measuring the kinase activity of activin receptor 2B. And it is disclosed that activin is a factor related to the differentiation of erythrocytes as an action exerted in vivo.
  • Patent Document 2 discloses a monoclonal antibody that recognizes an extracellular region of a mouse-derived activin receptor. It is also disclosed that activin is a protein that acts in the field of hematopoiesis.
  • Patent Document 3 discloses a method of increasing red blood cells using an activin receptor 2 antagonist. The use of such antagonists as a method for treating anemia is also disclosed.
  • Patent Document 4 discloses a bone marrow erythroid progenitor cell differentiation promoter comprising arginine as a main component and further containing activin.
  • a method useful for determining the cause of anemia is a cause of anemia. It is necessary to make a morphological diagnosis of the multi-blood cells. Therefore, it is very difficult to examine whether a patient who presents with anemia suffers from MDS in a general clinic where there is no facility capable of collecting a specimen from the bone marrow by bone marrow puncture and a hematologist.
  • a biomarker expressed in the process of differentiation from hematopoietic stem cells is used as an index in order to provide a method capable of easily diagnosing MDS by outsourcing a test even from a general clinic.
  • Development of a diagnostic method for MDS is required.
  • Sca-1, c-kit, CD71, Gata1, Ter119 etc. are expressed in mice, and CD34, CD71, GPA (Glycophorin A) etc. are expressed in humans.
  • ACVR2B which is one of the activin receptors known to be involved in hematopoiesis as described above, is involved in the process of hematopoietic differentiation. .
  • kits using mRNA encoding WT1 is not simple in that it is necessary to collect total RNA from peripheral blood.
  • the main problem of the present invention is to provide a method for determining the cause of anemia, and in particular, to determine by a simple method such as not performing bone marrow puncture or requiring the determination of a hematologist Is to provide a way to do. Furthermore, the subject of the present invention includes providing a simple method for determining whether anemia patients are prone to developing leukemia. The subject of the present invention also includes a method for predicting the necessity and frequency of blood transfusion in an anemic patient.
  • the present inventor isolates positively stained erythroblasts, reticulocytes, and erythrocytes, which are cell groups classified into differentiation stages from hematopoietic stem cells, by experiments using Gata1-GFP transgenic mice. succeeded in.
  • Item 1 A method for detecting an anemia factor in an anemia patient comprising the following steps 1 and 2; (1) Step 1 of measuring the expression level of activin receptor 2B (ACVR2B) in multiblood cells contained in peripheral blood obtained from the patient, (2) When the expression level of ACVR2B measured in step 1 is greater than the expression level of ACVR2B in the polycytic cells contained in peripheral blood obtained from a healthy person, it is determined that the cause of anemia in the patient is MDS Step 2 to do.
  • ACVR2B activin receptor 2B
  • Item 2 The method according to Item 1, wherein the multicytic cells are one or more selected from the group consisting of red blood cells, white blood cells, and platelets.
  • Item 3 The method according to Item 1 or 2, wherein the MDS is at least one selected from the group consisting of RA, RCMD, RCMD-RS, and RAEB.
  • Item 4 A method for determining the risk of developing leukemia in an anemia patient comprising the following steps 1 and 2; [1] Step 1 of measuring the expression level of ACVR2B in multicytic cells contained in peripheral blood obtained from the patient, [2] Step 2 for determining that the patient is at risk of developing leukemia when the expression level of ACVR2B measured in Step 1 is greater than the expression level of ACVR2B in polycytic cells contained in peripheral blood obtained from healthy subjects .
  • Item 5 The method according to Item 4, wherein the multicytic cells are one or more selected from the group consisting of red blood cells, white blood cells, and platelets.
  • Item 6 The method according to Item 4 or 5, wherein the leukemia is acute myeloid leukemia.
  • Item 7 A method for predicting the necessity or frequency of blood transfusion of an anemic patient comprising the following steps 1 and 2; ⁇ 1 ⁇ Step 1 of measuring the expression level of ACVR2B in multicytic cells contained in peripheral blood obtained from the patient, ⁇ 2 ⁇
  • Step 1 When the expression level of ACVR2B measured in step 1 is greater than the expression level of ACVR2B in the polycytic cells contained in peripheral blood obtained from healthy subjects, blood transfusion is required for the patient, and the frequency is Step 2 for determining that the frequency is higher than the blood transfusion frequency of the patient at the time of collection.
  • Item 8 The method according to Item 7, wherein the multicytic cells are any one selected from the group consisting of red blood cells, white blood cells, and platelets.
  • Item 9 The method according to Item 7 or 8, wherein the leukemia is acute myeloid leukemia.
  • Item 10 A diagnostic agent for detecting an anemia factor of an anemic patient as MDS, including an anti-ACVR2B antibody.
  • Item 11 The diagnostic agent according to Item 10, wherein the MDS is at least one selected from the group consisting of RA, RCMD, RCMD-RS, and RAEB.
  • Item 12 A kit for detecting an anemia factor of an anemic patient as MDS, including an anti-ACVR2B antibody.
  • Item 13 The kit according to Item 12, wherein the MDS is at least one selected from the group consisting of RA, RCMD, RCMD-RS, and RAEB.
