CN101500609A - Hematopoietic stem cell proliferation promoter - Google Patents

Abstract

The present inventors discovered that the administration of an agonistic minibody (VB22B sc(Fv)2) against the TPO receptor resulted in not only the induction of human megakaryocyte-specific differentiation (increase in platelet precursor cells), but also the engraftment of transplanted hematopoietic stem cells derived from human cord blood (CD34-positive cells) and significant increase in multi-lineage hematopoietic precursor cells. TPO and TPO receptor agonists can be used as agents for promoting the growth of CD34-positive hematopoietic cells or agents for promoting the engraftment of transplanted cells in the bone marrow, which can be effective when administered alone (without using G-CSF and erythropoietin in combination) after hematopoietic stem cell transplantation (in particular, cord blood transplantation). Furthermore, TPO and TPO receptor agonists can be used as agents for promoting the growth and/or differentiation of multilineage hematopoietic precursor cells and agents for promoting the recovery of multilineage hematopoiesis.

Description

Hematopoietic stem cell proliferation promoter

Technical field

The present invention relates to contain the hemopoietic stem cell proliferation promoter of TPO receptor (c-mpl) agonist as effective ingredient.The invention still further relates to contain TPO receptor (c-mpl) agonist as the propagation of the hemopoietic system CD34 positive cell of effective ingredient and/or differentiation accelerator, hemopoietic system CD34 positive cell in bone marrow give birth to (engraftment) give birth to hematopoietic function recovery promoter in promoter and the hematopoietic stem cell transplantation.

Background technology

Thrombopoietin (Thrombopoietin:TPO) is to promote the hematopoietic cell proliferation of megakaryocytic system and the cytokine of differentiation, and it is as the part of megakaryocyte colony stimulating factor (Megakaryocyte colony-stimulating factor) or c-mpl and known.Most of cytokine receptor forms dimer by combining with part to make between the receptor, transmit signal in cell.Report that TPO makes receptor form dimer by combining with its specific receptor c-mpl, thus in cell transmission information, show physiological action (with reference to non-patent literature 1).

Report, with the antibody of the receptors bind of the above-mentioned character of tool in, have the antibody that shows agonist activity.For example report, the antibody of erythropoietin (EPO) receptor replaces the erythropoietin function, when this antibody is converted into monovalence antibody (Fab), still keep active with combining of EPO receptor, but the loss signal propagation function thinks that thus be signal necessary (with reference to non-patent literature 2) by bivalence in conjunction with the dimer that forms erythropoietin receptor.

Equally, the somebody has reported and the antibody (with reference to non-patent literature 3～4, patent documentation 1～3) of the bonded tool TPO of c-mpl receptor agonist activity, has also attempted utilizing above-mentioned antibody to promote the propagation of hematopoietic stem cell.

For example, the someone has reported has the NOD/SCID mice of human cord blood cell (CD34 positive cell) to give TPO one time to transplanting, the experimental example (with reference to non-patent literature 5) of research cord blood cell propagation.But, according to this report do not confirm the CD34 positive cell give birth to strengthen.

In addition, the somebody has reported the TPO receptor stimulating agent (PEG-rHuMGDF) (with reference to non-patent literature 6) of Umbilical Cord Blood Transplantation model mice except that TPO.But, only reported the effect that promotes platelet recovery in the document, to the CD34 positive cell in bone marrow the effect of giving birth to report not fully then.

And the somebody has reported and transplanted the human cord blood cell in NOG mice (mice that immunodeficiency is higher than the NOD/SCID mice) body, given the experiment (with reference to non-patent literature 7) of c-mpl agonist after 2～6 months after transplanting.But, do not confirm the increase of CD34 positive cell in the bone marrow according to the document.

Report,, in fact can accelerate the recovery of hemocyte by after human cord blood is transplanted, giving TPO, G-CSF and three kinds of medicines of EPO simultaneously to 1 patient.But because record is, give above-mentioned three kinds of medicines and be the hemopoietic (the 198th page of You Lan stage casing) in order to promote three pedigrees (tri-lineage), it is individually dosed to the propagation of CD34 positive cell and the effect of differentiation (with reference to non-patent literature 8) not provide TPO.

On the other hand, report that by giving TPO to the TPO knock-out mice, efficiency of transplantation improves (with reference to non-patent literature 9).Hinted also in the document that TPO also plays a role to the cell lineage except that platelet.But do not report the CD34 positive cell whether give birth to, do not mention human cord blood yet.

As mentioned above, still there is not the report that in fact successfully activates the propagation of hematopoietic stem cell by the antibody that gives tool TPO receptor agonist activity up to now.

Look-ahead technique document of the present invention is as follows.

Patent documentation 1:WO2002/33072

Patent documentation 2:WO2005/056604

Patent documentation 3:WO2005/107784

Non-patent literature 1:Stem Cells.1996,1, the 124～132 page in the 14th volume supplementary issue

Work such as non-patent literature 2:Elliott S, J.Biol.Chem.,, the 271st volume (40), the 24691st～24697 page in 1996

Work such as non-patent literature 3:Abe, Immunol.Lett.1998, the 61st volume, the 73rd～78 page

Work such as non-patent literature 4:Bijia Deng, Blood,, the 92nd volume, the 1981st～1988 page in 1998

Non-patent literature 5:British Journal of Haematology, 122,837～846,2003

Non-patent literature 6:Japanese Journal of Transfusion Medicine, 46 (3), 311～316,2000

Non-patent literature 7:Blood 2006, the 107th volume, the 4300th～4307 page

Non-patent literature 8:Bone Marrow Transplantation 29,197～204 (2002)

Non-patent literature 9:The Journal of Clinical Investigation 110 (3), 389～394 (2002)

Summary of the invention

Invent problem to be solved

The present invention establishes in view of above-mentioned condition, and problem of the present invention is to provide and contains the hemopoietic stem cell proliferation promoter of TPO receptor stimulating agent as effective ingredient.Problem of the present invention also be to provide contain TPO receptor (c-mpl) agonist as the propagation of the hemopoietic system CD34 positive cell of effective ingredient and/or differentiation accelerator, hemopoietic system CD34 positive cell in bone marrow give birth to hematopoietic function recovery promoter in promoter and the hematopoietic stem cell transplantation.

Solve the method for problem

The inventor etc. further investigate in order to solve above-mentioned problem.Discoveries such as the inventor: by the exciting type antibody (VB22B sc (Fv) 2) of the low molecule that gives the TPO receptor, not only can induce people's megalokaryocyte specificity differentiation (increase of megakaryocytic series cell), can also promote hematopoietic stem cell (hemopoietic system CD34 positive cell) from human cord blood in bone marrow the remarkable propagation of giving birth to and causing multispectral system (multi-lineage) blood precursor.The inventor etc. expect thus: TPO or TPO receptor stimulating agent after hematopoietic stem cell transplantation (particularly Umbilical Cord Blood Transplantation) only individually dosed (not being used in combination G-CSF or erythropoietin) can be expected its effect, can be used as the enhancer of proliferation of hemopoietic system CD34 positive cell or in bone marrow give birth to give birth to promoter.The inventor etc. also expect: TPO or TPO receptor stimulating agent can be used as multispectral be the propagation of hemopoietic forebody cell and/or differentiation accelerator and multispectral be hematopoietic function recovery promoter.

More specifically, the invention provides following [1]～[42].

[1] hemopoietic stem cell proliferation promoter, it contains TPO receptor (c-mpl) agonist as effective ingredient.

[2] propagation of hemopoietic system CD34 positive cell and/or differentiation accelerator, it contains TPO receptor (c-mpl) agonist as effective ingredient.

[3] hemopoietic system CD34 positive cell in bone marrow give birth to give birth to dose, it contains TPO receptor (c-mpl) agonist as effective ingredient.

[4] hematopoietic function recovery promoter, it contains TPO receptor (c-mpl) agonist as effective ingredient.

[5] each described medicine in [1]～[4], this medicine uses in hematopoietic stem cell transplantation.

[6] each described medicine in [1]～[5], the administration after hematopoietic stem cell transplantation of this medicine.

[7] [5] or [6] described medicine, wherein hematopoietic stem cell transplantation is selected from bone marrow transplantation, the tip hemocytoblast is transplanted or Umbilical Cord Blood Transplantation.

[8] [7] described medicine, wherein Umbilical Cord Blood Transplantation is meant the human cord blood transplanting.

[9] [8] described medicine is characterized in that: carry out multiple dosing.

[10] enhancer of proliferation of lymphocyte and/or medullary cell, it contains TPO receptor (c-mpl) agonist as effective ingredient.

[11] to the differentiation accelerator of lymphocyte and/or medullary cell differentiation, it contains TPO receptor (c-mpl) agonist as effective ingredient.

[12] [5] described medicine is characterized in that: the patient to hemopoietic hypofunction of marrow carries out hematopoietic stem cell transplantation.

[13] [12] described medicine, wherein the patient is the patient who accepts behind radiotherapy or the chemotherapeutic treatment.

[14] [13] described medicine is characterized in that: carry out radiotherapy or the chemotherapy disease with treatment acute myelogenous leukemia (AML), acute lymphatic leukemia (ALL), chronic lymphocytic leukemia (CML), malignant lymphoma, adult T cell leukemia, myelodysplastic syndrome (MDS), aplastic anemia (AA) or other suitable hematopoietic stem cell transplantation.

[15] method of promotion hemopoietic stem cell proliferation, this method comprises being tried the step that body gives TPO receptor (c-mpl) agonist.

[16] promote hemopoietic system CD34 positive cell propagation and/or the method for breaking up, this method comprises being tried the step that body gives TPO receptor (c-mpl) agonist.

[17] increase hemopoietic system CD34 positive cell in bone marrow the method for giving birth to, this method comprises being tried the step that body gives TPO receptor (c-mpl) agonist.

[18] method of promotion hematopoietic function recovery, this method comprises being tried the step that body gives TPO receptor (c-mpl) agonist.

[19] each described method in [15]～[18], this method is carried out in hematopoietic stem cell transplantation.

[20] each described method in [15]～[19], this method is carried out after hematopoietic stem cell transplantation.

[21] [19] or [20] described method, wherein hematopoietic stem cell transplantation is selected from bone marrow transplantation, the tip hemocytoblast is transplanted or Umbilical Cord Blood Transplantation.

[22] [21] described method, wherein Umbilical Cord Blood Transplantation is meant the human cord blood transplanting.

[23] [22] described method is characterized in that: carry out multiple dosing.

[24] make the method for lymphocyte and/or proliferation of bone marrow cells, this method comprises being tried the step that body gives TPO receptor (c-mpl) agonist.

[25] promote that this method comprises being tried the step that body gives TPO receptor (c-mpl) agonist to the method for lymphocyte and/or medullary cell differentiation.

[26] [19] described method is characterized in that: the patient to hemopoietic hypofunction of marrow carries out hematopoietic stem cell transplantation.

[27] [26] described method, wherein the patient is the patient who accepts behind radiotherapy or the chemotherapeutic treatment.

[28] [27] described method is characterized in that: carry out radiotherapy or the chemotherapy disease with treatment acute myelogenous leukemia (AML), acute lymphatic leukemia (ALL), chronic lymphocytic leukemia (CML), malignant lymphoma, adult T cell leukemia, myelodysplastic syndrome (MDS), aplastic anemia (AA) or other suitable hematopoietic stem cell transplantation.

[29] application of TPO receptor (c-mpl) agonist in preparation hemopoietic stem cell proliferation promoter.

[30] TPO receptor (c-mpl) agonist is in the propagation of preparation hemopoietic system CD34 positive cell and/or the application in the differentiation accelerator.

[31] TPO receptor (c-mpl) agonist preparation hemopoietic system CD34 positive cell in bone marrow give birth to give birth to application in the dose.

[32] application of TPO receptor (c-mpl) agonist in preparation hematopoietic function recovery promoter.

[33] propagation of TPO receptor (c-mpl) agonist employed hemopoietic stem cell proliferation promoter, hemopoietic system CD34 positive cell in the preparation hematopoietic stem cell transplantation and/or differentiation accelerator, hemopoietic system CD34 positive cell in bone marrow give birth to give birth to application in dose or the hematopoietic function recovery promoter.

[34] TPO receptor (c-mpl) agonist the propagation of hemopoietic stem cell proliferation promoter that preparation is given after the hematopoietic stem cell transplantation, hemopoietic system CD34 positive cell and/or differentiation accelerator, hemopoietic system CD34 positive cell in bone marrow give birth to give birth to application in dose or the hematopoietic function recovery promoter.

[35] [33] or [34] described application, wherein hematopoietic stem cell transplantation is selected from bone marrow transplantation, the tip hemocytoblast is transplanted or Umbilical Cord Blood Transplantation.

[36] [35] described application, wherein Umbilical Cord Blood Transplantation is meant the human cord blood transplanting.

[37] [36] described application is characterized in that: carry out multiple dosing.

[38] application of TPO receptor (c-mpl) agonist in the enhancer of proliferation of preparation lymphocyte and/or medullary cell.

[39] TPO receptor (c-mpl) agonist is in the application of preparation in the differentiation accelerator of lymphocyte and/or medullary cell differentiation.

[40] [33] described application is characterized in that: the patient to hemopoietic hypofunction of marrow carries out hematopoietic stem cell transplantation.

[41] [40] described application, wherein the patient is the patient who accepts behind radiotherapy or the chemotherapeutic treatment.

[42] [41] described application is characterized in that: carry out radiotherapy or the chemotherapy disease with treatment acute myelogenous leukemia (AML), acute lymphatic leukemia (ALL), chronic lymphocytic leukemia (CML), malignant lymphoma, adult T cell leukemia, myelodysplastic syndrome (MDS), aplastic anemia (AA) or other suitable hematopoietic stem cell transplantation.

