WO2001079494A1 - Anticorps agonistes - Google Patents

Anticorps agonistes Download PDF

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Publication number
WO2001079494A1
WO2001079494A1 PCT/JP2001/003288 JP0103288W WO0179494A1 WO 2001079494 A1 WO2001079494 A1 WO 2001079494A1 JP 0103288 W JP0103288 W JP 0103288W WO 0179494 A1 WO0179494 A1 WO 0179494A1
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WIPO (PCT)
Prior art keywords
chain
receptor
antibody
region
cells
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PCT/JP2001/003288
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English (en)
Japanese (ja)
Inventor
Naoshi Fukushima
Masayuki Tsuchiya
Masayoshi Oheda
Shinsuke Uno
Yasufumi Kikuchi
Toshihiko Ohtomo
Original Assignee
Chugai Seiyaku Kabushiki Kaisha
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Priority claimed from PCT/JP2001/001912 external-priority patent/WO2001066737A1/fr
Application filed by Chugai Seiyaku Kabushiki Kaisha filed Critical Chugai Seiyaku Kabushiki Kaisha
Priority to AU2001246934A priority Critical patent/AU2001246934A1/en
Priority to US10/257,864 priority patent/US20040058393A1/en
Priority to RU2006120454/10A priority patent/RU2408606C2/ru
Priority to CA002424371A priority patent/CA2424371A1/fr
Priority to AU2002210918A priority patent/AU2002210918B2/en
Priority to KR1020037004605A priority patent/KR100870123B1/ko
Priority to PCT/JP2001/009260 priority patent/WO2002033073A1/fr
Priority to EP01978851A priority patent/EP1327680B1/fr
Priority to AU2002210917A priority patent/AU2002210917B2/en
Priority to KR10-2003-7004608A priority patent/KR20030055274A/ko
Priority to US10/399,585 priority patent/US20040242847A1/en
Priority to CN200410085664.9A priority patent/CN1721445B/zh
Priority to AU1091802A priority patent/AU1091802A/xx
Priority to PCT/JP2001/009259 priority patent/WO2002033072A1/fr
Priority to CNB018175449A priority patent/CN1308448C/zh
Priority to JP2002536442A priority patent/JP4261907B2/ja
Priority to CNB018174310A priority patent/CN1308447C/zh
Priority to EP10173971A priority patent/EP2351838A1/fr
Priority to AT01978851T priority patent/ATE391174T1/de
Priority to AU1091702A priority patent/AU1091702A/xx
Priority to RU2006120419/10A priority patent/RU2422528C2/ru
Priority to JP2002536441A priority patent/JPWO2002033072A1/ja
Priority to ES01978851T priority patent/ES2304235T3/es
Priority to EP01978852A priority patent/EP1327681A4/fr
Priority to DE60133479T priority patent/DE60133479T2/de
Priority to CA002424364A priority patent/CA2424364A1/fr
Priority to RU2006127049/10A priority patent/RU2430927C2/ru
Publication of WO2001079494A1 publication Critical patent/WO2001079494A1/fr
Priority to US10/645,085 priority patent/US20040258684A1/en
Priority to HK04103425A priority patent/HK1060372A1/xx
Priority to HK06106076A priority patent/HK1085932A1/xx
Priority to US12/497,131 priority patent/US20090311718A1/en
Priority to US13/225,999 priority patent/US8586039B2/en
Priority to US13/856,119 priority patent/US20130295096A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • 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
    • 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
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a modified antibody comprising two or more H chain V regions and two or more L chain V regions of a monoclonal antibody, which exhibits an agonist effect by crosslinking cell surface molecules.
  • the modified antibody has an agonist action capable of transmitting a signal into cells by cross-linking cell surface molecules, and is useful as various pharmaceuticals.
  • Japanese Patent Application Laid-Open No. 9-1959999 discloses an attempt to develop a monoclonal antibody using the spleen stromal cell line as a sensitizing antigen with the aim of developing a specific antibody capable of identifying spleen stromal cells. It describes the acquisition of a novel monoclonal antibody that recognizes mouse Integrin Associated Protein (mouse IAP).
  • Japanese Patent Application Laid-Open No. 9-299599 discloses that a monoclonal antibody has a property of inducing apoptosis in myeloid cells.
  • WO 99Z12973 describes a human Integrin Associated Protein (hereinafter referred to as human IAP; its amino acid sequence and nucleotide sequence are described in J. Cell Biol., 123, 485-496, 1993; Journal of Cell Science, 108, 3419- 3425, 1995), which is a monoclonal antibody having the property of inducing apoptosis in nucleated blood cells (myeloid cells and lymphocytes) having the human IAP.
  • Two antibodies, the hybridomas that produce it, MABL-1 (FERM BP-6100) and MABL-2 (FERM BP-6101) are described.
  • Japanese Patent Application No. 1-163557 discloses a single-chain FV region which has the property of inducing apoptosis in nucleated blood cells having human IAP from a monoclonal antibody using human IAP as an antigen. Discloses that a single-stranded Fv was obtained.
  • the present inventors have conducted intensive studies using a monoclonal antibody having human IAP as an antigen so as to be used as a therapeutic agent for the above-mentioned hematological disorders.
  • a single FV having a single-chain FV region having the following structure was obtained.
  • modified antibodies particularly low-molecular-weight antibodies, for example, single-chain Fvs
  • single-chain Fv dimers particularly heterodimers
  • monoclonal antibodies capable of cross-linking cell surface molecules and transmitting signals include, for example, an antibody against EPO receptor involved in cell differentiation and proliferation (Japanese Patent Application Laid-Open No. 2000-1985), Antibodies against MuSK receptor (Xie et al., Nature Biotech. 15, 768-771, 1997) are known. However, there are no reports on modified antibodies that have been reduced in molecular weight.
  • the present inventors first focused on the fact that the above-mentioned MABL-1 and MABL-2 antibodies and single-chain FV dimers derived therefrom induce apoptosis in cells having IAP.
  • cross-linking dimer-shading
  • the IAP receptor on the surface
  • a signal is transmitted to the cell, resulting in apoptosis.
  • I was found to have been guided. That is, the monospecific single-chain Fv dimer cross-links molecules (eg, receptors) on the cell surface, thereby transmitting a signal in the same manner as a ligand, and thus can exhibit an agonist action. Suggestive.
  • the present inventor has proposed that the antibody molecule (whoe IgG) can be modified into a single-chain Fv dimer or a single-chain bivalent antibody to reduce side effects such as cross-linking between cells.
  • the present inventors have found that the present invention can provide a novel pharmaceutical agent that can reduce the amount and crosslink a molecule on the cell surface to induce only a desired action on the cell, thereby completing the present invention.
  • the modified antibody of the present invention has a significantly higher activity than the original monoclonal antibody, and has a smaller molecular weight than the antibody molecule and has no constant region. It has the feature that migration is improved. Disclosure of the invention
  • An object of the present invention is to provide two or more H chain V regions and L chain V regions of a monoclonal antibody that can act as an agonist by transmitting a signal into a cell by binding to a cell surface molecule.
  • An object of the present invention is to provide a low-molecular-weight agonist-modified antibody containing two or more antibodies.
  • the present invention provides a monoclonal antibody that exhibits an agonist effect by cross-linking cell surface molecules, two or more H chain V regions and two or more L chain V regions, preferably 2 to 4, respectively. Particularly preferably, it relates to a modified antibody containing two each.
  • the modified antibody of the present invention preferably contains one H chain V region and one L chain V region.
  • the H chain V region and the L chain V region are preferably linked via a linker.
  • the dimer of the single-chain Fv may be a dimer formed by a non-covalent bond, a dimer formed by a covalent bond via a cross-linking group, or a cross-linking agent capable of binding to the single-chain Fv (an antibody, an antibody fragment, or a divalent (Modified antibodies).
  • Bridging group to form a dimer as possible out using a known crosslinking groups used in the crosslinking of peptides, for example, disulfide crosslinking by cysteine residue, other crosslinking radicals such as -C 1 0 alkylene (e.g.
  • a cross-linking agent capable of binding to the single-chain Fv is, for example, an antibody or a fragment thereof to an amino acid sequence, such as a FLAG sequence, which can be optionally introduced into Fv, or a modified antibody derived from the antibody, such as The main chain is Fv.
  • the present invention also provides a substance, which comprises administering a first ligand and a second ligand that bind to a cell surface molecule, further binding to the first and second ligands, and crosslinking the first and second ligands. And a method of inducing agonist action in cells.
  • the first and second ligands may be any as long as they can induce an agonist action by being cross-linked, but are preferably the same or different single-chain FV monomers, antibody fragments, etc. It is a multivalent modified antibody.
  • the substance that cross-links the ligand may be any substance that cross-links the first ligand and the second ligand to induce agonist action on cells, but is preferably an antibody, an antibody fragment, or (F ab) is a 2 or a divalent modified antibodies.
  • examples of the bivalent antibody include (F ab) 2 , a single-chain FV dimer containing one H chain V region and one L chain V region, or two H chains. V region and single-chain polypeptides containing two L chain V regions. This method is effective not only for searching for receptors that transmit signals to cells when cross-linked, but also for application to DDS for drug target molecules. It is useful as a drug administration system that can suppress side effects and exert the efficacy of a drug at a desired time and for a desired time.
  • the modified antibodies of the present invention also include L-chain V regions and H-chain V regions of monoclonal antibodies (eg, MABL-1 antibody, MABL-2 antibody, etc.), and include cell surface molecules, for example, proteins (receptors or signal transduction). Or any protein capable of specifically recognizing the sugar chain of the protein or cell membrane protein and cross-linking the surface molecule, thereby transmitting a signal into cells. Furthermore, a modified antibody obtained by modifying a part of the amino acid sequence of this V region is also included.
  • L-chain V regions and H-chain V regions of monoclonal antibodies eg, MABL-1 antibody, MABL-2 antibody, etc.
  • cell surface molecules for example, proteins (receptors or signal transduction). Or any protein capable of specifically recognizing the sugar chain of the protein or cell membrane protein and cross-linking the surface molecule, thereby transmitting a signal into cells.
  • a modified antibody obtained by modifying a part of the amino acid sequence of this V region is also included.
  • the present invention also relates to humanization of the modified antibody, wherein the humanized modified antibody comprises a humanized H chain V region and a humanized L chain V region.
  • the humanized modified antibody is a humanized L chain V region containing a framework region (F) of a human monoclonal antibody L chain V region and a CDR of a mouse monoclonal antibody L chain V region, or It is composed of humanized H chain V region including FR of H chain V region of human monoclonal antibody and CDR of H chain V region of mouse monoclonal antibody.
  • the amino acid sequences of CDR and FR may be partially modified (for example, deleted, substituted or added).
  • the present invention provides a polypeptide comprising a human monoclonal antibody L chain C region and a mouse monoclonal antibody L chain V region and / or a human monoclonal antibody H chain C region and a mouse monoclonal antibody H chain V region. Is also included.
  • the present invention also relates to a CDR derived from a monoclonal antibody of a mammal other than a mouse (for example, a human, a rat, a mouse, a hidge, a monkey, etc.) corresponding to the mouse CDR, or an H chain V containing the CDR.
  • a CDR derived from a monoclonal antibody of a mammal other than a mouse (for example, a human, a rat, a mouse, a hidge, a monkey, etc.) corresponding to the mouse CDR, or an H chain V containing the CDR.
  • TECHNICAL FIELD The present invention relates to a modified antibody comprising a region and an L chain V region, and capable of acting as an agonist by transmitting a signal inside a cell by binding to a cell surface molecule.
  • Such CDRs, H chain V regions and L chain V regions include, for example, CDRs derived from human monoclonal / reantibodies prepared from transgenic mice and the like, and human monoclonal antibodies containing the CDRs.
  • the H chain V region and the L chain V region are also included.
  • the present invention also provides DNAs encoding the above various modified antibodies, comprising the DNAs.
  • the present invention also relates to a host transformed with the recombinant vector.
  • the host is, for example, an animal cell such as a human cell or a mouse cell, or a microorganism such as Escherichia coli, Bacillus subtilis, or yeast.
  • the present invention also relates to a method for producing a modified antibody, which comprises culturing the above-mentioned host and collecting the modified antibody from the culture.
  • the present invention includes a single-chain FV dimer formed in the medium, wherein the host animal cell producing the single-chain FV is cultured in a serum-free medium to secrete the single-chain FV into the medium.
  • the present invention relates to a method for producing a single-chain FV dimer, which comprises purifying the culture medium supernatant.
  • the invention also relates to the use of the modified antibodies as agonists. That is, the present invention relates to a signaling agonist containing the obtained modified antibody as an active ingredient.
  • the modified antibody is capable of cross-linking a receptor on the cell surface and thereby inducing signal transmission. Therefore, the receptor binds to a ligand to form an oligomer, for example, 2 Any receptor can be used as long as it promotes the multimerization and consequently transmits a signal into cells.
  • Such receptors include, for example, hormone receptors and cytoforce receptors. Hormone receptors include, for example, estrogen receptors.
  • Cytokine receptors include hematopoietic factor receptors, lymphokine receptors, growth factor receptors and differentiation inhibitory factor receptors.
  • site force-in receptors include erythropoietin (EPO) receptor, thrombopoietin (TPO) receptor, granulocyte colony stimulating factor (G-CSF) receptor, and macrophage colony stimulating factor (M-CSF) receptor Body, granulocyte macrophage colony stimulating factor (GM-CSF) receptor, tumor necrosis factor (TNF) receptor, interleukin-1 (IL-1 1) receptor, interleukin-1 2 (IL-2).