  • Item 14 A diagnostic agent for determining the risk of developing leukemia in anemia patients, comprising an anti-ACVR2B antibody.
  • Item 15 The diagnostic agent according to Item 14, wherein the leukemia is acute myeloid leukemia.
  • Item 16 A kit for determining the risk of developing leukemia in anemia patients, comprising an anti-ACVR2B antibody.
  • Item 17 The kit according to Item 16, wherein the leukemia is acute myeloid leukemia.
  • Item 18 A diagnostic agent for the necessity and frequency of transfusion of anemia patients, including an anti-ACVR2B antibody.
  • Item 19 A kit for detecting the necessity and frequency of blood transfusion of an anemia patient, including an anti-ACVR2B antibody.
  • a biomarker comprising ACVR2B for detecting an anemia factor of an anemic patient as MDS.
  • Item 21 The biomarker according to Item 20, wherein the MDS is at least one selected from the group consisting of RA, RCMD, RCMD-RS, and RAEB.
  • Item 22 A biomarker comprising ACVR2B for determining the risk of developing leukemia in anemia patients.
  • Item 23 The biomarker according to Item 22, wherein the leukemia is acute myeloid leukemia.
  • Item 24 A biomarker for predicting the necessity and frequency of blood transfusion of an anemic patient, comprising ACVR2B.
  • diagnostic agent By using the method, diagnostic agent, kit, or biomarker according to the present invention, it is excellent in that it can be diagnosed without performing bone marrow puncture, which is conventionally performed in diagnosing MDS and burdens the patient. ing. And it is very excellent also in the point that the diagnosis of MDS is possible without using a hematologist with advanced technology.
  • the method, diagnostic agent, kit, or biomarker according to the present invention is particularly useful for determining the cause of anemia by MDS and a simple method. This is excellent in that it is possible to obtain sufficient and simple patient evidence necessary to introduce an outpatient in a general clinic to a specialist.
  • the method, diagnostic agent, kit, or biomarker of the present invention is effectively used for predicting progression from MDS to leukemia.
  • MDS is likely to progress to leukemia.
  • leukemia develops, leukemia cells proliferate violently in the bone marrow and cannot normally produce blood cells, and as a result, red blood cells can be made.
  • anemia is caused by disappearance, and blood transfusion is required for the treatment of MDS.
  • MDS is impaired in differentiation into red blood cells, maturation, etc., blood transfusion is also necessary for treatment of MDS itself.
  • the method, diagnostic agent, kit, or biomarker of the present invention is very useful in predicting the necessity and frequency of blood transfusion.
  • the figure which shows the result of having fractionated the blood cell of each differentiation stage The figure which shows the result of having measured the quantity of mRNA of Acvr2b (human protein homolog name: ACVR2B) expressed in the cell population of a normal erythroblast, a reticulocyte, and an erythrocyte.
  • Samples are peripheral blood obtained from healthy subjects, RA patients among MDS, and RCMD patients, and the expression level of ACVR2B in erythrocytes (marker is CD45 ⁇ / CD71 ⁇ / GPA + ) therein. Show.
  • FIG. 4 The figure which shows the result of having measured the quantity of ACVR2B expressed in the leukocyte contained in peripheral blood.
  • Samples are peripheral blood obtained from healthy subjects, RA patients among MDS, and RCMD patients, and the expression level of ACVR2B in white blood cells (marker is CD45 + / GPA ⁇ ) therein is shown. .
  • FIG. 5 The figure which shows the result of having measured the quantity of ACVR2B expressed in the erythrocyte contained in peripheral blood.
  • Samples are peripheral blood obtained from healthy individuals, RA patients among MDS, and RCMD patients, and healthy elderly and renal anemia patients as control experiments.
  • anemia means that blood hemoglobin concentration is 13 g / dL or less for men and 12 g / dL or less for women, as shown in the WHO guidelines.
  • the condition refers to symptoms such as palpitation, shortness of breath, dizziness, etc., but even if it is asymptomatic, it may be determined to be anemia if the blood hemoglobin concentration is as described above.
  • anemia patient is a person who suffers from the above-mentioned symptoms of “anemia”.
  • factor of anemia refers to the onset mechanism of the above-mentioned “anemia”, and can be rephrased as, for example, a disease in which anemia is cited as one of the symptoms.
  • Such diseases include MDS, renal anemia, aging-related anemia, iron deficiency anemia, thalassemia, ironblastic anemia, megaloblastic anemia (vitamin B12 deficiency, folate deficiency), primary Examples include bone marrow failure, inflammation, uremia, hemolytic anemia, aplastic anemia, sickle cell identification, and renal anemia.
  • MDS is a type of blood tumor, called myelodysplastic syndrome, a disease in which the quantity and quality of blood cells are abnormal, differentiation of multiblood cells including red blood cells, There is a disease in which a disorder is recognized in maturity.
  • MDS includes refractory anemia (RA), refractory cytopenias with multicytic dysplasia (RCMD), ironblastic refractory anemia with multicytic dysplasia (RCMD-RS), blasts Refractory anemia (RAEB) with an increase is included.