The accompanying drawing summary

Fig. 1 be expression sc (Fv) 2 (hVB22 u2-wz4) to the figure of the influence of each blood cell line cell number (meansigma methods with-represent; The SAS5.0 version, the wilcoxon check)

Fig. 2 be expression sc (Fv) 2 (hVB22 u2-wz4) to the figure of the influence of CFU-Meg colony number and photo (meansigma methods with-represent; The SAS5.0 version, the wilcoxon check).

Fig. 3 is the figure (meansigma methodss (-)+SD of expression sc (Fv) 2 (hVB22 u2-wz4) to the dose-dependent influence of each blood cell line cell number; The Jonckheere-Terpstra check).MAB among the figure (H), MAB (M), MAB (L) represent the high dose of hVB22sc (Fv) 2, middle dosage, low dosage administration group respectively.

The best mode that carries out an invention

The invention provides contain TPO receptor (c-mpl) agonist as the propagation of the hemopoietic stem cell proliferation promoter of effective ingredient, hemopoietic system CD34 positive cell and/or differentiation accelerator, hemopoietic system CD34 positive cell in bone marrow give birth to give birth to dose and hematopoietic function recovery promoter (below, sometimes said medicine is generically and collectively referred to as " medicine of the present invention " in this manual).

The invention provides and contain the hemopoietic stem cell proliferation promoter of TPO receptor (c-mpl) agonist as effective ingredient.In the present invention, agonist is meant and acts on receptor and show the material of identical functions with neurotransmitter or hormone etc.The example of agonist of the present invention has low molecular compound or antibody, but is not limited to this.Antibody of the present invention also comprises: low molecular antibody, humanized antibody or chimeric antibody etc. have changed the antibody of aminoacid sequence; Be combined with the modified antibodies of other molecule (for example macromolecule such as Polyethylene Glycol etc.); Any antibody such as antibody of sugar chain have been changed.

In the present invention, hematopoietic stem cell is meant the cell that can be divided into any lymphocyte or medullary cell.As long as the hematopoietic stem cell among the present invention has above-mentioned character, be not particularly limited, but can have the feature of hemopoietic system CD34 positive cell.Hemopoietic system CD34 positive cell is meant the allos cell colony that comprises positive hematopoietic stem cell of CD34 and the positive hemopoietic forebody cell of CD34, for example comprises pluripotent stem cell, lymphoid stem cell, CFU-GEMM, CFU-GM, BFU-E, CFU-MEG etc.Wherein, the positive hemopoietic forebody cell of CD34 is for expressing the cell of CD34, though be meant be in lymphocyte (B cell, T cell etc.) or medullary cell (neutrophil cell, mononuclear cell, erythrocyte, megalokaryocyte etc.) process of differentiation directed as yet towards each pedigree differentiation or be in can not the directed stages of cell of identification of cell differentiation in the morphology.Whether cell expresses CD34, this can according to those skilled in the art known method, for example document Journal of Hematotherapy 5, the method of record is judged among 213～226,1996 .The ISHAGE guidelines for CD34 celldetermination by Flow Cytometry such as () the Robert Sutherland.

Among the present invention, " promote propagation " meaning be meant with give medicine of the present invention before compare, the propagation of hematopoietic stem cell, hemopoietic system CD34 positive cell (the positive hematopoietic stem cell of CD34, the positive hemopoietic forebody cell of CD34) is activated.Whether the propagation of judging hematopoietic stem cell, hemopoietic system CD34 positive cell (the positive hematopoietic stem cell of CD34, the positive hemopoietic forebody cell of CD34) is activated, this can carry out according to the method that those skilled in the art adopt usually, for example can pass through the variation of the growth rate of detection hematopoietic stem cell, hemopoietic system CD34 positive cell (the positive hematopoietic stem cell of CD34, the positive hemopoietic forebody cell of CD34); The variation of the number (colony number) of hematopoietic stem cell, hemopoietic system CD34 positive cell (the positive hematopoietic stem cell of CD34, the positive hemopoietic forebody cell of CD34); Or participate in hematopoietic stem cell, hemopoietic system CD34 positive cell (the positive hematopoietic stem cell of CD34, the positive hemopoietic forebody cell of the CD34) proliferating cells variation of signal and wait and judge.

The present invention also provides and contains propagation and/or the differentiation accelerator of TPO receptor (c-mpl) agonist as the hemopoietic system CD34 positive cell of effective ingredient.In the present invention, " differentiation of hemopoietic system CD34 positive cell " meaning is meant that the hemopoietic system CD34 positive cell that can be divided into any lymphocyte or medullary cell breaks away from this state and determines to be divided into the process of certain particular cell types and the process of the cell type that cell differentiation becomes this decision.Wherein, directed differentiation cell type as hemopoietic system CD34 positive cell (the positive hematopoietic stem cell of CD34, the positive hemopoietic forebody cell of CD34), comprise medullary cells such as lymphocytes such as T cell, B cell, NK cell and erythrocyte, leukocyte, platelet, neutrophil cell, mononuclear cell, eosinophilic granulocyte, but be not limited to these.

In the present invention, " promote differentiation " meaning be meant with give medicine of the present invention before compare differentiation and be activated.Whether the differentiation of judging the positive hematopoietic stem cell of hemopoietic system CD34 is activated, this also can carry out according to method known in those skilled in the art, and the variation of variation that for example can be by detecting cell proliferation rate, the variation of cell number, the variation that participates in signal intensity in the cell of differentiation, differentiation marker, cellular morphology waits to be judged.

In hematopoietic stem cell transplantation, require transplanted hematopoietic stem cell in bone marrow give birth to.The invention provides contain TPO receptor (c-mpl) agonist as the hemopoietic system CD34 positive cell of effective ingredient in bone marrow give birth to give birth to dose.By giving medicine of the present invention, can promote hemopoietic system CD34 positive cell (the positive hematopoietic stem cell of CD34, the positive hemopoietic forebody cell of CD34) in bone marrow give birth to.Measure hemopoietic system CD34 positive cell whether in bone marrow give birth to or its degree of giving birth to (measure hemopoietic system CD34 positive cell in bone marrow give birth to rate) time, for example can be undertaken by measuring the absolute number of transplanting human hematopoietic system CD34 positive cell in the NOD/SCID mouse bone marrow cells that human hematopoietic cell is arranged.Can measure by people's cell that end user CD34 specificity fluorescent traget antibody detects in the bone marrow cells in mice.Specifically can according to following program determination hemopoietic system CD34 positive cell in bone marrow give birth to rate.At first, transplanting CD34 positive cell from human cord blood after 3 weeks, to NOD.CB17-Prkdc＜scid 〉/the J mice implements euthanasia, takes 2 Thigh bones.Next, excise femoral epiphysis part, with flushing of 26G syringe and collection medullary cell with shears.After the IMDM cleaning that contains 2%FBS, add the 5mL blood cytolysate, left standstill 5 minutes.Centrifugal remove supernatant after, be suspended among the IMDM that contains 2%FBS of 2mL, by the filter membrane of 70 μ m, the preparation medullary cell suspension (2mL/2 root Thigh bone/mice).Its dispensing in 100 μ L samples, was dyeed 15 minutes with fluorescent labeling people CD34 specific antibody and PI (final concentration is 2 μ g/ml).After the cleaning, add 50 μ L FLOW-COUNT fluorescent grains, with cell resolver EPICS XL measure people CD34 positive cell give birth to number.Measuring the mensuration of the cell absolute number that for example can carry out according to the Application Note 5:Flow-Count of use Beckman Coulter society system implements.Equally, by the antibody that change to detect, can instrumentation bone marrow in the absolute number of various hemocyte pedigree cells.Each one contained cell absolute number is expressed from the next in 2 Thigh bones.

People's cell absolute number=measured value (individual/μ l) * 1/2 (FLOW-COUNT addition (50)/sample addition (100)) * 2000 (2mL/2 root Thigh bone/mice)

(Barnett?D，Granger?V，Whitby?L，Storie?I，Reilly?JT.Absolute?CD4+T-lymphocyte?and?CD34+?stem?cell?counts?by?single-platform?flowcytometry：the?way?forward.Br.J.Haematol.，1999?Sep；106(4)：1059～62)

In addition, for example people's the hemopoietic system CD34 positive cell of having accepted hematopoietic stem cell transplantation in mensuration in bone marrow when giving birth to rate, monitor returning to form of hemocyte in the tip blood indirectly by utilizing method beyond above-mentioned, also can measure above-mentioned the rate of giving birth to.

The present invention also provides and contains the hematopoietic function recovery promoter of TPO receptor (c-mpl) agonist as effective ingredient.Usually, transplant the back and the time living, that leukocyte need to recover 1～3 week until hematopoietic stem cell.During this period because the blood middle leukocytes number is few, so there is the problem that the infection disease that pneumonia etc. causes by antibacterial or mycete is taken place easily.Usually, transplant the back and the time living, that platelet recovery needed for 2～10 weeks until hematopoietic stem cell.During this period owing to number of platelets in the blood is few, so there is the hemorrhage problem that takes place easily.The hematopoietic function recovery promoter of the application of the invention can solve after the above-mentioned transplanting and the active relevant problem of the hemopoietic function of stem cell.Among the present invention, " promotion hematopoietic function recovery " meaning be meant with give medicine of the present invention before the hemopoietic function compared in the bone marrow be activated.Whether the hemopoietic function in the bone marrow is activated, and this can judge by returning to form of hemocyte in the monitoring tip blood.

The present invention also provides and contains TPO receptor (c-mpl) agonist as the lymphocyte of effective ingredient and/or the enhancer of proliferation of medullary cell.Discoveries such as the inventor: use TPO receptor (c-mpl) agonist make hemopoietic system CD34 positive cell in bone marrow give birth to give birth to and increase, any cytophyletic cell such as lymphatic system and myeloid lineage also increases (with reference to embodiment) as a result.The enhancer of proliferation of lymphocyte of the present invention and/or medullary cell just is being based on above-mentioned discovery.Lymphocytic example among the present invention has: T cell, B cell, NK cell, but be not limited to these.The example of medullary cell has: erythrocyte, leukocyte, platelet, neutrophil cell, mononuclear cell, eosinophilic granulocyte etc., but be not limited to these.

Discoveries such as the inventor: TPO receptor (c-mpl) agonist can activate the differentiation of hemopoietic system CD34 positive cell (the positive hematopoietic stem cell of CD34, the positive hemopoietic forebody cell of CD34).Therefore, the invention provides and contain the differentiation accelerator to lymphocyte and/or medullary cell differentiation of TPO receptor (c-mpl) agonist as effective ingredient.Among the present invention, the directed differentiation cell as hemopoietic system CD34 positive cell (the positive hematopoietic stem cell of CD34, the positive hemopoietic forebody cell of CD34) for example has: lymphocytes such as T cell, B cell, NK cell; Medullary cells such as erythrocyte, leukocyte, platelet, neutrophil cell, mononuclear cell, eosinophilic granulocyte.

In the hematopoietic stem cell transplantation of carrying out for the disease for the treatment of acute leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome, acute lymphatic leukemia, adult T cell leukemia, aplastic anemia, malignant lymphoma or other suitable hematopoietic stem cell transplantation, medicine of the present invention can be used for promoting the propagation of hematopoietic stem cell, the propagation that promotes the positive hematopoietic stem cell of hemopoietic system CD34 and/or differentiation, the positive hematopoietic stem cell of increase hemopoietic system CD34 in bone marrow give birth to or promote the recovery of hemopoietic function.

Hematopoietic stem cell transplantation of the present invention comprises bone marrow transplantation, the transplanting of tip hemocytoblast and Umbilical Cord Blood Transplantation.Usually, when carrying out bone marrow transplantation, except that bone marrow transplantation, carry out extensively also that the tip hemocytoblast is transplanted, Umbilical Cord Blood Transplantation in order to treat leukemia etc.

C-mpl is the receptor of TPO, the gene order of people c-mpl resolved (Palacios etc., Cell,, the 41st volume, the 727th～734 page or Genbank:NM_005373 in 1985).In addition, machin c-mpl (nucleotide/SEQ ID NO:52; Aminoacid/SEQ ID NO:53) and the sequence of mice c-mpl (GenBank #NM_010823) also known.The aminoacid sequence of people c-mpl is seen SEQ ID NO:51.

In addition, the c-mpl among the present invention comprises that also aminoacid is substituted among the above-mentioned c-mpl, lacks, the sudden change c-mpl receptor of interpolation etc.The object lesson of sudden change c-mpl has: MatthiasBallmaier etc., BLOOD, (2001), the 97th volume, No.1, the sudden change c-mpl of record in the 139th page etc.

In the present invention, as long as the c-mpl agonist have the effect that promotes hemopoietic stem cell proliferation, the propagation that promotes hemopoietic system CD34 positive cell and/or differentiation effect, increase hemopoietic system CD34 positive cell in bone marrow the effect of giving birth to or the effect that promotes hematopoietic function recovery, unqualified.Whether candidate compound has above-mentioned effect, and this can utilize method known in those skilled in the art to confirm.

The agonist activity of c-mpl is meant, promote the activity of hemopoietic stem cell proliferation, the propagation that promotes hemopoietic system CD34 positive cell and/or differentiation activity, increase hemopoietic system CD34 positive cell in bone marrow the activity of giving birth to or the activity that promotes hematopoietic function recovery.The mensuration of agonist activity can utilize method known in those skilled in the art to carry out.In addition, the mensuration of agonist activity not only can be measured as index with original activity, can also measure as index with other activity.

For example, can measure agonist activity as index with cell proliferation.More specifically, in showing agonist dependency proliferating cells, add the antibody that desire is measured agonist activity, cultivate.Afterwards, can measure cell by using hematimeter; Utilize flow cytometry to measure cell number; Perhaps in cell, add tetrazolium WST-8 (colleague's chemistry institute) etc. and be the reagent of chromogenic reaction at specific wavelength after, measure its absorbance according to viable count, and with the gained absorbance as index etc., measure agonist activity.

