WO2005107784A1 - Remedy for thrombopenia - Google Patents

Remedy for thrombopenia Download PDF

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Publication number
WO2005107784A1
WO2005107784A1 PCT/JP2005/008592 JP2005008592W WO2005107784A1 WO 2005107784 A1 WO2005107784 A1 WO 2005107784A1 JP 2005008592 W JP2005008592 W JP 2005008592W WO 2005107784 A1 WO2005107784 A1 WO 2005107784A1
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Prior art keywords
antibody
seq
mpl
hvb22b
vb22b
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PCT/JP2005/008592
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French (fr)
Japanese (ja)
Inventor
Masayuki Tsuchiya
Tetsuro Orita
Yoshihiro Matsumoto
Original Assignee
Chugai Seiyaku Kabushiki Kaisha
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Priority to JP2006513039A priority Critical patent/JPWO2005107784A1/en
Publication of WO2005107784A1 publication Critical patent/WO2005107784A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/74—Inducing cell proliferation

Definitions

  • the present invention relates to a therapeutic agent for thrombocytopenia, which comprises an antibody that recognizes Mpl as an active ingredient. Further, the present invention relates to a platelet increasing agent containing an antibody recognizing Mpl as an active ingredient.
  • Thrombopoietin is a molecule that promotes the growth and differentiation of megakaryocytic hematopoietic cells, and is a site that plays a major role in regulating the number of platelets. It was also called megakaryocyte colony-stimulating factor (CJ) or c-MPL ligand. Human TPO is excised as a TPO precursor having 353 amino acids and becomes an active form.
  • Mpl is a receptor for TPO and is listed on CD110 as a Cluster of Differentiation (CD) antigen.
  • CD110 Cluster of Differentiation
  • the gene sequence of human Mpl has already been analyzed (see Non-Patent Document 1 or Genebank: NM_005373), and a signal peptide is cut out from a precursor consisting of 635 amino acids and expressed. Also, truncated forms shorter than 635 amino acids are known.
  • the receptors are dimerized by the binding of a ligand, and the signal is transmitted into the cell. It has also been reported that TPO also binds to its specific receptor, MPL, and dimerizes the receptor, thereby transmitting information into cells and exhibiting physiological actions! Reference 2).
  • Non-Patent Document 4 and 5 an antibody that binds to Mpl and has TPO agonist activity has been reported. This suggests that dimerization of the receptor is induced by binding of an antibody that is bivalent with respect to MPL.
  • Patent Document 1 US Patent No. 6342220
  • Patent Document 2 WO 01/79494
  • Patent Document 3 WO 02/33072
  • Patent Document 4 WO 02/33073
  • Non-Patent Document l Palacios et al., Cell, 1985, Vol.41, p.727-734
  • Non-Patent Document 2 Souyri et al., Cell, 1990, Vol. 63, p. 1137-1147
  • Non-Patent Document 3 Elliott S et al., J. Biol. Chem., 1996, Vol.271 (40), p.24691-24697
  • Non-Patent Document 4 Abe et al., Immunol. Lett. 1998, Vol. 61, p.73-78
  • Non-Patent Document 5 Bijia Deng et al., Blood, 1998, Vol. 92, p. 1981-1988.
  • the present invention has been made in view of such circumstances, and it is an object of the present invention to provide a therapeutic agent for thrombocytopenia and a thrombocytosis agent containing an anti-Mpl antibody having TPO receptor agonist activity as an active ingredient.
  • the task is to
  • the present inventors have conducted intensive studies in order to solve the above problems.
  • the present inventors obtained and purified the anti-human Mpl antibody VB22B, and constructed a single-chain antibody expression system using genetic engineering techniques. Specifically, first, the variable region of the anti-human Mpl antibody was cloned to prepare an anti-human Mpl antibody Diabody expression vector pCXND3-VB22Bdb. Further, using the vector pCXND3-VB22B db, an anti-human Mpl antibody sc (Fv) 2 expression vector pCXND3-VB22B sc (Fv) 2 was prepared.
  • the expression vector pCXND3-VB22B sc (Fv) 2 was expressed in CHO-DG44 cells, and the anti-human Mpl sc (Fv) 2 was purified from the culture supernatant. Also, TPO of VB22B sc (Fv) 2 Evaluation of similar agonist activity revealed that VB22B sc (Fv) 2 showed high agonist activity against VB22B IgG, and showed an activity equal to or higher than that of human TPO, which is a natural ligand. Was.
  • the present inventors have succeeded in producing five types of humanized VB22B sc (Fv) 2. In addition, it was found that no change in TPO-like agonist activity was observed when the humans were ridden.
  • VB22B sc (Fv) 2 and humanized VB22B sc (Fv) 2 have a platelet increasing effect in vivo.
  • the present invention more specifically relates to the following [1] to [22].
  • a therapeutic agent for thrombocytopenia comprising sc (Fv) 2 having binding activity to a TPO receptor (Mpl) as an active ingredient.
  • a therapeutic agent for thrombocytopenia comprising an antibody that binds to human Mpl and monkey Mpl as an active ingredient.
  • a therapeutic agent for thrombocytopenia comprising an antibody having an agonistic activity against human Mpl and monkey Mpl as an active ingredient.
  • a therapeutic agent for thrombocytopenia comprising, as an active ingredient, an antibody containing a heavy chain variable region having CDR1, 2, or 3 having the amino acid sequence described in SEQ ID NO: 1, 2, or 3.
  • a therapeutic agent for thrombocytopenia comprising, as an active ingredient, an antibody containing a light chain variable region having CDR1, 2, or 3 having the amino acid sequence described in SEQ ID NO: 4, 5, or 6.
  • a therapeutic agent for thrombocytopenia comprising as an active ingredient the antibody having the heavy chain variable region according to [6] and the light chain variable region according to [7].
  • a therapeutic agent for thrombocytopenia comprising as an active ingredient an antibody having the same activity as the antibody according to any one of the above.
  • a therapeutic agent for thrombocytopenia comprising, as an active ingredient, an antibody that recognizes amino acids 26 to 274 of human Mpl.
  • a platelet-increasing agent containing sc (Fv) 2 having binding activity to a TPO receptor (Mpl) as an active ingredient containing sc (Fv) 2 having binding activity to a TPO receptor (Mpl) as an active ingredient.
  • a platelet-increasing agent comprising, as an active ingredient, an antibody having agonist activity against human Mpl and monkey Mpl.
  • a platelet-increasing agent comprising, as an active ingredient, an antibody containing a heavy-chain variable region having CDRs 1, 2, and 3, which also has the amino acid sequence described in SEQ ID NOs: 1, 2, and 3.
  • a platelet-increasing agent comprising, as an active ingredient, an antibody containing a light chain variable region having CDR1, 2, or 3 having the amino acid sequence described in SEQ ID NO: 4, 5, or 6.
  • a platelet-increasing agent comprising, as an active ingredient, an antibody having the heavy chain variable region according to [17] and the light chain variable region according to [18].
  • one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence according to any of (17) to (19), and
  • a platelet-increasing agent comprising, as an active ingredient, an antibody having the same activity as the above antibody.
  • a platelet-increasing agent comprising, as an active ingredient, an antibody that recognizes an epitope recognized by the antibody according to any one of [17] to [19].
  • a platelet-increasing agent comprising, as an active ingredient, an antibody that recognizes amino acids 26 to 274 of human Mpl.
  • FIG. 2 is a diagram showing a continuation of FIG.
  • FIG. 2 is a graph showing the number of peripheral platelets in normal cynomolgus monkeys after administration of mouse VB22B.
  • FIG. 3 is a graph showing the effect of mouse VB22B administration on thrombocytopenia induced by ACNU.
  • FIG. 4 is a view showing a process of preparing a main chain antibody sc (Fv) 2.
  • FIG. 5 is a graph showing the results of evaluating the binding activity of VB22B sc (Fv) 2 using an Mpl-expressing CHO cell line. VB22B sc (Fv) 2 purified product was used.
  • FIG. 6 is a graph showing the results of evaluating the agonist activity of VB22B antibody using BaF-human Mpl. ⁇
  • FIG. 7 is a graph showing the results of evaluating the agonist activity of VB22B antibody using BaF-monkey Mpl.
  • FIG. 8 is a graph showing the results of evaluating the agonist activity of VB22B antibody using M_07e.
  • Figure 9 shows the evaluation of TPO-like agonist activity in BaF3-human Mpl using mouse VB22B sc (Fv) 2 and hVB22B esc (Fv) 2, hVB22B g-esc (Fv) 2 It is a figure which shows the result.
  • the vertical axis indicates absorbance (450 mn / 655 nni), and the horizontal axis indicates concentration.
  • Figure 10 shows mouse VB22B sc (Fv) 2 and WB22B p-z sc (Fv) 2, hVB22B u2-wz
  • FIG. 4 shows the results of evaluating TPO-like agonist activity in BaF3-human Mpl using 4sc (Fv) 2.
  • the vertical axis shows absorbance (450 nm / 655 nm), and the horizontal axis shows concentration.
  • FIG. 11 shows the results of BaF using mouse VB22B sc (Fv) 2 and hVB22B q-wz5 sc (Fv) 2.
  • FIG. 4 shows the results of evaluating TPO-like agonist activity in 3-human Mpl.
  • the vertical axis indicates the absorbance (450 nm / 655 nm), and the horizontal axis indicates the concentration.
  • Fig. 12 is a graph showing the platelet-increasing effect of hVB22B u2-wz4 sc (FV) 2 and hVB22B q-wz5 sc (FV) 2 in a single injection of normotensive quizzes.
  • the present invention provides a therapeutic agent for thrombocytopenia and an agent for increasing thrombocytopenia, which contain an antibody that binds to a TPO receptor (Mpl) as an active ingredient.
  • Replacement form (Rule 26)
  • Antibodies of the present invention include low-molecular-weight antibodies, antibodies with modified amino acid sequences such as humanized antibodies and chimerized antibodies, and other molecules (for example, polymers such as polyethylene glycol). Any antibody such as a modified antibody bound thereto, an antibody having a modified sugar chain, and the like are included.
  • Thrombocytopenia is a disease in which the number of platelets in peripheral blood decreases.
  • causes of thrombocytopenia include decreased bone marrow megakaryocyte count, congenital, ineffective hematopoiesis, increased destruction, increased consumption, increased stored calories, and dilution. More specifically, malignant tumors, myelofibrosis, granulomas, drugs, radiation, viruses, alcohol, Fanconi anemia, autosomal recessive thrombocytopenia, thrombocytopenia with radial loss, megaloblastic anemia , Myelodysplastic syndrome, autosomal dominant blood, J, platelet depletion;!
  • the therapeutic agent for thrombocytopenia of the present invention can also be used for treating thrombocytopenia by administering interferon. You. Therefore, the combination therapy of the therapeutic agent for thrombocytopenia of the present invention and interferon is clinically useful.
  • the thrombocytopenia of the present invention is not limited to thrombocytopenia due to the above-mentioned causes, and may be thrombocytopenia due to any cause.
  • Thrombocytopenia is generally determined by the number of platelets in peripheral blood. For example, in Japan, usually, when the platelet count is 100,000 ZL or less, thrombocytopenia is determined. However, in the present invention, even when the number of platelets in the peripheral blood exceeds the criterion, if the number of platelets is determined to need to be increased, It can be said that there is thrombocytopenia.
  • a platelet-increasing agent is a drug used for the purpose of increasing platelets, and may be used when platelets are in a reduced state, or may be used when platelets are not in a reduced state.
  • an antibody that recognizes Mpl to store its own platelets before surgery, etc. The body may be administered to increase platelets, or an antibody recognizing Mpl may be administered after donating platelet components to shorten the time required for platelet counts to return to normal values. Therefore, platelet augmenting agents can be used with or without thrombocytopenia.
  • Mpl is a receptor for TPO, and the gene sequence of human Mpl has already been analyzed! (Palacios et al, Cell, 1985, Vol. 41, p. 727—734 or Genebank: NM— 005373).
  • Rinichi-kumo Mpl base Z SEQ ID NO: 67, amino acid Z SEQ ID NO: 68
  • mouse Mpl
  • the Mpl in the present invention also includes a mutant Mpl receptor in which amino acids are substituted, deleted, added, etc. in the above-mentioned Mpl.
  • Specific examples of the mutant Mpl include, for example, the mutant Mpl described in Matthias Ballmaier et al., BLOOD, (2001), Vol. 97, No. 1, P139, and the like.
  • the antibody that recognizes Mpl is not particularly limited, but is preferably an antibody having a platelet increasing effect.
  • An antibody having a platelet-increasing action usually has an agonistic activity against Mpl.
  • the effect of increasing platelets can be confirmed by a method known to those skilled in the art. For example, the effect can be confirmed by the method described in Examples.
  • the agonist activity against Mpl is an activity of promoting differentiation into megakaryocytes or hematopoietic stem cells, which are parent cells thereof, into platelets, or an activity of increasing platelets.
  • the measurement of the agonist activity can be performed by a method known to those skilled in the art.
  • the agonist activity can be measured not only by using the original activity as an indicator but also by using other activities as indicators.
  • the determination can be made by a method of measuring agonist activity using cell proliferation as an index as described in Examples. More specifically, an antibody whose agonist activity is to be measured is added to cells exhibiting agonist-dependent growth, and cultured. Then tetrazolium salt
  • WST-8 Dojini Dani Kagaku Kenkyusho
  • Cells exhibiting an agonist-dependent proliferation can also be prepared by a method known to those skilled in the art.
  • the antigen is a receptor that emits a cell proliferation signal
  • cells expressing the receptor may be used.
  • the antigen is a receptor that does not emit a cell proliferation signal
  • a chimeric receptor comprising an intracellular region of a receptor that emits a cell proliferation signal and an extracellular region of a receptor that does not emit a cell proliferation signal is used.
  • Examples of the receptor that emits a cell proliferation signal include, for example, G-CSF receptor, mpl, neu, GM-CSF receptor, EPO receptor, c-kit, FLT-3 and the like.
  • Examples of cells that express the receptor include BaF3, NFS60, FDCP-1, FDCP-2, CTLL-2, DA-1, KT-3 and the like.
  • any quantitative and Z- or qualitative change can be used as long as it can be measured.
  • an index of a cell-free system (cell free assay), an index of a cell system (ceU-based assay), an index of a ligamentous system, and an index of a biological system can be used.
  • an enzymatic reaction or a quantitative and Z- or qualitative change of protein, DNA, or RNA can be used.
  • an enzymatic reaction for example, an amino acid transfer reaction, a sugar transfer reaction, a dehydration reaction, a dehydrogenation reaction, a substrate cleavage reaction and the like can be used.
  • protein phosphorylation, dephosphorylation, dimerization, multimerization, degradation, dissociation, etc., and amplification, cleavage, and extension of DNA and RNA can be used.
  • phosphorylation of a protein existing downstream of the signal transduction pathway can be used as a detection index.
  • Changes in cell phenotype, such as quantitative and Z or qualitative changes in product, changes in proliferation activity, changes in cell number, changes in morphology, changes in characteristics, etc. should be used as indicators of cell lines.
  • the produced substance include secretory proteins, surface antigens, intracellular proteins, mRNA, and the like.
  • Changes in morphology include protrusion formation and changes in the number of Z or protrusions, changes in flatness, changes in elongation Z, changes in aspect ratio, changes in cell size, changes in internal structure, abnormalities in cell populations Z uniformity, changes in cell density, etc. can be used. These morphological changes can be confirmed by observation under a microscope.
  • As the change in properties scaffold dependency, site force-in response, hormone dependency, drug resistance, cell motility, cell migration activity, pulsatility, changes in intracellular substances, and the like can be used.
  • Cell motility includes cell infiltration activity and cell migration activity.
  • changes in intracellular substances include, for example, enzyme activity, mRNA level, intracellular signal transduction substances such as Ca2 + and cAMP, and intracellular protein levels.
  • a change in cell proliferation activity induced by receptor stimulation can be used as an index.
  • a change in function according to the organization to be used can be used as a detection index.
  • Indices of the biological system include changes in tissue weight, changes in the blood system, such as changes in the number of blood cells, changes in protein levels, enzyme activities, and changes in electrolyzed mass, and changes in the circulatory system, such as blood pressure and heart rate. Can be used.
  • Methods for measuring these detection indices include, but are not particularly limited to, absorption, luminescence, color development, fluorescence, radioactivity, fluorescence polarization, surface plasmon resonance signal, time-resolved fluorescence, mass, absorption spectrum, light Scattering, fluorescence resonance energy transfer, and the like can be used. These measurement methods are well known to those skilled in the art, and can be appropriately selected according to the purpose. For example, the absorption spectrum can be measured by a commonly used photometer or plate reader, the luminescence can be measured by a luminometer, and the fluorescence can be measured by a fluorimeter. Mass can be measured using a mass spectrometer.
  • the radioactivity is measured using a measuring device such as a gamma force center according to the type of radiation.
  • the degree of fluorescence polarization is BEACON (Takara Shuzo)
  • the surface plasmon resonance signal is BIACORE
  • the time-resolved fluorescence is ARVO, etc.
  • a flow cytometer or the like can be used for the measurement. In these measurement methods, it is sufficient to measure two or more types of detection indices by one measurement method.If it is convenient, two or more types of measurement can be performed simultaneously and Z or consecutively. It is also possible to measure an index. For example, fluorescence and fluorescence resonance energy transfer can be measured simultaneously with a fluorimeter.
  • a preferred embodiment of the present invention includes a low molecular weight antibody.
  • the low-molecular-weight antibody is not particularly limited as long as it includes an antibody fragment in which a part of a full-length antibody (eg, whole IgG, etc.) is deleted and has an antigen-binding ability.
  • the low-molecular-weight antibody in the present invention has a significantly higher activity than the whole antibody.
  • the antibody fragment of the present invention is not particularly limited as long as it is a part of a full-length antibody, but preferably contains a heavy chain variable region (VH) or Z and a light chain variable region (VL).
  • VH heavy chain variable region
  • VL light chain variable region
  • the amino acid sequence of VH or VL may have substitutions, deletions, additions, and Z or insertions.
  • VH and / or VL may be partially deleted as long as they have the ability to bind to the antigen.
  • the variable region is chimerized. It may be converted to Specific examples of the antibody fragment include, for example, Fab, Fab ', F (ab') 2, Fv and the like.
  • Specific examples of the low molecular weight antibody include, for example, Fab, Fab ', F (ab') 2, Fv, scFv, single chain Fv), Diabody, sc (Fv) 2 (.single cnain (Fv ) And the like.
  • the "Fv” fragment is the minimum antibody fragment and contains a complete antigen recognition site and a binding site.
  • An “Fv” fragment is a dimer in which one VH and VL are tightly linked by non-covalent bonds (VH-VL dimer).
  • the three complementarity determining regions (CDRs) of each variable region interact to form an antigen-binding site on the surface of the VH-VL dimer.
  • Six CDRs confer an antigen-binding site on the antibody.
  • a single variable region or half of an Fv that contains only three CDRs specific for an antigen
  • scFv includes VH and VL of an antibody, and these regions are present in a single polypeptide chain.
  • Fv polypeptides further include a polypeptide linker between the VH and VL, which allows the scFv to form the necessary structure for antigen binding (for a review of scFv, see Pluckthun et al. The Pharmacology of Monoclonal Antibomes JVol.l lj (see Rosenburg and Mooreed (Springer Verlag, New York) pp.269-315, 1994).
  • the linker in the present invention is not particularly limited as long as it does not inhibit the expression of the antibody variable region linked to both ends.
  • Diabody refers to a bivalent antibody fragment constructed by gene fusion (Holliger
  • Diabody is a dimer composed of two polypeptide chains, and each of the polypeptide chains is usually short enough to be unable to bind to the VL and VH forces in the same chain, for example, by a linker of about 5 residues. Are combined. Since VL and VH encoded on the same polypeptide chain cannot form a single-chain variable region fragment due to the short linker between them and form a dimer, Diabody has two antigen-binding sites. Yes.
  • sc (Fv) 2 can be mentioned as a particularly preferred and embodiment of the antibody recognizing Mpl contained in the therapeutic agent for thrombocytopenia or the agent for increasing thrombocytopenia.
  • sc (Fv) 2 is a low molecular weight antibody in which two VHs and two VLs are linked to each other with a linker or the like to form a single chain (Hudson et al., J Immunol. Methods 1999; 231: 177-189). .
  • the present inventors have found that sc (Fv) 2 exhibits particularly high agonist activity as compared to full-length antibodies and other low molecular weight antibodies.
  • sc (Fv) 2 can be produced, for example, by linking scFv with a linker.
  • Antibodies characterized by being arranged in the order of VL, VH, VL are preferred.
  • the order of the two VHs and the two VLs is not particularly limited to the above arrangement, and may be arranged in any order. For example, the following arrangement can also be mentioned.
  • any peptide linker or synthetic compound linker that can be introduced by genetic engineering see, for example, Protein Engineering, 9 (3), 299-305, 1996)
  • a peptide linker is preferable.
  • the length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art according to the purpose, but is usually 1 to 100 amino acids, preferably 3 to 50 amino acids, and more preferably 5 to 30 amino acids. Particularly preferably, it is 12 to 18 amino acids (for example, 15 amino acids).
  • n is an integer of 1 or more.
  • the length and sequence of the peptide linker can be appropriately selected by those skilled in the art according to the purpose.
  • sc (Fv) 2 in the present invention include, for example, the following:
  • Synthetic chemical compound linkers are commonly used crosslinking agents for peptide crosslinking, for example, N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis (Sulfosuccinimidyl) suberate (BS 3 ), dithiobis (succinimidyl propionate) (DSP), dithiopis (sulfosuccinimidyl propionate) (DTSSP), ethylene glycol Bis (succinimidyl succinate) (EGS), ethylene glycol bis (sulfosuccinimidyl succinate) (sulfo EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (Sulfo DST), bis [2- (succinimidoxylcarboxy) ethyl] sulfone (BSOCOES), bis [2- (sulfosuccinimidoxylcarboxy) ethy
  • the preferred low molecular weight antibody in the present invention is Diabody or sc (Fv) 2, and particularly preferably sc (Fv) 2.
  • an enzyme such as papain or pepsin to generate an antibody fragment, or a DNA encoding these antibody fragments is constructed, and this is used as an expression vector. After introduction, expression may be carried out in an appropriate host cell (for example, Co, MS et al, J. Immunol. (1994) 152, 2968-2976; Better, M.
  • the anti-Mpl antibody sc (Fv) 2 has particularly high agonist activity against Mpl, and is therefore particularly useful as a therapeutic agent for thrombocytopenia or an agent for increasing thrombocytopenia.
  • preferred examples of the antibody that recognizes Mpl contained in the therapeutic agent for thrombocytopenia or the agent for increasing thrombocytopenia include modified antibodies such as chimeric antibodies and humanized antibodies. And especially humanized antibodies.
  • a chimeric antibody is an antibody produced by combining sequences derived from different animals.
  • the chimeric antibody is composed of the variable regions of the heavy and light chains of a mouse antibody and the constant regions of the heavy and light chains of a human antibody.
  • a chimeric antibody can be prepared by a known method.For example, a DNA encoding the antibody V region and a DNA encoding the human antibody C region are ligated, inserted into an expression vector, and introduced into a host. And produced.
  • the humanized antibody is also referred to as a reshaped human antibody, and is used to determine the complementarity determining region (CDR) of a non-human mammal, for example, a mouse antibody. It has been transplanted into a region, and its general genetic recombination technique is also known (see European Patent Application Publication No. EP 125023, WO 96/02576).
  • a CDR of a mouse antibody and a framework region of a human antibody (framework region;
  • a DNA sequence designed to ligate to FR is synthesized by a PCR method using as primers several oligonucleotides prepared to have overlapping portions in both CDR and FR terminal regions (W098 / 13388).
  • the framework region of the human antibody linked via the CDR has a favorable complementarity determining region. Those that form an antigen-binding site are selected. If necessary, amino acids in the framework region in the variable region of the antibody may be substituted so that the complementarity determining region of the reshaped human antibody forms an appropriate antigen-binding site (Sato, K. et al., CancerRes. (1993) 53, 851-856).
  • constant regions of chimeric and humanized antibodies those of human antibodies are used.
  • C ⁇ 1, C ⁇ 2, C ⁇ 3, and C ⁇ 4 are used for the H chain, and C ⁇ , C ⁇ can be used.
  • the constant region of the human antibody may be modified to improve the stability of the antibody or its production.
  • a chimeric antibody is composed of a variable region of an antibody derived from a mammal other than human and a constant region derived from a human antibody.
  • a humanized antibody is composed of a complementarity determining region of an antibody derived from a mammal other than human, a framework region and a constant region derived from a human antibody.
  • variable region eg, FR
  • constant region amino acids in the variable region or the constant region may be substituted with another amino acid.
  • the origin of the variable region in the chimeric antibody or the CDR in the humanized antibody is not particularly limited, and may be from any animal.
  • a sequence of a mouse antibody, a rat antibody, a rabbit antibody, a ratada antibody and the like can be used.
  • humanized antibodies that recognize Mpl include the humanized antibodies described in (9)-(19) below.
  • chimeric antibodies and humanized antibodies have reduced antigenicity in the human body, they are particularly useful when administered to humans, and are useful as therapeutic agents for thrombocytopenia or platelet increasing agents.
  • preferred examples of the antibody that recognizes Mpl contained in the therapeutic agent for thrombocytopenia or the agent for increasing thrombocytopenia include an antibody that binds to soluble Mpl.
  • the soluble Mpl refers to an Mpl other than the Mpl expressed on the cell membrane.
  • Specific examples of soluble Mpl include Mpl in which a part or all of the transmembrane region is missing. I can make it.
  • the transmembrane region corresponds to the portion from amino acid 492 to amino acid 513 in SEQ ID NO: 66.
  • Antibodies that bind to soluble recombinant Mpl can be used not only for detailed analysis of epitopes and for reaction kinetic analysis of binding, but also for evaluation of blood concentrations and pharmacokinetics in in vivo tests. Useful.
  • an antibody recognizing Mpl contained in a therapeutic agent for thrombocytopenia or an agent for increasing thrombocytopenia is preferred.
  • An antibody having activity can be mentioned.
  • Antibodies that have agonist activity against both human Mpl and monkey Mpl are very useful because monkeys can verify the pharmacokinetics and in vivo effects that are usually difficult to measure in humans. It is believed that there is .
  • These antibodies may further have binding activity 'active agonist activity' against Mpl of animals other than humans and monkeys (eg, mice).
  • An antibody having an EC50 of 10 nM or less can be mentioned.
  • the method for measuring the agonist activity can be performed by a method known to those skilled in the art, for example, the method described below.
  • ELISA enzyme-linked immunosorbent assay
  • EIA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • a sample containing a test antibody for example, a culture supernatant of a test antibody-producing cell or a purified antibody is added to a plate coated with an antigen to which the test antibody binds.
  • a secondary antibody labeled with an enzyme such as alkaline phosphatase
  • an enzyme substrate such as P-nitrophenyl phosphate
  • the upper limit of the binding activity is not particularly limited.
  • the upper limit of the range that can be technically prepared by those skilled in the art can be set.
  • the range that can be produced technically is expanded by technological advances.
  • the antibody according to any one of the following (1) to (19) is used. Can be listed.
  • the antibody according to any one of (1) to (19) is preferably a low-molecular-weight antibody.
  • CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, 2, 3 (VB22B: VH CDR1, 2, 3)
  • An antibody comprising VH having 2,3.
  • CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, 5, 6 (VB22B: VL CDR1, 2, 3)
  • An antibody comprising VL, having 2, 3.
  • An antibody comprising a VH having CDRs 1, 2, and 3, and a VL having CDRs 1, 2, and 3 having the amino acid sequences described in SEQ ID NOs: 4, 5, and 6 (VB22B: VL CDR1, 2, and 3).
  • An antibody comprising a VL consisting of the amino acid sequence of SEQ ID NO: 10 (VB22B: VL).
  • VH having the amino acid sequence described in SEQ ID NO: 8 (VB22B: VH), and SEQ ID NO:
  • a humanized antibody comprising a VL having FR1, 2, 3, or 4, comprising the amino acid sequence of (a) or (b) below.
  • a humanized antibody comprising VH and VL described in any of the following (a) to (c):
  • VH having FR1, 2, 3, or 4 consisting of the amino acid sequence of SEQ ID NO: 15, 16, 17, or 18, and the amino acid of IJ No. 27, 28, 29 or 30 VL with FR1, 2, 3 and 4
  • VH having FR1, 2, 3, 4 and amino acid described in SEQ ID NO: 81, 82, 83, 84, and IJ number: 86, 87, 88, 89 VL with FR1, 2, 3 and 4
  • VH having FR1, 2, 3, 4 comprising the amino acid sequence of SEQ ID NO: 81, 82, 85, 84, and the amino acid of IJ No. 86, 90, 91, 89 VL with FR1, 2, 3 and 4
  • a humanized antibody comprising a VL having CDRs 1, 2, and 3 consisting of the amino acid sequences of SEQ ID NOs: 4, 5, and 6.
  • VH having CDR1, 2, or 3 having the amino acid sequence described in SEQ ID NO: 1, 2, or 3, and CDR1 having the amino acid sequence described in SEQ ID NO: 4, 5, or 6 A humanized antibody containing VL having 2,3.
  • SEQ ID NO: 51 (hVB22B p-z: VH), SEQ ID NO: 53 (hVB22B g-e: VH), SEQ ID NO: 55 (hVB22B e: VH), SEQ ID NO: 92 (hVB22B u2-wz4: VH) or a humanized antibody comprising VH consisting of the amino acid sequence of SEQ ID NO: 93 (hVB22B q-wz5: VH).
  • SEQ ID NO: 57 (hVB22B p-z: VL), SEQ ID NO: 59 (hVB22B g-e: VL or hVB22B e: VL), SEQ ID NO: 94 (hVB22B u2-wz4: VL), Alternatively, a human antibody containing VL which also has the amino acid sequence power described in SEQ ID NO: 95 (hVB22B q-wz5: VH).
  • a humanized antibody comprising VH and VL described in any of the following (a) to (c):
  • VH consisting of the amino acid sequence described in SEQ ID NO: 51 (hVB22B p-z: VH), and VL consisting of the amino acid sequence described in SEQ ID NO: 57 (hVB22B p-z: VL)
  • VH consisting of the amino acid sequence of SEQ ID NO: 53 (hVB22Bg-e: VH), and VL consisting of the amino acid sequence of SEQ ID NO: 59 (hVB22Bg-e: VL or hVB22Be: VL)
  • VH having the amino acid sequence described in SEQ ID NO: 55 (hVB22B e: VH), and VL consisting of the amino acid sequence described in SEQ ID NO: 59 (hVB22B g-e: VL or hVB22B e: VL)
  • VH consisting of the amino acid sequence described in SEQ ID NO: 92 (hVB22Bu2-wz4: VH), and VL consisting of the amino acid sequence described in SEQ ID NO: 94 (hVB22Bu2-wz4: VL)
  • VH consisting of the amino acid sequence described in SEQ ID NO: 93 (hVB22B q-wz5: VH), and VL consisting of the amino acid sequence described in SEQ ID NO: 95 (hVB22B q-wz5: VL)
  • SEQ ID NO: 51 (hVB22B p-z: VH), SEQ ID NO: 53 (hVB22B g-e: VH), SEQ ID NO: 55 (hVB22B e: VH), SEQ ID NO: 92 (hVB22B u2-wz4) : VH) or the amino acid sequence of SEQ ID NO: 93 (hVB22B q-wz5: VH),
  • Amino acid positions: 31 to 35 are CDR1,
  • Amino acid positions: 1 to 30 are FR1,
  • Amino acid positions: 108 to 118 correspond to FR4.
  • SEQ ID NO: 57 (hVB22B p-z: VL) or SEQ ID NO: 59 (hVB22B g-e: VL or hVB22B e: VL), SEQ ID NO: 94 (hVB22B u2-wz4: VL), or In the amino acid sequence of SEQ ID NO: 95 (hVB22B q-wz5: VH),
  • Amino acid positions: 1 to 23 are FR1,
  • Amino acid positions: 103 to 112 correspond to FR4.
  • hVB22B pz VL CDR1Z SEQ ID NO: 4
  • hVB22B pz VL FR2 / SEQ ID NO: 28
  • hVB22B u2-wz4 VL CDR2Z SEQ ID NO: 5 hVB22B u2-wz4 VL: FR3 / SEQ ID NO: 88
  • FIG. 1 shows the correspondence between CDRs and FRs in the hVB22B p-z sequence, hVB22B g-e sequence, hVB22B e sequence, hVB22B u2-wz4 sequence, and hVB22B q-wz5 sequence.
  • the nucleotide sequence of VB22B VH is SEQ ID NO: 7
  • the nucleotide sequence of VB22B VL is SEQ ID NO: 9
  • nucleotide sequence of hVB22B pz VH is SEQ ID NO: 50
  • nucleotide sequence of hVB22Bge VH is SEQ ID NO: 52
  • nucleotide sequence of hVB22B e VH is SEQ ID NO: 54
  • nucleotide sequence of hVB22B u2-wz4 VH is SEQ ID NO: No .: 96 and hVB22B
  • the base sequence of q-wz5 VH is shown in SEQ ID NO: 98, hVB22B.
  • the nucleotide sequence of pz VL is SEQ ID NO: 56, the nucleotide sequence of hVB22Bge VL, the nucleotide sequence of hVB22B e VL is SEQ ID NO: 58, the nucleotide sequence of hVB22B u2-wz4 VL is the nucleotide sequence of SEQ ID NO: 97 and the nucleotide sequence of hVB22B q-wz5 VL SEQ ID NO: 99, base sequence of hVB22B pz sc (Fv) 2 is SEQ ID NO: 60, base sequence of hVB22B ge sc (Fv) 2 is SEQ ID NO: 62, base sequence of hVB22B esc (Fv) 2 No .: 64, the nucleotide sequence of hVB22B u2-wz4 sc (Fv) 2 is described in SEQ ID NO: 102, and the nucleotide sequence of hVB
  • SEQ ID NO: 61 (hVB22B p-z: sc (Fv) 2), SEQ ID NO: 63 (hVB22B g-e: sc (Fv) 2), SEQ ID NO: 65 (hVB22B e: sc ( Fv) 2), having the amino acid sequence of any of SEQ ID NO: 100 (hVB22B u2-wz4: sc (Fv) 2) or SEQ ID NO: 101 (hVB22B q-wz5: sc (Fv) 2) Humanized antibodies.
  • the antibody according to any one of the above (1) to (18) has an extremely high agonist activity against Mpl !, and is therefore particularly useful as a therapeutic agent for thrombocytopenia or an agent for increasing thrombocytopenia.
  • Antibody can be prepared. Amino acid mutations can also occur in nature. Thus, an antibody having an amino acid sequence in which one or more amino acids are mutated in the amino acid sequence of the antibody of the present invention and functionally equivalent to the antibody is also included in the antibody of the present invention.
  • the number of amino acids to be mutated in such a mutant is usually within 50 amino acids, preferably It is exempted from being within 30 amino acids, more preferably within 10 amino acids (eg, within 5 amino acids).
  • the properties of amino acid side chains include hydrophobic amino acids (A, I, M, F, P, W, Y, V) and hydrophilic 'amino acids (R, D, N, C, E, Q, G , H, K, S, T), an amino acid having a fatty acid side chain (G, A, V, L, I, P), an amino acid having a hydroxyl-containing side chain (S, T, ⁇ ), a sulfur atom
  • Amino acids with side chains containing C, M
  • amino acids with side chains containing carboxylic acids and amides D, N, E, Q
  • amino acids with side chains containing bases R, K, ⁇
  • aromatic Examples include amino acids having side chains (H, F, Y, W) (all brackets represent one letter of amino acids).
  • Antibodies in which a plurality of amino acid residues are added to the amino acid sequence of the antibodies of the present invention include fusion proteins containing these antibodies.
  • the fusion protein is obtained by fusing these antibodies with another peptide or protein, and is included in the present invention.
  • a method for producing a fusion protein is as follows: a polynucleotide encoding the antibody of the present invention and a polynucleotide encoding another peptide or polypeptide are ligated in frame so that they are introduced into an expression vector; A method known to those skilled in the art can be used as long as the expression is carried out.
  • peptides or polypeptides to be fused with the antibody of the present invention include, for example, FLAG (Hopp, TP et al., BioTechnology (1988) 6, 1204-1210), 6 His (histidine) ) Residues consisting of 6 X His, 10 X His, influenza agglutinin (HA), human c-myc fragment, VSV-GP fragment, pl8HIV fragment, T7-tag, HSV-tag, E-tag, Use known peptides such as SV40T antigen fragment, lck tag, a-tubulin fragment, B-tag and Protein C fragment. Can.
  • FLAG Hopp, TP et al., BioTechnology (1988) 6, 1204-1210
  • 6 His histidine
  • Residues consisting of 6 X His, 10 X His, influenza agglutinin (HA), human c-myc fragment, VSV-GP fragment, pl8HIV fragment, T7-tag, HSV
  • polypeptides to be fused with the antibody of the present invention include, for example, GST (daltathione-S transferase), HA (influenza agglutinin), immunoglobulin constant region, ⁇ -galactosidase, ⁇ (maltose binding) Protein) and the like.
  • a fusion polypeptide is prepared by fusing a polynucleotide encoding the peptide or polypeptide that is sold with a polynucleotide encoding the antibody of the present invention, and expressing the fusion polynucleotide prepared thereby. can do.
  • the antibody of the present invention may differ in amino acid sequence, molecular weight, isoelectric point, presence / absence and form of sugar chain, etc., depending on the cell or host producing the antibody or the purification method described below. However, as long as the obtained antibody has a function equivalent to that of the antibody of the present invention, it is included in the present invention.
  • the antibody of the present invention when expressed in a prokaryotic cell, for example, Escherichia coli, a methionine residue is added to the ⁇ -terminal of the amino acid sequence of the original antibody.
  • the antibodies of the present invention also include such antibodies.
  • the preferred U of the antibody recognizing Mpl contained in the therapeutic agent for thrombocytopenia or the agent for increasing thrombocytopenia is, in particular, an epitope recognized by the above-mentioned antibody (1) to (18). Can be mentioned.
  • An antibody that recognizes an epitope that is recognized by an antibody can be obtained by a method known to those skilled in the art. For example, a method for preparing an antibody using the polypeptide having the amino acid sequence contained in the epitope as an immunogen or a method for preparing an antibody epitope by an ordinary method is used to determine an epitope that is recognized by the above-mentioned antibody. Once determined, the antibody and the epitope can be obtained by a method of selecting the same antibody.
  • an antibody that recognizes an epitope that is recognized by an antibody having the amino acid sequence of SEQ ID NO: 100 is particularly preferable.
  • the antibody having the amino acid sequence of SEQ ID NO: 100 is a region from the 26th Glu to the 274th Leu of human Mpl, preferably the 189th Ala to the 245th Gly region, more preferably the 213th Gly region. It is expected to recognize the region from Gin to the 231st Ala. Therefore, an antibody that recognizes the 26th to 274th, or the 189th to 245th, or the 213th to 231st region of human Mpl is also included in the present invention. [0099]
  • An antibody recognizing the 245th or 213st to 231rd region can be obtained by a method known to those skilled in the art, for example, the 26th to 26th amino acids of human Mpl (SEQ ID NO: 66).
  • the 274th, or 189th to 245th, there are! / Determine the method of preparing antibodies using the 213st to 231st peptides as immunogens, and determine the epitope recognized by the antibody prepared by the usual method. It can be obtained by a method such as selecting an antibody that recognizes the same epitope as the antibody of the present invention.
  • Antibodies that bind to Mpl can be prepared by methods known to those skilled in the art.
  • a monoclonal antibody-producing hybridoma can be basically produced using a known technique as follows. That is, Mpl protein or Mpl-expressing cells are used as a sensitizing antigen and immunized according to a usual immunization method, and the obtained immunocytes are fused with a known parent cell by a usual cell fusion method to obtain a normal immunization cell. It can be prepared by screening monoclonal antibody-producing cells by a screening method.
  • a monoclonal antibody may be prepared as follows.
  • Mpl protein used as a sensitizing antigen for obtaining antibodies
  • the purified Mpl protein is used as a sensitizing antigen.
  • a partial peptide of Mpl can be used as a sensitizing antigen.
  • the partial peptide can be obtained by chemical synthesis from the amino acid sequence of human Mpl.
  • the epitope on the Mpl molecule recognized by the anti-MPL antibody of the present invention is not limited to a particular one, and any epitope present on the Mpl molecule may be recognized. Therefore, any fragment can be used as an antigen for producing the anti-Mpl antibody of the present invention, as long as it is a fragment containing an epitope present on the Mpl molecule.
  • the mammal to be immunized with the sensitizing antigen is not particularly limited. Generally, it is generally preferable to select a mammal in consideration of compatibility with the parent cell used for cell fusion. Bald Dental animals, for example, mice, rats, wild, musters, or egrets, monkeys, and the like are used.
  • Immunization of an animal with a sensitizing antigen is performed according to a known method.
  • the sensitizing antigen is injected intraperitoneally or subcutaneously into a mammal.
  • the sensitizing antigen is diluted and suspended in an appropriate amount with PBS (Phosphate-Buffered Saline) or physiological saline, and then mixed with an appropriate amount of a normal adjuvant, for example, Freund's complete adjuvant, if necessary, and emulsified.
  • PBS Phosphate-Buffered Saline
  • physiological saline physiological saline
  • an appropriate amount of a normal adjuvant for example, Freund's complete adjuvant, if necessary, and emulsified.
  • a suitable carrier can be used during immunization of the sensitizing antigen.
  • the mammal After immunizing a mammal in this manner and confirming that the desired antibody level is increased in the serum, the mammal is also harvested for immune cells and subjected to cell fusion. Preferred immune cells are preferred. And especially splenocytes.
  • Mammalian myeloma cells are used as the other parent cells to be fused with the immune cells.
  • the myeloma cells are known cell lines, for example, P3 (P3x63Ag8.653) (J. Immnol. (1979) 123, 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (Kohler. G. and Milstein, C. Eur. J.
  • the cell fusion between the immune cells and myeloma cells is basically performed by a known method, for example, the method of Kohler and Milstein, et al. (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46) can be performed.
  • the cell fusion is performed, for example, in a normal nutrient medium in the presence of a cell fusion promoter.
  • a cell fusion promoter for example, polyethylene glycol (PEG), Sendai virus (HVJ) and the like are used, and if necessary, an auxiliary agent such as dimethyl sulfoxide can be added to enhance the fusion efficiency.
  • the ratio of the use of the immune cells to the myeloma cells can be arbitrarily set. For example, Mi It is preferred that the number of immune cells be 1 to 10 times that of the emoma cells.
  • the culture solution used for the cell fusion for example, RPMI1640 culture solution, MEM culture solution, and other ordinary culture solutions used for this type of cell culture suitable for the growth of the myeloma cell line can be used.
  • a serum replacement solution such as fetal calf serum (FCS) can be used in combination.
  • a predetermined amount of the immune cells and the myeloma cells are mixed well in the culture solution, and the mixture is passed through a PEG solution (for example, an average molecular weight of about 1000 to 6000) which has been heated to about 37 ° C in advance.
  • the desired fusion cells are formed by adding and mixing always at a concentration of 30-60% (w / v). Subsequently, an appropriate culture solution is successively added, and the operation of removing the supernatant by centrifugation is repeated to remove a cell fusion agent or the like that is unfavorable for the growth of the hybridoma.
  • the hybridoma obtained in this manner is selected by culturing it in a normal selective culture medium, for example, a HAT culture medium (a culture medium containing hypoxanthine, aminopterin and thymidine).
  • a HAT culture medium a culture medium containing hypoxanthine, aminopterin and thymidine.
  • the culturing in the HAT culture solution is continued for a time (usually several days to several weeks) sufficient for killing cells (unfused cells) other than the target hybridoma.
  • a conventional limiting dilution method is performed, and screening and single cloning of hybridomas producing the desired antibody are performed.
  • human lymphocytes are sensitized to Mpl in vitro, and the sensitized lymphocytes are derived from a human-derived myeloma cell capable of permanent division. And a desired human antibody having a binding activity to Mpl can be obtained (see Japanese Patent Publication No. 1-59878).
  • transgenic animals having the entire repertoire of human antibody genes were administered with Mpl as an antigen to obtain anti-Mpl antibody-producing cells. Good (International Patent Application Publication No. WO 94/25585, WO 93/12227, WO 92/03918, WO
  • the thus-produced monoclonal antibody producing hybridomas can be subcultured in a normal culture solution, and can be stored for a long time in liquid nitrogen. .
  • the hybridoma is According to a usual method, a method of culturing and obtaining a culture supernatant thereof, or a method of administering a hybridoma to a mammal which is compatible with the hybridoma and proliferating the same to obtain as ascites fluid, and the like are employed.
  • the former method is suitable for obtaining high-purity antibodies, while the latter method is suitable for mass production of antibodies.
  • mRNA encoding the variable (V) region of the anti-Mpl antibody is isolated from the hybridoma producing the anti-Mpl antibody.
  • mRNA can be isolated by known methods, for example, guadin ultracentrifugation (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299), AGPC method (Chomczynski, P. et al., Anal. Prepare the total RNA by using Biochem. (1987) 162, 156-159) or the like, and prepare the target mRNA using the mRNA Purification Kit (Pharmacia). Alternatively, mRNA can be directly prepared by using the QuickPrep mRNA Purification Kit (Pharmacia).
  • cDNA of the antibody V region is synthesized using reverse transcriptase.
  • the cDNA is synthesized using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (manufactured by Iidaigaku Kogyo).
  • AMV Reverse Transcriptase First-strand cDNA Synthesis Kit manufactured by Iidaigaku Kogyo.
  • a 5'-1 ⁇ 5 method using 5'-Ampli FINDER RACE Kit manufactured by Clontech
  • PCR 1 "01 ⁇ 1 & MA et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002, Belyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932) and the like can be used.