  • EPO erythropoietin
  • TPO thrombopoietin
  • G-CSF granulocyte colony stimulating factor
  • M-CSF macrophage colony stimulating factor
  • interleukin-1 3 receptor
  • interleukin-4 IL-4
  • interleukin-5 IL-5
  • interleukin-6 IL-6
  • Receptor interleukin-1 7 (IL-7) receptor
  • interleukin-1 9 IL-19) receptor
  • interleukin-1 10 IL-10) receptor
  • Interleukin-11 IL—11
  • Interleukin-12 IL-12
  • Interleukin-13 IL-13
  • Interleukin-15 IL—15
  • interferon- ⁇ IFN- ⁇
  • interferon-J3 IFN-] 3
  • interferon- ⁇ IFN-y
  • growth hormone GH Receptor
  • insulin receptor blood stem cell growth factor (SCF) receptor
  • vascular epidermal growth factor (VEGF) receptor epithelial cell growth factor (EGF) receptor
  • nerve growth factor NGF
  • FGF fibroblast Growth factor
  • the modified antibody of the present invention contains two or more H chain V regions and two or more L chain V regions derived from a monoclonal antibody.
  • the modified antibody preferably comprises a single-chain FV dimer containing one H chain V region and one L chain V region or a polypeptide containing two H chain V regions and two L chain V regions. It can be.
  • the V regions of the H chain and the L chain are preferably connected via a peptide linker composed of one or more amino acids.
  • These modified antibodies contain the variable region of a monoclonal antibody, preserve the complementarity determining region (CDR), and provide the antigen with the same specificity as the original monoclonal antibody. Join.
  • the H chain V region derived from the monoclonal antibody recognizes a cell surface molecule, for example, a protein (a receptor or a protein involved in signal transduction), or a sugar chain on the protein or the cell membrane, and By cross-linking and oligomerizing the molecule, for example, by dimerization, the H chain V region in a monoclonal antibody capable of transmitting a signal into a cell capable of transmitting a signal inside the cell and acting as an agonist.
  • the present invention also includes H chain V regions derived from mammals (eg, human, mouse, rat, mouse, wedge, sanole, etc.) or H chain V regions obtained by partially modifying the amino acid sequence of the H chain V region.
  • a humanized H chain V region including FR of the H chain V region of a human monoclonal antibody and CDR of the H chain V region of a mouse monoclonal antibody is preferable.
  • an H chain V region derived from a human monoclonal antibody corresponding to the H chain V region of the mouse monoclonal antibody, which can be prepared using recombinant techniques, can also be used.
  • the H chain V region of the present invention also includes a region which is a fragment of the H chain V region and retains antigen binding.
  • the V region of the L chain in the present invention recognizes a cell surface molecule, for example, a protein (a receptor or a protein involved in signal transmission) or a sugar chain on the protein or the cell membrane, and crosslinks the molecule to form an oligomer.
  • mammals eg, human, mouse, rat, mouse, hidge, monkey, etc.
  • the L chain V region obtained by partially modifying the amino acid sequence of the L chain V region is also included in the L chain V region of the present invention.
  • a human ligated L chain V region containing the CDR of the L chain V region of a mouse monoclonal antibody containing the CDR of the L chain V region of a mouse monoclonal antibody.
  • an L chain V region derived from a human monoclonal antibody corresponding to the L chain V region of the mouse monoclonal antibody, which can be prepared using recombinant techniques, can also be used.
  • the L chain V region of the present invention also includes a fragment of the L chain V region that retains antigen binding.
  • CDR Complementarity determining region
  • Each V region of the L and H chains forms the antigen binding site, and the variable regions on the L and H chains share four relatively conserved frameworks and three hypervariable or complementary Linked by decision regions (CDRs) (Kabat, EA et al., "Sequences of Proteins of Immunological Interes s US Dept. Health and Human Services, 1983) 0
  • Most of the four framework regions (FR) are 3-sheet structures, As a result, the three CDRs form a loop, and the CDRs may optionally form part of a] 3-sheet structure.
  • the three CDRs are held sterically very close to each other by the FR, and together with the three CDRs in the paired region contribute to the formation of the antigen-binding site.
  • Single-chain Fv is a monomer of a polypeptide containing a linked H-chain V region and L-chain V region derived from a monoclonal antibody, and the resulting single-chain FV is variable from the original monoclonal antibody. It contains a region and conserves the complementarity-determining region, so it binds to an antigen with the same specificity as the original monoclonal antibody (Japanese Patent Application No. 11-6355 7.). Furthermore, in the single-chain FV of the present invention, a part of the variable region and / or the CDR or a part of the amino acid sequence thereof can be modified (for example, deleted, substituted or added).
  • the H chain V region and L chain V region constituting the single-chain Fv of the present invention are as described above, and the H chain V region and L chain V region are directly or preferably a linker, preferably a peptide linker.
  • the structure may be any of [H chain V region]-[L chain V region], [L chain V region]-[H chain V region].
  • these single-chain FVs form a dimer, trimer or tetramer, and can be used as the modified antibody of the present invention.
  • the single-chain modified antibody of the present invention comprising two or more H chain V regions and two or more L chain V regions, preferably 2 to 4, particularly preferably 2 each, has two or more as described above. It contains H chain V region and L chain V region respectively.
  • each region must be arranged so that the single-chain modified antibody can mimic a specific three-dimensional structure, specifically, a three-dimensional structure formed by a dimer of a single-chain FV.
  • the linker that links the H chain V region and the L chain V region includes any peptide linker that can be introduced by genetic engineering, or a synthetic compound linker, for example, Protein Engineering, 9 (3) , 299-305, 1996. These linkers may be the same or different in the same molecule. If a peptide linker is desired, examples of each linker include:
  • the preferred linker length varies depending on the receptor serving as the antigen, but it is usually preferably 1 to 20 amino acids in a single-chain FV.
  • the [H chain V region] one [L chain V region] (or the [L chain V region] one [H chain V region])
  • the length of the peptide linker for linking is 1 to 30 amino acids, preferably 1 to 20 amino acids, and more preferably 3 to 18 amino acids.
  • a peptide linker for linking [H chain V region]-[L chain V region] (or [L chain V region]-[H chain V region]) that does not form the same antigen binding site Has a length of 1 to 40 amino acids, preferably 3 to 30 amino acids, more preferably 5 to 20 amino acids. The method for introducing these linkers will be described in the description of the method for constructing the DNA encoding the modified antibody of the present invention.
  • the synthetic chemical linker (chemical cross-linking agent) in the present invention is a cross-linking agent commonly used for cross-linking of peptides, for example, N-hydroxysuccinimide (NHS) disuccinimidinoresverate (DSS), bis (sulfo Succinimidinolate) Subrate (BS 3 ), dithiobis (succinimidyl propionate) (DSP), dithiobis (sulfosuccinimidyl propionate) (DT SSP), ethylene glycol bis (Succinimidinoresuccinate) (EGS), Ethyleneglyconorebis (Snorrephosuccinimidyl succinate) (Sulfo-EGS), Disuccinimidyl tartrate (DST), Disulfosuccinimidyl tartrate (Sulfo DST), bis [2- (succinimidoxycarbonyloxy) ethyl] sulfone (B SOCOE S), bis [
  • the single-chain monomer produced in the host cell should be 20% or more, preferably 50 ° / 0 or more, and more preferably 80% or more in a solution such as a medium. It is most preferable to select a linker suitable for dimerization of 90% or more, specifically, 2 to 12 amino acids, more preferably 3 to 10 amino acids, or another linker equivalent thereto. preferable.
  • the modified antibody is obtained by linking the H chain V region and L chain V region derived from a known or novel monoclonal antibody that specifically binds to a cell surface molecule via the above-mentioned linker.
  • single-chain Fv include MAB L-1 antibody, MABL-2 antibody Those having an H chain V region and an L chain V region derived from are referred to as MAB L1-scFv and MABL2-scFv.
  • MAB L 1-sc ( F v) 2 and MAB L 2-sc (F v) 2 As an example of a single-chain polypeptide containing two H-chain V regions and two L-chain V regions.
  • a signal peptide can be added to its N-terminal.
  • a known sequence useful in polypeptide purification such as a FLAG sequence, can be inserted for efficient purification of the polypeptide.
  • a dimer can be formed using an anti-FLAG antibody.
  • a DNA encoding the same that is, a DNA encoding a single-stranded Fv or a DNA encoding a reconstituted single-stranded polypeptide.
  • These DNA for example MAB L 1- sc F v, MAB L 2- sc F v, MAB L 1 - sc (F v) 2 and Z or MAB L 2-sc said in the case of (Fv) 2 F
  • DNA encoding the H-chain V region and L-chain V region derived from v, or using these DNAs as type III the DNA portion encoding the desired amino acid sequence within the sequence is ligated at both ends. It can be obtained by amplification by a PCR method using a defined primer pair.
  • one or several amino acids have been modified by a known method using PCR, that is, one or several amino acids have been deleted, substituted, or substituted.
  • a V region having the added amino acid sequence can be obtained.
  • MABL-1 antibody and MABL-2 antibody MABL-1 antibody has ⁇ -type L chain and ⁇ 1 type ⁇ chain
  • MABL-2 antibody has ⁇ - type L chain and ⁇ 2a type ⁇ chain. It is clear that they have it (Japanese Patent Application No. 11-63557).
  • PCR polymerase chain reaction
  • 5'-terminal oligonucleotide primers and 3'-terminal oligonucleotide primers One is determined as described above.
  • a 5′-end primer and a 3′-end primer are determined for amplification of the H chain V region of the MAB L-1 antibody and the H chain V region of the MAB L-2 antibody, respectively.
  • the 5'-end primer contains the sequence GANTC which provides a restriction enzyme H inf I cleavage site near its 5'-end, and the 3'-end primer has its 5'-end Those containing the nucleotide sequence CCCGGG which provides a restriction enzyme Xma I cleavage site in the vicinity are used.
  • These restriction enzyme cleavage sites may be other restriction enzyme cleavage sites as long as they are used for subcloning a target DNA fragment encoding a variable region into a closing vector.
  • cDNAs encoding the respective V regions of the MABL-1 and MABL-2 antibodies were introduced with appropriate nucleotide sequences at their 5'- and 3'-ends, They were designed to be easily inserted into an expression vector and to function properly in the expression vector. (For example, in the present invention, it was devised to increase the transcription efficiency by introducing Kozak sequence. ing).
  • each V region of MAB L-1 and MAB L-12 antibodies obtained by PCR amplification using these primers was converted to a HEF expression vector (WO 92) already containing the desired human C region. -See 19759). Sequencing of the cloned DNA can be performed by any conventional method, for example, by inserting the DNA into an appropriate vector and using an automatic DNA sequencer (Applied Biosystems).
  • a linker for example, a peptide linker
  • a linker for example, a peptide linker
  • it has a sequence that is partially complementary to the above-described primers for the H chain V region and L chain V region, and that the N-terminal or C-terminal of the linker is
  • a primer so as to be loaded, and performing PCR using the primer
  • a DNA encoding a peptide linker having a desired amino acid sequence and length can be prepared.
  • DNA encoding the modified antibody of the present invention having a desired peptide linker can be obtained. it can.
  • DNA encoding one modified antibody can be obtained, the DNA is converted into a type II, primers for various linkers are designed, and PCR is performed using these primers. DNA encoding a modified antibody having the peptide phosphorus ⁇ "or a modified antibody having no linker can be easily obtained.
  • each chain V region of the modified antibody in the present invention can be converted into a human type by using a conventional technique (for example, see Sato, K. et al., Cancer Res., 53, 1-6 (1993)).
  • a conventional technique for example, see Sato, K. et al., Cancer Res., 53, 1-6 (1993)
  • the humanized single-chain Fv, humanized single-chain Fv fragment, and humanized Monoclonal antibodies or humanized monoclonal antibody fragments can be easily produced according to standard methods.
  • a part of the amino acid sequence of these V regions can be modified.
  • DNAs derived from other mammals corresponding thereto for example, human-derived DNA Of DNA can be obtained.
  • H-chain V region and L-chain V region derived from other mammals especially human, single-chain Fv derived from human and fragments thereof, and monoclonal antibody derived from human and fragments thereof are fragmented. Obtainable.
  • DNAs encoding the V regions of each chain of the modified antibody of interest and the V regions of each chain of the humanized modified antibody are prepared, an expression vector containing them, and an expression vector containing them.
  • the host transformed by the container can be obtained according to a conventional method.
  • the reconstituted single-chain Fv, reconstituted humanized single-chain Fv, humanized monoclonal antibody and humanized monoclonal antibody fragment produced by culturing the host according to a conventional method are intracellular or cellular. It can be separated from the outside and purified to homogeneity. In this case, usually The modified antibody of the present invention can be separated and purified by appropriately selecting and combining various purification methods, for example, various types of chromatography, ultrafiltration, salt filtration, dialysis, and the like. It is not limited.
  • the reconstituted single-chain FV is produced in animal cells, for example, COS 7 cells, cultured animal cells such as CHO cells, preferably CHO cells
  • the single-chain FV dimer can be efficiently formed in the medium.
  • the formed dimer can be stably recovered at a high rate and can be stored in a dimer state for a long period of time.
  • the serum-free medium that can be used in this case may be any medium that is usually used for producing a recombinant protein, and is not particularly limited.
  • any expression system e.g., eukaryotic cells, e.g., animal cells, e.g., established mammalian cell lines, eukaryotic fungal cells, and yeast cells, and prokaryotic cells, e.g., bacterial cells, For example, E. coli cells or the like can be used.
  • the modified antibodies of the invention are expressed in mammalian cells, such as COS 7 cells or CHO cells.
  • any expression system for example, a eukaryotic cell, for example, an animal cell, for example, an established mammalian cell line, or a filamentous fungal cell , And yeast cells, and prokaryotic cells, such as bacterial cells, such as E. coli cells, and the like.