Show that agonist dependency proliferating cells also can utilize method known in those skilled in the art to make, for example when receptor be when sending the receptor of cell proliferation signal, use the cell of expression this receptor to get final product.When receptor when not sending the receptor of cell proliferation signal, make the Chimerical receptor of extracellular domain that comprises born of the same parents' internal area of the receptor that sends the cell proliferation signal and do not send the receptor of cell proliferation signal, this Chimerical receptor is got final product at cell inner expression.The example that sends the receptor of cell proliferation signal has: G-CSF receptor, mpl, neu, GM-CSF receptor, EPO receptor, c-kit, FLT-3 etc.The example of the cell of expressed receptor has: BaF3, NFS60, FDCP-1, FDCP-2, CTLL-2, DA-1, KT-3 etc.

In addition, as the detection index that is used to measure agonist activity,, can use any material as long as can measure quantitative change and/or qualitative change.For example can use the index of acellular system (cell freeassay), the index of cell line (cell-based assay), the index of tissue system, the index of body system.The index of acellular system can be used: the quantitative change of enzyme reaction or protein, DNA, RNA and/or qualitative change.Enzyme reaction for example can be used: aminoacid transfer reaction, sugared transfer reaction, dehydration, dehydrogenation reaction, substrate cleavage reaction etc.Can also use proteinic phosphorylation, dephosphorylation, dimerization, multimerization, decompose, dissociate etc. or amplification, shearing, the elongation of DNA, RNA.For example can transmit the proteinic phosphorylation in pathway downstream as detecting index to be present in signal.The index of cell line can be used variation, metamorphosis, characteristic variations of variation, the cell number of the quantitative change of variation, for example product of cell phenotype and/or qualitative change, proliferation activity etc.Product can use secretory protein, surface antigen, intracellular protein, mRNA etc.Metamorphosis can use projection to form and/or the variation of protrusions number, the variation of flatness, the variation of elongation/aspect ratio, the variation of cell size, the variation of internal structure, the heteromorphism/uniformity of cell colony, the variation of cell density etc.Above-mentioned metamorphosis can be confirmed by examining under a microscope.Characteristic variations can be used the variation of the dependency that casts anchor, cytokine dependence responsiveness, hormonal dependent, drug resistance, cell mobility, cell migration activity, pulsation, intracellular organic matter etc.Cell mobility comprises cellular infiltration activity, cell migration activity.The variation of intracellular organic matter for example can be used: enzymatic activity, mRNA amount, Ca ²⁺With born of the same parents' internal information transmitter quality, intracellular protein amounts etc. such as cAMP.When using cell-membrane receptor, can with by the variation of the inductive cell-proliferation activity of receptor for stimulating as index.As the tissue system index, can with corresponding to the changes of function of used tissue as detecting index.Body means that mark can use: tissue weight changes; The variation of blood system, for example variation of the variation of blood cell count, protein content, enzymatic activity or amount of electrolyte; And the variation of blood circulation, for example variation of blood pressure, heart rate etc.

The method of measuring above-mentioned detection index is not particularly limited, can adopts extinction, luminous, colour developing, fluorescence, radioactivity, fluorescence degree of polarization, surperficial plasmon resonance signal, time-resolved fluorescence degree, quality, absorption spectrum, light scattering, FRET (fluorescence resonance energy transfer) etc.The said determination method is known by those skilled in the art, can suitably select according to purpose.For example, absorption spectrum can utilize common employed photometer or light absorption microplate reader mensuration such as (plate reader); Luminously can utilize mensuration such as luminometer; Fluorescence can be with mensuration such as exometers.Quality can be measured with mass spectrograph.Radioactivity can use determining instrument mensuration such as gamma counter according to the kind of lonizing radiation; The fluorescence degree of polarization can be measured with BEACON (precious wine is made); Surface plasmon resonance signal can be measured with BIACORE; Time-resolved fluorescence, FRET (fluorescence resonance energy transfer) etc. can be used mensuration such as ARVO.And flow cytometry etc. also can be used for measuring.Can measure two or more detection indexs with a kind of in the above-mentioned assay method, if easyly can also simultaneously and/or use two or more methods to measure a plurality of detection indexs continuously.For example, can measure fluorescence and FRET (fluorescence resonance energy transfer) simultaneously with exometer.

Agonist among the present invention can be that native compound also can be the chemical compound of synthetic.Agonist among the present invention can use known material.Can also use through said method and be judged to be new compound with agonist activity.

One of optimal way of agonist of the present invention is low molecular antibody.Low molecular antibody comprises the antibody fragment that the part of whole antibody (for example full IgG etc.) lacks, as long as have the antigen binding energy, is not particularly limited.Low molecular antibody among the present invention is compared with whole antibody has significantly high activity.Antibody fragment of the present invention is not particularly limited so long as the part of whole antibody gets final product, and preferably includes variable region of heavy chain (VH) and/or variable region of light chain (VL).The aminoacid sequence of VH or VL can be substituted, lacks, adds and/or insert.And VH and/or VL can excalations, as long as have the antigen binding energy.Can also be with the variable region chimeric or humanization.The object lesson of antibody fragment has: Fab, Fab ', F (ab ') 2, Fv etc.The object lesson of low molecular antibody has: Fab, Fab ', F (ab ') 2, Fv, scFv (strand Fv), double antibody, sc (Fv) 2 (strand (Fv) 2) etc.

Wherein, " Fv " fragment is minimum antibody fragment, comprises complete antigen recognition site and binding site." Fv " fragment is 1 VH and 1 VL dimer (VH-VL dimer) by the powerful be combined into of non-covalent bond.3 complementary strand determining area (CDR) of each variable region interact, and form antigen-binding site on the dimeric surface of VH-VL.6 CDR give the antigen-binding site of antibody.But, even 1 variable region (or only comprise 3 antigenic specificity CDR half of Fv) though to compare affinity low with full binding site, also has the ability of identification and conjugated antigen.

Comprise the VH and the VL of antibody among the scFv, these districts are present in the single polypeptide chain.Usually the Fv polypeptide also contains peptide linker between VH and VL, thus scFv can be formed for antigen in conjunction with necessary structure (about the summary of scFv can (Rosenburg and Moore compile (Springer Verlag with reference to Pluckthun " ThePharmacology of Monoclonal Antibody " the 113rd volume, New York) the 269th～315 page, 1994)).Joint among the present invention is not particularly limited as long as do not hinder the expression of the antibody variable region that is connected its two ends.

Double antibody is meant the bivalent antibody fragment that makes up by gene fusion (Holliger P etc., Proc.Natl.Acad.Sci.USA 90:6444～6448 (1993), EP404, No. 097, WO93/11161 number etc.).Double antibody is the dimer that is made of two polypeptide chains, usually each polypeptide chain in same chain VL and VH by be as short as the joint that can't mutually combine, for example the joint about 5 residues is connected.The VL and the VH that are coded on the same polypeptide chain can't form single chain variable fragment because joint therebetween is short, thereby have formed dimer, so double antibody has two antigen-binding sites.

The antibody of contained identification c-mpl in the medicine of the present invention, its particularly preferred mode for example has sc (Fv) 2.Sc (Fv) the 2nd, with two VH and two VL by joint etc. connect into strand low molecular antibody (Hudson etc., JImmunol.Methods 1999; 231:177～189).The applicant finds that sc (Fv) 2 compares with whole antibody or other low molecular antibody and demonstrate extra high agonist activity.Sc (Fv) 2 for example can prepare by connecting scFv with joint.

Preferably a kind of like this antibody is characterized in that: the N-terminal side with single chain polypeptide is a basic point, two VH and two sequence arrangement that VL presses VH, VL, VH, VL ([VH] joint [VL] joint [VH] joint [VL]).

The order of two VH and two VL is not particularly limited to above-mentioned arrangement, can be by any sequence arrangement.Can also enumerate as following arrangement:

[VL] joint [VH] joint [VH] joint [VL]

[VH] joint [VL] joint [VL] joint [VH]

[VH] joint [VH] joint [VL] joint [VL]

[VL] joint [VL] joint [VH] joint [VH]

[VL] joint [VH] joint [VL] joint [VH]

The joint that connects antibody variable region can be: any peptides joint that can import by genetic engineering or synthetic compound joint are (for example with reference to Protein Engineering, 9 (3), 299～305,1996) disclosed joint etc. in, but preferred peptide joint among the present invention.Length to peptide linker is not particularly limited, those skilled in the art can suitably select according to purpose, but are generally 1～100 aminoacid, preferred 3～50 aminoacid, further preferred 5～30 aminoacid are preferably 12～18 aminoacid (for example 15 aminoacid) especially.

When being peptide linker, can enumerate as:

Ser

Gly·Ser

Gly·Gly·Ser

Ser·Gly·Gly

Gly·Gly·Gly·Ser(SEQ?ID?NO：77)

Ser·Gly·Gly·Gly(SEQ?ID?NO：78)

Gly·Gly·Gly·Gly·Ser(SEQ?ID?NO：79)

Ser·Gly·Gly·Gly·Gly(SEQ?ID?NO：80)

Gly·Gly·Gly·Gly·Gly·Ser(SEQ?ID?NO：81)

Ser·Gly·Gly·Gly·Gly·Gly(SEQ?ID?NO：82)

Gly·Gly·Gly·Gly·Gly·Gly·Ser(SEQ?ID?NO：83)

Ser·Gly·Gly·Gly·Gly·Gly·Gly(SEQ?ID?NO：84)

(Gly·Gly·Gly·Gly·Ser(SEQ?ID?NO：79)) _n

(Ser·Gly·Gly·Gly·Gly(SEQ?ID?NO：80)) _n

[n is the integer more than 1] etc.But the length of peptide linker or sequence can suitably be selected according to purpose by those skilled in the art.

Therefore, the mode of particularly preferred sc (Fv) 2 for example has following sc (Fv) 2 among the present invention.

[VH] peptide linker (15 aminoacid) [VL] peptide linker (15 aminoacid) [VH] peptide linker (15 aminoacid) [VL].

Synthetic compound joint (chemical cross-linking agent) is to be generally used for the crosslinked cross-linking agent of peptide, for example has: N-hydroxy-succinamide (NHS), disuccinimidyl suberate (DSS), two (sulfosuccinimide the ester) (BS of suberic acid ³), dithio two (propanoic acid succinimide ester) (DSP), dithio two (propanoic acid sulfosuccinimide ester) (DTSSP), ethylene glycol bis (succinic acid succinimide ester) (EGS), ethylene glycol bis (succinic acid sulfosuccinimide ester) (Sulfo-EGS), two succinimido tartrates (DST), disulfo succinimido tartrate (Sulfo-DST), two [2-(succinimide oxygen base ketonic oxygen base) ethyl] sulfone (BSOCOES), two [2-(sulfosuccinic imide oxygen base ketonic oxygen base) ethyl] sulfones (Sulfo-BSOCOES) etc., above-mentioned cross-linking agent can have been bought on the market.

Usually need three joints when connecting four antibody variable regions, these joints can use identical also can use different.Preferred low molecular antibody is double antibody or sc (Fv) 2 among the present invention, is preferably sc (Fv) 2 especially.In order to obtain so low molecular antibody, can make the generation antibody fragment with processing antibody such as enzyme, for example papain, pepsin, perhaps make up the DNA of the above-mentioned antibody fragment of coding, it is imported in the expression vector, make it afterwards in appropriate host cell, to express and get final product (for example with reference to Co, M.S. etc., J.Immunol. (1994) 152, and 2968～2976; Better, M. and Horwitz, A.H., Methods Enzymol. (1989) 178, and 476～496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, and 497～515; Lamoyi, E., Methods Enzymol. (1986) 121, and 652～663; Rousseaux, J. etc., Methods Enzymol. (1986) 121, and 663～669; Bird, R.E. and Walker, B.W., Trends Biotechnol. (1991) 9, and 132～137).

Because the anti-c-mpl antibody that sc (Fv) 2 changes has extra high agonist activity to c-mpl, therefore as the propagation of the enhancer of proliferation of hematopoietic stem cell, hemopoietic system CD34 positive cell and/or differentiation accelerator, hemopoietic system CD34 positive cell in bone marrow give birth to the hematopoietic function recovery promoter of giving birth in dose or the hematopoietic stem cell transplantation particularly useful.

The antibody of contained identification c-mpl in the medicine of the present invention, its particularly preferred mode for example has modified antibodies such as chimeric antibody or humanized antibody, preferred especially humanized antibody.

Chimeric antibody is meant the antibody that will derive from the combined sequence of different animals and make, and for example has: the antibody of constant region that comprises heavy chain, the light chain of the variable region of heavy chain, light chain of mouse antibodies and people's antibody.The making of chimeric antibody can be adopted known method, and for example the DNA with the DNA in encoding antibody V district and coding people Antibody C region is connected, is inserted into expression vector and imports among the host, makes its generation chimeric antibody.

Humanized antibody structure (reshaped) the people antibody of also weighing, be will derive from beyond the people mammal for example the complementarity-determining region of mouse antibodies (CDR) be transplanted to the complementarity-determining region of people's antibody and the antibody that obtains, its common gene recombination method also known (with reference to European patent application publication No. EP125023 communique, WO96/02576 communique).

Particularly, have a plurality of oligonucleotide of lap as primer with the end region that is made into CDR and FR two sides, utilize PCR method synthetic DNA sequence, described DNA sequence is designed to connect the CDR of mouse antibodies and the framework region (FR) (with reference to the method for putting down in writing in the WO98/13388 communique) of people's antibody.

In the framework region of the people's antibody that connects via CDR, select complementarity-determining region to form the FR of good antigen-binding site.As required, can replace the aminoacid of framework region in the antibody variable region, make the complementarity-determining region of reconstructed hyman antibody form suitable antigen-binding site (CancerRes. (1993) 53 for Sato, K. etc., 851～856).