  • a target DNA fragment is purified from the obtained PCR product and ligated to a vector DNA. Further, a recombinant vector is prepared from this, introduced into E. coli, etc., and colonies are selected to prepare a desired recombinant vector. Then, the base sequence of the target DNA is confirmed by a known method, for example, a dideoxynucleotide chain termination method.
  • an antibody gene is usually expressed in an expression control region. Region, for example, an enhancer or a promoter.
  • host cells are transformed with this expression vector to express the antibody.
  • Antibody gene expression can be accomplished by co-transforming host cells by incorporating polynucleotides encoding the H or L chains separately into expression vectors, or by encoding the H and L chains.
  • the host cell may be transformed by incorporating the polynucleotide to be expressed into a single expression vector (see WO 94/11523).
  • Escherichia coli when Escherichia coli is used as a host, the vector is amplified in Escherichia coli (E. coli, for example, JM109, DH5a, HB101, XLlBlue), etc.
  • Escherichia coli for example, JM109, DH5a, HB101, XLlBlue
  • the restriction is not particularly limited as long as it has an "ori" for selection and a transformed gene of Escherichia coli (for example, a drug resistance gene that can be distinguished by any drug (ampicillin, tetracycline, kanamycin, chloramphenicol)). Absent.
  • the beta include M13-based vectors, pUC-based vectors, pBR322, pBluescript, pCR-Script, and the like.
  • the [0122] expression vector for example, if the purpose of expression in E. coli should have the above characteristics as the vector is amplified in E. coli, a host of JM109, DH5 a, HB101, XLl -Blue , etc.
  • a promoter that can be efficiently expressed in Escherichia coli such as the lacZ promoter (Ward et al., Nature (1989) 341, 544-546;
  • Such vectors include PGEX-5X-1 (Pharmacia), “QlAexpress system” (Qiagen), pEGFP, or pET (in this case, the host expresses T7 RNA polymerase in addition to the above vectors).
  • BL21 is preferred).
  • the vector contains a signal sequence for polypeptide secretion!
  • a signal sequence for protein secretion a pelB signal sequence (Lei, SP et al J. Bacteriol. (1987) 169, 4379) may be used for production in the periplasm of Escherichia coli.
  • the introduction of the vector into the host cell can be carried out, for example, by the calcium chloride method, It can be carried out by using the shion method.
  • mammalian-derived expression vectors for example, pcDNA3 (manufactured by Invitrogen), pEGF-BOS (Nucleic Acids. Res.
  • Insect cell-derived expression vectors eg, “Bac-to-BAC baculovairus expression system” (manufactured by Gibco BRL), pBacPAK8), plant-derived expression vectors (eg, ⁇ 1, pMH2), animal virus-derived expression vectors (for example, pHSV, pMV, pAdexLcw), retrovirus-derived expression vector (for example, pZIPneo), yeast-derived expression vector (for example, “Pichia Expression Kit” (manufactured by Invitrogen), pNVll, SP-Q01), Bacillus subtilis derived expression vector (e.g., P PL608, pKTH50) can be mentioned.
  • P PL608, pKTH50 Bacillus subtilis derived expression vector
  • promoters required for expression in cells such as the SV40 promoter (Muligan et al., Nature (1979) 277, 108), the MMTV-LTR promoter, the EF1 ⁇ promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), and the CMV promoter are essential, in order to select for transformation into cells.
  • SV40 promoter Muligan et al., Nature (1979) 277, 108
  • the MMTV-LTR promoter the EF1 ⁇ promoter
  • the CMV promoter are essential, in order to select for transformation into cells.
  • a drug resistance gene that can be distinguished by a drug neomycin, G418, etc.
  • Vectors having such properties include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, pOP13, and the like.
  • a DHFR gene that complements the gene in a CHO cell deficient in a nucleic acid synthesis pathway is used.
  • MTX methotrexate
  • a method of using a COS cell having a gene expressing the SV40 T antigen on the chromosome and transforming it with a vector having a replication origin of SV40 is used.
  • a replication origin of SV40 such as pcD
  • those derived from poliovirus, adenovirus, ⁇ papilloma virus (BPV) and the like can also be used.
  • the expression vector is selected as a selection marker for aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, Escherichia coli xanthinguanine phosphoribosyltransferase (Ecogpt) gene, It can contain the folate reductase (dhfr) gene and the like.
  • APH aminoglycoside transferase
  • TK thymidine kinase
  • Escherichia coli xanthinguanine phosphoribosyltransferase Escherichia coli xanthinguanine phosphoribosyltransferase
  • the host cell into which the vector is introduced is not particularly limited.
  • Escherichia coli and various animal cells can be used.
  • the host cell can be used, for example, as a
  • animal cells for example, animal cells, plant cells, and fungal cells can be used as hosts.
  • Animal cells include mammalian cells, for example, CH0 (J. Exp. Med. (1995) 108, 945), COS-3T3, myeloma, BHK (baby hamster kidney), HeLa, Vero, amphibian cells, for example, African Megafrog oocytes (Valle, et al., Nature (1981) 291, 358-340) or insect cells such as S19, Sf21, and Tn5 are known.
  • CH0 J. Exp. Med. (1995) 108, 945
  • myeloma myeloma
  • BHK baby hamster kidney
  • HeLa human hamster kidney
  • Vero amphibian cells
  • African Megafrog oocytes valle, et al., Nature (1981) 291, 358-340
  • insect cells such as S19, Sf21, and Tn5 are known
  • CHO cells are particularly preferred for the purpose of large-scale expression.
  • the vector can be introduced into a host cell by, for example, a calcium phosphate method, a DEAE dextran method, a method using a force ribosome DOTAP (manufactured by Boehringer Mannheim), an electoral poration method, or a lipofection method. .
  • Nicotiana tabacum As plant cells, for example, cells derived from Nicotiana tabacum (Nicotiana tabacum) are known as a protein production system, which may be callus cultured.
  • Fungal cells include yeast, for example, the genus Saccharomyces, for example, Saccharomyces cerevisiae, Saccharo; Saccharomyces pombe, filamentous fungi, for example, Aspergillus, for example, Aspergillus nigerj is known.
  • prokaryotic cells When prokaryotic cells are used, there is a production system using bacterial cells.
  • bacterial cells include Escherichia coli (E. coli), for example, JM109, DH5a, HB101, and the like, and Bacillus subtilis.
  • the above host cells are then cultured.
  • An antibody can be obtained by culturing cells transformed with the target polynucleotide in vitro.
  • Culture can be performed according to a known method. For example, as a culture solution of animal cells, for example, DMEM, MEM, RPMI1640, IMDM can be used. At that time, FBS, fetal bovine blood A serum replacement solution such as CIS (FCS) may be used in combination, or serum-free culture may be performed.
  • the pH during culturing is preferably about 6-8. Culture is usually performed at about 30 to 40 ° C for about 15 to 200 hours, and the medium is replaced, aerated, and agitated as necessary.
  • examples of a system for producing a polypeptide in vivo include a production system using animals and a production system using plants.
  • a polynucleotide of interest is introduced into these animals or plants, and the polypeptide is produced in the body of the animals or plants and collected.
  • the “host” in the present invention includes these animals and plants.
  • a polynucleotide of interest is prepared as a fusion gene with a gene encoding a polypeptide uniquely produced in milk, such as goat j8 casein. Then, a DNA fragment containing the fusion gene is injected into a goat embryo, and the embryo is transplanted into a female goat. Goats that have received the embryo Transgenic goats born or milk offspring produced by their progeny, and the desired antibody can be obtained. Transgeneic mosquitoes In order to increase the amount of milk containing antibodies produced, hormones may be used in transgenics as appropriate (Ebert, KM et al., Bio / Technology (1994). ) 12, 699-702).
  • silkworms can be used as insects.
  • the desired antibody can be obtained from the body fluid of the silkworm by infecting the silkworm with a baculovirus into which a polynucleotide encoding the antibody of interest has been inserted (Susumu, M. et al., Nature ( 1985) 315, 592-594).
  • a plant for example, tobacco can be used.
  • a polynucleotide encoding the antibody of interest is inserted into a plant expression vector, for example, pMON530, and this vector is inserted into an Agrobacterium tmmefaciens (
  • the bacteria are infected with tobacco, for example, Nicotiana tabacum, and the desired antibody can be obtained from the leaves of this tobacco (Julian K.-C. Ma et al., Eur. J. Immunol. (1994) 24, 131-138).
  • the antibody thus obtained can be isolated intracellularly or extracellularly (such as in a medium) and purified as a substantially pure and homogeneous antibody.
  • the separation and purification of the antibody is not limited at all, provided that the separation and purification methods used in ordinary purification of polypeptides are used. For example, select chromatography column, filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. If combined, the polypeptide can be separated and purified.
  • chromatography examples include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography, and the like (Strategies for Protein Purification and
  • an appropriate protein modification enzyme is allowed to act on the antibody to optionally modify the peptide or partially remove the peptide.
  • the protein modifying enzyme for example, trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, dalcosidase and the like are used.
  • the therapeutic agent for thrombocytopenia of the present invention may be administered after the onset of thrombocytopenia, or may be administered for a prophylactic purpose before the onset of thrombocytopenia.
  • the administration time of the platelet-increasing agent of the present invention is not limited, and it can be administered when it is necessary to increase platelets.
  • Antibodies that recognize Mpl can be formulated by methods known to those skilled in the art. For example, it can be used parenterally in the form of an injectable sterile solution with water or other pharmaceutically acceptable liquid, or a suspension.
  • a pharmacologically acceptable carrier or medium Body specifically, sterile water or physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, etc. It may be formulated by mixing in the unit dosage form required for accepted pharmaceutical practice. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.
  • a sterile composition for injection can be formulated using a vehicle such as distilled water for injection according to normal pharmaceutical practice.
  • Aqueous injection solutions include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mantol, and sodium chloride. It may be used in combination with an agent such as an alcohol, specifically ethanol, a polyalcohol such as propylene glycol, polyethylene glycol, a nonionic surfactant such as polysorbate 80 (TM) or HCO-50.
  • an agent such as an alcohol, specifically ethanol, a polyalcohol such as propylene glycol, polyethylene glycol, a nonionic surfactant such as polysorbate 80 (TM) or HCO-50.
  • oily liquid examples include sesame oil and soybean oil, and may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent. It may also be combined with a buffer such as a phosphate buffer and a sodium acetate buffer, a soothing agent such as proforce hydrochloride, and a stabilizer such as benzyl alcohol, phenol and an antioxidant.
  • a buffer such as a phosphate buffer and a sodium acetate buffer
  • a soothing agent such as proforce hydrochloride
  • a stabilizer such as benzyl alcohol, phenol and an antioxidant.
  • the prepared injection solution is usually filled in an appropriate ampoule.
  • Administration is preferably parenteral administration, and specific examples include injection, nasal, pulmonary, and transdermal administrations.
  • injection form include systemic or local administration by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection and the like.
  • the administration method can be appropriately selected depending on the age and symptoms of the patient.
  • the dose of the pharmaceutical composition containing the antibody or the polynucleotide encoding the antibody can be selected, for example, from O.OOOOmg to 100000 mg / kg body weight at a time. Or, for example, the ability to select a dose in the range of 0.001 to 100,000 mg / body per patient. These values are not necessarily limited.
  • the dose and administration method vary depending on the patient's body weight, age, symptoms, and the like, but can be appropriately selected by those skilled in the art. All prior art documents cited in this specification are incorporated herein by reference.
  • the platelet count was measured as follows.
  • test substance VB22B sc (Fv) 2 (SEQ ID NO: 14) was used as a solvent in 20 mmol / L Acetate buffer (pH 6.0) (150 mmol / L NaCl, 0.01% Tween 80) as a solvent.
  • Monkeys in the head were injected subcutaneously at 100 ⁇ g / kg body weight for 14 days (Dayl-14).
  • three monkeys in the control vehicle-administered group were injected subcutaneously with the solvent for 14 days so that the volume became the same as that of the VB22B-administered group.
  • VB22B sc (Fv) 2 or a solvent at intervals of 3 to 4 days during the experiment 1 mL of blood was collected from the saphenous vein and the platelet count was measured. If the platelet count exceeded the measurement limit, the blood was diluted appropriately and remeasured.
  • VB22B sc (Fv) 2 SEQ ID NO: 14
  • the test substance VB22B sc (Fv) 2 was used as a solvent in 20 mmol / L Acetate buffer (pH 6.0) (150 mmol / L NaCl, 0.01% Tween 80) to 14 days for 100 ⁇ g / kg body weight for monkey ( Dayl-14) Subcutaneous injection.
  • control vehicle-administered monkeys were injected subcutaneously with the solvent for 14 days so that the volume would be the same as that of the VB22B-administered group.
  • VB22B sc (Fv) 2 or a solvent at an interval of 3 to 4 days during the experiment 1 mL of blood was collected from the saphenous vein and the platelet count was measured.
  • peripheral blood platelets The measurement of the number of peripheral blood platelets was performed as follows. 0.5 mL of peripheral blood was collected from the saphenous vein of a monkey, immediately placed in MicroTina (Nippon Betaton 'Dickinson 365973), a blood collection tube coated with EDTA, and mixed with a rolling mixer (Sysmettas, Model RM-810). To prevent coagulation. Using the anticoagulated blood, the platelet count was measured with an automatic blood cell analyzer F-820 (Cismettas).
  • hVB22B u2-wz4 sc (FV) 2 (SEQ ID NO: 100) and hVB22B q-wz5 sc (FV) 2 (SEQ ID NO: 101) are 20 mmol / L sodium citrate buffer solution, 300 mmol / L NaCl, pH
  • iv intravenous injection was performed on Day 1 using 7.5, 0.01% Tween 80 as solvent. Thereafter, on Day 8, 11, 15, and 21, the platelet count was measured by the above method.
  • SMART RACE cDNA was extracted from total RNA extracted from force-monkey bone marrow cells.
  • force-quiz Mpl cDNA (SEQ ID NO: 67) was cloned. Insert the resulting force-quiz cDNA into pCOS2,
  • mouse Mpl cDNA (GenBank # NM_010823) was amplified by PCR and inserted into pCOS2 to construct pCOS2-mouseMplfoll.
  • Each vector was prepared (20 mu g) was mixed with BaF3 cells suspended in PBS (lxl0 7 cells / mL) , was added to the Gene Pulser cuvette, using a Gene Pulser II (Bio- manufactured Rad) Pulses were generated at a capacity of 0.33 kV, 950 ⁇ FD.
  • BaF3 cells transfected by electoral poration were transfected with Ing / mL mouse interleukin 3 (hereinafter, mIL-3, manufactured by Peprotech), 500 g / mL Geneticin (manufactured by Invitrogen), and 10% FBS (manufactured by Invitrogen).
  • the cells were selected and selected for human Mpl-expressing BaF3 cell line (hereinafter, BaF3-human Mpl), monkey Mpl-expressing BaF3 cell line (hereinafter, BaF3-monkey Mpl) and mouse Mpl-expressing BaF3 cell A strain (hereinafter, BaF3-mouse Mpl) was established. After the selection, the cells were cultured and maintained using RPMI1640 medium containing Ing / mL rhTPO (R & D) and 10% FBS.
  • RPMI1640 medium manufactured by Invitrogen
  • a CHO cell line expressing the full-length Mpl gene was established.
  • pCHOI was inserted into the Hindlll site of pCXN2 (Niwa et al., Gene 1991; 108: 193-199).
  • An expression vector pCXND3 was prepared by inserting a DHFR gene expression site.
  • pCOS2-hMpliulU pCOS2- monkeyMplfoll and pCOS2- mouseMplfoll in the ⁇ the claw each Mpl gene was amplified by PCR using primers with a His-tag sequence into pCXND3 - Ngushi, p CXND3-hMpl-His, pCXND3- monkey Mp His and
  • Each vector was prepared (25 mu g) were mixed in suspended CHO- DG44 cells (lxl0 7 cells / mL) into PBS, added to the Gene Pulser cuvette, the Gene Pulser II (Bio- manufactured Rad) Pulses were obtained at a capacity of 1.5 kV and 25 ⁇ FD.
  • CHO cells transfected by electoral poration were added to a CHO-S-SFMII medium (Invitrogen) containing 500 ⁇ g / mL Geneticin and lxHT (Invitrogen) and selected, and a human Mpl-expressing CHO cell line was selected.
  • CHO-human Mpl monkey Mpl-expressing CHO cell line
  • CHO-mouse Mpl mouse Mpl-expressing CHO cell line
  • a FLAG-tagged gene was prepared downstream of the extracellular region of human Mpl (Gln26 force Trp491), inserted into the Pstl-Smal site of pBACSurf-1 Transfer Plasmid (Novagen), and pBACSurfl-hMp FLAG was prepared. Subsequently, 4 g of pBACSurfl-hMp-to-FLAG was introduced into S19 cells using the Bac-N-Blue Transfection Kit (Invitrogen). After 3 days of culture, the culture supernatant was collected, and the recombinant virus was isolated by plaque assay. After preparing the virus stock, S19 cells were infected and the culture supernatant was collected.
  • a soluble human Mpl protein was purified as follows. After adsorbing the culture supernatant to Q Sepharose Fast Flow (manufactured by Amersham Biosciences), the culture supernatant was eluted using 50 mM Na-Phosphate Buffer, 0.01% (v / v) Tween 20, 500 mM NaCl (pH 7.2). After adsorbing the eluate to FLAG M2-Agarose (manufactured by SIGMA-ALDRICH), the eluate was eluted with lOOmM Glycine-HCl, 0.01% (v / v) Tween20 (pH 3.5).
  • the human MpHgG Fc fusion protein gene was prepared according to the method of Bennett et al. (Bennett et al., J. Biol. Chem. 1991; 266: 23060-23067).
  • the nucleotide sequence encoding the extracellular region of human Mpl (Gln26 to Trp491) is ligated to the nucleotide sequence encoding the Fc region of human IgG- ⁇ 1 (region downstream from Asp216), and the BstEII sequence is used as a fusion linker at the junction. (Amino acid VaKThr).
  • As the signal sequence 19 amino acids of the signal peptide of the human IgG heavy chain variable region were used.
  • the obtained human MpHgG Fc fusion protein gene was cloned into PCXND3 to construct pCXND3-hMpFc.
  • [0162] generated vector of (25 mu g) were combined mixed in suspended CHO- DG44 cells (lxl0 7 cells / mL) into PBS, added to the Gene Pulser cuvette, the Gene Pulser II (Bio- manufactured Rad) Pulses were obtained at a capacity of 1.5 kV and 25 ⁇ FD.
  • CHO cells transfected by electoral poration were added to a CHO-S-SFMII medium containing 500 g / mL Geneticin, ⁇ , and selected to establish a shMPL-Fc-expressing CHO cell line (CHO-hMpFc). did.
  • a human MpHgG Fc fusion protein was purified as follows.
  • MRL / MpJUmmCrj-lpr / lpr mice purchased from Nippon Charles' Riva.
  • MRL / lpr mice purchased from Nippon Charles' Riva.
  • 100 / zg / animal shMPL-FLAG was supplemented with Freund's complete adjuvant (H37Ra, manufactured by Betaton's Dickinson) and emulsified subcutaneously.
  • Booster immunization with 50 g / animal shMPL-FLAG Incomplete adjuvant (Bettaton 'Dickinson) was added, and the emulsion was subcutaneously administered.
  • the final immunization was performed by intravenously administering g / mouse of shMPL-FLAG to the three mice immunized a total of six times in the tail vein.
  • P3-X63Ag8Ul (P3U1, purchased from ATCC) and mouse spleen cells were mixed, and cell fusion was performed by mixing Polyethylene Glycol 1500 (manufactured by Roche Diagnostics) while mixing. From the next day, selection was performed using a HAT medium, and the culture supernatant was used as an indicator for ELISA using an immunoplate with immobilized shMp-FLAG or hMp-Fc and cell growth activity using BaF3-human Mpl as an index. Screening was performed.
  • the antibody concentration was determined by performing a mouse IgG sandwich ELISA using goat anti-mouse IgG (gamma) (manufactured by ZYMED) and alkaline phosphatase-goat anti-mouse IgG (manufactured by gammaXZYMED). A calibration curve was prepared using GraphPad Prism (GraphPad Software, USA), and the antibody concentration was converted.
  • the antibody isotype was determined by an antigen-dependent ELISA using an isotype-specific secondary antibody. Adjust the coating buffer (O.lmM NaHCO) so that the hMp Fc becomes 1 ⁇ g / mL.
  • the absorbance at 405 nm was measured with Benchmark Plus (manufactured by Bio-Rad) using the resulting data.
  • shMp FLAG and hMPL-Fc The binding activity to shMp FLAG and hMPL-Fc was evaluated by ELISA.
  • the purified shMp FLAG and hMPL-Fc were coated at a concentration of 1 ⁇ g / mL, and blocking treatment was performed with a Diluent buffer.
  • the culture supernatant of the hybridoma was dried and left at room temperature for 1 hour, and an anti-mouse IgG antibody (Zymed) labeled with Alkaline Phosphatase was added thereto, followed by color development in the same manner as described above. After coloring at room temperature for 1 hour, measure the absorbance at 405 And EC values were calculated using GraphPad Prism.
  • CHO- human Mp Fireflys is recovered CHO- monkey Mpl, and suspended in FACS Buffer (1% FBS / PBS ) so as to lxl0 6 cells / mL.
  • FACS Buffer 1% FBS / PBS
  • the solution was dispensed into Multiscreen (manufactured by Millipore) at 100 L / well, and the culture supernatant was removed by centrifugation.
  • the culture supernatant diluted to 5 g / mL was added and reacted on ice for 30 minutes.
  • the cells were washed once with FACS buffer, added with FITC-labeled anti-mouse IgG antibody (manufactured by Beckman Coulter), and allowed to react on ice for 30 minutes.
  • the mixture was centrifuged at 500 rpm for 1 minute, the supernatant was removed, the cells were suspended in 400 ⁇ l of FACS Buffer, and subjected to flow cytometry using EPICS ELITE ESP (Beckman Coulter). Gates were set on the live cell population using histograms of forward scatter and side scatter.
  • the agonist activity of the antibody is similar to that of BaF3-human Mp
  • Anti-human Mpl antibody was purified using the culture supernatant of hybridoma as described below.
  • a fusion protein of the partial sequence of human Mpl and GST was constructed and the epitope analysis of VB22B was performed.
  • the regions of MG1 (from Gln26 to Trp491) and MG2 (from Gln26 to Leu274) were each amplified by PCR and cloned into pGEX-4T-3 (Amersham) so as to be expressed as a GST fusion protein.
  • Plastic Transformants are obtained by introducing the plasmid DNA into DH5, and the expression of GST fusion protein is induced by adding IPTG to transformants in the logarithmic growth phase so as to become ImM. did.
  • the culture supernatant after centrifugation at 35,000 rpm for 30 minutes using XL-80 Ultracentrifoge (Beckman, Rotor 70.1 ⁇ ) was collected and purified using GST Purification Modules (Amersham). After separation by 10% -SDS-PAGE, the cells were transferred to a PVDF membrane and subjected to western blotting using a VB22B mouse antibody.
  • VB22B recognized MG-1 and MG-2, indicating that the epitope of VB22B was located in the region from Gln26 to Leu274.
  • a fusion protein of the region of MG3 (from Gln26 to Alal89), from MG426! 3 ⁇ 4 "0106), MG5 (from Gln26 to Glu259), and MG6 (from Gln26 to Gly245) and GST was prepared, and western blotting was similarly performed.
  • VB22B did not recognize MG5 and MG6, and did not recognize MG3 and MG4, so it was expected that the epitope of VB22B would exist around Alal89 to Gly245, and MG7 (from Gln26 to Ala231).
  • a fusion protein of MG8 (Gln26 to Pro217) and GST was prepared and evaluated.As a result, VB22B recognized MG7 but did not recognize MG8.Therefore, the VB22B epitope exists around Gln217 to Ala231. Furthermore, the binding between MG10 (Gln213 to Ala231) and the fusion protein of GST was confirmed, so that the epitope of VB22B was limited to 19 amino acids from Gln213 to Ala231.
  • VB22B IgG 1.25 to 20 g / mL of VB22B IgG was prepared using HBS-EP Buffer (manufactured by BIAcore), and VB22B IgG was added for 2 minutes to obtain a binding region. By adding for 2 minutes, a dissociation region was obtained.
  • VB22B IgG bound to the human MpHgG Fc fusion protein on the Sensor Chip was removed by adding 10 mM NaOH for 15 seconds, and the Sensor Chip was regenerated.
  • HBS-EP Buffer was used as the running buffer, and the flow rate was 20 ⁇ L / min.
  • a single chain antibody expression system was constructed by a genetic engineering technique for an antibody having high binding activity and agonist activity.
  • the following shows an example of preparing a single-chain antibody of the anti-human Mpl antibody VB22B.
  • dNTPs 0.2 mM dNTPs (dATP, dGTP, dCTP, dTTP),
  • reaction temperature conditions are as follows.
  • the PCR product was purified using a QIAquick Gel Extraction Kit (manufactured by QIAGEN), subjected to agarose gel force purification, and then cloned into a pGEM-T Easy vector (manufactured by Promega). Furthermore, the nucleotide sequence was determined using ABI 3700 DNA Analyzer (manufactured by Perkin Elmer).
  • VB22B-VH The nucleotide sequence of the cloned VB22B H chain variable region (hereinafter, VB22B-VH) is SEQ ID NO: 7, the amino acid sequence is SEQ ID NO: 8, and the nucleotide sequence of the L chain variable region (hereinafter, VB22B-VL) is SEQ ID NO: 9, and the amino acid sequence is shown in SEQ ID NO: 10.
  • VB22B Diabody a single-chain Fv (hereinafter referred to as “VB22B Diabody”) using a linker sequence consisting of 5 amino acids is the 3 ′ end of the gene encoding VB22B-VH and the gene encoding VB22B-VL. 'A linker with a terminal (Gly Ser) force
  • the genes to which the nucleotide sequence encoding 411 was added were amplified by PCR and ligated, respectively, and constructed.
  • the forward primer 70 • 115HF (SEQ ID NO: 71) of VB22B-VH was designed to have an EcoRI site, and the backward primer 33 • 115HR of VB22B-VH (SEQ ID NO: 72) was A linker that hybridizes to DNA encoding the C-terminus and has (Gly Ser) power
  • VB22B-VL forward primer 33-115LF (SEQ ID NO: 73) is a primer consisting of the nucleotide sequence encoding the N-terminus of VB22B-VL and (Gly Ser).
  • composition of the PCR reaction solution (50 L) is shown below.
  • pGEM-T Easy vector containing lOng VB22B-VH or VB22B-VL gene, lOpmole synthetic oligonucleotide 70 '115HF, 33115 HR or 33115LF, 33115LR
  • reaction temperature conditions are as follows.
  • reaction product was heated at 72 ° C for 5 minutes.
  • the agarose gel was also purified from the approximately 400 bp PCR product using the QIAquick Gel Extraction Kit (manufactured by QIAGEN), and a second PCR was performed using a part of each PCR product as follows.
  • composition of the PCR reaction solution (50 L) is shown below.
  • dNTPs dATP, dGTP, dCTP, dTTP
  • reaction temperature conditions are as follows.
  • the PCR product of about 800 bp was also purified using a QIAquick Gel Extraction Kit (manufactured by QIAGEN) for agarose gel, and then digested with the restriction enzymes EcoRI (manufactured by Takara Shuzo) and Notl (manufactured by Takara Shuzo). After that, the resultant was purified using a QIAquick PCR Purification Kit (manufactured by QIAGEN) and cloned into pCXND3 to prepare pCXND3-VB22B db.
  • VB22B-derived modified antibody containing two heavy chain variable regions and two light chain variable regions
  • FIG. 4 shows the construction process of the sc (Fv) 2 gene.
  • VB22B-VH forward primer VB22B_ft) vu (primer A, SEQ ID NO: 76) was designed to have an EcoRI site at the 5 'end and to convert Gln22 and Leu23 of VB22B db to a PvuII site.
  • the VB22B-VH rear primer SC-rL15 (Primer B, SEQ ID NO: 77) hybridizes to the DNA encoding the C-terminus of VB22B-VH and has a (Gly Ser) linker.
  • the forward primer SC-1L15 (primer C, SEQ ID NO: 78) of VB22B-VL is composed of a nucleotide sequence encoding the N-terminus of VB22B-VL and (Gly Ser)
  • composition of the PCR reaction solution (50 L) is shown below.
  • dNTPs dATP, dGTP, dCTP, dTTP
  • reaction temperature conditions are as follows.
  • reaction product was heated at 72 ° C for 5 minutes.
  • the agarose gel was also purified from the approximately 400 bp PCR product using the QIAquick Gel Extraction Kit (manufactured by QIAGEN), and a second PCR was performed using a part of each PCR product as follows.
  • composition of the PCR reaction solution (50 L) is shown below.
  • dNTPs dATP, dGTP, dCTP, dTTP
  • reaction temperature conditions are as follows.
  • reaction product was heated at 72 ° C for 5 minutes.
  • the forward primer Fv2-f (primer E, SEQ ID NO: 79) of the target fragment was designed to have a PvuII site at the 5 ′ end and a sequence at the 5 ′ end of VB22B-VH.
  • the primer Fv2-r (Primer F, SEQ ID NO: 80) after the target fragment is a linker that hybridizes to the DNA encoding the C-terminus of VB22B-VL and also has (Gly Ser) power.
  • composition of the PCR reaction solution (50 ⁇ L) is shown below.
  • dNTPs dATP, dGTP, dCTP, dTTP
  • reaction temperature conditions are as follows.
  • the approximately 800 bp PCR product was purified from agarose gel using the QIAquick Gel Extraction Kit (QIAGEN), and then cloned into a pGEM-T Easy vector (Promega). After determining the nucleotide sequence, the target fragment was recovered after digestion with the restriction enzyme PvuII (Takara Shuzo). After digestion with restriction pBacPAK9_scVB22B enzyme PvuII (manufactured by Takara Shuzo) was ligated to recovered fragment to prepare a P BacPAK9-VB22B sc (Fv) 2.
  • the production of a cell line stably expressing a single-chain antibody using CHO-DG44 cells was performed as follows.
  • the gene was introduced by an electoral-portation method using Gene Pulserll (BioRad).
  • a mixture of the expression vector (25 ⁇ g) and 0.75 mL of CHO-DG44 cells (1 ⁇ 10 7 cells / mL) suspended in PBS was cooled on ice for 10 minutes, transferred to a cuvette, and then transferred to a 1.5 kV, A pulse was given at the capacity of the FD.
  • electroporated cells should contain 500 ⁇ g / mL Geneticin (Invitrogen).
  • a CHO-S-SFMII medium (manufactured by Invitrogen) was selected for selection to establish an expression CHO cell line.
  • VB22B sc (Fv) 2 a cell line stably expressing and a culture supernatant thereof were prepared by this method.
  • Transient expression of a single-chain antibody using COS7 cells was performed as follows. A mixture of expression vector (10 ⁇ g) and 0.75 mL of COS7 cells (1 ⁇ 10 7 cells / mL) suspended in PBS was cooled on ice for 10 minutes, transferred to a cuvette, and then transferred to a 1.5 kV, 25 kV cell. Pulses were given with a ⁇ FD capacity. After a recovery period of 10 minutes at room temperature, the cells treated with the electoral port were treated with DMEM medium (manufactured by Invitrogen) containing 10% FBS, cultured for 1 hour, washed with PBS, and washed with CHO-S- The SFMII medium was cultured and cultured for about 3 days. The culture supernatant of VB22B Diabody was prepared by this method.
  • DMEM medium manufactured by Invitrogen
  • the concentration of the anti-human Mpl-chain antibody transiently expressed in COS cells in the culture supernatant was measured using surface plasmon resonance.
  • BIAcore2000 (Biacore) Insert Chip CM5 (Biacore), and insert ANTI-FLAG M2 Monoclonal Antibody
  • Anti-Flag M2 Affinity Gel (SIGMA-ALDRICH) column prepared by equilibrating the culture supernatant of VB22B Diabody expressing COS7 cells or CHO cells with 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05% Tween20 And eluted with 100 mM Glycine-HCl (pH 3.5). The eluted fraction was immediately neutralized with 1 M Tris-HCl (pH 8.0), and subjected to gel filtration chromatography using a HiLoad 26/60 Superdex200pg (Amersham-Bioscience) column. As a buffer for gel filtration chromatography, PBS and 0.01% Tween20 were used.
  • the culture supernatant of COS7 cells or CHO cells expressing VB22B sc (Fv) 2 was purified under the same conditions as for Diabody purification.
  • the CHO cell culture supernatant is applied to a Macro-Prep Ceramic Hydroxyapatite Type I (Bio-Rad) column equilibrated with 20 mM phosphate buffer (pH 6.8), and the Elution was performed stepwise with an acid buffer (pH 6.8).
  • the eluted fraction was concentrated using an ultrafiltration membrane, and then subjected to gel filtration chromatography using a HiLoad 26/60 Superdex 200pg column to collect a fraction having a molecular weight of about 40 kD to 70 kD. This fraction was adsorbed to an Anti-Flag M2 Affinity Gel column equilibrated with 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05% Tween20, and eluted with 100 mM Glycine-HCl (pH 3.5).
  • 50 mM Tris-HCl pH 7.4
  • 150 mM NaCl 150 mM NaCl
  • Tween20 0.05%
  • the eluted fraction was immediately neutralized with 1 M Tris-HCl (pH 8.0), and subjected to gel filtration chromatography using a HiLoad 26/60 Superdex200pg column.
  • As a buffer for gel filtration chromatography 20 mM acetate buffer (pH 6.0), 150 mM NaCl, and 0.01% Tween 80 were used.
  • Diabody and sc (Fv) 2 were confirmed by SDS-PAGE and Western Blotting using an anti-Flag antibody (SIGMA-ALDLICH) at each purification step. I did it.
  • CH0-human Mpl, CHO-monkey Mpl and CHO-mouse Mpl were collected and suspended in FACS Buffer (1% FBS / PBS) to lxlO 6 cells / mL.
  • FACS Buffer 1% FBS / PBS
  • the solution was dispensed into Multiscreen-HV Filter Plates (Millipore) at 100 L / well, and the supernatant was removed by centrifugation.
  • An appropriate concentration of Diabody or sc (Fv) 2 was added and allowed to react on ice for 30 minutes.
  • the cells were washed once with 200 ⁇ L of FACS buffer, added with 10 ⁇ g / mL ⁇ -FLAG M2 Monoclonal Antibody (manufactured by SIGMA-ALDRICH), and reacted on ice for 30 minutes.
  • the cells were washed once with 200 ⁇ L LOFACS buffer, and a 100-fold diluted FITC-labeled anti-mouse IgG antibody (manufactured by Beckman Coulter) was added, followed by reaction on ice for 30 minutes. Finally, the mixture was centrifuged to remove the supernatant, suspended in 400 ⁇ L of FACS Buffer, and subjected to Flow Cytometry using EPICS ELITE ESP (Beckman Coulter). Live cell populations were gated with histograms of forward scatter and side scatter.
  • FIG. 5 shows the results of evaluating the binding activity of various purified Mpl to CHO cells using the purified VB22B sc (Fv) 2. It showed no binding activity to CHO and CHO-mouse Mpl, which are host cells, and was confirmed to specifically bind to CHO-human and CHO-monkey Mpl. Since the tendency of the binding activity was not different from that of VB22B IgG, it was presumed that the binding site of the antibody was not changed by the reduction in molecular weight.
  • TPO-like agonistic activity was evaluated using M-07e (purchased from DSMZ), a human leukemia cell line showing TPO-dependent growth.
  • M-07e was washed twice with RPMI 1640 containing 1% Fetal Bovine Serum, then suspended in RPMI 1640 containing 10% Fetal Bovine Serum at 5xl0 5 cells / mL, and 96 ⁇ l plate at 50 ⁇ L / well was dispensed. Shake the concentration of rhTPO, COS7 culture supernatant or purified product, add 50 L to each well, 37 ° C, 5% CO for 48 hours
  • FIGS. 6, 7, and 8 The results of evaluating the TPO-like agonist activity of BaF3-human Mpl, BaF3-monkey Mpl, and M-07e are shown in FIGS. 6, 7, and 8, respectively. It is important for the agonist activity that the antigen-binding site is bivalent, but the distance and angle between the antigen-binding sites are also considered to be important factors (see WO02 / 33073 and WO02 / 33072). The same applies to the newly obtained anti-human Mpl antibody, in which VB22B IgG (BaF-human Mpl EC:> 30 nM) and VB22B Diabody and
  • VB22B sc (Fv) 2 has high agonist activity (BaF-human Mpl EC: 61pM, 27pM, respectively)
  • Humanized antibodies were prepared by transplanting a complementary antigen-determining region (hereinafter, CDR) into a framework region (hereinafter, FR) of these antibodies.
  • CDR complementary antigen-determining region
  • FR framework region
  • the humanized antibody sc (Fv) 2 was expressed in CHO-DG44 cells, and the agonist activity using BaF-human Mpl was evaluated. Using the agonist activity as an index, humanized VB22B sc (Fv) 2 having the same agonist activity as mouse VB22B sc (Fv) 2 was prepared by detecting amino acid substitution in FR.
  • a synthetic oligo DNA of about 50 bases was designed to hybridize about 20 bases, and these synthetic oligo DNAs were assembled by PCR to prepare a gene encoding each variable region.
  • sc (Fv) 2 was prepared and cloned into the expression vector pCXND3 in the same manner as in Reference Example 2.3, and each of the expression vectors pCXND3 was inserted with 35 types of humanized VB22B sc (Fv) 2. -hVB22B p-z sc (Fv) 2,
  • the nucleotide sequence of hVB22B pz sc (Fv) 2 contained in this plasmid is shown in SEQ ID NO: 60, the amino acid sequence is shown in SEQ ID NO: 61, the nucleotide sequence of hVB22Bge sc (Fv) 2 is shown in SEQ ID NO: 62, The sequence is represented by SEQ ID NO: 63, the nucleotide sequence of hVB22B esc (Fv) 2 is represented by SEQ ID NO: 64, the amino acid sequence is represented by SEQ ID NO: 65, and the nucleotide sequence of hVB22B u2-wz4 sc (Fv) 2 is represented by SEQ ID NO: No .: 102, the amino acid sequence is shown in SEQ ID NO: 100, the nucleotide sequence of hVB22B q-wz5 sc (Fv) 2 is shown in SEQ ID NO: 103, and the amino acid sequence is shown in SEQ ID NO:
  • the nucleotide sequence of mouse VB22B sc (Fv) 2 is shown in SEQ ID NO: 13, and the amino acid sequence is shown in SEQ ID NO: 14. Expression was carried out in CHO-DG44 cells in the same manner as in Reference Example 2.4, and the culture supernatant was recovered. Since humanized VB22B sc (Fv) 2 does not have a Flag tag, purification from culture supernatant was performed using GST fusion with MG10 (Gln213 to Ala231), an epitope recognized by VB22B described in Reference Example 1.8. This was performed using proteins. The MG10-GST fusion protein was purified using Glutathione Sepharose 4B (Amersham Biosciences) according to the manufacturer's protocol.
  • the purified MG10-GST fusion protein was immobilized on HiTrap NHS-activated HP (manufactured by Amersham Biosciences) according to the manufacturer's protocol to prepare an affinity column.
  • Humanized VB22B sc (Fv) 2 expression CHO cell culture supernatant was added to 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.01% Tween80
  • the column was passed through a MG10-GST fusion protein immobilized column equilibrated in step 2 to adsorb humanized VB22B sc (Fv) 2, and eluted with 100 mM Glycine-HCl (pH 3.5), 0.01% Tween80.
  • the eluted fraction was immediately neutralized with 1M Tris-HCl (pH 7.4), and subjected to gel filtration chromatography using HiLoad 16/60 Superdex200pg (manufactured by Amersham Biosciences).
  • As a buffer for the gel filtration chromatography 20 mM citrate buffer (pH 7.5), 300 mM NaCl, and 0.01% Tween 80 were used.
  • TPO-like agonist activity was evaluated in the same manner as in Reference Example 2.8.
  • a Biacore 3000 (manufactured by Biacore) was equipped with a Sensor Chip CM5 (manufactured by Biacore), and the MG10-GST fusion protein was immobilized by the amine coupling method.
  • HBS-EP Buffer (manufactured by Biacore) was used as the running buffer for the measurement, and the flow rate was 20 ⁇ L / min.
  • VB22B IgG prepared to a concentration of 5.5 to 175.0 nM with HBS-EP Buffer was added thereto for 2 minutes to obtain binding regions at each concentration, and then the dissociation region was measured for 2 minutes.
  • VB22B IgG bound to the MG10-GST fusion protein on the Sensor Chip was removed by adding 20 mM HC1 for 1 minute to regenerate the Sensor Chip.
  • 4.7 to 150.1 nM of VB22B sc (Fv) 2, 5.3 to 168.9 nM of hVB22B q-wz5 sc (Fv) 2, and 4.9 to 156.8 nM of hVB22Bu2-wz4 sc (Fv) 2 were prepared, and MG10- The GST fusion protein was added to the immobilized chip, and the measurement was performed.
  • KD dissociation constant
  • Recombinant human TPO has been tested in various forms as a therapeutic agent for thrombocytopenia caused by treatment with chemotherapeutic agents.
  • One of the major problems reported in the clinical trials was the emergence of anti- ⁇ antibodies due to ⁇ administration. Ounzhi Li, et. Al, Blood (2001) 98, 3241—324, Saroj Vandhan—Raj. Et. al. Ann. Intern. Med. (2000) 132, 364-368), specifically producing neutralizing antibodies that inhibit endogenous TPO activity, resulting in thrombocytopenia rather than restoring platelet counts It has been reported that the disease develops.
  • the administration of the anti-TPO receptor agonist low molecular weight antibody shown by the present invention does not induce the appearance of antibodies against endogenous TPO.
  • by lowering the molecular weight of the antibody high specific activity is exhibited and the half-life in blood can be shortened. Therefore, it is considered that the adjustment of the effective blood concentration becomes easy, which is advantageous for clinical application. Therefore, it is expected to be a therapeutic agent for thrombocytopenia that has properties superior to those of natural TPO and agonist antibody (IgG).
  • expression systems for mammals are not restricted, such as mammalian cell lines, yeast, insect cells, Escherichia coli, and plants. It can be produced in any expression system.

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Abstract

By obtaining and purifying antihuman Mp1 antibody, antihuman Mpl sv(Fv)2 is purified with the use of a genetic engineering technique. Further, antihuman Mp1 sc(FV)2 is successfully humanized. By evaluating the TPO-like agonistic activity of sc(Fv)2, it is found out that sc(Fv)2 shows a high agonistic activity to antihuman Mp1 antibody and the activity is comparable or even superior to the activity of human TPO which is a natural ligand. It is also found out that VB22B sc(Fv)2 and humanized VB22B sc(Fv)2 have an effect of increasing platelets in vivo.

Description

明 細 書  Specification
血小板減少症治療剤  Antithrombotic agent
技術分野  Technical field
[0001] 本発明は Mplを認識する抗体を有効成分として含有する血小板減少症治療剤に関 する。又、本発明は Mplを認識する抗体を有効成分として含有する血小板増加剤に 関する。  The present invention relates to a therapeutic agent for thrombocytopenia, which comprises an antibody that recognizes Mpl as an active ingredient. Further, the present invention relates to a platelet increasing agent containing an antibody recognizing Mpl as an active ingredient.
背景技術  Background art
[0002] トロンボポェチン（Thrombopoietin : TPO)は、巨核球系の造血細胞の増殖と分化を 促進する分子であり、血小板数の調節に主要な役割を担うサイト力インである。巨核 球コロニー朿 IJ激因子 (Megakaryocyte colony-stimulating factor)あるいは c— MPLリガ ンドとも呼ばれていた。ヒト TPOは、 353アミノ酸力 なる TPO前駆体力 切り出されて 活性体となる。  [0002] Thrombopoietin (TPO) is a molecule that promotes the growth and differentiation of megakaryocytic hematopoietic cells, and is a site that plays a major role in regulating the number of platelets. It was also called megakaryocyte colony-stimulating factor (CJ) or c-MPL ligand. Human TPO is excised as a TPO precursor having 353 amino acids and becomes an active form.
[0003] Mplは TPOの受容体であり、 Cluster of Differentiation (CD)抗原としては CD110にリ ストされる。ヒト Mplの遺伝子配列は既に解析されており（非特許文献 1又は、 Genebank:NM_005373参照）、 635アミノ酸からなる前駆体からシグナルペプチドが切 り出され、発現される。また、 635アミノ酸より短い truncated formが知られている。  [0003] Mpl is a receptor for TPO and is listed on CD110 as a Cluster of Differentiation (CD) antigen. The gene sequence of human Mpl has already been analyzed (see Non-Patent Document 1 or Genebank: NM_005373), and a signal peptide is cut out from a precursor consisting of 635 amino acids and expressed. Also, truncated forms shorter than 635 amino acids are known.
[0004] サイト力イン受容体の多くは、リガンドの結合により受容体同士が 2量体ィ匕し、シグナ ルが細胞内に伝達される。 TPOにおいても、その特異的レセプターである MPLと結合 し、受容体を 2量体化することにより、細胞内に情報を伝え、生理作用を示すことが報 告されて!/ヽる (非特許文献 2参照)。  [0004] In many of the site force-in receptors, the receptors are dimerized by the binding of a ligand, and the signal is transmitted into the cell. It has also been reported that TPO also binds to its specific receptor, MPL, and dimerizes the receptor, thereby transmitting information into cells and exhibiting physiological actions! Reference 2).
このような性質をもつ受容体に結合する抗体の中で、ァゴニスト活性を示す抗体が 存在することが報告されて！ヽる。  It has been reported that among antibodies that bind to receptors having such properties, there are antibodies that exhibit agonist activity! Puru.