  • a eukaryotic cell for example, an animal cell, for example, an established mammalian cell line, or a filamentous fungal cell , And yeast cells, and prokaryotic cells, such as bacterial cells, such as E. coli cells, and the like.
  • the reconstituted polypeptides of the invention are expressed in mammalian cells, such as COS 7 cells or CHO cells.
  • HCMV human cytomegalovirus
  • expression vectors containing the HCMV promoter ⁇ "include plasmid vectors such as HCMV-VH-HCyl and HCMV-VL-HCK, which are derived from PSV2neo (see WO 92/19759). ) Are included.
  • '' Promoters for gene expression include retroviruses, polioviruses, adenoviruses, simian virus 40 (SV40) and other viral motors, humans, polypeptide chains, and longation '' factor la (HEF-1a ) May be used.
  • SV40 simian virus 40
  • HEF-1a longation '' factor la
  • ori derived from SV40, poliovirus, adenovirus, bovine papilloma virus (BPV) and the like
  • the expression vector can be selected from phosphotransferase A PH ( 3 ') II or I (neo) gene, thymidine kinase (TK) gene, Escherichia coli xanthine-guanine phospholiposyltransferase (E cogpt) gene, dihydrofolate reductase (DHFR) gene and the like.
  • the antigen binding activity of the modified antibody prepared as described above can be evaluated using the binding inhibition ability of the original monoclonal antibody as an index. Specifically, the evaluation is performed using the presence or absence of a concentration-dependent inhibitory effect of the monoclonal antibody on the antigen as an index.
  • animal cells for example, COS 7 cells or CHO cells, which have been transformed with an expression vector containing DNA encoding the modified antibody of the present invention, are cultured, and the cultured cells and / or a culture supernatant thereof, or The binding to the antigen is measured using the modified antibodies purified therefrom.
  • As a control use the culture supernatant of cells transformed with the expression vector alone.
  • An antigen for example, in the case of the MABL-1 antibody and MABL-2 antibody, a human IAP-expressing mouse leukemia cell line L1210 cells, and a test sample such as the modified antibody of the present invention or a control culture supernatant are added. Perform flow cytometry to evaluate antigen binding activity.
  • the in vitro signal transduction-inducing effect (MABL-1 antibody and apoptosis-inducing effect in the case of MABL-2 antibody) can be achieved by applying the above-mentioned modified antibody test sample to antigen-expressing cells or cells transfected with the antigen gene. Signal transmission in the cells. (E.g., whether to induce cell death specifically for the human IAP antigen).
  • the in vivo evaluation test is performed as follows, for example, when the modified antibody recognizes human IAP (for example, a modified antibody derived from the MABL-1 or MABL-2 antibody), as an apoptosis-inducing effect.
  • human IAP for example, a modified antibody derived from the MABL-1 or MABL-2 antibody
  • a model mouse of human myeloma is prepared, and a monoclonal antibody that induces apoptosis in nucleated blood cells having EAP, the modified antibody of the present invention, is intravenously administered to the mouse.
  • the control group receives only PBS.
  • the induction of apoptosis is evaluated as an antitumor effect by the change in the amount of human IgG in mouse serum and the survival time.
  • the modified antibody of the present invention comprises two or more H chain V regions and two or more L chain V regions, preferably 2 to 4, and particularly preferably 2 each.
  • the modified antibody of the present invention achieves remarkable low molecular weight as compared with the antibody molecule (who1eIgG), and thus has excellent transferability to tissues and tumors. It has higher activity than agonist antibody molecules. Therefore, various signals can be transmitted into cells by appropriately selecting the monoclonal antibody that is the source of the modified antibody of the present invention. Therefore, pharmaceutical preparations containing it may be effective in inducing signaling to treat diseases, such as cancer, inflammation, hormonal abnormalities, and leukemia, malignant lymphoma, aplastic anemia, myelodysplastic syndrome and polymorphism It is expected to be used as a therapeutic drug for blood diseases such as hematosis. It is also expected to be used as a contrast agent by RI labeling, and its efficacy can be enhanced by binding to other compounds such as RI compounds and toxins.
  • diseases such as cancer, inflammation, hormonal abnormalities, and leukemia, malignant lymphoma, aplastic anemia, myelodysplastic syndrome and polymorphism
  • Hybridomas producing MAB L-11 and MAB L-2 antibodies against human IAP, MABL-1 and MAB L-12, which are used in the method for producing the modified antibody of the present invention are trade The Institute of Biotechnology, Industrial Technology Research Institute, Ministry of Industry. (Tsukuba, Higashi 1-3-1, Ibaraki Pref.), On September 11, 1997, accession numbers FERM BP-6100 and FERM BP, respectively. — Internationally deposited as 6101. '
  • MRNA from hybridomas MAB L-1 and MAB L-2 was prepared using mRNA Purification Kit (Pharmacia Biotech).
  • a double-stranded cDNA was synthesized from about 1 ⁇ g of mRNA using Marathon cDNA Amplification Kit (manufactured by CLONTECH), and an adapter was ligated.
  • the PCR method was performed using a Thermal Cycler (manufactured by PERKIN ELMER).
  • the primers used in the PCR method are the adapter primer 1 (manufactured by CLONTECH) shown in SEQ ID NO: 1 that hybridizes with the partial sequence of the adapter, and the sequence numbers that hybridize with the mouse L-type L chain C region sequence.
  • PCR solution 50M 1 was prepared by adding 5 ⁇ l of 10X PCR Buffer II, 2 mM MgCl 2 , 0.16 mM dNTPs (dATPdGTP, dCTP, dTTP), 2.5-unit DNA polymerase AmpliTaq Gold (all manufactured by PERKIN ELMER), 0.2 ⁇ ⁇ SEQ ID NO: 1 Containing 0.1 ⁇ g of the double-stranded cDNA derived from MAB L-1 and the adapter primer shown in SEQ ID NO: 2 and the MKC primer shown in SEQ ID NO: 2 in 0.2 / ⁇ at an initial temperature of 94 ° C. For 1 minute at 94 ° C, 1 minute at 60 ° C and 1 minute 20 seconds at 72 ° C. After repeating this temperature cycle 35 times, the reaction mixture was further heated at 72 ° C for 10 minutes.
  • the adapter primer 1 shown in SEQ ID NO: 1 and the MHC- ⁇ 1 (Mouse Heavy Constant) primer shown in SEQ ID NO: 3 were used as primers for PCR. .
  • the amplification of the cDNA was performed in the above-mentioned 1.3 (1) except that the amplification was carried out using the MHC- ⁇ 1 primer of 0.2% instead of the MKC primer of 0.2 / zM. Performed by the same method as described for gene amplification.
  • the adapter primer 1 shown in SEQ ID NO: 1 and the MKC primer shown in SEQ ID NO: 2 were used as primers for PCR.
  • the amplification of cDNA was performed using 0.1 ⁇ g of double-stranded cDNA derived from MAB L-2 instead of 0.1 ⁇ g of double-stranded cDNA derived from MAB L-1.
  • the amplification was performed in the same manner as described in 1.3 (1) above for the amplification of the MAB L-1 L chain V region gene.
  • the adapter primer 1 shown in SEQ ID NO: 1 and the MHC- ⁇ 2a primer shown in SEQ ID NO: 4 were used as primers for PCR.
  • the amplification of cDNA was carried out in 1.3 (3) above, except that amplification was performed using 0.2 / ⁇ ⁇ C-y2a primer instead of 0.2 ⁇ MKC primer. The amplification was performed in the same manner as described for the amplification of the region gene. 1.4 Purification of PCR product
  • the DNA fragment amplified by the PCR method as described above was subjected to QIAquick PCR.
  • the reaction was carried out at 5 ° C. for 3 hours for ligation.
  • the above ligation mixture of 11 was added to 50 ⁇ l of E. coli DH5 competent cells (manufactured by Toyobo), and the cells were incubated on ice for 30 minutes and at 42 ° C for 1 minute. And allowed to stand again on ice for 2 minutes. Then, 100 ⁇ l of SOC medium (GIBCO BRL) was added, and LB containing 100 ⁇ g Zm1 of ampicillin (SIGMA) was added.
  • the Escherichia coli was spread on an agar medium and cultured overnight at 37 ° C to obtain an Escherichia coli transformant.
  • the transformant was cultured overnight at 37 ° C in 3 ml of LB medium containing 50 ⁇ g / ml of ampicillin, and the QIAprep Spin Miniprep Kit
  • pGEM-MlL The thus obtained plasmid containing the gene encoding the mouse kappa L chain V region derived from the hybridoma MAB L-1 was named pGEM-MlL and
  • a plasmid containing the gene encoding the V region was made from the purified DNA fragment and named pGEM—MlH.
  • mouse kappa-type L chain V region derived from the hybridoma MAB L-2 was cloned.
  • a plasmid containing the gene to be loaded was prepared from the purified DNA fragment and named pGEM-M2L.
  • plasmid containing a gene encoding the mouse H chain V region derived from the hybridoma MAB L-2 was prepared from the purified DNA fragment; and named pGEM-M2H.
  • the nucleotide sequence of the cDNA coding region in the above plasmid was determined using an automated DNA sequencer (Applied Biosystem) and ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystem) using the manufacturer. This was performed according to the specified protocol.
  • SEQ ID NO: 5 shows the nucleotide sequence of the gene encoding the L chain V region of the mouse MAB L-1 antibody contained in plasmid p GEM-MIL.
  • the nucleotide sequence of the gene encoding the H chain V region of the mouse MAB L-1 antibody contained in the plasmid pGEM-MlH is shown in SEQ ID NO: 6.
  • SEQ ID NO: 7 shows the nucleotide sequence of the gene encoding the V region.
  • the nucleotide sequence of the gene encoding the H chain V region of the mouse MAB L-2 antibody contained in the plasmid pGEM-M2H is shown in SEQ ID NO: 8.
  • the four framework parts are linked by three hypervariable regions, the complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • the amino acid sequence of the framework is relatively well conserved, while the amino acid sequence of the CDR region has extremely high variability (Kabat, EA et al., “Equences of Proteins of Immunological Interest” US Dept. Health and Human Services). , 1983).
  • the amino acid sequence of the variable region of the mouse monoclonal antibody against human IAP was applied to the database of the amino acid sequence of the antibody prepared by Kabat et al. As shown Were determined.
  • the forward primer MLS (SEQ ID NO: 9) for the light chain V region and the forward primer MHS (SEQ ID NO: 10) for the heavy chain V region encode the beginning of the leader sequence for each V region. It was designed to hybridize to DNA and have a Kozak consensus sequence (J. mol. Biol., 196, 947-950, 1987) and a Hind III. Restriction site.
  • the rear primer MLAS for the L chain V region (SEQ ID NO: 11) and the rear primer MHAS for the H chain V region (SEQ ID NO: 12) hybridize to the DNA sequence encoding the end of the J region. It was designed to have a splice donor sequence and a BamHI restriction enzyme site.
  • PCR solution 100 / zl was added to ⁇ ⁇ of 10XPCR Buffer II, 2 mM Mg
  • the PCR product was purified using the QIAquick PGR Purification Kit (manufactured by QIAGEN), digested with HindIII and BamHI, and the L chain V region was added to the HEF expression vector HEF- ⁇ .
  • the chain V region was cloned into the HEF expression vector HEF-y.
  • the plasmids containing the _DNA fragment having the correct DNA sequence were named HEF-MlL and HEF-MlH, respectively.
  • Modification and cloning of cDNA was performed as described in 4. above, except that pGEM-M2L and pGEM-M2H were amplified to type I DNA instead of pGEM-MlL and pGEM-MlH. Amplification and cloning were performed in the same manner as described in 1. After DNA sequencing, plasmids containing DNA fragments having the correct DNA sequence were named HEF-M2L and HEF-M2H, respectively.
  • the expression vectors were tested in COS 7 cells to observe the transient expression of chimeric MABL-1 and chimeric MABL-2 antibodies.
  • HEF-MlL and HEF-M1H vectors were co-transformed into COS 7 cells by electoporation using a Gene Pulser device (BioRad). . Given each DNA (1 0 ⁇ g), added 0. 8m l of PB S in 1 X 1 0 7 cells 1 to the queue base dot and pulse in a volume of 1. 5 kV, 25 / z F was.
  • the electroporated cells were added to a DMEM culture solution (GIBC BRL) containing 10% V-globulin-free fetal serum. After culturing for 72 hours, the culture supernatant was collected, and cell debris was removed by centrifugation to obtain a recovered culture supernatant.
  • a DMEM culture solution GIBC BRL
  • the chimeric MABL-1 antibody and the chimeric MABL-2 antibody specifically bound to human IAP-expressing L1210 cells, and these chimeric antibodies were converted to the mouse monoclonal antibodies MAB L-1 and MAB. It was revealed that each V region of L-2 had the correct structure (Figs. 1-3).
  • Example 5 Preparation of reconstituted MA B L-1 antibody and reconstituted MA B L-2 antibody single chain Fv (sc Fv) region
  • Reconstituted MAB L-1 antibody single chain FV was prepared as follows.
  • the reconstituted MABL-1 antibody H chain V region, linker region, and reconstituted MABL-1 antibody L chain V region are each amplified by PCR and ligated to form the reconstituted MABL-1 antibody
  • the main chain Fv was prepared. This method is schematically shown in FIG.
  • Six PCR primers (AF) were used for the production of reconstituted MABL-1 antibody single chain FV.
  • Primers A, C and E have a sense sequence
  • primers B, D and F have an antisense sequence.
  • Forward primer VHS for H chain V region (Primer A, SEQ ID NO: 13) was designed to hybridize to DNA encoding the N-terminus of the H chain V region and to have an NcoI restriction enzyme recognition site.