In the constant region of constant region end user's antibody of chimeric antibody and humanized antibody, for example in the H chain, can use C γ 1, C γ 2, C γ 3, C γ 4, in the L chain, can use C κ, C λ.In order to improve antibody or its production stability, can modify people's antibody constant region.

Usually, chimeric antibody comprises variable region that derives from people's mammiferous antibody in addition and the constant region that derives from people's antibody.And humanized antibody comprises complementarity-determining region that derives from people's mammiferous antibody in addition and framework region and the constant region that derives from people's antibody.

Need to prove, make chimeric antibody or humanized antibody after, can be with the aminoacid in variable region (for example FR) or the constant region with other aminoacid replacement etc.

Source to variable region in the chimeric antibody or the CDR in the humanized antibody is not particularly limited, and can derive from any animal.For example can use the sequence of mouse antibodies, rat antibody, rabbit antibody, camel antibody etc.

The example of the humanized antibody of identification c-mpl has: the humanized antibody of record in aftermentioned (9)～(19).

Because chimeric antibody and humanized antibody are low in the intravital antigenicity of people, so particularly useful during to people's administration.Described antibody as the propagation of the enhancer of proliferation of hematopoietic stem cell, hemopoietic system CD34 positive cell and/or differentiation accelerator, hemopoietic system CD34 positive cell in bone marrow give birth to the hematopoietic function recovery promoter of giving birth in dose or the hematopoietic stem cell transplantation particularly useful.

The antibody of contained identification c-mpl in the medicine of the present invention, its optimal way for example have the bonded antibody with solvable type c-mpl.Here said solvable type c-mpl is meant except that the c-mpl the c-mpl that expresses on the cell membrane.The object lesson of solvable type c-mpl has: the c-mpl of part or all disappearance of membrane spaning domain.The membrane spaning domain of people c-mpl is equivalent to No. 492 aminoacid～No. 513 amino acid whose part among the SEQ ID NO:51.

The parsing of the response speed opinion that not only can be used for the detailed parsing of epi-position with the bonded antibody of solvable type reorganization c-mpl or combine also can be used for estimating in blood level in the in vivo test or the body dynamic.

The antibody of contained identification c-mpl in the medicine of the present invention, its optimal way for example have couple people c-mpl and monkey c-mpl two sides to have antibody in conjunction with activity or agonist activity.People c-mpl and monkey c-mpl two sides are had the antibody of agonist activity, can verify in the body that in human body, is difficult to measure usually dynamically or effect in the body with monkey, so think that above-mentioned antibody is very useful.Above-mentioned antibody can also have in conjunction with activity or agonist activity the c-mpl of the animal except that people and monkey (for example mice etc.).

And, the antibody of contained identification c-mpl in the medicine of the present invention, its optimal way for example has: TPO agonist activity (agonist activity of c-mpl) is the following antibody of EC50=100nM following, the further preferred EC50=10nM of following, preferred EC50=30nM.

And, the antibody of contained identification c-mpl in the medicine of the present invention, its optimal way for example has: with the active KD=10 of being of combining of solvable type c-mpl ^-6Following, the preferred KD=10 of M ^-7Following, the further preferred KD=10 of M ^-8The antibody that M is following.

In the present invention, whether antibody is KD=10 with the activity that combines of solvable type reorganization c-mpl ^-6Below the M, this can utilize method known in those skilled in the art to measure.For example can utilize and use the surperficial plasmon resonance of BIAcore to measure.That is, solvable type c-mpl-Fc proteopexy on sensor chip, can be calculated rate constant by interactional measured value between evaluation antibody and the solvable type c-mpl-Fc.Can adopt when estimating: ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay) or fluorescent antibody technique in conjunction with activity.When for example using enzyme immunoassay, will contain the sample of being examined antibody, for example be examined the culture supernatant of antibody produced cell or antibody purification and join and be coated with on the antigenic flat board, described antigen is used in conjunction with being examined antibody.Interpolation is bathed flat board with the secondary antibodies and the temperature of enzyme labellings such as alkali phosphatase, cleans the back and adds zymolytes such as p-nitrophenyl phosphoric acid, measures absorbance afterwards, thereby can estimate antigen-binding activity.

To being not particularly limited, for example can set the upper limit of the scope that those skilled in the art can make technically in conjunction with the active upper limit.But technical scope of making enlarges along with technological progress.

The antibody of contained identification c-mpl in the medicine of the present invention, its optimal way for example has: each antibody of putting down in writing in following (1)～(19).(1)～(19) each antibody of putting down in writing is preferably low molecular antibody in.

(1) comprise the antibody of VH, described VH has the CDR1,2,3 of the aminoacid sequence that comprises SEQ ID NO:1,2,3 (VB22B:VH CDR1,2,3) record respectively.

(2) comprise the antibody of VL, described VL has the CDR1,2,3 of the aminoacid sequence that comprises SEQ ID NO:4,5,6 (VB22B:VL CDR1,2,3) record respectively.

(3) comprise the antibody of VH and VL, described VH has the CDR1,2,3 of the aminoacid sequence that comprises SEQ ID NO:1,2,3 (VB22B:VH CDR1,2,3) record respectively, and described VL has the CDR1,2,3 of the aminoacid sequence that comprises SEQ ID NO:4,5,6 (VB22B:VL CDR1,2,3) record respectively.

(4) comprise the antibody of VH, described VH comprises the aminoacid sequence of SEQ ID NO:8 (VB22B:VH) record.

(5) comprise the antibody of VL, described VL comprises the aminoacid sequence of SEQ ID NO:10 (VB22B:VL) record.

(6) comprise the antibody of VH and VL, described VH comprises the aminoacid sequence of SEQ ID NO:8 (VB22B:VH) record, and described VL comprises the aminoacid sequence of SEQ ID NO:10 (VB22B:VL) record.

(7) antibody, this antibody have the aminoacid sequence of SEQ ID NO:12 (VB22B:scFv) record.

(8) antibody, this antibody have the aminoacid sequence of SEQ ID NO:14 (VB22B:sc (Fv) 2) record.

(9) comprise the humanized antibody of VH, described VH has the FR1,2,3,4 that comprises the aminoacid sequence of each record in following (a)～(e).

(a)SEQ?ID?NO：15、16、17、18(hVB22B?p-z：VH?FR1、2、3、4)

(b)SEQ?ID?NO：19、20、21、22(hVB22B?g-e：VH?FR1、2、3、4)

(c)SEQ?ID?NO：23、24、25、26(hVB22B?e：VH?FR1、2、3、4)

(d)SEQ?ID?NO：54、55、56、57(hVB22B?u2-wz4：VH?FR1、2、3、4)

(e)SEQ?ID?NO：54、55、58、57(hVB22B?q-wz5：VH?FR1、2、3、4)

(10) comprise the humanized antibody of VL, described VL has the FR1,2,3,4 that comprises the aminoacid sequence of each record in following (a)～(d).

(a)SEQ?ID?NO：27、28、29、30(hVB22B?p-z：VL?FR1、2、3、4)

(b) SEQ ID NO:31,32,33,34 (hVB22B g-e or hVB22B e:VL FR1,2,3,4)

(c)SEQ?ID?NO：59、60、61、62(hVB22B?u2-wz4：VL?FR1、2、3、4)

(d)SEQ?ID?NO：59、63、64、62(hVB22B?q-wz5：VL?FR1、2、3、4)

(11) humanized antibody, this antibody comprise the VL and the VH of each record in following (a)～(e).

(a) have the FR1,2,3 of the aminoacid sequence that comprises SEQ ID NO:15,16,17,18 records respectively, 4 VH and FR1,2,3,4 VL with the aminoacid sequence that comprises SEQ ID NO:27,28,29,30 records respectively.

(b) have the FR1,2,3 of the aminoacid sequence that comprises SEQ ID NO:19,20,21,22 records respectively, 4 VH and FR1,2,3,4 VL with the aminoacid sequence that comprises SEQ ID NO:31,32,33,34 records respectively.

(c) have the FR1,2,3 of the aminoacid sequence that comprises SEQ ID NO:23,24,25,26 records respectively, 4 VH and FR1,2,3,4 VL with the aminoacid sequence that comprises SEQ ID NO:31,32,33,34 records respectively.

(d) have the FR1,2,3 of the aminoacid sequence that comprises SEQ ID NO:54,55,56,57 records respectively, 4 VH and FR1,2,3,4 VL with the aminoacid sequence that comprises SEQ ID NO:59,60,61,62 records respectively.

(e) have the FR1,2,3 of the aminoacid sequence that comprises SEQ ID NO:54,55,58,57 records respectively, 4 VH and FR1,2,3,4 VL with the aminoacid sequence that comprises SEQ ID NO:59,63,64,62 records respectively.

(12) comprise the humanized antibody of VH, described VH has the CDR1,2,3 of the aminoacid sequences that comprise SEQ ID NO:1,2,3 records respectively.

(13) comprise the humanized antibody of VL, described VL has the CDR1,2,3 of the aminoacid sequences that comprise SEQ ID NO:4,5,6 records respectively.

(14) comprise the humanized antibody of VH and VL, described VH has the CDR1,2,3 of the aminoacid sequences that comprise SEQ ID NO:1,2,3 records respectively, and described VL has the CDR1,2,3 of the aminoacid sequences that comprise SEQ ID NO:4,5,6 records respectively.

(15) comprise the humanized antibody of VH, described VH comprises the aminoacid sequence of SEQ ID NO:36 (hVB22Bp-z:VH), SEQ ID NO:38 (hVB22B g-e:VH), SEQ ID NO:40 (hVB22Be:VH), SEQ ID NO:65 (hVB22B u2-wz4:VH) or SEQ ID NO:66 (hVB22B q-wz5:VH) record.

(16) comprise the humanized antibody of VL, described VL comprises the aminoacid sequence of SEQ ID NO:42 (hVB22Bp-z:VL), SEQ ID NO:44 (hVB22B g-e:VL or hVB22B e:VL), SEQ IDNO:67 (hVB22B u2-wz4:VL) or SEQ ID NO:68 (hVB22B q-wz5:VH) record.

(17) humanized antibody, this antibody comprise the VH and the VL of each record in following (a)～(e).

(a) comprise SEQ ID NO:36 (hVB22B p-z:VH) record aminoacid sequence VH and comprise the VL of the aminoacid sequence of SEQ ID NO:42 (hVB22B p-z:VL) record.

(b) comprise SEQ ID NO:38 (hVB22B g-e:VH) record aminoacid sequence VH and comprise the VL of the aminoacid sequence of SEQ ID NO:44 (hVB22B g-e:VL or hVB22B e:VL) record.

(c) comprise SEQ ID NO:40 (hVB22B e:VH) record aminoacid sequence VH and comprise the VL of the aminoacid sequence of SEQ ID NO:44 (hVB22B g-e:VL or hVB22B e:VL) record.

(d) comprise SEQ ID NO:65 (hVB22B u2-wz4:VH) record aminoacid sequence VH and comprise the VL of the aminoacid sequence of SEQ ID NO:67 (hVB22B u2-wz4:VL) record.

(e) comprise SEQ ID NO:66 (hVB22B q-wz5:VH) record aminoacid sequence VH and comprise the VL of the aminoacid sequence of SEQ ID NO:68 (hVB22B q-wz5:VL) record.

In the aminoacid sequence of SEQ ID NO:36 (hVB22B p-z:VH), SEQ ID NO:38 (hVB22Bg-e:VH), SEQ ID NO:40 (hVB22B e:VH), SEQ ID NO:65 (hVB22Bu2-wz4:VH) or SEQ ID NO:66 (hVB22B q-wz5:VH) record

The aminoacid position: 31～35 are equivalent to CDR1

The aminoacid position: 50～66 are equivalent to CDR2

The aminoacid position: 99～107 are equivalent to CDR3

The aminoacid position: 1～30 is equivalent to FR1

The aminoacid position: 36～49 are equivalent to FR2

The aminoacid position: 67～98 are equivalent to FR3

The aminoacid position: 108～118 are equivalent to FR4.

In the aminoacid sequence of SEQ ID NO:42 (hVB22B p-z:VL) or SEQ ID NO:44 (hVB22Bg-e:VL or hVB22B e:VL), SEQ ID NO:67 (hVB22B u2-wz4:VL) or SEQ ID NO:68 (hVB22B q-wz5:VH) record

The aminoacid position: 24～39 are equivalent to CDR1

The aminoacid position: 55～61 are equivalent to CDR2

The aminoacid position: 94～102 are equivalent to CDR3

The aminoacid position: 1～23 is equivalent to FR1

The aminoacid position: 40～54 are equivalent to FR2

The aminoacid position: 62～93 are equivalent to FR3

The aminoacid position: 103～112 are equivalent to FR4.