[0005] 例えば、エリスロポエチン (EPO)受容体に対する抗体がエリスロポエチン機能を代 替することが報告されており、この抗体を一価 (Fab)にすると EPO受容体への結合能 を維持したまま、シグナル伝達能を失うことから、二価の結合によるエリスロポエチン 受容体の二量体形成が必要と考えられる (非特許文献 3参照)。  [0005] For example, it has been reported that an antibody against the erythropoietin (EPO) receptor substitutes for the erythropoietin function. When this antibody is made monovalent (Fab), the signal remains unchanged while maintaining the binding ability to the EPO receptor. It is considered that dimerization of the erythropoietin receptor by divalent binding is necessary due to loss of transduction ability (see Non-Patent Document 3).
[0006] 又、 Mplに結合し、 TPOァゴニスト活性を有する抗体も報告されて 、る（非特許文献 4および 5参照）。これは、 MPLに関しても 2価である抗体の結合によるレセプターの 2 量体化の誘導を示唆して ヽる。 [0006] Also, an antibody that binds to Mpl and has TPO agonist activity has been reported (Non-Patent Document 4 and 5). This suggests that dimerization of the receptor is induced by binding of an antibody that is bivalent with respect to MPL.
[0007] 一方で、 TPOァゴ-スト活性を示す一本鎖抗体 (scFv)が報告されて 、る（特許文献On the other hand, a single-chain antibody (scFv) exhibiting TPO agonist activity has been reported (Patent Document
1参照）。し力しながら、 scFv力TPOァゴ-スト活性を示す機序として、 scFvの一部が 二量体（Diabody)化し、その Diabodyが活性本体であることが明らかになって!/、る（特 許文献 2〜4参照)。 1). As a mechanism to show the TPO agonist activity of scFv, it is revealed that a part of scFv is dimerized, and that the diabody is the active body! /, See Patent Documents 2-4).
[0008] 特許文献 1：米国特許第 6342220号 [0008] Patent Document 1: US Patent No. 6342220
特許文献 2：国際公開第 01/79494号  Patent Document 2: WO 01/79494
特許文献 3：国際公開第 02/33072号  Patent Document 3: WO 02/33072
特許文献 4:国際公開第 02/33073号  Patent Document 4: WO 02/33073
非特許文献 l : Palaciosら著、 Cell、 1985年、 Vol.41, p.727-734  Non-Patent Document l: Palacios et al., Cell, 1985, Vol.41, p.727-734
非特許文献 2 : Souyriら著、 Cell, 1990年、 Vol.63, p.1137- 1147  Non-Patent Document 2: Souyri et al., Cell, 1990, Vol. 63, p. 1137-1147
非特許文献 3 : Elliott Sら著、 J.Biol.Chem., 1996年、 Vol.271(40)、 p.24691-24697 非特許文献 4: Abeら著、 Immunol. Lett. 1998年、 Vol.61、 p.73-78  Non-Patent Document 3: Elliott S et al., J. Biol. Chem., 1996, Vol.271 (40), p.24691-24697 Non-Patent Document 4: Abe et al., Immunol. Lett. 1998, Vol. 61, p.73-78
非特許文献 5 : Bijia Dengら著、 Blood, 1998年、 Vol.92, p.1981- 1988  Non-Patent Document 5: Bijia Deng et al., Blood, 1998, Vol. 92, p. 1981-1988.
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0009] 本発明はこのような状況に鑑みて為されたものであり、その目的は TPOレセプター ァゴニスト活性を有する抗 Mpl抗体を有効成分として含有する血小板減少症治療剤、 および血小板増加剤を提供することを課題とする。 [0009] The present invention has been made in view of such circumstances, and it is an object of the present invention to provide a therapeutic agent for thrombocytopenia and a thrombocytosis agent containing an anti-Mpl antibody having TPO receptor agonist activity as an active ingredient. The task is to
課題を解決するための手段  Means for solving the problem
[0010] 本発明者らは上記課題を解決するために鋭意研究を行なった。本発明者らは、抗 ヒト Mpl抗体 VB22Bを取得 '精製し、遺伝子工学的手法を用いて一本鎖抗体の発現 系を構築した。具体的には、まず抗ヒト Mpl抗体の可変領域をクローユングし、抗ヒト Mpl抗体 Diabody発現ベクター pCXND3-VB22B dbを作製した。さらに該ベクター pCXND3-VB22B dbを用いて、抗ヒト Mpl抗体 sc(Fv)2発現ベクター pCXND3- VB22B sc(Fv)2を作製した。この発現ベクター pCXND3- VB22B sc(Fv)2を CHO- DG44細胞で 発現させ、培養上清力も抗ヒト Mpl sc(Fv)2を精製した。また、 VB22B sc(Fv)2の TPO 様ァゴ-スト活性を評価したところ、 VB22B IgGに対して、 VB22B sc(Fv)2は高いァゴ 二スト活性を示し、天然リガンドである human TPOと同等以上の活性を示すことが分 かった。 [0010] The present inventors have conducted intensive studies in order to solve the above problems. The present inventors obtained and purified the anti-human Mpl antibody VB22B, and constructed a single-chain antibody expression system using genetic engineering techniques. Specifically, first, the variable region of the anti-human Mpl antibody was cloned to prepare an anti-human Mpl antibody Diabody expression vector pCXND3-VB22Bdb. Further, using the vector pCXND3-VB22B db, an anti-human Mpl antibody sc (Fv) 2 expression vector pCXND3-VB22B sc (Fv) 2 was prepared. The expression vector pCXND3-VB22B sc (Fv) 2 was expressed in CHO-DG44 cells, and the anti-human Mpl sc (Fv) 2 was purified from the culture supernatant. Also, TPO of VB22B sc (Fv) 2 Evaluation of similar agonist activity revealed that VB22B sc (Fv) 2 showed high agonist activity against VB22B IgG, and showed an activity equal to or higher than that of human TPO, which is a natural ligand. Was.
[0011] また、本発明者らは 5種類のヒト化 VB22B sc(Fv)2を作製することに成功した。また、 ヒトイ匕することによる TPO様ァゴ-スト活性の変化は見られないことが分かった。  [0011] The present inventors have succeeded in producing five types of humanized VB22B sc (Fv) 2. In addition, it was found that no change in TPO-like agonist activity was observed when the humans were ridden.
さらに、本発明者らは、 in vivoにおいて VB22B sc(Fv)2およびヒト化 VB22B sc(Fv)2 力 血小板増加作用を有することを見出した。  Furthermore, the present inventors have found that VB22B sc (Fv) 2 and humanized VB22B sc (Fv) 2 have a platelet increasing effect in vivo.
[0012] 本発明はより具体的には、以下の〔1〕〜〔22〕に関する。 The present invention more specifically relates to the following [1] to [22].
〔1〕 TPO受容体 (Mpl)への結合活性を有する sc(Fv)2を有効成分として含有する血 小板減少症治療剤。  [1] A therapeutic agent for thrombocytopenia, comprising sc (Fv) 2 having binding activity to a TPO receptor (Mpl) as an active ingredient.
〔2〕 sc(Fv)2中の可変領域がリンカ一で結合されていることを特徴とする、〔1〕に記載 の血小板減少症治療剤。  [2] The therapeutic agent for thrombocytopenia according to [1], wherein the variable region in sc (Fv) 2 is linked with a linker.
〔3〕 リンカ一が 15アミノ酸であることを特徴とする、〔2〕に記載の血小板減少症治療 剤。  [3] The therapeutic agent for thrombocytopenia according to [2], wherein the linker has 15 amino acids.
〔4〕 ヒト Mpl及びサル Mplに結合する抗体を有効成分として含有する血小板減少症 治療剤。  [4] A therapeutic agent for thrombocytopenia, comprising an antibody that binds to human Mpl and monkey Mpl as an active ingredient.
〔5〕 ヒト Mpl及びサル Mplに対してァゴ-スト活性を有する抗体を有効成分として含 有する血小板減少症治療剤。  [5] A therapeutic agent for thrombocytopenia, comprising an antibody having an agonistic activity against human Mpl and monkey Mpl as an active ingredient.
〔6〕 配列番号： 1、 2、 3に記載のアミノ酸配列力 なる CDR1、 2、 3を有する重鎖可変 領域を含む抗体を有効成分として含有する血小板減少症治療剤。  [6] A therapeutic agent for thrombocytopenia comprising, as an active ingredient, an antibody containing a heavy chain variable region having CDR1, 2, or 3 having the amino acid sequence described in SEQ ID NO: 1, 2, or 3.
〔7〕 配列番号: 4、 5、 6に記載のアミノ酸配列力 なる CDR1、 2、 3を有する軽鎖可変 領域を含む抗体を有効成分として含有する血小板減少症治療剤。  [7] A therapeutic agent for thrombocytopenia comprising, as an active ingredient, an antibody containing a light chain variable region having CDR1, 2, or 3 having the amino acid sequence described in SEQ ID NO: 4, 5, or 6.
〔8〕 〔6〕に記載の重鎖可変領域、および〔7〕に記載の軽鎖可変領域を有する抗体 を有効成分として含有する血小板減少症治療剤。  [8] A therapeutic agent for thrombocytopenia, comprising as an active ingredient the antibody having the heavy chain variable region according to [6] and the light chain variable region according to [7].
〔9〕 〔6〕〜〔8〕の 、ずれかに記載のアミノ酸配列にぉ 、て 1又は複数のアミノ酸が置 換、欠失、付加および Zまたは挿入され、かつ〔6〕〜〔8〕のいずれかに記載の抗体と 同等の活性を有する抗体を有効成分として含有する血小板減少症治療剤。  (9) one or more amino acids are substituted, deleted, added and Z or inserted in the amino acid sequence according to any of (6) to (8), and (6) to (8) A therapeutic agent for thrombocytopenia, comprising as an active ingredient an antibody having the same activity as the antibody according to any one of the above.
〔10〕 〔6〕〜〔8〕のいずれかに記載の抗体が認識するェピトープを認識する抗体を 有効成分として含有する血小板減少症治療剤。 ' (10) an antibody that recognizes an epitope that is recognized by the antibody according to any of (6) to (8); A therapeutic agent for thrombocytopenia, which is contained as an active ingredient. '
〔11〕 ヒト Mplの 26番目から 274番目のアミノ酸部位を認識する抗体を有効成分として 含有する血小板減少症治療剤。  [11] A therapeutic agent for thrombocytopenia, comprising, as an active ingredient, an antibody that recognizes amino acids 26 to 274 of human Mpl.
〔 12〕 TPO受容体 (Mpl)への結合活性を有する sc(Fv)2を有効成分として含有する 血小板増加剤。  [12] A platelet-increasing agent containing sc (Fv) 2 having binding activity to a TPO receptor (Mpl) as an active ingredient.
〔13〕 sc(Fv)2中の可変領域力 Sリンカ一で結合されていることを特徴とする、〔12〕に 記載の血小板増加剤。  [13] The platelet-increasing agent according to [12], wherein the agent is bound by a variable region force S linker in sc (Fv) 2.
〔14〕 リンカ一が 15アミノ酸であることを特徴とする、〔13〕に記載の血小板増加剤。 〔15〕 ヒト Mpl及びサル Mplに結合する抗体を有効成分として含有する血小板増加剤  [14] the platelet increasing agent of [13], wherein the linker has 15 amino acids; (15) a platelet-increasing agent containing an antibody binding to human Mpl and monkey Mpl as an active ingredient
〔16〕 ヒト Mpl及ぴサル Mplに対してァゴニスト活性を有する抗体を有効成分として含 有する血小板増加：剤。 [16] A platelet-increasing agent comprising, as an active ingredient, an antibody having agonist activity against human Mpl and monkey Mpl.
〔17〕 配列番号： 1、 2、 3に記載のアミノ酸配列力もなる CDR1、 2、 3を有する重鎖可 変領域を含む抗体を有効成分として含有する血小板増加剤。  [17] A platelet-increasing agent comprising, as an active ingredient, an antibody containing a heavy-chain variable region having CDRs 1, 2, and 3, which also has the amino acid sequence described in SEQ ID NOs: 1, 2, and 3.
〔18〕 配列番号: 4、 5、 6に記載のアミノ酸配列力 なる CDR1、 2、 3を有する軽鎖可 変領域を含む抗体を有効成分として含有する血小板増加剤。  [18] A platelet-increasing agent comprising, as an active ingredient, an antibody containing a light chain variable region having CDR1, 2, or 3 having the amino acid sequence described in SEQ ID NO: 4, 5, or 6.
〔19〕 〔17〕に記載の重鎖可変領域、および〔18〕に記載の軽鎖可変領域を有する 抗体を有効成分として含有する血小板増加剤。  [19] A platelet-increasing agent comprising, as an active ingredient, an antibody having the heavy chain variable region according to [17] and the light chain variable region according to [18].
〔20〕 〔17〕〜〔19〕のいずれかに記載のアミノ酸配列において 1又は複数のアミノ酸 が置換、欠失、付加および/または揷入され、かつ〔17〕〜〔19〕のいずれかに記载 の抗体と同等の活性を有する抗体を有効成分として含有する血小板増加剤。  (20) one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence according to any of (17) to (19), and A platelet-increasing agent comprising, as an active ingredient, an antibody having the same activity as the above antibody.
〔21〕 〔17〕〜〔19〕のいずれかに記載の抗体が認識するェピトープを認識する抗体 を有効成分として含有する血小板増加剤。 [21] A platelet-increasing agent comprising, as an active ingredient, an antibody that recognizes an epitope recognized by the antibody according to any one of [17] to [19].
〔22〕 ヒト Mplの 26番目から 274番目のアミノ酸部位を認識する抗体を有効成分として 含有する血小板増加剤。  [22] A platelet-increasing agent comprising, as an active ingredient, an antibody that recognizes amino acids 26 to 274 of human Mpl.
図面の簡単な説明 Brief Description of Drawings
[図 1-1]ヒト化重鎖配列（hVB22B p- z、 hVB22B g- e、 hVB22B e、 hVB22B u2- wz4およ ぴ hVB22B q- wz5:VH)およぴヒト化軽鎖配列（ B22B p- z、 hVB22B g- e、 hVB22B [Figure 1-1] Humanized heavy chain sequence (hVB22B p-z, hVB22B g-e, hVB22Be, hVB22B u2-wz4 and ぴ hVB22B q-wz5: VH) and humanized light chain sequence (B22B p -z, hVB22B g- e, hVB22B
差替え用紙（規則 26) e、 hVB22B u2 - wz4および hVB22B q- wz5 :VL)、ならびに FRおよび CCiRの対応につ いて示す図である。 Replacement form (Rule 26) e, hVB22Bu2-wz4 and hVB22Bq-wz5: VL), and the correspondence between FR and CCiR.
[図卜 2]図 1一 1の続きを示す図である。  FIG. 2 is a diagram showing a continuation of FIG.
[図 2]正常力二クイザルにおけるマウス VB22B投与後の末梢血小板数を示すグラフで ある。  FIG. 2 is a graph showing the number of peripheral platelets in normal cynomolgus monkeys after administration of mouse VB22B.
[図 3]ACNUで誘導した血小板減少に対するマウス VB22B投与の効果を示すグラフで ある。  FIG. 3 is a graph showing the effect of mouse VB22B administration on thrombocytopenia induced by ACNU.
[図 4]—本鎖抗体 sc(Fv)2の作製過程を示す図である。  FIG. 4 is a view showing a process of preparing a main chain antibody sc (Fv) 2.
[図 5]Mpl発現 CHO細胞株を用いた VB22B sc(Fv)2の結合活性評価の結果を示すグ ラフである。 VB22B sc(Fv)2精製品を使用した。  FIG. 5 is a graph showing the results of evaluating the binding activity of VB22B sc (Fv) 2 using an Mpl-expressing CHO cell line. VB22B sc (Fv) 2 purified product was used.
[図 6]BaF- human Mplを用いた VB22B抗体のァゴニスト活性評価の結果を示すグラフ である。 ■■  FIG. 6 is a graph showing the results of evaluating the agonist activity of VB22B antibody using BaF-human Mpl. ■■
[図 7]BaF-monkey Mplを用レ、た VB22B抗体のァゴニスト活性評価の結果を示すグラ フである。  FIG. 7 is a graph showing the results of evaluating the agonist activity of VB22B antibody using BaF-monkey Mpl.
[図 8]M_07eを用レ、た VB22B抗体のァゴニスト活性評価の結果を示すグラフである。  FIG. 8 is a graph showing the results of evaluating the agonist activity of VB22B antibody using M_07e.
[図 9]図 9は、マウス型 VB22B sc(Fv)2および hVB22B e sc(Fv)2、 hVB22B g- e sc(Fv)2 を用いて、 BaF3- human Mplでの TPO様ァゴニスト活性を評価した結果を示す図であ る。縦軸は吸光度 (450mn/655nni)を示し、横軸は濃度を示す。  [Figure 9] Figure 9 shows the evaluation of TPO-like agonist activity in BaF3-human Mpl using mouse VB22B sc (Fv) 2 and hVB22B esc (Fv) 2, hVB22B g-esc (Fv) 2 It is a figure which shows the result. The vertical axis indicates absorbance (450 mn / 655 nni), and the horizontal axis indicates concentration.
[図 10]図 10は、マウス型 VB22B sc(Fv)2および WB22B p-z sc(Fv)2、 hVB22B u2-wz [Figure 10] Figure 10 shows mouse VB22B sc (Fv) 2 and WB22B p-z sc (Fv) 2, hVB22B u2-wz
4 sc(Fv)2を用いて、 BaF3-human Mplでの TPO様ァゴニスト活性を評価した結果をに 示す図である。縦軸は吸光度 (450nm/655nm)を示し、横軸は濃度を示す。 FIG. 4 shows the results of evaluating TPO-like agonist activity in BaF3-human Mpl using 4sc (Fv) 2. The vertical axis shows absorbance (450 nm / 655 nm), and the horizontal axis shows concentration.
[図 11]図 11は、マウス型 VB22B sc(Fv)2および hVB22B q-wz5 sc(Fv)2を用いて、 BaF [FIG. 11] FIG. 11 shows the results of BaF using mouse VB22B sc (Fv) 2 and hVB22B q-wz5 sc (Fv) 2.
3- human Mplでの TPO様ァゴニスト活性を評価した結果を示す図である。縦軸は吸 光度 (450nm/655nm)を示し、横軸は濃度を示す。 FIG. 4 shows the results of evaluating TPO-like agonist activity in 3-human Mpl. The vertical axis indicates the absorbance (450 nm / 655 nm), and the horizontal axis indicates the concentration.
[図 12]図 12は、正常力二クイサルにおける hVB22B u2-wz4 sc(FV)2ならびに hVB22B q-wz5 sc(FV)2の単回注射による血小板増多作用を示す図である。  [Fig. 12] Fig. 12 is a graph showing the platelet-increasing effect of hVB22B u2-wz4 sc (FV) 2 and hVB22B q-wz5 sc (FV) 2 in a single injection of normotensive quizzes.
発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION
本発明は、 TPO受容体 (Mpl)に結合する抗体を有効成分として含有する、血小板 減少症治療剤および血小板増加剤を提供する。 差替え用紙（規則 26) [0015] 本発明の抗体には、低分子化された抗体、ヒト化抗体やキメラ化抗体などのアミノ 酸配列が改変された抗体、他の分子 (例えば、ポリエチレングリコールなどの高分子 等)が結合した修飾抗体、糖鎖が改変された抗体、など如何なる抗体も含まれる。 The present invention provides a therapeutic agent for thrombocytopenia and an agent for increasing thrombocytopenia, which contain an antibody that binds to a TPO receptor (Mpl) as an active ingredient. Replacement form (Rule 26) [0015] Antibodies of the present invention include low-molecular-weight antibodies, antibodies with modified amino acid sequences such as humanized antibodies and chimerized antibodies, and other molecules (for example, polymers such as polyethylene glycol). Any antibody such as a modified antibody bound thereto, an antibody having a modified sugar chain, and the like are included.
[0016] 血小板減少症は末梢血中の血小板数が減少する疾患である。血小板減少症の原 因としては、骨髄巨核球数の減少、先天性、無効造血、破壊亢進、消費亢進、貯留 増カロ、希釈などが挙げられる。より具体的には、悪性腫瘍、骨髄線維症、肉芽腫、薬 剤、放射線、ウィルス、アルコール、 Fanconi貧血、常染色体劣性血小板減少症、橈 骨欠損を伴う血小板減少症、巨赤芽球性貧血、骨髄異形成症候群、常染色体優性 血, J、板減少;!正、 May— Hegglin異'吊正、 Alport ¾£候群、 Bernard— Soulie 正候群、 gray platelet症候群、 Wiskott-Aldrich症候群、特発性血小板減少性紫斑病、薬剤起因性 免疫性血小板減少症、二次性自己免疫性血小板減少症、 HIV感染、同種免疫性新 生児血小板減少性紫斑病、輸血後紫斑病、血栓性血小板減少性紫斑病、溶血性 尿毒症症候群、播種性欠陥内血液凝固、急性感染症、海綿状血管腫、人工肺バイ パス術、脾機能亢進症、低体温、希釈性血小板減少症、大量輸血、肝硬変，肝炎- 肝切除による血小板減少症、などを挙げることができる。又、例えば、インターフエ口 ンを投与した場合には血小板が減少することが知られているが、本発明の血小板減 少症治療剤はインターフェロン投与による血小板減少症の治療にも用いることができ る。従って、本発明の血小板減少症治療剤とインターフェロンの併用療法は臨床上、 有用である。しかしながら、本発明の血小板減少症は、上述の原因による血小板減 少症に限定されず、如何なる原因による血小板減少症でもよい。  [0016] Thrombocytopenia is a disease in which the number of platelets in peripheral blood decreases. Causes of thrombocytopenia include decreased bone marrow megakaryocyte count, congenital, ineffective hematopoiesis, increased destruction, increased consumption, increased stored calories, and dilution. More specifically, malignant tumors, myelofibrosis, granulomas, drugs, radiation, viruses, alcohol, Fanconi anemia, autosomal recessive thrombocytopenia, thrombocytopenia with radial loss, megaloblastic anemia , Myelodysplastic syndrome, autosomal dominant blood, J, platelet depletion;! Positive, May-Hegglin's positive, Alport 候, Bernard- Soulie positive, gray platelet syndrome, Wiskott-Aldrich syndrome, idiopathic Thrombocytopenic purpura, drug-induced immune thrombocytopenia, secondary autoimmune thrombocytopenia, HIV infection, alloimmune neonatal thrombocytopenic purpura, purpura after transfusion, thrombotic thrombocytopenia Purpura, hemolytic uremic syndrome, disseminated blood coagulation, acute infection, cavernous hemangioma, artificial lung bypass, hypersplenism, hypothermia, dilute thrombocytopenia, massive blood transfusion, cirrhosis , Hepatitis-thrombocytopenia due to hepatectomy Can be. Also, for example, it is known that platelets decrease when an interfering agent is administered, but the therapeutic agent for thrombocytopenia of the present invention can also be used for treating thrombocytopenia by administering interferon. You. Therefore, the combination therapy of the therapeutic agent for thrombocytopenia of the present invention and interferon is clinically useful. However, the thrombocytopenia of the present invention is not limited to thrombocytopenia due to the above-mentioned causes, and may be thrombocytopenia due to any cause.
[0017] 血小板減少症は、一般的には末梢血中の血小板の数により判断され、例えば、日 本においては通常、血小板数が 10万 Z L以下の場合に血小板減少症と判断され る。し力しながら、本発明においては、末梢血中の血小板の数が判断基準を上回つ ている場合であっても、血小板を増加させる必要があると判断される状態にある場合 には、血小板減少症にあるといえる。  [0017] Thrombocytopenia is generally determined by the number of platelets in peripheral blood. For example, in Japan, usually, when the platelet count is 100,000 ZL or less, thrombocytopenia is determined. However, in the present invention, even when the number of platelets in the peripheral blood exceeds the criterion, if the number of platelets is determined to need to be increased, It can be said that there is thrombocytopenia.
[0018] 血小板増加剤は、血小板を増加させる目的で使用される薬剤であり、血小板が減 少状態にある場合に使用されてもよいし、血小板が減少状態にない場合に使用され てもよい。例えば、手術前などに自己の血小板を貯蔵する目的で Mplを認識する抗 体を投与し、血小板を増加させてもよいし、血小板成分献血後に Mplを認識する抗体 を投与し、血小板数の正常値への回復の期間の短縮させたりしてもよい。従って、血 小板増加剤は、血小板減少症の有無に係わらず用いることができる。 [0018] A platelet-increasing agent is a drug used for the purpose of increasing platelets, and may be used when platelets are in a reduced state, or may be used when platelets are not in a reduced state. . For example, an antibody that recognizes Mpl to store its own platelets before surgery, etc. The body may be administered to increase platelets, or an antibody recognizing Mpl may be administered after donating platelet components to shorten the time required for platelet counts to return to normal values. Therefore, platelet augmenting agents can be used with or without thrombocytopenia.
[0019] Mplは TPOの受容体であり、ヒト Mplの遺伝子配列は既に解析されて!、る (Palacios et al、 Cell, 1985年、 Vol.41、 p.727— 734又は、 Genebank:NM— 005373)。又、力二クイ ザル Mpl (塩基 Z配列番号： 67、アミノ酸 Z配列番号： 68)、マウス Mpl [0019] Mpl is a receptor for TPO, and the gene sequence of human Mpl has already been analyzed! (Palacios et al, Cell, 1985, Vol. 41, p. 727—734 or Genebank: NM— 005373). In addition, Rinichi-kumo Mpl (base Z SEQ ID NO: 67, amino acid Z SEQ ID NO: 68), mouse Mpl
(GenBank#NM_010823)の配列も既に公知である。ヒト Mplのアミノ酸配列を配列番号： 66に示す。  The sequence of (GenBank # NM_010823) is already known. The amino acid sequence of human Mpl is shown in SEQ ID NO: 66.
[0020] 又、本発明における Mplには、上述の Mplにお 、てアミノ酸が置換、欠失、付加、等 された変異 Mpl受容体も含まれる。変異 Mplの具体例としては、例えば、 Matthias Ballmaier et al.， BLOOD, (2001)、 Vol.97, No.l、 P139等に記載された変異 Mplを挙 げることができる。  [0020] The Mpl in the present invention also includes a mutant Mpl receptor in which amino acids are substituted, deleted, added, etc. in the above-mentioned Mpl. Specific examples of the mutant Mpl include, for example, the mutant Mpl described in Matthias Ballmaier et al., BLOOD, (2001), Vol. 97, No. 1, P139, and the like.
[0021] 本発明においては Mplを認識する抗体は特に限定されないが、血小板増加作用を 有する抗体であることが好ましい。血小板増加作用を有する抗体は、通常、 Mplに対 してァゴ-スト活性を有する。血小板増加作用は当業者に公知の方法により確認す ることが可能である力 例えば、実施例記載の方法により確認することができる。  [0021] In the present invention, the antibody that recognizes Mpl is not particularly limited, but is preferably an antibody having a platelet increasing effect. An antibody having a platelet-increasing action usually has an agonistic activity against Mpl. The effect of increasing platelets can be confirmed by a method known to those skilled in the art. For example, the effect can be confirmed by the method described in Examples.
[0022] Mplに対するァゴニスト活性とは、巨核球又はその親細胞である造血幹細胞力 血 小板へと分化することを促進する活性、又は血小板を増加させる活性である。ァゴ- スト活性の測定は当業者に公知の方法により行うことが可能である。又、ァゴニスト活 性の測定は、本来の活性を指標に測定するだけでなぐ他の活性を指標に測定する ことも可能である。 [0022] The agonist activity against Mpl is an activity of promoting differentiation into megakaryocytes or hematopoietic stem cells, which are parent cells thereof, into platelets, or an activity of increasing platelets. The measurement of the agonist activity can be performed by a method known to those skilled in the art. The agonist activity can be measured not only by using the original activity as an indicator but also by using other activities as indicators.
[0023] 例えば、実施例に記載のように細胞増殖を指標にァゴニスト活性を測定する方法に より判定することが可能である。より具体的には、ァゴニスト依存性増殖を示す細胞に ァゴ-スト活性を測定したい抗体を添加し、培養する。その後、テトラゾリゥム塩 For example, the determination can be made by a method of measuring agonist activity using cell proliferation as an index as described in Examples. More specifically, an antibody whose agonist activity is to be measured is added to cells exhibiting agonist-dependent growth, and cultured. Then tetrazolium salt
WST-8 (同仁ィ匕学研究所)のような生細胞数に応じて特定の波長において発色反応 を呈する試薬を添加して吸光度を測定し、得られた吸光度を指標にァゴ-スト活性を 測定することが可能である。 Add a reagent that produces a color reaction at a specific wavelength according to the number of living cells, such as WST-8 (Dojini Dani Kagaku Kenkyusho), measure the absorbance, and use the resulting absorbance as an index to determine the agonist activity. Can be measured.
[0024] ァゴ-スト依存性増殖を示す細胞も当業者に公知の方法により作製することが可能 であり、例えば、抗原が細胞増殖シグナルを発する受容体である場合には、該受容 体を発現している細胞を用いればよい。又、抗原が細胞増殖シグナルを出さない受 容体である場合には、細胞増殖シグナルを発する受容体の細胞内領域と、細胞増殖 シグナルを出さな ヽ受容体の細胞外領域からなるキメラ受容体を作製し、該キメラ受 容体を細胞で発現させればよ!ヽ。細胞増殖シグナルを発する受容体の例としては、 例えば、 G- CSF受容体、 mpl、 neu、 GM- CSF受容体、 EPO受容体、 c-kit、 FLT-3等を 挙げることができる。受容体を発現させる細胞としては、例えば、 BaF3、 NFS60、 FDCP- 1、 FDCP- 2、 CTLL- 2、 DA- 1、 KT- 3等を挙げることができる。 [0024] Cells exhibiting an agonist-dependent proliferation can also be prepared by a method known to those skilled in the art. For example, when the antigen is a receptor that emits a cell proliferation signal, cells expressing the receptor may be used. When the antigen is a receptor that does not emit a cell proliferation signal, a chimeric receptor comprising an intracellular region of a receptor that emits a cell proliferation signal and an extracellular region of a receptor that does not emit a cell proliferation signal is used. Produce and express the chimeric receptor in cells! Examples of the receptor that emits a cell proliferation signal include, for example, G-CSF receptor, mpl, neu, GM-CSF receptor, EPO receptor, c-kit, FLT-3 and the like. Examples of cells that express the receptor include BaF3, NFS60, FDCP-1, FDCP-2, CTLL-2, DA-1, KT-3 and the like.
その他、ァゴ-スト活性を測定する為に用いる検出指標としては、量的及び Z又は 質的な変化が測定可能である限り使用することができる。例えば、無細胞系 (cell free assay)の指標、細胞系 (ceU-based assay)の指標、糸且織系の指標、生体系の指標を用 いることができる。無細胞系の指標としては、酵素反応やタンパク質、 DNA、 RNAの量 的及び Z又は質的な変化を用いることができる。酵素反応としては、例えば、アミノ酸 転移反応、糖転移反応、脱水反応、脱水素反応、基質切断反応等を用いることがで きる。また、タンパク質のリン酸化、脱リン酸化、二量化、多量化、分解、乖離等や、 DNA、 RNAの増幅、切断、伸長を用いることができる。例えばシグナル伝達経路の下 流に存在するタンパク質のリン酸ィ匕を検出指標とすることができる。細胞系の指標とし ては、細胞の表現型の変化、例えば、産生物質の量的及び Z又は質的変化、増殖 活性の変化、細胞数の変化、形態の変化、特性の変化等を用いることができる。産 生物質としては、分泌タンパク質、表面抗原、細胞内タンパク質、 mRNA等を用いるこ とができる。形態の変化としては、突起形成及び Z又は突起の数の変化、偏平度の 変化、伸長度 Z縦横比の変化、細胞の大きさの変化、内部構造の変化、細胞集団と しての異形性 Z均一性、細胞密度の変化等を用いることができる。これらの形態の変 化は検鏡下での観察で確認することができる。特性の変化としては、足場依存性、サ イト力イン依存応答性、ホルモン依存性、薬剤耐性、細胞運動性、細胞遊走活性、拍 動性、細胞内物質の変化等を用いることができる。細胞運動性としては、細胞浸潤活 性、細胞遊走活性がある。また、細胞内物質の変化としては例えば、酵素活性、 mRNA量、 Ca2+や cAMP等の細胞内情報伝達物質量、細胞内タンパク質量等を用い ることができる。また、細胞膜受容体の場合には、受容体の刺激によって誘導される 細胞の増殖活性の変化を指標とすることができる。組織系の指標としては、使用する 組織に応じた機能変化を検出指標とすることができる。生体系の指標としては組織重 量変化、血液系の変化、例えば血球細胞数の変化、タンパク質量や、酵素活性、電 解質量の変化、また、循環器系の変化、例えば、血圧、心拍数の変化等を用いること ができる。 In addition, as a detection index used for measuring an agonist activity, any quantitative and Z- or qualitative change can be used as long as it can be measured. For example, an index of a cell-free system (cell free assay), an index of a cell system (ceU-based assay), an index of a ligamentous system, and an index of a biological system can be used. As an indicator of a cell-free system, an enzymatic reaction or a quantitative and Z- or qualitative change of protein, DNA, or RNA can be used. As the enzymatic reaction, for example, an amino acid transfer reaction, a sugar transfer reaction, a dehydration reaction, a dehydrogenation reaction, a substrate cleavage reaction and the like can be used. Further, protein phosphorylation, dephosphorylation, dimerization, multimerization, degradation, dissociation, etc., and amplification, cleavage, and extension of DNA and RNA can be used. For example, phosphorylation of a protein existing downstream of the signal transduction pathway can be used as a detection index. Changes in cell phenotype, such as quantitative and Z or qualitative changes in product, changes in proliferation activity, changes in cell number, changes in morphology, changes in characteristics, etc. should be used as indicators of cell lines. Can be. Examples of the produced substance include secretory proteins, surface antigens, intracellular proteins, mRNA, and the like. Changes in morphology include protrusion formation and changes in the number of Z or protrusions, changes in flatness, changes in elongation Z, changes in aspect ratio, changes in cell size, changes in internal structure, abnormalities in cell populations Z uniformity, changes in cell density, etc. can be used. These morphological changes can be confirmed by observation under a microscope. As the change in properties, scaffold dependency, site force-in response, hormone dependency, drug resistance, cell motility, cell migration activity, pulsatility, changes in intracellular substances, and the like can be used. Cell motility includes cell infiltration activity and cell migration activity. In addition, changes in intracellular substances include, for example, enzyme activity, mRNA level, intracellular signal transduction substances such as Ca2 + and cAMP, and intracellular protein levels. Can. In the case of a cell membrane receptor, a change in cell proliferation activity induced by receptor stimulation can be used as an index. As an index of the organization system, a change in function according to the organization to be used can be used as a detection index. Indices of the biological system include changes in tissue weight, changes in the blood system, such as changes in the number of blood cells, changes in protein levels, enzyme activities, and changes in electrolyzed mass, and changes in the circulatory system, such as blood pressure and heart rate. Can be used.
[0026] これらの検出指標を測定する方法としては、特に制限はなぐ吸光、発光、発色、蛍 光、放射活性、蛍光偏光度、表面プラズモン共鳴シグナル、時間分解蛍光度、質量 、吸収スペクトル、光散乱、蛍光共鳴エネルギー移動、等を用いることができる。これ らの測定方法は当業者にとっては周知であり、目的に応じて、適宜選択することがで きる。例えば、吸収スペクトルは一般的に用いられるフォトメータやプレートリーダ等、 発光はルミノメータ等、蛍光はフルォロメータ等で測定することができる。質量は質量 分析計を用いて測定することができる。放射活性は、放射線の種類に応じてガンマ力 ゥンターなどの測定機器を用いて、蛍光偏光度は BEACON (宝酒造）、表面ブラズモ ン共鳴シグナルは BIACORE、時間分解蛍光、蛍光共鳴エネルギー移動などは ARVOなどにより測定できる。さらに、フローサイトメータなども測定に用いることができ る。これらの測定方法は、一つの測定方法で 2種以上の検出指標を測定しても良ぐ 簡便であれば、 2種以上の測定を同時及び Z又は連続して測定することによりさらに 多数の検出指標を測定することも可能である。例えば、蛍光と蛍光共鳴エネルギー 移動を同時にフルォロメータで測定することができる。  [0026] Methods for measuring these detection indices include, but are not particularly limited to, absorption, luminescence, color development, fluorescence, radioactivity, fluorescence polarization, surface plasmon resonance signal, time-resolved fluorescence, mass, absorption spectrum, light Scattering, fluorescence resonance energy transfer, and the like can be used. These measurement methods are well known to those skilled in the art, and can be appropriately selected according to the purpose. For example, the absorption spectrum can be measured by a commonly used photometer or plate reader, the luminescence can be measured by a luminometer, and the fluorescence can be measured by a fluorimeter. Mass can be measured using a mass spectrometer. The radioactivity is measured using a measuring device such as a gamma force center according to the type of radiation. The degree of fluorescence polarization is BEACON (Takara Shuzo), the surface plasmon resonance signal is BIACORE, the time-resolved fluorescence, the fluorescence resonance energy transfer is ARVO, etc. Can be measured. Further, a flow cytometer or the like can be used for the measurement. In these measurement methods, it is sufficient to measure two or more types of detection indices by one measurement method.If it is convenient, two or more types of measurement can be performed simultaneously and Z or consecutively. It is also possible to measure an index. For example, fluorescence and fluorescence resonance energy transfer can be measured simultaneously with a fluorimeter.
[0027] 本発明の好ま 、態様の一つとして、低分子化抗体が挙げられる。低分子化抗体 は、全長抗体 (whole antibody,例えば whole IgG等)の一部分が欠損している抗体断 片を含み、抗原への結合能を有していれば特に限定されない。本発明における低分 子化抗体は、 whole抗体と比較して、顕著に高い活性を有する。本発明の抗体断片 は、全長抗体の一部分であれば特に限定されないが、重鎖可変領域 (VH)又は Z及 び軽鎖可変領域 (VL)を含んで 、ることが好ま 、。 VHまたは VLのアミノ酸配列は、 置換、欠失、付加及び Z又は挿入がされていてもよい。さらに抗原への結合能を有 する限り、 VH又は/及び VLの一部を欠損させてもよい。又、可変領域はキメラ化ゃヒ ト化されていてもよい。抗体断片の具体例としては、例えば、 Fab、 Fab'、 F(ab')2、 Fv などを挙げることができる。また、低分子化抗体の具体例としては、例えば、 Fab, Fab' 、 F(ab')2、 Fv、 scFv、single chain Fv)、 Diabody、 sc(Fv)2 (.single cnain (Fvノ 2)などを举 げることができる。 [0027] A preferred embodiment of the present invention includes a low molecular weight antibody. The low-molecular-weight antibody is not particularly limited as long as it includes an antibody fragment in which a part of a full-length antibody (eg, whole IgG, etc.) is deleted and has an antigen-binding ability. The low-molecular-weight antibody in the present invention has a significantly higher activity than the whole antibody. The antibody fragment of the present invention is not particularly limited as long as it is a part of a full-length antibody, but preferably contains a heavy chain variable region (VH) or Z and a light chain variable region (VL). The amino acid sequence of VH or VL may have substitutions, deletions, additions, and Z or insertions. Furthermore, VH and / or VL may be partially deleted as long as they have the ability to bind to the antigen. The variable region is chimerized. It may be converted to Specific examples of the antibody fragment include, for example, Fab, Fab ', F (ab') 2, Fv and the like. Specific examples of the low molecular weight antibody include, for example, Fab, Fab ', F (ab') 2, Fv, scFv, single chain Fv), Diabody, sc (Fv) 2 (.single cnain (Fv ) And the like.
[0028] ここで、「Fv」断片は最小の抗体断片であり、完全な抗原認識部位と結合部位を含 む。「Fv」断片は 1つの VHおよび VLが非共有結合により強く連結されたダイマー（ VH- VLダイマー）である。各可変領域の 3つの相補鎖決定領域（complementarity determining region ; CDR)が相互作用し、 VH-VLダイマーの表面に抗原結合部位を 形成する。 6つの CDRが抗体に抗原結合部位を付与している。しかしながら、 1つの 可変領域 (または、抗原に特異的な 3つの CDRのみを含む Fvの半分)であっても、全 結合部位よりも親和性は低いが、抗原を認識し、結合する能力を有する。  [0028] Here, the "Fv" fragment is the minimum antibody fragment and contains a complete antigen recognition site and a binding site. An “Fv” fragment is a dimer in which one VH and VL are tightly linked by non-covalent bonds (VH-VL dimer). The three complementarity determining regions (CDRs) of each variable region interact to form an antigen-binding site on the surface of the VH-VL dimer. Six CDRs confer an antigen-binding site on the antibody. However, even a single variable region (or half of an Fv that contains only three CDRs specific for an antigen) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site .
[0029] scFvには、抗体の VHおよび VLが含まれ、これらの領域は単一のポリペプチド鎖中 に存在する。一般に、 Fvポリペプチドはさらに VHおよび VLの間にポリペプチドリンカ 一を含んでおり、これにより scFvは、抗原結合のために必要な構造を形成することが できる（scFvの総説については、 Pluckthun『The Pharmacology of Monoclonal AntibomesJVol.l lj (Rosenburg and Moore ed (Springer Verlag, New York) pp.269-315, 1994)を参照)。本発明におけるリンカ一は、その両端に連結された抗 体可変領域の発現を阻害するものでなければ特に限定されない。  [0029] scFv includes VH and VL of an antibody, and these regions are present in a single polypeptide chain. In general, Fv polypeptides further include a polypeptide linker between the VH and VL, which allows the scFv to form the necessary structure for antigen binding (for a review of scFv, see Pluckthun et al. The Pharmacology of Monoclonal Antibomes JVol.l lj (see Rosenburg and Mooreed (Springer Verlag, New York) pp.269-315, 1994). The linker in the present invention is not particularly limited as long as it does not inhibit the expression of the antibody variable region linked to both ends.
[0030] Diabodyは、遺伝子融合により構築された二価 (bivalent)の抗体断片を指す (Holliger  [0030] Diabody refers to a bivalent antibody fragment constructed by gene fusion (Holliger
P et al, Proc.Natl.Acad.Sci.USA 90: 6444-6448 (1993)、 EP404,097号、  USA et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), EP404,097,
W093/11161号等)。 Diabodyは、 2本のポリペプチド鎖から構成されるダイマーであり 、通常、ポリペプチド鎖は各々、同じ鎖中で VL及び VH力 互いに結合できない位に 短い、例えば、 5残基程度のリンカ一により結合されている。同一ポリペプチド鎖上に コードされる VLと VHとは、その間のリンカ一が短いため単鎖可変領域フラグメントを 形成することが出来ず二量体を形成するため、 Diabodyは 2つの抗原結合部位を有 することとなる。  W093 / 11161). Diabody is a dimer composed of two polypeptide chains, and each of the polypeptide chains is usually short enough to be unable to bind to the VL and VH forces in the same chain, for example, by a linker of about 5 residues. Are combined. Since VL and VH encoded on the same polypeptide chain cannot form a single-chain variable region fragment due to the short linker between them and form a dimer, Diabody has two antigen-binding sites. Yes.
[0031] 本発明にお 、て、血小板減少症治療剤又は血小板増加剤に含まれる Mplを認識 する抗体の特に好まし 、態様としては、 sc(Fv)2を挙げることができる。 sc(Fv)2は、 2つの VH及び 2つの VLをリンカ一等で結合して一本鎖にした低分子化 抗体である（Hudson et al、 J Immunol. Methods 1999 ;231 : 177- 189)。 sc(Fv)2は、全 長抗体や他の低分子化抗体と比較して、特に高 、ァゴ二スト活性を示すことが本発 明者らにより見出されている。 sc(Fv)2は、例えば、 scFvをリンカ一で結ぶことによって 作製できる。 [0031] In the present invention, sc (Fv) 2 can be mentioned as a particularly preferred and embodiment of the antibody recognizing Mpl contained in the therapeutic agent for thrombocytopenia or the agent for increasing thrombocytopenia. sc (Fv) 2 is a low molecular weight antibody in which two VHs and two VLs are linked to each other with a linker or the like to form a single chain (Hudson et al., J Immunol. Methods 1999; 231: 177-189). . The present inventors have found that sc (Fv) 2 exhibits particularly high agonist activity as compared to full-length antibodies and other low molecular weight antibodies. sc (Fv) 2 can be produced, for example, by linking scFv with a linker.
[0032] また 2つの VH及び 2つの VL力 一本鎖ポリペプチドの N末端側を基点として VH、 [0032] Also, two VHs and two VL forces VH, starting from the N-terminal side of the single-chain polypeptide,
VL、 VH、 VL ( [VH]リンカ一 [VL]リンカ一 [VH]リンカ一 [VL])の順に並んでいること を特徴とする抗体が好ま ヽ。 Antibodies characterized by being arranged in the order of VL, VH, VL ([VH] linker-1 [VL] linker-1 [VH] linker-1 [VL]) are preferred.
[0033] 2つの VHと 2つの VLの順序は特に上記配置に限定されず、どのような順序で並べら れていてもよい。例えば以下のような、配置も挙げることができる。  [0033] The order of the two VHs and the two VLs is not particularly limited to the above arrangement, and may be arranged in any order. For example, the following arrangement can also be mentioned.