  • the rear primer V HAS for the H chain V region (Primer B, SEQ ID NO: 14) was designed to hybridize to the DNA encoding the C-terminus of the H chain V region and overlap with the linker.
  • the forward primer LS (primer C, SEQ ID NO: 15) for the linker hybridizes to DNA encoding the N-terminus of the linker and overlaps with the DNA encoding the C-terminus of the H chain V region. Designed to.
  • the rear primer LAS (primer D, SEQ ID NO: 16) for the linker is designed to hybridize to the DNA encoding the C-terminus of the linker and to overlap with the DNA encoding the N-terminus of the light chain V region. did.
  • the forward primer VLS for the light chain V region hybridizes to DNA encoding the C-terminus of the linker and overlaps with the DNA encoding the N-terminus of the light chain V region Designed to be.
  • the rear primer VLAS-FLAG (primer F, SEQ ID NO: 18) for the L chain V region hybridizes to DNA encoding the C-terminus of the L chain V region and encodes the FLAG peptide (Hopp TP et al., Bio / Technology, 6, 1204-1210, 1988), designed to have two transcription stop codons and an EcoRI restriction enzyme recognition site.
  • three reactions A_B, CD and EF were performed, and each PCR product was purified.
  • plasmid p GEM—Ml H (see Example 2) encoding the reshaped MAB L-1 antibody H chain V region, G 1 y G 1 y G 1 y G 1 y A DNA sequence encoding a linker region consisting of SerG1yG1yGlyG1ySerG1yG1yGlyGlySer (SEQ ID NO: 19) (Huston, JS et al. USA, 85, 5879-5883, 1988), and plasmid pGEM—Ml encoding the V region of the reshaped MAB L-1 antibody L chain.
  • L (see Example 2) It was used as a type.
  • 50 ⁇ l of the solution of the first PCR step is composed of 51 10 XPCR Buffer II, 2 mM MgCl 2 , 0.16 mM dNTPs, and 2.5 units of DNA polymerase ArapliTaq Gold (all manufactured by PERKIN ELMER) , Containing 0.4 M of each primer and 5 ng of each type I DNA, at an initial temperature of 94 ° C for 9 minutes and then at 94 ° C for 1 minute, at 65 ° C for 1 minute and 72 minutes. Heated in this order for 1 minute and 20 seconds at ° C. After repeating this temperature cycle 35 times, the reaction mixture was further heated at 72 ° C for 7 minutes. -'
  • the 843 bp DNA fragment generated by the second PCR was purified, digested with NcoI and EcoRI, and the obtained DNA fragment was cloned into pSCFVT7 vector.
  • the expression vector: pSCFVT7 contains a pe IB signal sequence (Lei, SP et al., J. Bacteriology, 169, 4379-4383, 1987) suitable for the E. coli periplasmic secretion expression system.
  • the plasmid containing the DNA fragment encoding the correct amino acid sequence of the reshaped MABL-1 antibody single chain Fv was named pscMl (see Figure 5).
  • Reconstituted MAB contained in this plasmid ps cMl SEQ ID NO: 20 shows the nucleotide sequence and amino acid sequence of L-11 antibody single chain Fv.
  • the pscMl vector was modified by PCR to produce a vector that expresses the reconstituted MAB L-1 antibody single-chain FV in mammalian cells.
  • the obtained DNA fragment was introduced into a pCHO1 expression vector.
  • the expression vector p CHO 1 was obtained by deleting the antibody gene from DHFR- ⁇ -rvH-PM1- ( ⁇ ) (see W092 / 19759) by digestion with EcoRI and SmaI, and tl—A vector constructed by ligating BamHI Ad aptor (Takara Shuzo).
  • the primers used for the PCR were the 3 & 1-VHS primers shown in SEQ ID NO: 21 that hybridized to the DNA encoding the N-terminus of the V region of the H chain as the forward primer, and had the SEQ ID NO: 21 recognition site.
  • a FRH1 anti primer shown in SEQ ID NO: 22 ′ that hybridizes to DNA encoding the end of the first framework sequence was used.
  • the PCR product is purified using the QIAquick PCR Purification Kit (manufactured by QIAGEN), digested with Sa1I and MboII, and the DNA encoding the N-terminal reconstituted MAB L-1 antibody single-chain FV A fragment was obtained.
  • the pscMl vector was digested with MboII and EcoRI to obtain a DNA fragment encoding the C-terminal reconstituted MABL-1 antibody single chain Fv. Then, the Sa1I-MboII DNA fragment and the MboII-EcoRI DNA fragment were cloned into the pCHO1-Igs vector.
  • the expression vector pCHO 1-Igs is a mouse IgG1 signal sequence (Nature, 332, 323-327, 1988). This plasmid! )
  • the nucleotide sequence and amino acid sequence of the reconstituted MAB L-1 antibody single chain FV contained in CHOM 1 are shown in SEQ ID NO: 23.
  • a CHO M2 vector for mammalian cell expression containing a DNA fragment encoding the correct amino acid sequence of the reconstituted MAB L-2 antibody single-chain FV was obtained by modifying the pscM2 vector.
  • the nucleotide sequence and the amino acid sequence of the reconstituted MAB L-2 antibody single chain FV contained in this plasmid pCHOM2 are shown in SEQ ID NO: 25.
  • the P CHOM2 vector was transformed into COS 7 cells by electoporation using a Gene Pulser apparatus (BioRad). DNA (10 g) and P
  • the electroporated cells were added to an IMDM culture (GIBC BRL) containing 10% fetal calf serum.
  • the antigen-binding activity of the reshaped MABL-2 antibody single-chain FV was measured using the inhibitory activity of the mouse monoclonal antibody against antigen binding as an index.
  • the anti-FLAG antibody adjusted to 1 ⁇ g / ml was added to each well of a 96-well plate, and incubated at 37 ° C for 2 hours. After washing, 1 ° /. Blocking was performed at BSA-PBS. After incubating and washing at room temperature, the culture supernatant of COS 7 cells transfected with the secreted human IAP antigen-gene (SEQ ID NO: 26) was diluted 2-fold with PBS and added to each well. added.
  • the reconstituted MAB L-2 antibody single-chain FV (MAB L2-sc Fv) was clearly concentration-dependent compared to the control supernatant of pCHOI-introduced COS 7 cell culture supernatant. It inhibited the binding of mouse MAB L-2 antibody to human IAP antigen (Fig. 12). This suggested that the reshaped MAB L-2 antibody single-chain FV had the correct structure of each V region of the mouse monoclonal antibody MAB L-2. 5.7 Apoptosis-inducing effect in vitro
  • FIGS. 13 to 18 The results of analysis by Annexin-V staining are shown in FIGS. 13 to 18, respectively.
  • the dots in the lower left area of the figure indicate live cells
  • the lower right area indicates cells in the early stage of apoptosis
  • the upper right area indicates cells in the late apoptosis.
  • MAB L2-scFv reshaped MABL-2 antibody single-chain Fv
  • FIGS. 13 to 16 induced marked cell death in LI210 cells specifically for the human IAP antigen
  • CCR F-.C EM cells also induced remarkable cell death as compared to the control (FIGS. 17 to 18).
  • the pCHOM2 vector was transformed into CHO cells by electoporation using a Gene Pulser apparatus (BioRad).
  • a mixture of DNA (10 / ig) and 0.7 ml of CHO cells ( ⁇ ⁇ ! ⁇ A cells !! ⁇ ) suspended in PBS is added to the cuvette, and the volume is 1.5 kV and 25 ⁇ F.
  • a pulse was given at.
  • the cells subjected to electoral poration were added to a nucleic acid-free MEM medium (GIBCO BRL) containing 10% fetal bovine serum and cultured.
  • GEBCO BRL nucleic acid-free MEM medium
  • the culture supernatant of the single-chain FV-expressing CHO-producing strain obtained in 5.8 was concentrated approximately 20-fold using a cartridge for artificial dialysis (PAN130 SF, Asahi Medical). The concentrate was stored at 20 ° C and thawed during purification. Purification of single-chain Fv from CHO cell culture supernatant was performed by three types of chromatography: blue-sepharose, hydroxyapatite and gel filtration.
  • the concentrated solution of the culture supernatant was diluted 10-fold with 2 OmM acetic acid buffer ( ⁇ 6.0), and insolubles were removed by centrifugation (10000 rpm x 30 minutes).
  • the supernatant was added to a Blue-sepharose column (20 ml) equilibrated with the same buffer, and the column was washed with the same buffer. After that, the NaC1 concentration in the buffer was adjusted to 0.1, 0.2, The protein was gradually increased to 0.3, 0.5 and 1.0 M, and the protein adsorbed on the column was eluted.
  • the concentrated solution of (1) was diluted 10-fold with 1 OmM phosphate buffer (pH 7.0), and added to a hydroxypropyl apatite column (20 ml, BioRad). After washing the column with 60 ml of 10 mM phosphate buffer (pH 7.0), the concentration of the phosphate buffer was increased linearly to 200 mM, and the protein adsorbed on the column was eluted (FIG. 19). As a result of analyzing each fraction by SDS-PAGE, single-stranded FV was confirmed in fraction A and fraction B.
  • the force was applied to a TSKge1G3000 SWG column (21.5 ⁇ 600 mm) equilibrated with a 2 OmM acetate buffer (pH 6.0) containing NaC1.
  • the chromatogram is shown in FIG.
  • the fractions obtained were analyzed by SDS-PAGE.
  • the major peaks were the single-stranded Fv of interest, and analyzed by gel filtration.
  • the purified single-chain Fv (AI, BI) was analyzed using a 15% -SDS-polyacrylamide gel.
  • Fraction AI and BI were gel filtration using a TSKgel G3000 SW force ram (7.5 x 60 mm) showed that Fraction AI detected only the monomer peak and Fraction BI detected only the dimer peak (See Figure 22).
  • the dimer fraction (fraction BI) was about 4% of the total single-stranded FV. 90% or more of the dimers in one fraction of the dimer were stably maintained at 4 ° C for 1 month or more.
  • the pscM2 vector was modified by PCR. The obtained DNA fragment! It was introduced into SCFVT7 expression vector.
  • the primers used for PCR include the Nde-VHSmO2 primer shown in SEQ ID NO: 27 that hybridizes to DNA encoding the N-terminus of the H chain V region as a forward primer and has an initiation codon and an NdeI restriction enzyme recognition site.
  • a VLAS primer shown in SEQ ID NO: 28 that hybridized to DNA encoding the C-terminus of the L chain V region and had two stop codons and an EcoRI restriction enzyme recognition site was used.
  • the forward primer N de-VHSmO 2 contains 5 point mutations in the portion that hybridizes to the DNA encoding the N-terminus of the V region of the H chain in order to be efficiently expressed in E. coli cells. .
  • the PCR product was purified using the QIAquick PCR Purification Kit (manufactured by QIAGE), digested with NdeI and EcoRI, and the resulting DNA fragment was pSCFVT7-based. It was cloned into Kuta.
  • the expression vector pSCFVT7 was digested with NdeI and EcoRI, and the pe1B signonole sequence was deleted.
  • the plasmid containing the DNA fragment with the correct DNA sequence was named pscM2DEmO2 (see Figure 23).
  • the nucleotide sequence and amino acid sequence of the single-chain Fv derived from the MAB L-2 antibody contained in the plasmid pscM2DEmO2 are shown in SEQ ID NO: 29.
  • the p sc M2 D Em 02 vector was transformed into E. coli BL 21 (DE 3) p Lys S
  • the clones obtained were examined for the expression of the target protein by SDS-PAGE, and clones having high expression levels were selected as strains producing single-chain FV polypeptides derived from the MABL-12 antibody.
  • the culture was centrifuged (10000 Xg, 10 minutes), and the cells collected as a precipitate were added to a 5 OmM Tris-HCl buffer (pH 8) containing 5 mM EDTA, 0.1 M NaCl, and 1% Triton X-100. 0) was added, and the cells were crushed by ultrasonic waves (out put: 4, duty cycle: 70%, 1 minute ⁇ 10 times).
  • This suspension was centrifuged (12000 X g s for 10 minutes), and the inclusion body collected as a precipitate was added to 5 OmM Tris-HCl buffer containing 5 mM EDTA, 0.1 M NaCl, and 4% Triton X-100.
  • the inclusion body containing the target protein is dissolved in 50 mM Tris-HCl buffer (pH 8.0) containing 6 M Urea, 5 mM EDTA and O. IM NaCl, and 4M Urea, 5 mM EDTA, O. IM Sephacryl S-300 (5 ⁇ 90 cm, manufactured by AMERSHAM PHARMACIA) gel equilibrated with 50 mM Tris-HCl buffer (pH 8.0) containing NaCl and 10 mM mercaptoethanol. Per minute to remove associated high molecular weight single stranded Fv.
  • antibody sample _ was added at a final concentration of 3 ⁇ g / 1 to 5 ⁇ 10 4 h IAP / L 1210 cells, and after 2 hours of culture, the anti-FLAG antibody (SIGMA was added at a final concentration of 15 ⁇ g / ml and the cells were further cultured for 22 hours.
  • the CHO cell-derived MABL2-single-chain FV monomer obtained in 5.9 and a mouse IgG antibody as a control were examined. After the culture, Annexin-V staining was performed, and the fluorescence intensity was measured using a FACS can device.
  • FIGS. 25 to 31 The results of analysis by AnneXin-V staining are shown in FIGS. 25 to 31 respectively.
  • the dimer of single-chain FV polypeptide derived from MAB L-2 antibody produced by CH ⁇ cells and Escherichia coli cells induced marked cell death as compared to the control (Fig. 25) (Figs. 26, 27).