  • RA refractory anemia
  • RCMD refractory cytopenias with multicytic dysplasia
  • RCMD-RS ironblastic refractory anemia with multicytic dysplasia
  • RAEB blasts Refractory anemia
  • These diseases can be diagnosed by examining blood cells obtained from bone marrow and / or peripheral blood, respectively, using morphological means.
  • a specific diagnostic method may be a known method and is not particularly limited.
  • RAEB is most likely to progress to leukemia.
  • leukemia is classified into, for example, acute leukemia, chronic leukemia, etc., depending on its symptoms, pathological conditions, and the like. Moreover, according to another classification method, it is classified into myeloid leukemia and lymphocytic leukemia. Among the above-mentioned leukemias, acute myeloid leukemia is particularly preferable.
  • M0 acute undifferentiated myeloid leukemia (most undifferentiated type); M1: acute undifferentiated myeloblastic leukemia; M2: acute Differentiated myeloblastic leukemia; M3 acute promyelocytic leukemia; M4: acute myelomonocytic leukemia; M5: acute monocytic leukemia; M6: erythroleukemia; M7: acute megakaryocytic leukemia.
  • risk of onset refers to the risk of developing a certain disease, and is not influenced by the result of diagnosis of whether or not the disease actually develops. .
  • the term “frequency” used in this specification is used as a blood transfusion frequency, and indicates the degree of whether the required number of blood transfusions is large or small. This is because the amount that is possible for a single blood transfusion has been determined, so if a large amount of blood transfusion is required, increase the number of blood transfusions and perform the necessary amount of blood transfusion.
  • the number of blood transfusions is determined based on the fact that the period during which the effect is exerted is a maximum of 120 days.
  • the hemoglobin level in blood is 7 g / dL or less in a patient diagnosed with myelodysplastic syndrome clinically (body weight 60 kg)
  • 1 transfusion can be performed as an upper limit of 2 units (280 mL).
  • the hemoglobin value of about 3 to 1.4 g / dL increases. If the blood hemoglobin level still does not reach the desired blood hemoglobin level, or the deterioration of symptoms, i.e., red blood cell differentiation or maturation, is not improved, and the blood hemoglobin level has decreased, the period again The upper limit of 2 units of blood transfusion will be performed.
  • activin receptor 2B is a receptor protein having an extracellular domain containing a cysteine-rich region from the N-terminus, a transmembrane domain, and an intracellular domain containing a serine / threonine-type kinase domain.
  • Activin receptor 2B includes Bone Morphogenetic Protein 7 (BMP7), Growth / Differentialation factor 5 (GDF5), Myostatin (MSTN), Inhibin ⁇ (INHA), Inhibin ⁇ A subunit (INHBA), Inhibin ⁇ B A serine / threonine kinase type receptor having inhibin ⁇ C subunit (INHBC), NODAL, TDGF and the like as ligands, and BMP receptor 1A (BMPR1A), BMP receptor 1B (BMPR1B) in response to stimulation of the ligand, A receptor that forms a complex with activin receptor 1 (ACVR1) etc. and plays a part in the function of activating the intracellular Smad cascade etc. It is over protein.
  • BMP7 Bone Morphogenetic Protein 7
  • GDF5 Growth / Differentialation factor 5
  • MSTN Myostatin
  • Inhibin ⁇ Inhibin ⁇
  • IHBA Inhibin ⁇ A subunit
  • Activin receptor 2B specifically includes, for example, http: // www. uniprot. It is a protein consisting of the amino acid sequence (SEQ ID NO: 1) shown in org / uniprot / Q13705 and the like. Among such proteins, it is particularly preferable to have an extracellular domain consisting of at least the first to 119th amino acid sequences in SEQ ID NO: 1.
  • the ACVR2B targeted by the present invention is not limited to the above protein itself, and may be a protein with mutation (substitution, deletion, addition, etc.).
  • expression level means not the expression level of mRNA but the expression level of protein translated based on mRNA.
  • biomarker used in the present specification refers to substances derived from living bodies such as carbohydrates, lipids, proteins, and the like, which serve as an index for quantitatively grasping biological changes in the living body. .
  • the biomarker is a role as an index for diagnosing or judging the current state used for grasping the current state of diseases and the like, and a predictive diagnosis for predicting the prevention of diseases and the transition of disease states. Or, it has a role as an index for estimation.
  • the method of the present invention includes ⁇ a method for detecting an anemia factor>, ⁇ a method for determining the risk of developing leukemia>, and ⁇ a method for predicting the necessity and frequency of blood transfusion>. .
  • the method for detecting an anemia factor of an anemic patient according to the present invention includes the following steps 1 and 2.
  • Step 1 of measuring the expression level of ACVR2B in multicytic cells contained in peripheral blood obtained from the patient (2) When the expression level of ACVR2B measured in step 1 is greater than the expression level of ACVR2B in the polycytic cells contained in peripheral blood obtained from a healthy person, it is determined that the cause of anemia in the patient is MDS Step 2 to do.
  • Step 1 is a step of measuring the expression level of ACVR2B in multicytic cells contained in peripheral blood obtained from an anemic patient.