[VL]リンカ一 [VH]リンカ一 [VH]リンカ一 [VL]  [VL] Linker [VH] Linker [VH] Linker [VL]
[VH]リンカ一 [VL]リンカ一 [VL]リンカ一 [VH]  [VH] Linker [VL] Linker [VL] Linker [VH]
[VH]リンカ一 [VH]リンカ一 [VL]リンカ一 [VL]  [VH] Linker [VH] Linker [VL] Linker [VL]
[VL]リンカ一 [VL]リンカ一 [VH]リンカ一 [VH]  [VL] Linker [VL] Linker [VH] Linker [VH]
[VL]リンカ一 [VH]リンカ一 [VL]リンカ一 [VH]  [VL] Linker [VH] Linker [VL] Linker [VH]
[0034] 抗体の可変領域を結合するリンカ一としては、遺伝子工学により導入し得る任意の ペプチドリンカ一、又は合成化合物リンカ一（例えば、 Protein Engineering, 9(3), 299-305, 1996参照）に開示されるリンカ一等を用いることができる力 本発明におい てはペプチドリンカ一が好ましい。ペプチドリンカ一の長さは特に限定されず、 目的に 応じて当業者が適宜選択することが可能であるが、通常、 1〜100アミノ酸、好ましくは 3〜50アミノ酸、更に好ましくは 5〜30アミノ酸、特に好ましくは 12〜18アミノ酸 (例えば 、 15アミノ酸)である。  [0034] As the linker that binds to the variable region of the antibody, any peptide linker or synthetic compound linker that can be introduced by genetic engineering (see, for example, Protein Engineering, 9 (3), 299-305, 1996) In the present invention, a peptide linker is preferable. The length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art according to the purpose, but is usually 1 to 100 amino acids, preferably 3 to 50 amino acids, and more preferably 5 to 30 amino acids. Particularly preferably, it is 12 to 18 amino acids (for example, 15 amino acids).
[0035] 例えば、ペプチドリンカ一の場合：  For example, in the case of a peptide linker:
Ser  Ser
GlySer  GlySer
GlyGlySer  GlyGlySer
Sef ulyuly  Sef ulyuly
Gly GlyGlySer Ser'GlyGlyGly Gly GlyGlySer Ser'GlyGlyGly
Gly · Gly · Gly · Gly · Ser  Gly · Gly · Gly · Gly · Ser
Ser · Gly · Gly · Gly · Gly  Ser · Gly · Gly · Gly · Gly
Gly · Gly · Gly · Gly · Gly · Ser  Gly · Gly · Gly · Gly · Gly · Ser
Ser · Gly · Gly · Gly · Gly · Gly  Ser · Gly · Gly · Gly · Gly · Gly
Gly · Gly · Gly · Gly · Gly · Gly · Ser  Gly · Gly · Gly · Gly · Gly · Gly · Ser
Ser · Gly · Gly · Gly · Gly · Gly · Gly  Ser · Gly · Gly · Gly · Gly · Gly · Gly
(Gly · Gly · Gly · Gly · Ser)n  (GlyGlyGlyGlySer) n
(Ser · Gly · Gly · Gly · Gly)n  (SerGlyGlyGlyGly) n
[nは 1以上の整数である]等を挙げることができる。但し、ペプチドリンカ一の長さや 配列は目的に応じて当業者が適宜選択することができる。  [n is an integer of 1 or more]. However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art according to the purpose.
[0036] よって本発明において特に好ましい sc(Fv)2の態様としては、例えば、以下の  [0036] Therefore, particularly preferred embodiments of sc (Fv) 2 in the present invention include, for example, the following:
sc(Fv)2を挙げることができる。  sc (Fv) 2.
[VH]ペプチドリンカ一 (15アミノ酸) [VL]ペプチドリンカ一 (15アミノ酸) [VH]ペプチド リンカ一 (15アミノ酸) [VL]  [VH] Peptide linker (15 amino acids) [VL] Peptide linker (15 amino acids) [VH] Peptide linker (15 amino acids) [VL]
[0037] 合成化学物リンカ一 (ィ匕学架橋剤）は、ペプチドの架橋に通常用いられている架橋 剤、例えば N-ヒドロキシスクシンイミド（NHS)、ジスクシンイミジルスべレート（DSS)、ビ ス（スルホスクシンィミジル)スべレート（BS3)、ジチォビス（スクシンィミジルプロビオネ ート）（DSP)、ジチオピス（スルホスクシンィミジルプロピオネート）（DTSSP)、エチレン グリコールビス（スクシンイミジルスクシネート）（EGS)、エチレングリコールビス（スルホ スクシンイミジルスクシネート）（スルホー EGS)、ジスクシンィミジル酒石酸塩（DST)、 ジスルホスクシンィミジル酒石酸塩 (スルホー DST)、ビス [2- (スクシンイミドォキシカ ルポ-ルォキシ）ェチル]スルホン（BSOCOES)、ビス [2- (スルホスクシンイミドォキシ カルボ-ルォキシ）ェチル]スルホン (スルホ- BSOCOES)などであり、これらの架橋剤 は市販されている。 [0037] Synthetic chemical compound linkers (crosslinking agents) are commonly used crosslinking agents for peptide crosslinking, for example, N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis (Sulfosuccinimidyl) suberate (BS 3 ), dithiobis (succinimidyl propionate) (DSP), dithiopis (sulfosuccinimidyl propionate) (DTSSP), ethylene glycol Bis (succinimidyl succinate) (EGS), ethylene glycol bis (sulfosuccinimidyl succinate) (sulfo EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (Sulfo DST), bis [2- (succinimidoxylcarboxy) ethyl] sulfone (BSOCOES), bis [2- (sulfosuccinimidoxylcarboxy-loxy) ethyl] sulfo (Sulfo - BSOCOES) and the like, these crosslinking agents are commercially available.
[0038] 4つの抗体可変領域を結合する場合には、通常、 3つのリンカ一が必要となる力 全 て同じリンカ一を用いてもよいし、異なるリンカ一を用いてもよい。本発明において好 ましい低分子化抗体は Diabody又は sc(Fv)2であり、特に好ましくは sc(Fv)2である。こ のような低分子化抗体を得るには、抗体を酵素、例えば、パパイン、ペプシンなどで 処理し、抗体断片を生成させるか、又はこれら抗体断片をコードする DNAを構築し、 これを発現ベクターに導入した後、適当な宿主細胞で発現させればよい（例えば、 Co, M. S. et al, J. Immunol. (1994) 152, 2968-2976； Better, M. and Horwitz, A. H., Methods Enzymol. (1989) 178, 476 - 496； Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515； Lamoyi, E.， Methods Enzymol. (1986) 121, 652-663； Rousseaux, J. et al" Methods Enzymol. (1986) 121, 663—669； Bird, R. E. and Walker, B. W" Trends Biotechnol. (1991) 9, 132- 137参照）。 [0038] When four antibody variable regions are to be bound, usually, all linkers that require three linkers may be the same or different linkers may be used. The preferred low molecular weight antibody in the present invention is Diabody or sc (Fv) 2, and particularly preferably sc (Fv) 2. This In order to obtain a low molecular weight antibody such as described above, the antibody is treated with an enzyme such as papain or pepsin to generate an antibody fragment, or a DNA encoding these antibody fragments is constructed, and this is used as an expression vector. After introduction, expression may be carried out in an appropriate host cell (for example, Co, MS et al, J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, AH, Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663; Rousseaux, J. et al "Methods Enzymol. (1986) 121, 663-669; Bird, RE and Walker, B. W" Trends Biotechnol. (1991) 9, 132-137).
[0039] sc(Fv)2ィ匕された抗 Mpl抗体は、 Mplに対して特に高いァゴニスト活性を有するので、 血小板減少症治療剤又は血小板増加剤として特に有用である。  The anti-Mpl antibody sc (Fv) 2 has particularly high agonist activity against Mpl, and is therefore particularly useful as a therapeutic agent for thrombocytopenia or an agent for increasing thrombocytopenia.
[0040] 本発明にお ヽて、血小板減少症治療剤又は血小板増加剤に含まれる Mplを認識 する抗体の好まし ヽ態様としては、キメラ抗体又はヒト化抗体等の改変抗体を挙げる ことができ、特にヒト化抗体を挙げることができる。  [0040] In the present invention, preferred examples of the antibody that recognizes Mpl contained in the therapeutic agent for thrombocytopenia or the agent for increasing thrombocytopenia include modified antibodies such as chimeric antibodies and humanized antibodies. And especially humanized antibodies.
[0041] キメラ抗体は、異なる動物由来の配列を組み合わせて作製される抗体であり、例え ば、マウス抗体の重鎖、軽鎖の可変領域とヒト抗体の重鎖、軽鎖の定常領域からなる 抗体などである。キメラ抗体の作製は公知の方法を用いて行うことができ、例えば、抗 体 V領域をコードする DNAとヒト抗体 C領域をコードする DNAとを連結し、これを発現 ベクターに組み込んで宿主に導入し産生させることにより得られる。  [0041] A chimeric antibody is an antibody produced by combining sequences derived from different animals. For example, the chimeric antibody is composed of the variable regions of the heavy and light chains of a mouse antibody and the constant regions of the heavy and light chains of a human antibody. Antibodies. A chimeric antibody can be prepared by a known method.For example, a DNA encoding the antibody V region and a DNA encoding the human antibody C region are ligated, inserted into an expression vector, and introduced into a host. And produced.
[0042] ヒト化抗体は、再構成 (reshaped)ヒト抗体とも称され、これは、ヒト以外の哺乳動物、 例えばマウス抗体の相補性決定領域（CDR; complementarity determining region)を ヒト抗体の相補性決定領域へ移植したものであり、その一般的な遺伝子組換え手法 も知られている（欧州特許出願公開番号 EP 125023号公報、 WO 96/02576号公報 参照)。  [0042] The humanized antibody is also referred to as a reshaped human antibody, and is used to determine the complementarity determining region (CDR) of a non-human mammal, for example, a mouse antibody. It has been transplanted into a region, and its general genetic recombination technique is also known (see European Patent Application Publication No. EP 125023, WO 96/02576).
[0043] 具体的には、マウス抗体の CDRとヒト抗体のフレームワーク領域（framework region ;  Specifically, a CDR of a mouse antibody and a framework region of a human antibody (framework region;
FR)とを連結するように設計した DNA配列を、 CDR及び FR両方の末端領域にオーバ 一ラップする部分を有するように作製した数個のオリゴヌクレオチドをプライマーとして 用いて PCR法により合成する (W098/13388号公報に記載の方法を参照)。  A DNA sequence designed to ligate to FR) is synthesized by a PCR method using as primers several oligonucleotides prepared to have overlapping portions in both CDR and FR terminal regions (W098 / 13388).
[0044] CDRを介して連結されるヒト抗体のフレームワーク領域は、相補性決定領域が良好 な抗原結合部位を形成するものが選択される。必要に応じ、再構成ヒト抗体の相補 性決定領域が適切な抗原結合部位を形成するように、抗体の可変領域におけるフレ ームワーク領域のアミノ酸を置換してもよい（Sato, K.etal., CancerRes. (1993) 53, 851-856)。 [0044] The framework region of the human antibody linked via the CDR has a favorable complementarity determining region. Those that form an antigen-binding site are selected. If necessary, amino acids in the framework region in the variable region of the antibody may be substituted so that the complementarity determining region of the reshaped human antibody forms an appropriate antigen-binding site (Sato, K. et al., CancerRes. (1993) 53, 851-856).
[0045] キメラ抗体及びヒト型化抗体の定常領域には、ヒト抗体のものが使用され、例えば H 鎖では、 C γ 1、 C γ 2、 C γ 3、 C γ 4を、 L鎖では C κ、 C λを使用することができる。ま た、抗体またはその産生の安定性を改善するために、ヒト抗体定常領域を修飾しても よい。  For the constant regions of chimeric and humanized antibodies, those of human antibodies are used. For example, C γ 1, C γ 2, C γ 3, and C γ 4 are used for the H chain, and C κ, C λ can be used. In addition, the constant region of the human antibody may be modified to improve the stability of the antibody or its production.
[0046] 一般的に、キメラ抗体は、ヒト以外の哺乳動物由来抗体の可変領域とヒト抗体由来 の定常領域とからなる。一方、ヒト化抗体は、ヒト以外の哺乳動物由来抗体の相補性 決定領域と、ヒト抗体由来のフレームワーク領域および定常領域とからなる。  [0046] In general, a chimeric antibody is composed of a variable region of an antibody derived from a mammal other than human and a constant region derived from a human antibody. On the other hand, a humanized antibody is composed of a complementarity determining region of an antibody derived from a mammal other than human, a framework region and a constant region derived from a human antibody.
[0047] なお、キメラ抗体やヒト化抗体を作製した後に、可変領域 (例えば、 FR)や定常領域 中のアミノ酸を他のアミノ酸で置換等してもょ 、。 [0047] After producing a chimeric antibody or a humanized antibody, amino acids in the variable region (eg, FR) or the constant region may be substituted with another amino acid.
キメラ抗体における可変領域、又はヒト化抗体における CDRの由来は特に限定され ず、どのような動物由来でもよい。例えば、マウス抗体、ラット抗体、ゥサギ抗体、ラタ ダ抗体などの配列を用いることが可能である。  The origin of the variable region in the chimeric antibody or the CDR in the humanized antibody is not particularly limited, and may be from any animal. For example, a sequence of a mouse antibody, a rat antibody, a rabbit antibody, a ratada antibody and the like can be used.
[0048] 抗体のキメラ化ゃヒト化において、通常、由来となった抗体のァゴニスト活性を維持 したままキメラ化やヒト化を行うことは困難であるが、本発明においては、マウス抗体と 同等のァゴニスト活性を有するヒト化抗体の取得に成功した。 In antibody chimerization ゃ humanization, it is usually difficult to carry out chimerization or humanization while maintaining the agonist activity of the derived antibody, but in the present invention, it is as effective as a mouse antibody. A humanized antibody having agonist activity was successfully obtained.
[0049] Mplを認識するヒト化抗体の例としては、後述の（9)一（19)に記載のヒト化抗体を挙 げることができる。  [0049] Examples of humanized antibodies that recognize Mpl include the humanized antibodies described in (9)-(19) below.
キメラ抗体やヒト化抗体はヒト体内における抗原性が低下しているため、ヒトに投与 する場合に特に有用であり、血小板減少症治療剤又は血小板増加剤として有用で ある。  Since chimeric antibodies and humanized antibodies have reduced antigenicity in the human body, they are particularly useful when administered to humans, and are useful as therapeutic agents for thrombocytopenia or platelet increasing agents.
[0050] 本発明にお ヽて、血小板減少症治療剤又は血小板増加剤に含まれる Mplを認識 する抗体の好まし 、態様としては、可溶型 Mplに結合する抗体を挙げることができる。 ここでいう可溶型 Mplとは、細胞膜上に発現している Mpl以外の Mplのことをいう。可溶 型 Mplの具体的な例としては、膜貫通領域の一部又は全部が欠損して 、る Mplを挙 げることができる。ヒト Mplの場合、膜貫通領域は配列番号： 66において 492番目のァ ミノ酸〜 513番目のアミノ酸の部分が相当する。 [0050] In the present invention, preferred examples of the antibody that recognizes Mpl contained in the therapeutic agent for thrombocytopenia or the agent for increasing thrombocytopenia include an antibody that binds to soluble Mpl. Here, the soluble Mpl refers to an Mpl other than the Mpl expressed on the cell membrane. Specific examples of soluble Mpl include Mpl in which a part or all of the transmembrane region is missing. I can make it. In the case of human Mpl, the transmembrane region corresponds to the portion from amino acid 492 to amino acid 513 in SEQ ID NO: 66.
[0051] 可溶型組換え Mplに結合する抗体は、ェピトープの詳細な解析や結合における反 応速度論的解析に利用できるだけでなぐ in vivo試験における血中濃度や体内動態 を評価することにも有用である。  [0051] Antibodies that bind to soluble recombinant Mpl can be used not only for detailed analysis of epitopes and for reaction kinetic analysis of binding, but also for evaluation of blood concentrations and pharmacokinetics in in vivo tests. Useful.
[0052] 本発明にお ヽて、血小板減少症治療剤又は血小板増加剤に含まれる Mplを認識 する抗体の好まし 、態様としては、ヒト Mplとサル Mplの両方に対して結合活性又はァ ゴニスト活性を有する抗体を挙げることができる。ヒト Mplとサル Mplの両方に対してァ ゴニスト活性を有する抗体は、通常、ヒトにおいて測定することが困難な体内動態や in vivoでの効果を、サルを用いて検証できることから、非常に有用であると考えられる 。これらの抗体は、さらに、ヒト及びサル以外の動物（例えば、マウスなど）の Mplに対 して、結合活'性ゃァゴニスト活'性を有していてもよい。  [0052] In the present invention, an antibody recognizing Mpl contained in a therapeutic agent for thrombocytopenia or an agent for increasing thrombocytopenia is preferred. An antibody having activity can be mentioned. Antibodies that have agonist activity against both human Mpl and monkey Mpl are very useful because monkeys can verify the pharmacokinetics and in vivo effects that are usually difficult to measure in humans. It is believed that there is . These antibodies may further have binding activity 'active agonist activity' against Mpl of animals other than humans and monkeys (eg, mice).
[0053] さらに本発明にお 、て、血小板減少症治療剤又は血小板増加剤に含まれる Mplを 認識する抗体の好ま 、態様としては、 TPOァゴ-スト活性 (Mplに対するァゴ-スト 活性）力 ¾C50=100nM以下、好ましくは EC50=30nM以下、さらに好ましくは  [0053] Further, in the present invention, an antibody recognizing Mpl contained in a therapeutic agent for thrombocytopenia or a thrombocytopenic agent is preferred, and as an embodiment, TPO agonist activity (agonist activity against Mpl) Force ¾C50 = 100 nM or less, preferably EC50 = 30 nM or less, more preferably
EC50=10nM以下である抗体を挙げることができる。  An antibody having an EC50 of 10 nM or less can be mentioned.
[0054] ァゴ-スト活性の測定方法は、当業者に公知の方法により行うことが可能であり、例 えば、後述する方法により行うことが可能である。  [0054] The method for measuring the agonist activity can be performed by a method known to those skilled in the art, for example, the method described below.
さらに本発明にお 、て、血小板減少症治療剤又は血小板増加剤に含まれる Mplを 認識する抗体の好ま 、態様としては、可溶型 Mplへの結合活性力 D=10— 6M以下、 好ましくは KD=10— 7M以下、さらに好ましくは KD=10— 8M以下の抗体を挙げることができ る。 Further Te you, the present invention, 'preferred Mpl recognize antibodies contained in thrombocytopenia therapeutic or platelet-increasing agent, as the embodiment, the binding activity force D = 10- 6 M or less to soluble Mpl, preferably the KD = 10- 7 M or less, more preferably Ru can be mentioned KD = 10- 8 M following antibodies.
[0055] 本発明において、可溶型組換え Mplへの結合活性が KD=10—6M以下の抗体である か否かは、当業者に公知の手段を使用して測定することができる。例えば、 BIAcore を用いた表面プラズモン共鳴を利用して測定することが可能である。すなわち Sensor Chip上に可溶型 MPL-Fc蛋白質を固定させ、抗体と可溶型 Mp卜 Fcの相互作用を測 定値カも反応速度定数として算出することができる。また、結合活性の評価には、 ELISA (酵素結合免疫吸着検定法)、 EIA (酵素免疫測定法)、 RIA (放射免疫測定法 )あるいは蛍光抗体法を用いることができる。例えば、酵素免疫測定法を用いる場合 、被験抗体が結合する抗原をコーティングしたプレートに、被験抗体を含む試料、例 えば、被験抗体産生細胞の培養上清や精製抗体を加える。アルカリフォスファターゼ 等の酵素で標識した二次抗体を添加し、プレートをインキュベートし、洗浄した後、 P- ニトロフ ニル燐酸などの酵素基質を加えて吸光度を測定することで抗原結合活性 を評価することができる。 [0055] In the present invention, whether binding activity to soluble recombinant Mpl is KD = 10- 6 M or less of an antibody can be measured using means known to those skilled in the art. For example, it can be measured using surface plasmon resonance using BIAcore. That is, the soluble MPL-Fc protein is immobilized on the Sensor Chip, and the interaction between the antibody and the soluble Mpt-Fc can be measured as a reaction rate constant. In addition, ELISA (enzyme-linked immunosorbent assay), EIA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay) ) Or the fluorescent antibody method. For example, when using an enzyme immunoassay, a sample containing a test antibody, for example, a culture supernatant of a test antibody-producing cell or a purified antibody is added to a plate coated with an antigen to which the test antibody binds. After adding a secondary antibody labeled with an enzyme such as alkaline phosphatase, incubating the plate, washing, and adding an enzyme substrate such as P-nitrophenyl phosphate, the antigen binding activity can be evaluated by measuring the absorbance. it can.
[0056] 結合活性の上限は特に限定されないが、例えば、当業者が技術的に作製可能な 範囲の上限を設定することができる。しかしながら、技術的に作製可能な範囲は、技 術の進歩により拡大される。  [0056] The upper limit of the binding activity is not particularly limited. For example, the upper limit of the range that can be technically prepared by those skilled in the art can be set. However, the range that can be produced technically is expanded by technological advances.
[0057] 本発明にお ヽて、血小板減少症治療剤又は血小板増加剤に含まれる Mplを認識 する抗体の好ましい態様としては、以下の（1)〜（19)のいずれかに記載の抗体を挙 げることができる。（1)〜（19)のいずれかに記載の抗体は好ましくは低分子化抗体 である。  In a preferred embodiment of the antibody that recognizes Mpl contained in the therapeutic agent for thrombocytopenia or the agent for increasing thrombocytopenia according to the present invention, the antibody according to any one of the following (1) to (19) is used. Can be listed. The antibody according to any one of (1) to (19) is preferably a low-molecular-weight antibody.
[0058] (1)配列番号1、 2、 3 (VB22B :VH CDRl, 2、 3)に記載のアミノ酸配列からなる CDRl (1) CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, 2, 3 (VB22B: VH CDR1, 2, 3)
、 2、 3を有する VHを含む抗体。 2. An antibody comprising VH having 2,3.
[0059] (2)配列番号 4、 5、 6 (VB22B :VL CDRl, 2、 3)に記載のアミノ酸配列からなる CDRl(2) CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, 5, 6 (VB22B: VL CDR1, 2, 3)
、 2、 3を有する VLを含む抗体。 An antibody comprising VL, having 2, 3.
[0060] (3)配列番号： 1、 2、 3 (VB22B :VH CDRl, 2、 3)に記載のアミノ酸配列からなる(3) Consists of the amino acid sequence described in SEQ ID NO: 1, 2, 3 (VB22B: VH CDR1, 2, 3)
CDR1、 2、 3を有する VH、および配列番号： 4、 5、 6 (VB22B :VL CDRl, 2、 3)に記載 のアミノ酸配列力 なる CDR1、 2、 3を有する VLを含む抗体。 An antibody comprising a VH having CDRs 1, 2, and 3, and a VL having CDRs 1, 2, and 3 having the amino acid sequences described in SEQ ID NOs: 4, 5, and 6 (VB22B: VL CDR1, 2, and 3).
[0061] (4)配列番号： 8 (VB22B :VH)に記載のアミノ酸配列力 なる VHを含む抗体。 (4) An antibody comprising a VH having an amino acid sequence described in SEQ ID NO: 8 (VB22B: VH).
[0062] (5)配列番号： 10 (VB22B :VL)に記載のアミノ酸配列からなる VLを含む抗体。 (5) An antibody comprising a VL consisting of the amino acid sequence of SEQ ID NO: 10 (VB22B: VL).
[0063] (6)配列番号： 8 (VB22B： VH)に記載のアミノ酸配列力 なる VH、および配列番号：(6) VH having the amino acid sequence described in SEQ ID NO: 8 (VB22B: VH), and SEQ ID NO:
10 (VB22B :VL)に記載のアミノ酸配列からなる VLを含む抗体。 10 (VB22B: VL).
[0064] (7)配列番号： 12 (VB22B： scFv)に記載のアミノ酸配列を有する抗体。 (7) An antibody having the amino acid sequence of SEQ ID NO: 12 (VB22B: scFv).
[0065] (8)配列番号： 14 (VB22B： sc(Fv)2)に記載のアミノ酸配列を有する抗体。 (8) An antibody having the amino acid sequence of SEQ ID NO: 14 (VB22B: sc (Fv) 2).
[0066] (9)以下の (a)〜(c)のいずれかに記載のアミノ酸配列からなる、 FR1、 2、 3、 4を有する(9) having FR1, 2, 3, or 4 consisting of the amino acid sequence of any one of the following (a) to (c):
VHを含むヒト化抗体。 (a)配列番号： 15、 16、 17、 18 (hVB22B p- z :VH FR1、 2、 3、 4) Humanized antibodies containing VH. (a) SEQ ID NO: 15, 16, 17, 18 (hVB22B p-z: VH FR1, 2, 3, 4)
(b)配列番号： 19、 20、 21、 22 (hVB22B g- e :VH FR1、 2、 3、 4)  (b) SEQ ID NO: 19, 20, 21, 22 (hVB22B g-e: VH FR1, 2, 3, 4)
(c)配列番号： 23、 24、 25、 26 (hVB22B e:VH FR1、 2、 3、 4)  (c) SEQ ID NOS: 23, 24, 25, 26 (hVB22B e: VH FR1, 2, 3, 4)
(d)配列番号： 81、 82、 83、 84 (hVB22B u2-wz4:VH FR1、 2、 3、 4)  (d) SEQ ID NO: 81, 82, 83, 84 (hVB22B u2-wz4: VH FR1, 2, 3, 4)
(e)配列番号： 81、 82、 85、 84 (hVB22B q- wz5 :VH FR1、 2、 3、 4)  (e) SEQ ID NO: 81, 82, 85, 84 (hVB22B q-wz5: VH FR1, 2, 3, 4)
[0067] (10)以下の (a)または (b)に記載のアミノ酸配列からなる、 FR1、 2、 3、 4を有する VLを 含むヒト化抗体。  (10) A humanized antibody comprising a VL having FR1, 2, 3, or 4, comprising the amino acid sequence of (a) or (b) below.
(a)配列番号: 27、 28、 29、 30 (hVB22B p-z :VL FR1、 2、 3、 4)  (a) SEQ ID NO: 27, 28, 29, 30 (hVB22B p-z: VL FR1, 2, 3, 4)
(b)配列番号: : 31、 32、 33、 34 (hVB22B g- eまたは hVB22B e :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)配列番号: 86、 87、 88、 89 (hVB22B u2-wz4:VL FR1、 2、 3、 4) (C) SEQ ID NO: 86, 87, 88, 89 (hVB22B u2-wz4: VL FR1, 2, 3, 4)
(d)配列番号: : 86、 90、 91、 89 (hVB22B q-wz5 :VL FR1、 2、 3、 4)  (d) SEQ ID NO :: 86, 90, 91, 89 (hVB22B q-wz5: VL FR1, 2, 3, 4)
[0068] (11)以下の (a)〜(c)のいずれかに記載の VHおよび VLを含むヒト化抗体。  (11) A humanized antibody comprising VH and VL described in any of the following (a) to (c):
(a)配列番号： 15、 16、 17、 18に記載のアミノ酸配列からなる、 FR1、 2、 3、 4を有する VH、および酉己歹 IJ番号： 27, 28, 29, 30に記載のアミノ酸酉己歹 IJ力らなる、 FR1、 2、 3、 4 を有する VL  (a) VH having FR1, 2, 3, or 4 consisting of the amino acid sequence of SEQ ID NO: 15, 16, 17, or 18, and the amino acid of IJ No. 27, 28, 29 or 30 VL with FR1, 2, 3 and 4
(b)配列番号： 19、 20、 21、 22に記載のアミノ酸配列力もなる、 FR1、 2、 3、 4を有する VH、および配列番号： 31、 32、 33、 34に記載のアミノ酸配列からなる、 FR1、 2、 3、 4 を有する VL  (b) consisting of VH having FR1, 2, 3, and 4 and amino acid sequence described in SEQ ID NOs: 31, 32, 33, and 34, which also have the amino acid sequence described in SEQ ID NOs: 19, 20, 21, and 22 VL with, FR1,2,3,4
(c)配列番号： 23、 24、 25、 26に記載のアミノ酸配列からなる、 FR1、 2、 3、 4を有する VH、および配列番号： 31、 32、 33、 34に記載のアミノ酸配列からなる、 FR1、 2、 3、 4 を有する VL  (c) VH having FR1, 2, 3, 4 consisting of the amino acid sequence of SEQ ID NO: 23, 24, 25, 26, and consisting of the amino acid sequence of SEQ ID NO: 31, 32, 33, 34 VL with, FR1,2,3,4
(d)配列番号： 81、 82、 83、 84に記載のアミノ酸配列力もなる、 FR1、 2、 3、 4を有する VH、および酉己歹 IJ番号： 86、 87、 88、 89に記載のアミノ酸酉己歹 IJ力らなる、 FR1、 2、 3、 4 を有する VL  (d) VH having FR1, 2, 3, 4 and amino acid described in SEQ ID NO: 81, 82, 83, 84, and IJ number: 86, 87, 88, 89 VL with FR1, 2, 3 and 4
(e)配列番号： 81、 82、 85、 84に記載のアミノ酸配列からなる、 FR1、 2、 3、 4を有する VH、および酉己歹 IJ番号： 86、 90、 91、 89に記載のアミノ酸酉己歹 IJ力らなる、 FR1、 2、 3、 4 を有する VL  (e) VH having FR1, 2, 3, 4 comprising the amino acid sequence of SEQ ID NO: 81, 82, 85, 84, and the amino acid of IJ No. 86, 90, 91, 89 VL with FR1, 2, 3 and 4
[0069] (12)配列番号： 1、 2、 3に記載のアミノ酸配列力 なる CDR1、 2、 3を有する VHを含 むヒト化抗体。 (12) VH having CDR1, 2, and 3 having the amino acid sequences described in SEQ ID NOs: 1, 2, and 3 Humanized antibodies.
[0070] (13)配列番号: 4、 5、 6に記載のアミノ酸配列からなる CDR1、 2、 3を有する VLを含 むヒト化抗体。  (13) A humanized antibody comprising a VL having CDRs 1, 2, and 3 consisting of the amino acid sequences of SEQ ID NOs: 4, 5, and 6.
[0071] (14)配列番号： 1、 2、 3に記載のアミノ酸配列力 なる CDR1、 2、 3を有する VH、およ び配列番号: 4、 5、 6に記載のアミノ酸配列力 なる CDR1、 2、 3を有する VL含むヒト 化抗体。  (14) VH having CDR1, 2, or 3 having the amino acid sequence described in SEQ ID NO: 1, 2, or 3, and CDR1 having the amino acid sequence described in SEQ ID NO: 4, 5, or 6 A humanized antibody containing VL having 2,3.
[0072] (15)配列番号： 51 (hVB22B p- z :VH)、配列番号： 53 (hVB22B g- e:VH)、配列番 号： 55 (hVB22B e :VH)、配列番号： 92 (hVB22B u2- wz4:VH)、あるいは配列番号： 93 (hVB22B q-wz5 :VH)に記載のアミノ酸配列からなる VHを含むヒト化抗体。  (15) SEQ ID NO: 51 (hVB22B p-z: VH), SEQ ID NO: 53 (hVB22B g-e: VH), SEQ ID NO: 55 (hVB22B e: VH), SEQ ID NO: 92 (hVB22B u2-wz4: VH) or a humanized antibody comprising VH consisting of the amino acid sequence of SEQ ID NO: 93 (hVB22B q-wz5: VH).
[0073] (16)配列番号： 57 (hVB22B p- z :VL)、配列番号： 59 (hVB22B g- e:VLあるいは hVB22B e :VL)、配列番号： 94 (hVB22B u2- wz4:VL)、あるいは配列番号： 95 ( hVB22B q-wz5 :VH)に記載のアミノ酸配列力もなる VLを含むヒトイ匕抗体。  (16) SEQ ID NO: 57 (hVB22B p-z: VL), SEQ ID NO: 59 (hVB22B g-e: VL or hVB22B e: VL), SEQ ID NO: 94 (hVB22B u2-wz4: VL), Alternatively, a human antibody containing VL which also has the amino acid sequence power described in SEQ ID NO: 95 (hVB22B q-wz5: VH).
[0074] (17)以下の (a)〜(c)のいずれかに記載の VHおよび VLを含むヒト化抗体。  (17) A humanized antibody comprising VH and VL described in any of the following (a) to (c):
(a)配列番号： 51 (hVB22B p-z :VH)に記載のアミノ酸配列からなる VH、及び配列番 号： 57 (hVB22B p-z :VL)に記載のアミノ酸配列からなる VL  (a) VH consisting of the amino acid sequence described in SEQ ID NO: 51 (hVB22B p-z: VH), and VL consisting of the amino acid sequence described in SEQ ID NO: 57 (hVB22B p-z: VL)
(b)配列番号： 53 (hVB22B g-e :VH)に記載のアミノ酸配列からなる VH、及び配列番 号： 59 (hVB22B g- e:VLあるいは hVB22B e :VL)に記載のアミノ酸配列からなる VL (b) VH consisting of the amino acid sequence of SEQ ID NO: 53 (hVB22Bg-e: VH), and VL consisting of the amino acid sequence of SEQ ID NO: 59 (hVB22Bg-e: VL or hVB22Be: VL)
(c)配列番号： 55 (hVB22B e:VH)に記載のアミノ酸配列力 なる VH、及び配列番号: 59 (hVB22B g- e:VLあるいは hVB22B e :VL)に記載のアミノ酸配列からなる VL(c) VH having the amino acid sequence described in SEQ ID NO: 55 (hVB22B e: VH), and VL consisting of the amino acid sequence described in SEQ ID NO: 59 (hVB22B g-e: VL or hVB22B e: VL)
(d)配列番号： 92 (hVB22B u2-wz4:VH)に記載のアミノ酸配列からなる VH、及び配 列番号： 94 (hVB22B u2-wz4:VL)に記載のアミノ酸配列からなる VL (d) VH consisting of the amino acid sequence described in SEQ ID NO: 92 (hVB22Bu2-wz4: VH), and VL consisting of the amino acid sequence described in SEQ ID NO: 94 (hVB22Bu2-wz4: VL)
(e)配列番号： 93 (hVB22B q-wz5 :VH)に記載のアミノ酸配列からなる VH、及び配列 番号： 95 (hVB22B q-wz5 :VL)に記載のアミノ酸配列からなる VL  (e) VH consisting of the amino acid sequence described in SEQ ID NO: 93 (hVB22B q-wz5: VH), and VL consisting of the amino acid sequence described in SEQ ID NO: 95 (hVB22B q-wz5: VL)
[0075] 配列番号： 51 (hVB22B p- z： VH)、配列番号： 53 (hVB22B g- e： VH)、配列番号： 5 5 (hVB22B e :VH)、配列番号： 92 (hVB22B u2- wz4:VH)、または配列番号： 93 ( hVB22B q-wz5 :VH)に記載のアミノ酸配列において、  [0075] SEQ ID NO: 51 (hVB22B p-z: VH), SEQ ID NO: 53 (hVB22B g-e: VH), SEQ ID NO: 55 (hVB22B e: VH), SEQ ID NO: 92 (hVB22B u2-wz4) : VH) or the amino acid sequence of SEQ ID NO: 93 (hVB22B q-wz5: VH),
アミノ酸部位： 31〜35が CDR1、  Amino acid positions: 31 to 35 are CDR1,
アミノ酸咅 立： 50〜66力 ^CDR2、 アミノ酸咅 M立： 99〜107力 ^CDR3、 Amino acids: 50-66 ^ CDR2, Amino acid 咅 M standing: 99-107 force ^ CDR3,
アミノ酸部位： 1〜30が FR1、  Amino acid positions: 1 to 30 are FR1,
アミノ酸部位： 36〜49力 FR2、  Amino acid site: 36-49 force FR2,
アミノ酸部位： 67〜98力 FR3、  Amino acid site: 67-98 force FR3,
アミノ酸部位： 108〜118が FR4に相当する。  Amino acid positions: 108 to 118 correspond to FR4.
[0076] 又、配列番号： 57(hVB22B p- z:VL)または配列番号： 59(hVB22B g- e:VLまたは hVB22B e:VL)、配列番号： 94(hVB22B u2- wz4:VL)、あるいは配列番号： 95 ( hVB22B q-wz5:VH)に記載のアミノ酸配列において、 [0076] Also, SEQ ID NO: 57 (hVB22B p-z: VL) or SEQ ID NO: 59 (hVB22B g-e: VL or hVB22B e: VL), SEQ ID NO: 94 (hVB22B u2-wz4: VL), or In the amino acid sequence of SEQ ID NO: 95 (hVB22B q-wz5: VH),
アミノ酸部位： 24〜39が CDR1、  Amino acid positions: 24-39 for CDR1,
アミノ酸咅 M立： 55〜61力 ^CDR2、  Amino acid 咅 M standing: 55 ~ 61 force ^ CDR2,
アミノ酸部位： 94〜102力 SCDR3、  Amino acid site: 94-102 force SCDR3,
アミノ酸部位： 1〜23が FR1、  Amino acid positions: 1 to 23 are FR1,
ア 酸部位： 40〜54力 FR2、  A acid part: 40-54 force FR2,
アミノ酸部位： 62〜93力 FR3、  Amino acid site: 62-93 force FR3,
アミノ酸部位： 103〜112が FR4に相当する。  Amino acid positions: 103 to 112 correspond to FR4.
[0077] 本発明において、 hVB22B p-z VH配列における CDRおよび FRと配列番号との対 応は以下の通りである。 [0077] In the present invention, the correspondence between CDR and FR in the hVB22B p-z VH sequence and SEQ ID NOs is as follows.
hVB22B p-z VH: FRIZ配列番号： 15  hVB22B p-z VH: FRIZ SEQ ID NO: 15
hVB22B p-z VH:CDRlZ配列番号： 1  hVB22B p-z VH: CDRlZ SEQ ID NO: 1
hVB22B p-z VH:FR2Z配列番号： 16  hVB22B p-z VH: FR2Z SEQ ID NO: 16
hVB22B p-z VH： CDR2/配列番号： 2  hVB22B p-z VH: CDR2 / SEQ ID NO: 2
hVB22B p-z VH:FR3Z配列番号： 17  hVB22B p-z VH: FR3Z SEQ ID NO: 17
hVB22B p-z VH： CDR3/配列番号： 3  hVB22B p-z VH: CDR3 / SEQ ID NO: 3
hVB22B p-z VH:FR4Z配列番号： 18  hVB22B p-z VH: FR4Z SEQ ID NO: 18
[0078] 本発明において、 hVB22B p-z VL配列における CDRおよび FRと配列番号との対応 は以下の通りである。 [0078] In the present invention, the correspondence between CDR and FR in the hVB22B p-z VL sequence and SEQ ID NOs is as follows.
hVB22B p-z VL: FRIZ配列番号： 27  hVB22B p-z VL: FRIZ SEQ ID NO: 27
hVB22B p-z VL:CDR1Z配列番号： 4 hVB22B p-z VL： FR2/配列番号： 28 hVB22B pz VL: CDR1Z SEQ ID NO: 4 hVB22B pz VL: FR2 / SEQ ID NO: 28
hVB22B p-z VL: CDR2Z配列番号： 5  hVB22B p-z VL: CDR2Z SEQ ID NO: 5
hVB22B p-z VL: FR3Z配列番号： 29  hVB22B p-z VL: FR3Z SEQ ID NO: 29
hVB22B p-z VL: CDR3Z配列番号： 6  hVB22B p-z VL: CDR3Z SEQ ID NO: 6
hVB22B p-z VL： FR4/配列番号： 30  hVB22B p-z VL: FR4 / SEQ ID NO: 30
[0079] 本発明において、 hVB22B g-e VH配列における CDRおよび FRと配列番号との対応 は以下の通りである。 [0079] In the present invention, the correspondence between CDRs and FRs in the hVB22B g-e VH sequence and SEQ ID NOs is as follows.
hVB22B g-e VH : FRlZ配列番号： 19  hVB22B g-e VH: FRlZ SEQ ID NO: 19
hVB22B g-e VH : CDRlZ配列番号： 1  hVB22B g-e VH: CDRlZ SEQ ID NO: 1
hVB22B g-e VH： FR2/配列番号： 20  hVB22B g-e VH: FR2 / SEQ ID NO: 20
hVB22B g-e VH： CDR2/配列番号： 2  hVB22B g-e VH: CDR2 / SEQ ID NO: 2
hVB22B g-e VH： FR3/配列番号： 21  hVB22B g-e VH: FR3 / SEQ ID NO: 21
hVB22B g-e VH： CDR3/配列番号： 3  hVB22B g-e VH: CDR3 / SEQ ID NO: 3
hVB22B g-e VH： FR4/配列番号： 22  hVB22B g-e VH: FR4 / SEQ ID NO: 22
[0080] 本発明において、 hVB22B g-e VL配列における CDRおよび FRと配列番号との対応 は以下の通りである。 [0080] In the present invention, the correspondence between CDRs and FRs in the hVB22B g-e VL sequence and SEQ ID NOs is as follows.
hVB22B g-e VL: FRlZ配列番号： 31  hVB22B g-e VL: FRlZ SEQ ID NO: 31
hVB22B g-e VL: CDRlZ配列番号： 4  hVB22B g-e VL: CDRlZ SEQ ID NO: 4
hVB22B g-e VL： FR2/配列番号： 32  hVB22B g-e VL: FR2 / SEQ ID NO: 32
hVB22B g-e VL： CDR2/配列番号： 5  hVB22B g-e VL: CDR2 / SEQ ID NO: 5
hVB22B g-e VL： FR3/配列番号： 33  hVB22B g-e VL: FR3 / SEQ ID NO: 33
hVB22B g-e VL: CDR3Z配列番号： 6  hVB22B g-e VL: CDR3Z SEQ ID NO: 6
hVB22B g-e VL： FR4/配列番号： 34  hVB22B g-e VL: FR4 / SEQ ID NO: 34
[0081] 本発明において、 hVB22B e VH配列における CDRおよび FRと配列番号との対応は 以下の通りである。 [0081] In the present invention, the correspondence between CDRs and FRs in the hVB22B e VH sequence and SEQ ID NOs is as follows.
hVB22B e VH : FRlZ配列番号： 23  hVB22B e VH: FRlZ SEQ ID NO: 23
hVB22B e VH : CDR1/配列番号： 1  hVB22B e VH: CDR1 / SEQ ID NO: 1
hVB22B e VH： FR2/配列番号： 24 hVB22B e VH:CDR2Z配列番号： 2 hVB22B e VH: FR2 / SEQ ID NO: 24 hVB22B e VH: CDR2Z SEQ ID NO: 2
hVB22B e VH： FR3/配列番号： 25  hVB22B e VH: FR3 / SEQ ID NO: 25
hVB22B e VH:CDR3Z配列番号： 3  hVB22B e VH: CDR3Z SEQ ID NO: 3
hVB22B e VH： FR4/配列番号： 26  hVB22B e VH: FR4 / SEQ ID NO: 26
[0082] 本発明にお!/、て、 hVB22B e VL配列における CDRおよび FRと配列番号との対応は 以下の通りである。 [0082] In the present invention, the correspondence between CDR and FR in the hVB22B e VL sequence and the SEQ ID NOs is as follows.
hVB22B e VL:FRlZ配列番号： 31  hVB22B e VL: FRlZ SEQ ID NO: 31
hVB22B e VL:CDRlZ配列番号： 4  hVB22B e VL: CDRlZ SEQ ID NO: 4
hVB22B e VL:FR2 配列番号： 32  hVB22B e VL: FR2 SEQ ID NO: 32
hVB22B e VL： CDR2Z配列番号： 5  hVB22B e VL: CDR2Z SEQ ID NO: 5
hVB22B e VL:FR3Z配列番号： 33  hVB22B e VL: FR3Z SEQ ID NO: 33
hVB22B e VL:CDR3Z配列番号： 6  hVB22B e VL: CDR3Z SEQ ID NO: 6
hVB22B e VL： FR4/配列番号： 34  hVB22B e VL: FR4 / SEQ ID NO: 34
[0083] 本発明にお!/、て、 hVB22B u2-wz4 VH配列における CDRおよび FRと配列番号との 対応は以下の通りである。 [0083] In the present invention, the correspondence between CDRs and FRs in the hVB22B u2-wz4 VH sequence and SEQ ID NOs is as follows.
hVB22B u2-wz4 VH:FRlZ配列番号： 81  hVB22B u2-wz4 VH: FRlZ SEQ ID NO: 81
hVB22B u2-wz4 VH:CDRlZ配列番号： 1  hVB22B u2-wz4 VH: CDRlZ SEQ ID NO: 1
hVB22B u2-wz4 VH:FR2Z配列番号： 82  hVB22B u2-wz4 VH: FR2Z SEQ ID NO: 82
hVB22B u2-wz4 VH:CDR2Z配列番号： 2  hVB22B u2-wz4 VH: CDR2Z SEQ ID NO: 2
hVB22B u2-wz4 VH:FR3Z配列番号： 83  hVB22B u2-wz4 VH: FR3Z SEQ ID NO: 83
hVB22B u2-wz4 VH： CDR3Z配列番号： 3  hVB22B u2-wz4 VH: CDR3Z SEQ ID NO: 3
hVB22B u2-wz4 VH： FR4Z配列番号： 84  hVB22B u2-wz4 VH: FR4Z SEQ ID NO: 84
[0084] 本発明にお！/、て、 hVB22B u2-wz4 VL配列における CDRおよび FRと配列番号との 対応は以下の通りである。 [0084] In the present invention! The correspondence between CDRs and FRs in the hVB22B u2-wz4 VL sequence and the SEQ ID NOs is as follows.
hVB22B u2-wz4 VL:FRlZ配列番号： 86  hVB22B u2-wz4 VL: FRlZ SEQ ID NO: 86
hVB22B u2-wz4 VL:CDRlZ配列番号： 4  hVB22B u2-wz4 VL: CDRlZ SEQ ID NO: 4
hVB22B u2-wz4 VL:FR2Z配列番号： 87  hVB22B u2-wz4 VL: FR2Z SEQ ID NO: 87
hVB22B u2-wz4 VL:CDR2Z配列番号： 5 hVB22B u2-wz4 VL: FR3/配列番号： 88 hVB22B u2-wz4 VL: CDR2Z SEQ ID NO: 5 hVB22B u2-wz4 VL: FR3 / SEQ ID NO: 88
hVB22B u2-wz4 VL: CDR3,配列番号： 6  hVB22B u2-wz4 VL: CDR3, SEQ ID NO: 6
hVB22B u2-wz4 VL: FR4Z配列番号： 89  hVB22B u2-wz4 VL: FR4Z SEQ ID NO: 89
[0085] 本発明にお 、て、 hVB22B q-wz5 VH配列における CDRおよび FRと配列番号との 対応は以下の通りである。 [0085] In the present invention, the correspondence between CDRs and FRs in the hVB22B q-wz5 VH sequence and SEQ ID NOs is as follows.
hVB22B q-wz5 VH : FRlZ配列番号： 81  hVB22B q-wz5 VH: FRlZ SEQ ID NO: 81
hVB22B q-wz5 VH : CDRlZ配列番号： 1  hVB22B q-wz5 VH: CDRlZ SEQ ID NO: 1
hVB22B q-wz5 VH : FR2Z配列番号： 82  hVB22B q-wz5 VH: FR2Z SEQ ID NO: 82
hVB22B q-wz5 VH： CDR2/配列番号： 2  hVB22B q-wz5 VH: CDR2 / SEQ ID NO: 2
hVB22B q-wz5 VH : FR3Z配列番号： 83  hVB22B q-wz5 VH: FR3Z SEQ ID NO: 83
hVB22B q-wz5 VH： CDR3/配列番号： 3  hVB22B q-wz5 VH: CDR3 / SEQ ID NO: 3
hVB22B q-wz5 VH : FR4Z配列番号： 84  hVB22B q-wz5 VH: FR4Z SEQ ID NO: 84
[0086] 本発明にお 、て、 hVB22B q-wz5 VL配列における CDRおよび FRと配列番号との対 応は以下の通りである。 [0086] In the present invention, the correspondence between CDRs and FRs in the hVB22B q-wz5 VL sequence and SEQ ID NOs is as follows.
hVB22B q-wz5 VL: FRlZ配列番号： 86  hVB22B q-wz5 VL: FRlZ SEQ ID NO: 86
hVB22B q-wz5 VL: CDRlZ配列番号： 4  hVB22B q-wz5 VL: CDRlZ SEQ ID NO: 4
hVB22B q-wz5 VL: FR2Z配列番号： 90  hVB22B q-wz5 VL: FR2Z SEQ ID NO: 90
hVB22B q-wz5 VL: CDR2Z配列番号： 5  hVB22B q-wz5 VL: CDR2Z SEQ ID NO: 5
hVB22B q-wz5 VL: FR3Z配列番号： 91  hVB22B q-wz5 VL: FR3Z SEQ ID NO: 91
hVB22B q-wz5 VL: CDR3Z配列番号： 6  hVB22B q-wz5 VL: CDR3Z SEQ ID NO: 6
hVB22B q-wz5 VL: FR4Z配列番号： 89  hVB22B q-wz5 VL: FR4Z SEQ ID NO: 89
[0087] なお、 hVB22B p- z配列、 hVB22B g- e配列、 hVB22B e配列、 hVB22B u2- wz4配 列、および hVB22B q-wz5配列における CDRおよび FRの対応を、図 1に示した。 なお、 VB22B VHの塩基配列を配列番号： 7、 VB22B VLの塩基配列を配列番号： 9Note that FIG. 1 shows the correspondence between CDRs and FRs in the hVB22B p-z sequence, hVB22B g-e sequence, hVB22B e sequence, hVB22B u2-wz4 sequence, and hVB22B q-wz5 sequence. The nucleotide sequence of VB22B VH is SEQ ID NO: 7, and the nucleotide sequence of VB22B VL is SEQ ID NO: 9
、 VB22B scFvの塩基配列を配列番号： 11、 VB22B sc(Fv)2の塩基配列を配列番号：SEQ ID NO: 11, the nucleotide sequence of VB22B scFv, SEQ ID NO: 11, the nucleotide sequence of VB22B sc (Fv) 2
13、 hVB22B p-z VHの塩基配列を配列番号： 50、 hVB22B g-e VHの塩基配列を配 列番号： 52、 hVB22B e VHの塩基配列を配列番号： 54、 hVB22B u2-wz4 VHの塩基 配列を配列番号： 96および hVB22B q-wz5 VHの塩基配列を配列番号： 98、 hVB22B p-z VLの塩基配列を配列番号： 56、 hVB22B g-e VL、 hVB22B e VLの塩基配列を 配列番号： 58、 hVB22B u2-wz4 VLの塩基配列を配列番号： 97および hVB22B q-wz5 VLの塩基配列を配列番号： 99、 hVB22B p-z sc(Fv)2の塩基配列を配列番号 : 60、 hVB22B g-e sc(Fv)2の塩基配列を配列番号： 62、 hVB22B e sc(Fv)2の塩基配 列を配列番号： 64、 hVB22B u2-wz4 sc(Fv)2の塩基配列を配列番号： 102および hVB22B q-wz5 sc(Fv)2の塩基配列を配列番号： 103に記載する。 13, the nucleotide sequence of hVB22B pz VH is SEQ ID NO: 50, the nucleotide sequence of hVB22Bge VH is SEQ ID NO: 52, the nucleotide sequence of hVB22B e VH is SEQ ID NO: 54, and the nucleotide sequence of hVB22B u2-wz4 VH is SEQ ID NO: No .: 96 and hVB22B The base sequence of q-wz5 VH is shown in SEQ ID NO: 98, hVB22B. The nucleotide sequence of pz VL is SEQ ID NO: 56, the nucleotide sequence of hVB22Bge VL, the nucleotide sequence of hVB22B e VL is SEQ ID NO: 58, the nucleotide sequence of hVB22B u2-wz4 VL is the nucleotide sequence of SEQ ID NO: 97 and the nucleotide sequence of hVB22B q-wz5 VL SEQ ID NO: 99, base sequence of hVB22B pz sc (Fv) 2 is SEQ ID NO: 60, base sequence of hVB22B ge sc (Fv) 2 is SEQ ID NO: 62, base sequence of hVB22B esc (Fv) 2 No .: 64, the nucleotide sequence of hVB22B u2-wz4 sc (Fv) 2 is described in SEQ ID NO: 102, and the nucleotide sequence of hVB22B q-wz5 sc (Fv) 2 is described in SEQ ID NO: 103.