  • the apoptosis-inducing effect of the monomer of the single-chain FV polypeptide produced by 3 ⁇ 4, CHO cells and E. coli cells was not observed (FIGS. 28 and 29).
  • the addition of the anti-FLAG antibody caused the monomer of the single-chain FV polypeptide derived from the MABL-2 antibody produced by CHO cells to induce marked cell death (FIG. 31) as compared to the control (FIG. 30).
  • the monomer and dimer of the scFvZCHO polypeptide were prepared using filtration-sterilized PBS (-) to be 0.4 mg / m1 and O.S SmgZml, respectively, and used as administration samples. .
  • the human myeloma mouse model was prepared as follows. KP MM2 cells (published in Japanese Patent Application Laid-Open No. 7-236475) passaged in vivo using SCID mice (Japan Tare) were RPMI 1640 containing 10% fetal serum (GIBC0 BRL). A medium (GIBC0 BRL) was used to prepare 3 ⁇ 10 7 cells / ml.
  • the serum human IgG (M protein) level was increased to about 8500 gZm1
  • the serum human IgG (M protein) level was 0% in the control group.
  • the values were remarkably low as follows, indicating that the sc FV // CHO dimer very strongly suppressed the growth of KPMM2 cells (Fig. 32).
  • the survival time was significantly prolonged in the scFvZCHO dimer administration group as compared with the PBS (-) administration group.
  • the scFvZCHO dimer was shown to have an antitumor effect on a human myeloma mouse model.
  • the antitumor effect of scFvZCHO dimer which is the modified antibody of the present invention, is considered to be based on the apoptosis-inducing action of the modified antibody.
  • the hemagglutination test and the determination method of hemagglutination were carried out in accordance with the immunobiochemical research method of the Sequel Chemistry Laboratory Course (edited by The Biochemical Society of Japan, Tokyo Chemical Dojin).
  • the final antibody concentration is mouse I g G, MAB L-2 antibody: 0.01, 0.1, 1, 10, 100 ⁇ gZm1, single-chain Fv: 0.004, 0.04, 0.4, 4
  • a single-chain FV polypeptide-dimer-produced dimer produced by Escherichia coli at 40, 80 ⁇ g Zm 1 was further dosed at 160 g / m 1.
  • Table 2 shows that, as shown in Table 2 below, the MABL-2 antibody showed hemagglutination at 0.1 / zg / m1 or higher, whereas the single-chain FV polypeptide showed both the monomer and dimer. Hemagglutination was not observed.
  • Hemagglutination test control 0.01 0.1 1 10 100 ( ⁇ / ⁇ )
  • Example 6 scFv / E. coli timer
  • Example 6 Modified antibody sc (Fv) 2 containing two H chain V regions and two L chain V regions and MAB L-2 antibody having peptide linkers of various lengths sc FV
  • p CHOMS MAB L (Including DNA encoding scFV derived from -2 antibody) by the PCR method as shown below, and the obtained DNA fragment was introduced into pCHOM2.
  • Primers used for P CR is, EF 1 primer (SEQ ID NO: 3 0) to High Priestess soybean EF 1 alpha to DNA you code as sense primer using,
  • EF 1 primer SEQ ID NO: 3 0
  • antisense primer a DNA sequence that hybridizes to DNA encoding the C-terminus of the L chain V region and encodes a linker region (SEQ ID NO: 19) and a VL LAS primer having a S a1 I restriction enzyme recognition site ( SEQ ID NO: 3 1) was used.
  • PC R The solution 100 / il, the 10 1 of 10 XPCR Buffer # 1, 1 mM M gC l 2, 0. 2mM dNTP s (dATP, dGTP, dCTP, dTTP) ⁇ 5 units KOD DNA polymerase (all manufactured by Toyobo Co., Ltd. of ), 1 M of each primer. And 100 ng of type I DNA (pCHOM2).
  • the PCR solution was heated in this order for 30 seconds at 94 ° C, 30 seconds at 50 ° C, and 1 minute at 74 ° C. This temperature cycle was repeated 30 times.
  • the PCR product was purified using the QIAquick PCR Purification Kit (QIAGEN), digested with Sail, and the obtained DNA fragment was cloned into pBluescript KS + Vector (Toyobo). After DNA sequencing, the plasmid containing the DNA fragment with the correct DNA sequence was digested with Sa1I, and the resulting DNA fragment was digested with Sa1I! ) It was ligated to CHOM2 using Rapid DNA Ligation Kit (manufactured by BOEHRINGER MANNHEIM). After DNA sequencing, replace the plasmid containing the DNA fragment with the correct DNA sequence! It was named CHOM2 (Fv) 2 (see Figure 34). This plasmid! The nucleotide sequence and the amino acid sequence of the MAB L-2 antibody sc (Fv) 2 region contained in CHOM2 (Fv) 2 are shown in SEQ ID NO: 32.
  • pCHOM2 (Fv) 2 is used as type II for CFHL-F1 (SEQ ID NO: 33) and CFHL- R2 (SEQ ID NO: 34) primer, CFHL- F2 (SEQ ID NO: 35) and CFHL—R1 primer (SEQ ID NO: 036) with KOD polymerase at 94 ° C for 30 seconds, 60 ° C30
  • a PCR reaction in which the reaction was repeated 30 times at 72 ° C for 1 minute for 30 times was performed to prepare a cDNA gene for an H chain containing a leader sequence on the 5 ′ side and an L chain containing a FLAG sequence on the 3 ′ side.
  • H-chain and L-chain cDNAs were mixed as type III, and a PCR reaction was performed using KOD polymerase to repeat the reaction at 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 1 minute five times.
  • F1 and CFHL—R1 primers were added, and the mixture was further reacted for 30 cycles to prepare a linker-free HL-0 type cDNA.
  • LH-type scFv In order to prepare LH-type scFv, first, plasmids pGEM-M2L and pGEM-M2H containing the cDNA of the L chain of MAB L-2 and the H region V region (Japanese Patent Application No. 11-63557) ), T7 (SEQ ID NO: 37) and CF LH-R2 (SEQ ID NO: 38) primers, CFLH-F2 (SEQ ID NO: 39) and CFLH-R1 (SEQ ID NO: 40) Using primers, perform a PCR reaction with KOD polymerase (Toyobo) for 30 times at 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 1 minute 30 times.
  • KOD polymerase Toyobo
  • CDNA genes for the L chain containing the sequence and the H chain containing the FLAG sequence on the 3 ′ side were prepared.
  • the obtained L-chain and H-chain cDNAs were mixed as type ⁇ , and a PCR reaction in which KOD polymerase was repeated 5 times at 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 1 minute was performed, and T7 and T7 were performed.
  • CFLH—R 1 primer was added for a further 30 cycles of reaction. This reaction product is designated as ⁇ , C
  • the linker was obtained by performing a PCR reaction in which the reaction at 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 1 minute was repeated 30 times using FLH-F4 (SEQ ID NO: 41) and CFLH-R1 primer.
  • An LH-0 type cDNA was prepared without it.
  • the LH-0 and HL-0 type cDNAs thus prepared are treated with the restriction enzymes EcoRI and BamHI (Takara Shuzo), and the mammalian expression plasmid INPEP4 that does not contain the XhoI restriction enzyme cleavage site is ligated to Ligation High. (Toyobo), and transformed into Competent E. coli JM109 (Futan Gene). Plasmids were purified from the transformed E. coli using QIAGEN Plasraid Maxi Kit (QIAGEN). Thus, plasmids: CF2LH-0 and pCF2HL-0 were prepared.
  • the HL type was changed to CFHL-X3 (SEQ ID NO: 42) and CFHL- X4 (SEQ ID NO: 43), CFHL-X5 (SEQ ID NO: 44), CFHL-X6 (SEQ ID NO: 45), or CFHL-X7 (SEQ ID NO: 46) as sense and antisense primers in the vector sequence 30 seconds at 94 ° C for 30 seconds with KOD polymerase using the complementary BGH-1 (SEQ ID NO: 47) primer.
  • pCF 2HL- 0, p CF 2HL- 3 N p CF 2HL- 4, p CF 2HL- 5, p CF 2 HL- 6 And pCF2HL-7 were treated with restriction enzymes EcoRI and BamHI (Takara Shuzo), and a fragment of about 800 bp was purified from the gel by agarose gel electrophoresis. The obtained fragment was introduced into the EcoRI and BamHI sites of a mammalian cell expression plasmid: COSl using Ligation High to transform Competent E. coli DH5 ⁇ (Toyobo). The plasmid was purified from the transformed E.
  • CF LH—X3 (SEQ ID NO: 49), CFLH—X4 (SEQ ID NO: 50), CFLH-X5 (SEQ ID NO: 51), CFLH-X6 (sequence SEQ ID NO: 52) or CFLH-X7 (SEQ ID NO: 53) sense and antisense primers to vector sequences; BGH-1 primer with KOD polymerase at 94 ° C for 30 seconds, 60 ° C at 30 ° C A PCR reaction in which a reaction at 72 ° C. for 1 second was repeated 30 times was performed, and the obtained reaction product was treated with restriction enzymes XhoI and BamHI.
  • the obtained fragment was introduced into the Xh0I, BamHI site of pCF2LH-0 using Ligation High to transform Competent E. coli DH5 ⁇ (Toyobo). Plasmid was purified from the transformed E. coli using QIAGEN Plasmid Maxi Kit. Thus, expression plasmids ⁇ 2 «-3, pCF2LH-4, pCF2LH-5, pCF2LH-6 and pCF2LH-7 were prepared.
  • PCF2LH-0, pCF2LH-3, pCF2LH-4, pCF2LH-5, pCF2LH —6 and pCF2LH-7 were treated with restriction enzymes EcoRI and BamHI (Takara Shuzo), and a fragment of about 800 bp was purified from the gel by agarose gel electrophoresis. The obtained fragment was introduced into the EcHIRI and BamHI sites of the mammalian cell expression plasmid pC ⁇ Sl using Ligation High to transform Competent E. coli DH5 ⁇ (Toyobo). Plasmids were purified from the transformed E.
  • COS7 cells For expression of HL type, LH type sc FV and sc (Fv) 2 , transient expression was performed in COS7 cells (JCRB9127, Human Science Foundation). COS 7 cells were prepared in DMEM medium (GIBC0) containing 10% fetal bovine serum (HyClone). (Manufactured by BRL) in a CO 2 constant temperature bath at 37 ° C.
  • CM culture supernatant
  • the cells transfected in the same manner as in (1) above are added to a DMEM (10% FBS) medium, cultured in a 75 cm 3 flask overnight, the culture supernatant is discarded, and the cells are washed with PBS and washed with CHO- S—SFM II medium (GIBCO BRL) was added. After culturing for 72 hours, the culture supernatant was collected, cell debris was removed by centrifugation, and the mixture was further filtered through a 0.22: m Potonore Top filter to obtain CM.
  • MABL 2 COS 7 cell culture supernatant prepared in 6.3 (1) above to measure the binding of sc Fv and sc (Fv) 2 to human Integrin Assosiated Protein (IAP) antigen was used for flow cytometry.
  • IAP Integrin Assosiated Protein
  • the 001001 1-8 amHI fragment of pCF2HL-0, 3 to 7 and pCF2LH-0, 3 to 7 prepared in 1.2 above was transcribed into the CHO cell expression vector pCHO1 Eco
  • the DNA was introduced into the RI and BamHI sites using Ligation High to transform Competent E. coli DH5 ⁇ .
  • the plasmid was purified from the transformed E. coli using the QIAGEN Plasmid Midi Kit (QIAGEN).
  • QIAGEN QIAGEN Plasmid Midi Kit
  • the expression plasmids p CHOM2HL-5 and p CHOM2 (Fv) 2 were digested into a linear form by digestion with a restriction enzyme P vu, and these were electroporated using a Gene Pulser apparatus (BioRad) to CHO cells. I had a transfection. DNA (10 ⁇ g) and 0.75 ml of 1 ⁇ 10 7 cells Zm1 in PBS were added to the cuvette and pulsed at 1.5 kV, 25 F volume. After a recovery period of 10 minutes at room temperature, the cells subjected to electoral poration were added to a nucleic acid-containing ⁇ -MEM medium (GIBCO BRL) containing 10% fetal bovine serum and cultured.
  • GEBCO BRL nucleic acid-containing ⁇ -MEM medium
  • the culture supernatant was removed, rinsed with PBS, and a nucleic acid-free ⁇ -MEM medium (GIBCO BRL) containing 10% fetal bovine serum was added and cultured. After culturing for about 2 weeks, the cells were further cultured in a medium containing methotrexate (manufactured by SIGMA) at a final concentration of 10 nM. Thereafter, the concentration was increased to 50 nM and then to 100 nM, and the culture was continued.
  • a nucleic acid-free ⁇ -MEM medium GIBCO BRL
  • methotrexate manufactured by SIGMA
  • the cells obtained in this way are placed in a roller bottle in a serum-free medium CHO—S—SFM II (GIBCO After culturing with BRL, the culture supernatant was collected, cell debris was removed by centrifugation, and further filtered through a 0.20 ⁇ filter to obtain each CM.
  • SIGMA anti-Flag M2 Affinity gel
  • the obtained fraction was analyzed by SDSZPAGE, and the elution of scFv was confirmed.
  • the sc FV fraction was added with Tween 20 to a final concentration of 0.01%, and concentrated with Centricon-10 (MILLIP0RE). 20 mM acetate buffer containing 1 50 mM Na C l ⁇ Beauty 0.01 0 / oTwe en 20
  • the concentrate was subjected to TSKg e 1 G3000 SW column equilibrated with H6.0 (7. 5 X 600 mm) . At a flow rate of 0.4 mI in, sc FV was detected by absorption at 280 nm.