  • Peripheral blood means blood existing in blood vessels. Such peripheral blood may be collected by inserting an injection needle or the like into a human blood vessel. For example, a metal needle may be used as the injection needle or the like, and an indwelling material such as SURFLOW (registered trademark) may be used. Peripheral blood may be collected by inserting a plastic injection needle into a blood vessel together with a metal needle and placing it. The collected peripheral blood may be subjected to a step of preventing coagulation as appropriate. Specifically, an anticoagulant such as heparin or EDTA may be used, but it is preferable to use EDTA which is inexpensive and highly versatile.
  • an anticoagulant such as heparin or EDTA may be used, but it is preferable to use EDTA which is inexpensive and highly versatile.
  • the polycytic cells are not particularly limited, and examples include red blood cells, white blood cells, and platelets. More preferred are red blood cells, white blood cells and the like.
  • ACVR2B can measure by using the flow cytometry method using the antibody with respect to ACVR2B. As other methods, measurement can also be performed by using ELISA method using an antibody against ACVR2B, Western blotting method, or the like.
  • an antibody against ACVR2B a commercially available antibody or an antibody prepared based on a conventional method may be used as it is, or a modified antibody obtained by adding a labeling substance to the antibody.
  • the origin of the antibody is not particularly limited, and examples include antibodies derived from animal species such as humans, mice, rats, chickens, goats, sheep, donkeys, llamas, ostriches, alpaca and horses.
  • the antibody may be a monoclonal antibody or a polyclonal antibody.
  • the ACVR2B antibody is not particularly limited as long as it is a molecule that specifically binds to ACVR2B. Examples thereof include immunoglobulins (Ig) or antibody fragments such as scFv, F (ab ′) 2 , minibody, and the like. Affibodies are also included in the above-described antibodies. Antisera are also encompassed by the antibodies described above.
  • the antibody when the antibody is an immunoglobulin, its subtype is not particularly limited, and may be any of IgA, IgD, IgE, IgG, IgM, IgY, IgW, and the like. Further, the subclass of the antibody is not limited to a specific subclass, and may be any of IgG1 to IgG as long as it is human IgG, for example.
  • the anti-ACVR2B antibody used in Step 1 exhibits the ability to specifically bind to ACVR2B, the above-mentioned several types of antibodies may be mixed and used.
  • the multiblood cells are identified using an antibody against a marker that is specifically expressed in the multiblood cells. This makes it possible to measure the amount of ACVR2B in specific blood cells.
  • Such markers include CD3, CD4, CD5, CD10, CD13, CD19, CD20, CD23, CD33, CD34, CD38, CD45, CD45RA, CD90, IgM, HLA-DR and other leukocyte markers; CD71, GPA, etc. Red blood cell markers; platelet markers such as CD41, CD42b, CD61, CD62P, PAC-1, and the like, and these antibodies may be combined as appropriate.
  • antibodies against these markers are not limited to specific origin species, and may be the same as the antibodies against the above-mentioned ACVR2B, and may be appropriately labeled with a labeling substance.
  • the specific method for using the antibody against the marker is not particularly limited as long as it is used in accordance with a known method such as the flow cytometry method, ELISA method, Western blotting method or the like.
  • step 2 the expression level of ACVR2B in the polycytic cells contained in the peripheral blood of the anemic patient measured in the above (step 1) is larger than the expression level of ACVR2B in the polycytic cells contained in the peripheral blood obtained from a healthy subject
  • the step of determining that the anemia factor of the patient is MDS.
  • a healthy person is a person who is not in an anemic state as described in the term “anemia”, and whether or not he / she complains of symptoms of anemia as described above is not particularly limited in defining a healthy person. Moreover, the race, age, and gender of a healthy person are not ask
  • the “peripheral blood” obtained from healthy subjects is as described above.
  • the “multiple blood cell” is defined in the same manner as the multiple blood cell contained in the peripheral blood obtained from the patient to be measured in (Step 1).
  • the expression level of ACVR2B in multicytic cells contained in peripheral blood obtained from healthy subjects can be determined by the same method as the ACVR2B measurement method in (Step 1) described above.
  • the comparison between the expression level of ACVR2B in the polycytic cells contained in the peripheral blood obtained from the anemia patient measured in (Step 1) and the expression level of ACVR2B in the polycytic cells contained in the peripheral blood obtained from healthy subjects is as follows. Although not particularly limited, for example, the following comparison method may be employed.
  • Step 1 the same method for measuring a multiblood cell sample contained in peripheral blood obtained from a healthy person as the multiblood cell sample contained in peripheral blood obtained from an anemia patient measured in (Step 1), and There is a method in which the measurement is performed under the same measurement conditions and the amount of the obtained ACVR2B is relatively compared.
  • the amount of ACVR2B in a multi-blood cell sample contained in peripheral blood obtained from a healthy subject is measured in advance, and is obtained from an anemia patient measured in (Step 1) rather than the measured value.
  • the cause of anemia in an anemic patient can be determined as MDS.
  • the ACVR2B measurement method and measurement conditions in peripheral blood polycytic cells obtained from an anemic patient are the same as in the first method described above. It is the same as the measurement method and measurement conditions of ACVR2B in multiblood cells contained in blood.