[0088] (18)配列番号： 61 (hVB22B p- z : sc(Fv)2)、配列番号： 63 (hVB22B g- e： sc(Fv)2)、 配列番号： 65 (hVB22B e： sc(Fv)2)、配列番号： 100 (hVB22B u2- wz4： sc(Fv)2)また は配列番号： 101 (hVB22B q-wz5 : sc(Fv)2)のいずれかに記載のアミノ酸配列を有す るヒト化抗体。 (18) SEQ ID NO: 61 (hVB22B p-z: sc (Fv) 2), SEQ ID NO: 63 (hVB22B g-e: sc (Fv) 2), SEQ ID NO: 65 (hVB22B e: sc ( Fv) 2), having the amino acid sequence of any of SEQ ID NO: 100 (hVB22B u2-wz4: sc (Fv) 2) or SEQ ID NO: 101 (hVB22B q-wz5: sc (Fv) 2) Humanized antibodies.
[0089] (19)上記（1)〜（18)のいずれかに記載のアミノ酸配列において 1又は複数のァミノ 酸が置換、欠失、付加および Zまたは挿入され、かつ上記抗体と同等の活性を有す る抗体。ここで、上記抗体と同等の活性を有するとは、血小板増加作用において同 等の活性を有することを意味する。  (19) In the amino acid sequence according to any one of the above (1) to (18), one or more amino acids are substituted, deleted, added and Z or inserted, and have an activity equivalent to that of the above antibody. Antibodies. Here, having the same activity as the above antibody means having the same activity in the platelet increasing effect.
[0090] 上記（1)〜（18)のいずれかに記載の抗体は Mplに対するァゴニスト活性が非常に 高!、為、血小板減少症治療剤又は血小板増加剤として特に有用である。  [0090] The antibody according to any one of the above (1) to (18) has an extremely high agonist activity against Mpl !, and is therefore particularly useful as a therapeutic agent for thrombocytopenia or an agent for increasing thrombocytopenia.
[0091] あるポリペプチドと機能的に同等なポリペプチドを調製するための、当業者によく知 られた方法としては、ポリペプチドに変異を導入する方法が知られている。例えば、 当業者であれば、部位特異的変異誘発法（Hashimoto-Gotoh, T. et al. (1995) Gene 152, 271-275, Zoller, MJ, and Smith, M.(1983) Methods Enzymol. 100, 468-500、 Kramer, W. et al. (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)などを用いて 、本発明の抗体に適宜変異を導入することにより、該抗体と機能的に同等な抗体を 調製することができる。また、アミノ酸の変異は自然界においても生じうる。このように 、本発明の抗体のアミノ酸配列において 1もしくは複数のアミノ酸が変異したアミノ酸 配列を有し、該抗体と機能的に同等な抗体もまた本発明の抗体に含まれる。このよう な変異体における、変異するアミノ酸数は、通常、 50アミノ酸以内であり、好ましくは 30アミノ酸以内であり、さらに好ましくは 10アミノ酸以内（例えば、 5アミノ酸以内）であ ると考免られる。 [0091] As a method well known to those skilled in the art for preparing a polypeptide functionally equivalent to a certain polypeptide, a method for introducing a mutation into a polypeptide is known. For example, those skilled in the art can use a site-directed mutagenesis method (Hashimoto-Gotoh, T. et al. (1995) Gene 152, 271-275, Zoller, MJ, and Smith, M. (1983) Methods Enzymol. 100). , 468-500, Kramer, W. et al. (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, 2762-2766), etc. Antibody can be prepared. Amino acid mutations can also occur in nature. Thus, an antibody having an amino acid sequence in which one or more amino acids are mutated in the amino acid sequence of the antibody of the present invention and functionally equivalent to the antibody is also included in the antibody of the present invention. The number of amino acids to be mutated in such a mutant is usually within 50 amino acids, preferably It is exempted from being within 30 amino acids, more preferably within 10 amino acids (eg, within 5 amino acids).
[0092] 変異するアミノ酸残基にお!、ては、アミノ酸側鎖の性質が保存されて 、る別のアミノ 酸に変異されることが望ましい。例えばアミノ酸側鎖の性質としては、疎水性アミノ酸（ A、 I、し、 M、 F、 P、 W、 Y、 V)、親水'性アミノ酸（R、 D、 N、 C、 E、 Q、 G、 H、 K、 S、 T)、月旨 肪族側鎖を有するアミノ酸 (G、 A、 V、 L、 I、 P)、水酸基含有側鎖を有するアミノ酸 (S、 T、 Υ)、硫黄原子含有側鎖を有するアミノ酸 (C、 M)、カルボン酸及びアミド含有側鎖 を有するアミノ酸 (D、 N、 E、 Q)、塩基含有側鎖を有するアミノ酸 (R、 K、 Η)、芳香族 含有側鎖を有するアミノ酸 (H、 F、 Y、 W)を挙げることができる (括弧内はいずれもアミ ノ酸の一文字標記を表す)。  [0092] In the amino acid residue to be mutated, it is desirable that the properties of the amino acid side chain are preserved and the amino acid residue is mutated to another amino acid. For example, the properties of amino acid side chains include hydrophobic amino acids (A, I, M, F, P, W, Y, V) and hydrophilic 'amino acids (R, D, N, C, E, Q, G , H, K, S, T), an amino acid having a fatty acid side chain (G, A, V, L, I, P), an amino acid having a hydroxyl-containing side chain (S, T, Υ), a sulfur atom Amino acids with side chains containing (C, M), amino acids with side chains containing carboxylic acids and amides (D, N, E, Q), amino acids with side chains containing bases (R, K, Η), aromatic Examples include amino acids having side chains (H, F, Y, W) (all brackets represent one letter of amino acids).
[0093] あるアミノ酸配列に対する 1又は複数個のアミノ酸残基の欠失、付加及び Z又は他 のアミノ酸による置換により修飾されたアミノ酸配列を有するポリペプチドがその生物 学的活性を維持することはすでに知られている（Mark, D. F. et al.， Proc. Natl.  [0093] It is already known that a polypeptide having an amino acid sequence modified by deletion or addition of one or more amino acid residues to a certain amino acid sequence and substitution with Z or another amino acid maintains its biological activity. Known (Mark, DF et al., Proc. Natl.
Acad. Sci. USA (1984) 81, 5662—5666、 Zoller, M. J. & Smith, M. Nucleic Acids Research (1982) 10, 6487-6500、 Wang, A. et al, Science 224, 1431-1433、 Dalbadie- McFarland, G. et al" Proc. Natl. Acad. Sci. USA (1982) 79, 6409—6413 )  Acad. Sci. USA (1984) 81, 5662-5666, Zoller, MJ & Smith, M. Nucleic Acids Research (1982) 10, 6487-6500, Wang, A. et al, Science 224, 1431-1433, Dalbadie -McFarland, G. et al "Proc. Natl. Acad. Sci. USA (1982) 79, 6409—6413)
[0094] 本発明の抗体のアミノ酸配列に複数個のアミノ酸残基が付加された抗体には、これ ら抗体を含む融合タンパク質が含まれる。融合タンパク質は、これら抗体と他のぺプ チド又はタンパク質とが融合したものであり、本発明に含まれる。融合タンパク質を作 製する方法は、本発明の抗体をコードするポリヌクレオチドと他のペプチド又はポリべ プチドをコードするポリヌクレオチドをフレームが一致するように連結してこれを発現 ベクターに導入し、宿主で発現させればよぐ当業者に公知の手法を用いることがで きる。本発明の抗体との融合に付される他のペプチド又はポリペプチドとしては、例え ば、 FLAG (Hopp, T. P. et al., BioTechnology (1988) 6, 1204- 1210 )、 6個の His (ヒス チジン）残基からなる 6 X His、 10 X His,インフルエンザ凝集素（HA)、ヒト c-mycの断 片、 VSV- GPの断片、 pl8HIVの断片、 T7- tag、 HSV- tag、 E- tag、 SV40T抗原の断片 、 lck tag, a -tubulinの断片、 B-tag、 Protein Cの断片等の公知のペプチドを使用す ることができる。また、本発明の抗体との融合に付される他のポリペプチドとしては、 例えば、 GST (ダルタチオン一 S トランスフェラーゼ）、 HA (インフルエンザ凝集素）、 ィムノグロブリン定常領域、 β ガラクトシダーゼ、 ΜΒΡ (マルトース結合タンパク質） 等が挙げられる。巿販されて 、るこれらペプチドまたはポリペプチドをコードするポリヌ クレオチドを、本発明の抗体をコードするポリヌクレオチドと融合させ、これにより調製 された融合ポリヌクレオチドを発現させることにより、融合ポリペプチドを調製すること ができる。 [0094] Antibodies in which a plurality of amino acid residues are added to the amino acid sequence of the antibodies of the present invention include fusion proteins containing these antibodies. The fusion protein is obtained by fusing these antibodies with another peptide or protein, and is included in the present invention. A method for producing a fusion protein is as follows: a polynucleotide encoding the antibody of the present invention and a polynucleotide encoding another peptide or polypeptide are ligated in frame so that they are introduced into an expression vector; A method known to those skilled in the art can be used as long as the expression is carried out. Other peptides or polypeptides to be fused with the antibody of the present invention include, for example, FLAG (Hopp, TP et al., BioTechnology (1988) 6, 1204-1210), 6 His (histidine) ) Residues consisting of 6 X His, 10 X His, influenza agglutinin (HA), human c-myc fragment, VSV-GP fragment, pl8HIV fragment, T7-tag, HSV-tag, E-tag, Use known peptides such as SV40T antigen fragment, lck tag, a-tubulin fragment, B-tag and Protein C fragment. Can. Other polypeptides to be fused with the antibody of the present invention include, for example, GST (daltathione-S transferase), HA (influenza agglutinin), immunoglobulin constant region, β-galactosidase, ΜΒΡ (maltose binding) Protein) and the like. A fusion polypeptide is prepared by fusing a polynucleotide encoding the peptide or polypeptide that is sold with a polynucleotide encoding the antibody of the present invention, and expressing the fusion polynucleotide prepared thereby. can do.
[0095] 本発明の抗体は、後述するそれを産生する細胞や宿主あるいは精製方法により、 アミノ酸配列、分子量、等電点又は糖鎖の有無や形態などが異なり得る。しかしなが ら、得られた抗体が、本発明の抗体と同等の機能を有している限り、本発明に含まれ る。例えば、本発明の抗体を原核細胞、例えば大腸菌で発現させた場合、本来の抗 体のアミノ酸配列の Ν末端にメチォニン残基が付加される。本発明の抗体はこのよう な抗体も包含する。  [0095] The antibody of the present invention may differ in amino acid sequence, molecular weight, isoelectric point, presence / absence and form of sugar chain, etc., depending on the cell or host producing the antibody or the purification method described below. However, as long as the obtained antibody has a function equivalent to that of the antibody of the present invention, it is included in the present invention. For example, when the antibody of the present invention is expressed in a prokaryotic cell, for example, Escherichia coli, a methionine residue is added to the Ν-terminal of the amino acid sequence of the original antibody. The antibodies of the present invention also include such antibodies.
[0096] さらに、本発明にお ヽて、血小板減少症治療剤又は血小板増加剤に含まれる Mpl を認識する抗体の好ま U、態様としては、上記 (1)〜(18)抗体が認識するェピトープを 認識する抗体を挙げることができる。  [0096] Further, in the present invention, the preferred U of the antibody recognizing Mpl contained in the therapeutic agent for thrombocytopenia or the agent for increasing thrombocytopenia is, in particular, an epitope recognized by the above-mentioned antibody (1) to (18). Can be mentioned.
[0097] 抗体が認識するェピトープを認識する抗体は当業者に公知の方法により得ることが 可能である。例えば、上述の抗体が認識するェピトープを通常の方法により決定し、 該ェピトープに含まれるアミノ酸配列を有するポリペプチドを免疫原として抗体を作製 する方法や、通常の方法で作製された抗体のェピトープを決定し、上述の抗体とェ ピトープが同じ抗体を選択する方法などにより得ることができる。  [0097] An antibody that recognizes an epitope that is recognized by an antibody can be obtained by a method known to those skilled in the art. For example, a method for preparing an antibody using the polypeptide having the amino acid sequence contained in the epitope as an immunogen or a method for preparing an antibody epitope by an ordinary method is used to determine an epitope that is recognized by the above-mentioned antibody. Once determined, the antibody and the epitope can be obtained by a method of selecting the same antibody.
[0098] 本発明においては、配列番号： 100に記載のアミノ酸配列を有する抗体が認識する ェピトープを認識する抗体が特に好ましい。配列番号： 100に記載のアミノ酸配列を 有する抗体は、ヒト Mplの 26番目の Gluから 274番目の Leuまでの領域、好ましくは 189 番目の Alaから 245番目の Glyの領域、さらに好ましくは 213番目の Ginから 231番目の Alaまでの領域を認識していると予想される。従って、ヒト Mplの 26番目〜274番目、あ るいは 189番目〜245番目、あるいは 213番目〜231番目の領域を認識する抗体も本 発明に含まれる。 [0099] ヒト Mplのアミノ酸配列（配列番号： 66)の 26番目〜 274番目、あるいは 189番目〜[0098] In the present invention, an antibody that recognizes an epitope that is recognized by an antibody having the amino acid sequence of SEQ ID NO: 100 is particularly preferable. The antibody having the amino acid sequence of SEQ ID NO: 100 is a region from the 26th Glu to the 274th Leu of human Mpl, preferably the 189th Ala to the 245th Gly region, more preferably the 213th Gly region. It is expected to recognize the region from Gin to the 231st Ala. Therefore, an antibody that recognizes the 26th to 274th, or the 189th to 245th, or the 213th to 231st region of human Mpl is also included in the present invention. [0099] The amino acid sequence of human Mpl (SEQ ID NO: 66) at positions 26 to 274, or at positions 189 to 189
245番目、あるいは 213番目〜231番目の領域を認識する抗体は、当業者に公知の方 法により得ることが可能であり、例えば、ヒト Mplのアミノ酸配列（配列番号： 66)の 26番 目〜274番目、あるいは 189番目〜245番目、ある!/、は 213番目〜231番目のペプチド を免疫原として抗体を作製する方法や、通常の方法で作製した抗体が認識するェピ トープを決定し、本発明の抗体と同じェピトープを認識する抗体を選択する方法など により得ることが可能である。 An antibody recognizing the 245th or 213st to 231rd region can be obtained by a method known to those skilled in the art, for example, the 26th to 26th amino acids of human Mpl (SEQ ID NO: 66). The 274th, or 189th to 245th, there are! /, Determine the method of preparing antibodies using the 213st to 231st peptides as immunogens, and determine the epitope recognized by the antibody prepared by the usual method. It can be obtained by a method such as selecting an antibody that recognizes the same epitope as the antibody of the present invention.
[0100] Mplに結合する抗体は当業者に公知の方法により作成することができる。  [0100] Antibodies that bind to Mpl can be prepared by methods known to those skilled in the art.
例えば、モノクローナル抗体産生ハイプリドーマは、基本的には公知技術を使用し 、以下のようにして作製できる。すなわち、 Mplタンパク質又は Mpl発現細胞を感作抗 原として使用して、これを通常の免疫方法にしたがって免疫し、得られる免疫細胞を 通常の細胞融合法によって公知の親細胞と融合させ、通常のスクリーニング法により 、モノクローナルな抗体産生細胞をスクリーニングすることによって作製できる。  For example, a monoclonal antibody-producing hybridoma can be basically produced using a known technique as follows. That is, Mpl protein or Mpl-expressing cells are used as a sensitizing antigen and immunized according to a usual immunization method, and the obtained immunocytes are fused with a known parent cell by a usual cell fusion method to obtain a normal immunization cell. It can be prepared by screening monoclonal antibody-producing cells by a screening method.
[0101] 具体的には、モノクローナル抗体を作製するには次のようにすればよい。  [0101] Specifically, a monoclonal antibody may be prepared as follows.
まず、抗体取得の感作抗原として使用される Mplタンパク質を、  First, Mpl protein used as a sensitizing antigen for obtaining antibodies
Genebank:NM_005373に開示された Mpl遺伝子 Zアミノ酸配列を発現することによつ て得る。すなわち、 Mplをコードする遺伝子配列を公知の発現ベクター系に挿入して 適当な宿主細胞を形質転換させた後、その宿主細胞中または培養上清中から目的 のヒト Mplタンパク質を公知の方法で精製する。  Obtained by expressing the Z amino acid sequence of the Mpl gene disclosed in Genebank: NM_005373. That is, after inserting the gene sequence encoding Mpl into a known expression vector system and transforming an appropriate host cell, the target human Mpl protein is purified from the host cell or culture supernatant by a known method. I do.
[0102] 次に、この精製 Mplタンパク質を感作抗原として用いる。あるいは、 Mplの部分ぺプ チドを感作抗原として使用することもできる。この際、部分ペプチドはヒト Mplのァミノ 酸配列より化学合成により得ることも可能である。  Next, the purified Mpl protein is used as a sensitizing antigen. Alternatively, a partial peptide of Mpl can be used as a sensitizing antigen. At this time, the partial peptide can be obtained by chemical synthesis from the amino acid sequence of human Mpl.
[0103] 本発明の抗 MPL抗体の認識する Mpl分子上のェピトープは特定のものに限定され ず、 Mpl分子上に存在するェピトープならばどのェピトープを認識してもよい。従って 、本発明の抗 Mpl抗体を作製するための抗原は、 Mpl分子上に存在するェピトープを 含む断片ならば、如何なる断片も用いることが可能である。  [0103] The epitope on the Mpl molecule recognized by the anti-MPL antibody of the present invention is not limited to a particular one, and any epitope present on the Mpl molecule may be recognized. Therefore, any fragment can be used as an antigen for producing the anti-Mpl antibody of the present invention, as long as it is a fragment containing an epitope present on the Mpl molecule.
[0104] 感作抗原で免疫される哺乳動物としては、特に限定されるものではないが、細胞融 合に使用する親細胞との適合性を考慮して選択するのが好ましぐ一般的にはげつ 歯類の動物、例えば、マウス、ラット、ノ、ムスター、あるいはゥサギ、サル等が使用され る。 [0104] The mammal to be immunized with the sensitizing antigen is not particularly limited. Generally, it is generally preferable to select a mammal in consideration of compatibility with the parent cell used for cell fusion. Bald Dental animals, for example, mice, rats, wild, musters, or egrets, monkeys, and the like are used.
[0105] 感作抗原を動物に免疫するには、公知の方法にしたがって行われる。例えば、一 般的方法として、感作抗原を哺乳動物の腹腔内または皮下に注射することにより行 われる。具体的には、感作抗原を PBS (Phosphate-Buffered Saline)や生理食塩水等 で適当量に希釈、懸濁したものに所望により通常のアジュバント、例えばフロイント完 全アジュバントを適量混合し、乳化後、哺乳動物に 4-21日毎に数回投与する。また、 感作抗原免疫時に適当な担体を使用することもできる。  [0105] Immunization of an animal with a sensitizing antigen is performed according to a known method. For example, as a general method, the sensitizing antigen is injected intraperitoneally or subcutaneously into a mammal. Specifically, the sensitizing antigen is diluted and suspended in an appropriate amount with PBS (Phosphate-Buffered Saline) or physiological saline, and then mixed with an appropriate amount of a normal adjuvant, for example, Freund's complete adjuvant, if necessary, and emulsified. The mammal is administered several times every 4-21 days. In addition, a suitable carrier can be used during immunization of the sensitizing antigen.
[0106] このように哺乳動物を免疫し、血清中に所望の抗体レベルが上昇するのを確認した 後に、哺乳動物力も免疫細胞を採取し、細胞融合に付されるが、好ましい免疫細胞 としては、特に脾細胞が挙げられる。  [0106] After immunizing a mammal in this manner and confirming that the desired antibody level is increased in the serum, the mammal is also harvested for immune cells and subjected to cell fusion. Preferred immune cells are preferred. And especially splenocytes.
[0107] 前記免疫細胞と融合される他方の親細胞として、哺乳動物のミエローマ細胞を用い る。このミエローマ細胞は、公知の種々の細胞株、例えば、 P3 (P3x63Ag8.653) (J. Immnol. (1979) 123, 1548-1550)、 P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1—7)、 NS— 1 (Kohler. G. and Milstein, C. Eur. J.  [0107] Mammalian myeloma cells are used as the other parent cells to be fused with the immune cells. The myeloma cells are known cell lines, for example, P3 (P3x63Ag8.653) (J. Immnol. (1979) 123, 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. et al., Cell (1976) 8, 405- 415)、 SP2/0 (Shulman, M. et al., Nature (1978) 276, 269- 270)、 FO (deSt. Groth, S. F. et al., J. Immunol. Methods (1980) 35, 1—21)、 S194 (Trowbridge, I. S. J. Exp. Med. (1978) 148, 313— 323)、 R210 (Galfre, G. et al., Nature (1979) 277, 131-133)等が好適に使用される。  Immunol. (1976) 6, 511-519), MPC-11 (Margulies.DH et al., Cell (1976) 8, 405-415), SP2 / 0 (Shulman, M. et al., Nature (1978) 276, 269-270), FO (deSt. Groth, SF et al., J. Immunol. Methods (1980) 35, 1-21), S194 (Trowbridge, ISJ Exp. Med. (1978) 148, 313-323) ), R210 (Galfre, G. et al., Nature (1979) 277, 131-133) and the like are preferably used.
[0108] 前記免疫細胞とミエローマ細胞との細胞融合は、基本的には公知の方法、たとえば 、ケーラーとミルスティンらの方法（Kohler. G. and Milstein, C.、 Methods Enzymol. ( 1981) 73, 3-46)等に準じて行うことができる。  The cell fusion between the immune cells and myeloma cells is basically performed by a known method, for example, the method of Kohler and Milstein, et al. (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46) can be performed.
より具体的には、前記細胞融合は、例えば細胞融合促進剤の存在下に通常の栄 養培養液中で実施される。融合促進剤としては、例えばポリエチレングリコール (PEG )、センダイウィルス (HVJ)等が使用され、更に所望により融合効率を高めるためにジ メチルスルホキシド等の補助剤を添加使用することもできる。  More specifically, the cell fusion is performed, for example, in a normal nutrient medium in the presence of a cell fusion promoter. As the fusion promoter, for example, polyethylene glycol (PEG), Sendai virus (HVJ) and the like are used, and if necessary, an auxiliary agent such as dimethyl sulfoxide can be added to enhance the fusion efficiency.
[0109] 免疫細胞とミエローマ細胞との使用割合は任意に設定することができる。例えば、ミ エローマ細胞に対して免疫細胞を 1-10倍とするのが好ましい。前記細胞融合に用い る培養液としては、例えば、前記ミエローマ細胞株の増殖に好適な RPMI1640培養液 、 MEM培養液、その他、この種の細胞培養に用いられる通常の培養液が使用可能 であり、さらに、牛胎児血清 (FCS)等の血清補液を併用することもできる。 [0109] The ratio of the use of the immune cells to the myeloma cells can be arbitrarily set. For example, Mi It is preferred that the number of immune cells be 1 to 10 times that of the emoma cells. As the culture solution used for the cell fusion, for example, RPMI1640 culture solution, MEM culture solution, and other ordinary culture solutions used for this type of cell culture suitable for the growth of the myeloma cell line can be used. Furthermore, a serum replacement solution such as fetal calf serum (FCS) can be used in combination.
[0110] 細胞融合は、前記免疫細胞とミエローマ細胞との所定量を前記培養液中でよく混 合し、予め 37°C程度に加温した PEG溶液 (例えば平均分子量 1000-6000程度）を通 常 30-60% (w/v)の濃度で添加し、混合することによって目的とする融合細胞 (ハイブ リドーマ）を形成する。続いて、適当な培養液を逐次添加し、遠心して上清を除去す る操作を繰り返すことによりハイプリドーマの生育に好ましくない細胞融合剤等を除去 する。 [0110] In the cell fusion, a predetermined amount of the immune cells and the myeloma cells are mixed well in the culture solution, and the mixture is passed through a PEG solution (for example, an average molecular weight of about 1000 to 6000) which has been heated to about 37 ° C in advance. The desired fusion cells (hybridomas) are formed by adding and mixing always at a concentration of 30-60% (w / v). Subsequently, an appropriate culture solution is successively added, and the operation of removing the supernatant by centrifugation is repeated to remove a cell fusion agent or the like that is unfavorable for the growth of the hybridoma.
[Oil 1] このようにして得られたノヽイブリドーマは、通常の選択培養液、例えば HAT培養液（ ヒポキサンチン、アミノプテリンおよびチミジンを含む培養液)で培養することにより選 択される。上記 HAT培養液での培養は、目的とするハイプリドーマ以外の細胞 (非融 合細胞）が死滅するのに十分な時間（通常、数日〜数週間)継続する。ついで、通常 の限界希釈法を実施し、目的とする抗体を産生するハイプリドーマのスクリーニング および単一クローユングを行う。  [Oil 1] The hybridoma obtained in this manner is selected by culturing it in a normal selective culture medium, for example, a HAT culture medium (a culture medium containing hypoxanthine, aminopterin and thymidine). The culturing in the HAT culture solution is continued for a time (usually several days to several weeks) sufficient for killing cells (unfused cells) other than the target hybridoma. Then, a conventional limiting dilution method is performed, and screening and single cloning of hybridomas producing the desired antibody are performed.
[0112] また、ヒト以外の動物に抗原を免疫して上記ハイプリドーマを得る他に、ヒトリンパ球 を in vitroで Mplに感作し、感作リンパ球をヒト由来の永久分裂能を有するミエローマ 細胞と融合させ、 Mplへの結合活性を有する所望のヒト抗体を得ることもできる（特公 平 1-59878号公報参照)。さらに、ヒト抗体遺伝子の全てのレパートリーを有するトラン スジヱニック動物に抗原となる Mplを投与して抗 Mpl抗体産生細胞を取得し、これを不 死化させた細胞力も Mplに対するヒト抗体を取得してもよい（国際特許出願公開番号 WO 94/25585号公報、 WO 93/12227号公報、 WO92/03918号公報、 WO  [0112] In addition to obtaining the above-mentioned hybridoma by immunizing an animal other than a human with an antigen, human lymphocytes are sensitized to Mpl in vitro, and the sensitized lymphocytes are derived from a human-derived myeloma cell capable of permanent division. And a desired human antibody having a binding activity to Mpl can be obtained (see Japanese Patent Publication No. 1-59878). In addition, transgenic animals having the entire repertoire of human antibody genes were administered with Mpl as an antigen to obtain anti-Mpl antibody-producing cells. Good (International Patent Application Publication No. WO 94/25585, WO 93/12227, WO 92/03918, WO
94/02602号公報参照）。  94/02602).
[0113] このようにして作製されるモノクローナル抗体を産生するノ、イブリドーマは、通常の 培養液中で継代培養することが可能であり、また、液体窒素中で長期保存することが 可能である。  [0113] The thus-produced monoclonal antibody producing hybridomas can be subcultured in a normal culture solution, and can be stored for a long time in liquid nitrogen. .
[0114] 当該ノ、イブリドーマ力 モノクローナル抗体を取得するには、当該ハイプリドーマを 通常の方法にしたがい培養し、その培養上清として得る方法、あるいはハイプリドー マをこれと適合性がある哺乳動物に投与して増殖させ、その腹水として得る方法など が採用される。前者の方法は、高純度の抗体を得るのに適しており、一方、後者の方 法は、抗体の大量生産に適している。 [0114] In order to obtain the monoclonal antibody of the hybridoma, the hybridoma is According to a usual method, a method of culturing and obtaining a culture supernatant thereof, or a method of administering a hybridoma to a mammal which is compatible with the hybridoma and proliferating the same to obtain as ascites fluid, and the like are employed. The former method is suitable for obtaining high-purity antibodies, while the latter method is suitable for mass production of antibodies.
[0115] 抗体遺伝子をノヽイブリドーマ力 クローユングし、適当なベクターに組み込んで、こ れを宿主に導入し、遺伝子組換え技術を用いて産生させた組換え型の抗体を作製 することも可能である（例えば、 Vandamme, A. M. et al., Eur. J. Biochem. (1990) 192, 767-775, 1990参照）。  [0115] It is also possible to produce a recombinant antibody produced by subjecting the antibody gene to a hybridoma force-cloning method, incorporating the antibody gene into an appropriate vector, introducing this into a host, and producing the same using a gene recombination technique. (See, for example, Vandamme, AM et al., Eur. J. Biochem. (1990) 192, 767-775, 1990).
[0116] 具体的には、抗 Mpl抗体を産生するハイブリドーマから、抗 Mpl抗体の可変 (V)領域 をコードする mRNAを単離する。 mRNAの単離は、公知の方法、例えば、グァ-ジン超 遠心法（Chirgwin, J. M. et al., Biochemistry (1979) 18, 5294— 5299)、 AGPC法（ Chomczynski, P.et al., Anal. Biochem. (1987) 162, 156- 159)等により行って全 RNAを 調製し、 mRNA Purification Kit (Pharmacia製）等を使用して目的の mRNAを調製する 。また、 QuickPrep mRNA Purification Kit (Pharmacia製）を用いることにより mRNAを 直接調製することもできる。  [0116] Specifically, mRNA encoding the variable (V) region of the anti-Mpl antibody is isolated from the hybridoma producing the anti-Mpl antibody. mRNA can be isolated by known methods, for example, guadin ultracentrifugation (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299), AGPC method (Chomczynski, P. et al., Anal. Prepare the total RNA by using Biochem. (1987) 162, 156-159) or the like, and prepare the target mRNA using the mRNA Purification Kit (Pharmacia). Alternatively, mRNA can be directly prepared by using the QuickPrep mRNA Purification Kit (Pharmacia).
[0117] 得られた mRNAから逆転写酵素を用いて抗体 V領域の cDNAを合成する。 cDNAの 合成は、 AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (生ィ匕学工業 社製）等を用いて行う。また、 cDNAの合成および増幅を行うには、 5'-Ampli FINDER RACE Kit (Clontech製）および PCRを用いた 5'-1^じ5法 1"01^1& M. A. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998—9002、 Belyavsky, A.et al., Nucleic Acids Res. (1989) 17, 2919- 2932)等を使用することができる。  [0117] From the obtained mRNA, cDNA of the antibody V region is synthesized using reverse transcriptase. The cDNA is synthesized using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (manufactured by Iidaigaku Kogyo). In addition, in order to synthesize and amplify cDNA, a 5'-1 ^ 5 method using 5'-Ampli FINDER RACE Kit (manufactured by Clontech) and PCR 1 "01 ^ 1 & MA et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002, Belyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932) and the like can be used.
[0118] 得られた PCR産物から目的とする DNA断片を精製し、ベクター DNAと連結する。さら に、これより組換えベクターを作製し、大腸菌等に導入してコロニーを選択して所望 の組換えベクターを調製する。そして、目的とする DNAの塩基配列を公知の方法、例 えば、ジデォキシヌクレオチドチェインターミネーシヨン法等により確認する。  [0118] A target DNA fragment is purified from the obtained PCR product and ligated to a vector DNA. Further, a recombinant vector is prepared from this, introduced into E. coli, etc., and colonies are selected to prepare a desired recombinant vector. Then, the base sequence of the target DNA is confirmed by a known method, for example, a dideoxynucleotide chain termination method.
目的とする抗 Mpl抗体の V領域をコードする DNAを得たのち、これを、所望の抗体定 常領域 (C領域)をコードする DNAを含有する発現ベクターへ組み込む。  After obtaining the DNA encoding the V region of the desired anti-Mpl antibody, this is incorporated into an expression vector containing the DNA encoding the desired antibody constant region (C region).
[0119] 本発明で使用される抗 Mpl抗体を製造するには、通常、抗体遺伝子を発現制御領 域、例えば、ェンハンサー、プロモーターの制御のもとで発現するよう発現ベクターに 組み込む。次に、この発現ベクターにより、宿主細胞を形質転換し、抗体を発現させ る。 [0119] To produce the anti-Mpl antibody used in the present invention, an antibody gene is usually expressed in an expression control region. Region, for example, an enhancer or a promoter. Next, host cells are transformed with this expression vector to express the antibody.
[0120] 抗体遺伝子の発現は、 H鎖または L鎖をコードするポリヌクレオチドを別々に発現べ クタ一に組み込んで宿主細胞を同時形質転換させてもょ 、し、あるいは H鎖および L 鎖をコードするポリヌクレオチドを単一の発現ベクターに組み込んで宿主細胞を形質 転換させてもよい (WO 94/11523号公報参照)。  [0120] Antibody gene expression can be accomplished by co-transforming host cells by incorporating polynucleotides encoding the H or L chains separately into expression vectors, or by encoding the H and L chains. The host cell may be transformed by incorporating the polynucleotide to be expressed into a single expression vector (see WO 94/11523).
[0121] ベクターとしては、例えば、大腸菌を宿主とする場合には、ベクターを大腸菌（例え ば、 JM109、 DH5 a、 HB101、 XLlBlue)などで大量に増幅させ大量調製するために、 大腸菌で増幅されるための「ori」をもち、さらに形質転換された大腸菌の選抜遺伝子 (例えば、なんらかの薬剤（アンピシリンやテトラサイクリン、カナマイシン、クロラムフエ 二コール）により判別できるような薬剤耐性遺伝子)を有すれば特に制限はない。ベタ ターの例としては、 M13系ベクター、 pUC系ベクター、 pBR322、 pBluescript、 pCR-Scriptなどが挙げられる。また、 cDNAのサブクローユング、切り出しを目的とした 場合、上記ベクターの他に、例えば、 pGEM- T、 pDIRECT、 pT7などが挙げられる。  [0121] As a vector, for example, when Escherichia coli is used as a host, the vector is amplified in Escherichia coli (E. coli, for example, JM109, DH5a, HB101, XLlBlue), etc. The restriction is not particularly limited as long as it has an "ori" for selection and a transformed gene of Escherichia coli (for example, a drug resistance gene that can be distinguished by any drug (ampicillin, tetracycline, kanamycin, chloramphenicol)). Absent. Examples of the beta include M13-based vectors, pUC-based vectors, pBR322, pBluescript, pCR-Script, and the like. When subcloning or excision of cDNA is intended, in addition to the above vectors, for example, pGEM-T, pDIRECT, pT7 and the like can be mentioned.
[0122] 発現ベクターとしては、例えば、大腸菌での発現を目的とした場合は、ベクターが 大腸菌で増幅されるような上記特徴を持つほかに、宿主を JM109、 DH5 a、 HB101、 XLl-Blueなどの大腸菌とした場合にぉ 、ては、大腸菌で効率よく発現できるようなプ 口モーター、例えば、 lacZプロモーター（Wardら， Nature (1989) 341, 544-546 ; The [0122] expression vector, for example, if the purpose of expression in E. coli should have the above characteristics as the vector is amplified in E. coli, a host of JM109, DH5 a, HB101, XLl -Blue , etc. In the case of Escherichia coli, a promoter that can be efficiently expressed in Escherichia coli, such as the lacZ promoter (Ward et al., Nature (1989) 341, 544-546;
FASEB J. (1992) 6, 2422— 2427)、 araBプロモーター（Betterら， Science (1988) 240, 1041-1043 )、または T7プロモーターなどを持っていることが不可欠である。このよう なベクターとしては、上記ベクターの他に PGEX-5X- 1 (フアルマシア社製）、「 QlAexpress system」（キアゲン社製）、 pEGFP、または pET (この場合、宿主は T7 RNA ポリメラーゼを発現している BL21が好ましい)などが挙げられる。  It is essential to have FASEB J. (1992) 6, 2422-2427), araB promoter (Better et al., Science (1988) 240, 1041-1043), or T7 promoter. Such vectors include PGEX-5X-1 (Pharmacia), “QlAexpress system” (Qiagen), pEGFP, or pET (in this case, the host expresses T7 RNA polymerase in addition to the above vectors). BL21 is preferred).
[0123] また、ベクターには、ポリペプチド分泌のためのシグナル配列が含まれて!/、てもよ!/ヽ 。蛋白質分泌のためのシグナル配列としては、大腸菌のペリブラズムに産生させる場 合、 pelBシグナル配列（Lei, S. P. et al J. Bacteriol. (1987) 169, 4379)を使用すれば よい。宿主細胞へのベクターの導入は、例えば塩化カルシウム法、エレクト口ポレー シヨン法を用いて行うことができる。 [0123] Also, the vector contains a signal sequence for polypeptide secretion! As a signal sequence for protein secretion, a pelB signal sequence (Lei, SP et al J. Bacteriol. (1987) 169, 4379) may be used for production in the periplasm of Escherichia coli. The introduction of the vector into the host cell can be carried out, for example, by the calcium chloride method, It can be carried out by using the shion method.
[0124] 大腸菌以外にも、例えば、哺乳動物由来の発現ベクター（例えば、 pcDNA3 (インビ トロゲン社製）や、 pEGF— BOS (Nucleic Acids. Res.1990, 18(17),p5322)、 pEF、 pCDM8)、昆虫細胞由来の発現ベクター（例えば「Bac- to- BAC baculovairus expression system] (ギブコ BRL社製）、 pBacPAK8)、植物由来の発現ベクター（例え ば ρΜΗ1、 pMH2)、動物ウィルス由来の発現ベクター（例えば、 pHSV、 pMV、 pAdexLcw)、レトロウイルス由来の発現ベクター（例えば、 pZIPneo)、酵母由来の発現 ベクター（例えば、「Pichia Expression Kit」（インビトロゲン社製）、 pNVll、 SP- Q01)、 枯草菌由来の発現ベクター（例えば、 PPL608、 pKTH50)が挙げられる。 [0124] In addition to Escherichia coli, for example, mammalian-derived expression vectors (for example, pcDNA3 (manufactured by Invitrogen), pEGF-BOS (Nucleic Acids. Res. 1990, 18 (17), p5322), pEF, pCDM8 ), Insect cell-derived expression vectors (eg, “Bac-to-BAC baculovairus expression system” (manufactured by Gibco BRL), pBacPAK8), plant-derived expression vectors (eg, ρΜΗ1, pMH2), animal virus-derived expression vectors ( For example, pHSV, pMV, pAdexLcw), retrovirus-derived expression vector (for example, pZIPneo), yeast-derived expression vector (for example, “Pichia Expression Kit” (manufactured by Invitrogen), pNVll, SP-Q01), Bacillus subtilis derived expression vector (e.g., P PL608, pKTH50) can be mentioned.
[0125] CHO細胞、 COS細胞、 NIH3T3細胞等の動物細胞での発現を目的とした場合には 、細胞内で発現させるために必要なプロモーター、例えば SV40プロモーター（ Mulliganら， Nature (1979) 277, 108)、 MMTV-LTRプロモーター、 EF1 αプロモータ 一（Mizushimaら， Nucleic Acids Res. (1990) 18, 5322)、 CMVプロモーターなどを持 つていることが不可欠であり、細胞への形質転換を選抜するための遺伝子 (例えば、 薬剤 (ネオマイシン、 G418など）により判別できるような薬剤耐性遺伝子)を有すれば さらに好ましい。このような特性を有するベクターとしては、例えば、 pMAM、 pDR2、 pBK- RSV、 pBK-CMV, pOPRSV、 pOP13などが挙げられる。  [0125] When expression in animal cells such as CHO cells, COS cells, and NIH3T3 cells is intended, promoters required for expression in cells, such as the SV40 promoter (Muligan et al., Nature (1979) 277, 108), the MMTV-LTR promoter, the EF1 α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), and the CMV promoter are essential, in order to select for transformation into cells. (Eg, a drug resistance gene that can be distinguished by a drug (neomycin, G418, etc.)) is more preferable. Vectors having such properties include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, pOP13, and the like.
[0126] さらに、遺伝子を安定的に発現させ、かつ、細胞内での遺伝子のコピー数の増幅を 目的とする場合には、核酸合成経路を欠損した CHO細胞にそれを相補する DHFR遺 伝子を有するベクター（例えば、 pCHOIなど）を導入し、メトトレキセート (MTX)により 増幅させる方法が挙げられ、また、遺伝子の一過性の発現を目的とする場合には、 [0126] Furthermore, when aiming for stable expression of a gene and amplification of the copy number of the gene in a cell, a DHFR gene that complements the gene in a CHO cell deficient in a nucleic acid synthesis pathway is used. A method of introducing a vector (e.g., pCHOI etc.) and amplifying it with methotrexate (MTX).
