WO2001079494A1 - Agonist antibodies - Google Patents

Agonist antibodies 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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WO
WIPO (PCT)
Prior art keywords
chain
receptor
antibody
region
cells
Prior art date
Application number
PCT/JP2001/003288
Other languages
French (fr)
Japanese (ja)
Inventor
Naoshi Fukushima
Masayuki Tsuchiya
Masayoshi Oheda
Shinsuke Uno
Yasufumi Kikuchi
Toshihiko Ohtomo
Original Assignee
Chugai Seiyaku Kabushiki Kaisha
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority claimed from PCT/JP2001/001912 external-priority patent/WO2001066737A1/en
Application filed by Chugai Seiyaku Kabushiki Kaisha filed Critical Chugai Seiyaku Kabushiki Kaisha
Priority to US10/257,864 priority Critical patent/US20040058393A1/en
Priority to AU2001246934A priority patent/AU2001246934A1/en
Priority to EP01978851A priority patent/EP1327680B1/en
Priority to CA002424364A priority patent/CA2424364A1/en
Priority to RU2006127049/10A priority patent/RU2430927C2/en
Priority to RU2006120454/10A priority patent/RU2408606C2/en
Priority to AU2002210918A priority patent/AU2002210918B2/en
Priority to KR10-2003-7004608A priority patent/KR20030055274A/en
Priority to DE60133479T priority patent/DE60133479T2/en
Priority to ES01978851T priority patent/ES2304235T3/en
Priority to EP10173971A priority patent/EP2351838A1/en
Priority to PCT/JP2001/009259 priority patent/WO2002033072A1/en
Priority to JP2002536442A priority patent/JP4261907B2/en
Priority to AU2002210917A priority patent/AU2002210917B2/en
Priority to PCT/JP2001/009260 priority patent/WO2002033073A1/en
Priority to CA002424371A priority patent/CA2424371A1/en
Priority to JP2002536441A priority patent/JPWO2002033072A1/en
Priority to CN200410085664.9A priority patent/CN1721445B/en
Priority to AU1091802A priority patent/AU1091802A/en
Priority to CNB018175449A priority patent/CN1308448C/en
Priority to CNB018174310A priority patent/CN1308447C/en
Priority to AT01978851T priority patent/ATE391174T1/en
Priority to US10/399,585 priority patent/US20040242847A1/en
Priority to RU2006120419/10A priority patent/RU2422528C2/en
Priority to KR1020037004605A priority patent/KR100870123B1/en
Priority to EP01978852A priority patent/EP1327681A4/en
Priority to AU1091702A priority patent/AU1091702A/en
Publication of WO2001079494A1 publication Critical patent/WO2001079494A1/en
Priority to US10/645,085 priority patent/US20040258684A1/en
Priority to HK04103425A priority patent/HK1060372A1/en
Priority to HK06106076A priority patent/HK1085932A1/en
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.

Abstract

Modified antibodies containing 2 or more H chain V domains and 2 or more L chain V domains of a monoclonal antibody which can transmit a signal into cells by crosslinking a cell surface molecule, thereby serving as an agonist. Because of being usable as agonists for signal transmission, these modified antibodies are useful as, for example, preventives and/or remedies for various diseases such as cancer, inflammation, hormone disorders and blood diseases.

Description

明 細 書  Specification
ァゴニスト抗体 技術分野  Agonist antibody technical field
本発明は、 細胞表面分子を架橋することによりァゴニスト作用を示す、 モノク 口ーナル抗体の H鎖 V領域を 2つ以上及ぴ L鎖 V領域を 2つ以上含む改変抗体に 関する。 当該改変抗体は、 細胞表面分子を架橋することにより細胞内にシグナル を伝 しうるァゴニスト作用を有しており、 種々の医薬として有用である。 背景技術  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. Background art
特開平 9一 29 5999号公報は、 脾臓間質細胞を識別し得る特定の抗体を開 発することを目標として当該脾臓間質細胞株を感作抗原とするモノクローナル抗 体の作製を試み、 抗原としてマウス Integrin Associated Protein (マウス I A P) を認識する新規モノクローナル抗体の取得を記載している。 また、 特開平 9 - 29 599 9号公報は、 モノクローナル抗体が骨髄系細胞にアポトーシスを誘 起する特性を有することを開示している。  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は、 ヒ トの Integrin Associated Protein (以下ヒ ト I APとする ； J. Cell Biol., 123, 485-496, 1993 にアミノ酸配列及び塩基配列 が記載； Journal of Cell Science, 108, 3419-3425, 1995) を抗原とするモノク ローナル抗体であって、 当該ヒト I APを有する有核血液細胞 （骨髄系細胞及び リンパ球） にアポトーシスを誘起させる特性を有するモノクローナル MAB L— 1抗体、 MABL— 2抗体、 これを産生するハイプリ ドーマ、 MABL— 1 (F ERM BP— 6 1 00) 及び MABL— 2 (FERM BP— 6101) を記 载している。  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.
特願平 1 1— 6 3557号は、 ヒ ト I A Pを抗原とするモノクローナル抗体か ら、 ヒ ト I APを有する有核血液細胞にアポトーシスを誘起する特性を有する一 本鎖の F V領域を有する一本鎖 Fvを得たことを開示している。 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.
しかし、 I APを抗原とするモノクローナル抗体の投与は、 I APを有する有 核血液細胞にアポトーシスを誘起するものの、 in vitro で赤血球の凝集作用をも たらす。 これは、 I A Pを抗原とするモノクローナル抗体を多量に生体内に投与 した場合、 赤血球の凝集という弊害が生じる可能性があることを示唆する ので ある。 However, administration of monoclonal antibodies using IAP as an antigen It induces apoptosis in nuclear blood cells, but has the effect of agglutinating red blood cells in vitro. This suggests that when a large amount of a monoclonal antibody using IAP as an antigen is administered to a living body, the adverse effect of red blood cell aggregation may occur.
本発明者らは、 ヒ ト I A Pを抗原とするモノクローナル抗体を用いて、 上記の 血液疾患の治療薬等として利用するべく鋭意研究した結果、 ヒト I A Pを有する 有核血液細胞にアポトーシスを誘起する特性を有する一本鎖の F V領域を有する 一本 F Vを得た。  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.
一方、 改変抗体、 特に低分子化抗体、 例えば一本鎖 F vは、 その低分子化によ り組織、 腫瘍等への移行性を改善し、 遺伝子工学的に調製する目的で開発された ものであるが、 近年、 一本鎖 F vのダイマー、 特にへテロダイマーを細胞同士を 架橋させる目的で使用されている。 これは、 二重特異性 [bispedfic] の改変抗 体であり、 代表的には癌細胞抗原と N K細胞や好中球など宿主細胞抗原を認識す る一本鎖 F Vのへテロダイマーが知られている （Kipriyanov et al. , Int. J. Cancer, 77, 9763-9772, 1998) 0 これらは、 細胞間架橋を誘導させることにより 癌を治療するためのより効率的な改変抗体として、 一本鎖 F Vの構築技術から作 成されたものである。 このため、 抗体およびその断片 （例えば F a b断片など） および二重特異性の改変抗体、 さらには単一特異性である一本鎖 F Vのダイマー でも細胞間の架橋が誘導されると考えられていた。 On the other hand, modified antibodies, particularly low-molecular-weight antibodies, for example, single-chain Fvs, have been developed for the purpose of improving their translocation to tissues, tumors, etc. by reducing their molecular weight and preparing them by genetic engineering. However, recently, single-chain Fv dimers, particularly heterodimers, have been used for the purpose of cross-linking cells. This is a modified bispecific [bispedfic] antibody, typically a single-chain FV heterodimer that recognizes cancer cell antigens and host cell antigens such as NK cells and neutrophils. (Kipriyanov et al., Int. J. Cancer, 77, 9763-9772, 1998) 0 These are single-chain, more efficient modified antibodies for treating cancer by inducing intercellular cross-links. It was created from FV construction technology. For this reason, it is thought that cross-linking between cells is induced even by antibodies and their fragments (for example, Fab fragments) and bispecific modified antibodies, and even dimers of monospecific single-chain FV. Was.
また、 細胞表面分子を架橋してシグナルを伝達しうるモノクローナル抗体とし て、 例えば細胞の分化 ·増殖に関与する E P O受容体に対する抗体 （特開 2 0 0 0— 9 5 8 0 0号公報）、 M u S K受容体に対する抗体 （Xie et al. , Nature Biotech. 15, 768-771, 1997) などが知られている。 しカゝし、 低分子化した改変 抗体については報告はない。  Examples of 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.
そこで、 先ず本発明者は上記 MA B L— 1および MA B L - 2抗体並びにこれ らに由来する一本鎖 F Vのダイマーが I A Pを有する細胞に対してアポトーシス を誘導することに注目し、 これらが細胞表面上の I A P受容体を架橋 （2量体 ィ匕） することにより当該細胞にシグナルが伝達されて、 その結果アポトーシスが 誘導されたことを突き止めた。 即ち、 これは、 単一特異性の一本鎖 F vダイマー が細胞表面上の分子 （例えば受容体) を架橋することにより、 リガンドと同様に シグナルを伝達し、 これによりァゴニスト作用を示し得ること示唆するものであ る。 Therefore, 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. By 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.
次に細胞間の架橋形成に注目したところ、 前記モノク口ーナル抗体は赤血球凝 集を引き起こすが、 前記一本鎖 F Vのダイマーは赤血球凝集を起こさないことを 見出した。 同様の結果は、 一本鎖 2価抗体 （2っの1^鎖¥領域及び2っの 鎖¥ 領域^含む一本鎖ポリペプチド） でも観察された。 即ち、 これはモノクローナル 抗体では細胞間で架橋が形成される可能性があるのに対して、 一本鎖 F vダイマ 一または一本鎖 2価抗体等の改変抗体では、 細胞表面上の分子を架橋するが、 細 胞間の架橋を形成しないことを示唆するものである。 Next, focusing on the formation of cross-links between cells, it was found that the monoclonal antibody causes red blood cell aggregation, but the single-chain FV dimer does not cause red blood cell aggregation. Similar results were also observed for single-chain bivalent antibodies (single-chain polypeptides containing two 1 ^ chain regions and two chain ^ regions ^). That is, this is against the potentially crosslinking between cells is formed by a monoclonal antibody, a modified antibody such as a single chain F v dimer one or single chain bivalent antibodies, the molecules on the cell surface Crosslinks are indicated, but do not form crosslinks between cells.
故に、 本発明者は、 抗体分子 （w h o 1 e I g G) を一本鎖 F vダイマーま たは一本鎖 2価抗体などの改変抗体にすることにより、 細胞間の架橋などによる 副作用を軽減し、 且つ細胞表面上の分子を架橋して、 細胞に所望の作用のみを誘 起しうる新規な医薬品を提供しうることを見出し、 本発明を完成させた。 また、 本発明の改変抗体は元のモノクローナノレ抗体と比較して顕著に高い活·生を有して おり、 さらに抗体分子に比べ分子量が小さく、 定常領域を有しないという特徴か ら、 組織移行性が向上しているという特徴を有している。 発明の開示  Therefore, 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. In addition, 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
本発明の課題は、 本発明は、 細胞表面分子と結合することにより細胞内にシグ ナルを伝達してァゴニストとして作用しうる、 モノクローナル抗体の H鎖 V領域 を 2つ以上及び L鎖 V領域を 2つ以上含む低分子化ァゴニスト改変抗体を提供す ることである。  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.
従って、 本発明は、 細胞表面分子を架橋することによりァゴニス ト作用を示す、 モノクローナル抗体の H鎖 V領域を 2つ以上及び L鎖 V領域を 2つ以上、 好まし くは各々 2〜4、 特に好ましくは各々 2つ含む改変抗体に関する。  Therefore, 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.
本発明の改変抗体は、 好ましくは 1つの H鎖 V領域及び 1つの L鎖 V領域を含 む一本鎖 F vのダイマーであるか、 又は 2つの H鎖 V領域及び 2つの L鎖 V領域 を含む一本鎖ポリペプチドである。 該改変抗体中において、 H鎖 V領域及び L鎖 V領域は、 好ましくはリンカ一を介して連結されている。 The modified antibody of the present invention preferably contains one H chain V region and one L chain V region. A single-chain Fv dimer, or a single-chain polypeptide comprising two H-chain V regions and two L-chain V regions. In the modified antibody, the H chain V region and the L chain V region are preferably linked via a linker.
前記一本鎖 F vのダイマーは、 非共有結合によるダイマー、 架橋基を介した共 有結合によるダイマー、 さらに前記一本鎖 F vと結合しうる架橋剤 （抗体、 抗体 断片、 又は 2価の改変抗体） を介したダイマーが包含される。 ダイマーを形成さ せる架橋基は、 ぺプチドの架橋に用いられている公知の架橋基を用いることがで きる 、 例えばシスティン残基によるジスルフィド架橋、 他の架橋基、 例えば 〜C 1 0アルキレン (例えば、 テトラメチレン、 ペンタメチレン、 へキサメチレン、 ヘプタメチレンおよびオタタメチレンなど） または C 4〜C 1 0ァノレケニレン （cis Ztrans— 3—ブテュレン、 cis/ trans— 2—ペンテ二レン、 cis " trans— 3—ぺ ンテニレンおよび ciS//trans— 3—へキセニレンなど） である。 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. , tetramethylene, pentamethylene, to Kisamechiren and heptamethylene and Otatamechiren) or C 4 -C 1 0 Anorekeniren (cis Ztrans- 3- Buteyuren, cis / trans- 2-pent two Ren, cis "trans- 3- Bae Nteniren And ci S / / trans-3-hexenylene).
また、 一本鎖 F vと結合しうる架橋剤は、 例えば F v中に随意に導入しうるァ ミノ酸配列、 例えば F L A G配列等に対する抗体もしくはその断片、 またはその 抗体由来の改変抗体、 例えば一本鎖 F vである。  Further, 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.
本発明はまた、 細胞表面分子に結合する第 1のリガンドと第 2のリガンドを投 与し、 さらに第 1及び第 2のリガンドに結合して、 前記第 1及び第 2のリガンド を架橋する物質を投与することを特徴とする、 細胞にァゴニスト作用を誘導する 方法に関する。 ここで、 第 1及び第 2のリガンドは架橋されることによりァゴニ スト作用を誘導しうるものであればいかなるものでもよいが、 好ましくは同一又 は異なる一本鎖 F Vモノマー、 抗体断片等の一価の改変抗体である。 また、 前記 リガンドを架橋する物質は、 第 1のリガンドと第 2のリガンドを架橋して細胞に ァゴニスト作用を誘導する物質であればいかなるものでもよいが、 好ましくは抗 体、 抗体断片、 （F a b ) 2又は 2価の改変抗体である。 ここで、 2価の抗体の例と しては、 （F a b ) 2、 1つの H鎖 V領域及び 1つの L鎖 V領域を含む一本鎖 F Vの ダイマーであるか、 又は 2つの H鎖 V領域及ぴ 2つの L鎖 V領域を含む一本鎖ポ リペプチドが挙げられる。 本方法は、 架橋されてシグナルを細胞に伝達する受容 体の探索に有効なだけでなく、 薬剤のターゲット分子への D D Sへの応用も期待 でき、 副作用の抑制や、 所望の時期に所望の時間薬剤の効力を発揮させうる薬剤 投与システムとして有用である。 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. Here, 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. Here, 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.
本発明の改変抗体はまた、 モノクローナル抗体 （例えば、 MABL— 1抗体、 MABL— 2抗体など） の L鎖 V領域及び H鎖 V領域を含み、 細胞表面分子、 例 えば蛋白質 （受容体またはシグナル伝達に関与する蛋白質)、 または前記蛋白質も しくは細胞膜タンパク質の糖鎖を特異的に認識して当該表面分子を架橋し、 これ により細胞内にシグナルを伝達しうるものであればいかなるものでもよく、 さら には、 ·この V領域のアミノ酸配列の一部を改変した改変抗体も包含される  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.