  • the amount (measured value) of ACVR2B in the peripheral blood polycytic cells obtained from a healthy person measured in advance is generalized, and based on the generalized numerical value (for example, called a cut-off value) (steps). You may compare whether the amount of ACVR2B in the multiblood cell contained in the peripheral blood obtained from the anemia patient measured in 1) is large or small.
  • the generalized numerical value is a statistical analysis of the amount of ACVR2B in peripheral blood multi-blood cells obtained from a certain number of different or more healthy subjects, and is generally used as a value per unit peripheral blood volume or per unit multi-blood cell volume.
  • the method is not limited to the above-described method, and any known analysis technique, statistical technique, or the like may be used.
  • the method for determining the risk of developing leukemia in an anemic patient of the present invention includes the following steps 1 and 2. [1] Step 1 of measuring the expression level of ACVR2B in multicytic cells contained in peripheral blood obtained from the patient, [2] Step 2 in which when the expression level of ACVR2B measured in Step 1 is greater than the expression level of ACVR2B in polycytic cells contained in peripheral blood obtained from healthy subjects, the patient is determined to be at risk for leukemia.
  • Step 1 is a step of measuring the expression level of ACVR2B in multicytic cells contained in peripheral blood obtained from an anemic patient.
  • This step may be performed in the same manner as (Step 1) in the above ⁇ Method for detecting anemia factor>.
  • step 2 when the expression level of ACVR2B measured in the above [step 1] is larger than the expression level of ACVR2B in the multiblood cells contained in peripheral blood obtained from a healthy person, the patient is at risk of developing leukemia. It is a step of determining.
  • the healthy person is as described in (Step 1) in the above ⁇ Method for detecting anemia factor>.
  • the “multiple blood cell” is defined in the same manner as the multiblood cell contained in the peripheral blood obtained from the patient to be measured in [Step 1].
  • the expression level of ACVR2B in multicytic cells contained in peripheral blood obtained from healthy subjects can be determined by the same method as the measurement method of ACVR2B in [Step 1] described above.
  • the comparison between the expression level of ACVR2B in the polycytic cells contained in the peripheral blood obtained from the anemia patient measured in [Step 1] and the expression level of ACVR2B in the polycytic cells contained in the peripheral blood obtained from healthy subjects is as follows: What is necessary is just to employ
  • the greater the amount of ACVR2B the stronger the degree of disorder such as differentiation and maturation of the above-mentioned multiblood cells, and thus it can be determined that anemia patients are more likely to develop leukemia.
  • the amount of ACVR2B in the polycytic cells contained in the peripheral blood obtained from the anemia patient measured in 1) is greater than the amount of ACVR2B in the polycytic cells contained in the peripheral blood obtained from a healthy subject, the anemia patient is leukemia. It can be determined that there is a risk. .
  • the amount of ACVR2B in a multiblood cell sample contained in peripheral blood obtained from a healthy person is measured in advance, and the obtained value is obtained from an anemia patient measured in [Step 1].
  • Anemia patients can be determined to be at risk of developing leukemia when the amount of ACVR2B in the multicytic cells in the peripheral blood obtained is high.
  • the ACVR2B measurement method and measurement conditions in peripheral blood polycytic cells obtained from an anemic patient are the same as in the first method described above.
  • the measurement method and measurement conditions for ACVR2B in multiblood cells contained in blood are the same.
  • the target numerical value to be compared with the amount of ACVR2B in the peripheral blood polycytic cells obtained from the anemia patient measured in [Step 1] is the above ⁇ Method for detecting anemia factor>.
  • the cut-off value described in the above may be used similarly.
  • the method for predicting the necessity and frequency of blood transfusion of an anemic patient includes the following steps 1 and 2. ⁇ 1 ⁇ Step 1 of measuring the expression level of ACVR2B in multicytic cells contained in peripheral blood obtained from the patient, ⁇ 2 ⁇ If the expression level of ACVR2B measured in step 1 is greater than the expression level of ACVR2B in the multiblood cells contained in peripheral blood obtained from a healthy subject, blood transfusion is required for the patient, and the frequency is Step 2 for determining that the blood transfusion frequency is higher than that of the patient at the time of blood collection.
  • Step 1 is a step of measuring the expression level of ACVR2B in multicytic cells contained in peripheral blood obtained from the patient.
  • This step may be performed in the same manner as (Step 1) in the above ⁇ Method for detecting anemia factor>.
  • Step 2 determines that transfusion is necessary for the patient when the expression level of ACVR2B measured in Step 1 above is greater than the expression level of ACVR2B in the multiblood cells contained in peripheral blood obtained from a healthy subject In both cases, the transfusion frequency is determined to be higher than the transfusion frequency of the patient at the time of collecting peripheral blood.
  • An anemia patient who has been determined to have a high transfusion frequency according to Step 2 and who has received 0 transfusions at the time of peripheral blood collection (no transfusion measures have been taken for anemia).
  • Patients who have been determined to be necessary and who have received at least one transfusion treatment for anemia at the time of peripheral blood collection are determined to require more transfusions.
  • the healthy person is as described in (Step 1) in the above ⁇ Method for detecting anemia factor>.
  • the “multiple blood cell” is defined in the same manner as the multiple blood cell contained in the peripheral blood obtained from the patient to be measured in ⁇ Step 1 ⁇ .