SV40 T抗原を発現する遺伝子を染色体上に持つ COS細胞を用いて SV40の複製起 点を持つベクター (pcDなど)で形質転換する方法が挙げられる。複製開始点として は、また、ポリオ一マウィルス、アデノウイルス、ゥシパピローマウィルス（BPV)等の由 来のものを用いることもできる。さらに、宿主細胞系で遺伝子コピー数増幅のため、発 現ベクターは選択マーカーとして、アミノグリコシドトランスフェラーゼ (APH)遺伝子、 チミジンキナーゼ (TK)遺伝子、大腸菌キサンチングァニンホスホリボシルトランスフエ ラーゼ (Ecogpt)遺伝子、ジヒドロ葉酸還元酵素（dhfr)遺伝子等を含むことができる。 [0127] ベクターが導入される宿主細胞としては特に制限はなぐ例えば、大腸菌や種々の 動物細胞などを用いることが可能である。宿主細胞は、例えば、本発明の抗体の製 造や発現のための産生系として使用することができる。ポリペプチド製造のための産 生系は、 in vitroおよび in vivoの産生系がある。 in vitroの産生系としては、真核細胞 を使用する産生系や原核細胞を使用する産生系が挙げられる。 A method of using a COS cell having a gene expressing the SV40 T antigen on the chromosome and transforming it with a vector having a replication origin of SV40 (such as pcD) is used. As the origin of replication, those derived from poliovirus, adenovirus, ゥ papilloma virus (BPV) and the like can also be used. Furthermore, the expression vector is selected as a selection marker for aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, Escherichia coli xanthinguanine phosphoribosyltransferase (Ecogpt) gene, It can contain the folate reductase (dhfr) gene and the like. [0127] The host cell into which the vector is introduced is not particularly limited. For example, Escherichia coli and various animal cells can be used. The host cell can be used, for example, as a production system for producing or expressing the antibody of the present invention. Production systems for polypeptide production include in vitro and in vivo production systems. Examples of the in vitro production system include a production system using eukaryotic cells and a production system using prokaryotic cells.
[0128] 真核細胞を使用する場合、例えば、動物細胞、植物細胞、真菌細胞を宿主に用い ることができる。動物細胞としては、哺乳類細胞、例えば、 CH0 (J. Exp. Med. (1995) 108, 945)、 COSゝ 3T3、ミエローマ、 BHK (baby hamster kidney)、 HeLa、 Vero、両生 類細胞、例えばアフリカッメガエル卵母細胞（Valle, et al.， Nature (1981) 291, 358-340)、あるいは昆虫細胞、例えば、 S19、 Sf21、 Tn5が知られている。本発明にお いては、 CHO-DG44, CH0-DXB11、 COS7細胞、 BHK細胞が好適に用いられる。動 物細胞において、大量発現を目的とする場合には特に CHO細胞が好ましい。宿主 細胞へのベクターの導入は、例えば、リン酸カルシウム法、 DEAEデキストラン法、力 チォニックリボソーム DOTAP (ベーリンガーマンハイム社製）を用いた方法、エレクト口 ポーレーシヨン法、リポフエクシヨンなどの方法で行うことが可能である。  [0128] When using eukaryotic cells, for example, animal cells, plant cells, and fungal cells can be used as hosts. Animal cells include mammalian cells, for example, CH0 (J. Exp. Med. (1995) 108, 945), COS-3T3, myeloma, BHK (baby hamster kidney), HeLa, Vero, amphibian cells, for example, African Megafrog oocytes (Valle, et al., Nature (1981) 291, 358-340) or insect cells such as S19, Sf21, and Tn5 are known. In the present invention, CHO-DG44, CH0-DXB11, COS7 cells, and BHK cells are preferably used. In animal cells, CHO cells are particularly preferred for the purpose of large-scale expression. The vector can be introduced into a host cell by, for example, a calcium phosphate method, a DEAE dextran method, a method using a force ribosome DOTAP (manufactured by Boehringer Mannheim), an electoral poration method, or a lipofection method. .
[0129] 植物細胞としては、例えば、ニコチアナ 'タパカム（Nicotiana tabacum)由来の細胞 が蛋白質生産系として知られており、これをカルス培養すればよい。真菌細胞として は、酵母、例えば、サッカロミセス（Saccharomyces)属、例えば、サッカロミセス'セレビ ンェ (Saccharomyces cerevisiae)、サッカロ；セス-ホンへ (Saccharomyces pombe)、 糸状菌、例えば、ァスペルギルス（Aspergillus)属、例えば、ァスペルギルス '二ガー（ Aspergillus nigerjが知られている。  [0129] As plant cells, for example, cells derived from Nicotiana tabacum (Nicotiana tabacum) are known as a protein production system, which may be callus cultured. Fungal cells include yeast, for example, the genus Saccharomyces, for example, Saccharomyces cerevisiae, Saccharo; Saccharomyces pombe, filamentous fungi, for example, Aspergillus, for example, Aspergillus nigerj is known.
[0130] 原核細胞を使用する場合、細菌細胞を用いる産生系がある。細菌細胞としては、大 腸菌（E. coli)、例えば、 JM109, DH5 a、 HB101等が挙げられ、その他、枯草菌が知 られている。  [0130] When prokaryotic cells are used, there is a production system using bacterial cells. Examples of bacterial cells include Escherichia coli (E. coli), for example, JM109, DH5a, HB101, and the like, and Bacillus subtilis.
[0131] 本方法においては次いで上記宿主細胞を培養する。 目的とするポリヌクレオチドに より形質転換された細胞を in vitroで培養することにより、抗体が得られる。培養は、公 知の方法に従い行うことができる。例えば、動物細胞の培養液として、例えば、 DMEM、 MEM, RPMI1640, IMDMを使用することができる。その際、 FBS、牛胎児血 清 (FCS)等の血清補液を併用することもできるし、無血清培養してもよい。培養時の pHは、約 6〜8であるのが好ましい。培養は、通常、約 30〜40°Cで約 15〜200時間行 い、必要に応じて培地の交換、通気、攪拌を加える。 [0131] In the present method, the above host cells are then cultured. An antibody can be obtained by culturing cells transformed with the target polynucleotide in vitro. Culture can be performed according to a known method. For example, as a culture solution of animal cells, for example, DMEM, MEM, RPMI1640, IMDM can be used. At that time, FBS, fetal bovine blood A serum replacement solution such as CIS (FCS) may be used in combination, or serum-free culture may be performed. The pH during culturing is preferably about 6-8. Culture is usually performed at about 30 to 40 ° C for about 15 to 200 hours, and the medium is replaced, aerated, and agitated as necessary.
[0132] 一方、 in vivoでポリペプチドを産生させる系としては、例えば、動物を使用する産生 系や植物を使用する産生系が挙げられる。これらの動物又は植物に目的とするポリ ヌクレオチドを導入し、動物又は植物の体内でポリペプチドを産生させ、回収する。 本発明における「宿主」とは、これらの動物、植物を包含する。  [0132] On the other hand, examples of a system for producing a polypeptide in vivo include a production system using animals and a production system using plants. A polynucleotide of interest is introduced into these animals or plants, and the polypeptide is produced in the body of the animals or plants and collected. The “host” in the present invention includes these animals and plants.
[0133] 動物を使用する場合、哺乳類動物、昆虫を用いる産生系がある。哺乳類動物として は、ャギ、ブタ、ヒッジ、マウス、ゥシを用いることができる（Vicki Glaser, SPECTRUM Biotechnology Applications, 1993)。また、哺乳類動物を用いる場合、トランスジェ- ック動物を用いることができる。  [0133] When animals are used, there are production systems using mammals and insects. Goats, pigs, sheep, mice, and pests can be used as mammals (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). When a mammal is used, a transgenic animal can be used.
[0134] 例えば、目的とするポリヌクレオチドを、ャギ j8カゼインのような乳汁中に固有に産 生されるポリペプチドをコードする遺伝子との融合遺伝子として調製する。次いで、こ の融合遺伝子を含む DNA断片をャギの胚へ注入し、この胚を雌のャギへ移植する。 胚を受容したャギカ 生まれるトランスジエニックャギ又はその子孫が産生する乳汁 力 、目的の抗体を得ることができる。トランスジヱニックャギカ 産生される抗体を含 む乳汁量を増加させるために、適宜ホルモンをトランスジエニックャギに使用してもよ い（Ebert, K.M. et al.， Bio/Technology (1994) 12, 699—702)。  For example, a polynucleotide of interest is prepared as a fusion gene with a gene encoding a polypeptide uniquely produced in milk, such as goat j8 casein. Then, a DNA fragment containing the fusion gene is injected into a goat embryo, and the embryo is transplanted into a female goat. Goats that have received the embryo Transgenic goats born or milk offspring produced by their progeny, and the desired antibody can be obtained. Transgeneic mosquitoes In order to increase the amount of milk containing antibodies produced, hormones may be used in transgenics as appropriate (Ebert, KM et al., Bio / Technology (1994). ) 12, 699-702).
[0135] また、昆虫としては、例えばカイコを用いることができる。カイコを用いる場合、目的 の抗体をコードするポリヌクレオチドを挿入したバキュロウィルスをカイコに感染させる ことにより、このカイコの体液から目的の抗体を得ることができる（Susumu, M. et al.， Nature (1985) 315, 592-594)。  [0135] As insects, for example, silkworms can be used. When a silkworm is used, the desired antibody can be obtained from the body fluid of the silkworm by infecting the silkworm with a baculovirus into which a polynucleotide encoding the antibody of interest has been inserted (Susumu, M. et al., Nature ( 1985) 315, 592-594).
[0136] さらに、植物を使用する場合、例えばタバコを用いることができる。タバコを用いる場 合、目的とする抗体をコードするポリヌクレオチドを植物発現用ベクター、例えば pMON 530に挿入し、このベクターをァグロバタテリゥム'ッメファシエンス（  [0136] Further, when a plant is used, for example, tobacco can be used. When tobacco is used, a polynucleotide encoding the antibody of interest is inserted into a plant expression vector, for example, pMON530, and this vector is inserted into an Agrobacterium tmmefaciens (
Agrobacterium tumefaciens)のようなバクテリアに導入する。このバクテリアをタバコ、 例えば、ニコチアナ 'タパカム（Nicotiana tabacum)に感染させ、本タバコの葉より所 望の抗体を得ることができる（Julian K.-C. Ma et al.， Eur. J. Immunol. (1994) 24, 131-138)。 (Agrobacterium tumefaciens). The bacteria are infected with tobacco, for example, Nicotiana tabacum, and the desired antibody can be obtained from the leaves of this tobacco (Julian K.-C. Ma et al., Eur. J. Immunol. (1994) 24, 131-138).
[0137] これにより得られた抗体は、宿主細胞内または細胞外 (培地など)力 単離し、実質 的に純粋で均一な抗体として精製することができる。抗体の分離、精製は、通常のポ リペプチドの精製で使用されている分離、精製方法を使用すればよぐ何ら限定され るものではない。例えば、クロマトグラフィーカラム、フィルター、限外濾過、塩析、溶 媒沈殿、溶媒抽出、蒸留、免疫沈降、 SDS-ポリアクリルアミドゲル電気泳動、等電点 電気泳動法、透析、再結晶等を適宜選択、組み合わせればポリペプチドを分離、精 製することができる。  [0137] The antibody thus obtained can be isolated intracellularly or extracellularly (such as in a medium) and purified as a substantially pure and homogeneous antibody. The separation and purification of the antibody is not limited at all, provided that the separation and purification methods used in ordinary purification of polypeptides are used. For example, select chromatography column, filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. If combined, the polypeptide can be separated and purified.
[0138] クロマトグラフィーとしては、例えばァフィ二ティークロマトグラフィー、イオン交換クロ マトグラフィー、疎水性クロマトグラフィー、ゲル濾過、逆相クロマトグラフィー、吸着ク 口マトグラフィ一等が挙げられる（Strategies for Protein Purification and  [0138] Examples of the chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography, and the like (Strategies for Protein Purification and
Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al" Cold Spring Harbor Laboratory Press, 1996)。これらのクロマトグラフィーは、液ネ目クロマト グラフィー、例えば HPLC、 FPLC等の液相クロマトグラフィーを用いて行うことができる 。ァフィユティークロマトグラフィーに用いるカラムとしては、プロテイン Aカラム、プロテ イン Gカラムが挙げられる。例えば、プロテイン Aを用いたカラムとして、 Hyper D, POROS, Sepharose F. F. (Pharmacia)等が挙げられる。  Ed Daniel R. Marshak et al "Cold Spring Harbor Laboratory Press, 1996). These chromatographies are performed using liquid chromatography such as liquid phase chromatography such as HPLC and FPLC. Columns used for affinity chromatography include protein A columns and protein G columns, for example, columns using protein A include Hyper D, POROS, Sepharose FF (Pharmacia) and the like. Can be
[0139] なお、抗体の精製前又は精製後に適当な蛋白質修飾酵素を作用させることにより、 任意に修飾を加えたり部分的にペプチドを除去することもできる。蛋白質修飾酵素と しては、例えば、トリプシン、キモトリブシン、リシルエンドべプチダーゼ、プロテインキ ナーゼ、ダルコシダーゼなどが用いられる。  [0139] In addition, before or after purification of the antibody, an appropriate protein modification enzyme is allowed to act on the antibody to optionally modify the peptide or partially remove the peptide. As the protein modifying enzyme, for example, trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, dalcosidase and the like are used.
[0140] 本発明の血小板減少症治療剤は、血小板減少症の発症後に投与してもよ!/ヽし、血 小板減少症が発症する前に予防目的で投与されてもよい。本発明の血小板増加剤 の投与時期は限定されず、血小板を増加させる必要のある場合に投与することがで きる。  The therapeutic agent for thrombocytopenia of the present invention may be administered after the onset of thrombocytopenia, or may be administered for a prophylactic purpose before the onset of thrombocytopenia. The administration time of the platelet-increasing agent of the present invention is not limited, and it can be administered when it is necessary to increase platelets.
[0141] Mplを認識する抗体は、当業者に公知の方法で製剤化することが可能である。例え ば、水もしくはそれ以外の薬学的に許容し得る液との無菌性溶液、又は懸濁液剤の 注射剤の形で非経口的に使用できる。例えば、薬理学上許容される担体もしくは媒 体、具体的には、滅菌水や生理食塩水、植物油、乳化剤、懸濁剤、界面活性剤、安 定剤、香味剤、賦形剤、べヒクル、防腐剤、結合剤などと適宜組み合わせて、一般に 認められた製薬実施に要求される単位用量形態で混和することによって製剤化する ことが考えられる。これら製剤における有効成分量は指示された範囲の適当な容量 が得られるようにするものである。 [0141] Antibodies that recognize Mpl can be formulated by methods known to those skilled in the art. For example, it can be used parenterally in the form of an injectable sterile solution with water or other pharmaceutically acceptable liquid, or a suspension. For example, a pharmacologically acceptable carrier or medium Body, specifically, sterile water or physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, etc. It may be formulated by mixing in the unit dosage form required for accepted pharmaceutical practice. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.
[0142] 注射のための無菌組成物は注射用蒸留水のようなべヒクルを用いて通常の製剤実 施に従って処方することができる。  [0142] A sterile composition for injection can be formulated using a vehicle such as distilled water for injection according to normal pharmaceutical practice.
注射用の水溶液としては、例えば生理食塩水、ブドウ糖やその他の補助薬を含む 等張液、例えば D-ソルビトール、 D-マンノース、 D-マン-トール、塩化ナトリウムが挙 げられ、適当な溶解補助剤、例えばアルコール、具体的にはエタノール、ポリアルコ ール、例えばプロピレングリコール、ポリエチレングリコール、非イオン性界面活性剤 、例えばポリソルベート 80 (TM)、 HCO- 50と併用してもよい。  Aqueous injection solutions include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mantol, and sodium chloride. It may be used in combination with an agent such as an alcohol, specifically ethanol, a polyalcohol such as propylene glycol, polyethylene glycol, a nonionic surfactant such as polysorbate 80 (TM) or HCO-50.
[0143] 油性液としてはゴマ油、大豆油があげられ、溶解補助剤として安息香酸ベンジル、 ベンジルアルコールと併用してもよい。また、緩衝剤、例えばリン酸塩緩衝液、酢酸 ナトリウム緩衝液、無痛化剤、例えば、塩酸プロ力イン、安定剤、例えばべンジルアル コール、フエノール、酸ィ匕防止剤と配合してもよい。調製された注射液は通常、適当 なアンプルに充填させる。  [0143] Examples of the oily liquid include sesame oil and soybean oil, and may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent. It may also be combined with a buffer such as a phosphate buffer and a sodium acetate buffer, a soothing agent such as proforce hydrochloride, and a stabilizer such as benzyl alcohol, phenol and an antioxidant. The prepared injection solution is usually filled in an appropriate ampoule.
[0144] 投与は好ましくは非経口投与であり、具体的には、注射剤型、経鼻投与剤型、経肺 投与剤型、経皮投与型などが挙げられる。注射剤型の例としては、例えば、静脈内 注射、筋肉内注射、腹腔内注射、皮下注射などにより全身または局部的に投与する ことができる。  [0144] Administration is preferably parenteral administration, and specific examples include injection, nasal, pulmonary, and transdermal administrations. Examples of the injection form include systemic or local administration by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection and the like.
[0145] また、患者の年齢、症状により適宜投与方法を選択することができる。抗体または 抗体をコードするポリヌクレオチドを含有する医薬組成物の投与量としては、例えば、 一回につき体重 lkgあたり O.OOOlmgから lOOOmgの範囲で選ぶことが可能である。ある いは、例えば、患者あたり 0.001〜100000mg/bodyの範囲で投与量を選ぶことができ る力 これらの数値に必ずしも制限されるものではない。投与量、投与方法は、患者 の体重や年齢、症状などにより変動するが、当業者であれば適宜選択することが可 能である。 なお本明細書において引用された全ての先行技術文献は、参照として本明細書に 組み入れられる。 [0145] The administration method can be appropriately selected depending on the age and symptoms of the patient. The dose of the pharmaceutical composition containing the antibody or the polynucleotide encoding the antibody can be selected, for example, from O.OOOOmg to 100000 mg / kg body weight at a time. Or, for example, the ability to select a dose in the range of 0.001 to 100,000 mg / body per patient. These values are not necessarily limited. The dose and administration method vary depending on the patient's body weight, age, symptoms, and the like, but can be appropriately selected by those skilled in the art. All prior art documents cited in this specification are incorporated herein by reference.
実施例  Example
[0146] 以下、本発明を実施例により詳細に説明するが、本発明はこれら実施例に制限さ れるものではない。  Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples.
[0147] 〔実施例 1〕 正常力二クイサルにおける血小板増多作用 [Example 1] [0147] Platelet-increasing effect in normotensive quixar
血小板数の測定は以下のように行った。  The platelet count was measured as follows.
サルの伏在静脈力も末梢血 lmLを採取し、直ちに EDTAカップに入れ、ローリングミ キサー (型式 RM-810)にて混和して、凝固を防止した。その抗凝固処理した血液を 用いて、シスメッタス F-820で各血球パラメーターを測定し、血小板数を算出した。 DayOで予め血小板数を測定した 6頭のサルを各 3頭の二群に分け、 VB22B投与群と 溶媒投与群を設定した。被験物質である VB22B sc(Fv)2 (配列番号： 14)を、 20 mmol/L Acetate buffer (pH6.0)(150 mmol/L NaCl, 0.01%Tween 80)を溶媒として、 VB22B投与群の 3頭のサルに対して 100 μ g/kg体重となるように 14日間（Dayl〜14) 皮下注射した。一方、対照の溶媒投与群とした 3頭のサルに対しては、体重あたり VB22B投与群と同一容量となるように溶媒を 14日間皮下注射した。尚、実験期間中 3 —4日の間隔で VB22B sc(Fv)2あるいは溶媒を投与する前に伏在静脈から lmL採血 し血小板数を測定した。血小板数が測定限界を超えて ヽる場合は適宜血液を希釈し て再測定した。  As for the saphenous vein force of monkeys, 1 mL of peripheral blood was collected, immediately placed in an EDTA cup, and mixed with a rolling mixer (model RM-810) to prevent coagulation. Using the anticoagulated blood, each blood cell parameter was measured with Cismetas F-820, and the platelet count was calculated. Six monkeys whose platelet count was measured in advance on DayO were divided into two groups of three each, and a VB22B administration group and a solvent administration group were set. The test substance VB22B sc (Fv) 2 (SEQ ID NO: 14) was used as a solvent in 20 mmol / L Acetate buffer (pH 6.0) (150 mmol / L NaCl, 0.01% Tween 80) as a solvent. Monkeys in the head were injected subcutaneously at 100 μg / kg body weight for 14 days (Dayl-14). On the other hand, three monkeys in the control vehicle-administered group were injected subcutaneously with the solvent for 14 days so that the volume became the same as that of the VB22B-administered group. Before the administration of VB22B sc (Fv) 2 or a solvent at intervals of 3 to 4 days during the experiment, 1 mL of blood was collected from the saphenous vein and the platelet count was measured. If the platelet count exceeded the measurement limit, the blood was diluted appropriately and remeasured.
[0148] その結果、 VB22B sc(Fv)2投与群では、投与後 4日目以降に、血小板数の顕著な 増加がみられ 14日で最大値を示した（図 2)。すなわち、 in vitroアツセィで示したァゴ 二スト活性は in vivoでも示されることが確認された。  [0148] As a result, in the VB22B sc (Fv) 2 administration group, the number of platelets was remarkably increased from day 4 onward after administration, and reached a maximum value on day 14 (Fig. 2). That is, it was confirmed that the agonist activity shown in the in vitro assay was also exhibited in vivo.
[0149] 〔実施例 2〕 血小板減少力-クイサルにおける血小板減少回復作用  [Example 2] Thrombocytopenia-thrombocytopenia recovery effect in quisars
DayOで予め血小板数を測定した 5頭のサルに、化学療法剤のひとつである塩酸二 ムスチン (ACNU)を 15 mg/kg体重となるように静脈内注射した。これらを二群に分け 、 VB22B投与群 2頭と溶媒投与群 3頭を設定した。被験物質である VB22B sc(Fv)2 (配 列番号： 14)を、 20 mmol/L Acetate buffer (pH6.0)(150 mmol/L NaCl, 0.01%Tween 80)を溶媒として、 VB22B投与群のサルに対して 100 μ g/kg体重となるように 14日間（ Dayl〜14)皮下注射した。一方、対照の溶媒投与群としたサルに対しては、体重あ たり VB22B投与群と同一容量となるように溶媒を 14日間皮下注射した。尚、実験期間 中 3—4日の間隔で VB22B sc(Fv)2あるいは溶媒を投与する前に伏在静脈から lmL 採血し血小板数を測定した。 Five monkeys whose platelet count was measured in advance on DayO were intravenously injected with dimustine hydrochloride (ACNU), one of the chemotherapeutic agents, at a dose of 15 mg / kg body weight. These were divided into two groups, and two VB22B administration groups and three solvent administration groups were set. The test substance VB22B sc (Fv) 2 (SEQ ID NO: 14) was used as a solvent in 20 mmol / L Acetate buffer (pH 6.0) (150 mmol / L NaCl, 0.01% Tween 80) to 14 days for 100 μg / kg body weight for monkey ( Dayl-14) Subcutaneous injection. On the other hand, the control vehicle-administered monkeys were injected subcutaneously with the solvent for 14 days so that the volume would be the same as that of the VB22B-administered group. Prior to administration of VB22B sc (Fv) 2 or a solvent at an interval of 3 to 4 days during the experiment, 1 mL of blood was collected from the saphenous vein and the platelet count was measured.
[0150] その結果、溶媒投与群では、 ACNU投与後 10日後から、末梢血小板数の顕著な減 少がみられ 18日で極小値 (Nadir)を示した（図 3)。一方、 VB22B sc(Fv)2投与群では 、投与後 4日以降に血小板数の増加がみられ 11日で最大値を示し血小板減少を回 避した（図 3)。すなわち、正常サルのみならず、血小板減少症に対しても血小板増 多作用が示されることが確認された。  [0150] As a result, in the vehicle-administered group, a marked decrease in the number of peripheral platelets was observed from 10 days after the administration of ACNU, and showed a minimum value (Nadir) at 18 days (Fig. 3). On the other hand, in the VB22B sc (Fv) 2 administration group, the number of platelets increased 4 days after administration and reached the maximum value on the 11th day, avoiding thrombocytopenia (FIG. 3). That is, it was confirmed that a thrombocytosis effect was exhibited not only for normal monkeys but also for thrombocytopenia.
[0151] 〔実施例 3〕 正常力-クイサルにおける hVB22B u2~wz4 sc(FV)2ならびに hVB22B q-wz5 sc(FV)2の単回投与による血小板増多作用  Example 3 Platelet-Increasing Effect of Normal Administration of hVB22B u2-wz4 sc (FV) 2 and hVB22B q-wz5 sc (FV) 2 in Normal-Quizal Monkeys
末梢血血小板数の測定は以下のように行った。サルの伏在静脈から末梢血 0.5 mL を採取し、直ちに EDTAを塗布した採血チューブであるマイクロティナ（日本べタトン' ディッキンソン 365973)に入れ、ローリングミキサー（シスメッタス、型式 RM- 810)にて 混和して、凝固を防止した。その抗凝固処理した血液を用いて、自動血球分析装置 F-820 (シスメッタス)で血小板数を測定した。  The measurement of the number of peripheral blood platelets was performed as follows. 0.5 mL of peripheral blood was collected from the saphenous vein of a monkey, immediately placed in MicroTina (Nippon Betaton 'Dickinson 365973), a blood collection tube coated with EDTA, and mixed with a rolling mixer (Sysmettas, Model RM-810). To prevent coagulation. Using the anticoagulated blood, the platelet count was measured with an automatic blood cell analyzer F-820 (Cismettas).
[0152] DayOで予め血小板数を測定した 5頭のサルを二群に分け、 hVB22B u2~wz4  [0152] The five monkeys whose platelet count was measured in advance on DayO were divided into two groups, and hVB22Bu2 to wz4
sc(FV)2 (N=3)ならびに hVB22B q- wz5 sc(FV)2 (N=2)の投与量を 1 mg/kg体重に設 定した。 hVB22B u2-wz4 sc(FV)2 (配列番号： 100)ならびに hVB22B q- wz5 sc(FV)2 ( 配列番号： 101)は、 20 mmol/Lクェン酸ナトリウム緩衝溶液、 300 mmol/L NaCl, pH 7.5, 0.01% Tween 80を溶媒として使用し、 Day 1に単回静脈内 (i.v.)注射した。その後 、 Day 8, 11, 15, 21に上記の方法で血小板数を測定した。静脈内 (i.v.)注射のおよそ 一月後、再び DayOを設定し、同一個体を用いて、同一の投与量で Day 1に単回皮下 (s. )注射をした。その後、 Day 8, 11, 14, 22に上記の方法で血小板数を測定した。  The dose of sc (FV) 2 (N = 3) and hVB22B q-wz5 sc (FV) 2 (N = 2) was set to 1 mg / kg body weight. hVB22B u2-wz4 sc (FV) 2 (SEQ ID NO: 100) and hVB22B q-wz5 sc (FV) 2 (SEQ ID NO: 101) are 20 mmol / L sodium citrate buffer solution, 300 mmol / L NaCl, pH A single intravenous (iv) injection was performed on Day 1 using 7.5, 0.01% Tween 80 as solvent. Thereafter, on Day 8, 11, 15, and 21, the platelet count was measured by the above method. Approximately one month after the intravenous (i.v.) injection, DayO was set again, and a single subcutaneous (s.) Injection was performed on Day 1 at the same dose using the same individual. Thereafter, on Day 8, 11, 14, and 22, the platelet count was measured by the above method.
[0153] その結果、 hVB22B u2- wz4 sc(FV)2ならびに hVB22B q- wz5 sc(FV)2は、静脈内 (i.v.)注射ならびに皮下 (s.c.)注射の両投与経路で用量に依存した血小板数の増加 がみられた（図 12)。すなわち、 hVB22B u2-wz4 sc(FV)2ならびに hVB22B q-wz5 sc(FV)2の活性は in vivoでも良好に示されることが確認された。 [0154] 〔参考例 1〕 抗ヒト Mpl抗体の作製 [0153] As a result, hVB22B u2-wz4 sc (FV) 2 and hVB22B q-wz5 sc (FV) 2 showed a dose-dependent platelet count in both the intravenous (iv) injection and subcutaneous (sc) injection routes. Increased (Figure 12). That is, it was confirmed that the activities of hVB22B u2-wz4 sc (FV) 2 and hVB22B q-wz5 sc (FV) 2 are well exhibited in vivo. [Reference Example 1] Production of anti-human Mpl antibody
1.1 Mpl発現 BaF3細胞株の榭立  1.1 Establishment of Mpl-expressing BaF3 cell line
TPO依存増殖性細胞株を得るために、全長 Mpl遺伝子を発現する BaF3細胞株の 榭立を行った。  To obtain a TPO-dependent proliferating cell line, a BaF3 cell line expressing the full-length Mpl gene was established.
全長ヒト Mpl cDNA (Palaciosら、 Cell 1985 ;41 : 727-734) (GenBank#NM— 005373)を PCRにより増幅し、 pCHOI(Hirataら、 FEBS Letter 1994 ;356 : 244- 248)の DHFR遺伝 子発現部位を除去し、 HEF-VH-g y l(Satoら、 Mol Immunol. 1994;31 : 371- 381)の Neomycin耐性遺伝子発現部位を挿入した発現ベクター pCOS2にクローユングし、 pCOS2-hMplfollを構築した。  Full-length human Mpl cDNA (Palacios et al., Cell 1985; 41: 727-734) (GenBank # NM-005373) was amplified by PCR, and DHFR gene expression of pCHOI (Hirata et al., FEBS Letter 1994; 356: 244-248) was performed. The site was removed and cloned into the expression vector pCOS2 into which the Neomycin resistance gene expression site of HEF-VH-gyl (Sato et al., Mol Immunol. 1994; 31: 371-381) was inserted, to construct pCOS2-hMplfoll.
[0155] また、力-クイザル骨髄細胞から抽出した Total RNAから SMART RACE cDNA[0155] In addition, SMART RACE cDNA was extracted from total RNA extracted from force-monkey bone marrow cells.
Amplification Kit (Clontech社製)を用いて、力-クイザル Mpl cDNA (配列番号： 67) をクロー-ングした。得られた力-クイザル cDNAを pCOS2に挿入し、 Using the Amplification Kit (manufactured by Clontech), force-quiz Mpl cDNA (SEQ ID NO: 67) was cloned. Insert the resulting force-quiz cDNA into pCOS2,
pCOS2- monkeyMplfollを構築した。  pCOS2-monkeyMplfoll was constructed.
さらに、全長マウス Mpl cDNA(GenBank#NM_010823)を PCRにより増幅し、 pCOS2に 挿入し、 pCOS2- mouseMplfollを構築した。  Furthermore, full-length mouse Mpl cDNA (GenBank # NM_010823) was amplified by PCR and inserted into pCOS2 to construct pCOS2-mouseMplfoll.
[0156] 作製した各ベクター (20 μ g)を PBSに懸濁した BaF3細胞 (lxl07cells/mL)に混合し、 Gene Pulserキュベットに加え、 Gene Pulser II (Bio- Rad社製)を用いて 0.33kV, 950 ^ FDの容量でパルスをカ卩えた。エレクト口ポーレーシヨン処理により遺伝子導入した BaF3細胞を Ing/mLマウスインターロイキン 3 (以下、 mIL- 3、 Peprotech社製）、 500 g/mL Geneticin(Invitrogen社製)、 10% FBS(Invitrogen社製)を含む RPMI1640培地（ Invitrogen社製）に加えて選抜し、ヒト Mpl発現 BaF3細胞株（以下、 BaF3-human Mpl) 、サル Mpl発現 BaF3細胞株（以下、 BaF3- monkey Mpl)およびマウス Mpl発現 BaF3細 胞株（以下、 BaF3-mouse Mpl)を榭立した。選抜後は、 Ing/mL rhTPO(R&D社製)、 10% FBSを含む RPMI1640培地を用いて培養、維持した。 [0156] Each vector was prepared (20 mu g) was mixed with BaF3 cells suspended in PBS (lxl0 7 cells / mL) , was added to the Gene Pulser cuvette, using a Gene Pulser II (Bio- manufactured Rad) Pulses were generated at a capacity of 0.33 kV, 950 ^ FD. BaF3 cells transfected by electoral poration were transfected with Ing / mL mouse interleukin 3 (hereinafter, mIL-3, manufactured by Peprotech), 500 g / mL Geneticin (manufactured by Invitrogen), and 10% FBS (manufactured by Invitrogen). In addition to the RPMI1640 medium (manufactured by Invitrogen), the cells were selected and selected for human Mpl-expressing BaF3 cell line (hereinafter, BaF3-human Mpl), monkey Mpl-expressing BaF3 cell line (hereinafter, BaF3-monkey Mpl) and mouse Mpl-expressing BaF3 cell A strain (hereinafter, BaF3-mouse Mpl) was established. After the selection, the cells were cultured and maintained using RPMI1640 medium containing Ing / mL rhTPO (R & D) and 10% FBS.
[0157] 1.2 Mpl発現 CHO細胞株の榭立 [0157] 1.2 Establishment of CHO cell line expressing Mpl
Flow Cytometryを用いた結合活性評価用の細胞株を得るために、全長 Mpl遺伝子 を発現する CHO細胞株の榭立を行った。  To obtain a cell line for evaluating binding activity using Flow Cytometry, a CHO cell line expressing the full-length Mpl gene was established.
はじめに、 pCXN2(Niwaら、 Gene 1991 ; 108 : 193- 199)の Hindlll部位に pCHOIの DHFR遺伝子発現部位を挿入して、発現ベクター pCXND3を作製した。 pCOS2-hMpliulU pCOS2- monkeyMplfollおよび pCOS2- mouseMplfollを铸型にして、 His-tag配列を含むプライマーを用いて PCRにより増幅した各 Mpl遺伝子を pCXND3 にクロー-ングし、 pCXND3-hMpl-His, pCXND3- monkey Mp Hisおよび First, pCHOI was inserted into the Hindlll site of pCXN2 (Niwa et al., Gene 1991; 108: 193-199). An expression vector pCXND3 was prepared by inserting a DHFR gene expression site. pCOS2-hMpliulU pCOS2- monkeyMplfoll and pCOS2- mouseMplfoll in the铸型the claw each Mpl gene was amplified by PCR using primers with a His-tag sequence into pCXND3 - Ngushi, p CXND3-hMpl-His, pCXND3- monkey Mp His and
pCXND3- mouse Mp卜 Hisを構築した。  pCXND3- mouse Mp His was constructed.
[0158] 作製した各ベクター (25 μ g)を PBSに懸濁した CHO- DG44細胞 (lxl07cells/mL)に 混合し、 Gene Pulserキュベットに加え、 Gene Pulser II (Bio- Rad社製)を用いて 1.5kV, 25 μ FDの容量でパルスをカ卩えた。エレクト口ポーレーシヨン処理により遺伝子導入し た CHO細胞を 500 μ g/mL Geneticin、 lxHT (Invitrogen社製)を含む CHO- S- SFMII 培地 (Invitrogen社製)に加えて選抜し、ヒト Mpl発現 CHO細胞株（以下、 CHO-human Mpl)およびサル Mpl発現 CHO細胞株（以下、 CHO- monkey Mpl)およびマウス Mpl発 現 CHO細胞株（以下、 CHO-mouse Mpl)を榭立した。 [0158] Each vector was prepared (25 mu g) were mixed in suspended CHO- DG44 cells (lxl0 7 cells / mL) into PBS, added to the Gene Pulser cuvette, the Gene Pulser II (Bio- manufactured Rad) Pulses were obtained at a capacity of 1.5 kV and 25 μFD. CHO cells transfected by electoral poration were added to a CHO-S-SFMII medium (Invitrogen) containing 500 μg / mL Geneticin and lxHT (Invitrogen) and selected, and a human Mpl-expressing CHO cell line was selected. (Hereinafter, CHO-human Mpl), monkey Mpl-expressing CHO cell line (hereinafter, CHO-monkey Mpl) and mouse Mpl-expressing CHO cell line (hereinafter, CHO-mouse Mpl) were established.
[0159] 1.3 可溶型ヒト Mplタンパク質の調製  [0159] 1.3 Preparation of soluble human Mpl protein
可溶型ヒト Mplタンパク質を調製するため、昆虫細胞 S19細胞で分泌産生する発現 系を以下のように構築した。  To prepare soluble human Mpl protein, an expression system secreted and produced in insect cell S19 cells was constructed as follows.
ヒト Mplの細胞外領域（Gln26力 Trp491)の下流に FLAGタグを付カ卩した遺伝子を 作製し、 pBACSurf- 1 Transfer Plasmid (Novagen社製）の Pstl- Smal部位に挿入し、 pBACSurfl- hMp卜 FLAGを作製した。続いて、 Bac- N- Blue Transfection Kit ( Invitrogen)を用いて、 4 gの pBACSurfl- hMpト FLAGを S19細胞に導入した。培養 3 日後に培養上清を回収し、プラークアツセィにより組換えウィルスを単離した。ウィル スストックを調製後に S19細胞に感染させて培養上清を回収した。  A FLAG-tagged gene was prepared downstream of the extracellular region of human Mpl (Gln26 force Trp491), inserted into the Pstl-Smal site of pBACSurf-1 Transfer Plasmid (Novagen), and pBACSurfl-hMp FLAG Was prepared. Subsequently, 4 g of pBACSurfl-hMp-to-FLAG was introduced into S19 cells using the Bac-N-Blue Transfection Kit (Invitrogen). After 3 days of culture, the culture supernatant was collected, and the recombinant virus was isolated by plaque assay. After preparing the virus stock, S19 cells were infected and the culture supernatant was collected.
[0160] 得られた培養上清を用いて、以下のように可溶型ヒト Mplタンパク質を精製した。培 養上清を Q Sepharose Fast Flow (Amersham Biosciences社製)に吸着させた後に、 50mM Na- Phosphate Buffer, 0.01%(v/v) Tween20, 500mM NaCl (pH 7.2)を用いて溶 出した。溶出液を FLAG M2 -Agarose (SIGMA-ALDRICH社製)に吸着させた後に、 lOOmM Glycine- HCl, 0.01%(v/v) Tween20 (pH 3.5)を用いて溶出した。溶出後、直ち に 1M Tris-Cl (pH8.0)により中和し、 PD- 10 column (Amersham Biosciences社製)を 用いて、 PBS(-), 0.01% (v/v) Tween20に置換を行った。精製した可溶型 Mplタンパク 質を shMp卜 FLAGと称する。 Using the obtained culture supernatant, a soluble human Mpl protein was purified as follows. After adsorbing the culture supernatant to Q Sepharose Fast Flow (manufactured by Amersham Biosciences), the culture supernatant was eluted using 50 mM Na-Phosphate Buffer, 0.01% (v / v) Tween 20, 500 mM NaCl (pH 7.2). After adsorbing the eluate to FLAG M2-Agarose (manufactured by SIGMA-ALDRICH), the eluate was eluted with lOOmM Glycine-HCl, 0.01% (v / v) Tween20 (pH 3.5). Immediately after elution, neutralize with 1M Tris-Cl (pH8.0), and replace with PBS (-), 0.01% (v / v) Tween20 using PD-10 column (Amersham Biosciences). went. Purified soluble Mpl protein The quality is called shMp FLAG.
[0161] 1.4 ヒト MpHgG Fc融合タンパク質の調製  [0161] 1.4 Preparation of human MpHgG Fc fusion protein
ヒト MpHgG Fc融合タンパク質遺伝子は、 Bennettらの方法 (Bennettら、 J.Biol.Chem. 1991 ;266 : 23060-23067)に従って作製した。ヒト Mplの細胞外領域 (Gln26から Trp491)をコードする塩基配列をヒト IgG- γ 1の Fc領域 (Asp216よりの下流 の領域）をコードする塩基配列に連結し、連結部に Fusion Linkerとして BstEII配列（ アミノ酸 VaKThr)を付カ卩した。シグナル配列は、ヒト IgG H鎖可変領域のシグナルぺ プチド 19アミノ酸を使用した。得られたヒト MpHgG Fc融合タンパク質遺伝子を PCXND3にクロー-ングし、 pCXND3- hMp卜 Fcを構築した。  The human MpHgG Fc fusion protein gene was prepared according to the method of Bennett et al. (Bennett et al., J. Biol. Chem. 1991; 266: 23060-23067). The nucleotide sequence encoding the extracellular region of human Mpl (Gln26 to Trp491) is ligated to the nucleotide sequence encoding the Fc region of human IgG-γ1 (region downstream from Asp216), and the BstEII sequence is used as a fusion linker at the junction. (Amino acid VaKThr). As the signal sequence, 19 amino acids of the signal peptide of the human IgG heavy chain variable region were used. The obtained human MpHgG Fc fusion protein gene was cloned into PCXND3 to construct pCXND3-hMpFc.
[0162] 作製したベクター (25 μ g)を PBSに懸濁した CHO- DG44細胞 (lxl07cells/mL)に混 合し、 Gene Pulserキュベットに加え、 Gene Pulser II (Bio- Rad社製)を用いて 1.5kV, 25 μ FDの容量でパルスをカ卩えた。エレクト口ポーレーシヨン処理により遺伝子導入し た CHO細胞を 500 g/mL Geneticin, ΙχΗΤを含む CHO- S- SFMII培地に加えて選抜 し、 shMPL- Fc発現 CHO細胞株（CHO- hMp卜 Fc)を榭立した。 [0162] generated vector of (25 mu g) were combined mixed in suspended CHO- DG44 cells (lxl0 7 cells / mL) into PBS, added to the Gene Pulser cuvette, the Gene Pulser II (Bio- manufactured Rad) Pulses were obtained at a capacity of 1.5 kV and 25 μFD. CHO cells transfected by electoral poration were added to a CHO-S-SFMII medium containing 500 g / mL Geneticin, ΙχΗΤ, and selected to establish a shMPL-Fc-expressing CHO cell line (CHO-hMpFc). did.
[0163] 得られた培養上清を用いて、以下のようにヒト MpHgG Fc融合タンパク質を精製し た。  [0163] Using the obtained culture supernatant, a human MpHgG Fc fusion protein was purified as follows.
培養上清を Q Sepharose Fast Flow (Amersham Biosciences社製)に吸着させた後 に、 50mM Na- Phosphate Buffer, 0.01%(v/v) Tween20, 1M NaCl (pH 7.6)を用いて溶 出した。溶出液を HiTrap proteinG HPカラム（Amersham Biosciences社製）に吸着さ せた後に、 0.1 M Glycine— HC1, 150 mM NaCl, 0.01%(v/v) Tween20 (pH 2.7)を用い て溶出した。溶出後、直ちに 1M Tris-Cl (pH8.0)により中和し、 PD- 10 column (Amersham Biosciences社製)を用いて、 PBS (-) , 0.01% (v/v) Tween20に置換を行つ た。精製した可溶型 Mplタンパク質を hMpト Fcと称する。  After adsorbing the culture supernatant to Q Sepharose Fast Flow (manufactured by Amersham Biosciences), it was eluted using 50 mM Na-Phosphate Buffer, 0.01% (v / v) Tween20, 1M NaCl (pH 7.6). After adsorbing the eluate on a HiTrap protein G HP column (manufactured by Amersham Biosciences), the eluate was eluted using 0.1 M Glycine-HC1, 150 mM NaCl, 0.01% (v / v) Tween20 (pH 2.7). Immediately after elution, neutralize with 1 M Tris-Cl (pH 8.0), and replace with PBS (-), 0.01% (v / v) Tween20 using PD-10 column (Amersham Biosciences). Was. The purified soluble Mpl protein is called hMp to Fc.
[0164] 1.5 shMp卜 FLAGの免疫およびハイプリドーマの選抜 [0164] 1.5 shMp FLAG immunization and selection of hybridomas
MRL/MpJUmmCrj- lpr/lprマウス（以下、 MRL/lprマウス、 日本チヤ一ルス'リバ一よ り購入)を用いて、 8週齢より免疫を開始した。初回免疫は 100 /z g/匹の shMPL-FLAG にフロイント完全アジュバント（H37 Ra、ベタトン'ディッキンソン社製）をカ卩え、ェマル ジョン化したものを皮下に投与した。追加免疫は 50 g/匹の shMPL-FLAGにフロイン ト不完全アジュバント（ベタトン'ディッキンソン社製）を加え、ェマルジヨン化したものを 皮下に投与した。合計 6回免疫を行ったマウス 3匹に対し、 g/匹の shMPL-FLAG を尾静脈内投与することにより最終免疫を行った。マウスミエローマ細胞 Immunization was started from 8 weeks of age using MRL / MpJUmmCrj-lpr / lpr mice (hereinafter, MRL / lpr mice, purchased from Nippon Charles' Riva). For the first immunization, 100 / zg / animal shMPL-FLAG was supplemented with Freund's complete adjuvant (H37Ra, manufactured by Betaton's Dickinson) and emulsified subcutaneously. Booster immunization with 50 g / animal shMPL-FLAG Incomplete adjuvant (Bettaton 'Dickinson) was added, and the emulsion was subcutaneously administered. The final immunization was performed by intravenously administering g / mouse of shMPL-FLAG to the three mice immunized a total of six times in the tail vein. Mouse myeloma cells
P3-X63Ag8Ul (P3U1、 ATCCより購入）とマウス脾臓細胞を混合し、 Polyethylene Glycol 1500 (Roche Diagnostics社製）をカ卩えながら混合することにより細胞融合を行 つた。翌日より HAT培地を用いて選抜を行 、、培養上清を用いて shMp卜 FLAGまた は hMpト Fcを固相化したィムノプレートを用いた ELISAおよび BaF3-human Mplを用い た細胞増殖活性を指標としたスクリーニングを実施した。  P3-X63Ag8Ul (P3U1, purchased from ATCC) and mouse spleen cells were mixed, and cell fusion was performed by mixing Polyethylene Glycol 1500 (manufactured by Roche Diagnostics) while mixing. From the next day, selection was performed using a HAT medium, and the culture supernatant was used as an indicator for ELISA using an immunoplate with immobilized shMp-FLAG or hMp-Fc and cell growth activity using BaF3-human Mpl as an index. Screening was performed.