本発明はまた、 前記改変抗体のヒト型化に関するものであり、 ヒ ト型化改変抗 体はヒト型化 H鎖 V領域及ぴ 又はヒト型化 L鎖 V領域を含む。 詳細には、 ヒト 型化改変抗体は、 ヒトモノクローナル抗体 L鎖 V領域のフレームワーク領域 （F ) とマウスモノクローナル抗体の L鎖 V領域の CDRを含むヒト型化 L鎖 V領 域及ぴノ又はヒトモノクローナル抗体 H鎖 V領域の FRとマウスモノクローナル 抗体の H鎖 V領域の CDRを含むヒト型化 H鎖 V領域から構成される。 この場合、 CDRおよび FRのアミノ酸配列を一部改変 （例えば、 欠失、 置換又は付加） し てもよい。  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. Specifically, 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. In this case, the amino acid sequences of CDR and FR may be partially modified (for example, deleted, substituted or added).
さらに本発明は、 ヒ トモノクローナル抗体 L鎖 C領域とマウスモノクローナル 抗体の L鎖 V領域及び/又はヒ トモノクローナル抗体 H鎖 C領域とマウスモノク ローナノレ抗体の H鎖 V領域を含んで成る、 ポリペプチドをも包含する。  Furthermore, 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.
本発明はまた、 上記マウス CD Rに相当する、 マウス以外の哺乳動物 （例えば、 ヒ ト、 ラット、 ゥシ、 ヒッジ、 サルなど） のモノクローナル抗体由来の CDR、 又は当該 CDRを含有する H鎖 V領域及び L鎖 V領域を含んで成る、 細胞表面分 子と結合することにより細胞内にシグナル伝達してァゴニストとして作用しうる 改変抗体に関する。 そのような CDR、 H鎖 V領域及ぴ L鎖 V領域には、 例えば トランスジエニックマウス等から作製されたヒトモノクローナ /レ抗体由来の CD R、 該 CDRを含有するヒ トモノクローナル抗体由来の H鎖 V領域及ぴ L鎖 V領 域も包含される。  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. 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.
本発明はまた、 前記種々の改変抗体をコードする DNA、 該 DNAを含んで成 ； ' る組換えベクターを製造する遺伝子工学的方法に関する。 The present invention also provides DNAs encoding the above various modified antibodies, comprising the DNAs. A genetic engineering method for producing a recombinant vector.
本発明はまた、 該組換えベクターにより形質転換された宿主に関する。 宿主は、 例えばヒト細胞、 マウス細胞などの動物細胞、 又は大腸菌、 枯草菌、 酵母などの 微生物である。  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.
本発明はまた、 上記の宿主を培養し、 培養物から改変抗体を採取することを特 徴とする、 改変抗体の製造方法に関する。  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.
さらに本発明は、 一本鎖 F Vを産生する宿主動物細胞を無血清培地で培養して 該培 中に一本鎖 F Vを分泌させ、 該培地中で形成された一本鎖 Fvのダイマー を含む該培地上清を精製することを特徴とする一本鎖 F Vのダイマーの製造方法 に関する。  Further, 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.
本発明はまた、 改変抗体のァゴニストとしての使用に関する。 即ち、 前記得ら れた改変抗体を有効成分として含有するシグナル伝達ァゴニストに関する。 本発 明において改変抗体は、 細胞表面上の受容体を架橋して、 これによりシグナル伝 達を誘起しうるものであるため、 当該受容体は、 リガンドと結合して、 オリゴマ 一化、 例えば 2量体化が促進され、 その結果シグナルを細胞内に伝達しうる受容 体であればいかなるものもよい。 そのような当該受容体には、 例えばホルモン受 容体やサイト力イン受容体が包含される。 ホルモン受容体には、 例えばエストロ ゲン受容体等が包含される。 サイト力イン受容体等には、 造血因子受容体、 リン ホカイン受容体、 増殖因子受容体および分化抑制因子受容体等が包含される。 サ イト力イン受容体の例としては、 エリスロポエチン （EPO) 受容体、 トロンポ ポェチン （TPO) 受容体、 顆粒球コロニー刺激因子 （G— CSF) 受容体、 マ クロファージコロニー刺激因子 （M— CSF) 受容体、 顆粒球マクロファージコ ロニー刺激因子 （GM—CSF) 受容体、 腫瘍壌死因子 （TNF) 受容体、 イン ターロイキン一 1 ( I L一 1) 受容体、 インターロイキン一 2 (I L— 2).受容 体、 インターロイキン一 3 ( I L- 3) 受容体、 インターロイキン一 4 ( I L- 4) 受容体、 インターロイキン一 5 ( I L- 5) 受容体、 インターロイキン一 6 (I L-6) 受容体、 インターロイキン一 7 (I L-7) 受容体、 インターロイ キン一 9 ( I L一 9) 受容体、 インターロイキン一 10 ( I L— 10) 受容体、 インターロイキン一 1 1 ( I L— 1 1 ) 受容体、 インターロイキン一 1 2 ( I L - 1 2) 受容体、 ィンターロイキン一 1 3 ( I L— 1 3) 受容体、 インターロイ キン一 1 5 ( I L— 1 5) 受容体、 インターフェロン一α ( I FN-α) 受容体、 インターフェロン一J3 ( I FN- ]3) 受容体、 インターフェロン一γ ( I FN- y) 受容体、 成長ホルモン （GH) 受容体、 インスリン受容体、 血液幹細胞増殖 因子 （S C F) 受容体、 血管上皮増殖因子 （VEGF) 受容体、 上皮細胞増殖因 子 （EGF) 受容体、 神経成長因子 （NGF) 受容体、 線維芽細胞増殖因子 （F GF)'受容体、 血小板由来増殖因子 （PDGF) 受容体、 トランスフォーミング 増殖因子一 (TGF-β) 受容体、 白血球遊走阻止因子 （L I F) 受容体、 毛 様体神経栄養因子 （CNTF) 受容体、 オンコスタチン M (O SM) 受容体およ び N o t c hファミリー受容体等を挙げることができる。 故に、 当該ァゴニス ト 改変抗体を有効成分として含有する医薬製剤は、 癌、 炎症、 ホルモン異常および 血液疾患などの治療及び Z又は予防に有用である。 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. In the present invention, 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. Examples of 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). Body, interleukin-1 3 (IL-3) receptor, interleukin-4 (IL-4) receptor, interleukin-5 (IL-5) receptor, 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) receptor, Interleukin-12 (IL-12) receptor, Interleukin-13 (IL-13) receptor, Interleukin-15 ( IL—15) receptor, interferon-α (IFN-α) receptor, interferon-J3 (IFN-] 3) receptor, interferon- γ (IFN-y) receptor, 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) receptor, fibroblast Growth factor (FGF) 'receptor, platelet-derived growth factor (PDGF) receptor, transforming growth factor-1 (TGF-β) receptor, leukocyte migration inhibitory factor (LIF) receptor, ciliary neurotrophic factor ( CNTF) receptor, Oncostatin M (OSM) receptor, Notch family receptor, etc. Can be mentioned. Therefore, a pharmaceutical preparation containing the agonist-modified antibody as an active ingredient is useful for treating and preventing or preventing cancer, inflammation, hormonal abnormalities, and blood diseases.
本発明の改変抗体は、 モノクローナル抗体に由来する H鎖 V領域を 2つ以上及 び L鎖 V領域を 2つ以上含む。 当該改変抗体の構成としては、 好ましくは 1つの H鎖 V領域及び 1つの L鎖 V領域を含む一本鎖 F Vのダイマー又は 2つの H鎖 V 領域及び 2つの L鎖 V領域を含むポリぺプチドとすることができる。 該改変抗体 中において、 H鎖および L鎖の V領域は、 1個以上のアミノ酸からなるペプチド リンカ一を介して連結されているのが好ましい。 とれらの改変抗体は、 モノクロ ーナル抗体の可変領域を含有し、 且つ相補性決定領域 （complementarity determining region；以下 CD Rとする） を保存し、 もとのモノクローナル抗体 と同一の特異性をもって抗原に結合する。  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. In the modified antibody, 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.
H鎖 V領域 H chain V region
本発明において、 モノクローナル抗体に由来する H鎖 V領域には、 細胞表面分 子、 例えば蛋白質 （受容体またはシグナル伝達に関与する蛋白質)、 または前記蛋 白質もしくは細胞膜上の糖鎖を認識し、 且つ前記分子を架橋してオリゴマー化、 例えば 2量体化することにより、 細胞内にシグナルを伝達しうる細胞内にシグナ ルを伝達してァゴニストとして作用しうるモノクローナル抗体中の H鎖 V領域で あって、 哺乳動物 （例えば、 ヒ ト、 マウス、 ラット、 ゥシ、 ヒッジ、 サノレなど） に由来する H鎖 V領域又は前記 H鎖 V領域のアミノ酸配列を一部改変した H鎖 V 領域も本発明における H鎖 V領域に包含されるが、 ヒトモノクローナル抗体 H鎖 V領域の F Rとマウスモノクローナル抗体の H鎖 V領域の C D Rを含むヒト型化 H鎖 V領域が好ましい。 さらに、 組換え技術を使用して作成し得る、 前記マウス モノクローナノレ抗体の H鎖 V領域に相当するヒトモノクローナル抗体由来の H鎖 V領域も用いることができる。 また、 本発明の H鎖 V領域には、 前記 H鎖 V領域 の断^であって、 抗原結合性を保持する領域も包含される。 In the present invention, 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. In addition, 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. Although included in the H chain V region in the present invention, 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. Furthermore, 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.
L鎖 V領域 L chain V region
本発明における L鎖 V領域には、 細胞表面分子、 例えば蛋白質 （受容体または シグナノレ伝達に関与する蛋白質)、 または前記蛋白質もしくは細胞膜上の糖鎖を認 識し、 且つ前記分子を架橋してオリゴマー化、 例えば 2量体化することにより、 細胞内にシグナルを伝達しうるモノクローナル抗体中の L鎖 V領域であって、 哺 轧動物 （例えば、 ヒト、 マウス、 ラット、 ゥシ、 ヒッジ、 サルなど） に由来する L鎖 V領域又は前記 L鎖 V領域のアミノ酸配列を一部改変した L鎖 V領域も本発 明における L鎖 V領域に包含されるが、 ヒトモノクローナル抗体 L鎖 V領域の F Rとマウスモノクローナル抗体の L鎖 V領域の C D Rを含むヒト型ィヒ L鎖 V領域 が好ましい。 さらに、 組換え技術を使用して作成し得る、 前記マウスモノクロ一 ナル抗体の L鎖 V領域に相当するヒトモノクローナル抗体由来の L鎖 V領域も用 いることができる。 また、 本発明の L鎖 V領域には、 前記 L鎖 V領域の断片であ つて、 抗原結合性を保持する領域も包含される。  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. L-chain V region in a monoclonal antibody that can transmit a signal into cells by conversion into a cell, for example, by dimerization, and can be performed in mammals (eg, human, mouse, rat, mouse, hidge, monkey, etc.). ) Or 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. And a human ligated L chain V region containing the CDR of the L chain V region of a mouse monoclonal antibody. Further, 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.
相補性決定領域 （C D R) Complementarity determining region (CDR)
L鎖及び H鎖の各 V領域は抗原結合部位を形成し、 L鎖及び H鎖上の可変領域 は共通性のある比較的保存された 4個のフレームワークと 3個の超可変又は相補 佐決定領域 （C D R ) により連結されている （Kabat, E. A.ら、 「Sequences of Proteins of Immunological Interes s US Dept. Health and Human Services, 1983) 0 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
前記 4個のフレームワーク領域 （F R) の多くの部分は 3—シート構造をとり、 その結果 3個の C D Rはループを形成し、 C D Rは場合により ]3—シート構造の 一部分を形成することもある。 3個の C D Rは F Rによつて相互に立体的に非常 に近い位置に保持され、 そして対をなす領域の 3個の C D Rと共に抗原結合部位 の形成に寄与する。 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.
これらの C D R領域は、 得られた抗体の V領域のアミノ酸配列と既知抗体の V 領域の既知アミノ酸配列とを照合することによって、 Kabat, E. A. ら、 I Sequences of Proteins of Immunological IrrterestJ の経験則から見出すこと ができる。  By comparing the amino acid sequence of the V region of the obtained antibody with the known amino acid sequence of the V region of the known antibody, these CDR regions are found from the rules of thumb of Kabat, EA et al., I Sequences of Proteins of Immunological IrrterestJ. be able to.
一本鎖 F V Single-strand F V
一本鎖 F vは、 モノクローナル抗体に由来する、 連結した H鎖 V領域及ぴ L鎖 V領域を含むポリぺプチドのモノマーであり、 得られる一本鎖 F Vはもとのモノ クローナル抗体の可変領域を含有し、 相補性決定領域を保存するため、 もとのモ ノクローナル抗体と同一の特異性をもって抗原に結合する （特願平 1 1—6 3 5 5 7.号)。 さらに、 本発明の一本鎖 F Vにおいて、 前記可変領域および/または C D Rの一部またはそのアミノ酸配列の一部を改変 （例えば、 欠失、 置換又は付 カロ） することができる。 本発明の一本鎖 F vを構成する H鎖 V領域及び L鎖 V領 域は上述したものであり、 H鎖 V領域と L鎖 V領域を直接又はリンカ一、 好まし くはペプチドリンカ一を介して連結することができ、 その構成としては、 [H鎖 V 領域] ― [ L鎖 V領域]、 [ L鎖 V領域] 一 [H鎖 V領域] のいずれでもよい。 本 発明においては、 これら一本鎖 F Vはダイマー、 トリマー又はテトラマーを形成 させ、 本発明の改変抗体とすることができる。  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]. In the present invention, these single-chain FVs form a dimer, trimer or tetramer, and can be used as the modified antibody of the present invention.
一本鎖改変抗体 Single chain modified antibody
本発明の 2つ以上の H鎖 V領域及び 2つ以上の L鎖 V領域、 好ましくは各々 2 〜 4、 特に好ましくは各々 2つ含む一本鎖改変抗体は、 上述のような 2つ以上の H鎖 V領域と L鎖 V領域をそれぞれ含有する。 このポリペプチドにおいて各領域 は、 該一本鎖改変抗体が特定の立体構造、 具体的には一本鎖 F Vのダイマーが構 成する立体構造を模倣し得るよう配置させる必要があり、 例えば  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. In the polypeptide, 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.
[H鎖 V領域] 一 [L鎖 V領域] - [H鎖 V領域] ― [ L鎖 V領域] 又は [H chain V region] 1 [L chain V region]-[H chain V region]-[L chain V region] Or
[乙鎖 領域] 一 [H鎖 V領域] ― [し鎖 領域] 一 [H鎖 V領域]  [O chain region]-[H chain V region]-[H chain region]-[H chain V region]
の順序で各領域が配置され、 これらの領域はリンカ一を介して連結される。 Are arranged in this order, and these regions are connected via a linker.