  • the expression level of ACVR2B in multicytic cells contained in peripheral blood obtained from healthy subjects can be determined by the same method as the measurement method of ACVR2B in ⁇ Step 1 ⁇ described above.
  • the comparison of the expression level of ACVR2B in the polycytic cells contained in the peripheral blood obtained from the anemia patient measured in ⁇ Step 1 ⁇ and the expression level of ACVR2B in the polycytic cells contained in the peripheral blood obtained from healthy subjects is as follows: What is necessary is just to employ
  • the amount of ACVR2B in the included polycytic cells is greater than the amount of ACVR2B in the polycytic cells contained in peripheral blood obtained from a healthy person, it may be determined that blood transfusion is necessary for anemia patients and the transfusion frequency is high it can.
  • the amount of ACVR2B in a multiblood cell sample contained in peripheral blood obtained from a healthy person is measured in advance, and the obtained value is obtained from an anemia patient measured in ⁇ Step 1 ⁇ .
  • the amount of ACVR2B in the polycytic cells in the peripheral blood obtained is high, it can be determined that blood transfusion is required for anemia patients and the frequency is high.
  • the ACVR2B measurement method and measurement conditions in peripheral blood polycytic cells obtained from an anemic patient are the same as in the first method described above. It is the same as the measurement method and measurement conditions of ACVR2B in multiblood cells contained in blood.
  • the target numerical value to be compared with the amount of ACVR2B in the peripheral blood polycytic cells obtained from the anemia patient measured in ⁇ Step 1 ⁇ is the above ⁇ Method for detecting anemia factor>.
  • the cut-off value described in the above may be used similarly.
  • High transfusion frequency means more transfusions compared to the number of transfusions (including 0) at the time of peripheral blood collection, in other words, the future transfusion frequency of anemia patients Means more than the number of blood transfusions at the time of blood collection.
  • the diagnostic agent of the present invention is a ⁇ diagnostic agent for detecting that the cause of anemia is MDS>, ⁇ diagnostic agent for determining the risk of developing leukemia>, and ⁇ necessity of blood transfusion> And diagnostic agents of sex and frequency thereof.
  • the diagnostic agent of the present invention contains an anti-ACVR2B antibody.
  • the anti-ACVR2B antibody is the same as the antibody against ACVR2B described in detail in (Step 1) of ⁇ Method for detecting anemia factor> described above.
  • the diagnostic agent itself is an anti-ACVR2B antibody, it may contain other components.
  • the content of the anti-ACVR2B antibody in the diagnostic agent is not particularly limited, but is usually about 0.0001 to 99.99 parts by weight per diagnostic agent.
  • the other components contained in the diagnostic agent are known components used in the field of diagnostic agents as long as they exhibit the ability of the anti-ACVR2B antibody to specifically bind to ACVR2B, and are not particularly limited. .
  • the diagnostic agent for detecting an anemia factor as MDS is preferably used as one of the components of ⁇ kit for detecting an anemia factor as MDS> described later. And it is usefully used according to the method described in the above ⁇ Method for detecting anemia factor>.
  • the diagnostic agent for determining the risk of developing leukemia is suitably used as one of the components of ⁇ kit for determining the risk of developing leukemia> described later. And it is usefully used according to the method described in the above ⁇ Method for determining leukemia risk>.
  • the diagnostic agent for the necessity and frequency of blood transfusion is suitably used as one of the constituent elements of ⁇ kit for predicting the necessity and frequency of blood transfusion> described later. And it is usefully used according to the method described in the above ⁇ Method for predicting the necessity and frequency of blood transfusion>.
  • Kit of the Present Invention comprises ⁇ a kit for detecting an anemia factor as MDS>, ⁇ a kit for determining the risk of developing leukemia>, and ⁇ necessity and frequency of blood transfusion in anemia patients> A kit for predicting ⁇ a kit for detecting an anemia factor as MDS>. ⁇ a kit for determining the risk of developing leukemia>, and ⁇ necessity and frequency of blood transfusion in anemia patients> A kit for predicting
  • the kit of the present invention contains an anti-ACVR2B antibody.
  • the anti-ACVR2B antibody may be the same as the antibody against ACVR2B described in detail in (Step 1) of ⁇ Method for detecting anemia factor>.
  • the anti-ACVR2B antibody included in the kit of the present invention may include two or more types of antibodies as long as the antibody exhibits an ability to specifically bind to ACVR2B.
  • each antibody may be filled in a separate container, or may be filled in the same container.
  • the above-mentioned anti-ACVR2B antibody may be included in the kit of the present invention in a mode of being adsorbed on the bottom surface of an instrument that may be included in the kit of the present invention described later.
  • the kit of the present invention may further contain an antibody against a marker that is specifically expressed in multiblood cells.
  • the multiblood cell is as described in (Step 1) of ⁇ Method for detecting an anemia factor>, and such an antibody is specifically described in (Method 1 for detecting an anemia factor) (Step 1). It is described in.
  • the kit of the present invention may include instruments mainly used for appropriate biochemical experiments.
  • a plate, a multiwell plate, a seal, a dish, a dropper, a capillary, a tube and the like can be mentioned.