[0165] 1.6 抗ヒト Mpl抗体の解析  [0165] 1.6 Analysis of anti-human Mpl antibody
抗体濃度はャギ抗マウス IgG (gamma) (ZYMED社製)とアルカリフォスファターゼ - ャギ抗マウス IgG (gammaXZYMED社製)を用いたマウス IgGサンドイッチ ELISAを行い 、 Isotypeの等しい巿販抗体をスタンダードにして、 GraphPad Prism (GraphPad Software, USA)を用いて検量線を作成し、抗体濃度の換算を行った。  The antibody concentration was determined by performing a mouse IgG sandwich ELISA using goat anti-mouse IgG (gamma) (manufactured by ZYMED) and alkaline phosphatase-goat anti-mouse IgG (manufactured by gammaXZYMED). A calibration curve was prepared using GraphPad Prism (GraphPad Software, USA), and the antibody concentration was converted.
[0166] 抗体のアイソタイプは、アイソタイプ特異的な二次抗体を用いた抗原依存的 ELISA にて決定した。 hMp卜 Fcを 1 μ g/mLとなるように coating buffer (O.lmM NaHCO  [0166] The antibody isotype was determined by an antigen-dependent ELISA using an isotype-specific secondary antibody. Adjust the coating buffer (O.lmM NaHCO) so that the hMp Fc becomes 1 μg / mL.
3 Three
(pH9.6), 0.02%(w/v) NaN )で希釈したものを加え、 4°Cにてー晚反応し、コーティング (pH 9.6), 0.02% (w / v) NaN), and react at 4 ° C.
3  Three
した。 Diluent buffer (50mM Tris- HCl(pH8.1), ImM MgCl , 150mM NaCl, 0.05%(v/v)  did. Diluent buffer (50 mM Tris-HCl (pH8.1), ImM MgCl, 150 mM NaCl, 0.05% (v / v)
2  2
Tween20, 0.02%(w/v) NaN , l%(w/v) BSA)にてブロッキング処理を行った後、ハイブ  Tween20, 0.02% (w / v) NaN, l% (w / v) BSA)
3  Three
リドーマの培養上清をカ卩え、室温で 1時間放置した。 Rinse buffer (0.05%(v/v) Tween20, PBS)にて洗浄した後、 Alkaline phosphatase標識したアイソタイプ特異的二 次抗体を加え、室温で 1時間放置した。発色は SIGMA104(SIGMA-ALDRICH社製)を lmg/mLとなるように Substrate Buffer (50mM NaHCO (pH9.8), lOmM MgCl )に希釈  The culture supernatant of the lidoma was collected and left at room temperature for 1 hour. After washing with Rinse buffer (0.05% (v / v) Tween20, PBS), an Alkaline phosphatase-labeled isotype-specific secondary antibody was added and left at room temperature for 1 hour. For color development, dilute SIGMA104 (manufactured by SIGMA-ALDRICH) in Substrate Buffer (50mM NaHCO (pH9.8), lOmM MgCl) to lmg / mL.
3 2 したものを用い、 405nmの吸光度を Benchmark Plus (Bio-Rad社製）にて測定した。  The absorbance at 405 nm was measured with Benchmark Plus (manufactured by Bio-Rad) using the resulting data.
[0167] shMp卜 FLAGおよび hMPL-Fcに対する結合活性は、 ELISAにより評価した。精製し た shMp卜 FLAGおよび hMPL- Fcを 1 μ g/mLになるようにコーティングし、 Diluent bufferにてブロッキング処理を行った。ハイプリドーマの培養上清をカ卩え、室温で 1時 間放置した後、 Alkaline Phosphatase標識した抗マウス IgG抗体 (Zymed社製）を加え、 上記方法と同様に発色を行った。室温で 1時間発色させた後に 405應の吸光度を測 定し、 GraphPad Prismを用いて EC 値を算出した。 [0167] The binding activity to shMp FLAG and hMPL-Fc was evaluated by ELISA. The purified shMp FLAG and hMPL-Fc were coated at a concentration of 1 μg / mL, and blocking treatment was performed with a Diluent buffer. The culture supernatant of the hybridoma was dried and left at room temperature for 1 hour, and an anti-mouse IgG antibody (Zymed) labeled with Alkaline Phosphatase was added thereto, followed by color development in the same manner as described above. After coloring at room temperature for 1 hour, measure the absorbance at 405 And EC values were calculated using GraphPad Prism.
50  50
[0168] CHO- human Mpほたは CHO- monkey Mplを回収し、 lxl06cells/mLになるように FACS Buffer (1% FBS/ PBS)に懸濁した。 100 L/wellとなるように Multiscreen (Millipore社製)に分注し、遠心操作にて培養上清を除去した。 5 g/mLになるように 希釈した培養上清を加え、氷上にて 30分間反応させた。細胞を FACS bufferにて 1回 洗浄し、 FITC標識抗マウス IgG抗体（Beckman Coulter社製）を添カ卩し、氷上にて 30分 間反応させた。反応後、 500rpmで 1分間遠心し、上清を除き、 FACS Buffer 400 μしに 懸濁し、 EPICS ELITE ESP (Beckman Coulter)を用いてフローサイトメトリーを行った。 前方散乱光（forward scatter)及び側方散乱光（side scatter)のヒストグラムにて生細 胞集団にゲートを設定した。 [0168] CHO- human Mp Fireflys is recovered CHO- monkey Mpl, and suspended in FACS Buffer (1% FBS / PBS ) so as to lxl0 6 cells / mL. The solution was dispensed into Multiscreen (manufactured by Millipore) at 100 L / well, and the culture supernatant was removed by centrifugation. The culture supernatant diluted to 5 g / mL was added and reacted on ice for 30 minutes. The cells were washed once with FACS buffer, added with FITC-labeled anti-mouse IgG antibody (manufactured by Beckman Coulter), and allowed to react on ice for 30 minutes. After the reaction, the mixture was centrifuged at 500 rpm for 1 minute, the supernatant was removed, the cells were suspended in 400 μl of FACS Buffer, and subjected to flow cytometry using EPICS ELITE ESP (Beckman Coulter). Gates were set on the live cell population using histograms of forward scatter and side scatter.
[0169] 抗体のァゴニスト活性は、 TPO依存性増殖を示す BaF3-human Mpほたは  [0169] The agonist activity of the antibody is similar to that of BaF3-human Mp
BaF3-monkey Mplを用いて評価した。各細胞をそれぞれ 4xl05cells/mLとなるように 10% Fetal Bovine Serum(Invitrogen社製)を含む RPMI1640(Invitrogen社製)に懸濁し、 60 μ L/wellで 96well plateに分注した。 rhTPO (R&D社製)およびハイプリドーマ培養 上清の濃度を振り、各 wellに 40 L加え、 37°C、 5%CO条件下で、 24時間培養した。 Evaluation was performed using BaF3-monkey Mpl. Each cell was suspended in RPMI1640 (manufactured by Invitrogen) containing 10% Fetal Bovine Serum (manufactured by Invitrogen) at a concentration of 4 × 10 5 cells / mL, and dispensed into a 96-well plate at 60 μL / well. The concentrations of rhTPO (manufactured by R & D) and the hybridoma culture supernatant were varied, and 40 L was added to each well, followed by culturing at 37 ° C and 5% CO for 24 hours.
2  2
10 μ L/wellで Cell Count Reagent SF (ナカライテスタ社製)をカ卩え、 2時間培養後に、 450 nmの吸光度 (対照 655nm)を Benchmark Plusにて測定し、 GraphPad Prismを用い て EC 値を算出した。  After washing Cell Count Reagent SF (manufactured by Nacalai Tester) at 10 μL / well and culturing for 2 hours, the absorbance at 450 nm (control 655 nm) was measured with Benchmark Plus, and the EC value was calculated using GraphPad Prism. Calculated.
50  50
[0170] 1.7 抗ヒト Mpl抗体の精製  [0170] 1.7 Purification of anti-human Mpl antibody
ノ、イブリドーマの培養上清を用いて、以下のように抗ヒト Mpl抗体を精製した。  (4) Anti-human Mpl antibody was purified using the culture supernatant of hybridoma as described below.
培養上清を HiTrap proteinG HPカラム（Amersham Biosciences社製）に吸着させた 後に、 0.1 M Glycine-HCl (pH 2.7)を用いて溶出した。溶出後、直ちに 1M Tris-Cl (PH9.0)により直ちに中和し、 PBSで一昼夜透析を行い、バッファー置換を行った。  After adsorbing the culture supernatant to a HiTrap protein G HP column (manufactured by Amersham Biosciences), elution was performed using 0.1 M Glycine-HCl (pH 2.7). Immediately after the elution, the mixture was immediately neutralized with 1 M Tris-Cl (PH9.0), dialyzed against PBS for 24 hours, and replaced with a buffer.
[0171] 1.8抗ヒト Mpl抗体 VB22Bのェピトープの決定 [0171] 1.8 Determination of Epitope of Anti-Human Mpl Antibody VB22B
抗ヒト Mpl抗体 VB22Bが western blottingに使用可能である性質を利用し、ヒト Mplの 部分配列と GSTの融合蛋白質を構築し VB22Bのェピトープ解析を行った。 MG1 ( Gln26から Trp491)、 MG2 (Gln26から Leu274)の領域をそれぞれ PCR増幅し、 GST融 合蛋白質として発現されるように pGEX-4T-3 (Amersham社）へクローユングした。プラ スミド DNAを DH5ひへ導入し形質転換体を得、対数増殖期にある形質転換体に ImM となるように IPTGを加えることにより GST融合蛋白質の発現を誘導し、 2時間培養後に 菌体を回収した。 Sonicationにより破碎後、 XL- 80 Ultracentrifoge (Beckman, Rotor 70.1Ή)を用い 35,000rpmで 30分遠心後の培養上清を回収し、 GST Purification Modules (Amersham社)を用いて精製した。 10%- SDS- PAGEにより分離後、 PVDF膜に トランスファーし、 VB22Bマウス抗体を用いた western blottingを行った。 VB22Bは MG- 1、 MG- 2を認識した事から、 VB22Bのェピトープは Gln26から Leu274の領域にあ ることが判明した。 Utilizing the property that the anti-human Mpl antibody VB22B can be used for western blotting, a fusion protein of the partial sequence of human Mpl and GST was constructed and the epitope analysis of VB22B was performed. The regions of MG1 (from Gln26 to Trp491) and MG2 (from Gln26 to Leu274) were each amplified by PCR and cloned into pGEX-4T-3 (Amersham) so as to be expressed as a GST fusion protein. Plastic Transformants are obtained by introducing the plasmid DNA into DH5, and the expression of GST fusion protein is induced by adding IPTG to transformants in the logarithmic growth phase so as to become ImM. did. After disruption by Sonication, the culture supernatant after centrifugation at 35,000 rpm for 30 minutes using XL-80 Ultracentrifoge (Beckman, Rotor 70.1Ή) was collected and purified using GST Purification Modules (Amersham). After separation by 10% -SDS-PAGE, the cells were transferred to a PVDF membrane and subjected to western blotting using a VB22B mouse antibody. VB22B recognized MG-1 and MG-2, indicating that the epitope of VB22B was located in the region from Gln26 to Leu274.
[0172] 次に、 MG3 (Gln26から Alal89)、 MG4 26から!¾"0106)、 MG5 (Gln26から Glu259) 、 MG6 (Gln26から Gly245)の領域と GSTの融合蛋白を作製し同様に western blotting を行った結果、 VB22Bは MG5、 MG6を認識した力 MG3、 MG4を認識しなかった事か ら、 VB22Bのェピトープは Alal89から Gly245の周辺に存在する事が予想された。さら に MG7 (Gln26から Ala231)、 MG8 (Gln26から Pro217)と GSTの融合蛋白質を作製し評 価した結果、 VB22Bは MG7を認識した力 MG8は認識しなかった事から、 VB22Bのェ ピトープは Gln217から Ala231の周辺に存在することが示された。さらに MG10 (Gln213 から Ala231)と GSTの融合蛋白質との結合が確認されたことから、 VB22Bのェピトープ は Gln213から Ala231の 19アミノ酸に限定された。  [0172] Next, a fusion protein of the region of MG3 (from Gln26 to Alal89), from MG426! ¾ "0106), MG5 (from Gln26 to Glu259), and MG6 (from Gln26 to Gly245) and GST was prepared, and western blotting was similarly performed. As a result, VB22B did not recognize MG5 and MG6, and did not recognize MG3 and MG4, so it was expected that the epitope of VB22B would exist around Alal89 to Gly245, and MG7 (from Gln26 to Ala231). ), A fusion protein of MG8 (Gln26 to Pro217) and GST was prepared and evaluated.As a result, VB22B recognized MG7 but did not recognize MG8.Therefore, the VB22B epitope exists around Gln217 to Ala231. Furthermore, the binding between MG10 (Gln213 to Ala231) and the fusion protein of GST was confirmed, so that the epitope of VB22B was limited to 19 amino acids from Gln213 to Ala231.
[0173] 1.9 抗ヒト Mpl抗体 VB22Bの抗原抗体反応の反応速度論的解析  [0173] 1.9 Kinetic analysis of antigen-antibody reaction of anti-human Mpl antibody VB22B
抗ヒト Mpl抗体 VB22Bが可溶型組換え Mplに結合する性質を利用し、参考例 1.4で 示したヒト MpHgG Fc融合タンパク質と VB22B IgGとの抗原抗体反応における速度論 的な解析を行った。 BIAcore2000(BIAcore社製)に Sensor Chip CM5(BIAcore社製)を セットし、ァミンカップリング法にてヒト MpHgG Fc融合タンパク質を固定ィ匕した。次に 、 HBS-EP Buffer(BIAcore社製)を用いて 1.25〜20 g/mLの VB22B IgGを調製し、 VB22B IgGを 2分間添カ卩し、結合領域を得た後に、 HBS- EP Bufferを 2分間添加する ことで解離領域を得た。 Sensor Chip上のヒト MpHgG Fc融合タンパク質に結合した VB22B IgGは、 10mM NaOHを 15秒間添カ卩して除去し、 Sensor Chipを再生した。ラン ユングバッファ一として HBS-EP Bufferを用い、流速は 20 μ L/minとした。  Utilizing the property that the anti-human Mpl antibody VB22B binds to soluble recombinant Mpl, the kinetic analysis of the antigen-antibody reaction between the human MpHgG Fc fusion protein and VB22B IgG shown in Reference Example 1.4 was performed. A Sensor Chip CM5 (manufactured by BIAcore) was set on BIAcore2000 (manufactured by BIAcore), and the human MpHgG Fc fusion protein was immobilized by an amine coupling method. Next, 1.25 to 20 g / mL of VB22B IgG was prepared using HBS-EP Buffer (manufactured by BIAcore), and VB22B IgG was added for 2 minutes to obtain a binding region. By adding for 2 minutes, a dissociation region was obtained. VB22B IgG bound to the human MpHgG Fc fusion protein on the Sensor Chip was removed by adding 10 mM NaOH for 15 seconds, and the Sensor Chip was regenerated. HBS-EP Buffer was used as the running buffer, and the flow rate was 20 μL / min.
BIAevaluation ver.3.1ソフトウェア (BIAcore社製)を用いて、各濃度で得られたセンサ 一グラムより反応速度定数を算出した。その結果、 VB22B IgGの解離定数 (KD)は 1.67 ±0.713 X 10— 9Mであった。 Using BIAevaluation ver.3.1 software (BIAcore), sensors obtained at each concentration The reaction rate constant was calculated from one gram. As a result, the dissociation constant of VB22B IgG (KD) was 1.67 ± 0.713 X 10- 9 M.
[0174] 〔参考例 2〕 抗ヒト Mpl—本鎖抗体の作製 [Reference Example 2] Production of anti-human Mpl—main chain antibody
取得した抗ヒト Mpl抗体の中で、結合活性およびァゴニスト活性が高カゝつた抗体に ついて、遺伝子工学的手法により一本鎖抗体の発現系を構築した。以下に抗ヒト Mpl 抗体 VB22Bの一本鎖抗体作製例につ 、て示す。  Among the obtained anti-human Mpl antibodies, a single chain antibody expression system was constructed by a genetic engineering technique for an antibody having high binding activity and agonist activity. The following shows an example of preparing a single-chain antibody of the anti-human Mpl antibody VB22B.
[0175] 2.1 抗ヒト Mpl抗体可変領域のクローニング [0175] 2.1 Cloning of variable region of anti-human Mpl antibody
抗ヒト Mpl抗体を産生するハイブリドーマより抽出した Total RNAを用いて、 RT- PCR 法によって増幅した。 Total RNAは、 RNeasy Plant Mini Kits (QIAGEN社製）を用いて lxlO7細胞のハイプリドーマより抽出した。 Amplification was performed by RT-PCR using total RNA extracted from hybridomas producing anti-human Mpl antibody. Total RNA was extracted from hybridomas of lxlO 7 cells using RNeasy Plant Mini Kits (QIAGEN).
[0176] 1 μ gの Total RNAを使用して、 SMART RACE cDNA Amplification Kit ( [0176] Using 1 μg of total RNA, use the SMART RACE cDNA Amplification Kit (
CLONTECH社製)を用いて、マウス IgG2b定常領域配列に相補的な合成オリゴヌタレ ォチド MHC-IgG2b (配列番号: 69)またはマウス κ鎖定常領域塩基配列に相補的な 合成オリゴヌクレオチド kappa (配列番号： 70)を用いて、 5'末端側遺伝子断片を増幅 した。逆転写反応は 42°Cで 1時間 30分間反応させた。  CLONTECH), using synthetic oligonucleotide MHC-IgG2b (SEQ ID NO: 69) complementary to the mouse IgG2b constant region sequence or kappa (SEQ ID NO: 70) complementary to the mouse κ chain constant region base sequence ) Was used to amplify the 5 'terminal gene fragment. The reverse transcription reaction was performed at 42 ° C for 1 hour and 30 minutes.
[0177] PCR反応溶液 (50 μ L)の組成を次に示す。 [0177] The composition of the PCR reaction solution (50 µL) is shown below.
5 μ Lの 10 X Advantage 2 PCR Buffer,  5 μL of 10X Advantage 2 PCR Buffer,
5 μ Lの 10 X Universal Primer A Mix、  5 μL of 10X Universal Primer A Mix,
0.2mM dNTPs (dATP, dGTP, dCTP, dTTP)、  0.2 mM dNTPs (dATP, dGTP, dCTP, dTTP),
1 μ Lの Advantage 2 Polymerase Mix  1 μL Advantage 2 Polymerase Mix
(以上の成分は!、ずれも CLONTECH社製）、  (The above ingredients are!, The gap is also made by CLONTECH),
2.5 Lの逆転写反応産物、  2.5 L reverse transcription reaction product,
1 Opmoleの合成オリゴヌクレオチド MHC- IgG2bまたは kappa  1 Opmole synthetic oligonucleotide MHC-IgG2b or kappa
[0178] また反応温度条件は次のとおりである。 [0178] The reaction temperature conditions are as follows.
94°Cの初期温度にて 30秒間、  30 seconds at an initial temperature of 94 ° C,
94°C/5秒間、 72°C/3分間のサイクルを 5回反復、  94 ° C / 5 seconds, 72 ° C / 3 minutes cycle 5 times,
94°C/5秒間、 70°C/10秒間、 72°C/3分間のサイクルを 5回反復、  5 cycles of 94 ° C / 5 seconds, 70 ° C / 10 seconds, 72 ° C / 3 minutes
94°C/5秒間、 68°C/10秒間、 72°C/3分間のサイクルを 25回反復、 最後に反応産物を 72°Cで 7分間加熱した。 25 cycles of 94 ° C / 5 seconds, 68 ° C / 10 seconds, 72 ° C / 3 minutes Finally, the reaction product was heated at 72 ° C for 7 minutes.
[0179] PCR産物は QIAquick Gel Extraction Kit (QIAGEN社製）を用いて、ァガロースゲル 力 精製した後、 pGEM- T Easyベクター（Promega社製）へクローユングした。さらに、 ABI 3700 DNA Analyzer (Perkin Elmer社製)を用いて塩基配列を決定した。 [0179] The PCR product was purified using a QIAquick Gel Extraction Kit (manufactured by QIAGEN), subjected to agarose gel force purification, and then cloned into a pGEM-T Easy vector (manufactured by Promega). Furthermore, the nucleotide sequence was determined using ABI 3700 DNA Analyzer (manufactured by Perkin Elmer).
クロー-ングした VB22B H鎖可変領域（以下、 VB22B-VH)の塩基配列を配列番号 : 7、アミノ酸配列を配列番号: 8、および L鎖可変領域 (以下、 VB22B-VL)の塩基配 列を配列番号： 9、アミノ酸配列を配列番号： 10に示す。  The nucleotide sequence of the cloned VB22B H chain variable region (hereinafter, VB22B-VH) is SEQ ID NO: 7, the amino acid sequence is SEQ ID NO: 8, and the nucleotide sequence of the L chain variable region (hereinafter, VB22B-VL) is SEQ ID NO: 9, and the amino acid sequence is shown in SEQ ID NO: 10.
[0180] 2.2 抗ヒト Mpl抗体 Diabody発現ベクターの作製 [0180] 2.2 Construction of Antibody Mpl Antibody Diabody Expression Vector
5アミノ酸からなるリンカ一配列を用いた VB22B—本鎖 Fv (以下、 VB22B Diabody)を コードする遺伝子は、 VB22B-VHをコードする遺伝子の 3'末端および VB22B-VLをコ ードする遺伝子の 5'末端に（Gly Ser)力も成るリンカ  The gene encoding VB22B—a single-chain Fv (hereinafter referred to as “VB22B Diabody”) using a linker sequence consisting of 5 amino acids is the 3 ′ end of the gene encoding VB22B-VH and the gene encoding VB22B-VL. 'A linker with a terminal (Gly Ser) force
4 1 一をコードする塩基配列を付カロ させた遺伝子について、それぞれ PCR法を用いて増幅し、連結することにより構築し た。  The genes to which the nucleotide sequence encoding 411 was added were amplified by PCR and ligated, respectively, and constructed.
[0181] VB22B-VHの前方プライマー 70· 115HF (配列番号： 71)は、 EcoRI部位を有するよ うに設計し、 VB22B-VHの後方プライマー 33 · 115HR (配列番号： 72)は、 VB22B-VH の C末端をコードする DNAにハイブリダィズし、かつ（Gly Ser)力 成るリンカ  [0181] The forward primer 70 • 115HF (SEQ ID NO: 71) of VB22B-VH was designed to have an EcoRI site, and the backward primer 33 • 115HR of VB22B-VH (SEQ ID NO: 72) was A linker that hybridizes to DNA encoding the C-terminus and has (Gly Ser) power
4 1 一をコー ドする塩基配列ならびに VB22B-VLの N末端をコードする DNAにハイブリダィズする 塩基配列を有するように設計した。 VB22B-VLの前方プライマー 33 - 115LF (配列番 号： 73)は、 VB22B-VLの N末端をコードする塩基配列ならびに（Gly Ser)から成るリ  It was designed to have a nucleotide sequence encoding 411 and a nucleotide sequence that hybridizes to DNA encoding the N-terminus of VB22B-VL. VB22B-VL forward primer 33-115LF (SEQ ID NO: 73) is a primer consisting of the nucleotide sequence encoding the N-terminus of VB22B-VL and (Gly Ser).
4 1 ンカーをコードする塩基配列、 VB22B-VHの C末端をコードする塩基配列を有するよ うに設計した。 VB22B-VLの後方プライマー 33 - 115LR (配列番号： 74)は、  It was designed to have a nucleotide sequence encoding the 41 linker and a nucleotide sequence encoding the C-terminal of VB22B-VH. VB22B-VL rear primer 33-115LR (SEQ ID NO: 74)
VB22B- VLの C末端をコードする DNAにハイブリダィズし、かつ FLAGタグ  VB22B- Hybridizes to the DNA encoding the C-terminus of VL and has a FLAG tag
(AspTyrLysAspAsp AspAspLysZ配列番号： 75)をコードする塩基配列を有し、さらに Notl部位を有するように設計した。  (AspTyrLysAspAsp AspAspLysZ SEQ ID NO: 75) and was designed to have a Notl site.
[0182] 第一 PCRにおいて、 VB22B- VHおよびリンカ一配列と VB22B- VLおよびリンカ一配 列を含む 2つの PCR反応物を以下のように合成した。 [0182] In the first PCR, two PCR products containing the VB22B-VH and linker sequences and the VB22B-VL and linker sequences were synthesized as follows.
PCR反応溶液 (50 L)の組成を次に示す。  The composition of the PCR reaction solution (50 L) is shown below.
S ^ LOIO X PCR Bufferゝ 0.4mM dNTPs (dATP, dGTP, dCTP, dTTP)、 S ^ LOIO X PCR Buffer ゝ 0.4 mM dNTPs (dATP, dGTP, dCTP, dTTP),
2.5ユニットの DNAポリメラーゼ TaKaRa Ex Taq  2.5 units of DNA polymerase TaKaRa Ex Taq
(以上の成分は!、ずれも宝酒造社製)、  (The above ingredients are from Takara Shuzo)
lOngの VB22B- VHまたは VB22B- VL遺伝子を含む pGEM- T Easyベクター、 lOpmoleの合成オリゴヌクレオチド 70' 115HF、 33 · 115HRまたは 33 · 115LF、 33 · 115LR  pGEM-T Easy vector containing lOng VB22B-VH or VB22B-VL gene, lOpmole synthetic oligonucleotide 70 '115HF, 33115 HR or 33115LF, 33115LR
[0183] また反応温度条件は次のとおりである。  [0183] The reaction temperature conditions are as follows.
94°Cの初期温度にて 30秒間、  30 seconds at an initial temperature of 94 ° C,
94°C/15秒間、 72°C/2分間のサイクルを 5回反復、  94 ° C / 15 seconds, 72 ° C / 2 minutes cycle 5 times,
94°C/15秒間、 70°C/2分間のサイクルを 5回反復、  94 ° C / 15 seconds, 70 ° C / 2 minutes cycle 5 times,
94°C/15秒間、 68°C/2分間のサイクルを 28回反復、  94 ° C / 15 seconds, 68 ° C / 2 minutes cycle 28 times,
最後に反応産物を 72°Cで 5分間加熱した。  Finally, the reaction product was heated at 72 ° C for 5 minutes.
[0184] 約 400bpの PCR産物を QIAquick Gel Extraction Kit (QIAGEN社製）を用いて、ァガ ロースゲル力も精製した後、各 PCR産物の一部を用いて以下のように第二 PCRを行 つた o [0184] The agarose gel was also purified from the approximately 400 bp PCR product using the QIAquick Gel Extraction Kit (manufactured by QIAGEN), and a second PCR was performed using a part of each PCR product as follows.
PCR反応溶液 (50 L)の組成を次に示す。  The composition of the PCR reaction solution (50 L) is shown below.
5 ;z L 10 X PCR Bufferゝ  5; z L 10 X PCR Buffer ゝ
0.4mM dNTPs (dATP, dGTP, dCTP, dTTP)、  0.4 mM dNTPs (dATP, dGTP, dCTP, dTTP),
2.5ユニットの DNAポリメラーゼ TaKaRa Ex Taq  2.5 units of DNA polymerase TaKaRa Ex Taq
(以上の成分は!、ずれも宝酒造社製)、  (The above ingredients are from Takara Shuzo)
1 μ Lの第一 PCR産物（2種類)、  1 μL of the first PCR product (2 types),
lOpmoleの合成オリゴヌクレオチド 70 · 115HF、 33 - 115LR  lOpmole synthetic oligonucleotide 70 115HF, 33-115LR
[0185] また反応温度条件は次のとおりである。 [0185] The reaction temperature conditions are as follows.
94°Cの初期温度にて 30秒間、  30 seconds at an initial temperature of 94 ° C,
94°C/15秒間、 72°C/2分間のサイクルを 5回反復、  94 ° C / 15 seconds, 72 ° C / 2 minutes cycle 5 times,
94°C/15秒間、 70°C/2分間のサイクルを 5回反復、  94 ° C / 15 seconds, 70 ° C / 2 minutes cycle 5 times,
94°C/15秒間、 68°C/2分間のサイクルを 28回反復、  94 ° C / 15 seconds, 68 ° C / 2 minutes cycle 28 times,
最後に反応産物を 72°Cで 5分間加熱した。 [0186] 約 800bpの PCR産物を QIAquick Gel Extraction Kit (QIAGEN社製）を用いて、ァガ ロースゲル力も精製した後、制限酵素 EcoRI (宝酒造社製)および制限酵素 Notl (宝 酒造社製）で消化した後に、 QIAquick PCR Purification Kit (QIAGEN社製）を用いて 精製し、 pCXND3にクローユングし、 pCXND3- VB22B dbを作製した。 Finally, the reaction product was heated at 72 ° C for 5 minutes. [0186] The PCR product of about 800 bp was also purified using a QIAquick Gel Extraction Kit (manufactured by QIAGEN) for agarose gel, and then digested with the restriction enzymes EcoRI (manufactured by Takara Shuzo) and Notl (manufactured by Takara Shuzo). After that, the resultant was purified using a QIAquick PCR Purification Kit (manufactured by QIAGEN) and cloned into pCXND3 to prepare pCXND3-VB22B db.
[0187] 2.3 抗ヒト Mpl抗体 sc(Fv)2発現ベクターの作製  [0187] 2.3 Preparation of anti-human Mpl antibody sc (Fv) 2 expression vector
VB22B由来の 2つの H鎖可変領域および 2つの L鎖可変領域を含む改変抗体  VB22B-derived modified antibody containing two heavy chain variable regions and two light chain variable regions
[sc(Fv)2]を発現するプラスミドを作製するために、前述の pCXND3-VB22B dbを用い て以下のように PCR法により修飾した。 sc(Fv)2遺伝子の構築過程について、図 4に示 した。  In order to prepare a plasmid expressing [sc (Fv) 2], the above-described pCXND3-VB22Bdb was modified by the PCR method as follows. FIG. 4 shows the construction process of the sc (Fv) 2 gene.
[0188] はじめに、 VB22B-VHをコードする遺伝子の 3'末端および VB22B-VLをコードする 遺伝子の 5'末端に 15アミノ酸力も成るリンカ一（Gly Ser)をコードする塩基配列を付  [0188] First, a nucleotide sequence encoding a linker (Gly Ser) having 15 amino acids was added to the 3 'end of the gene encoding VB22B-VH and the 5' end of the gene encoding VB22B-VL.
4 3  4 3
カロさせた遺伝子について、それぞれ PCR法を用いて増幅し、連結することにより構築 した。この構築過程において、 3種類のプライマーを新たに設計した。 VB22B-VHの 前方プライマー VB22B_ft)vu (プライマー A,配列番号： 76)は、 5'末端に EcoRI部位を 有し、 VB22B dbの Gln22および Leu23が PvuII部位に変換するように設計した。  Each of the excreted genes was amplified by PCR and ligated. During this construction process, three new primers were designed. VB22B-VH forward primer VB22B_ft) vu (primer A, SEQ ID NO: 76) was designed to have an EcoRI site at the 5 'end and to convert Gln22 and Leu23 of VB22B db to a PvuII site.
VB22B- VHの後方プライマー SC- rL15 (プライマー B,配列番号： 77)は、 VB22B- VH の C末端をコードする DNAにハイブリダィズし、かつ（Gly Ser)力 成るリンカ  The VB22B-VH rear primer SC-rL15 (Primer B, SEQ ID NO: 77) hybridizes to the DNA encoding the C-terminus of VB22B-VH and has a (Gly Ser) linker.
4 3 一をコー ドする塩基配列ならびに VB22B-VLの N末端をコードする DNAにハイブリダィズする 塩基配列を有するように設計した。 VB22B-VLの前方プライマー SC-1L15 (プライマー C,配列番号： 78)は、 VB22B-VLの N末端をコードする塩基配列ならびに（Gly Ser)  It was designed to have a nucleotide sequence encoding 433 and a nucleotide sequence hybridizing to DNA encoding the N-terminus of VB22B-VL. The forward primer SC-1L15 (primer C, SEQ ID NO: 78) of VB22B-VL is composed of a nucleotide sequence encoding the N-terminus of VB22B-VL and (Gly Ser)
4 3 力 成るリンカ一をコードする塩基配列、 VB22B-VHの C末端をコードする塩基配列 を有するように設計した。  It was designed to have a nucleotide sequence encoding a linker, a nucleotide sequence encoding the C-terminal of VB22B-VH.
[0189] 第一 PCRにおいて、 VB22B- VHおよびリンカ一配列と VB22B- VLおよびリンカ一配 列を含む 2つの PCR反応物を以下のように合成した。 [0189] In the first PCR, two PCR products containing the VB22B-VH and linker sequences and the VB22B-VL and linker sequences were synthesized as follows.
PCR反応溶液 (50 L)の組成を次に示す。  The composition of the PCR reaction solution (50 L) is shown below.
5 ;z L 10 X PCR Bufferゝ  5; z L 10 X PCR Buffer ゝ
0.4mM dNTPs (dATP, dGTP, dCTP, dTTP)、  0.4 mM dNTPs (dATP, dGTP, dCTP, dTTP),
2.5ユニットの DNAポリメラーゼ TaKaRa Ex Taq (以上の成分は!、ずれも宝酒造社製)、 2.5 units of DNA polymerase TaKaRa Ex Taq (The above ingredients are from Takara Shuzo)
10ngの pCXND3- VB22B db、  10 ng of pCXND3-VB22B db,
lOpmoleの合成オリゴヌクレオチド VB22B- ft)vu、 sc- rL15または sc- 1L15、 33 · 115LR (プライマー D)  lOpmole synthetic oligonucleotide VB22B-ft) vu, sc-rL15 or sc-1L15, 33115LR (primer D)
[0190] また反応温度条件は次のとおりである。 [0190] The reaction temperature conditions are as follows.
94°Cの初期温度にて 30秒間、  30 seconds at an initial temperature of 94 ° C,
94°C/15秒間、 72°C/2分間のサイクルを 5回反復、  94 ° C / 15 seconds, 72 ° C / 2 minutes cycle 5 times,
94°C/15秒間、 70°C/2分間のサイクルを 5回反復、  94 ° C / 15 seconds, 70 ° C / 2 minutes cycle 5 times,
94°C/15秒間、 68°C/2分間のサイクルを 28回反復、  94 ° C / 15 seconds, 68 ° C / 2 minutes cycle 28 times,
最後に反応産物を 72°Cで 5分間加熱した。  Finally, the reaction product was heated at 72 ° C for 5 minutes.
[0191] 約 400bpの PCR産物を QIAquick Gel Extraction Kit (QIAGEN社製）を用いて、ァガ ロースゲル力も精製した後、各 PCR産物の一部を用いて以下のように第二 PCRを行 つた o [0191] The agarose gel was also purified from the approximately 400 bp PCR product using the QIAquick Gel Extraction Kit (manufactured by QIAGEN), and a second PCR was performed using a part of each PCR product as follows.
PCR反応溶液 (50 L)の組成を次に示す。  The composition of the PCR reaction solution (50 L) is shown below.
5 ;z L 10 X PCR Bufferゝ  5; z L 10 X PCR Buffer ゝ
0.4mM dNTPs (dATP, dGTP, dCTP, dTTP)、  0.4 mM dNTPs (dATP, dGTP, dCTP, dTTP),
2.5ユニットの DNAポリメラーゼ TaKaRa Ex Taq  2.5 units of DNA polymerase TaKaRa Ex Taq
(以上の成分は!、ずれも宝酒造社製)、  (The above ingredients are from Takara Shuzo)
1 μ Lの第一 PCR産物（2種類)、  1 μL of the first PCR product (2 types),
lOpmoleの合成オリゴヌクレオチド 70 · 115HF、 33 - 115LR  lOpmole synthetic oligonucleotide 70 115HF, 33-115LR
[0192] また反応温度条件は次のとおりである。 [0192] The reaction temperature conditions are as follows.
94°Cの初期温度にて 30秒間、  30 seconds at an initial temperature of 94 ° C,
94°C/15秒間、 72°C/2分間のサイクルを 5回反復、  94 ° C / 15 seconds, 72 ° C / 2 minutes cycle 5 times,
94°C/15秒間、 70°C/2分間のサイクルを 5回反復、  94 ° C / 15 seconds, 70 ° C / 2 minutes cycle 5 times,
94°C/15秒間、 68°C/2分間のサイクルを 28回反復、  94 ° C / 15 seconds, 68 ° C / 2 minutes cycle 28 times,
最後に反応産物を 72°Cで 5分間加熱した。  Finally, the reaction product was heated at 72 ° C for 5 minutes.
[0193] 約 800bpの PCR産物を QIAquick Gel Extraction Kit (QIAGEN社製）を用いて、ァガ ロースゲル力も精製した後、制限酵素 EcoRI (宝酒造社製)および制限酵素 Notl (宝 酒造社製）で消化した後に、 QIAquick PCR Purification Kit (QIAGEN社製）を用いて 精製し、 pBacPAK9(CLONTECH社製)にクローユングし、 pBacPAK9- scVB22Bを作 製した。 [0193] After the PCR product of about 800 bp was purified for agarose gel using the QIAquick Gel Extraction Kit (QIAGEN), the restriction enzymes EcoRI (Takara Shuzo) and Notl (Takara Shuzo) were used. After digestion with Shuzo Co., Ltd., it was purified using QIAquick PCR Purification Kit (QIAGEN) and cloned into pBacPAK9 (CLONTECH) to produce pBacPAK9-scVB22B.
[0194] 次に、 pBacPAK9-scVB22Bの PvII部位に挿入す片を作製した。すなわち N末端が 欠けた VB22B-VHと VB22B-VLを（Gly Ser)力 成るリンカ一で連結したアミノ酸をコ  [0194] Next, a piece to be inserted into the PvII site of pBacPAK9-scVB22B was prepared. In other words, amino acids obtained by linking VB22B-VH and VB22B-VL lacking the N-terminus with a linker (Gly Ser) are used.
4 3  4 3
ードする遺伝子をさらに VB22B-VHの N末端をコードする遺伝子と（Gly Ser)カゝら成る  Gene that encodes the N-terminus of VB22B-VH and (Gly Ser)
4 3 リンカ一をコードする塩基配列で連結する断片で、両末端が PvuII認識配列となる断 片である。 2種類のプライマーを新たに設計し、 PCR法を用いて、この断片を作製した 。 目的断片の前方プライマー Fv2-f (プライマー E,配列番号： 79)は、 5'末端に PvuII 部位を有し、 VB22B-VHの 5'末端側の配列を持つように設計した。目的断片の後方 プライマー Fv2-r (プライマー F,配列番号： 80)は、 VB22B-VLの C末端をコードする DNAにハイブリダィズし、かつ（Gly Ser)力も成るリンカ  43 Fragment that is linked by a linker-encoding base sequence and has a PvuII recognition sequence at each end. Two types of primers were newly designed, and this fragment was prepared using PCR. The forward primer Fv2-f (primer E, SEQ ID NO: 79) of the target fragment was designed to have a PvuII site at the 5 ′ end and a sequence at the 5 ′ end of VB22B-VH. The primer Fv2-r (Primer F, SEQ ID NO: 80) after the target fragment is a linker that hybridizes to the DNA encoding the C-terminus of VB22B-VL and also has (Gly Ser) power.
4 3 一をコードする塩基配列なら びに VB22B-VHの N末端をコードする DNAにハイブリダィズする塩基配列、さらに PvuII部位を有するように設計した。 pBacPAK9-scVB22Bを铸型にして、以下のように PCRを行った。  It was designed to have a base sequence that codes for 43, a base sequence that hybridizes to DNA that codes for the N-terminus of VB22B-VH, and a PvuII site. Using pBacPAK9-scVB22B as type III, PCR was performed as follows.
[0195] PCR反応溶液 (50 μ L)の組成を次に示す。 [0195] The composition of the PCR reaction solution (50 µL) is shown below.
5 ;z L 10 X PCR Bufferゝ  5; z L 10 X PCR Buffer ゝ
0.4mM dNTPs (dATP, dGTP, dCTP, dTTP)、  0.4 mM dNTPs (dATP, dGTP, dCTP, dTTP),
2.5ユニットの DNAポリメラーゼ TaKaRa Ex Taq  2.5 units of DNA polymerase TaKaRa Ex Taq
(以上の成分は!、ずれも宝酒造社製)、  (The above ingredients are from Takara Shuzo)
10 μ gの pBacPAK9— scVB22B、  10 μg of pBacPAK9—scVB22B,
lOpmoleの合成オリゴヌクレオチド Fv2-f、 Fv2-r  lOpmole synthetic oligonucleotides Fv2-f, Fv2-r
[0196] また反応温度条件は次のとおりである。 [0196] The reaction temperature conditions are as follows.
94°Cの初期温度にて 30秒間、  30 seconds at an initial temperature of 94 ° C,
94°C/15秒間、 72°C/2分間のサイクルを 5回反復、  94 ° C / 15 seconds, 72 ° C / 2 minutes cycle 5 times,
94°C/15秒間、 70°C/2分間のサイクルを 5回反復、  94 ° C / 15 seconds, 70 ° C / 2 minutes cycle 5 times,
94°C/15秒間、 68°C/2分間のサイクルを 28回反復、  94 ° C / 15 seconds, 68 ° C / 2 minutes cycle 28 times,
最後に反応産物を 72°Cで 5分間加熱した。 [0197] 約 800bpの PCR産物を QIAquick Gel Extraction Kit (QIAGEN社製）を用いて、ァガ ロースゲルから精製した後、 pGEM- T Easyベクター（Promega社製）へクローユングし た。塩基配列の決定後、制限酵素 PvuII (宝酒造社製)で消化した後に、 目的断片を 回収した。 pBacPAK9_scVB22Bを制限酵素 PvuII (宝酒造社製）で消化した後に、回 収した断片を連結し、 PBacPAK9-VB22B sc(Fv)2を作製した。作製したベクターを制 限酵素 EcoRI (宝酒造社製)および制限酵素 Notl (宝酒造社製)で消化した後に、 QIAquick Gel Extraction Kit (QIAGEN社製）を用いて、約 1600bpの断片をァガロー スゲルから精製し、発現ベクター pCXND3にクローユングし、 pCXND3- VB22B sc(Fv)2を作製した。 Finally, the reaction product was heated at 72 ° C for 5 minutes. [0197] The approximately 800 bp PCR product was purified from agarose gel using the QIAquick Gel Extraction Kit (QIAGEN), and then cloned into a pGEM-T Easy vector (Promega). After determining the nucleotide sequence, the target fragment was recovered after digestion with the restriction enzyme PvuII (Takara Shuzo). after digestion with restriction pBacPAK9_scVB22B enzyme PvuII (manufactured by Takara Shuzo) was ligated to recovered fragment to prepare a P BacPAK9-VB22B sc (Fv) 2. After digesting the prepared vector with the restriction enzymes EcoRI (Takara Shuzo) and restriction enzyme Notl (Takara Shuzo), about 1600 bp fragment was purified from agarose gel using QIAquick Gel Extraction Kit (QIAGEN). Then, the expression vector pCXND3 was cloned to prepare pCXND3-VB22B sc (Fv) 2.
[0198] 2.4 動物細胞を用いた抗ヒト Mpl—本鎖抗体の発現  [0198] 2.4 Expression of anti-human Mpl-chain antibody using animal cells
CHO-DG44細胞を用いた一本鎖抗体の安定発現細胞株の作製は次のようにして 行った。 Gene Pulserll (BioRad社製）を用いたエレクト口ポレーシヨン法により遺伝子 導入した。発現ベクター (25 μ g)と PBSに懸濁した CHO- DG44細胞（1 X 107細胞/ mL )の 0.75mLを混合したものを氷上で 10分間冷却し、キュベットに移した後に 1.5kV、 25 FDの容量にてパルスを与えた。室温にて 10分間の回復期間の後、エレクトロボレ ーシヨン処理された細胞を、 500 μ g/mL Geneticin (Invitrogen社製）を含む The production of a cell line stably expressing a single-chain antibody using CHO-DG44 cells was performed as follows. The gene was introduced by an electoral-portation method using Gene Pulserll (BioRad). A mixture of the expression vector (25 μg) and 0.75 mL of CHO-DG44 cells (1 × 10 7 cells / mL) suspended in PBS was cooled on ice for 10 minutes, transferred to a cuvette, and then transferred to a 1.5 kV, A pulse was given at the capacity of the FD. After a 10-minute recovery period at room temperature, electroporated cells should contain 500 μg / mL Geneticin (Invitrogen).
CHO-S-SFMII培地（Invitrogen社製）に加えて選抜し、発現 CHO細胞株を榭立した。 VB22B sc(Fv)2は、この方法で安定発現細胞株およびその培養上清を調製した。  A CHO-S-SFMII medium (manufactured by Invitrogen) was selected for selection to establish an expression CHO cell line. For VB22B sc (Fv) 2, a cell line stably expressing and a culture supernatant thereof were prepared by this method.