リンカー Linker
本発明において、 H鎖 V領域と L鎖 V領域とを連結するリンカ一としては、 遺 伝子工学により導入し得る任意のぺプチドリンカー、 又は合成化合物リンカー、 例えば、 Protein Engineering, 9(3), 299-305, 1996 に開示されるリンカ一を用 いる.ことができる。 これらのリンカ一は同一分子内で同じ又は異なっていてもよ い。 ペプチドリンカ一を所望する場合、 各々のリンカ一の例としては：  In the present invention, 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:
S e r  S e r
G 1 y ■ S e r G 1 y ■ S e r
Figure imgf000011_0001
Figure imgf000011_0001
S e r · G 1 y · G 1 y  S e rG 1 yG 1 y
G l y - G l y - G l y - S e r  G l y-G l y-G l y-S e r
S e r - G l y - G l y - G l y  S e r-G l y-G l y-G l y
G l y - G l y - G l y - G l y - S e r  G l y-G l y-G l y-G l y-S e r
S e r - G l y - G l y - G l y - G l y  S e r-G l y-G l y-G l y-G l y
G l y - G l y - G l y - G l y - G l y - S e r  G l y-G l y-G l y-G l y-G l y-S e r
S e r - G l y - G l y - G l y - G l y - G l y  S e r-G l y-G l y-G l y-G l y-G l y
G】 y ' G ] y ' G】 y ' G l y ' G】 y ' G ;i y ' S e r  G】 y 'G] y' G】 y 'G l y' G] y 'G; i y' S e r
S e r - G l y - G l y - G l y - G l y - G l y - G l y  S e r-G l y-G l y-G l y-G l y-G l y-G l y
(G l y - G l y - G l y - G l y - S e r)n  (Gly-Gly-Gly-Gly-Ser) n
(S e r ' G l y ' G l y ' G l y ' G l y)n  (S e r 'G l y' G l y 'G l y' G l y) n
[nは 1以上の整数である] を挙げることができる。 好ましいリンカ^ iプチド の長さは抗原となる受容体によって異なるが、 一本鎖 F Vにおいては通常 1〜2 0アミノ酸であるのが好ましい。 2つ以上の H鎖 V領域及び 2つ以上の L鎖 V領 域を含む一本鎖改変抗体においては、 [H鎮 V領域] 一 [L鎖 V領域] (又は 〔L 鎖 V領域] 一 [H鎖 V領域]) 力 らなる同一の抗原結合部位を形成するもの同士を 連結するためのペプチドリンカーの長さは 1〜 30アミノ酸、 好ましくは 1〜2 0アミノ酸、 さらに好ましくは 3〜18アミノ酸である。 また、 [H鎖 V領域] ― [L鎖 V領域] (又は [L鎖 V領域] 一 [H鎖 V領域]) からなる同一の抗原結合 部位を形成しないもの同士を連結するためのペプチドリンカーの長さは 1〜40 アミノ酸、 好ましくは 3〜30アミノ酸、 さらに好ましくは 5〜20アミノ酸で ある。 これらのリンカ一を導入する方法は本発明の改変抗体をコードする DNA の構築方法の説明において述べる。 [n is an integer of 1 or more]. 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. In a single-chain modified antibody containing two or more H chain V regions and two or more L chain V regions, the [H chain V region] one [L chain V region] (or the [L chain V region] one [H chain V region]) Those that form the same antigen-binding site 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. Also, 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.
本 明における合成化学物リンカ一 （化学架橋剤） は、 ペプチドの架橋に通常 用いられている架橋剤、 例えば N—ヒドロキシスクシンイミド （NHS) ジスクシンイミジノレスべレート （DS S)、 ビス （スルホスクシンイミジノレ） スべ レート （BS3)、 ジチォビス （スクシンィミジルプロピオネート） （DS P)、 ジ チォビス （スルホスクシンィミジルプロピオネート） （DT S S P)、 エチレング リコールビス (スクシンイミジノレスクシネート） （EGS)、 エチレングリコーノレ ビス （スノレホスクシンィミジルスクシネート） （スルホ一 EGS)、 ジスクシンィ ミジル酒石酸塩 （DST)、 ジスルホスクシンィミジル酒石酸塩 （スルホー D S T)、 ビス [2— (スクシンイミ ドォキシカルボニルォキシ) ェチル] スルホン (B SOCOE S)、 ビス [2— （スルホスクシンイミ ドォキシカルポ二ルォキ シ） ェチル] スルホン （スルホー B S OCOE S) などであり、 これらの架橋剤 は市販されている。 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 [2-—sulfosucci And the like Imi Dokishikarupo two Ruoki Shi) Echiru] sulfone (Suruho BS OCOE S), These crosslinking agents are commercially available.
特に、 一本鎖 F Vのダイマーを形成させる場合、 宿主細胞で産生された一本鎖 モノマーを培地等の溶液中で、 20%以上、 好ましくは 50 °/0以上、 さらに好ま しくは 80%以上、 最も好ましくは 90%以上ダイマー化するのに適したリンカ 一を選択することが好ましく、 具体的には 2〜12アミノ酸、 より好ましくは 3 〜10アミノ酸、 またはこれに相当する他のリンカ一が好ましい。 In particular, when a single-chain FV dimer is formed, 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.
改変抗体の製造 Production of modified antibodies
改変抗体は、 細胞表面分子に特異的に結合する既知または新規なモノクローナ ル抗体由来の H鎖 V領域と L鎖 V領域とを前述のリンカ一を介して連結すること により得られる。 一本鎖 Fvの例として、 MAB L— 1抗体、 MABL— 2抗体 に由来する H鎖 V領域と L鎖 V領域を有するものを MAB L 1— s c F v、 MA B L 2- s c F vとする。 2つの H鎖 V領域及び 2つの L鎖 V領域を含む一本鎖 ポリべプチドの例としては、 前記モノクローナル抗体由来の H鎖 V領域と L鎖 V 領域を有するものを MAB L 1— s c (F v)2、 MAB L 2 - s c (F v)2とする。 これらポリペプチドを製造するにあたり、 該ポリペプチドが分泌性であること を所望する場合は、 その N—末端にシグナルペプチドを付加することができる。 また、 該ポリペプチドの効率的精製等のために、 ポリペプチド精製において有用 であ.る公知の配列、 例えば FLAG配列などを揷入することができる。 この場合、 抗 F LAG抗体を用いてダイマー形成させることもできる。 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. Examples of 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. As an example of a single-chain polypeptide containing two H-chain V regions and two L-chain V regions, MAB L 1-sc ( F v) 2 and MAB L 2-sc (F v) 2 . In producing these polypeptides, if it is desired that the polypeptide is secretory, a signal peptide can be added to its N-terminal. In addition, a known sequence useful in polypeptide purification, such as a FLAG sequence, can be inserted for efficient purification of the polypeptide. In this case, a dimer can be formed using an anti-FLAG antibody.
本発明の改変を作製するためには、 これをコードする DNA、 即ち一本鎖 F v をコードする DN A又は再構成一本鎖ポリぺプチドをコ一ドする DNAを得る必 要がある。 これらの DNAは、 例えば MAB L 1— s c F v、 MAB L 2— s c F v、 MAB L 1 - s c (F v) 2及び Z又は MAB L 2— s c (Fv)2の場合には 前記 F v由来の H鎖 V領域及び L鎖 V領域をコードする DN Aを用いて、 又はこ れらの DNAを铸型とし、 その配列内の所望のアミノ酸配列をコードする DNA 部分を、 その両端を規定するプライマー対を用いる P C R法により増幅すること により得ることができる。 In order to produce the modification of the present invention, it is necessary to obtain 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 Using 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.
各 V領域について、 アミノ酸配列の一部改変を所望する場合には、 PCR法を 用いる公知の方法によって 1又は数個のアミノ酸が改変された、 即ち 1もしくは 数個のアミノ酸が欠失、 置換もしくは付加されたアミノ酸配列を有する V領域を 得ることができる。 特定の抗原に対して十分に活性がある改変抗体を作製するた めに、 PCR法を用いる公知の方法によって前記 V領域のアミノ酸配列の一部を 改変することが望ましい。  When partial modification of the amino acid sequence of each V region is desired, 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. In order to prepare a modified antibody having sufficient activity against a specific antigen, it is desirable to modify a part of the amino acid sequence of the V region by a known method using PCR.
PCRに用いるプライマーを決定するにあたり、 所望のモノクローナル抗体由 来の H鎖及び L鎖のタイピングをして両鎖の型を決める必要がある。 MABL— 1抗体、 MABL— 2抗体の場合、 MABL— 1抗体は κ型 L鎖及び γ 1型の Η 鎖を有し、 MABL— 2抗体は κ型 L鎖及び γ 2 a型の Η鎖を有することが明ら かになつている （特願平 11ー63557号)。 前記 MABL— 1抗体及び 又は MAB L— 2抗体の H鎖 V領域及ぴ L鎖 V領域をコ—ドする DNAをポリメラー ゼ連鎖反応 （ P C R ) 法を用いて増幅するには、 Jones, S. T. ら、 Bio/Technology, 9, 88 - 89, 1991 に記載されているプライマーを用いることがで さる。 To determine the primers used for PCR, it is necessary to determine the type of both chains by typing the H and L chains derived from the desired monoclonal antibody. In the case of MABL-1 antibody and MABL-2 antibody, MABL-1 antibody has κ-type L chain and γ1 type 鎖 chain, and MABL-2 antibody has κ- type L chain and γ2a type Η chain. It is clear that they have it (Japanese Patent Application No. 11-63557). The MABL-1 antibody and / or To amplify the DNA encoding the H chain V region and L chain V region of the MAB L-2 antibody by polymerase chain reaction (PCR), Jones, ST et al., Bio / Technology, 9, It is better to use the primers described in 88-89, 1991.
' 次に、 ポリメラーゼ連鎖反応 （PCR) 法を用いて MABL— 1抗体及び MA B L— 2抗体の L鎖 V領域を増幅するため、 5 ' —末端オリゴヌクレオチドプライ マー及び 3'—末端オリゴヌクレオチドプライマ一を上述のように決定する。 同様 にし _て、 MAB L— 1抗体の H鎖 V領域及び MAB L— 2抗体の H鎖 V領域の増 幅のため、 それぞれ 5 '—末端プライマー及び 3 '—末端プライマーを決定する。 その例として本発明においては、 5'—末端プライマーはその 5 ' —末端近傍に 制限酵素 H i n f I切断部位を提供する配列 GANTCを含有し、 そして 3'—末 端プライマーはその 5 '—末端近傍に制限酵素 Xm a I切断部位を提供するヌクレ ォチド配列 C C C G G Gを含有するものを使用している。 これらの制限酵素切断 部位は可変領域をコードする目的の DNA断片をクローユングベクターにサブク ローニングするために用いられる限り、 他の制限酵素切断部位でもよい。 'Next, to amplify the light chain V region of MABL-1 and MABL-2 antibodies using the polymerase chain reaction (PCR) method, 5'-terminal oligonucleotide primers and 3'-terminal oligonucleotide primers One is determined as described above. Similarly, 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. By way of example, in the present invention, 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.
特に設計された P CRプライマーを用いて、 MABL— 1、 MABL— 2抗体 の各 V領域をコードする c DN Aをそれらの 5'—及び 3'—末端において適当な 塩基配列を導入して、 それらが発現ベクターに容易に挿入されるように、 且つそ れらが該発現ベクター中で適切に機能するようにした （例えば、 本発明では Ko z a k配列の導入により転写効率を上げるように工夫されている）。 次に、 これら のプライマーを用いて P CRにより増幅して得た MAB L— 1、 MAB L一 2抗 体の各 V領域を、 所望のヒ ト C領域をすでに含有する HEF発現ベクター （WO 92- 19759参照） に挿入した。 クローン化された DN Aの配列決定は任意 の常法、 例えば、 適当なベクターに挿入し、 自動 DNAシークェンサ一 （Applied Biosystems社製） を用いて行うことができる。  Using a specially designed PCR primer, 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). Next, 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).
本発明の改変抗体において、 リンカ一、 例えばペプチドリンカ一は次のように 導入することができる。 即ち、 上述の H鎖 V領域及び L鎖 V領域のためのプライ マーと一部相補的な配列を有し、 且つ該リンカーの N—末端または C一末端をコ ードするようにプライマーを設計し、 これを用いて P C Rを行うことによって所 望のアミノ酸配列および長さを有するぺプチドリンカ一をコードする D NAを作 成することができる。 そして、 該 D NAを介して H鎖 V領域及ぴ L鎖 V領域をコ ードする D N Aを連結すれば、 所望のペプチドリンカーを有する本発明の改変抗 体をコードする D NAを得ることができる。 さらに、 1つの改変抗体をコードす る D NAを得ることができれば、 前記 D NAを铸型にして、 そして種々のリンカ 一用のプライマーを設計し、 これを用いて P C Rを実施すれば、 所望のペプチド リン ^ "を有する改変抗体又はリンカ一を有さない改変抗体をコードする D NA は容易に得ることができる。 In the modified antibody of the present invention, a linker, for example, a peptide linker can be introduced as follows. That is, 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 By designing 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. Then, by linking DNAs encoding the H chain V region and the L chain V region via the DNA, DNA encoding the modified antibody of the present invention having a desired peptide linker can be obtained. it can. Furthermore, if 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.
また、 本発明における改変抗体の各鎖 V領域は、 従来の技術 （例えば、 Sato, K. ら、 Cancer Res.， 53， 1-6 (1993)を参照のこと） を用いることによって、 ヒト型 化することが可能であり、 また一旦ヒト型化 F V領域をコードする D NAが作製 されれば、 ヒト型化一本鎖 F v、 ヒ ト型化一本鎖 F v断片、 ヒ ト型化モノクロ一 ナル抗体あるいはヒト型化モノクローナル抗体断片は、 常法に従って容易に作出 する事が可能である。 さらに、 必要な場合、 これらの V領域のアミノ酸配列の一 部を改変することも可能である。  In addition, 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)). Once the DNA encoding the humanized FV region has been produced, 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. Furthermore, if necessary, a part of the amino acid sequence of these V regions can be modified.
さらに、 遺伝子工学における慣用技術を用いて上述のマウス由来の H鎮 V領域 及び L鎖 V領域をコードする D NAと同様に、 これらに相当する他の哺乳動物由 来の D NA、 例えばヒト由来の D NAを得ることができる。 得られた D NAを用 いて、 他の哺乳動物、 特にヒト由来の H鎖 V領域及び L鎖 V領域、 ヒト由来の一 本鎖 F v及びその断片、 並びにヒト由来のモノクローナル抗体及びその断片を得 ることができる。  Furthermore, similar to the above-described DNA encoding the H-protein V region and L-chain V region derived from the mouse using the conventional techniques in genetic engineering, DNAs derived from other mammals corresponding thereto, for example, human-derived DNA Of DNA can be obtained. Using the obtained DNA, 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.
以上のように、 目的とする改変抗体の各鎖 V領域、 ヒト型化改変抗体の各鎖 V 領域をコードする D NAが作製されれば、 それらを含有する発現ベクター、. 及ぴ 該発現べクタ一により形質転換された宿主を常法に従って得ることができる。 ま た、 常法に従って宿主を培養し、 産生した再構成一本鎖 F v、 再構成ヒト型化一 本鎖 F v、 ヒト型化モノクローナル抗体及びヒト型化モノクローナル抗体断片は、 細胞内又は細胞外から分離し均一にまで精製することができる。 この場合、 通常 の蛋白質で用いられる分離.精製方法、 例えば各種クロマトグラフィー、 限外濾 過、 塩祈、 透析等を適宜選択、 組合せて、 本発明の改変抗体を分離■精製するこ とができるが、 これらに限定されるものではない。 As described above, if 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. In addition, 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.
再構成一本鎖 F Vを動物細胞、 例えば、 COS 7細胞、 CHO細胞などの動物 培養細胞、 好ましくは CHO細胞で産生する場合、 無血清培地で該再構成一本鎖 F vを産生させると、 培地中で効率よく該一本鎖 F Vのダイマーを形成すること ができる。 さらに、 該ダイマーを精製する際には、 形成されたダイマーを安定的 に高^率で回収することができると共に長期間、 ダイマーの状態で保存すること ができる。 この場合に用いることができる無血清培地は、 通常組み換えタンパク 質の産生に用いられている培地であればいかなるものでもよく、 特に限定される ものではない。  When 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, when the reconstituted single-chain FV is produced in a serum-free medium, The single-chain FV dimer can be efficiently formed in the medium. Furthermore, when purifying the dimer, 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.
本発明の改変抗体の製造のために任意の発現系、 例えば真核細胞、 例えば動物 細胞、 例えば樹立された哺乳類細胞系、 真糸状菌細胞、 及び酵母細胞、 並びに原 核細胞、 例えば細菌細胞、 例えば大腸菌細胞等を使用することができる。 好まし くは、 本発明の改変抗体は哺乳類細胞、 例えば CO S 7細胞又は CHO細胞中で 発現される。  For the production of the modified antibodies of the present invention, 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. Preferably, the modified antibodies of the invention are expressed in mammalian cells, such as COS 7 cells or CHO cells.