  • HL-5 was eluted at the dimer position as the main peak, and sc (FV) 2 was eluted at the monomer position.
  • Purification method 2> HL-5 and sc (Fv) 2 were purified in three steps: ion exchange chromatography, hydroxyapatite and gel filtration.
  • ion exchange chromatograms Fi one, the HL- 5 in Q Sepharose fast flo column (Fanoremashia> using sc (F v) 2
  • the SP- sepharose fast flow column, the second step and subsequent HL-5 and S c (Fv 2 ) The same conditions were used in 2 .
  • CM for HL-5 is 2 OmM Tris HCl containing 0.02% Tween 20
  • pH 9.0 the pH was adjusted to 9.0 with 1 M Tris.
  • it is applied to a Q Sepharose fast flow column equilibrated with 2 OmM Tris hydrochloric acid buffer containing 0.02% Tween 20 and H8.5, and NaC1 of 0.1 M to 0.55 M in the same buffer is applied.
  • the polypeptide adsorbed on the column was eluted with a linear concentration gradient.
  • the obtained fraction was analyzed by SDSZPAGE, and the fraction containing HL-5 was collected and subjected to the second step of hydroxyapatite.
  • the C [of sc (FV) 2 was diluted 2-fold with 20 mM acetate buffer containing 0.02% Tween 20, pH 5.5, and then adjusted to pH 5.5 with 1 M acetic acid.
  • 20 mM acetate buffer containing 0.02% Tween 20 was applied to a SP-Sepharose fast flow column equilibrated with H5.5, and the NaCl concentration was increased linearly from 0 to 0.5 M in the same buffer.
  • the polypeptide adsorbed on the column was eluted.
  • the obtained fractions were analyzed by SDS / PAGE, and fractions containing sc (FV) 2 were collected and subjected to a second step of hydroxyapatite.
  • a hydroxyapatite column (BioRad) in which the HL-5 fraction and the sc (FV) 2 fraction obtained in the first step were equilibrated with 10 mM phosphate buffer containing 0.02% Tween 20 and H 7.0, respectively. After washing the column with the same buffer, the phosphate buffer concentration was increased linearly to 0.5 M, and the polypeptide adsorbed on the column was eluted. Each fraction was analyzed by SDS-PAGE, and fractions containing the desired polypeptide were collected.
  • Flow cytometry was performed to measure the binding of the purified dimer of MABL2-scFv ⁇ HL5> and the binding of sc (Fv) 2 to the human Integrin Assosiated Protein (IAP) antigen.
  • a FITC-labeled anti-mouse IgG antibody (BECTON DICKINSON) was added. After the incubation and washing again, the fluorescence intensity was measured using a FAC Scan apparatus (manufactured by BECTON DICKINSON).
  • L1210 cells (hIAP / L1210) transfected with human IAP and human leukemia cell line CCRF-CEM for purified MABL2-scFv / HL5> dimer and sc (Fv) 2
  • MABL2-scFv / HL5> dimer and sc (Fv) 2 The apoptosis-inducing effect on was examined by Ann e Xin-V (B0EHRINGER MANNHEIM) staining.
  • the dimer of MAB L 2 -sc FV ⁇ HL 5> and MAB L 2 -sc (Fv) 2 caused concentration-dependent cell death of both hIAPZL1210 and CCRF-CEM cells. Induced ( Figure 43).
  • the dimer of MABL2-scFv ⁇ HL5> and MABL2-sc (Fv) 2 have an improved apoptosis-inducing effect as compared with the original monoclonal antibody MABL-2. It was shown that. 6. 1 2 Purified sc FV HL-5> dimer and sc (FV hemagglutination test
  • Example 5.15 a dimer of purified scFv ⁇ HL-5> at various concentrations and a blood agglutination test of sc (Fv) 2 were performed.
  • the antitumor effect of scFv ⁇ HL-5> dimer and sc (Fv) 2 produced and purified in Examples 6.8 and 6.9 was tested. Specifically, using the human myeloma mouse model prepared in Example 5.14 (3), the amount of ⁇ ⁇ protein produced by human myeloma cells in mouse serum was quantified by ELISA, and The number of surviving days was recorded. The scFV and HL-5> dimers and the anti-tumor effect of sc (FV) 2 were evaluated based on the change in the amount of M protein in the serum and the survival time.
  • the dose is 0.1, 1 or 10 mg / Mice were dosed to give kg.
  • Serum was collected on day 26 after human myeloma cell transplantation, and the amount of M protein in the serum was measured by ELISA according to Example 5.14.
  • the amount of serum M protein was reduced in a dose-dependent manner (see FIG. 44).
  • the HL- 5 administration group (Fig. 45) and the extension of the sc (F v) 2 administration group (Fig. 46) both a significant survival compared to control (V EHIC 1 e-administered group) was observed.
  • Example 7 D encoding the V region of human monoclonal antibody 12B5 against single-chain Fv-human MPL containing the H chain V region and L chain V region of human antibody 12B5 against human MPL
  • NA was constructed as follows.
  • the gene encoding the human antibody 12B5 heavy chain V region that binds to human MPL is Using the nucleotide sequence of the gene (SEQ ID NO: 55), a leader sequence derived from a human antibody gene (SEQ ID NO: 56) (Eur. J. Immunol. 1996; 26: 63-69) is ligated to its 5 'end. It was designed by letting it go.
  • Four oligonucleotides (12B5VH—1, 12B5VH—2, 12B5VH—3, and 12B5VH—4) were designed so that each designed nucleotide sequence had a 15 bp overlap sequence.
  • 12B5VH-1 (SEQ ID NO: 57) and 1 2B5VH-3 (SEQ ID NO: 59) are sense directions, and 12B5VH-2 (SEQ ID NO: 58) and 12B5VH-4 (SEQ ID NO: 60) was synthesized in the antisense direction.
  • outer primers (12B5VH-S and 12B5VH-A) were added to amplify the full-length gene.
  • 12B5VH-S (sequence number: 61) hybridizes to the 5 'end of the leader sequence with a forward primer, and has a Kozak sequence in ⁇ if it is a HindIII restriction enzyme recognition sequence.
  • 2B5VH-A hybridizes to the base sequence encoding the C-terminus of the H chain V region with a rear primer, and has a splice donor sequence and a BamHI restriction enzyme recognition sequence. Designed.
  • the PCR product was purified using 1.5% low melting point agarose gel (manufactured by Sigma), digested with the restriction enzymes BamHI and HindIII, and cloned into the human H chain expression vector HEF-g ⁇ 1. did.
  • the plasmid containing the D ⁇ fragment with the correct DNA sequence was named HEF-12 ⁇ 5 ⁇ -g ⁇ 1.
  • HEF-12B5H-g71 with restriction enzymes EcoRI and BamHI to prepare a gene encoding 12B5VH
  • a human Fab H chain expression vector pCOS Insertion into -Fd gave pFd-12B5H.
  • the human Fab H chain expression vector contains a DNA (including an intron region existing between the human antibody H chain V region and the gene encoding the constant region) and a gene encoding a part of the human H chain constant region.
  • This is a vector constructed by amplifying SEQ ID NO: 63) using the PCR method and then inserting it into the animal cell expression vector pCOS1.
  • the human H chain constant region is HE F.-.gy 1 type ⁇ , amplifies the gene under the same conditions as above, hybridizes with the sequence at the 5 'end of intron 1 as a forward primer, and G 1 CH 1—S (SEQ ID NO: 64) designed to have Eco RI and BamHI restriction enzyme recognition sequences was used as a rear primer, and the DNA at the 3 ′ end of the human H chain constant region CHI domain was used as a rear primer.
  • G1CH1-A SEQ ID NO: 65
  • designed to have two stop codons, a sequence encoding a part of the hinge region, and a Bg1II restriction enzyme recognition site was used as a rear primer.
  • SEQ ID NO: 66 shows the nucleotide sequence and amino acid sequence of the variable region of reconstituted 12B5 H chain contained in plasmid HEF-12B5H-g ⁇ 1 and pFd-12B5H.
  • the gene encoding the human antibody 12B5 light chain V region that binds to human MPL was obtained using the nucleotide sequence of the gene (SEQ ID NO: 67), and the human antibody gene 3D6 (Nuc. Acid Res. 1990: 18; 4927) was designed by ligating a leader sequence (SEQ ID NO: 68).
  • the designed base sequence was combined with four oligonucleotides (12B5VL-1, 12B5VL-2, 12B5VL-3, 12B5VL-4) so that each had a 15 bp overlap sequence in the same manner as above. Divided and synthesized.
  • 12B5VL—1 (SEQ ID NO: 69) and 12B5VL—3 (SEQ ID NO: 71) are sense sequences
  • 12B5VL—2 (SEQ ID NO: 70) and 12B5VL-4 (SEQ ID NO: : 72) has an antisense sequence.
  • Each synthetic oligonucleotide is assembled by its own complementarity, and then the outer primers (12B5 VL-S and 12B5 VL-A) are added to add the full-length gene. Amplified.
  • 12 B 5 VL-S (SEQ ID NO: 73) hybridizes to the 5 'end of the leader sequence with a forward primer, and has a Kozak sequence if it is a Hind III restriction enzyme recognition sequence.
  • A (SEQ ID NO: 74) was designed to hybridize to the base sequence encoding the C-terminus of the L chain V region with a rear primer, and to have a splice donor sequence and a BamHI restriction
  • HEF-12B5L-g ⁇ The nucleotide sequence and amino acid sequence of the reconstituted 12B5 light chain V region contained in this plasmid HEF-12B5L-g ⁇ are shown in SEQ ID NO: 75.
  • the reconstituted 12B5 antibody single-chain FV has the order of 12B5VH—linker 12B5VL, and has a FLAG sequence at its C-terminus for easy detection and purification (SEQ ID NO: 76). It was designed by adding. Further, the linker one sequence was constructed (G 1 y 4 S er) using linker one sequence consisting of 3 to 15 amino acids, reconstruction 12 B 5-stranded FV (sc 12B 5).
  • the gene encoding the single-chain FV of the 12B5 antibody uses the 12B5 H chain V region, the linker region, and the 12B5 L chain V region by PCR. Amplified using and ligated. This method is schematically shown in FIG. Six PCR primers (AF) were used for the production of reconstituted 12B 5--stranded FV. Primers A, C and E have a sense sequence and primers B, D and F have an antisense sequence.
  • the forward primer 12B5-S (primer A, SEQ ID NO: 77) for the H chain V region is designed to hybridize to the 5 'end of the H chain leader sequence and to have an EcoRI restriction enzyme recognition site. did.
  • Back primer HuV for H chain V region HJ3 (primer B, SEQ ID NO: 78) was designed to hybridize to DNA encoding the C-terminus of the H chain V region.
  • the forward primer RHu J H3 (Primer C, SEQ ID NO: 79) for the linker hybridizes to DNA encoding the N-terminus of the linker and binds to the DNA encoding the C-terminus of the H chain V region. Designed to overlap.
  • the rear primer RHu VK1 (primer D, SEQ ID NO: 80) for the linker hybridizes to the DNA encoding the C-terminus of the linker and overlaps with the DNA encoding the N-terminus of the light chain V region. Designed to.
  • the forward primer Hu VK1.2 (primer E, SEQ ID NO: 81) for the light chain V 'region was designed to hybridize to DNA encoding the N-terminus of the light chain V region.
  • the rear primer 12B5F—A (primer F, SEQ ID NO: 82) for the L chain V region hybridizes to the DNA encoding the C-terminus of the L chain V region and encodes the FLAG peptide (Hopp, TP et al., Bio / Technology, 6, 1204-1210, 1988), designed to have two transcription stop codons and a Not I restriction site.
  • the plasmid HEF-12B5H-g ⁇ 1 encoding the V region of the reconstructed 12B5H chain see Example 7.1
  • GlyG1yG1yGly Ser GlyGlyGlyGlyGlyGlySerG1yGlyGlyGlyGlySer DNA sequence encoding linker region SEQ ID NO: 83
  • Plasmid p SCFVT 7—hM21 human type ONS—M21 antibody
  • the 50 ⁇ l solution of the first PCR stage was prepared using 51 1 OxPCR Gold Buffer II, 1.5 mM MgCl 2 , 0.08 mM dNT Ps, and 5 units of DNA polymerase AmpliTaq Gold (all PERKIN ELMER), containing 100 pmo 1 e of each primer and 100 ng of each type I DNA, at an initial temperature of 94 ° C for 9 minutes and then 94 ° After 30 cycles of 30 seconds at C, 30 seconds at 55 ° C and 1 minute at 72 ° C, the reaction mixture was further heated at 72 ° C for 5 minutes. .
  • the PCR products AB, CD, and EF were assembled in a second PCR.
  • 1 ⁇ l of the first PCR reactant A— ⁇ 1 ⁇ l of the first PCR reactant A— ⁇
  • 0.5 ⁇ l of the? ⁇ 1 reactant 0-0 and 1 ⁇ l of the PCR reactant E—F 1 0 ⁇ 1 l O xP CR Gold Buffer II, 1. 5 mM Mg C 1 2, 0.
  • the DNA fragment generated by the second PCR was 1.5. /. It was purified using a low-melting point agarose gel, digested with EcoRI and NotI, and the obtained DNA fragment was subjected to pCHO1 vector and pCOS1 vector (Japanese Patent Application No. 8-255, 1919). 6) Crawling on.
  • the expression vector pCHO1 was obtained by deleting the antibody gene from DHFR- ⁇ -rvH-PM1-f (see W092 / 197959) by digestion with EcoRI and SmaI. And EcoRI—Notl—BamHIA dator (manufactured by Takara Shuzo).
  • SEQ ID NO: 84 shows the nucleotide sequence and amino acid sequence of reconstituted 12B5 single-stranded FV contained in the present plasmids CHO-scl2B5 and: COS-scl2B5.