  • the kit of the present invention may contain an appropriate biochemical reagent.
  • an appropriate biochemical reagent for example, a washing solution, a staining solution, a coloring solution, a sensitizing solution, a reaction stopping solution containing a buffer solution, an appropriate surfactant and the like.
  • Avidinized compounds biotinylated compounds; anti-immunoglobulin antibodies, labeled anti-immunoglobulin antibodies; stabilizers, preservatives, preservatives, proteolytic enzyme inhibitors; ACVR2B standard substances and the like.
  • a kit for detecting the cause of anemia as MDS includes the above-mentioned anti-ACVR2B antibody.
  • the kit for detecting that the cause of anemia is MDS can be particularly suitably used in the above-described ⁇ Method for detecting anemia factor of anemia patient>.
  • the kit may include a protocol described for the method.
  • any one or more of the components included in other kits for detecting that an anemia factor is MDS may be included.
  • the component is not particularly limited.
  • a kit for determining the risk of developing leukemia contains the anti-ACVR2B antibody described above.
  • the kit for determining the risk of developing leukemia can be particularly suitably used for the above-mentioned ⁇ Method for determining risk of developing leukemia>.
  • the kit may include a protocol described for the method.
  • any one or more of the components included in another kit for determining the risk of developing leukemia may be included.
  • examples of such a component include anti-Tim-3 antibody.
  • the method for determining the risk of developing leukemia using such components may be in accordance with a known protocol when using the other kits described above.
  • the kit for determining the risk of developing leukemia may include components of the kit for determining the type of leukemia.
  • anti-CD1a antibody, anti-CD2 antibody, anti-CD3 antibody, anti-CD4 Antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD10 antibody, anti-CD13 antibody, anti-CD14 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD33 antibody, anti-CD34 antibody examples include anti-CD38 antibody, anti-CD41 antibody, anti-HLA-DR antibody, anti-CD56 antibody, anti-CD235a: GPA antibody.
  • the method of determining leukemia typing by using such components may follow a known protocol when using the above-described kit for determining leukemia typing.
  • a kit for predicting the need for transfusion and its frequency comprises the anti-ACVR2B antibody described above.
  • the kit for predicting the necessity and frequency of blood transfusion can be particularly suitably used in the above-described ⁇ Method for predicting the necessity and frequency of blood transfusion>.
  • the kit may include a protocol describing the method, and further includes any one or more of the components included in other kits for predicting the need and frequency of transfusion. Also good.
  • a component For example, an anti- ferritin antibody, an antifolate antibody, an anti- vitamin B12 antibody etc. are mentioned.
  • the biomarker of the present invention comprises ⁇ a biomarker for determining that the cause of anemia is MDS>, ⁇ a biomarker for determining the risk of developing leukemia>, and ⁇ necessity of blood transfusion> Biomarkers for predicting sex and its frequency.
  • the biomarker for determining the cause of anemia as MDS consists of ACVR2BACVR2B.
  • ACVR2B is as described above. Such ACVR2B is present in peripheral blood multi-cells. Peripheral blood is as described above.
  • the biomarker for detecting an anemia factor as MDS is usefully used according to the method described in the above ⁇ Method for detecting an anemia factor>.
  • the biomarker for determining the risk of developing leukemia consists of ACVR2B.
  • ACVR2B is as described above. Such ACVR2B is present in peripheral blood multi-cells. Peripheral blood is as described above.
  • the biomarker for determining the risk of developing leukemia is usefully used according to the method described in the above ⁇ Method for determining risk of developing leukemia>.
  • a biomarker for predicting the need and frequency of transfusions consists of ACVR2B.
  • ACVR2B is as described above. Such ACVR2B is present in peripheral blood multi-cells. Peripheral blood is as described above.
  • the biomarker for predicting the necessity and frequency of blood transfusion is usefully used according to the method described in the above ⁇ Method for predicting the necessity and frequency of blood transfusion>.
  • human ACVR2B-APC was prepared using human ACVR2B (abcam) and APC conjugation kit (Dojindo) according to a conventional method.
  • Liver tissue was collected from a 16.5 week-old fetus of a Gata1-GFP transgenic mouse prepared according to a conventional method, and then dispersed into a cell group according to a conventional method. The obtained cell group was washed with 3 mL of PBS and then subjected to blocking treatment with 3 mL of MACS buffer on ice for 30 minutes.
  • mouse CD45-PE mouse Sca1-PE
  • mouse cKit mouse CD71-PE
  • mouse Ter119-PB mouse Ter119-PB
  • the cell groups subjected to these treatments were analyzed using a flow cytometry apparatus manufactured by BD Bioscience, and then fractionated into cells of various hematopoietic differentiation stages using a cell sorter apparatus. The results are shown in FIG.
  • the region gated with A was a cell population of positive erythroblasts
  • the region gated with B was a cell population of reticuloblasts.
  • the lower left of the figure is a cell population of erythrocytes.
  • cell populations of positively dyed erythroblasts and reticulocytes were sorted using a cell sorter device.