ヒト I APを有する細胞に結合する本発明の再構成ポリペプチドの製造のため に任意の発現系、 例えば真核細胞、 例えば動物細胞、 例えば樹立された哺轧類細 胞系、 真糸状菌細胞、 及び酵母細胞、 並びに原核細胞、 例えば細菌細胞、 例えば 大腸菌細胞等を使用することができる。 好ましくは、 本発明の再構成ポリべプチ ドは哺乳類細胞、 例えば COS 7細胞又は CHO細胞中で発現される。  For the production of the reconstituted polypeptide of the present invention that binds to a cell having human IAP, 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. Preferably, the reconstituted polypeptides of the invention are expressed in mammalian cells, such as COS 7 cells or CHO cells.
これらの場合、 哺乳類細胞での発現のために有用な常用のプロモーターを用い ることができる。 例えば、 ヒ ト 'サイ トメガロウィルス （Human cytomegalovirus： HCMV) 前期 （immediate early) プロモーターを使用するのが好まし い。 HCMVプロモータ^"を含有する発現ベクターの例には、 HCMV— VH— HCy l、 HCMV— VL— HCK等であって、 P S V 2 n e oに由来するプラ スミドベクター （国際公開公報 WO 92/1 9759参照） が包含される。  In these cases, conventional promoters useful for expression in mammalian cells can be used. For example, it is preferred to use the human cytomegalovirus (HCMV) immediate early promoter. Examples of 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.
また、 その他に、 本発明のために用いることのできる哺乳動物細胞における遺 ' 伝子発現のプロモーターとしてはレトロウイルス、 ポリオ一マウィルス、 アデノ ウィルス、 シミアンウィルス 40 (S V40) などのウィルスフ口モーターゃヒ ト ·ポリべプチドチェーン ·ェロンゲーション 'ファクター l a (HEF- 1 a) などの哺乳動物細胞由来のプロモーターを用いればよい。 例えば SV40の プロモーターを使用する場合は、 Mulligan, R. C.らの方法 （Nature, 277, 108 - 114, (1979))、 また、 HE F— 1 αプロモーターを使用する場合は、 Mizushima， S.らの方法 （Nucleic Acids Research, 18, 5322, (1990)) に従えば容易に実施 する _ことができる。 Also, in addition to the above, in mammalian cells that can be used for the present invention. '' 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. For example, when using the SV40 promoter, the method of Mulligan, RC et al. (Nature, 277, 108-114, (1979)), and when using the HEF-1α promoter, Mizushima, S. et al. The method can be easily carried out according to the method (Nucleic Acids Research, 18, 5322, (1990)).
複製起原 （o r i) としては、 SV40、 ポリオ一マウィルス、 アデノウィル ス、 牛パピローマウィルス （BPV) 等の由来の o r iを用いることができ、 さ らに発現ベクターは選択マーカーとして、 ホスホトランスフェラーゼ A PH (3') II あるいは I (n e o) 遺伝子、 チミジンキナーゼ （TK) 遺伝子、 大 腸菌キサンチンーグァニンホスホリポシルトランスフェラーゼ （E c o g p t) 遺伝子、 ジヒドロ葉酸還元酵素 （DHFR) 遺伝子等を含むことができる。  As the origin of replication (ori), ori derived from SV40, poliovirus, adenovirus, bovine papilloma virus (BPV) and the like can be used. In addition, 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.
上述のように作成した改変抗体の抗原結合活性は、 元のモノクローナル抗体の 結合阻害能を指標にして評価することができる。 具体的には、 該モノクローナル 抗体のその抗原への濃度依存的阻害作用の有無を指標にして評価する。  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.
具体的には、 本発明の改変抗体をコードする D N Aを包含する発現ベクターで 形質転換した動物細胞、 例えば COS 7細胞又は CHO細胞を培養し、 前記培養 した細胞及び/又はその培養上清、 又はこれらから精製した改変抗体を用いて抗 原への結合を測定する。 対照として発現ベクターのみで形質転換した細胞の培養 上清などを用いる。 抗原、 例えば MABL— 1抗体、 MABL— 2抗体の場合に はヒト I A Pを発現するマウス白血病細胞株 L 1210細胞に、 本発明の改変抗 体などの試験試料又は対照の培養上清を加え、 例えばフローサイトメトリーを実 施して抗原結合活性を評価する。  Specifically, 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.
in vitro でのシグナル伝達誘起効果 （MABL— 1抗体、 MABL— 2抗体の 場合はアポトーシス誘導効果） は、 抗原を発現する細胞又は該抗原遺伝子を導入 した細胞に、 前述の改変抗体の試験試料を添加し、 当該細胞においてシグナル伝 達による変化 （例えば、 ヒト I A P抗原特異的に細胞死を誘導するか否か） を評 価する。 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).
in vivo での評価試験は、 例えば改変抗体がヒ ト I A Pを認識する場合 （例え ば MAB L—1抗体、 MA B L— 2抗体由来の改変抗体）、 アポトーシス誘起効果 として、 次の通りに行う。 先ずヒト骨髄腫のモデルマウスを作成し、 当該マウス に I A Pを有する有核血液細胞にアポトーシスを誘起するモノクローナル抗体、 本発明の改変抗体を静脈投与する。 対照群には P B Sのみを投与する。 そして、 アポトーシス誘起を、 抗腫瘍効果としてマウス血清中のヒト I g Gの量の変化及 び生存期間によつて評偭する。  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. First, 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.
本発明の改変抗体は、 2つ以上の H鎖 V領域及ぴ 2つ以上の L鎖 V領域、 好ま しくは各々 2〜4、 特に好ましくは各々 2つ含むものであり、 1つの H鎖 V領域 及び 1つの L鎖 V領域を含む一本鎖 F Vのダイマー、 又は 2つ以上の H鎖 V領域 及び 2つ以上の L鎖 V領域を連結した一本鎖ポリペプチドである。 このような構 成をとることで、 もとのモノクローナル抗体の抗原結合部位の立体構造を模倣し て、 優れた抗原結合性を保挣するものと考えられる。  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. A single-chain FV dimer containing a region and one L chain V region, or a single chain polypeptide in which two or more H chain V regions and two or more L chain V regions are linked. By adopting such a configuration, it is considered that excellent three-dimensional structure of the antigen-binding site of the original monoclonal antibody is imitated and excellent antigen-binding properties are maintained.
本発明の改変抗体は、 抗体分子 （w h o 1 e I g G) と比較して顕著な低分 子化が達成させているため、 組織、 腫瘍への移行性に優れており、 さらにもとの ァゴニスト抗体分子よりも高い活性を有する。 このため、 本発明の改変抗体の元 となるモノクローナル抗体を適宜選択することによって、 種々のシグナルを細胞 内に伝達することがでる。 故に、 これを含有する医薬製剤は、 シグナル伝達の誘 起が疾病の治療に有効である、 例えば癌、 炎症、 ホルモン異常、 並びに白血病、 悪性リンパ腫、 再生不良性貧血、 骨髄異形成症候群および真性多血症などの血液 疾患の治療薬としての利用が期待される。 また、 R I標識による造影剤としての 利用も期待され、 R I化合物やトキシン等の他の化合物と結合させることにより、 効力を増強させることも可能である。  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.
次に本発明を、 ヒ ト I A Pに結合するモノクローナル抗体 （MA B L— 1抗体、 MA B L— 2抗体） 由来の改変抗体を例にして、 下記の実施例により具体的に説 明するが、 これにより本発明の範囲が限定されるものではない。 発明を実施するための最良の形態 Next, the present invention will be described in detail with reference to the following examples using modified antibodies derived from monoclonal antibodies (MABL-1 antibody and MABL-2 antibody) that bind to human IAP. Does not limit the scope of the present invention. BEST MODE FOR CARRYING OUT THE INVENTION
次に、 本発明を下記の実施例により具体的に説明するが、 これにより本発明の 範囲が限定されるものではない。  Next, the present invention will be specifically described with reference to the following examples, but the scope of the present invention is not limited thereto.
本発明の改変抗体の製造方法を、 下記の一本鎖 F Vの作製を例にして説明する。 本発明の改変抗体の製造方法において用いる、 ヒト I APに対するマウス MAB L一 1、 MAB L— 2抗体を産生するハイブリ ドーマ、 MABL— 1及び MAB L一 2は、 公的微生物寄託機関である通商産業省工業技術院生命工学工業技術研 究所. (茨城県つくば巿東一丁目 1番 3号） に、 1 997年 9月 1 1日に、 受託番 号それぞれ FERM BP— 6 100、 FERM B P— 6101として国際寄 託されている。 '  The method for producing the modified antibody of the present invention will be described using the production of the following single-chain FV as an example. 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. '
実施例 1 (ヒ ト I APに対するマウスモノクローナノレ抗体の V領域をコードす る DNAのクローン化） Example 1 (Cloning of DNA Encoding V Region of Mouse Monoclonal Antibody Against Human IAP)
ヒト I APに対するマウスモノクローナノレ抗体 MAB L— 1及ぴ MAB L— 2 の可変領域をコードする D N Aを次のようにしてクローン化した。  The DNA encoding the variable region of mouse monoclonal antibody MAB L-1 and MAB L-2 against human IAP was cloned as follows.
1. 1 メッセンジャー RNA (mRNA) の調製  1.1 Preparation of messenger RNA (mRNA)
ハイブリ ドーマ MAB L— 1及び MAB L— 2からの mRNAを、 mRNA Purification Kit (Pharmacia Biotech社製） を用いて調製した。  MRNA from hybridomas MAB L-1 and MAB L-2 was prepared using mRNA Purification Kit (Pharmacia Biotech).
1. 2 二本鎖 cDNAの合成  1.2 Double-stranded cDNA synthesis
約 1 μ gの mRNAより Marathon cDNA Amplification Kit (CLONTECH社製） を用いて二本鎖 cDNAを合成し、 アダプターを連結した。  A double-stranded cDNA was synthesized from about 1 μg of mRNA using Marathon cDNA Amplification Kit (manufactured by CLONTECH), and an adapter was ligated.
1. 3 抗体可変領域をコードする遺伝子の PC R法による増幅  1.3 Amplification of antibody variable region-encoding gene by PCR method
Thermal Cycler (PERKIN ELMER社製） を用いて P C R法を行った。  The PCR method was performed using a Thermal Cycler (manufactured by PERKIN ELMER).
(1) MABL— 1 L鎖 V領域をコードする遺伝子の増幅  (1) MABL—amplification of gene encoding 1 L chain V region
PCR法に使用するプライマーは、 アダプターの部分配列とハイプリダイズす る配列番号： 1に示すアダプタープライマー 1 (CLONTECH社製）、 及びマウス力 ッパ型 L鎖 C領域配列とハイブリダイズする配列番号： 2に示す MK C (Mouse Kappa Constant) プライマー （Bio/Technology, 9， 88-89, 1991) を用いた。  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. The MKC (Mouse Kappa Constant) primer shown in 2 (Bio / Technology, 9, 88-89, 1991) was used.
P C R溶液 50M 1は、 5 μ 1の 10XPCR Buffer II、 2 mM M g C 12、 0. 1 6mM dNTP s (d AT P d GTP、 d CTP、 d TTP)、 2. 5ュ 二ットの DNAポリメラーゼ AmpliTaq Gold (以上 PERKIN ELMER社製）、 0. 2 μ Μの配列番号： 1に示すアダプタープライマーと 0. 2 / Μの配列番号： 2に示す MKCプライマー及び MAB L— 1由来の二本鎖 c DNA 0. 1 μ gを含有し、 9 4 °Cの初期温度にて 9分間そして次に 94°Cにて 1分間、 6 0°Cにて 1分間及 び 7 2°Cにて 1分 2 0秒間、 この順序で加熱した。 この温度サイクルを 3 5回反 復した後、 反応混合物を更に 7 2 °Cで 1 0分間加熱した。 PCR solution 50M 1 was prepared by adding 5 μl 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 μ μ SEQ ID NO: 1 Containing 0.1 μg 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 / Μ 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.
(2) MAB L— 1 H鎖 V領域をコードする c DNAの増幅  (2) Amplification of cDNA encoding MAB L-1 H chain V region
P CRのためのプライマーとして配列番号： 1に示すアダプタープライマー 1、 及び配列番号： 3に示す MHC— γ 1 (Mouse Heavy Constant) プライマー (Bio/Technology, 9， 88-89， 1991) を用いた。  The adapter primer 1 shown in SEQ ID NO: 1 and the MHC-γ1 (Mouse Heavy Constant) primer shown in SEQ ID NO: 3 (Bio / Technology, 9, 88-89, 1991) were used as primers for PCR. .
c DNAの増幅は、 0. 2 /zMの MKCプライマーの代わりに 0. 2 Μの MH C一 γ 1プライマーを用いて増幅した点を除いて、 前記 1. 3 ( 1) において L 鎖 V領域遺伝子の増幅について記載したのと同じ方法により行った。  The amplification of the cDNA was performed in the above-mentioned 1.3 (1) except that the amplification was carried out using the MHC-γ1 primer of 0.2% instead of the MKC primer of 0.2 / zM. Performed by the same method as described for gene amplification.
(3) MAB L- 2 L鎖 V領域をコードする c DNAの増幅  (3) Amplification of cDNA encoding MAB L-2 L chain V region
P CRのためのプライマーとして配列番号： 1に示すアダプタープライマー 1、 及び配列番号： 2に示す MKCプライマーを用いた。  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.
c DNAの増幅は、 MAB L— 1由来の二本鎖 c DNA 0. 1 μ gの代わりに MAB L— 2由来の二本鎖 c DNA 0. 1 μ gを用いて増幅した点を除いて、 前 記 1. 3 ( 1 ) において MAB L— 1 L鎖 V領域遺伝子の増幅について記載した のと同じ方法により行った。  The amplification of cDNA was performed using 0.1 μg of double-stranded cDNA derived from MAB L-2 instead of 0.1 μg 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.
(4) MAB L— 2H鎖 V領域をコードする c DNAの増幅  (4) Amplification of cDNA encoding MAB L—2H chain V region
P CRのためのプライマーとして配列番号： 1に示すアダプタープライマー 1、 及び配列番号： 4に示す MHC— γ 2 aプライマー （Bio/Technology, 9, 88 - 89， 1991) を用いた。  The adapter primer 1 shown in SEQ ID NO: 1 and the MHC-γ2a primer shown in SEQ ID NO: 4 (Bio / Technology, 9, 88-89, 1991) were used as primers for PCR.
c DNAの増幅は、 0. 2 μΜの MKCプライマーの代わりに 0. 2 / Μの ΜΗ C— y 2 aプライマーを用いて増幅した点を除いて、 前記 1. 3 (3) において L鎖 V領域遺伝子の増幅について記載したのと同じ方法により行った。 1. 4 P CR生成物の精製 The amplification of cDNA was carried out in 1.3 (3) above, except that amplification was performed using 0.2 / Μ ΜΗC-y2a primer instead of 0.2 μΜ 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
前記のようにして P C R法により増幅した D NA断片を QIAquick PCR The DNA fragment amplified by the PCR method as described above was subjected to QIAquick PCR.
Purification Kit (QIAGEN社製） を用いて精製し、 1 mM EDTAを含有するPurify using Purification Kit (QIAGEN) and contain 1 mM EDTA
1 OmM T r i s—HC l ( p H 8. 0 ) に溶解した。 It was dissolved in 1 OmM Tris-HCl (pH 8.0).
1. 5 連結及び形質転換  1.5 Ligation and transformation
上記のようにして調製した MAB L— 1由来マウスカッパ型 L鎖 V領域をコー ドす 遺伝子を含んで成る DN A断片約 1 4 0 n gを p GEM— T E a s yベ クタ—一 （Promega社製） 5 0 n gと、 3 0 mM T r i s—HC l (p H 7. 8)、 Approximately 140 ng of a DNA fragment containing the gene encoding the mouse kappa-type L chain V region derived from MAB L-1 prepared as described above was ligated with pGEM-TE asy vector (Promega). ) 50 ng and 30 mM Tris-HCl (pH 7.8),
1 OmM Mg C l 2、 1 OmM ジチオスレィトール、 1 mM AT P及び 3ュ ニット T 4 DNAリガーゼ （Promega社製） を含有する反応混合液中で、 1In a reaction mixture containing 1 OmM MgCl 2 , 1 OmM dithiothreitol, 1 mM ATP and 3-unit T4 DNA ligase (Promega), 1
5 °Cにて 3時間反応させ連結した。 The reaction was carried out at 5 ° C. for 3 hours for ligation.