Among the present invention, CDR in the hVB22B p-z VH sequence and FR are corresponding as follows with serial number.

hVB22B?p-z?VH：FR1/SEQ?ID?NO：15

hVB22B?p-z?VH：CDR1/SEQ?ID?NO：1

hVB22B?p-z?VH：FR2/SEQ?ID?NO：16

hVB22B?p-z?VH：CDR2/SEQ?ID?NO：2

hVB22B?p-z?VH：FR3/SEQ?ID?NO：17

hVB22B?p-z?VH：CDR3/SEQ?ID?NO：3

hVB22B?p-z?VH：FR4/SEQ?ID?NO：18

Among the present invention, CDR in the hVB22B p-z VL sequence and FR are corresponding as follows with serial number.

hVB22B?p-z?VL：FR1/SEQ?ID?NO：27

hVB22B?p-z?VL：CDR1/SEQ?ID?NO：4

hVB22B?p-z?VL：FR2/SEQ?ID?NO：28

hVB22B?p-z?VL：CDR2/SEQ?ID?NO：5

hVB22B?p-z?VL：FR3/SEQ?ID?NO：29

hVB22B?p-z?VL：CDR3/SEQ?ID?NO：6

hVB22B?p-z?VL：FR4/SEQ?ID?NO：30

Among the present invention, CDR in the hVB22B g-e VH sequence and FR are corresponding as follows with serial number.

hVB22B?g-e?VH：FR1/SEQ?ID?NO：19

hVB22B?g-e?VH：CDR1/SEQ?ID?NO：1

hVB22B?g-e?VH：FR2/SEQ?ID?NO：20

hVB22B?g-e?VH：CDR2/SEQ?ID?NO：2

hVB22B?g-e?VH：FR3/SEQ?ID?NO：21

hVB22B?g-e?VH：CDR3/SEQ?ID?NO：3

hVB22B?g-e?VH：FR4/SEQ?ID?NO：22

Among the present invention, CDR in the hVB22B g-e VL sequence and FR are corresponding as follows with serial number.

hVB22B?g-e?VL：FR1/SEQ?ID?NO：31

hVB22B?g-e?VL：CDR1/SEQ?ID?NO：4

hVB22B?g-e?VL：FR2/SEQ?ID?NO：32

hVB22B?g-e?VL：CDR2/SEQ?ID?NO：5

hVB22B?g-e?VL：FR3/SEQ?ID?NO：33

hVB22B?g-e?VL：CDR3/SEQ?ID?NO：6

hVB22B?g-e?VL：FR4/SEQ?ID?NO：34

Among the present invention, CDR in the hVB22B e VH sequence and FR are corresponding as follows with serial number.

hVB22B?e?VH：FR1/SEQ?ID?NO：23

hVB22B?e?VH：CDR1/SEQ?ID?NO：1

hVB22B?e?VH：FR2/SEQ?ID?NO：24

hVB22B?e?VH：CDR2/SEQ?ID?NO：2

hVB22B?e?VH：FR3/SEQ?ID?NO：25

hVB22B?e?VH：CDR3/SEQ?ID?NO：3

hVB22B?e?VH：FR4/SEQ?ID?NO：26

Among the present invention, CDR in the hVB22B e VL sequence and FR are corresponding as follows with serial number.

hVB22B?e?VL：FR1/SEQ?ID?NO：31

hVB22B?e?VL：CDR1/SEQ?ID?NO：4

hVB22B?e?VL：FR2/SEQ?ID?NO：32

hVB22B?e?VL：CDR2/SEQ?ID?NO：5

hVB22B?e?VL：FR3/SEQ?ID?NO：33

hVB22B?e?VL：CDR3/SEQ?ID?NO：6

hVB22B?e?VL：FR4/SEQ?ID?NO：34

Among the present invention, CDR in the hVB22B u2-wz4 VH sequence and FR are corresponding as follows with serial number.

hVB22B?u2-wz4?VH：FR1/SEQ?ID?NO：54

hVB22B?u2-wz4?VH：CDR1/SEQ?ID?NO：1

hVB22B?u2-wz4?VH：FR2/SEQ?ID?NO：55

hVB22B?u2-wz4?VH：CDR2/SEQ?ID?NO：2

hVB22B?u2-wz4?VH：FR3/SEQ?ID?NO：56

hVB22B?u2-wz4?VH：CDR3/SEQ?ID?NO：3

hVB22B?u2-wz4?VH：FR4/SEQ?ID?NO：57

Among the present invention, CDR in the hVB22B u2-wz4 VL sequence and FR are corresponding as follows with serial number.

hVB22B?u2-wz4?VL：FR1/SEQ?ID?NO：59

hVB22B?u2-wz4?VL：CDR1/SEQ?ID?NO：4

hVB22B?u2-wz4?VL：FR2/SEQ?ID?NO：60

hVB22B?u2-wz4?VL：CDR2/SEQ?ID?NO：5

hVB22B?u2-wz4?VL：FR3/SEQ?ID?NO：61

hVB22B?u2-wz4?VL：CDR3/SEQ?ID?NO：6

hVB22B?u2-wz4?VL：FR4/SEQ?ID?NO：62

Among the present invention, CDR in the hVB22B q-wz5 VH sequence and FR are corresponding as follows with serial number.

hVB22B?q-wz5?VH：FR1/SEQ?ID?NO：54

hVB22B?q-wz5?VH：CDR1/SEQ?ID?NO：1

hVB22B?q-wz5?VH：FR2/SEQ?ID?NO：55

hVB22B?q-wz5?VH：CDR2/SEQ?ID?NO：2

hVB22B?q-wz5?VH：FR3/SEQ?ID?NO：56

hVB22B?q-wz5?VH：CDR3/SEQ?ID?NO：3

hVB22B?q-wz5?VH：FR4/SEQ?ID?NO：57

Among the present invention, CDR in the hVB22B q-wz5 VL sequence and FR are corresponding as follows with serial number.

hVB22B?q-wz5?VL：FR1/SEQ?ID?NO：59

hVB22B?q-wz5?VL：CDR1/SEQ?ID?NO：4

hVB22B?q-wz5?VL：FR2/SEQ?ID?NO：63

hVB22B?q-wz5?VL：CDR2/SEQ?ID?NO：5

hVB22B?q-wz5?VL：FR3/SEQ?ID?NO：64

hVB22B?q-wz5?VL：CDR3/SEQ?ID?NO：6

hVB22B?q-wz5?VL：FR4/SEQ?ID?NO：62

Need to prove, the nucleotide sequence of VB22B VH is documented among the SEQ ID NO:7, the nucleotide sequence of VB22B VL is documented among the SEQ ID NO:9, the nucleotide sequence of VB22B scFv is documented among the SEQ ID NO:11, the nucleotide sequence of VB22B sc (Fv) 2 is documented among the SEQ ID NO:13, the nucleotide sequence of hVB22B p-z VH is documented among the SEQ ID NO:35, the nucleotide sequence of hVB22B g-e VH is documented among the SEQ ID NO:37, the nucleotide sequence of hVB22B e VH is documented among the SEQ ID NO:39, the nucleotide sequence of hVB22Bu2-wz4 VH is documented among the SEQ ID NO:69, the nucleotide sequence of hVB22B q-wz5VH is documented among the SEQ ID NO:71, the nucleotide sequence of hVB22B p-z VL is documented among the SEQ ID NO:41, the nucleotide sequence of hVB22B g-e VL and hVB22B e VL is documented among the SEQ ID NO:43, the nucleotide sequence of hVB22B u2-wz4 VL is documented among the SEQ ID NO:70, the nucleotide sequence of hVB22B q-wz5 VL is documented among the SEQ ID NO:72, the nucleotide sequence of hVB22B p-z sc (Fv) 2 is documented among the SEQID NO:45, the nucleotide sequence of hVB22B g-e sc (Fv) 2 is documented among the SEQ ID NO:47, the nucleotide sequence of hVB22B e sc (Fv) 2 is documented among the SEQ ID NO:49, the nucleotide sequence of hVB22B u2-wz4 sc (Fv) 2 is documented among the SEQ ID NO:75, and the nucleotide sequence of hVB22B q-wz5 sc (Fv) 2 is documented among the SEQ ID NO:76.(18) humanized antibody, this antibody have the aminoacid sequence of each record among SEQ ID NO:46 (hVB22B p-z:sc (Fv) 2), SEQ ID NO:48 (hVB22B g-e:sc (Fv) 2), SEQ ID NO:50 (hVB22B e:sc (Fv) 2), SEQ ID NO:73 (hVB22B u2-wz4:sc (Fv) 2) or the SEQ ID NO:74 (hVB22B q-wz5:sc (Fv) 2).(19) antibody, wherein in above-mentioned (1)～(18) in the aminoacid sequence of each record one or more aminoacid be substituted, lack, add and/or insert and have same isoreactivity with above-mentioned antibody.Wherein, have with the isoreactivity meaning with above-mentioned antibody and to be meant, this sudden change antibody the propagation of the propagation facilitation of hematopoietic stem cell, hemopoietic system CD34 positive cell and/or differentiation facilitation, hemopoietic system CD34 positive cell in bone marrow give birth to give birth to aspect the hematopoietic function recovery facilitation in increase effect and the hematopoietic stem cell transplantation and original antibody has equal activity.

Because each antibody is very high to the agonist activity of c-mpl in above-mentioned (1)～(19), thus as the propagation of the enhancer of proliferation of hematopoietic stem cell, hemopoietic system CD34 positive cell and/or differentiation accelerator, hemopoietic system CD34 positive cell in bone marrow give birth to the hematopoietic function recovery promoter of giving birth in dose or the hematopoietic stem cell transplantation particularly useful.

In order to prepare the polypeptide identical,, import the method for sudden change in the known oriented polypeptide as method well-known to those skilled in the art with certain peptide species function.For example, those skilled in the art by utilize the site-specific mutagenesis method (Hashimoto-Gotoh, T etc. (1995) Gene 152,271～275; Zoller, MJ, and Smith, M. (1983) Methods Enzymol.100,468～500; Kramer, W. etc. (1984) Nucleic Acids Res.12,9441～9456; Kramer, W. and Fritz HJ, (1987) Methods Enzymol.154,350～367; Kunkel, TA, (1985) Proc.Natl.Acad.Sci.USA.82,488～492; Kunkel, (1988) Methods Enzymol.85,2763～2766) etc. in antibody of the present invention, import suitable sudden change, can prepare the antibody identical with this antibody function.Amino acid whose sudden change also can spontaneous generation.The aminoacid sequence that so in the aminoacid sequence of antibody of the present invention, contains one or more amino acid mutations, and be also contained in the antibody of the present invention with antibody that this antibody has a same function.Mutating acid number in the said mutation body be generally 50 aminoacid with interior, be preferably 30 aminoacid with interior, more preferably 10 aminoacid is with interior (for example 5 aminoacid in).

In the amino acid residue of undergoing mutation, preferably be mutated into the another kind of aminoacid of the character of preserving amino acid side chain.For example, the character of amino acid side chain comprises: hydrophobic amino acid (A, I, L, M, F, P, W, Y, V), hydrophilic amino acid (R, D, N, C, E, Q, G, H, K, S, T), aminoacid (G with aliphatic lateral chain, A, V, L, I, P), aminoacid (S with side chain of hydroxyl, T, Y), aminoacid (C with side chain of sulfur atom-containing, M), aminoacid (D with the side chain that contains carboxylic acid and amide, N, E, Q), aminoacid (R with the side chain that contains base, K, H) and have an aminoacid (H of the side chain that contains aromatics, F, Y, W) (all represent an amino acid whose word marking in the parantheses).

Known contain to certain aminoacid sequence by disappearance, add one or more amino acid residues and/or by other aminoacid replacement and the polypeptide of adorned aminoacid sequence can be kept its original biologic activity (Mark, D.F. etc., Proc.Natl.Acad.Sci.USA (1984) 81, and 5662～5666; Zoller, M.J.﹠amp; Smith, M.Nucleic Acids Research (1982) 10,6487～6500; Wang, A. etc., Science224,1431～1433; Dalbadie-McFarland, G. etc., Proc.Natl.Acad.Sci.USA (1982) 79, and 6409～6413).

The antibody that has added a plurality of amino acid residues in the aminoacid sequence of antibody of the present invention comprises the fusion rotein that contains these antibody.Fusion rotein is meant the product that above-mentioned antibody and other peptide or albumen merge, and it comprises in the present invention.The method of making fusion rotein can adopt method known in those skilled in the art, the polynucleotide that for example connect code book invention antibody make framework consistent with the polynucleotide of other peptide of coding or polypeptide, afterwards it is imported in the expression vector, make it in the host, to express getting final product.As other peptide or the polypeptide that merge with antibody of the present invention, for example can use: FLAG (Hopp, T.P. etc., BioTechnology (1988) 6,1204～1210) the known peptide such as 6 * His, 10 * His, influenza hemagglutinin (HA), people c-myc fragment, VSV-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, alpha-tubulin fragment, B-tag, PROTEIN C fragment that, comprises 6 His (histidine) residue.In addition, other polypeptide with antibody fusion of the present invention for example has: GST (glutathione-S-transferase), HA (influenza hemagglutinin), constant region for immunoglobulin, beta galactosidase, MBP (maltose-binding protein) etc.The polynucleotide of these commercially available peptides of coding or polypeptide and the polynucleotide of code book invention antibody are merged, and the fusion polynucleotides of preparation is thus expressed, can prepare fused polypeptide thus.

Antibody of the present invention produces the cell of this antibody or host or purification process according to aftermentioned and in aminoacid sequence, molecular weight, isoelectric point, IP or not have aspects such as sugar chain or conformation different.But gained antibody promptly is included in the antibody of the present invention as long as have identical functions with antibody of the present invention.For example, make antibody of the present invention at prokaryotic cell for example during expression in escherichia coli, add methionine residues at the N-terminal of the aminoacid sequence of original antibody.Antibody of the present invention also comprises such antibody.

As the optimal way of the antibody of identification c-mpl contained in the medicine of the present invention, also comprise the antibody of the epi-position that the antibody of discerning above-mentioned (1)～(19) is discerned.

The antibody of the epi-position that identification antibody is discerned can utilize method known in those skilled in the art to obtain.For example utilize following method to obtain, that is: utilize usual way to determine the epi-position that above-mentioned antibody is discerned, made the method for antibody with polypeptide as immunity originally with aminoacid sequence contained in this epi-position; Perhaps determine to select the method for the antibody identical etc. with above-mentioned antibody epitope according to the epi-position of the antibody of usual way making.

In the present invention, preferred especially identification contains the antibody of the epi-position that the antibody of the aminoacid sequence of SEQ ID NO:73 record discerns.Anticipation contains the district, the district of preferred Ala 189～Gly245, the district of further preferred Gln 213～Ala 231 of Glu 26～Leu 274 of antibody recognition people c-mpl of the aminoacid sequence of SEQ ID NO:73 record.Therefore, the antibody in 26～274 or 189～245 or 213～231 of identification people c-mpl district is also contained among the present invention.

