WO2005107784A1 - Remède à la thrombopénie - Google Patents

Remède à la thrombopénie 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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Japanese (ja)
Inventor
Masayuki Tsuchiya
Tetsuro Orita
Yoshihiro Matsumoto
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Chugai Pharmaceutical Co Ltd
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Chugai Pharmaceutical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IG], 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

En obtenant et en purifiant un anticorps antihumain Mp1, du Mp1 sv(Fv)2 antihumain est purifié grâce à l'utilisation d'une technique d'ingénierie génétique. En outre le Mp1 sc(FV)2 antihumain est humanisé avec succès. En évaluant l'activité agoniste de type TPO (thrombopoïétine ) du sc(FV)2, on a trouvé que le sc(FV)2 montre une activité agoniste élevée sur un anticorps antihumain Mp1 et que l'activité est comparable ou même supérieure à l'activité de la thrombopoïétine qui est un ligand naturel. On a également trouvé que le VB22B sc(Fv)2 et le VB22B sc(Fv)2 humanisés présentent l'effet d'augmenter in vivo les plaquettes.
PCT/JP2005/008592 2004-05-11 2005-05-11 Remède à la thrombopénie Ceased WO2005107784A1 (fr)

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006132352A1 (fr) * 2005-06-10 2006-12-14 Chugai Seiyaku Kabushiki Kaisha Composition pharmaceutique contenant sc(fv)2
WO2007145227A1 (fr) 2006-06-14 2007-12-21 Chugai Seiyaku Kabushiki Kaisha Promoteur de la prolifération des cellules souches hématopoïétiques
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 (zh) * 2005-06-10 2015-04-01 中外制药株式会社 含有sc(Fv)2的药物组合物
JP2016518455A (ja) * 2013-05-24 2016-06-23 昆明聖火薬業（集団）有限公司 血小板減少症治療薬の製造におけるデンシチンの使用
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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JPH09289892A (ja) * 1996-04-25 1997-11-11 Yamanouchi Pharmaceut Co Ltd ｃ−ＭＰＬリガンドアゴニスト抗体
JP2001513999A (ja) * 1997-08-25 2001-09-11 ジェネンテク・インコーポレイテッド トロンボポエチン受容体に対するアゴニスト抗体、及びそれらの治療学的使用
WO2002033073A1 (fr) * 2000-10-20 2002-04-25 Chugai Seiyaku Kabushiki Kaisha Anticorps agoniste degrade
WO2002033072A1 (fr) * 2000-10-20 2002-04-25 Chugai Seiyaku Kabushiki Kaisha Anticorps degrade, agoniste de tpo
WO2005056604A1 (fr) * 2003-12-12 2005-06-23 Chugai Seiyaku Kabushiki Kaisha Anticorps anti-mpl
WO2005056798A1 (fr) * 2003-12-12 2005-06-23 Chugai Seiyaku Kabushiki Kaisha Procede permettant de renforcer l'activite d'un anticorps

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09289892A (ja) * 1996-04-25 1997-11-11 Yamanouchi Pharmaceut Co Ltd ｃ−ＭＰＬリガンドアゴニスト抗体
JP2001513999A (ja) * 1997-08-25 2001-09-11 ジェネンテク・インコーポレイテッド トロンボポエチン受容体に対するアゴニスト抗体、及びそれらの治療学的使用
WO2002033073A1 (fr) * 2000-10-20 2002-04-25 Chugai Seiyaku Kabushiki Kaisha Anticorps agoniste degrade
WO2002033072A1 (fr) * 2000-10-20 2002-04-25 Chugai Seiyaku Kabushiki Kaisha Anticorps degrade, agoniste de tpo
WO2005056604A1 (fr) * 2003-12-12 2005-06-23 Chugai Seiyaku Kabushiki Kaisha Anticorps anti-mpl
WO2005056798A1 (fr) * 2003-12-12 2005-06-23 Chugai Seiyaku Kabushiki Kaisha Procede permettant de renforcer l'activite d'un anticorps

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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 (zh) * 2005-06-10 2015-04-01 中外制药株式会社 含有sc(Fv)2的药物组合物
US9777066B2 (en) 2005-06-10 2017-10-03 Chugai Seiyaku Kabushiki Kaisha Pharmaceutical compositions containing sc(Fv)2
WO2006132352A1 (fr) * 2005-06-10 2006-12-14 Chugai Seiyaku Kabushiki Kaisha Composition pharmaceutique contenant 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 (fr) 2006-06-14 2007-12-21 Chugai Seiyaku Kabushiki Kaisha Promoteur de la prolifération des cellules souches hématopoïétiques
US10336820B2 (en) 2008-02-20 2019-07-02 Amgen Inc. Antibodies directed to angiopoietin-1 and angiopoietin-2 and uses thereof
JP2016518455A (ja) * 2013-05-24 2016-06-23 昆明聖火薬業（集団）有限公司 血小板減少症治療薬の製造におけるデンシチンの使用
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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