次に、 1 1の上記連結混合液を大腸菌 DH 5ひのコンビテント細胞 （東洋紡 社製） 5 0 μ 1に加え、 そしてこの細胞を氷上で 3 0分間、 4 2°Cにて 1分間そ して再び氷上で 2分間静置した。 次いで 1 00 μ 1の S O C培地 （GIBCO BRL社 製） を加え、 1 00 μ gZm 1のアンピシリン （SIGMA社製） を含有する L B Next, the above ligation mixture of 11 was added to 50 μl 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 μl of SOC medium (GIBCO BRL) was added, and LB containing 100 μg Zm1 of ampicillin (SIGMA) was added.
(Molecular Cloning： A Laboratory Manual, Sambrook ら、 Cold Spring Harbor(Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor
Laboratory Press, 1989) 寒天培地上にこの大腸菌を塗布し、 3 7 °Cにて終夜培 養して大腸菌形質転換体を得た。 Laboratory Press, 1989) The Escherichia coli was spread on an agar medium and cultured overnight at 37 ° C to obtain an Escherichia coli transformant.
この形質転換体を、 50 μ g/m 1のアンピシリンを含有する LB培地 3m 1 中で 3 7°Cにて終夜培養し、 そしてこの培養物から QIAprep Spin Miniprep Kit The transformant was cultured overnight at 37 ° C in 3 ml of LB medium containing 50 μg / ml of ampicillin, and the QIAprep Spin Miniprep Kit
(QIAGEN社製） を用いてプラスミド DNAを調製した。 (Manufactured by QIAGEN) to prepare plasmid DNA.
こうして得られた、 ハイブリ ドーマ MAB L— 1に由来するマウスカッパ型 L 鎖 V領域をコードする遺伝子を含有するプラスミドを p GEM— Ml Lと命名し 卞一  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
上記の同じ方法に従って、 ハイブリ ドーマ MAB L— 1に由来するマウス H鎖 Mouse H chain derived from hybridoma MAB L-1 according to the same method described above
V領域をコードする遺伝子を含有するプラスミ ドを精製 D N A断片から作製し、 p GEM— Ml Hと命名した。 A plasmid containing the gene encoding the V region was made from the purified DNA fragment and named pGEM—MlH.
また、 ハイプリ ドーマ MAB L— 2に由来するマウスカッパ型 L鎖 V領域をコ ードする遺伝子を含有するプラスミ ドを精製 DNA断片から作製し、 pGEM— M2 Lと命名した。 In addition, the 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.
また、 ハイプリ ドーマ MAB L— 2に由来するマウス H鎖 V領域をコードする 遺伝子を含有するプラスミドを精製 DN A断片から作製し、 ； pGEM— M2Hと 命名した。  In addition, a 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.
実施例 2 (D N Aの塩基配列の決定） Example 2 (Determination of DNA base sequence)
前記のプラスミド中の cDN Aコード領域の塩基配列の決定は、 自動 DNAシ ーケ-ンサー （Applied Biosystem社製） 及び ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystem社製) ¾用いて、 メーカー 指定のプロトコールに従って行った。  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.
プラスミド p GEM— Ml Lに含まれるマウス MAB L— 1抗体の L鎖 V領域 をコードする遺伝子の塩基配列を配列番号： 5に示す。  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.
また、 プラスミド p GEM— Ml Hに含まれるマウス MAB L— 1抗体の H鎖 V領域をコードする遺伝子の塩基配列を配列番号： 6に示す。  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.
また、 プラスミ ド p GEM— M2 Lに含まれるマウス MAB L— 2抗体の L鎖 In addition, the light chain of mouse MAB L-2 antibody contained in plasmid p GEM-M2 L
V領域をコードする遺伝子の塩基配列を配列番号： 7に示す。 SEQ ID NO: 7 shows the nucleotide sequence of the gene encoding the V region.
また、 プラスミ ド p GEM— M2 Hに含まれるマウス MAB L— 2抗体の H鎖 V領域をコードする遺伝子の塩基配列を配列番号： 8に示す。  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.
実施例 3 (CDRの決定） Example 3 (Determination of CDR)
L鎖及び H鎖の V領域の全般的構造は、 互いに類似性を有しており、 それぞれ The overall structures of the V regions of the L and H chains are similar to each other,
4つのフレームワーク部分が 3つの超可変領域、 即ち相補性決定領域 （CDR) により連結されている。 フレームワークのアミノ酸配列は、 比較的良く保存され ているが、 一方、 CDR領域のアミノ酸配列の変異性は極めて高い （Kabat， E. A. ら、 「 equences of Proteins of Immunological Interest] US Dept. Health and Human Services, 1983)。 The four framework parts are linked by three hypervariable regions, the complementarity determining regions (CDRs). 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).
このような事実に基づき、 ヒト I APに対するマウスモノクローナル抗体の可 変領域のアミノ酸配列を K a b a tらにより作製された抗体のアミノ酸配列のデ ータベースにあてはめ、 相同性を調べることにより CDR領域を表 1に示す如く 決定した。 Based on these facts, 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.
プラスミド 配列番号 CDR(l) CDR(2) CDR(3) p GEM-MI L 5 43-58 74-80 113-121 p GEM-MI H 6 50-54 69-85 118-125 p GEM-M2 L 7 43-58 74-80 113-121 Plasmid SEQ ID NO: CDR (l) CDR (2) CDR (3) p GEM-MI L 5 43-58 74-80 113-121 p GEM-MI H 6 50-54 69-85 118-125 p GEM-M2 L 7 43-58 74-80 113-121
GEM-M2H 8 50-54 69-85 118-125 実施例 4 (クローン化 c DNAの発現の確認 （キメラ MAB L— 1抗体及びキ メラ MABL— 2抗体の作製)）  GEM-M2H 8 50-54 69-85 118-125 Example 4 (Confirmation of Expression of Cloned cDNA (Preparation of Chimeric MABL-1 Antibody and Chimeric MABL-2 Antibody))
4. 1 キメラ MAB L— 1抗体発現ベクターの作製  4.1 Construction of chimeric MAB L-1 antibody expression vector
キメラ MABL— 1抗体を発現するベクターを作製するため、 それぞれマウス MAB L- 1 L鎖及ぴ H鎖 V領域をコードする c DNAクローン; GEM— M 1 L及び p GEM— Ml Hを P CR法により修飾した。 そして HEF発現ベクター (国際公開公報 WO 92/19759参照） に導入した。  To prepare a vector expressing the chimeric MABL-1 antibody, cDNA clones encoding mouse MAB L-1 L chain and H chain V region, respectively; GEM-M1L and pGEM-MlH were subjected to PCR method. Modified by Then, it was introduced into a HEF expression vector (see International Publication WO92 / 19759).
L鎖 V領域のための前方プライマー ML S (配列番号： 9) 及ぴ H鎖 V領域の ための前方プライマー MHS (配列番号： 10) は、 各々の V領域のリーダー配 列の最初をコードする DN Aにハイブリダイズし且つ K o z a kコンセンサス配 列 （J. mol. Biol. , 196, 947-950， 1987) 及び H i n d III.制限酵素部位を有 するように設計した。 L鎖 V領域のための後方プライマー MLAS (配列番号： 11) 及び H鎖 V領域のための後方プライマー MHAS (配列番号： 12) は、 J領域の末端をコ一ドする D N A配列にハイブリダイズし且つスプライスドナー 配列及び B a mH I制限酵素部位を有するように設計した。  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.
PC R溶液 100 /z lは、 Ι Ομ Ιの 10XPCR Buffer II、 2 mM M g PCR solution 100 / zl was added to 、 ΙμΙ of 10XPCR Buffer II, 2 mM Mg
C 12、 0. 16mM dNTP s (dATP、 dGTP、 dCTP、 dTTP)、 5ユニットの DN Aポリメラーゼ AmpliTaq Gold, 0.4 Mずつの各プライマー、 及び 8 n gの铸型 DNA (p GEM— Ml L及び P GEM— Ml H) を含有し、 94°Cの初期温度にて 9分間そして次に 94°Cにて 1分間、 6 0°Cにて 1分間及 び 72 °Cにて 1分 2 0秒間、 この順序で加熱した。 この温度サイクルを 3 5回反 復した後、 反応混合物を更に 72 °Cで 1 0分間加熱した。 C 1 2, 0. 16mM dNTP s (dATP, dGTP, dCTP, dTTP), DN A polymerase AmpliTaq Gold 5 units, 0.4 of each primer by M, and 8 ng of铸型DNA (p GEM- Ml L and P GEM—Ml H) Heating was performed in this order at an initial temperature of 94 ° C for 9 minutes and then at 94 ° C for 1 minute, at 60 ° C for 1 minute and at 72 ° C for 1 minute and 20 seconds. After repeating this temperature cycle 35 times, the reaction mixture was further heated at 72 ° C for 10 minutes.
PCR生成物を QIAquick PGR Purification Kit (QIAGEN社製） を用いて精製 し、 H i n d III 及び B a mH Iで消化し、 そして L鎖 V領域については、 HE F発現ベクター HE F— κに、 H鎖 V領域については HE F発現ベクター HE F 一 yにそれぞれクローニングした。 DN A配列決定の後、 正しい DN A配列を有 する _DN A断片を含むプラスミドをそれぞれ HE F— Ml L、 HE F— Ml Hと 命名した。  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-κ. The chain V region was cloned into the HEF expression vector HEF-y. After the DNA sequencing, the plasmids containing the _DNA fragment having the correct DNA sequence were named HEF-MlL and HEF-MlH, respectively.
4. 2 キメラ MAB L— 2抗体努現ベクターの作製 4.2 Construction of chimeric MAB L-2 antibody antibody vector
c DN Aの修飾及びクローニングは、 p GEM— Ml L及ぴ p GEM— Ml H の代わりに p GEM— M2 L及ぴ p GEM— M2Hを铸型 DNAに増幅した点を 除いて、 前記 4. 1において記載したのと同じ方法により増幅及ぴクローニング を行い、 DN A配列決定の後、 正しい DNA配列を有する DNA断片を含むプラ スミドをそれぞれ HEF— M2 L、 HEF— M2Hと命名した。  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.
4. 3 COS 7細胞への遺伝子導入 4.3 Gene transfer into COS 7 cells
キメラ MA B L— 1抗体及びキメラ MAB L— 2抗体の一過性発現を観察する ため、 前記発現ベクターを COS 7細胞において試験した。  The expression vectors were tested in COS 7 cells to observe the transient expression of chimeric MABL-1 and chimeric MABL-2 antibodies.
(1) キメラ MAB L— 1抗体の遺伝子導入  (1) Gene transfer of chimeric MAB L-1 antibody
HE F— Ml Lと HE F—M 1 Hベクターを、 Gene Pulser 装置 （BioRad社 製） を用いてエレクト口ポレーシヨンにより COS 7細胞に同時形質転換した。 . 各 DNA (1 0 μ g) と、 PB S中 1 X 1 07細胞 1の 0. 8m lをキュべッ トに加え、 1. 5 kV、 25 /z Fの容量にてパルスを与えた。 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 μ 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.
室温にて 1 0分間の回復期間の後、 エレクトロポレーシヨン処理された細胞を、 1 0 %の V—グロブリンフリーゥシ胎児血清を含有する D M E M培養液 （GIBC0 BRL社製） に加えた。 72時間培養の後、 培養上清を集め、 遠心分離により細胞 破片を除去して回収培養上清を得た。  After a 10-minute recovery period at room temperature, 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.
J 2) キメラ MAB L— 2抗体の遺伝子導入 キメラ MAB L— 2抗体遺伝子の導入は、 HEF— Ml Lと HEF— M1Hベ クタ一の代わりに HE F—M2 Lと HEF— M2Hベクターを用いた点を除いて、 前記 4. 3 (1) に記載したのと同じ方法により COS 7細胞に同時形質転換し、 回収培養上 '清を得た。 J 2) Gene transfer of chimeric MAB L-2 antibody The introduction of the chimeric MAB L-2 antibody gene was performed according to the procedure described in 4.3 (1) except that the HEF-M2L and HEF-M2H vectors were used instead of the HEF-M1L and HEF-M1H vectors. COS 7 cells were co-transformed by the same method as described above, and the supernatant was recovered and cultured.
4. 4 フローサイトメトリー 4.4 Flow cytometry
抗原への結合を測定するため、 前記 COS 7細胞培養上清を用いてフローサイ トメトリーを行った。 ヒト I APを発現するマウス白血病細胞株 L 1210細胞 4 X.l 05個に、 キメラ MAB L— 1抗体を発現させた COS 7細胞の培養上清あ るいはキメラ MAB L— 2抗体を発現させた COS 7細胞の培養上清あるいはコ ントロールとしてヒト I g G 1抗体 （SIGMA社製） を加え、 氷上にてインキュべ ーシヨン及ぴ洗浄の後、 F I TC標識した抗ヒト I gG抗体 （Cappel 社製） を加 えた。 インキュベーショ ン及び洗浄の後、 FAC S c a n装置 （BECT0N DICKINSON社製） にて蛍光強度を測定した。 In order to measure the binding to the antigen, flow cytometry was performed using the COS 7 cell culture supernatant. To 4 Xl 0 5 or mouse leukemia cell line L 1210 cells expressing human I AP, culture UeKiyoshia Rui COS 7 cells expressing chimeric MAB L-1 antibody was expressed chimeric MAB L-2 antibody A human IgG1 antibody (manufactured by SIGMA) was added as a culture supernatant or control of COS 7 cells, and the mixture was incubated and washed on ice, followed by FITC-labeled anti-human IgG antibody (manufactured by Cappel). ) Was added. After the incubation and washing, the fluorescence intensity was measured with a FAC Scan device (BECT0N DICKINSON).
その結果、 キメラ MABL— 1抗体及ぴキメラ MABL—2抗体は、 ヒト I A Pを発現する L 1 210細胞に特異的に結合したことにより、 これらのキメラ抗 体がマウスモノクローナル抗体 MAB L— 1及び MAB L— 2のそれぞれの V領 域の正しい構造を有することが明らかとなった （図 1〜3)。  As a result, 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).
実施例 5 (再構成 MA B L— 1抗体及び再構成 MA B L- 2抗体一本鎖 F v (s c Fv) 領域の作製） Example 5 (Preparation of reconstituted MA B L-1 antibody and reconstituted MA B L-2 antibody single chain Fv (sc Fv) region)
5. 1 再構成 MAB L— 1抗体一本鎖 F Vの作製 5.1 Preparation of reconstituted MAB L-1 antibody single-chain FV
再構成 MAB L― 1抗体一本鎖 F Vを次の様にして作製した。 再構成 MAB L 一 1抗体 H鎖 V領域、 リンカ一領域、 及び再構成 MABL— 1抗体 L鎖 V領域を それぞれ PCR法を用いて増幅し、 連結することにより、 再構成 MABL— 1抗 体一本鎖 F vを作製した。 この方法を図 4に模式的に示す。 再構成 MABL— 1 抗体一本鎖 F Vの作製のために 6個の P CRプライマー （A〜F) を使用した。 プライマー A、 C及ぴ Eはセンス配列を有し、 プライマー B、 D及ぴ Fはアンチ センス配列を有する。  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, and primers B, D and F have an antisense sequence.