The antibody in 26～274 or 189～245 or 213～231 district can utilize method known in those skilled in the art to obtain in the aminoacid sequence (SEQ ID NO:51) of identification people c-mpl, for example utilize following method to obtain, that is: make the method for antibody with 26～274 or 189～245 or 213～231 peptide in the aminoacid sequence (SEQ ID NO:51) of people c-mpl as immunogen; Perhaps determine the epi-position discerned according to the antibody that usual way is made, select with the method for the antibody of the identical epi-position of antibody recognition of the present invention etc.

Can utilize method known in those skilled in the art to make with the bonded antibody of c-mpl.For example, the hybridoma that produces monoclonal antibody can utilize the following making of known technology basically.Promptly, use c-mpl albumen or c-mpl express cell as sensitization antigen, according to common immunization method it is carried out immunity, utilize common cell fusion method that gained immunocyte and known parental cell are merged again, utilize common screening technique screening monoclonal antibody to produce cell, can make above-mentioned hybridoma.

Particularly, making monoclonal antibody can followingly carry out.At first, by expressing disclosed c-mpl gene/aminoacid sequence among the GenBank:NM_005373, obtain as the antigenic c-mpl albumen of sensitization of obtaining antibody.That is, the gene order of coding c-mpl is inserted in the known expression vector and transformed appropriate host cell, utilize known method purification of target people c-mpl albumen in its host cell or in the culture supernatant afterwards.

Next, with this purification c-mpl albumen as sensitization antigen.Perhaps, can also be with the partial peptide of c-mpl as sensitization antigen.At this moment, partial peptide can obtain by chemosynthesis according to the aminoacid sequence of people c-mpl.

Epi-position on the c-mpl molecule that anti-c-mpl antibody of the present invention is discerned is not limited to defined epitope, so long as be present in the epi-position on the c-mpl molecule, can discern any epi-position.Therefore, be used to make the antigen of anti-c-mpl antibody of the present invention,, can use any fragment so long as contain the fragment that is present in the epi-position on the c-mpl molecule.

Mammal through the sensitization antigen immune is not particularly limited, selects after the preferred fitness of considering with the parental cell that is used for cell fusion, use rodent, for example mice, rat, hamster, rabbit or monkey etc. usually.

With sensitization antigen animal being carried out immunity can carry out according to known method.For example, usual way is: by mammal being carried out intraperitoneal or subcutaneous injection sensitization antigen carries out immunity.Particularly, sensitization antigen with suitably dilution and suspending such as phosphate buffer (PBS) or normal saline, is mixed common adjuvant, for example Freund's complete adjuvant to wherein an amount of as required, carry out emulsifying, mammal given for several times in per then 4～21 days.Can also use appropriate carriers when carrying out the sensitization antigen immune.

So mammal is carried out immunity, after required antibody horizontal rises in the affirmation serum, take immunocyte from mammal, this immunocyte is used for cell fusion, particularly preferred immunocyte is a splenocyte.

Can use mammiferous myeloma cell as the opposing party-parental cell with above-mentioned immunocyte fusion.This myeloma cell can suitably use known various cell strain, for example (J.Immnol. (1979) 123 for P3 (P3x63Ag8.653), 1548～1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81,1～7), NS-1 (Kohler, G. and Milstein, C.Eur.J.Immunol. (1976) 6,511～519), MPC-11 (Margulies, D.H. etc., Cell, (1976) 405～415), SP2/0 (Shulman, M. etc., Nature (1978) 276,269～270), FO (deSt.Groth, S.F. etc., J.Immunol.Methods (1980) 35, and 1～21), S194 (Trowbridge, I.S., J.Exp.Med. (1978) 148,313～323), R210 (Galfre, G. etc., Nature (1979) 277, and 131～133) etc.

Above-mentioned immunocyte and myeloma cell's cell fusion can merge according to people's such as known method, for example Kohler and Milstein method (Kohler, G. and Milstein, C., Methods Enzymol. (1981) 73,3～46) basically.

More specifically, above-mentioned cell fusion for example can be implemented in common nutrient medium in the presence of cell fusion promoter.As merging promoter, for example use Polyethylene Glycol (PEG), Sendai virus (HVJ) etc., as required,, can also further add adjuvant such as using dimethyl sulfoxide in order to improve fusion efficiencies.

Immunocyte and myeloma cell's usage ratio can be set arbitrarily.For example preferred immunocyte is 1～10 times of myeloma cell.The culture fluid that is used for above-mentioned cell fusion for example can use the RPMI RPMI-1640 that is suitable for above-mentioned myeloma cell strain propagation, MEM culture fluid, other is used for the common culture fluid of this cell culture, can also be used in combination additional liquid such as hyclone (FCS) serum of etc.ing.

Cell fusion can followingly carry out: the above-mentioned immunocyte and the myeloma cell of scheduled volume are fully mixed in above-mentioned culture fluid, usually add the PEG solution (for example mean molecule quantity is about 1000～6000) that is heated to 37 ℃ in advance by the concentration of 30～60% (w/v) and mix formation Target Fusion cell (hybridoma).Then, add suitable culture fluid one by one, centrifugal, remove supernatant, repeat aforesaid operations to remove the cell fusion agent that is unfavorable for the hybridoma growth etc.

So operation and the hybridoma that obtains are selected by cultivating in the selection culture fluid of routine, for example HAT culture fluid (culture fluid that contains hypoxanthine, aminopterin and thymidine).Cultivation in the above-mentioned HAT culture fluid continues to the required long enough time of killing except that the target hybridoma (a couple of days～several weeks usually) of cell (non-fused cell).Implement conventional limiting dilution assay then, screening can produce the hybridoma of target antibody and carry out single cell clone.

Obtain beyond the above-mentioned hybridoma except the animal the people being carried out antigen immune, at external use c-mpl sensitization human lymphocyte, primed lymphocyte and the myeloma cell with permanent break-up energy who derives from the people are merged, also can obtain having c-mpl in conjunction with active required people's antibody (with reference to the special fair 1-59878 communique of Japan).Can also give transgenic animal as antigenic c-mpl with fully human antibodies gene bank, obtain anti-MPL antibody produced cell, obtain people's antibody (with reference to International Patent Application Publication No. WO94/25585 communique, WO93/12227 communique, WO92/03918 communique, WO94/02602 communique) of anti-c-mpl by the cell that makes its immortalization.

So the hybridoma of operation and the generation monoclonal antibody of making can be in conventional culture fluid successive transfer culture, also can preserve in that liquid nitrogen is medium-term and long-term.

When obtaining monoclonal antibody, can adopt following method: cultivate this hybridoma according to the method for routine, obtain the method for its culture supernatant by this hybridoma; Perhaps to have with it adaptive mammal give hybridoma make it propagation, obtain the method for its ascites etc.Preceding a kind of method is suitable for obtaining highly purified antibody, and then a kind of method is suitable for producing in a large number antibody.

Recombinant antibodies also can followingly be made: by hybridoma clonal antibody gene, this gene is inserted in the appropriate carriers, again it is imported among the host, utilize gene recombination technology to produce recombinant antibodies (for example with reference to Vandamme, A.M. etc., Eur.J.Biochem. (1990) 192,767～775,1990).

Particularly, the mRNA that from the hybridoma that produces anti-c-mpl antibody, separates the anti-c-mpl antibody variable of coding (V) district.Utilize known method, for example guanidine ultracentrifugation (Chirgwin, J.M. etc., Biochemistry (1979) 18,5294～5299), AGPC method (Chomczynski, P. etc., Anal.Biochem. (1987) 162,156～159) etc. separate mRNA, prepare total RNA, re-use preparation target mRNA such as mRNA Purification Kit (Pharmacia system).Can also directly prepare mRNA by using QuickPrep mRNA Purification Kit (Pharmacia system).

Use the cDNA of reverse transcriptase by gained mRNA synthetic antibody V district.The synthetic of cDNA can use AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (biochemical industrial society system) to wait and carry out.In addition, when carrying out the synthetic of cDNA and amplification, can adopt 5 '-RACE method of having used 5 '-Ampli FINDER RACE Kit (Clontech system) and PCR (Proc.Natl.Acad.Sci.USA (1988) 85 for Frohman, M.A. etc., 8998～9002; Belyavsky, A. etc., Nucleic Acids Res. (1989) 17,2919～2932) etc.

By gained PCR product purification target dna fragment, be connected with carrier DNA afterwards.Make recombinant vector more thus, in the importing escherichia coli etc., select bacterium colony afterwards, prepare required recombinant vector.Utilize known method, for example dideoxy nucleotide chain cessation method etc. to confirm the nucleotide sequence of target dna then.

Obtain encoding behind the DNA in the anti-c-mpl antibody of target V district, be inserted in the expression vector of the DNA that contains the required antibody constant region of coding (C district).

When preparing anti-c-mpl antibody used in the present invention,, usually antibody gene is inserted in the expression vector in order for example to express under the adjusting of enhancer, promoter expressing regulatory region.Next, utilize this expression vector transformed host cell, make antibody expression.

For the expressing antibodies gene, the polynucleotide of coding H chain and L chain can be inserted in the expression vector common transfection host cell respectively; The polynucleotide of H chain and L chain of perhaps will encoding insert in the single expression vector, and transfection host cell (with reference to WO94/11523).

For example with escherichia coli during as the host, for amplification in a large number in escherichia coli (for example JM109, DH5 α, HB101, XL1Blue) etc., prepare carrier in a large number, as long as carrier (for example has the marker gene that is used for " ori " that increases escherichia coli and has screening escherichia coli transformant, the drug resistance gene that available any medicine (ampicillin, tetracycline, kanamycin, chloromycetin) is differentiated) gets final product, be not particularly limited.The example of carrier has: M13 is that carrier, pUC are carrier, pBR322, pBluescript, pCR-Script etc.During for sub-clone, excision cDNA, except that above-mentioned carrier, for example also have carriers such as pGEM-T, pDIRECT, pT7.

As expression vector, for example for when the expression in escherichia coli, carrier is except having the above-mentioned feature that increases in escherichia coli, also must have can be in escherichia coli hosts such as JM109, DH5 α, HB101, XL1-Blue expression promoter expeditiously, lacZ promoter (Ward etc. for example, Nature (1989) 341, and 544～546; FASEB J. (1992) 6,2422～2427), araB promoter (Better etc., Science (1988) 240,1041～1043) or T7 promoter etc.As such carrier, except that above-mentioned carrier, also comprise pGEX-5X-1 (Pharmacia system), " QIAexpress system " (Quiagen system), pEGFP or pET (BL21 of host's preferred expression T7 RNA polymerase at this moment) etc.

Can also comprise in the carrier and be used for the excretory signal sequence of polypeptide.As the signal sequence that is used for protein excretion, when secretory protein in colibacillary pericentral siphon, can use pelB signal sequence (J.Bacteriol. (1987) 169 for Lei, S.P. etc., 4379).For example can utilize Calcium Chloride Method, electroporation that carrier is imported in the host cell.

Except that escherichia coli, for example also have: derive from mammiferous expression vector (for example pcDNA3 (Invitrogen society system) or pEF-BOS (Nucleic Acids.Res.1990, the 18 (17), the 5322nd page), pEF, pCDM8); Derive from the expression vector (for example " Bac-to-BAC baculovirus expression system " (Gibco-BRL system), pBacPAK8) of insect cell; Derive from the expression vector (for example pMH1, pMH2) of plant; Derive from the expression vector (for example pHSV, pMV, pAdexLcw) of animal virus; Derive from the expression vector (for example pZIPneo) of retrovirus retrovirus; Derive from zymic expression vector (for example " Pichia Expression Kit " (Invitrogen system), pNV11, SP-Q01); Derive from the expression vector (for example pPL608, pKTH50) of hay bacterium.

In order in zooblasts such as Chinese hamster ovary celI, COS cell, NIH3T3 cell, to express, carrier must have at the necessary promoter of cell inner expression, for example SV40 promoter (Mulligan etc., Nature (1979) 277,108), MMLV-LTR promoter, EF1 α promoter (Mizushima etc., Nucleic Acids Res. (1990) 18,5322), CMV promoter etc., further preferred vector has the gene (drug resistant gene that for example available medicine (neomycin, G418 etc.) is differentiated) that is used to screen transformant.Example with carrier of above-mentioned characteristic has: pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, pOP13 etc.

And, during for the gene copy number in stably express gene and the amplifying cells, can enumerate following method, promptly in the Chinese hamster ovary celI that lacks the nucleic acid synthesis path, import the carrier with DHFR gene (for example pCHOI etc.) in this path of compensation, the increase method of this carrier of reuse methotrexate (MTX).In addition, during for the transient expression gene, can enumerate following method, promptly use to have the COS cell of expressing the antigenic gene of SV40T on the chromosome, the method that transforms with the carrier (pcD etc.) of origin of replication with SV40.Origin of replication can use the starting point that derives from polyoma virus, adenovirus, bovine papilloma virus (BPV) etc.For amplification gene copy number in host cell system, expression vector can further contain aminoglycoside transferring enzyme (APH) gene, thymidine kinase (TK) gene, escherichia coli xanthine-guanine phosphoribosyl transferase (Ecogpt) gene, dihydrofolate reductase (dhfr) gene etc. as selection markers.

There is the host cell of carrier to be not particularly limited to importing, for example can uses escherichia coli and various zooblasts etc.Host cell can be as for example being used for preparation and expression production of antibodies of the present invention system.The generation system that is used to prepare polypeptide comprises: (invivo) generation system in external (in vitro) and the body.External generation system for example has: use generation system that eukaryotic cell carries out and the generation system that uses prokaryotic cell to carry out.