  • the 12B5 antibody (IgG, Fab) and the single-chain Fv (polypeptide) derived from the 12B5 antibody were expressed using COS-7 cells or CHO cells.
  • Transient expression using COS-17 cells was performed as follows. That is, the gene was introduced by an electoral-portation method using a Gene Pulser device (manufactured by BioRad).
  • the expression vectors HEF-12B5H-g71 and HEF-12B5L-gK described above were used at 10 / zg each.
  • the cells were washed once with PBS, and a serum-free medium CHO-S-SFMII medium was further added, and the cells were further cultured for 2 days.
  • the culture supernatant was centrifuged to remove cell debris, and then prepared by passing through a 0.22 ⁇ . Filter.
  • p CHO—sc 12 ⁇ 5 expression vector was transferred into C ⁇ cells as follows. Introduced.
  • the expression vector was introduced into CHO cells by an electroporation method using a Gene Pulser apparatus (manufactured by BioRad). Restriction enzyme PV u I digested to queue a mixture of 0. 8m 1 of a straight shape with the DNA (1 0 0 ig) and CHO cells were suspended in PBS (1> ⁇ 1 0 7 cells 7 ml) After adding to the bet and allowing to stand on ice for 10 minutes, a pulse was applied at 1.5 kV and a volume of 25 ⁇ FD. After a 10-minute recovery period at room temperature, the cells treated with Erect Mouth Pole: / Yon were transferred to CHO—S—S FM II (GIBC0 BRL) containing 10% fetal bovine serum.
  • CHO—S—S FM II GIBC0 BRL
  • the cells were cultured. Two days after the culture, the cells were cultured in 5 M methotrexate (SIGMA) and CH OSS FM II (GIBC0 BRL) containing 10% fetal serum. About the obtained clone A clone with a high expression level was selected as a cell line producing 12B 5-single-chain FV. After culturing in a serum-free medium CHO—S—SFM II (GIBCO BRL) containing 10 nM methotrexate (SIGMA), collect the culture supernatant, remove cell debris by centrifugation, and perform culturing. I got Qing.
  • SIGMA methotrexate
  • GIBC0 BRL CH OSS FM II
  • the culture supernatant was added to an anti-FLAG M2 affinity gel (SIGMA) equilibrated with PBS. After washing the column with the same buffer, the protein adsorbed on the column was eluted with a 0.1 M glycine hydrochloride buffer (pH 3.5). Immediately after elution, the eluted fraction was neutralized by adding 1 M Tris-HCl buffer (pH 8.0). The eluted fraction was analyzed by SDS-PAGE, and the fraction in which ⁇ main-chain FV was confirmed was concentrated using Centricon-10 (MILLIPORE).
  • SIGMA anti-FLAG M2 affinity gel
  • the concentrated solution of (1) was applied to a Superdex200 column (10 ⁇ 300 mm, manufactured by AMERSHAM PHARMACIA) equilibrated with PBS containing 0.01% Tween20.
  • fraction A had an apparent molecular weight of approximately 44 kD. In fraction B, it was eluted at 22 kD (see FIGS. 50 a and b). From the above results, fraction A is a non-covalent dimer of sc12B5-single-chain FV, and B is a monomer. 7.6 Measurement of TP ⁇ -like agonist activity of various single-chain Fvs
  • the TPO-like activity of the anti-MPL-chain antibody was evaluated by measuring the proliferation activity on Ba / F3 cells (BaF / rapl) expressing human TPO receptor (MPL).
  • Ba FZMp1 cells were washed twice with RPMI1640 medium (GIBC0) containing 10% fetal calf serum (HyClone), and the cell density of 5 ⁇ 105 cells 1 was suspended in the medium.
  • Anti-MPL This antibody or human TPO (manufactured by R & D Systems) is appropriately diluted with the medium, and 50 ⁇ l of the cell suspension is added with antibody or human ⁇ P p_ diluent 501 and added to the suspension.
  • the agonist activity against MPL was measured.
  • the antigen-binding site was bivalent.
  • B5IgG showed a concentration-dependent increase in absorbance, indicating TPO-like agonist activity (ED50; 29 nM), whereas the antigen-binding site was monovalent
  • the agonist activity of '12B5Fab was very weak (ED50; 34, 724 nM).
  • sc12B5 single-chain Fv having a monovalent antigen-binding site as in the case of Fab, a strong agonist activity with an ED50 value of 75 nM was observed.
  • each variable region dissociates in solution and intervenes with the variable region of another molecule to form a dimer. It is known to form multimers such as bodies.
  • the molecular weight of purified sc12B5 was measured using gel filtration. As a result, molecules that could be considered as monomers and dimers were confirmed (see Fig. 48). .
  • monomer and die The sc12B5 monomers were isolated (see FIG. 50), and their agonist activity against MPL was measured. As shown in FIGS. 51 and 52, the sc12B5 monomer had an ED50 value of 4438.
  • FIG. 1 Flow cytometry results showing that human IgG1 antibody does not bind to L1210 cells expressing human IAP (hIAP / L1210).
  • FIG. 2 Flow cytometry results showing that the chimeric MAB L-1 antibody specifically binds to L1210 cells expressing human IAP (hIAP / L1210).
  • FIG. 1 Flow cytometry results showing that the chimeric MAB L-2 antibody specifically binds to L1210 cells expressing human IAP (hIAP / L1210). .
  • FIG. 4 is a diagram schematically showing a method for producing a single-stranded Fv according to the present invention.
  • FIG. 6 shows the structure of an example of an expression plasmid used for expressing a DNA encoding the single-stranded FV of the present invention in a mammalian cell.
  • FIG. 7 is a photograph showing the result of the Western plot obtained in Example 5.4. From the left, molecular weight markers (indicating 97.4, 66, 45, 31, 21.5, 14.5 kDa from the top), pCHO1-introduced COS 7 cell culture supernatant, and pCHOM2-introduced cell culture supernatant. Reconstituted in the culture supernatant of p-CHOM2 transfected cells MAB L-2 antibody single chain Fv (Arrow) indicates that it is explicitly included.
  • Figure 8 Flow cytometry results showing that antibodies from the culture supernatant of CHO lZCO S7 cells do not bind to COS1 / L1210 cells as control.
  • FIG. 9 shows the results of flow cytometry showing that the antibody of the culture supernatant of MABL2-scFvZCO S7 cells does not bind to pCOS1 / L1210 cells as a control.
  • FIG. 1_0 is a view showing the results of flow cytometry showing that the antibody of the culture supernatant of pCOS1 / COS7 cells as a control does not bind to hIAP / L1210 cells.
  • FIG. 11 is a diagram showing the results of oral and cytometric analysis showing that the antibody of the culture supernatant of MAB L 2 — sc F VNO COS 7 cells specifically binds to hI APZL 1210 cells.
  • FIG. 12 is a view showing the results of the Compet et Ve ELISA shown in Example 5.6, showing that the antigen-binding activity of the single-chain Fv (MABL2-scFv) of the present invention was controlled. compared to the p CHO 1 / COS 7 cell culture supernatant as a diagram showing as an index the inhibition of antigen-binding of the mouse monoclonal antibody MAB L-2.
  • FIG. 13 shows the results of the apoptosis-inducing effect of Example 5.7, as control; in pCO S1 "L 1210 cells, as control; on CHO 1 / COS 7 cell culture. Shows that the clear antibody does not induce apoptosis.
  • FIG. 14 shows the results of the apoptosis-inducing effect of Example 5.7.
  • COS1 / L1210 cells were cultured on MAB L2-scFv / COS7 cell culture. Shows that the clear antibody does not induce apoptosis.
  • FIG. 15 shows the results of the apoptosis-inducing effect of Example 5.7, which shows that h.I AP ZL1210 cells do not induce apoptosis in pCHO1 / COS7 cell culture supernatant antibody as a control. Show.
  • FIG. 16 is a graph showing the results of the apoptosis-inducing effect of Example 5.7, in which MAB L 2 — sc F v / COS 7 cell culture supernatant antibody was used for hlAP / L 1210 cells. It shows that apoptosis is specifically induced.
  • FIG. 17 shows the results of the apoptosis-inducing effect of Example 5.7.
  • pCH ⁇ 1 / COS7 cell culture supernatant antibody as a control does not induce apoptosis. (Final concentration 50%) 0
  • FIG. 18 This figure shows the results of the apoptosis-inducing effect of Example 5.7.
  • MABL2-scFv / COS7 cell culture supernatant antibody specifically induces apoptosis. (Final concentration 50%).
  • FIG. 3 is a view showing a chromatogram at the time, showing that fractions A and B were obtained as main peaks.
  • Fig. 20 shows the results of purification by gel filtration of fraction A and fraction B obtained in Example 5.9 (2).
  • Fraction A had an apparent molecular weight of about 36 kD
  • fraction B the major peak (AI and BI) eluted at the position of 76 kD.
  • Figure 21 SDS-PAGE analysis of fractions obtained during the purification process of single-chain FV derived from MAB L-2 antibody produced by CHO cells in Example 5.9, all of which have a molecular weight of about 35 kD. Shows that there is only a single band.
  • FIG. 22 shows the results of gel filtration of fractions AI and BI obtained in the purification of single-chain FV derived from MAB L-2 antibody produced by CHO cells. Fraction BI indicates that it consists of dimers.
  • FIG. 23 shows the structure of an example of an expression plasmid that can be used to express the DNA encoding the single-stranded FV of the present invention in Escherichia coli. :
  • FIG. 26 shows the results of the apoptosis-inducing effect of Example 5.13, in which MAB L 2 -scF v dimer produced by CHO cells significantly induced apoptosis in h IAP / L 1 210 cells (Final concentration 3 g / ml).
  • FIG. 27 shows the results of the apoptosis-inducing effect of Example 5.13, in which MAB L2-scF v dimer produced by E. coli cells remarkably induces apoptosis in h IAP / L 1210 cells. (Final concentration 3 g / ml).
  • FIG. 28 is a graph showing the results of the apoptosis-inducing effect of Example 5.13.
  • h IAP / L1210 cells the apoptosis-inducing effect of MAB L 2—sc FV monomer produced by CHO cells
  • Figure 29 shows the results of the apoptosis-inducing effect of Example 5.13.
  • H IAP / L 1210 cells contained MAB L produced by E. coli cells. This indicates that the apoptosis-inducing effect of the 2-scFv monomer is comparable to that of the control (final concentration: 3 g / ml).
  • FIG. 30 shows the results of the apoptosis-inducing effect of Example 5.13.
  • the mouse IgG antibody as a control exhibited apoptosis in h IAP / L1210 cells even when an anti-FLAG antibody was added. Not to induce (final concentration 3 ⁇ gZ ml).
  • FIG. 31 shows the results of the apoptosis-inducing effect of Example 5.13, in which MAB L 2 — sc Fv monomer produced by CHO cells was added to h IAP / L 1210 cells by the addition of anti-FLAG antibody. connexion significantly indicating that induce apoptosis (final concentration 3 ⁇ ⁇ ⁇ 1).
  • - Figure 32 Quantification of the amount of human IgG in the serum of mice transplanted with human myeloma cell line ⁇ 2, showing the results of measuring the amount of human IgG produced by human myeloma in mice Indicating that the scFvZCHO dimer very strongly inhibits the growth of KPMM2 cells.
  • FIG. 33 Survival days of mice after tumor implantation, showing that the survival time is significantly prolonged in the sc Fv / CHO dimer single administration group.
  • FIG. 34 shows the structure of an example of a plasmid that expresses a modified antibody [sc (Fv) 2 ] containing two H chain V regions and two L chain V regions derived from the MAB L-2 antibody.
  • FIG. 35 Shows the structure of an example of a plasmid that expresses scFv (HL type) that does not contain peptide linkers by connecting V regions so that [H chain]-[L chain].
  • FIG. 36 shows the structure of the HL type polypeptide and the amino acid sequence of the peptide linker.
  • Figure 37 Shows the structure of an example of a plasmid that expresses scFv (LH type) that does not contain peptide linkers by connecting V regions so that [L chain]-[H chain].
  • Figure 38 Structure of LH-type polypeptide and amino acid sequence of peptide linker.
  • FIG. 39 shows the results of Western blotting in Example 6.4, in which the modified antibody sc (Fv) 2 containing two H chain V regions and two L chain V regions and peptides of various lengths are shown. This indicates that the MAB L-2 antibody scFv having a linker is expressed.
  • Fig. 40a and b Fig. 40 shows the results of flow cytometry using the COS 7 cell culture supernatant prepared in Example 6.3 (1), showing that MABL 2-- having peptide linkers of various lengths was used.
  • sc FV and sc (Fv) 2 show high affinity for human IAP.
  • FIG. 41 shows the results of the apoptosis-inducing effect of Example 6.6, where scFv ⁇ HL3, 4, 6, 7, LH3, 4, 6, 7> and sc (Fv) 2 indicate hIA. It shows that PZL 1210 cells induce remarkable cell death.
  • FIG. 42 is a diagram showing the results of antigen binding evaluation of Example 6.10, showing that a dimer of sc Fv ⁇ HL 5> and sc (Fv) 2 have high affinity for human IAP. You.
  • FIG. 43 shows the results of the in vitro apoptosis-inducing effect of Example 6.11, wherein the dimer of MAB L 2-sc F v ⁇ HL 5> and the MAB L 2 -sc (F v) 2 3 ⁇ 4h I It shows that both AP / L 1210 and CCRF-CEM cells induce cell death in a concentration-dependent manner.
  • Figure 44 shows the results of measuring the amount of M protein in serum produced by human myeloma in mice transplanted with the human myeloma cell line KPMM2, showing sc F v ⁇ HL-5> and sc (Fv) 2 shows that KPMM2 cell proliferation is very strongly inhibited.