H鎖 V領域のための前方プライマー VHS (プライマー A、 配列番号： 13) は、 H鎖 V領域の N末端をコードする DN Aにハイブリダィズし且つ N c o I制 限酵素認識部位を有するように設計した。 H鎖 V領域のための後方プライマー V HAS (プライマー B、 配列番号： 14) は、 H鎖 V領域の C末端をコードする D N Aにハイブリダイズし且つリンカ一とオーバーラップするように設計した。 リンカ一のための前方プライマー L S (プライマー C、 配列番号： 1 5) は、 リンカーの N末端をコードする DN Aにハイブリダィズし且つ H鎖 V領域の C末 端をコードする DNAとオーバーラップするように設計した。 リンカーのための 後方プライマー LAS (プライマー D、 配列番号： 16) は、 リンカ一の C末端 をコードする DNAにハイブリダィズし且つ L鎖 V領域の N末端をコードする D NAとオーバーラップするように設計した。 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.
L鎖 V領域のための前方プライマー VLS (プライマー E、 配列番号： 1 7) は、 リンカーの C末端をコードする DN Aにハイブリダィズし且つ L鎖 V領域の N末端をコードする DNAにオーバーラップするように設計した。 L鎖 V領域の ための後方プライマー VLAS— F LAG (プライマー F、 配列番号： 18) は、 L鎖 V領域の C末端をコードする DN Aにハイブリダィズし且つ FLAGぺプチ ドをコードする配列 （Hopp, T. P.ら、 Bio/Technology， 6， 1204-1210， 1988)、 2個の転写停止コドン及び E c oR I制限酵素認識部位を有するように設計した。 第一 P CR段階において 3つの反応 A_B、 C— D及び E— Fを行い、 そして 各 PCR生成物を精製した。 第一 PCRから得られた 3つの PCR生成物をそれ ら自体の相補性によりアッセンブルさせた。 次に、 プライマー A及び Fを加えて、 再構成 M A B L- 1抗体一本鎖 F Vをコードする全長 D N Aを増幅した （第二 P CR)。 なお、 第一 PCRにおいては、 再構成 MAB L— 1抗体 H鎖 V領域をコー ドするプラスミ ド p GEM— Ml H (実施例 2を参照）、 G 1 y G 1 y G 1 y G 1 y S e r G 1 y G 1 y G l y G 1 y S e r G 1 y G 1 y G l y G l y S e r (配列番号： 1 9) からなるリンカ一領域をコードする DNA配列 （Huston, J. S. ら、 Proc. Natl. Acad. Sci. USA, 85, 5879-5883， 1988) を含んで成るプラスミ ド p S C— DP 1、 及び再構成 MAB L— 1抗体 L 鎖 V領域をコードするプラスミ ド pGEM— Ml L (実施例 2を参照） をそれぞ れ鐯型として用いた。 The forward primer VLS for the light chain V region (Primer E, SEQ ID NO: 17) 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. In the first PCR stage, three reactions A_B, CD and EF were performed, and each PCR product was purified. The three PCR products from the first PCR were assembled by their own complementarity. Next, primers A and F were added to amplify the full-length DNA encoding the reshaped MAB L-1 antibody single-chain FV (second PCR). In the first PCR, 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.
第一 P C R段階の溶液 50 μ 1は、 5 1の 10 XPCR Buffer II、 2 mM Mg C l 2、 0. 1 6 mM dNTP s、 2. 5ユニットの D N Aポリメラーゼ ArapliTaq Gold (以上 PERKIN ELMER社製）、 0.4 Mずつの各プライマー及び 5 n gの各铸型 DNAを含有し、 94°Cの初期温度にて 9分間そして次に 94°Cに て 1分間、 6 5 °Cにて 1分間及び 72 °Cにて 1分 20秒間、 この順序で加熱した。 この温度サイクルを 35回反復した後、 反応混合物を更に 72°Cで 7分間加熱し た。 -' 50 μl 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. -'
?〇1 生成物 —；8 (371 b p)、 C-D (63 b ), 及び E— F (384 b p) を QIAquick PCR Purification Kit (QIAGEN社製） を用いて精製し、 第二 PCRでアッセンブルした。 第二 PC Rにおいて、 铸型として 120 n gの第一 ? 1 生成物 —8、 20 n gの PCR生成物 C— D及び 120 n gの PCR生 成物 E— F、 10 μ 1の 10 X P CR Buffer II、 2 mM MgC l 2、 0. 16 mM dNT P s、 5ユニッ トの D N Aポリメラーゼ AmpliTaq Gold (以上 PERKIN ELMER社製） を含有する 98 μ 1の P CR混合液を、 94°Cの初期温度に て 8分間そして次に 94 °Cにて 2分間、 65 °Cにて 2分間及ぴ 72 °Cにて 2分間、 この順序でカ卩熱した。 この温度サイクルを 2回反復した後、 それぞれ 0.4 μ の プライマー Α及び Fを加えた。 そして 94°Cの初期温度にて 1分間そして次に 9 4 °Cにて 1分間、 6 5 °Cにて 1分間及び 72 °Cにて 1分 20秒間、 この順序で加 熱し、 この温度サイクルを 35回反復した後、 反応混合物を 72 °Cにて 7分間加 熱した。 ? 〇1 product—; 8 (371 bp), CD (63 b), and E—F (384 bp) were purified using QIAquick PCR Purification Kit (QIAGEN) and assembled in a second PCR. In the second PCR, type I: 120 ng of the first to first product—8, 20 ng of PCR product C—D and 120 ng of PCR product E—F, 10 μl of 10 XP CR Buffer II, 2 mM MgC l 2, 0. 16 mM dNT P s, 5 units bets DNA polymerase AmpliTaq Gold (or manufactured by PERKIN ELMER) 98 mu 1 of P CR mixture containing the initial temperature of 94 ° C The potatoes were heated in this order for 8 minutes and then at 94 ° C for 2 minutes, at 65 ° C for 2 minutes and at 72 ° C for 2 minutes. After this temperature cycle was repeated twice, 0.4 μm of each of primers Α and F was added. Heat for 1 minute at an initial temperature of 94 ° C and then for 1 minute at 94 ° C, 1 minute at 65 ° C and 1 minute 20 seconds at 72 ° C, in this order. After 35 cycles, the reaction mixture was heated at 72 ° C for 7 minutes.
第二 PCRにより生じた 843 b pの DNA断片を精製し、 Nc o I及ぴ E c o R Iで消化し、 得られた DNA断片を p SCFVT7ベクターにクローニング した。 なお、 本発現ベクター: p S CFVT 7は、 大腸菌ペリブラズム分泌発現系 に適する p e I Bシグナル配列 （Lei, S. P.ら、 J. Bacteriology, 169, 4379- 4383, 1987) を含んでいる。 DNA配列決定の後、 再構成 M A B L— 1抗体一本 鎖 Fvの正しいアミノ酸配列をコードする DN A断片を含むプラスミドを p s c Mlと命名した （図 5を参照）。 本プラスミド p s cMlに含まれる再構成 MAB L一 1抗体一本鎖 F vの塩基配列及びァミノ酸配列を配列番号： 20に示す。 次に、 哺乳動物細胞にて再構成 MAB L— 1抗体一本鎖 F Vを発現するべクタ 一を作製するため、 p s cMlベクターを PCR法により修飾した。 そして得ら れた DNA断片を p CHO 1発現ベクターに導入した。 なお、 本発現ベクター p CHO 1は、 DHFR— ΔΕ— r vH— PM1— ί (W092/19759参 照） から、 E c o R I及ぴ Sma I消化により抗体遺伝子を削除し、 E c oR I — No t l— B amHI Ad a p t o r (宝酒造社製） を連結することにより 構築.したベクターである。 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. After DNA sequencing, 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. Next, 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. Then, 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-ΔΕ-rvH-PM1- (ί) (see W092 / 19759) by digestion with EcoRI and SmaI, and tl—A vector constructed by ligating BamHI Ad aptor (Takara Shuzo).
PCRに使用するプライマーは、 前方プライマーとして H鎖 V領域の N末端を コードする DNAにハイブリダィズし且つ S a 1 I制限酵素認識部位を有する配 列番号： 21に示す3 & 1— VHSプライマー及び後方プライマーとして第一フ レームワーク配列の最後をコードする DNAにハイブリダィズする配列番号： 2 2'に示す FRH 1 a n t iプライマーを用いた。  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. As a primer, a FRH1 anti primer shown in SEQ ID NO: 22 ′ that hybridizes to DNA encoding the end of the first framework sequence was used.
P C R溶液 Ι Ο Ο μ Ιは、 Ι Οίχ Ιの 10XPCR Buffer II、 2 mM M g C 12、 0. 16 mM dNTP s、 5ユニッ トの D N Aポリメラーゼ AmpliTaq Gold, ◦.4 xMずつの各プライマー、 及び 8 n gの鎵型 DNA (p s cMl) を 含有し、 95 °Cの初期温度にて 9分間そして次に 95°Cにて 1分間、 60°Cにて 1分間及ぴ 72 °Cにて 1分 20秒間、 この順序で加熱した。 この温度サイクルを 35回反復した後、 反応混合物を更に 72 °Cで 7分間加熱した。 The PCR solution Ι Ο Ο μ Ι, Ι Οίχ 10XPCR Buffer II of Ι, 2 mM M g C 1 2, 0. 16 mM dNTP s, 5 units of DNA polymerase AmpliTaq Gold, each primer by ◦.4 xM, And 8 ng of type I DNA (pscMl) at an initial temperature of 95 ° C for 9 minutes and then at 95 ° C for 1 minute, at 60 ° C for 1 minute and at 72 ° C. Heated in this order for 1 minute and 20 seconds. After repeating this temperature cycle 35 times, the reaction mixture was further heated at 72 ° C for 7 minutes.
PCR生成物を QIAquick PCR Purification Kit (QIAGEN社製） を用いて精製 し、 S a 1 I及ぴ Mb o II で消化し、 N末端側再構成 MAB L— 1抗体一本鎖 F Vをコードする DNA断片を得た。 また、 p s cMlベクターを Mb o II 及 び E c o R Iで消化し、 C末端側再構成 MAB L— 1抗体一本鎖 F vをコードす る DN A断片を得た。 そして、 S a 1 I— Mb o II DNA断片及び Mb o II一 Ec oR I DNA断片を p CHO 1— I g sベクターにクローニングした。 D NA配列決定の後、 正しい DNA配列を有する DNA断片を含むプラスミドを p CHOM1と命名した （図 6を参照）。 なお、 本発現ベクター p CHO 1— I g s は、 哺乳動物細胞分泌発現系に適するマウス I g G lシグナル配列 （Nature, 332, 323-327, 1988) を含んでいる。 本プラスミド！） C HOM 1に含まれる再構 成 M A B L- 1抗体一本鎖 F Vの塩基配列及びァミノ酸配列を配列番号： 2 3に 示す。 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. In addition, 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. After DNA sequencing, the plasmid containing the DNA fragment with the correct DNA sequence was named p CHOM1 (see FIG. 6). 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.
5. 2 再構成 MAB L— 2抗体一本鎖 F Vの作製  5.2 Preparation of reconstituted MAB L-2 antibody single chain FV
再構成 MAB L— 2抗体一本鎖 F Vを前記 5. 1に従って作製した。 第一 P C Reconstituted MAB L-2 antibody single chain FV was prepared according to 5.1 above. 1st PC
Rにおいては、 ； GEM— Ml Hの代わりに再構成 MAB L— 2抗体 H鎖 V領域 をコードするプラスミド p GEM— M2H (実施例 2を参照）、 及び p GEM— MIn R: GEM—plasmid pGEM—M2H encoding the reshaped MAB L-2 antibody H chain V region instead of MlH (see Example 2); and pGEM—M
1 Lの代わりに再構成 MA B L— 2抗体 L鎖 V領域をコ一ドするプラスミド] p GPlasmid coding for the reshaped MA B L-2 antibody L chain V region instead of 1 L] p G
EM-M2 L (実施例 2を参照） を使用し、 再構成 MAB L— 2抗体一本鎖 F V の正しいアミノ酸配列をコードする DNA断片を含むプラスミド p s cM2を得 た。 本プラスミ ド p s cM2に含まれる再構成] VIAB L— 2抗体一本鎖 F Vの塩 基配列及びァミノ酸配列を配列番号： 24に示す。 Using EM-M2 L (see Example 2), a plasmid p scM2 containing a DNA fragment encoding the correct amino acid sequence of the reshaped MAB L-2 antibody single chain F V was obtained. Reconstitution included in this plasmid pscM2] The base sequence and amino acid sequence of single chain FV of VIAB L-2 antibody are shown in SEQ ID NO: 24.
また、 p s cM2ベクターの修飾により再構成 MAB L— 2抗体一本鎖 F Vの 正しいァミノ酸配列をコードする D N A断片を含む哺乳動物細胞発現用 CHO M2ベクターを得た。 本プラスミド p CHOM2に含まれる再構成 MAB L— 2 抗体一本鎖 F Vの塩基配列及びァミノ酸配列を配列番号： 2 5に示す。  In addition, 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.
5. 3 CO S 7細胞への遺伝子導入  5.3 Gene transfer into COS 7 cells
再構成 MAB L— 2抗体一本鎖 F Vの一過性発現を観察するため、 p CHOM To observe the transient expression of the reconstituted MAB L-2 antibody single-chain FV, p CHOM
2ベクターを CO S 7細胞において試験した。 Two vectors were tested in COS 7 cells.
P CHOM2ベクターを、 Gene Pulser装置 （BioRad社製） を用いてエレクト 口ポレーシヨンにより CO S 7細胞に形質転換した。 DNA ( 1 0 g) と、 P The P CHOM2 vector was transformed into COS 7 cells by electoporation using a Gene Pulser apparatus (BioRad). DNA (10 g) and P
B S中 1 X 1 07細胞ノ m 1の 0. 8m 1をキュベットに加え、 1. 5 kV、 2 5 μThe 0. 8m 1 of BS in 1 X 1 0 7 cells Roh m 1 was added to the cuvette, 1. 5 kV, 2 5 μ
Fの容量にてパ^/スを与えた。 I gave a pass at the capacity of F.
室温にて 1 0分間の回復期間の後、 エレクトロポレーシヨン処理された細胞を、 1 0%のゥシ胎児血清を含有する I MDM培養液 （GIBC0 BRL社製） に加えた。 After a 10 minute recovery period at room temperature, the electroporated cells were added to an IMDM culture (GIBC BRL) containing 10% fetal calf serum.
7 2時間培養の後、 培養上清を集め、 遠心分離により細胞破片を除去して回収培 養上清を得た。 After culturing for 72 hours, the culture supernatant was collected, and cell debris was removed by centrifugation to obtain a recovered culture supernatant.
5^_4 CO S 7細胞培養上清 Φの再構成 MA B L- 2抗体一本 F vの検出 CHOM2ベクターを遺伝子導入した COS 7細胞培養上清中における再構 成 MA B L- 2抗体一本鎖 F Vをウェスタンブロッテイング法により確認した。 p CHOM 2ベクターを遺伝子導入した COS 7細胞培養上清及びコントロー ルとして p CHO 1ベクターを遺伝子導入した CO S 7細胞培養上清について S DS電気泳動を行い、 RE I NFORCED NC膜 （Schleicher & Schuell 社製） に転写した。 5%スキムミルク （森永乳業社製） にてブロッキングを行い、 0.05%Twe e n 20— PBSにて洗浄後、 抗 FLAG抗体 （SIGMA社製） を 加.え _た。 室温にてインキュベーション及び洗浄の後、 アルカリフォスファターゼ 結合抗マウス I gG抗体 （Zymed社製） を加え、 室温にてインキュベーション及 ぴ洗浄後、 基質溶液 （Kirkegaard Perry Laboratories 社製） を添加し、 発色さ せた （図 7)。 Reconstruction of 5 ^ _4 CO S 7 cell culture supernatant Φ Detection of single MA B L-2 antibody Fv The reconstituted MAB L-2 antibody single chain FV in the culture supernatant of COS 7 cells transfected with the CHOM2 vector was confirmed by Western blotting. SDS electrophoresis was performed on the COS 7 cell culture supernatant transfected with the pCHOM2 vector and the COS 7 cell culture supernatant transfected with the pCHO1 vector as a control, and the REINFORCED NC membrane (Schleicher & Schuell) was used. Co., Ltd.). After blocking with 5% skim milk (manufactured by Morinaga Milk Industry Co., Ltd.) and washing with 0.05% Tween 20-PBS, anti-FLAG antibody (manufactured by SIGMA) was added. After incubation and washing at room temperature, an alkaline phosphatase-conjugated anti-mouse IgG antibody (Zymed) is added. After incubation and washing at room temperature, a substrate solution (Kirkegaard Perry Laboratories) is added to allow color development. (Figure 7).