When using eukaryotic cell, can use for example zooblast, plant cell, fungal cell among the host.Known zooblast comprises: mammalian cell, for example CHO (J.Exp.Med. (1995) 108,945), COS, 3T3, myeloma cell, BHK (young hamster kidney), HeLa, Vero; Amphibian cell, for example Africa xenopus oocyte (Valle etc., Nature (1981) 291:338～340); Perhaps insect cell, for example Sf9, Sf21, Tn5.Be fit to use CHO-DG44, CHO-DX11B, COS7 cell, bhk cell among the present invention.In the zooblast, when being purpose with the great expression, preferred especially Chinese hamster ovary celI.About in host cell, importing carrier, for example can carry out by the following method: calcium phosphate method, DEAE-glucosan method, the method for using cationic-liposome DOTAP (Boehringer Mannheim society system), electroporation, lipofection etc.

Plant cell for example has the cell that derives from Nicotiana tabacum L., and it produces system and as everyone knows, can carry out callus culture to it as protein.The fungal cell is known to be had: yeast, and for example yeast (Saccharomyces) belongs to (for example beer yeast (Saccharomyces cerevisiae), schizosaccharomyces pombe (Saccharmyces pombe)); Filamentous bacteria, for example aspergillosis (Aspergillus) belongs to (for example aspergillus niger (Aspergillus niger)).

When using prokaryotic cell, the generation system that uses bacterial cell is arranged.The example of bacterial cell has escherichia coli (E.coli) for example JM109, DH5 α, HB101 etc., and known in addition have a bacillus subtilis.

In the method, next cultivate above-mentioned host cell.By obtaining antibody through the herbicide-tolerant polynucleotide cell transformed at In vitro culture.Can cultivate according to known method.For example can use DMEM, MEM, RPMI1640, IMDM culture fluid as zooblast.At this moment, FBS, the additional liquid of hyclone serum such as (FCS) can also be used in combination, also serum-free culture can be carried out.PH during cultivation is preferably about 6～8.Normally cultivated about 15～200 hours down, can exchange culture medium as required or ventilate, stir at about 30～40 ℃.

On the other hand, for example have as the system that produces polypeptide in vivo: use the generation system of animal or use the generation system of plant.In these animal or plants, import herbicide-tolerant polynucleotide, make and in the animal or plant body, produce polypeptide and recovery." host " of the present invention comprises above-mentioned animal and plant.

When using animal, the generation system that uses mammal and insecticide is arranged.Mammal can be used: goat, pig, sheep, mice, cattle (Vicki Glaser, SPECTRUM BiotechnologyApplications, 1993).When using mammal, can use transgenic animal.

For example, the preparation herbicide-tolerant polynucleotide, with its as with milk such as goat β-casein in the gene Fusion gene of coded polypeptide of intrinsic generation.Then, will comprise that the dna fragmentation of this fusion gene injects the goat embryo, this embryo transfer will be arrived in the arnee body.Accept embryo's goat output transgenic goat, from the milk of this transgenic goat or its offspring generation, can obtain target antibody.The milk amount that contains antibody that produces for the render transgenic goat increases, and can give the suitable hormone of above-mentioned transgenic goat (Ebert, K.M. etc., Bio/Technology (1994) 12:699-702).

As insecticide, for example can use silkworm.When using silkworm, be inserted with the baculovirus of the polynucleotide of coding target antibody, can from the body fluid of this silkworm, obtain target antibody (Susumu, M. etc., Nature (1985) 315:592～594) by making the silkworm infection.

And, when using plant, for example can use Nicotiana tabacum L. (tabacoo).When using Nicotiana tabacum L., the polynucleotide of coding target antibody are inserted into expression of plants for example among the pMON 530, this carrier are imported in the Agrobacterium tumdfaciens antibacterials such as (Agrobacterium tumefaciens) with carrier.With this bacterial infection Nicotiana tabacum L. Nicotiana tabacum L. (Nicotiana tabacum) for example, can from the leaf of this Nicotiana tabacum L., obtain required antibody (Julian K.-C.Ma etc., Eur.J.Immunol. (1994) 24:131～138).

So operation and the antibody that obtains, can with its in host cell or extracellular (culture medium etc.) separate, and purification becomes pure in fact and uniform antibody.The separation of antibody, purification can use employed separation in the purification of common polypeptide, purification process, but without any restriction.For example, can suitably select with make up chromatographic column, filter, ultrafiltration, saltout, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric point, IP electrophoresis method, dialysis, recrystallization wait and separate and purified polypeptide.

Chromatography for example has: (Strategies for Protein Purification andCharacterization (protein purification and identification experiment guide): A Laboratory CourseManual.Ed Daniel R.Marshark etc. such as affinity chromatography, ion-exchange chromatography, hydrophobic chromatography, gel filtration, reversed phase chromatography, adsorption chromatography, Cold Spring Harbor Laboratory Press, 1996).Above-mentioned chromatography can use liquid chromatography (LC), and for example HPLC, FPLC wait and carry out.The post that is used for affinity chromatography has: protein A post, Protein G post.The protein A post for example has: Hyper D, POROS, Sepharose F.F. (Pharmacia) etc.

Need to prove, can also before or after antibody purification, make suitable protein modified enzyme and antibody effect, at random remove peptide in modified antibodies or part ground by this processing.Protein modified enzyme for example uses: trypsin, Chymotrypsin, lysyl endopeptidase, protein kinase, glycosidase etc.

In addition, TPO receptor of the present invention (c-mpl) agonist can be a low molecular compound.As long as low molecular compound of the present invention has the effect that promotes hemopoietic stem cell proliferation, promote the propagation of hemopoietic system CD34 positive cell and/or the effect of differentiation, increase hemopoietic system CD34 positive cell in bone marrow the effect of giving birth to, or promote the effect of hematopoietic function recovery to get final product, it can be any low molecular compound, but optimal way is the chemical compound shown in the following chemical formula: { 5-[(2-{1-[5-(3, the 4-Dichlorobenzene base)-and 4-hydroxyl-3-thienyl] ethylidene } diazanyl) carbonyl-2-Thiophene Carboxylic Acid (Blood First Edition Paper, prepublished online on February 16,2006; DOI10.1182/blood-2005-11-4433).

The example of TPO receptor of the present invention (c-mpl) agonist also has: Amgen AMG-531 (the reorganization megalokaryocyte generates stimulatory protein(SP)), SB297115/Eltrombopag (Gsk ' the oral TPO-R agonist compound of s), peg-TPOmp, YM477, NIP004.Amgen AMG-531 be contain Fc domain and peptide receptors bind domain, molecular weight is 60, the protein of 000D has character (the Clinical Pharmacology ﹠amp of following table 1 record; Therapeutics.76 (6), 628,2004, Blood, the 104th volume, abstract #511,2004).

(table 1)

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·MW＝60,000D

The 4Mpl binding site

There is not the sequence homology with TPO

At expression in escherichia coli

---------------------------------------------

SB297115/Eltrombopag is the chemical compound shown in the following chemical formula, has the character (Blood, the 104th volume, abstract # 2909,2004) of following table 2 record.

(table 2)

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Binding site: hu c-mpl membrane spaning domain (His499, Thr496)

The species specificity height

No cross reaction: machin, cat, mice etc.

Cross reactivity: chimpanzee

External activity

Hu BM CD34 fractional analysis: EC50～100nM

T _1/2=12 hours

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Peg-TPOmp is the PEGization peptide of finding from phage display combined peptide library, by following chemical formulation.Character (Blood, the 106th volume, Number11, abstract # 1249,2005) with following table 4 record.

(table 3)

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The peptide of 14mer links together through Lys, and the two ends of 29mer peptide are by PEGization

There is not homology with hTPO

Cross reaction takes place with mice, rat, Canis familiaris L.

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The example of TPO receptor of the present invention (c-mpl) agonist also has YM477.The details of relevant YM477 is disclosed in Blood, the 106th volume, and number 11, and abstract #2298 is in 2005.

Can make preparation according to the antibody that method known in those skilled in the art will be discerned c-mpl.For example, can carry out parenteral with the form of injections such as the sterile solution of the pharmaceutically acceptable liquid beyond antibody and water or the water or suspension.Think by going up acceptable carrier or medium with for example pharmacology, appropriate combination such as aquesterilisa or normal saline, vegetable oil, emulsifying agent, suspending agent, surfactant, stabilizing agent, flavouring agent, excipient, solvent, antiseptic, binding agent are particularly arranged, be mixed and made into preparation according to the desired unit of the pharmacy of common approval rule consumption form.In above-mentioned preparation, the amount of effective ingredient is the amount of wishing to get the suitable capacity of required scope.

The aseptic composite that is used to inject can use solvents such as distilled water for injection, fills a prescription according to common preparation rule.

The example of aqueous solution for injection has: normal saline and contain isosmotic solution, for example D-sorbitol, D-mannose, D-mannitol, the sodium chloride of glucose or other adjuvant, and can be with suitable cosolvent, for example alcohol (ethanol is specifically arranged), polyhydric alcohol (for example propylene glycol, Polyethylene Glycol), nonionic surfactant (for example polyoxyethylene sorbitan monoleate (TM), HCO-50) are used in combination.

Oil-based liquid for example has Oleum sesami, soybean oil, and it can be used in combination with benzyl benzoate, benzyl alcohol as cosolvent.Oil-based liquid can also mix with buffer agent (for example phosphate buffer, sodium acetate buffer), analgesic (for example procaine hydrochloride), stabilizing agent (for example benzyl alcohol, phenol), antioxidant.Prepared injection is filled in the suitable ampoule usually.

Preferred parenteral during administration specifically has injection type, nose administration dosage form, through lung form of administration, percutaneous dosing dosage form etc.As the example of injection type, for example can carry out whole body or topical by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection etc.

In addition, can select suitable medication according to patient's age, symptom.As the dosage of the medical composition of the polynucleotide that contain antibody or encoding antibody, for example can 0.0001mg～1000mg/kg body weight/time scope in select.Perhaps, for example can in 0.001～100000mg/lkg body weight/people's scope, select dosage, but may not be limited to above-mentioned numerical value.Dosage, medication change according to patient's body weight, age or symptom etc., and those skilled in the art can suitably select.

Medicine-feeding period to medicine of the present invention is unqualified, for example can be when hope promotes the propagation of hematopoietic stem cell, wish to promote the propagation of hemopoietic system CD34 positive cell and/or differentiation phase, hope increase hemopoietic system CD34 positive cell in bone marrow when giving birth to or administration when wishing to recover hemopoietic function.Administration when medicine of the present invention for example can carry out hematopoietic stem cell transplantation the patient to hemopoietic hypofunction of marrow.When only giving medicine of the present invention, not only strengthen hematopoietic stem cell in bone marrow give birth to and also medullary cell and/or lymphocyte in bone marrow give birth to and also demonstrate the dosage dependence potential.

By giving the medicine of the present invention of higher dosage in during just transplanting certain that the back rises, can obtain the effect (with reference to embodiment 3) that promotes hematopoietic stem cell in bone marrow, deciding.Those skilled in the art can consider to give the patient's of medicine of the present invention symptom, age etc. suitably decision during the administration of just transplanting back medicine of the present invention and dosage.For example, giving during medicine of the present invention certain (medicine-feeding period) can be from transplanting the same day or second day administration more than 3 days, preferred 7 days～28 days or longer time, but is not limited to this.Dosage can be more than 10 times of TPO concentration in bone marrow transplantation patient's the inherent blood, preferred more than 5 times, the more preferably amount more than 2 times, but be not limited to this, can or be used to keep for more time the necessary amount of blood Chinese medicine concentration during giving necessarily after the transplanting.

In addition, the administration number of times of medicine of the present invention in hematopoietic stem cell transplantation is unqualified, can distinguish administration when hematopoietic stem cell transplantation and after the hematopoietic stem cell transplantation arbitrarily time.The medicine-feeding period of medicine of the present invention, dosage and administration number of times can suitably be judged according to the patient's who accepts hematopoietic stem cell transplantation symptom, for example can be above-mentioned medicine-feeding period and dosages.

Medicine of the present invention uses in hematopoietic stem cell transplantation.Medicine of the present invention also uses after hematopoietic stem cell transplantation.In the present invention, hematopoietic stem cell transplantation comprises that bone marrow transplantation, tip hemocytoblast are transplanted, Umbilical Cord Blood Transplantation, but is not limited to this.Use the special optimal way behaviour Umbilical Cord Blood Transplantation of the hematopoietic stem cell transplantation of medicine of the present invention.

Medicine-feeding part to medicine of the present invention is not particularly limited, and subcutaneous injection, intravenous injection, oral administration etc. are for example arranged.Preferred especially intravenous drip administration among the present invention.All right and the together administration of hematopoietic stem cell of medicine of the present invention.With hematopoietic stem cell together during administration, medicine of the present invention and hematopoietic stem cell can be simultaneously in the administrations of same position, also can be at different times respectively in the different parts administration.When the administration of same position, for example can be through intravenously administrable.In addition, medicine-feeding period can be selected period identical when giving medicine of the present invention separately.

On the other hand, medicine of the present invention and hematopoietic stem cell can give any earlier among both when the different times administration.Dosing interval to both is also unqualified.

Together during administration, hematopoietic stem cell can also can be provided (heteroplastic transplantation) by other people from I (autotransplantation) for medicine of the present invention and hematopoietic stem cell.Can according to those skilled in the art known method obtain hematopoietic stem cell, for example can obtain according to the method for putting down in writing in the following document.

Heike, T etc., Biochimica et Biophysica Acta 2002, the 1592nd volume, the 313rd～321 page of .Ex vivo expansion of hematopoietic stem cells by cytokines., Yvette van Hensbergen etc., Experimental Hematology 2006, the 34th volume, the 943rd～950 page of .Ex vivo culture of human CD34+ cord blood cell withthrombopoietin (TPO) accelerates platelet engraftment in a NOD/SCIDmouse model.

Among the present invention, to the disease of carrying out hematopoietic stem cell transplantation without any qualification, preferred acute myelogenous leukemia (AML), acute lymphatic leukemia (ALL), chronic lymphocytic leukemia (CML), myelodysplastic syndrome (MDS) or aplastic anemia (AA), malignant lymphoma, adult T cell leukemia.