  • ⁇ Search for differentiation marker candidates> The sorted positive erythroblasts and reticulocytes were subjected to the DNA array method (Agilent Technology), and the genes expressed in each cell population were analyzed. Table 2 shows genes that are expressed less in reticulocytes than in normal erythroblasts.
  • Acvr2b was selected from these genes, and the amount of mRNA of Acvr2b expressed in the cell populations of positive erythroblasts, reticulocytes, and erythrocytes was measured according to a conventional method. The results are shown in FIG.
  • ⁇ About the expression level of ACVR2B in human peripheral blood Peripheral blood from healthy individuals, RA patients, and RCMD patients was collected at 1 to 2 mL, and treated with EDTA-2Na to prevent coagulation. Next, 1 ⁇ 10 6 cells were taken into a tube, the peripheral blood was washed 3 times with 3 mL of PBS, and subjected to blocking treatment with 3 mL of MACS buffer for 30 minutes on ice. Thereafter, human GPA-FITC, human CD71-PE, human CD45-APC-Cy7, and human ACTR-IIB-APC were added to the peripheral blood on the peripheral blood, and an immune reaction was performed for 30 minutes. It was.
  • peripheral blood treated with these treatments was analyzed using a flow cytometry apparatus manufactured by BD Bioscience. The results are shown in FIGS.
  • MDS patients (RA and RCMD) clearly had higher expression levels of ACVR2B in peripheral blood erythrocytes than in healthy individuals. This was also the same for leukocytes.
  • MDS patients have a high expression level of ACVR2B compared to elderly healthy individuals who are likely to become anemic, and anemia is caused by other factors (renal anemia). It became clear that it was higher than

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Abstract

La présente invention aborde les problèmes de fournir un procédé pour identifier la cause de l'anémie en tant que MDS, et en particulier de fournir un procédé pour identifier MDS comme cause d'une manière simple sans réaliser un prélèvement de moelle osseuse, sans avoir besoin de la décision d'un spécialiste du sang, ou similaire. Les problèmes sont résolus par un procédé de détection d'un facteur pour l'anémie chez un patient anémique, le procédé comprenant les étapes (1) et (2), l'étape (1) comprenant la mesure du taux d'expression de ACVR2B dans des cellules sanguines polycythémiques contenues dans le sang périphérique obtenu à partir du patient, et l'étape (2) comprenant la détermination qu'un facteur de l'anémie du patient est MDS si le taux d'expression de ACVR2B mesuré dans l'étape (1) est supérieur au taux d'expression de ACVR2B dans les cellules sanguines polycythémiques contenues dans le sang périphérique obtenu à partir d'un individu sain.
PCT/JP2013/072335 2012-08-21 2013-08-21 Biomarqueur pour la détection d'un facteur pour l'anémie chez un patient anémique WO2014030683A1 (fr)

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JP2015228843A (ja) * 2014-06-06 2015-12-21 公益財団法人神奈川科学技術アカデミー 生体内の鉄量に関する状態を予測する方法
CN115524490A (zh) * 2022-10-24 2022-12-27 广州市第一人民医院（广州消化疾病中心、广州医科大学附属市一人民医院、华南理工大学附属第二医院） Hla-dr+cd14+cd56+单核细胞在aa或hlh中诊断的应用
WO2024051675A1 (fr) * 2022-09-05 2024-03-14 广州市第一人民医院（广州消化疾病中心、广州医科大学附属市一人民医院、华南理工大学附属第二医院） Utilisation de cellules cd38+hla-dr+cd8+t dans le diagnostic précoce de l'agvhd

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JPH11335399A (ja) * 1998-05-20 1999-12-07 Seibutsu Bunshi Kogaku Kenkyusho:Kk 新規モノクローナル抗体
JP2003250537A (ja) * 2002-02-28 2003-09-09 Pfizer Prod Inc ＡｃｔＲＩＩＢキナーゼの活性に関するアッセイ
JP2006516886A (ja) * 2002-10-25 2006-07-13 ワイエス Ａｃｔｒｉｉｂ融合ポリペプチドおよびその使用
WO2006115274A1 (fr) * 2005-04-26 2006-11-02 Ajinomoto Co., Inc. Inducteur de differenciation des progeniteurs myeloerythroides
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Publication number Priority date Publication date Assignee Title
JP2015228843A (ja) * 2014-06-06 2015-12-21 公益財団法人神奈川科学技術アカデミー 生体内の鉄量に関する状態を予測する方法
WO2024051675A1 (fr) * 2022-09-05 2024-03-14 广州市第一人民医院（广州消化疾病中心、广州医科大学附属市一人民医院、华南理工大学附属第二医院） Utilisation de cellules cd38+hla-dr+cd8+t dans le diagnostic précoce de l'agvhd
CN115524490A (zh) * 2022-10-24 2022-12-27 广州市第一人民医院（广州消化疾病中心、广州医科大学附属市一人民医院、华南理工大学附属第二医院） Hla-dr+cd14+cd56+单核细胞在aa或hlh中诊断的应用
CN115524490B (zh) * 2022-10-24 2023-08-22 广州市第一人民医院（广州消化疾病中心、广州医科大学附属市一人民医院、华南理工大学附属第二医院） Hla-dr+cd14+cd56+单核细胞在aa或hlh中诊断的应用

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