その結果、 p CHOM2ベクター導入 COS 7細胞培養上清中にのみ F LAG ぺプチド特異的なタンパク質が検出され、 この培養上清中に再構成 MAB L— 2 抗体一本鎖 F Vが分泌されていることが明らかとなった。  As a result, a FLAG peptide-specific protein was detected only in the culture supernatant of the COS 7 cell transfected with the p CHOM2 vector, and the reconstituted MAB L-2 antibody single-chain FV was secreted into the culture supernatant. It became clear.
5. 5 フローサイトメ トリー  5.5 Flow cytometry
抗原への結合を測定するため、 前記 COS 7細胞培養上清を用いてフローサイ 卜メ 卜ジーを行った。 ヒ卜 Integrin Associated Protein (I AP) を発現する マウス白血病細胞株 L 1210細胞、 あるいはコントロールとして p CO S 1ベ クタ一を形質転換した L 1210細胞 2X 105個に、 再構成 MABL— 2抗体一 本鎖 F Vを発現させた COS 7細胞の培養上清あるいはコントロールとして p C H〇 1ベクターを形質転換した COS 7細胞の培養上清を加え、 氷上にてインキ ュベーシヨン及び洗浄の後、 マウス抗 F LAG抗体 （SIGMA社製） を加えた。 ィ ンキュベーション及ぴ洗浄の後、 F I TC標識した抗マウス I gG抗体 （BECTON DICKINSON社製） を加えた。 再度インキュベーション及び洗浄の後、 FACS c a n装置 （BECTON DICKINSON社製） にて蛍光強度を測定した。 In order to measure the binding to the antigen, flow cytometry was performed using the COS 7 cell culture supernatant. Reconstituted MABL-2 antibody was expressed in mouse leukemia cell line L1210 cells expressing human Integrin Associated Protein (IAP) or 2X10 5 L1210 cells transformed with pCOS1 vector as control. Add the culture supernatant of COS 7 cells expressing the main chain FV or the culture supernatant of COS 7 cells transformed with pCH〇1 vector as a control, and incubate and wash on ice. Antibody (manufactured by SIGMA) was added. After incubation and washing, a FITC-labeled anti-mouse IgG antibody (BECTON DICKINSON) was added. After the incubation and washing again, the fluorescence intensity was measured using a FACS can device (manufactured by BECTON DICKINSON).
その結果、 再構成 MA BL-2抗体一本鎖 F Vは、 ヒト I A Pを発現する L 1 210細胞に特異的に結合したことにより、 この再構成 MAB L— 2抗体一本鎖 F Vがヒト Integrin Associated Protein に対するァフィ二ティーを有すること が明らかとなった （図 8〜1 1)。 As a result, the reconstituted MABL-2 antibody single-chain FV specifically bound to human IAP-expressing L1210 cells, and this reconstituted MAB L-2 antibody single-chain FV was converted to human Integrin Associated Have affinity for Protein (Figs. 8-11).
5. 6 C omp e t i t i v e E L I S A  5.6 C omp e t i t i v e E L I S A
マウスモノクローナノレ抗体の抗原結合に対する阻害活性を指標に、 再構成 MA B L- 2抗体一本鎖 F Vの抗原結合活性を測定した。  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.
1 μ g/m 1に調整した抗 F LAG抗体を 9 6ゥエルプレートの各ゥエルに加 え、 3 7°Cにて 2時間インキュベートした。 洗浄後、 1 °/。B S A— P B Sにてプ ロッキングを行った。 室温にてインキュベート及ぴ洗浄後、 分泌型ヒト I AP抗 原遺-伝子 （配列番号： 2 6) を導入した CO S 7細胞培養上清を P B Sにて 2倍 希釈したものを各ゥエルに加えた。 室温にてインキュベート及び洗浄後、 1 0 0 n g /m 1に調整したピオチン化 M A B L— 2抗体 5 0 1及び順次希釈した再 構成 MAB L— 2抗体一本鎖 F V発現 CO S 7細胞培養上清 5 0 μ 1を混和した ものを各ゥエルに加えた。 室温にてインキュベート及び洗浄後、 アルカリフォス ファターゼ結合ストレプトアビジン （Zymed社製） を加えた。 室温にてインキュ ベート及び洗浄後、 基質溶液 （SIGMA社製） を加え、 次に 40 5 nmでの吸光度 を ^定した。  The anti-FLAG antibody adjusted to 1 μg / 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. After incubation and washing at room temperature, 500 ng / ml of biotinylated MABL-2 antibody 501 and serially diluted reconstituted MABL-2 antibody single-chain FV-expressing COS 7 cell culture supernatant A mixture of 50 μl was added to each well. After incubation and washing at room temperature, alkaline phosphatase-conjugated streptavidin (Zymed) was added. After incubation and washing at room temperature, a substrate solution (manufactured by SIGMA) was added, and then the absorbance at 405 nm was determined.
その結果、 再構成 MAB L— 2抗体一本鎖 F V (MAB L 2— s c F v) は、 コントローノレの p CHO I導入 CO S 7細胞培養上清に比較して明らかに濃度依 存的にマウス MAB L— 2抗体のヒト I A P抗原への結合を阻害した （図 1 2)。 このことから、 再構成 MAB L— 2抗体一本鎖 F Vは、 マウスモノクローナル抗 体 MAB L— 2のそれぞれの V領域の正しい構造を有することが示唆された。 5. 7 in vitro でのアポトーシス誘起効果  As a result, 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
ヒト I A Pを遺伝子導入した L 1 2 1 0細胞、 及びコントロールとして p C O S 1ベクターを遺伝子導入した L 1 2 1 0細胞、 及び CCRF— CEM細胞を用 い、 再構成 MAB L— 2抗体一本鎖 F Vのアポトーシス誘起作用を An n .e X i n-V (B0EHRINGER MANNHEIM社製） 染色により検討した。  Reconstituted MAB L-2 antibody single chain using L1210 cells transfected with human IAP, L120 cells transfected with pCOS1 vector as a control, and CCRF-CEM cells The apoptosis-inducing effect of FV was examined by Ann.eXinV (B0EHRINGER MANNHEIM) staining.
各細胞 1 X 1 05個に、 再構成 MA B L- 2抗体一本鎖 F v発現 C O S 7細胞培 養上清あるいはコントロールとして; p CHO 1ベクター導入 CO S 7細胞培養上 清を終濃度 5 0 %で添加し、 24時間培養した。 その後、 An n e x i n— V染 色を行い、 FACS c a n装置 （BECTON DICKINSON社製） にて蛍光強度を測定し た。 Each cell 1 X 1 0 5 pieces, reconstruction MA B L-2 as the antibody single-chain F v expression COS 7 fine胞培Youe supernatant or control; p CHO 1 vector introduced CO S 7 final concentration on cell culture supernatant It was added at 50% and cultured for 24 hours. After that, An nexin—V dyeing The color was measured, and the fluorescence intensity was measured with a FACS can device (BECTON DICKINSON).
An n e x i n— V染色による解析の結果を図 1 3〜18にそれぞれ示した。 ここで、 図の左下の領域にあるドットは生細胞を、 右下の領域はアポトーシス初 期の細胞を、 右上の領域はアポトーシス後期の細胞を示す。 その結果、 再構成 M ABL— 2抗体一本鎖 Fv (MAB L 2 - s c F v) は L I 210細胞において ヒト I AP抗原特異的に著しい細胞死を誘導した （図 13〜16)。 また、 CCR F-.C EM細胞においてもコントロールに比較して著しい細胞死を誘導した （図 1 7〜18)。  The results of analysis by Annexin-V staining are shown in FIGS. 13 to 18, respectively. Here, 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, and the upper right area indicates cells in the late apoptosis. As a result, the reshaped MABL-2 antibody single-chain Fv (MAB L2-scFv) induced marked cell death in LI210 cells specifically for the human IAP antigen (FIGS. 13 to 16). In addition, CCR F-.C EM cells also induced remarkable cell death as compared to the control (FIGS. 17 to 18).
5.— 8— CHO細胞における MAB L— 2抗体由来の一本鎖 F Vポリぺプチドの  5.— 8— Single-chain F V polypeptide derived from MAB L-2 antibody in CHO cells
MAB L— 2抗体由来の一本鎖 F V (ポリペプチド） の恒常的発現 CHO細胞 株を樹立するため、 p CHOM2ベクターを CHO細胞に遺伝子導入した。 Constitutive Expression of Single-chain FV (Polypeptide) Derived from MAB L-2 Antibody To establish a CHO cell line, the pCHOM2 vector was transfected into CHO cells.
p CHOM2ベクターを、 Gene Pulser装置 （BioRad社製） を用いてエレクト 口ポレーシヨンにより CHO細胞に形質転換した。 DNA (10 /i g) と PB S に懸濁した CHO細胞 （丄 丄 ！^ァ細胞 !!^ ) の 0.7m 1を混合したものをキ ュベットに加え、 1. 5 kV、 25 μ Fの容量にてパルスを与えた。 室温にて 10 分間の回復期間の後、 エレクト口ポレーシヨン処理された細胞を、 10%のゥシ 胎児血清を含有する核酸不含ひ—MEM培地 （GIBCO BRL社製） に加え培養した。 得られたクローンについて、 SD S— PAGEにて目的とするタンパク質の発現 を確認し、 発現量の高いクローンを MAB L— 2抗体由来の一本鎖 F Vの産生細 胞株として選択した。 10 nM methotrexate (SIGMA社製） を含む無血清培地 CHO-S-SFM II (GIBCO BRL社製） にて培養後、 培養上清を集め、 遠心分 離により細胞破片を除去して回収培養上清を得た。.. - 5. 9 〇 0細胞産生の?^[ ；61^—2抗体由来の一本鎖 の精製  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 (丄 丄! ^ A cells !! ^) suspended in PBS is added to the cuvette, and the volume is 1.5 kV and 25 μF. A pulse was given at. After a recovery period of 10 minutes at room temperature, the cells subjected to electoral poration were added to a nucleic acid-free MEM medium (GIBCO BRL) containing 10% fetal bovine serum and cultured. With respect to the obtained clone, expression of the target protein was confirmed by SDS-PAGE, and a clone having a high expression level was selected as a cell line producing a single-chain FV derived from the MABL-2 antibody. 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 recover culture supernatant I got ..-5.9 Purification of single chain derived from? ^ [; 61 ^ -2 antibody produced by 〇0.9 cells
5. 8で得た一本鎖 F V発現 CHO産生株の培養上清を人工透析用カートリツ ジ （PAN1 30 SF、 旭メディカル） を用いて約 20倍まで濃縮した。 濃縮液 は一 20 °Cで保存し、 精製時解凍して用いた。 C HO細胞培養上清から一本鎖 F vの精製は、 Blue- sepharose、 ハイドロキシ ァパタイト及ぴゲル濾過の三種のクロマトグラフィーにより行った。 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.
上) Blue— sepharoseカラムクロマトグラフィー  Above) Blue— Sepharose column chromatography
培養上清の濃縮液を 2 OmM酢酸緩德 ί液 (ρΗ6.0) にて 10倍希釈し、 遠 心分離 （10000 r pmX 30分） により不溶物を除去した。 上清を同緩衝液 で平衡化した Blue- sepharoseカラム （20m l) に添加し、 同緩衝液で力ラムを 洗浄後、 同緩衝液中 N a C 1濃度を 0. 1、 0. 2、 0. 3、 0. 5及ぴ 1.0Mまで 段階.的に上げ、 カラムに吸着した蛋白質を溶出した。 SDS— PAGEで素通り 及び各溶出画分を分析し、 一本鎖 F Vが確認された画分 （0. 1〜0. 3M Na C I溶出画分） をプールし、 Centriprep- 10 (アミコン） を用いて約 20倍濃縮し た。  The concentrated solution of the culture supernatant was diluted 10-fold with 2 OmM acetic acid buffer (ρΗ6.0), 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 flow-through and each eluted fraction were analyzed by SDS-PAGE, and fractions in which single-chain FV was confirmed (0.1-0.3M NaCI eluted fraction) were pooled and centrifuged using Centriprep-10 (Amicon). And concentrated about 20 times.
(2) ハイドロキシァパタイト  (2) Hydroxyapatite
(1) の濃縮液を 1 OmM リン酸緩衝液 (pH7. 0) にて 10倍希釈し、 ノヽ ィドロキシァパタイトカラム ( 20 m 1、 BioRad) に添加した。 60 m 1の 10 mM リン酸緩衝液 （ p H 7.0 ) でカラムを洗浄後、 リン酸緩衝液濃度を 200 mMまで直線的に上げ、 カラムに吸着した蛋白質を溶出した （図 19)。 SDS- P AG Eにより各画分を分析した結果、 画分 A及び画分 Bに一本鎖 F Vが確認さ れた。  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.
(3) ゲル濾過  (3) Gel filtration
(2) の画分 A及ぴ Bをそれぞれ Centriprep- 10 を用いて濃縮し、 0. 15M Fractions A and B of (2) were concentrated using Centriprep-10, respectively.
N a C 1を含む 2 OmM酢酸緩衝液 (pH6. 0) で平衡化した TSKg e 1 G 3000 SWGカラム （21. 5 X 600mm) に添力 flした。 クロマトグラムを図 20に示す。 得られた画分を SD S— PAGEで分析した結果、 いずれも主要ピ ーク （A I、 B I) が目的の一本鎖 Fvであり、 ゲル濾過で分析した結果、. 画分 Aでは見かけ上の分子量約 36 kD、 画分 Bでは同 76 kDに溶出された。 精製 した一本鎖 F v (A I、 B I ) を 1 5%— SD S—ポリアクリルアミドゲルを用 いて分析した。 サンプルを還元剤添加、 非添加で処理し、 L a emml iの方法 に準じて電気泳動を行い、 泳動後蛋白質をクマシ一プリリアントブルー染色した。 図 21に示すように、 A I、 B Iいずれも還元剤の添加の有無に関わらず、 見か け上の分子量約 3 5 kDに単一パンドを与えた。 以上の結果から、 AIは一本鎖 F vのモノマーで、 B Iは一本鎖 F Vの非共有結合性ダイマーと考えられる。 画 分 A I及び B Iを TSKg e l G3000 S W力ラム （7. 5 X 60 mm) を用 いたゲル濾過により分析した結果、 画分 A Iはモノマーのピークのみ、 画分 B I はダイマーのピークのみ検出された （図 22を参照）。 また、 ダイマー画分 （画分 B I ) は、 全一本鎖 F Vの約 4%であった。 該ダイマ一画分中のダイマーは、 そ の 90 %以上が 4 °Cで 1ヶ月以上安定的に維持された。 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. As a result, the major peaks (AI, BI) were the single-stranded Fv of interest, and analyzed by gel filtration. Was eluted at a molecular weight of about 36 kD and in fraction B at 76 kD. The purified single-chain Fv (AI, BI) was analyzed using a 15% -SDS-polyacrylamide gel. The sample was treated with or without the addition of a reducing agent, subjected to electrophoresis according to the method of Laemmli, and after the electrophoresis, the protein was stained with Coomassie brilliant blue. As shown in FIG. 21, both AI and BI gave a single band with an apparent molecular weight of about 35 kD regardless of whether a reducing agent was added or not. These results suggest that AI is a monomer of single-chain Fv and BI is a non-covalent dimer of single-chain FV. Analysis of Fractions AI and BI by 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.