The present invention is based on the inventor's etc. discovery, promptly contacts with TPO receptor (c-mpl) agonist by hematopoietic stem cell, and the cell-lymphocyte of directed differentiation and/or the number of medullary cell increase.Therefore, the present invention relates to contain the medicine of TPO receptor (c-mpl) agonist, increase the directed differentiation cell-lymphocyte of hematopoietic stem cell and/or the number of medullary cell by this agonist and contacting of hematopoietic stem cell as effective ingredient.The invention still further relates to and contain the medicine of TPO receptor (c-mpl) agonist, by this medicine and hematopoietic stem cell intravenous drip simultaneously administration are increased the directed differentiation cell-lymphocyte of hematopoietic stem cell and/or the number of medullary cell as effective ingredient.The explanation of relevant agonist, hematopoietic stem cell, lymphocyte, medullary cell, medicine-feeding period, dosage etc. is the same.

Need to prove that all look-ahead technique documents of quoting in this description are all included in this description as reference.

Embodiment

Below, further specify the present invention by embodiment, but the present invention is not limited to these embodiment.

[embodiment 1] TPO receptor stimulating agent human blood cell in the human hematopoietic system reconstruct mouse bone marrow cells Give birth to the influence of number

In order to study from the initial stage after the hematopoietic stem cell transplantation of human cord blood, sc (Fv) 2 antibody (hVB22 u2-wz4:sc (Fv) 2) that comprise the aminoacid sequence of SEQ ID NO:73 record the influence of giving birth to number to human blood cell in the human hematopoietic system reconstruct mouse bone marrow cells, experimentize by the following method.Need to prove that sc (Fv) 2 antibody that comprise the aminoacid sequence of SEQ ID NO:73 record can be prepared according to the method for WO2005/056604 record.

Method

Mice is used the male NOD.CB17-Prkdc＜scid in 6 ages in week of domestication 〉/J.Behind the X ray to mice total irradiation 3.0Gy, give anti-asialo-GM1 antibody 1 time from irradiation intraperitoneal on per 10 days of the same day.Shine back second day to every mice through 5 * 104 CD34 positive cells of tail vein transplantation from human cord blood.Transplant and played sc (Fv) 2 antibody that gave SEQ IDNO:73 record in continuous 10 days in back second day, carry out administration by administration 5 days, the mode of being interrupted 2 days afterwards.The administering mode of this antibody is a subcutaneous administration, dosage be morning 0.25mg/5ml/kg, evening 2mg/5ml/kg, dosing interval is 8 hours, every day 2 times, 3 weeks of administration (n=10) altogether.As solvent, carry out administration equally with the citrate buffer of the 20mmol/L that contains 0.02%Tween 80 with sc (Fv) 2 antibody of SEQID NO:73 record.Transplant the back and the 3rd week taked bone marrow, carry out FACS with EPIX XL and resolve.Use streaming counting device (Beckman) to measure the absolute number of people's cell.

Result, investigation

Parsing is present in the people's cell number in the Thigh bone of the mice left and right sides.In sc (Fv) the 2 antibody administration groups of SEQ ID NO:73 record, not only also significantly increased (Fig. 1) statistically as the people CD34 positive cell number of target but also CD45 positive cell number, CD41 positive cell number, CD19 positive cell number, CD33 positive cell number originally than the solvent group.The above results shows: sc (Fv) 2 antibody that give c-mpl agonist-SEQ ID NO:73 record, not only induce the Megakaryocytic specificity differentiation of people, also help to increase the survival number of hemopoietic system CD34 positive cell and the increase of each blood cell line cell number of appearance thereupon.

[embodiment 2] TPO receptor stimulating agent is to people CFU-Meg in the human hematopoietic system reconstruct mouse bone marrow cells The influence of colony number

In order to study after the hematopoietic stem cell transplantation from human cord blood, sc (Fv) 2 antibody of SEQ ID NO:73 record carry out following experiment to the influence of people CFU-Meg colony number.

Method

Mice is used the male NOD.CB17-Prkdc＜scid in 6 ages in week of domestication 〉/J.Behind the X ray to mice total irradiation 3.0Gy, give anti-asialo-GM1 antibody 1 time from irradiation intraperitoneal on per 10 days of the same day.Shine back second day to every mice through tail vein transplantation 5 * 10 ⁴Individual CD34 positive cell from human cord blood.Transplant to rise in back second day and gave SEQ IDNO:73 sc (Fv) 2 antibody of record in continuous 10 days, give above-mentioned antibody by administration 5 days, 2 days mode of interruption afterwards, administering mode is a subcutaneous administration, the dosage in morning is 0.25mg/5ml/kg, dosing interval is 8 hours, the dosage in evening is 2mg/5ml/kg, every day 2 times, 3 weeks of administration (n=10) altogether.As solvent, carry out administration equally with the citrate buffer of the 20mmol/L that contains 0.02%Tween 80 with sc (Fv) 2 antibody of SEQ ID NO:73 record.Transplant the back and the 3rd week taked medullary cell, use MegaCult-C (StemCell Technologies) to cultivate sc (Fv) 2 antibody 13 days of medullary cell contained in the every mice Thigh bone and SEQ ID NO:73 record simultaneously.By the number of counting CD41 male 50 cells under optical microscope/more than the colony, measure people CFU-Meg colony number.

Result, person examine

The result shows: organize with solvent and compare, the CFU-Meg colony number that exists in the bone marrow of sc (Fv) the 2 antibody administration groups of SEQ ID NO:73 record significantly increases (Fig. 2) statistically.

[embodiment 3] TPO receptor stimulating agent human blood cell in the human hematopoietic system reconstruct mouse bone marrow cells Give birth to the dose-dependent effect of number

In order to study after the hematopoietic stem cell transplantation from human cord blood, sc (Fv) 2 antibody of SEQ ID NO:73 record the dose-dependent effect of giving birth to number to human blood cell in the human hematopoietic system reconstruct mouse bone marrow cells, carry out following experiment.

Method

Mice is used the male NOD.CB17-Prkdc＜scid in 6 ages in week of domestication 〉/J.Behind the X ray to mice total irradiation 3.0Gy, the same day and 8 days give anti-asialo-GM1 antibody in the posterior peritoneum in irradiation.Shine back second day to every mice through tail vein transplantation 5 * 10 ⁴Individual CD34 positive cell from human cord blood.Transplant and played sc (Fv) 2 antibody (subcutaneous administration, respectively organize n=9) that gave SEQ ID NO:73 record in continuous 10 days by following 3 kinds of consumptions in back second day.

High dose administration group (early 0.25mg/kg, 8 hours, late 2mg/kg at interval, every day)

In dosed administration group (early 0.05mg/kg, 8 hours, late 0.2mg/kg at interval, every day)

Low dosage administration group (early 0.01mg/kg, 8 hours, late 0.02mg/kg at interval, every day)

In high dose, middle dosage, each administration group of low dosage, during the administration in the tip blood minimum drug level be made as 50ng/ml, 10ng/ml, 2ng/ml respectively.

As solvent, similarly carry out administration with sodium citrate/150mM NaCl buffer (pH6.5) of the 20mmoL/L that contains 0.02%Tween 80 with sc (Fv) 2 antibody of SEQ ID NO:73 record.Transplant the back and the 2nd week taked medullary cell, carry out FACS and resolve.Use streaming counting device (Beckman) to measure the absolute number of people's cell.

Result, investigation

Parsing is present in the people's cell number in the Thigh bone of the mice left and right sides.Find not only CD34 positive cell number, and CD45 positive cell number, CD41 positive cell number, CD19 positive cell number, CD33 positive cell number, CD38 positive cell number also has the tendency (Fig. 3) that dose dependent increases.The above results shows: give SEQ ID NO:73 sc (Fv) 2 antibody of record, may more directly bring into play drug effect.Also show and to control drug effect by changing dosage.

The inherent TPO concentration of mice is (literature value and indoor (in house) measured value) about about 1ng/ml, through about 5～10 times (indoor data of mice of x-ray bombardment rising.Known people's clinically TPO concentration rises to about 1～3ng/ml from about normal value 80pg/ml similarly).In the present embodiment, by sc (Fv) 2 antibody that the SEQ ID NO:73 that is higher than inherent TPO concentration puts down in writing, confirm the collaborative drug effect of dose dependent.

Need to prove that in the present embodiment, shown in the method part, administering mode is designed so that sc (Fv) 2 antibody concentration of the SEQ ID NO:73 record in the blood maintain floor level.

Industrial applicability

According to the present invention, provide novel hemopoietic stem cell proliferation promoter, hemopoietic system CD34 The propagation of positive cell and/or differentiation accelerator, hemopoietic system CD34 positive cell are in marrow Give birth to give birth to dose and hematopoietic function recovery promoter. Medicine of the present invention contains TPO acceptor (c-mpl) activator is as active ingredient.

Novel hemopoietic stem cell proliferation promoter of the present invention, hemopoietic system CD34 positive cell Propagation and/or differentiation accelerator, hemopoietic system CD34 positive cell in marrow give birth to Giving birth to dose and hematopoietic function recovery promoter is characterised in that: in HSCT It is only individually dosed after (particularly Umbilical Cord Blood Transplantation) that (not being used in combination G-CSF or promoting erythrocyte gives birth to Cheng Su) can take effect.

Said medicine provided by the present invention is in order to treat for example acute leukemia, chronic bone Myelogenous leukemia, myelodysplastic syndrome, acute lymphatic leukemia, adult T cell The disease of the suitable HSCTs such as leukaemia, alpastic anemia, malignant lymphoma And carry out HSCT (bone-marrow transplantation, peripheral blood stem cell transplantation, Umbilical Cord Blood Transplantation) The time, can be used for promoting candidate stem cell propagation, promote that the positive Hematopoietic Stem of hemopoietic system CD34 is thin Born of the same parents' propagation and/or differentiation, promotion hemopoietic system CD34 positive candidate stem cell is in marrow Give birth to or promote the recovery of hematopoiesis function.

Particularly in Umbilical Cord Blood Transplantation, there is the rear slow problem of platelet recovery of transplanting. Short When advancing to transplant candidate stem cell in marrow give birth to the macronucleus that this and c-mpl activator are intrinsic The Cell Differentiation proliferation activity is coordinated mutually, promotes hematoblastic recovery, and therefore medicine of the present invention is .

In addition, in HSCT in the past, use G-CSF, but the G-CSF existence Problem is only neutrophil cell to be worked. With respect to this, think that TPO is not only thin to macronucleus Born of the same parents are worked and also stem cell are worked, thereby more kinds of cell series are recovered. And, Expect very that also TPO and G-CSF or the EPO of now approval produce synergy. From above-mentioned sight Point considers that medicine of the present invention also is useful.

Sequence table

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Claims (23)

1. hemopoietic stem cell proliferation promoter, it contains TPO receptor (c-mpl) agonist as effective ingredient.

2. the propagation of hemopoietic system CD34 positive cell and/or differentiation accelerator, it contains TPO receptor (c-mpl) agonist as effective ingredient.

Hemopoietic system CD34 positive cell in bone marrow give birth to give birth to dose, it contains TPO receptor (c-mpl) agonist as effective ingredient.

4. hematopoietic function recovery promoter, it contains TPO receptor (c-mpl) agonist as effective ingredient.

5. each described medicine in the claim 1～4, this medicine uses in hematopoietic stem cell transplantation.

6. each described medicine in the claim 1～5, the administration after hematopoietic stem cell transplantation of this medicine.

7. claim 5 or 6 described medicines, wherein hematopoietic stem cell transplantation is selected from bone marrow transplantation, the tip hemocytoblast is transplanted or Umbilical Cord Blood Transplantation.

8. the described medicine of claim 7, wherein Umbilical Cord Blood Transplantation is meant that human cord blood transplants.

9. the described medicine of claim 8 is characterized in that: carry out multiple dosing.

10. the enhancer of proliferation of lymphocyte and/or medullary cell, it contains TPO receptor (c-mpl) agonist as effective ingredient.

11. the differentiation accelerator to lymphocyte and/or medullary cell differentiation wherein contains TPO receptor (c-mpl) agonist as effective ingredient.

12. promote the method for hemopoietic stem cell proliferation, this method comprises being tried the step that body gives TPO receptor (c-mpl) agonist.

13. promote the method for hemopoietic system CD34 positive cell propagation and/or differentiation, this method comprises being tried the step that body gives TPO receptor (c-mpl) agonist.

14. increase hemopoietic system CD34 positive cell in bone marrow the method for giving birth to, this method comprises being tried the step that body gives TPO receptor (c-mpl) agonist.

15. promote the method for hematopoietic function recovery, this method comprises being tried the step that body gives TPO receptor (c-mpl) agonist.

16. make the method for lymphocyte and/or proliferation of bone marrow cells, this method comprises being tried the step that body gives TPO receptor (c-mpl) agonist.

17. promote that this method comprises being tried the step that body gives TPO receptor (c-mpl) agonist to the method for lymphocyte and/or medullary cell differentiation.

18.TPO the application of receptor (c-mpl) agonist in preparation hemopoietic stem cell proliferation promoter.

19.TPO receptor (c-mpl) agonist is in the propagation of preparation hemopoietic system CD34 positive cell and/or the application in the differentiation accelerator.

20.TPO receptor (c-mpl) agonist preparation hemopoietic system CD34 positive cell in bone marrow give birth to give birth to application in the dose.

21.TPO the application of receptor (c-mpl) agonist in preparation hematopoietic function recovery promoter.

22.TPO the application of receptor (c-mpl) agonist in the enhancer of proliferation of preparation lymphocyte and/or medullary cell.

23.TPO receptor (c-mpl) agonist is in the application of preparation in the differentiation accelerator of lymphocyte and/or medullary cell differentiation.

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