[0199] COS7細胞を用いた一本鎖抗体の一過性発現は次のようにして行った。発現べクタ 一 (10 μ g)と PBSに懸濁した COS7細胞（1 X 107細胞/ mL)の 0.75mLを混合したものを 氷上で 10分間冷却し、キュベットに移した後に 1.5kV、 25 μ FDの容量にてパルスを与 えた。室温にて 10分間の回復期間の後、エレクト口ポレーシヨン処理された細胞を、 10% FBSを含む DMEM培地（Invitrogen社製）にカロえ、ー晚培養した後に、 PBSで洗浄 後に CHO-S-SFMII培地をカ卩えて約 3日間培養した。 VB22B Diabodyは、この方法で 培養上清を調製した。 [0199] Transient expression of a single-chain antibody using COS7 cells was performed as follows. A mixture of expression vector (10 μg) and 0.75 mL of COS7 cells (1 × 10 7 cells / mL) suspended in PBS was cooled on ice for 10 minutes, transferred to a cuvette, and then transferred to a 1.5 kV, 25 kV cell. Pulses were given with a μFD capacity. After a recovery period of 10 minutes at room temperature, the cells treated with the electoral port were treated with DMEM medium (manufactured by Invitrogen) containing 10% FBS, cultured for 1 hour, washed with PBS, and washed with CHO-S- The SFMII medium was cultured and cultured for about 3 days. The culture supernatant of VB22B Diabody was prepared by this method.
[0200] 2.5 培養上清中の抗ヒト Mpl—本鎖抗体の定量  [0200] 2.5 Quantification of anti-human Mpl—single-chain antibody in culture supernatant
COS細胞に一過性発現させた抗ヒト Mpl—本鎖抗体の培養上清中の濃度は、表面 プラズモン共鳴を利用して測定した。すなわち BIAcore2000(Biacore社製)に Sensor Chip CM5 (Biacore社製）をセットし、 ANTI- FLAG M2 Monoclonal Antibody The concentration of the anti-human Mpl-chain antibody transiently expressed in COS cells in the culture supernatant was measured using surface plasmon resonance. In other words, BIAcore2000 (Biacore) Insert Chip CM5 (Biacore), and insert ANTI-FLAG M2 Monoclonal Antibody
(SIGMA-ALDRICH社製)を結合した。流速 5mL/secで適濃度のサンプルを流し、 50mMジェチルァミンを流して結合した抗体を解離させた。サンプルを流したときの質 量変化を測定し、標準品の質量変化に基づいて作成した検量線を用いて、濃度を 算出した。 Diabodyについての標準品は、 dbl2E10(WO02/33073、 WO02/33072参照 )を使用し、 sc(Fv)2についての標準品は同じ遺伝子構造を持つ 12E10 sc(Fv)2を使用 した。  (Made by SIGMA-ALDRICH). An appropriate concentration sample was flowed at a flow rate of 5 mL / sec, and 50 mM getylamine was flowed to dissociate the bound antibody. The change in mass when the sample was flowed was measured, and the concentration was calculated using a calibration curve created based on the change in mass of the standard product. As a standard for Diabody, dbl2E10 (see WO02 / 33073 and WO02 / 33072) was used, and as a standard for sc (Fv) 2, 12E10 sc (Fv) 2 having the same gene structure was used.
[0201] 2.6 抗ヒト Mpl Diabodyおよび一本鎖抗体の精製  [0201] 2.6 Purification of anti-human Mpl Diabody and single chain antibody
VB22B Diabody発現 COS7細胞あるいは CHO細胞の培養上清を、 50 mM Tris-HCl(pH7.4), 150 mM NaCl, 0.05% Tween20で平衡化した Anti- Flag M2 Affinity Gel (SIGMA- ALDRICH社製)カラムに吸着させ、 100 mM Glycine- HCl(pH 3.5)で溶 出させた。溶出画分は、直ちに 1M Tris-HCl (pH8.0)で中和を行い、 HiLoad 26/60 Superdex200pg (Amersham- Bioscience社製)カラムを用いてゲルろ過クロマトグラフィ 一を行った。ゲルろ過クロマトグラフィーのバッファ一は、 PBS、 0.01% Tween20を使用 した。  Anti-Flag M2 Affinity Gel (SIGMA-ALDRICH) column prepared by equilibrating the culture supernatant of VB22B Diabody expressing COS7 cells or CHO cells with 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05% Tween20 And eluted with 100 mM Glycine-HCl (pH 3.5). The eluted fraction was immediately neutralized with 1 M Tris-HCl (pH 8.0), and subjected to gel filtration chromatography using a HiLoad 26/60 Superdex200pg (Amersham-Bioscience) column. As a buffer for gel filtration chromatography, PBS and 0.01% Tween20 were used.
[0202] VB22B sc(Fv)2発現 COS7細胞あるいは CHO細胞の培養上清を、 Diabody精製と同 一条件で精製を行った。また、大量に調製する場合には、 CHO細胞の培養上清を 20mMリン酸緩衝液（pH6.8)で平衡化した Macro- Prep Ceramic Hydroxyapatite Type I (Bio-Rad社製)カラムにかけ、 250mMリン酸緩衝液 (pH6.8)で段階的に溶出し た。溶出画分は、限外ろ過膜を用いて濃縮後、 HiLoad 26/60 Superdex200pgカラム を用いてゲルろ過クロマトグラフィーを行い、分子量が約 40kD〜70kDに相当する画 分を分取した。この画分を、 50 mM Tris-HCl(pH7.4), 150 mM NaCl, 0.05% Tween20 で平衡化した Anti- Flag M2 Affinity Gelカラムに吸着させ、 100 mM Glycine- HCl(pH 3.5)で溶出させた。溶出画分は、直ちに 1M Tris-HCl (pH8.0)で中和を行い、 HiLoad 26/60 Superdex200pgカラムを用いてゲルろ過クロマトグラフィーを行った。ゲルろ過 クロマトグラフィーのバッファ一は、 20 mM酢酸緩衝液 (pH6.0), 150 mM NaCl, 0.01% Tween 80を使用した。各精製ステップにおいて、 Diabodyおよび sc(Fv)2の確認は、 SDS- PAGEおよび抗 Flag抗体（SIGMA- ALDLICH社）を用いた Western Blottingを用 いて行なった。 [0202] The culture supernatant of COS7 cells or CHO cells expressing VB22B sc (Fv) 2 was purified under the same conditions as for Diabody purification. When preparing a large amount, the CHO cell culture supernatant is applied to a Macro-Prep Ceramic Hydroxyapatite Type I (Bio-Rad) column equilibrated with 20 mM phosphate buffer (pH 6.8), and the Elution was performed stepwise with an acid buffer (pH 6.8). The eluted fraction was concentrated using an ultrafiltration membrane, and then subjected to gel filtration chromatography using a HiLoad 26/60 Superdex 200pg column to collect a fraction having a molecular weight of about 40 kD to 70 kD. This fraction was adsorbed to an Anti-Flag M2 Affinity Gel column equilibrated with 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05% Tween20, and eluted with 100 mM Glycine-HCl (pH 3.5). Was. The eluted fraction was immediately neutralized with 1 M Tris-HCl (pH 8.0), and subjected to gel filtration chromatography using a HiLoad 26/60 Superdex200pg column. As a buffer for gel filtration chromatography, 20 mM acetate buffer (pH 6.0), 150 mM NaCl, and 0.01% Tween 80 were used. Diabody and sc (Fv) 2 were confirmed by SDS-PAGE and Western Blotting using an anti-Flag antibody (SIGMA-ALDLICH) at each purification step. I did it.
[0203] 2.7 Flow Cytometryによる抗ヒト Mpl—本鎖抗体の結合活性の評価  [0203] 2.7 Evaluation of binding activity of anti-human Mpl-single-chain antibody by Flow Cytometry
CH0— human Mpl、 CHO— monkey Mplおよび CHO— mouse Mplを回収し、 lxlO6 cells/mLになるように FACS Buffer (1% FBS/ PBS)に懸濁した。 100 L/wellとなるよう に Multiscreen - HV Filter Plates (Millipore社製）に分注し、遠心操作にて上清を除 去した。適濃度の Diabodyまたは sc(Fv)2を加え、氷上にて 30分間反応させた。細胞を 200 μ Lの FACS bufferにて 1回洗浄し、 10 μ g/mLの ΑΝΉ- FLAG M2 Monoclonal Antibody(SIGMA-ALDRICH社製）を添カ卩し、氷上にて 30分間反応させた。次に 200 μ LOFACS bufferにて細胞を 1回洗浄した後、 100倍希釈した FITC標識抗マウス IgG 抗体 (Beckman Coulter社製)を添加し、氷上にて 30分間反応させた。最後に遠心し 上清を除き、 FACS Buffer 400 μ Lに懸濁し、 EPICS ELITE ESP (Beckman Coulter社 )を用いて Flow Cytometryに供した。前方散乱光 (forward scatter)及び側方散乱光（ side scatter)のヒストグラムにて生細胞集団にゲートを設定した。 CH0-human Mpl, CHO-monkey Mpl and CHO-mouse Mpl were collected and suspended in FACS Buffer (1% FBS / PBS) to lxlO 6 cells / mL. The solution was dispensed into Multiscreen-HV Filter Plates (Millipore) at 100 L / well, and the supernatant was removed by centrifugation. An appropriate concentration of Diabody or sc (Fv) 2 was added and allowed to react on ice for 30 minutes. The cells were washed once with 200 μL of FACS buffer, added with 10 μg / mL ΑΝΉ-FLAG M2 Monoclonal Antibody (manufactured by SIGMA-ALDRICH), and reacted on ice for 30 minutes. Next, the cells were washed once with 200 μL LOFACS buffer, and a 100-fold diluted FITC-labeled anti-mouse IgG antibody (manufactured by Beckman Coulter) was added, followed by reaction on ice for 30 minutes. Finally, the mixture was centrifuged to remove the supernatant, suspended in 400 μL of FACS Buffer, and subjected to Flow Cytometry using EPICS ELITE ESP (Beckman Coulter). Live cell populations were gated with histograms of forward scatter and side scatter.
[0204] 精製した VB22B sc(Fv)2を用いて、各種 Mplを発現させた CHO細胞に対する結合 活性を評価した結果を図 5に示す。宿主細胞である CHOおよび CHO- mouse Mplに 対しては結合活性を示さず、 CHO-human Mplおよび CHO- monkey Mplに特異的に 結合することが確認された。この結合活性の傾向は、 VB22B IgGと変わらないことから 、低分子化により抗体の結合部位は変化していないことが推測された。  [0204] FIG. 5 shows the results of evaluating the binding activity of various purified Mpl to CHO cells using the purified VB22B sc (Fv) 2. It showed no binding activity to CHO and CHO-mouse Mpl, which are host cells, and was confirmed to specifically bind to CHO-human and CHO-monkey Mpl. Since the tendency of the binding activity was not different from that of VB22B IgG, it was presumed that the binding site of the antibody was not changed by the reduction in molecular weight.
[0205] 2.8 抗ヒト Mpl—本鎖抗体の TPO様ァゴニスト活性の評価  [0205] 2.8 Evaluation of TPO-like agonist activity of anti-human Mpl-chain antibody
TPO依存性増殖を示す BaF3-human Mpほたは BaF3-monkey Mplを用いて TPO様 ァゴ-スト活性を評価した。  BaF3-human Mp showing TPO-dependent proliferation was evaluated for TPO-like activity using BaF3-monkey Mpl.
各細胞を 1% Fetal Bovine Serum(Invitrogen社製)を含む RPMI1640 (Invitrogen社製) で 2回洗浄した後、 4xl05cells/mLとなるように 10% Fetal Bovine Serumを含む Wash each cell twice with RPMI1640 (Invitrogen) containing 1% Fetal Bovine Serum (Invitrogen), then 10% Fetal Bovine Serum to 4xl0 5 cells / mL
RPMI1640に懸濁し、 60 μ L/wellで 96well plateに分注した。 rhTPO (R&D社製)、 COS7培養上清または精製品の濃度を振り、各 wellに 40 Lカ卩え、 37°C、 5%CO条件  It was suspended in RPMI1640 and dispensed into a 96-well plate at 60 μL / well. Shake the concentration of rhTPO (manufactured by R & D), COS7 culture supernatant or purified product, and add 40 L to each well, 37 ° C, 5% CO.
2 下で、 24時間培養した。 10 L/wellで WST- 8試薬（Cell Count Reagent SF、ナカライ テスタ社製）をカ卩え、直後に Benchmark Plusを用いて 450nmの吸光度 (対照 655nm)を 測定し、 2時間培養後に、再度 450 nmの吸光度 (対照 655nm)を測定した。 WST-8試 薬は生細胞数に応じて 450nmの発色反応を呈することから、 2時間の吸光度変化を 指標に TPO様ァゴ-スト活性を評価した。また、 GraphPad Prismを用いて EC 値を算 2 and cultured for 24 hours. After culturing WST-8 reagent (Cell Count Reagent SF, manufactured by Nacalai Tester) at 10 L / well, immediately measure the absorbance at 450 nm (control: 655 nm) using Benchmark Plus. The absorbance at nm (control 655 nm) was measured. WST-8 trial Since the drug exhibited a color development reaction at 450 nm according to the number of living cells, TPO-like agonist activity was evaluated using the change in absorbance over 2 hours as an index. Calculate EC value using GraphPad Prism.
50 出した。  50 issued.
[0206] また、 TPO依存性増殖を示すヒト白血病細胞株である M-07e(DSMZより購入)を使用 して、 TPO様ァゴ-スト活性を評価した。 M- 07eを 1% Fetal Bovine Serumを含む RPMI 1640で 2回洗浄した後、 5xl05cells/mLとなるように 10% Fetal Bovine Serumを含 む RPMI1640に懸濁し、 50 μ L/wellで 96well plateに分注した。 rhTPO、 COS7培養上 清または精製品の濃度を振り、各 wellに 50 Lカ卩え、 37°C、 5%CO条件下で、 48時間 [0206] Further, TPO-like agonistic activity was evaluated using M-07e (purchased from DSMZ), a human leukemia cell line showing TPO-dependent growth. M-07e was washed twice with RPMI 1640 containing 1% Fetal Bovine Serum, then suspended in RPMI 1640 containing 10% Fetal Bovine Serum at 5xl0 5 cells / mL, and 96 μl plate at 50 μL / well Was dispensed. Shake the concentration of rhTPO, COS7 culture supernatant or purified product, add 50 L to each well, 37 ° C, 5% CO for 48 hours
2  2
培養した。 10 L/wellで WST- 8試薬（Cell Count Reagent SF、ナカライテスタ社製）を 加え、直後に Benchmark Plusを用いて 450 nmの吸光度 (対照 655nm)を測定し、 4時間 培養後に、再度 450 nmの吸光度 (対照 655nm)を測定した。 4時間の吸光度変化を指 標に TPO様ァゴ-スト活性を評価した。  Cultured. Add WST-8 reagent (Cell Count Reagent SF, manufactured by Nacalai Tester) at 10 L / well, measure absorbance at 450 nm (control: 655 nm) using Benchmark Plus immediately, and after culturing for 4 hours, re-drain at 450 nm again. Was measured for absorbance (control: 655 nm). TPO-like agonist activity was evaluated using the change in absorbance over 4 hours as an indicator.
[0207] 精製した VB22B IgG、 VB22B Diabodyおよび VB22B sc(Fv)2を用いて、 [0207] Using purified VB22B IgG, VB22B Diabody and VB22B sc (Fv) 2,
BaF3- human Mpl、 BaF3- monkey Mpl、 M-07eでの TPO様ァゴ-スト活性を評価した 結果をそれぞれ図 6、図 7、図 8に示す。ァゴニスト活性は、抗原結合部位が二価で あることが重要であるが、抗原結合部位間の距離や角度も重要な要素であると考えら れる（WO02/33073、 WO02/33072参照）。新たに取得した抗ヒト Mpl抗体も同様であり 、 VB22B IgG (BaF- human Mpl EC ： >30nM)に対して、 VB22B Diabodyおよび  The results of evaluating the TPO-like agonist activity of BaF3-human Mpl, BaF3-monkey Mpl, and M-07e are shown in FIGS. 6, 7, and 8, respectively. It is important for the agonist activity that the antigen-binding site is bivalent, but the distance and angle between the antigen-binding sites are also considered to be important factors (see WO02 / 33073 and WO02 / 33072). The same applies to the newly obtained anti-human Mpl antibody, in which VB22B IgG (BaF-human Mpl EC:> 30 nM) and VB22B Diabody and
50  50
VB22B sc(Fv)2は高いァゴ-スト活性 (BaF- human Mpl EC ：それぞれ 61pM, 27pM)  VB22B sc (Fv) 2 has high agonist activity (BaF-human Mpl EC: 61pM, 27pM, respectively)
50  50
であり、天然リガンドである human TPO(BaF- human Mpl EC : 76pM)と同等以上の活  And its activity is equal to or higher than that of the natural ligand human TPO (BaF-human Mpl EC: 76 pM).
50  50
性を示した。 VB22B Diabodyと比較して VB22B sc(Fv)2が高い活性を示したことから、 一本鎖抗体はリンカ一配列の長さや分子形状によって構造が大きく変化し、ァゴニス ト活性も変化することが推測される。  Showed sex. Since VB22B sc (Fv) 2 showed higher activity than VB22B Diabody, it is speculated that the structure of single-chain antibodies greatly changes depending on the length and molecular shape of the linker sequence, and that agonist activity also changes. Is done.
[0208] 2.9 抗ヒト Mpl—本鎖抗体のヒトイ匕 [0208] 2.9 Anti-human Mpl—Human chain ligation of main chain antibody
VB22B sc(Fv)2のヒト化を実施するために、公開されている Kabat Database (ftp://ftp.ebi.ac.uk/pub/databases/kabat/)より抗体の配列データを入手し、 H鎖可 変領域、 L鎖可変領域に分けてホモロジ一検索を行った。その結果、 H鎖可変領域 は DN13(Smithsonら、 Mol Immunol. 1999 ; 36 : 113- 124)と高い相同性を持つことが分 かった。また、 L鎖可変領域は ToP027(Hougsら、 J. Immunol. 1999 ; 162 : 224-237)と高 い相同性を持つことが分力つた。これらの抗体のフレームワーク領域 (以下、 FR)に相 補性抗原決定領域 (以下、 CDR)を移植したヒト化抗体を作製した。ヒト化抗体 sc(Fv)2 を CHO- DG44細胞にて発現させ、 BaF-human Mplを用いたァゴ-スト活性を評価し た。ァゴ-スト活性を指標に、 FR内にアミノ酸置換をカロえ、マウス型 VB22B sc(Fv)2と 同等のァゴ-スト活性を有するヒト化 VB22B sc(Fv)2を作製した。 To carry out humanization of VB22B sc (Fv) 2, obtain antibody sequence data from the published Kabat Database (ftp://ftp.ebi.ac.uk/pub/databases/kabat/), A homology search was performed for the H chain variable region and the L chain variable region. As a result, the heavy chain variable region was found to have high homology to DN13 (Smithson et al., Mol Immunol. 1999; 36: 113-124). won. It was also implicit that the L chain variable region had high homology to ToP027 (Hougs et al., J. Immunol. 1999; 162: 224-237). Humanized antibodies were prepared by transplanting a complementary antigen-determining region (hereinafter, CDR) into a framework region (hereinafter, FR) of these antibodies. The humanized antibody sc (Fv) 2 was expressed in CHO-DG44 cells, and the agonist activity using BaF-human Mpl was evaluated. Using the agonist activity as an index, humanized VB22B sc (Fv) 2 having the same agonist activity as mouse VB22B sc (Fv) 2 was prepared by detecting amino acid substitution in FR.
具体的には、 50base程度の合成オリゴ DNAを約 20base程度ハイブリダィズするよう に設計し、これらの合成オリゴ DNAを PCR法によりアッセンプリさせて各可変領域をコ ードする遺伝子を作製した。これらの遺伝子を用いて、参考例 2.3の方法と同様に、 sc(Fv)2を作製し、発現ベクター pCXND3にクローユングし、 35種類のヒト化 VB22B sc(Fv)2を挿入した各発現ベクター pCXND3- hVB22B p- z sc(Fv)2、  Specifically, a synthetic oligo DNA of about 50 bases was designed to hybridize about 20 bases, and these synthetic oligo DNAs were assembled by PCR to prepare a gene encoding each variable region. Using these genes, sc (Fv) 2 was prepared and cloned into the expression vector pCXND3 in the same manner as in Reference Example 2.3, and each of the expression vectors pCXND3 was inserted with 35 types of humanized VB22B sc (Fv) 2. -hVB22B p-z sc (Fv) 2,
pCXND3-hVB22B g— e sc(Fv)2、 pCXND3— hVB22B e sc(Fv)2、 pCXND3— hVB22B u2-wz4 sc(Fv)2、 pCXND3- hVB22B q- wz5 sc(Fv)2を作製した。本プラスミドに含まれ る hVB22B p-z sc(Fv)2の塩基配列を配列番号： 60に、アミノ酸配列を配列番号： 61 に、 hVB22B g-e sc(Fv)2の塩基配列を配列番号： 62に、アミノ酸配列を配列番号： 6 3に、 hVB22B e sc(Fv)2の塩基配列を配列番号： 64に、アミノ酸配列を配列番号： 65 に、 hVB22B u2-wz4 sc(Fv)2の塩基配列を配列番号： 102に、アミノ酸配列を配列番 号： 100に、 hVB22B q-wz5 sc(Fv)2の塩基配列を配列番号： 103に、アミノ酸配列を 配列番号： 101に示す。またマウス型 VB22B sc(Fv)2の塩基配列を配列番号： 13に、 アミノ酸配列を配列番号： 14に示す。参考例 2.4の方法と同様に CHO-DG44細胞に 発現させ、培養上清を回収した。ヒト化 VB22B sc(Fv)2は Flagタグを付加していないこ とから、培養上清からの精製は、参考例 1.8に記載した VB22Bが認識するェピトープ である MG10 (Gln213から Ala231)と GST融合蛋白質を利用して行った。 MG10と GST 融合蛋白質の精製は、 Glutathione Sepharose 4B (Amersham Biosciences社製）を用 いて、メーカーのプロトコールに従って精製した。さらに、精製した MG10と GST融合 蛋白質をメーカーのプロトコールに従って、 HiTrap NHS- activated HP (Amersham Biosciences社製）に固定化し、ァフィ-ティカラムを作製した。ヒト化 VB22B sc(Fv)2発 現 CHO細胞の培養上清を、 50mM Tris- HCl(pH7.4), 150mM NaCl, 0.01% Tween80 で平衡ィ匕した MG10-GST融合蛋白質固定ィ匕カラムに流し、ヒト化 VB22B sc(Fv)2を吸 着させ、 lOOmM Glycine-HCl(pH3.5),0.01% Tween80で溶出させた。溶出画分は直ち に 1M Tris- HCl(pH7.4)で中和を行い、 HiLoad 16/60 Superdex200pg (Amersham Biosciences社製）を用いてゲルろ過クロマトグラフィーを行った。ゲルろ過クロマトグラ フィ一の緩衝液は、 20mMクェン酸緩衝液 (pH7.5) , 300mM NaCl, 0.01% Tween 80 を使用した。得られた精製品を用いて、参考例 2.8の方法と同様に TPO様ァゴニスト 活性を評価した。精製したマウス型 VB22B sc(Fv)2、 hVB22B p- z sc(Fv)2、 hVB22B u2-wz4 sc(Fv)2、 hVB22B q- wz5 sc(Fv)2およびヒト化した hVB22B e sc(Fv)2、 hVB22B g-e sc(Fv)2を用いて、 BaF3- human Mplでの TPO様ァゴ-スト活性を評価し た結果を図 9、 10、 11に示す。各種ヒト化 VB22B sc(Fv)2のァゴニスト活性はほぼ同 等であり、ヒト化による活性変化は無いことが示された。 pCXND3-hVB22B g-esc (Fv) 2, pCXND3-hVB22B esc (Fv) 2, pCXND3-hVB22B u2-wz4 sc (Fv) 2, pCXND3-hVB22B q-wz5 sc (Fv) 2 were prepared. The nucleotide sequence of hVB22B pz sc (Fv) 2 contained in this plasmid is shown in SEQ ID NO: 60, the amino acid sequence is shown in SEQ ID NO: 61, the nucleotide sequence of hVB22Bge sc (Fv) 2 is shown in SEQ ID NO: 62, The sequence is represented by SEQ ID NO: 63, the nucleotide sequence of hVB22B esc (Fv) 2 is represented by SEQ ID NO: 64, the amino acid sequence is represented by SEQ ID NO: 65, and the nucleotide sequence of hVB22B u2-wz4 sc (Fv) 2 is represented by SEQ ID NO: No .: 102, the amino acid sequence is shown in SEQ ID NO: 100, the nucleotide sequence of hVB22B q-wz5 sc (Fv) 2 is shown in SEQ ID NO: 103, and the amino acid sequence is shown in SEQ ID NO: 101. The nucleotide sequence of mouse VB22B sc (Fv) 2 is shown in SEQ ID NO: 13, and the amino acid sequence is shown in SEQ ID NO: 14. Expression was carried out in CHO-DG44 cells in the same manner as in Reference Example 2.4, and the culture supernatant was recovered. Since humanized VB22B sc (Fv) 2 does not have a Flag tag, purification from culture supernatant was performed using GST fusion with MG10 (Gln213 to Ala231), an epitope recognized by VB22B described in Reference Example 1.8. This was performed using proteins. The MG10-GST fusion protein was purified using Glutathione Sepharose 4B (Amersham Biosciences) according to the manufacturer's protocol. Furthermore, the purified MG10-GST fusion protein was immobilized on HiTrap NHS-activated HP (manufactured by Amersham Biosciences) according to the manufacturer's protocol to prepare an affinity column. Humanized VB22B sc (Fv) 2 expression CHO cell culture supernatant was added to 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.01% Tween80 The column was passed through a MG10-GST fusion protein immobilized column equilibrated in step 2 to adsorb humanized VB22B sc (Fv) 2, and eluted with 100 mM Glycine-HCl (pH 3.5), 0.01% Tween80. The eluted fraction was immediately neutralized with 1M Tris-HCl (pH 7.4), and subjected to gel filtration chromatography using HiLoad 16/60 Superdex200pg (manufactured by Amersham Biosciences). As a buffer for the gel filtration chromatography, 20 mM citrate buffer (pH 7.5), 300 mM NaCl, and 0.01% Tween 80 were used. Using the purified product obtained, TPO-like agonist activity was evaluated in the same manner as in Reference Example 2.8. Purified mouse VB22B sc (Fv) 2, hVB22B p-z sc (Fv) 2, hVB22B u2-wz4 sc (Fv) 2, hVB22B q-wz5 sc (Fv) 2 and humanized hVB22B e sc (Fv) 2. The results of evaluating the TPO-like agonist activity in BaF3-human Mpl using hVB22Bgesc (Fv) 2 are shown in FIGS. The agonist activity of various humanized VB22B sc (Fv) 2 was almost the same, indicating that there was no change in activity due to humanization.
[0210] 2.10 抗ヒト Mpl抗体 VB22B IgG, VB22B sc(Fv)2およびヒト化 VB22B sc(Fv)2の抗原 抗体反応における反応速度論的解析  [0210] 2.10 Kinetic analysis of antigen-antibody reaction of anti-human Mpl antibodies VB22B IgG, VB22B sc (Fv) 2 and humanized VB22B sc (Fv) 2
抗ヒト Mpl抗体 VB22Bが可溶型組換え Mplに結合する性質を利用し、参考例 1.8で 示した MG10 (Gln213から Ala231) - GST融合蛋白質と VB22B IgG, VB22B sc(Fv)2お よびヒト化 VB22B sc(Fv)2との抗原抗体反応における速度論的解析を行った。  Utilizing the property that anti-human Mpl antibody VB22B binds to soluble recombinant Mpl, MG10 (Gln213 to Ala231) -GST fusion protein and VB22B IgG, VB22B sc (Fv) 2 and humanized as shown in Reference Example 1.8 Kinetic analysis on the antigen-antibody reaction with VB22B sc (Fv) 2 was performed.
Biacore 3000(Biacore社製)に Sensor Chip CM5(Biacore社製)を装着し、ァミンカツプリ ング法にて MG10-GST融合蛋白質を固定ィ匕した。測定のランニングバッファ一は HBS-EP Buffer(Biacore社製)を使用し、流速は 20 μ L/minとした。ここに HBS- EP Bufferで 5.5〜175.0nMの濃度に調製した VB22B IgGをそれぞれ 2分間添カ卩して、各 濃度での結合領域を得た後に、解離領域を 2分間測定した。 Sensor Chip上の MG10-GST融合蛋白質に結合した VB22B IgGは、 20mM HC1を 1分間添カ卩して除去 し、 Sensor Chipを再生した。同様に、 4.7〜150.1nMの VB22B sc(Fv)2、 5.3〜168.9nM の hVB22B q-wz5 sc(Fv)2、 4.9〜156.8nMの hVB22B u2- wz4 sc(Fv)2を調製し、 MG10-GST融合蛋白質を固定ィ匕したチップに添加して、測定を実施した。  A Biacore 3000 (manufactured by Biacore) was equipped with a Sensor Chip CM5 (manufactured by Biacore), and the MG10-GST fusion protein was immobilized by the amine coupling method. HBS-EP Buffer (manufactured by Biacore) was used as the running buffer for the measurement, and the flow rate was 20 μL / min. VB22B IgG prepared to a concentration of 5.5 to 175.0 nM with HBS-EP Buffer was added thereto for 2 minutes to obtain binding regions at each concentration, and then the dissociation region was measured for 2 minutes. VB22B IgG bound to the MG10-GST fusion protein on the Sensor Chip was removed by adding 20 mM HC1 for 1 minute to regenerate the Sensor Chip. Similarly, 4.7 to 150.1 nM of VB22B sc (Fv) 2, 5.3 to 168.9 nM of hVB22B q-wz5 sc (Fv) 2, and 4.9 to 156.8 nM of hVB22Bu2-wz4 sc (Fv) 2 were prepared, and MG10- The GST fusion protein was added to the immobilized chip, and the measurement was performed.
[0211] いずれも二価抗体であるため、各濃度で得られたセンサーグラムでは、一価と二価 の両方の結合が混在した状態になる。このため、 BIAevaluation ver.3.1ソフトウェア (Biacore社製)の Bivalent analyte modelを適用して解析することにより、一価の状態で の反応速度定数を算出した。以上の解析は全ての抗体について 3回実施した。この ようにして算出された、いずれも一価での結合速度定数 (ka)と解離速度定数 (kd)およ び解離定数 (KD)を表 1に示した。 VB22B IgG、 VB22B sc(Fv)2、 hVB22B q-wz5 sc(Fv)2、 hVB22B u2- wz4 sc(Fv)2の解離定数 (KD)は、それぞれ 1.15x10— 8M、 1.17x10— 8M、 1.36 xlO— 8M、 1.02 xlO— 8Mであり、 MG10- GST融合蛋白質に対して、ほ ぼ同等の結合活性を持つことが確認された。 [0211] Since both are bivalent antibodies, sensorgrams obtained at each concentration show a state in which both monovalent and bivalent bonds are mixed. For this reason, BIAevaluation ver.3.1 software (manufactured by Biacore) Bivalent analyte model is applied for analysis to obtain a monovalent state. Was calculated. The above analysis was performed three times for all antibodies. Table 1 shows the monovalent binding rate constant (ka), dissociation rate constant (kd), and dissociation constant (KD) calculated in this way. VB22B IgG, VB22B sc (Fv) 2, hVB22B q-wz5 sc (Fv) 2, hVB22B u2- wz4 sc (Fv) 2 of the dissociation constant (KD) respectively 1.15x10- 8 M, 1.17x10- 8 M, 1.36 xlO— 8 M and 1.02 xlO— 8 M, confirming that they have almost the same binding activity to the MG10-GST fusion protein.
[表 1]  [table 1]
抗ヒト Mp l抗体の抗原抗体反応における速度論的解析結果
Figure imgf000057_0001
産業上の利用の可能性 Kinetic analysis results of antigen-antibody reaction of anti-human Mpl antibody
Figure imgf000057_0001
Industrial potential
組換え型ヒト TPOは、化学療法剤等の治療による血小板減少症の治療薬として、さ まざまな形で、臨床試験が行われてきた。その臨床試験において、一つの大きな問 題として、 ΤΡΟの投与による抗 ΤΡΟ抗体の出現が報告されており Ounzhi Li, et. al, Blood (2001) 98, 3241—324、 Saroj Vandhan— Raj. et. al. Ann. Intern. Med. (2000) 132, 364-368)、特に内在性の TPO活性を阻害する中和抗体が産生され、その結果 として、血小板数を回復させるのではなぐ逆に血小板減少症を発症することが報告 されて 、る。本発明によって示される抗 TPO受容体ァゴニスト低分子化抗体の投与 によって、内在性 TPOに対する抗体の出現を誘導することはない。また抗体を低分 子化することにより、高い比活性を示し、また血中半減期を短くできることから、有効 血中濃度の調節が容易となり、臨床応用上有利となると考えられる。従って、天然型 TPOやァゴニスト抗体 (IgG)よりも優れた性質をもつ、血小板減少症の治療薬となるこ とが期待される。また、低分子化抗体は、糖鎖が結合していないことから、組換え型タ ンパクの発現においても、その発現系に制限はなぐ哺乳動物由来の細胞株、酵母 、昆虫細胞、大腸菌、植物いずれの発現系においても、作製することが可能である。  Recombinant human TPO has been tested in various forms as a therapeutic agent for thrombocytopenia caused by treatment with chemotherapeutic agents. One of the major problems reported in the clinical trials was the emergence of anti-ΤΡΟ antibodies due to ΤΡΟ administration. Ounzhi Li, et. Al, Blood (2001) 98, 3241—324, Saroj Vandhan—Raj. Et. al. Ann. Intern. Med. (2000) 132, 364-368), specifically producing neutralizing antibodies that inhibit endogenous TPO activity, resulting in thrombocytopenia rather than restoring platelet counts It has been reported that the disease develops. The administration of the anti-TPO receptor agonist low molecular weight antibody shown by the present invention does not induce the appearance of antibodies against endogenous TPO. In addition, by lowering the molecular weight of the antibody, high specific activity is exhibited and the half-life in blood can be shortened. Therefore, it is considered that the adjustment of the effective blood concentration becomes easy, which is advantageous for clinical application. Therefore, it is expected to be a therapeutic agent for thrombocytopenia that has properties superior to those of natural TPO and agonist antibody (IgG). In addition, since low-molecular-weight antibodies do not have sugar chains attached thereto, even in the expression of recombinant proteins, expression systems for mammals are not restricted, such as mammalian cell lines, yeast, insect cells, Escherichia coli, and plants. It can be produced in any expression system.

Claims

請求の範囲  The scope of the claims
[I] TPO受容体 (Mpl)への結合活性を有する sc(Fv)2を有効成分として含有する血小板 減少症治療剤。  [I] A therapeutic agent for thrombocytopenia, comprising sc (Fv) 2 having binding activity to a TPO receptor (Mpl) as an active ingredient.
[2] sc(Fv)2中の可変領域カ^ンカ一で結合されていることを特徴とする、請求項 1に記載 の血小板減少症治療剤。  [2] The therapeutic agent for thrombocytopenia according to claim 1, wherein the therapeutic agent is bound by a variable region protein in sc (Fv) 2.
[3] リンカ一が 15アミノ酸であることを特徴とする、請求項 2に記載の血小板減少症治療 剤。 [3] The therapeutic agent for thrombocytopenia according to claim 2, wherein the linker is 15 amino acids.
[4] ヒト Mpl及びサル Mplに結合する抗体を有効成分として含有する血小板減少症治療 剤。  [4] A therapeutic agent for thrombocytopenia, comprising an antibody that binds to human Mpl and monkey Mpl as an active ingredient.
[5] ヒト Mpl及びサル Mplに対してァゴニスト活性を有する抗体を有効成分として含有する 血小板減少症治療剤。  [5] A therapeutic agent for thrombocytopenia, comprising as an active ingredient an antibody having agonist activity against human Mpl and monkey Mpl.
[6] 配列番号： 1、 2、 3に記載のアミノ酸配列力 なる CDR1、 2、 3を有する重鎖可変領域 を含む抗体を有効成分として含有する血小板減少症治療剤。  [6] A therapeutic agent for thrombocytopenia, comprising, as an active ingredient, an antibody containing a heavy chain variable region having CDRs 1, 2, and 3 having the amino acid sequences of SEQ ID NOs: 1, 2, and 3.
[7] 配列番号: 4、 5、 6に記載のアミノ酸配列力 なる CDR1、 2、 3を有する軽鎖可変領域 を含む抗体を有効成分として含有する血小板減少症治療剤。 [7] A therapeutic agent for thrombocytopenia comprising, as an active ingredient, an antibody containing a light chain variable region having CDR1, 2, or 3 having the amino acid sequence of SEQ ID NO: 4, 5, or 6.
[8] 請求項 6に記載の重鎖可変領域、および請求項 7に記載の軽鎖可変領域を有する 抗体を有効成分として含有する血小板減少症治療剤。 [8] A therapeutic agent for thrombocytopenia, comprising as an active ingredient the antibody having the heavy chain variable region according to claim 6 and the light chain variable region according to claim 7.
[9] 請求項 6〜8のいずれかに記載のアミノ酸配列において 1又は複数のアミノ酸が置換[9] The amino acid sequence according to any one of claims 6 to 8, wherein one or more amino acids are substituted.
、欠失、付加および Zまたは挿入され、かつ請求項 6〜8のいずれかに記載の抗体と 同等の活性を有する抗体を有効成分として含有する血小板減少症治療剤。 A therapeutic agent for thrombocytopenia comprising, as an active ingredient, an antibody having the same activity as the antibody according to any one of claims 6 to 8, wherein the antibody is deleted, added, Z- or inserted.
[10] 請求項 6〜8の ヽずれかに記載の抗体が認識するェピトープを認識する抗体を有効 成分として含有する血小板減少症治療剤。 [10] A therapeutic agent for thrombocytopenia, comprising as an active ingredient an antibody recognizing an epitope recognized by the antibody according to any one of claims 6 to 8.
[I I] ヒト Mplの 26番目から 274番目のアミノ酸部位を認識する抗体を有効成分として含有 する血小板減少症治療剤。  [II] A therapeutic agent for thrombocytopenia, comprising, as an active ingredient, an antibody that recognizes amino acids 26 to 274 of human Mpl.
[12] TPO受容体 (Mpl)への結合活性を有する sc(Fv)2を有効成分として含有する血小板 増加剤。  [12] A platelet-increasing agent comprising sc (Fv) 2 having binding activity to a TPO receptor (Mpl) as an active ingredient.
[13] sc(Fv)2中の可変領域カ^ンカ一で結合されていることを特徴とする、請求項 12に記 載の血小板増加剤。 [13] The platelet-increasing agent according to claim 12, which is bound by a variable region protein in sc (Fv) 2.
[14] リンカ一が 15アミノ酸であることを特徴とする、請求項 13に記載の血小板増加剤。 14. The platelet increasing agent according to claim 13, wherein the linker has 15 amino acids.
[15] ヒト Mpl及びサル Mplに結合する抗体を有効成分として含有する血小板増加剤。 [15] A platelet-increasing agent comprising as an active ingredient an antibody that binds to human Mpl and monkey Mpl.
[16] ヒト Mpl及びサル Mplに対してァゴニスト活性を有する抗体を有効成分として含有する 血小板増加剤。 [16] A platelet increasing agent comprising, as an active ingredient, an antibody having agonist activity against human Mpl and monkey Mpl.
[17] 配列番号： 1、 2、 3に記載のアミノ酸配列力 なる CDR1、 2、 3を有する重鎖可変領域 を含む抗体を有効成分として含有する血小板増加剤。  [17] A platelet-increasing agent comprising, as an active ingredient, an antibody containing a heavy chain variable region having CDRs 1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3.
[18] 配列番号: 4、 5、 6に記載のアミノ酸配列力 なる CDR1、 2、 3を有する軽鎖可変領域 を含む抗体を有効成分として含有する血小板増加剤。 [18] A platelet-increasing agent comprising, as an active ingredient, an antibody containing a light chain variable region having CDRs 1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6.
[19] 請求項 17に記載の重鎖可変領域、および請求項 18に記載の軽鎖可変領域を有す る抗体を有効成分として含有する血小板増加剤。 [19] A platelet-increasing agent comprising as an active ingredient the antibody having the heavy chain variable region according to claim 17 and the light chain variable region according to claim 18.
[20] 請求項 17〜 19のいずれかに記載のアミノ酸配列において 1又は複数のアミノ酸が置 換、欠失、付加および Zまたは挿入され、かつ請求項 17〜 19のいずれかに記載の 抗体と同等の活性を有する抗体を有効成分として含有する血小板増加剤。 [20] The antibody according to any one of claims 17 to 19, wherein one or more amino acids are substituted, deleted, added and Z or inserted in the amino acid sequence according to any one of claims 17 to 19, and A platelet-increasing agent containing an antibody having an equivalent activity as an active ingredient.
[21] 請求項 17〜 19のいずれかに記載の抗体が認識するェピトープを認識する抗体を有 効成分として含有する血小板増加剤。  [21] A platelet-increasing agent comprising, as an active ingredient, an antibody that recognizes an epitope that is recognized by the antibody according to any one of claims 17 to 19.
[22] ヒト Mplの 26番目から 274番目のアミノ酸部位を認識する抗体を有効成分として含有 する血小板増加剤。 [22] A platelet-increasing agent containing, as an active ingredient, an antibody that recognizes amino acids 26 to 274 of human Mpl.
PCT/JP2005/008592 2004-05-11 2005-05-11 Remedy for thrombopenia WO2005107784A1 (en)

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WO2007145227A1 (en) 2006-06-14 2007-12-21 Chugai Seiyaku Kabushiki Kaisha Hematopoietic stem cell proliferation promoter
US7658924B2 (en) 2001-10-11 2010-02-09 Amgen Inc. Angiopoietin-2 specific binding agents
US8597911B2 (en) 2003-06-11 2013-12-03 Chugai Seiyaku Kabushiki Kaisha Process for producing antibodies
US8945543B2 (en) 2005-06-10 2015-02-03 Chugai Seiyaku Kabushiki Kaisha Stabilizer for protein preparation comprising meglumine and use thereof
CN101262885B (en) * 2005-06-10 2015-04-01 中外制药株式会社 Pharmaceutical compositions containing sc(Fv)2
JP2016518455A (en) * 2013-05-24 2016-06-23 昆明聖火薬業（集団）有限公司 Use of dencithin in the manufacture of thrombocytopenia therapeutics
US9493569B2 (en) 2005-03-31 2016-11-15 Chugai Seiyaku Kabushiki Kaisha Structural isomers of sc(Fv)2
US9670269B2 (en) 2006-03-31 2017-06-06 Chugai Seiyaku Kabushiki Kaisha Methods of modifying antibodies for purification of bispecific antibodies
US10011858B2 (en) 2005-03-31 2018-07-03 Chugai Seiyaku Kabushiki Kaisha Methods for producing polypeptides by regulating polypeptide association
US10336820B2 (en) 2008-02-20 2019-07-02 Amgen Inc. Antibodies directed to angiopoietin-1 and angiopoietin-2 and uses thereof
US11124576B2 (en) 2013-09-27 2021-09-21 Chungai Seiyaku Kabushiki Kaisha Method for producing polypeptide heteromultimer
US11649262B2 (en) 2015-12-28 2023-05-16 Chugai Seiyaku Kabushiki Kaisha Method for promoting efficiency of purification of Fc region-containing polypeptide

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US7658924B2 (en) 2001-10-11 2010-02-09 Amgen Inc. Angiopoietin-2 specific binding agents
US8597911B2 (en) 2003-06-11 2013-12-03 Chugai Seiyaku Kabushiki Kaisha Process for producing antibodies
US9493569B2 (en) 2005-03-31 2016-11-15 Chugai Seiyaku Kabushiki Kaisha Structural isomers of sc(Fv)2
US11168344B2 (en) 2005-03-31 2021-11-09 Chugai Seiyaku Kabushiki Kaisha Methods for producing polypeptides by regulating polypeptide association
US10011858B2 (en) 2005-03-31 2018-07-03 Chugai Seiyaku Kabushiki Kaisha Methods for producing polypeptides by regulating polypeptide association
US8945543B2 (en) 2005-06-10 2015-02-03 Chugai Seiyaku Kabushiki Kaisha Stabilizer for protein preparation comprising meglumine and use thereof
US9241994B2 (en) 2005-06-10 2016-01-26 Chugai Seiyaku Kabushiki Kaisha Pharmaceutical compositions containing sc(Fv)2
CN101262885B (en) * 2005-06-10 2015-04-01 中外制药株式会社 Pharmaceutical compositions containing sc(Fv)2
US9777066B2 (en) 2005-06-10 2017-10-03 Chugai Seiyaku Kabushiki Kaisha Pharmaceutical compositions containing sc(Fv)2
WO2006132352A1 (en) * 2005-06-10 2006-12-14 Chugai Seiyaku Kabushiki Kaisha Pharmaceutical composition containing sc(fv)2
US9670269B2 (en) 2006-03-31 2017-06-06 Chugai Seiyaku Kabushiki Kaisha Methods of modifying antibodies for purification of bispecific antibodies
US10934344B2 (en) 2006-03-31 2021-03-02 Chugai Seiyaku Kabushiki Kaisha Methods of modifying antibodies for purification of bispecific antibodies
WO2007145227A1 (en) 2006-06-14 2007-12-21 Chugai Seiyaku Kabushiki Kaisha Hematopoietic stem cell proliferation promoter
US10336820B2 (en) 2008-02-20 2019-07-02 Amgen Inc. Antibodies directed to angiopoietin-1 and angiopoietin-2 and uses thereof
JP2016518455A (en) * 2013-05-24 2016-06-23 昆明聖火薬業（集団）有限公司 Use of dencithin in the manufacture of thrombocytopenia therapeutics
US11124576B2 (en) 2013-09-27 2021-09-21 Chungai Seiyaku Kabushiki Kaisha Method for producing polypeptide heteromultimer
US11649262B2 (en) 2015-12-28 2023-05-16 Chugai Seiyaku Kabushiki Kaisha Method for promoting efficiency of purification of Fc region-containing polypeptide

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