  • Figure 45 Survival days of mice after tumor implantation, showing that the survival time was significantly prolonged in the scFv and HL-5> administration groups.
  • FIG. 46 Survival days of mice after transplantation of fl severe ulcer, showing that the survival time was significantly prolonged in the sc (Fv) 2 administration group.
  • Figure 47 Schematic representation of the construction and structure of a DNA fragment encoding a reconstructed 12B5-single-chain FV containing a 15 amino acid linker sequence.
  • Figure 48 This figure shows the results of gel filtration of each of the 12B5-single-chain FVs obtained in Example 7.5 (1). In sc12B5, two peaks (fraction A , B).
  • FIG 50 In Example 7.5 (2), the results of analysis of each fraction A and B using a Superde X 200 column are shown.
  • Figure 5 1. The measurement results of TPO-like agonist activity of sc12B5 and 12B5 antibodies (IgG, Fab) are shown, showing that 12B5IgG and single-chain FV (sc 12 B 5) shows that it has TPO-like agonist activity in a concentration-dependent manner.
  • FIG 52 The results of measuring the TPO-like agonist activity of sc12B5 monomer and dimer.
  • Single-chain FV having a divalent antigen-binding site (sc12B5 dimer) was converted to monovalent scl2B5. It shows about 400 times stronger agonist activity than that of human TPO.
  • the modified antibody of the present invention has an agonist action capable of transmitting a signal into a cell by cross-linking a molecule on the cell surface, and has a lower molecular weight than an antibody molecule (who 1 e IG). Because of this, it has the feature of being superior to transfer to tissues and tumors. Furthermore, the modified antibody of the present invention has significantly higher activity than the original monoclonal antibody, which is a form in which the modified antibody of the present invention is closer to the ligand than the antibody molecule. It is thought that it is.
  • the modified antibody can be used as a signal transduction agent, and by making the antibody molecule the modified antibody of the present invention, side effects such as cross-linking between cells are reduced, and molecules on the cell surface are cross-linked.
  • Pharmaceutical preparations containing the modified antibody of the present invention as an active ingredient include cancer, inflammation, hormonal abnormalities, and prevention of blood diseases such as leukemia, malignant lymphoma, aplastic anemia, myelodysplastic syndrome, and polycythemia vera and z or Useful as a therapeutic.

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Abstract

L'invention concerne des anticorps modifiés contenant au moins deux domaines V à chaîne H et au moins deux domaines V à chaîne L d'un anticorps monoclonal qui peut transmettre un signal dans des cellules par réticulation d'une molécule de surface cellulaire, agissant ainsi en tant qu'agoniste. Etant donné que ces anticorps modifiés peuvent être utilisés en tant qu'agonistes dans la transmission de signal, on peut les employer, par exemple en tant que médicament, pour prévenir et/ou traiter diverses maladies telles que le cancer, l'inflammation, les dérèglements hormonaux et les maladies du sang.
PCT/JP2001/003288 2000-04-17 2001-04-17 Anticorps agonistes WO2001079494A1 (fr)

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AU2001246934A AU2001246934A1 (en) 2000-04-17 2001-04-17 Agonist antibodies
US10/257,864 US20040058393A1 (en) 2000-04-17 2001-04-17 Agonist antibodies
RU2006127049/10A RU2430927C2 (ru) 2000-10-20 2001-10-22 Агонистическое соединение, способное специфически узнавать и поперечно сшивать молекулу клеточной поверхности или внутриклеточную молекулу
CNB018174310A CN1308447C (zh) 2000-10-20 2001-10-22 低分子化的激动剂抗体
EP10173971A EP2351838A1 (fr) 2000-10-20 2001-10-22 Anticorps agonistiques qui réticuler
AU2002210918A AU2002210918B2 (en) 2000-10-20 2001-10-22 Degraded agonist antibody
KR1020037004605A KR100870123B1 (ko) 2000-10-20 2001-10-22 저분자화 아고니스트 항체
PCT/JP2001/009260 WO2002033073A1 (fr) 2000-10-20 2001-10-22 Anticorps agoniste degrade
EP01978851A EP1327680B1 (fr) 2000-10-20 2001-10-22 Anticorps modifie, agoniste de tpo
AU2002210917A AU2002210917B2 (en) 2000-10-20 2001-10-22 Degraded TPO agonist antibody
KR10-2003-7004608A KR20030055274A (ko) 2000-10-20 2001-10-22 저분자화 트롬보포에틴 아고니스트 항체
US10/399,585 US20040242847A1 (en) 2000-10-20 2001-10-22 Degraded agonist antibody
CN200410085664.9A CN1721445B (zh) 2000-10-20 2001-10-22 低分子化的tpo激动剂抗体
AU1091802A AU1091802A (en) 2000-10-20 2001-10-22 Degraded agonist antibody
PCT/JP2001/009259 WO2002033072A1 (fr) 2000-10-20 2001-10-22 Anticorps degrade, agoniste de tpo
CNB018175449A CN1308448C (zh) 2000-10-20 2001-10-22 低分子化的tpo激动剂抗体
JP2002536442A JP4261907B2 (ja) 2000-10-20 2001-10-22 低分子化アゴニスト抗体
RU2006120454/10A RU2408606C2 (ru) 2000-10-20 2001-10-22 Соединение - агонист тро
CA002424371A CA2424371A1 (fr) 2000-10-20 2001-10-22 Fragments d'anticorps monoclonal agoniste
AT01978851T ATE391174T1 (de) 2000-10-20 2001-10-22 Modifizierter tpo-agonisten antikörper
AU1091702A AU1091702A (en) 2000-10-20 2001-10-22 Degraded tpo agonist antibody
RU2006120419/10A RU2422528C2 (ru) 2000-10-20 2001-10-22 Днк, кодирующая модифицированное антитело или соединение с активностью агониста тро, способ их получения и животная клетка или микроорганизм, их продуцирующие
JP2002536441A JPWO2002033072A1 (ja) 2000-10-20 2001-10-22 低分子化ｔｐｏアゴニスト抗体
ES01978851T ES2304235T3 (es) 2000-10-20 2001-10-22 Anticuerpo agonista de tpo modificado.
EP01978852A EP1327681A4 (fr) 2000-10-20 2001-10-22 Anticorps agoniste degrade
DE60133479T DE60133479T2 (de) 2000-10-20 2001-10-22 Modifizierter tpo-agonisten antikörper
CA002424364A CA2424364A1 (fr) 2000-10-20 2001-10-22 Anticorps degrade, agoniste de tpo
US10/645,085 US20040258684A1 (en) 2000-04-17 2002-10-07 Agonist antibodies
HK04103425A HK1060372A1 (en) 2000-10-20 2004-05-14 Degraded agonist antibody
HK06106076A HK1085932A1 (en) 2000-10-20 2006-05-26 Degraded agonist antibody
US12/497,131 US20090311718A1 (en) 2000-10-20 2009-07-02 Degraded agonist antibody
US13/225,999 US8586039B2 (en) 2000-10-20 2011-09-06 Degraded TPO agonist antibody
US13/856,119 US20130295096A1 (en) 2000-10-20 2013-04-03 Degraded agonist antibody

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PCT/JP2001/001912 WO2001066737A1 (fr) 2000-03-10 2001-03-12 Polypeptide provoquant l'apoptose
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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003091424A1 (fr) 2002-04-26 2003-11-06 Chugai Seiyaku Kabushiki Kaisha Procede de criblage d'un anticorps agoniste
WO2005056798A1 (fr) * 2003-12-12 2005-06-23 Chugai Seiyaku Kabushiki Kaisha Procede permettant de renforcer l'activite d'un anticorps
WO2005056602A1 (fr) * 2003-12-12 2005-06-23 Chugai Seiyaku Kabushiki Kaisha Procede de criblage d'anticorps modifies ayant une activite agoniste
WO2006132341A1 (fr) * 2005-06-10 2006-12-14 Chugai Seiyaku Kabushiki Kaisha MUTANT DIRIGE SUR LE SITE sc(Fv)2
US7531643B2 (en) * 1997-09-11 2009-05-12 Chugai Seiyaku Kabushiki Kaisha Monoclonal antibody inducing apoptosis
US7691588B2 (en) 2003-03-13 2010-04-06 Chugai Seiyaku Kabushiki Kaisha Ligand having agonistic activity to mutated receptor
US7696325B2 (en) 1999-03-10 2010-04-13 Chugai Seiyaku Kabushiki Kaisha Polypeptide inducing apoptosis
US8034903B2 (en) 2000-10-20 2011-10-11 Chugai Seiyaku Kabushiki Kaisha Degraded TPO agonist antibody
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
US9241994B2 (en) 2005-06-10 2016-01-26 Chugai Seiyaku Kabushiki Kaisha Pharmaceutical compositions containing sc(Fv)2
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
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

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4946778A (en) * 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
WO1999012973A1 (fr) * 1997-09-11 1999-03-18 Chugai Seiyaku Kabushiki Kaisha Anticorps monoclonal induisant l'apoptose
WO2000053634A1 (fr) * 1999-03-10 2000-09-14 Chugai Seiyaku Kabushiki Kaisha Fv monocatenaire induisant l'apoptose

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4946778A (en) * 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
WO1999012973A1 (fr) * 1997-09-11 1999-03-18 Chugai Seiyaku Kabushiki Kaisha Anticorps monoclonal induisant l'apoptose
WO2000053634A1 (fr) * 1999-03-10 2000-09-14 Chugai Seiyaku Kabushiki Kaisha Fv monocatenaire induisant l'apoptose

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
BAZZONI F. ET AL.: "Chimeric tumor necrosis factor receptors with constitutive signaling activity", PROC. NATL. ACAD. SCI. USA, vol. 92, no. 12, 6 June 1995 (1995-06-06), pages 5376 - 5380, XP002940661 *
GRELL M. ET AL.: "TR60 and TR80 tumor necrosis factor (TNF)-receptors can independently mediate cytolysis", LYMPHOKINE AND CYTOKINE RESEARCH, vol. 12, no. 3, June 1993 (1993-06-01), pages 143 - 148, XP002940662 *
MATEO V. ET AL.: "CD47 ligation induces caspase-independent cell death in chronic lymphocytic leukemia", NATURE MEDICINE, vol. 5, no. 11, November 1999 (1999-11-01), pages 1277 - 1284, XP002940658 *
O'BRIEN RICHARD M. ET AL.: "Monoclonal antibodies for the human insulin receptor stimulate intrinsic receptor-kinase activity", BIOCHEM. SOC. TRANS., vol. 14, no. 6, 1986, pages 1021 - 1023, XP002940663 *
PETTERSEN R.D. ET AL.: "CD47 signals T cell death", J. IMMUNOL., vol. 162, no. 12, 15 June 1999 (1999-06-15), pages 7031 - 7040, XP002940659 *
ROZSNYAY Z. ET AL.: "Phenylarsine oxide (PAO) blocks antigen receptor-induced calcium response and tyrosine phosphorylation of a distinct group of proteins", IMMUNOLOGY LETT., vol. 37, no. 2-3, August 1993 (1993-08-01), pages 197 - 205, XP002940660 *
YANABU M. ET AL.: "Tyrosine phosphorylation and p72syk activation by an anti-glycoprotein Ib monoclonal antibody", BLOOD, vol. 89, no. 5, 1997, pages 1590 - 1598, XP002940665 *
YARDEN Y., SCHLESSINGER J.: "Self-phosphorylation of epidermal growth factor receptor: evidence for a model of intermolecular allosteric activation", BIOCHEMISTRY, vol. 26, no. 5, 1987, pages 1434 - 1442, XP002940664 *

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US7696325B2 (en) 1999-03-10 2010-04-13 Chugai Seiyaku Kabushiki Kaisha Polypeptide inducing apoptosis
US8586039B2 (en) 2000-10-20 2013-11-19 Chugai Seiyaku Kabushiki Kaisha Degraded TPO agonist antibody
US8034903B2 (en) 2000-10-20 2011-10-11 Chugai Seiyaku Kabushiki Kaisha Degraded TPO agonist antibody
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US7691588B2 (en) 2003-03-13 2010-04-06 Chugai Seiyaku Kabushiki Kaisha Ligand having agonistic activity to mutated receptor
US8597911B2 (en) 2003-06-11 2013-12-03 Chugai Seiyaku Kabushiki Kaisha Process for producing antibodies
JPWO2005056602A1 (ja) * 2003-12-12 2008-03-06 中外製薬株式会社 アゴニスト活性を有する改変抗体のスクリーニング方法
WO2005056602A1 (fr) * 2003-12-12 2005-06-23 Chugai Seiyaku Kabushiki Kaisha Procede de criblage d'anticorps modifies ayant une activite agoniste
WO2005056798A1 (fr) * 2003-12-12 2005-06-23 Chugai Seiyaku Kabushiki Kaisha Procede permettant de renforcer l'activite d'un anticorps
JP4634305B2 (ja) * 2003-12-12 2011-02-16 中外製薬株式会社 抗体の活性を増強させる方法
JPWO2005056798A1 (ja) * 2003-12-12 2007-12-06 中外製薬株式会社 抗体の活性を増強させる方法
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
US9777066B2 (en) 2005-06-10 2017-10-03 Chugai Seiyaku Kabushiki Kaisha Pharmaceutical compositions containing sc(Fv)2
US9241994B2 (en) 2005-06-10 2016-01-26 Chugai Seiyaku Kabushiki Kaisha Pharmaceutical compositions containing sc(Fv)2
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JP5224580B2 (ja) * 2005-06-10 2013-07-03 中外製薬株式会社 ｓｃ（Ｆｖ）２部位特異的変異体
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