5. 10 大腸菌細胞での MAB L— 2抗体由来の一本鎖 F Vポリペプチド発現 ベクターの構築  5.10 Construction of single-chain FV polypeptide expression vector derived from MAB L-2 antibody in E. coli cells
MAB L— 2抗体由来の一本鎖 F Vを大腸菌菌体内にて効率的に発現するべク ターを作製するため、 p s cM2ベクターを PCR法により修飾した。 得られた DNA断片を！ SCFVT7発現べクタ一に導入した。  In order to prepare a vector that efficiently expresses a single-chain FV derived from the MAB L-2 antibody in E. coli cells, the pscM2 vector was modified by PCR. The obtained DNA fragment! It was introduced into SCFVT7 expression vector.
PCRに使用するプライマーは、 前方プライマーとして H鎖 V領域の N末端を コードする DNAにハイブリダィズし且つ開始コドン及び N d e I制限酵素認識 部位を有する配列番号： 27に示す Nd e— VHSmO 2プライマー及び後方プ ライマーとして L鎖 V領域の C末端をコードする DN Aにハイプリダイズし且つ 2個の停止コドン及び E c o R I制限酵素認識部位を有する配列番号： 28に示 す VLASプライマーを用いた。 なお、 前方プライマーの N d e—VHSmO 2 は大腸菌菌体内にて効率的に発現するため、 H鎖 V領域の N末端をコードする D N Aにハイブリダイズする部分に 5力所の点変異を含んでいる。  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. As a rear primer, 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. .
P C R溶液 Ι Ο Ο μ Ιは、 Ι Ο μ Ιの 10 XPCR Buffer # 1、 1 mM M g C l 2、 0. 2mM dNTP s、 5ユニットの KOD DNAポリメラーゼ (以上東洋紡社製）、 1 Mずつの各プライマー、 及び 100 n gの鑤型 DNA (p s cM2) を含有し、 98°Cにて 1 5秒間、 6 5 °Cにて 2秒間及び 74 °Cに て 30秒間、 この順序で加熱した。 この温度サイクノレを 25回反復した。 The PCR solution Ι Ο Ο μ Ι, 10 XPCR Buffer # 1 of Ι Ο μ Ι, 1 mM M g C l 2, ( all manufactured by Toyobo Co., Ltd.) 0. 2mM dNTP s, 5 units of KOD DNA polymerase, one by 1 M And 100 ng of type I DNA (pscM2) and heated in this order for 15 seconds at 98 ° C, 2 seconds at 65 ° C, and 30 seconds at 74 ° C. . This temperature cycle was repeated 25 times.
PCR生成物を QIAquick PCR Purification Kit (QIAGE 社製） を用いて精製 し、 Nd e I及び E c oR Iで消化し、 得られた DNA断片を p SCFVT 7ベ クタ一にクローニングした。 なお、 本発現ベクター p SCFVT 7は Nd e I及 び E c oR Iで消化したことにより； p e 1 Bシグナノレ配列が削除されている。 D N A配列決定の後、 正しい DN A配列を有する DN A断片を含むプラスミドを p s cM2DEmO 2と命名した （図 23を参照のこと）。 本プラスミド p s cM2 DEmO 2に含まれる MAB L— 2抗体由来の一本鎖 F vの塩基配列及びアミノ 酸配列を配列番号： 29に示す。 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. After DNA sequencing, 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.
5. 1 1 大腸菌細胞における MABL— 2抗体由来の一本鎖 F Vポリペプチド の発現  5.11 Expression of single-chain FV polypeptide derived from MABL-2 antibody in E. coli cells
MABL-2抗体由来の一本鎖 F Vポリぺプチドを発現する大腸菌株を得るた め、 p s c M2 D Em 0 2ベクターを大腸菌 B L 2 1 (D E 3 ) p L y s S In order to obtain an E. coli strain that expresses a single-chain FV polypeptide derived from the MABL-2 antibody, the p sc M2 D Em 02 vector was transformed into E. coli BL 21 (DE 3) p Lys S
(STRATAGENE社製） に形質転換した。 得られたクローンについて、 SDS— PA GEにて目的とするタンパク質の発現を検討し、 発現量の高いクローンを MAB L一 2抗体由来の一本鎖 F Vポリぺプチドの産生株として選択した。 (Manufactured by STRATAGENE). 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.
5. 1 2 大腸菌細胞産生の MAB L— 2抗体由来の一本鎖 F Vポリペプチドの 形質転換して得られた大腸菌のシングルコロニーを LB培地 3m 1にて 28°C で 7時間培養し、 これを 70 m 1の L B培地に植え継ぎ、 28 °Cにて一夜培養を 行った。 この！） r e— c u l t u r eを 7 の LB培地に植え,權ぎ、 ジャーファ ーメンターを用いて 28°C, 攪拌速度 300 r pmにて培養した。 O.D. = l. 5 のときに ImM I PTGで誘導をかけ、 その後 3時間培養を行った。  5.12 A single colony of Escherichia coli obtained by transforming a single-chain FV polypeptide derived from MABL-2 antibody produced by E. coli cells was cultured in 3 ml of LB medium at 28 ° C for 7 hours. Was inoculated into 70 ml of LB medium and cultured at 28 ° C overnight. this! ) R e-c u lt tur was inoculated in an LB medium of No. 7, cultivated, and cultured at 28 ° C using a jar fermenter at a stirring speed of 300 rpm. When O.D. = l.5, induction was performed with ImM I PTG, followed by culturing for 3 hours.
培養液を遠心分離 （10000 X g、 10分） し、 沈殿として回収した菌体に 5mM EDTA、 0. 1M Na C l、 1% T r i t on X— 100を含む 5 OmM トリス塩酸緩衝液 （pH8. 0) を加え、 超音波 （out put： 4、 duty cycle： 70%, 1分 X 10回） により菌体を石皮碎した。 この懸濁液を遠心分離 （1 2000 X gs 10分） にかけ、 沈殿として回収した封入体に 5 mM EDTA、 0. 1M Na C l、 4%T r i t o n X— 100を含む 5 OmM トリス塩酸 緩衝液 （pH8. 0) を加え、 再度超音波処理 (out put： 4、 duty cycle： 50%、 30秒 X 2) を行い、 遠心分離 （12000 X g、 10分） により目的蛋白質を沈 殿として回収し、 上清にくる夾雑蛋白質を除去した。 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. Solution (pH 8.0), sonicate again (out put: 4, duty cycle: 50%, 30 seconds X 2), and centrifuge (12000 X g, 10 minutes) to precipitate the target protein. And the contaminating proteins in the supernatant were removed.
目的蛋白質を含んだ封入体を 6 M Ur e a, 5 mM EDTA、 O. IM N a C lを含む 50mM トリス塩酸緩衝液 (pH8. 0) に溶解し、 4M Ur e a、 5mM EDTA、 O. IM Na C l、 10 mM メルカプトエタノールを 含む 50 mM トリス塩酸緩衝液 （p H 8.0) で平衡化した S e p h a c r y l S— 300 (5 X 90 cm、 AMERSHAM PHARMACIA社製） ゲル濾過カラムに、 流速 5 m l/分で添加し、 会合している高分子量の一本鎖 Fvを除去した。 各画分を SDS— P AGEで分析し、 純度の高い画分について、 O. D 280= 0. 25にな るようにゲル濾過で用いた溶媒で希釈後、 5 mM EDTA、 0. 1M Na C l、 0. 5M Ar g、 2 mM 還元型グルタチオン、 0. 2 mM酸化型ダルタチオン を含む 50mM トリス塩酸緩衝液 （pH8.0) に対して透析を 3回行うことに より、 卷き戻し操作を行った。 さらに 0. 15M N a C 1を含む 2 OmM酢酸 緩衝液 (pH6.0) に対して 3回透析し、 溶媒交換を行った。 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. Each fraction was analyzed by SDS-PAGE, and the highly purified fraction was diluted with the solvent used for gel filtration so that OD 280 = 0.25, and then diluted with 5 mM EDTA, 0.1 M NaOH. Unwinding is performed by performing dialysis three times against 50 mM Tris-HCl buffer (pH 8.0) containing Cl, 0.5 M Ar g, 2 mM reduced glutathione, and 0.2 mM oxidized daltathione. Was done. Furthermore, dialysis was performed three times against 2 OmM acetate buffer (pH 6.0) containing 0.15 M NaC1, and the solvent was exchanged.
わずかに含まれる分子間で S— S結合で架橋された高分子を分離除去するため、 0. 15M N a C 1を含む 2 OmM酢酸緩衝液 （pH6.0) で平衡化した S u p e r d e X 200 p g (2. 6 X 60 cm, AMERSHAM PHARMACIA社製） ゲル濾 過カラムに添加した。 図 24に示すように、 高分子量の会合体と考えられるプロ ードなピークのあと、 主要ピークとサブピークの 2つのピ^ "クが検出された。 S DS— PAGEによる分析 （図 21参照） 及びゲル濾過の溶出位置から、 主要ピ ' ークは一本鎖 F Vポリペプチドのモノマーであり、 サブピークは非共有結合性の ダイマーと考えられる。 なお、 形成された非共有結合性のダイマーは、 全一本鎖 F Vポリぺプチドの約 4 %であつた。  Superde X 200 equilibrated with 2 OmM acetate buffer (pH 6.0) containing 0.15M NaC1 to separate and remove macromolecules cross-linked by S—S bonds between a few contained molecules pg (2.6 × 60 cm, manufactured by AMERSHAM PHARMACIA) was added to a gel filtration column. As shown in Fig. 24, two peaks, a main peak and a sub-peak, were detected after the prone peak which is considered to be a high molecular weight aggregate. Analysis by SDS-PAGE (see Fig. 21) From the elution position and gel filtration, the main peak is considered to be a monomer of the single-chain FV polypeptide, and the sub-peak is considered to be a non-covalent dimer. It was about 4% of all single-chain FV polypeptides.
5. 1 3 MAB L— 2抗体由来の精製一本鎖 F Vポリペプチドの in vitro で のアポトーシス誘起効果  5.1 In vitro apoptosis-inducing effect of purified single-chain FV polypeptide derived from 13 MABL-2 antibody
ヒ ト I APを遺伝子導入した L 1210細胞 （h I AP/L 1210) を用い、 C H O細胞及び大腸菌細胞産生の M A B L-2抗体由来の一本鎖 F vポリぺプチ ド （MABL 2— s c Fv) のアポトーシス誘起作用を、 次の 2つのプロトコ一 ルにて An n e x i n— V (BOEHRINGER MANNHEIM社製） 染色により検討した。 第一のプロトコ一ノレは、 h I AP/L 1 2 1 0細胞 5 X 1 04個に、 抗体試料を 終濃度 3 g /m 1で添加し、 24時間培養した。 抗体試料として、 5. 9で得 た CH〇細胞由来 MAB L 2—本鎖 F Vのモノマー及びダイマー、 さらに 5. 1 2で得た大腸菌細胞由来の同モノマー及びダイマー、 そしてコントロールとして マウス I g G抗体について検討した。 培養後、 An n e X i n— V染色を行い、 FAC S e a n装置 （BECTON DICKINSON社製） にて蛍光強度を測定した。 Using L1210 cells transfected with human IAP (hIAP / L1210), single-chain Fv polypeptide (MABL2-sc) derived from MAB L-2 antibody produced by CHO cells and E. coli cells The apoptosis-inducing effect of Fv) was examined by Annexin-V (manufactured by BOEHRINGER MANNHEIM) staining with the following two protocols. In the first protocol, an antibody sample was added to 5 × 10 4 hIAP / L1210 cells at a final concentration of 3 g / ml and cultured for 24 hours. As antibody samples, the monomer and dimer of MAB L2-single-chain FV derived from CH〇 cells obtained in 5.9, the same monomer and dimer derived from Escherichia coli cells obtained in 5.12, and mouse IgG as a control Antibodies were studied. After the culture, Anne X in-V staining was performed, and the fluorescence intensity was measured with a FAC Sean device (manufactured by BECTON DICKINSON).
また、 第二のプロトコールは、 h I AP/L 1 2 1 0細胞 5 X 104個に、 抗体 試料 _を終濃度 3 μ g / 1で添加し、 2時間培養後に抗 F L AG抗体 （SIGMA社 製） を終濃度 1 5 μ g/m 1で添加し、 更に 2 2時間培養した。 抗体試料として、 5. 9で得た CHO細胞由来 MAB L 2—本鎖 F Vのモノマー及びコントロール としてマウス I g G抗体について検討した。 培養後、 An n e x i n— V染色を 行い、 FACS c a n装置にて蛍光強度を測定した。 In the second protocol, antibody sample _ was added at a final concentration of 3 μg / 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 μg / ml and the cells were further cultured for 22 hours. As antibody samples, 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.
An n e X i n— V染色による解析の結果を図 2 5〜3 1にそれぞれ示した。 その結果、 CH〇細胞及ぴ大腸菌細胞産生の MAB L— 2抗体由来一本鎖 F Vポ リペプチドのダイマーはコントロール （図 25) と比較して著しい細胞死を誘導 した （図 2 6、 2 7)'カ¾、 CHO細胞及び大腸菌細胞産生の一本鎖 F Vポリぺプ チドのモノマーのアポトーシス誘導作用は認められなかった （図 28、 2 9)。 ま た、 抗 FLAG抗体の添加により、 CHO細胞産生の MABL— 2抗体由来一本 鎖 F Vポリペプチドのモノマーはコントロール （図 3 0) と比較して著しい細胞 死を誘導した （図 3 1)。  The results of analysis by AnneXin-V staining are shown in FIGS. 25 to 31 respectively. As a result, the dimer of single-chain FV polypeptide derived from MAB L-2 antibody produced by CH〇 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 ¾, CHO cells and E. coli cells was not observed (FIGS. 28 and 29). In addition, 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).
5. 1 4 s c F vZCHOポリペプチドのモノマー及びダイマーのヒ ト骨髄腫 マウスモデルに対する抗腫瘍効果  5. Anti-tumor effect of 14 scF vZCHO polypeptide monomer and dimer on human myeloma mouse model
(1) マウス血？冑ヒト I gG定量法  (1) Mouse blood? Armor human IgG assay
マウス血清中における、 ヒト骨髄腫細胞が産生するヒ ト I g G (Mタンパク 質） の定量は、 以下の EL I S Aで行った。 0. 1%重炭酸緩衝液 (p H9. 6) で 1 μ gZm 1に希釈したャギ抗ヒト I g G抗体 （BIOSOURCE社製、 L o t # 7 902) Ι Ο Ο μ Ιを 96ゥエルプレート （Nunc社製） に加え、 4°Cでー晚ィン キュベーシヨンし、 抗体を固相化した。 ブロッキングの後、 段階希釈したマウス 血清あるいは標品としてヒ ト I g G (Cappel 社製、 Lo t # 00915) 100 μ 1を添加し、 室温にて 2時間インキュベーションした。 洗浄後、 5000倍希 釈したアルカリフォスファターゼ標識抗ヒト I g G抗体 （BI0S0URCE社製、 L o t # 6202) 100 μ 1を加え、 室温にて 1時間インキュベーションした。 洗 浄後、 基質溶液を加え、 インキュベーショ ンの後、 MICROPLATE READER Model 3550 (BioRad社製） を用いて 405 nmの吸光度を測定し、 標品のヒ ト I g Gの 吸光度より得られた検量線から、 マウス血清中のヒト I gG (Mタンパク質） 濃 度を算出した。 Quantification of human IgG (M protein) produced by human myeloma cells in mouse serum was performed by the following ELISA. Goat anti-human IgG antibody (BIOSOURCE, Lot # 7 902) diluted to 1 μg Zm1 with 0.1% bicarbonate buffer (pH 9.6) 96 μl of ャ Ο Ο μ Ι The plate was added to a plate (manufactured by Nunc) and subjected to vacuum convection at 4 ° C to immobilize the antibody. Serially diluted mice after blocking Serum or 100 μl of human IgG (Lot # 00915, manufactured by Cappel) as a standard was added, and the mixture was incubated at room temperature for 2 hours. After washing, 100 μl of 5,000-fold diluted alkaline phosphatase-labeled anti-human IgG antibody (Lot # 6202, manufactured by BI0S0URCE) was added, and the mixture was incubated at room temperature for 1 hour. After washing, the substrate solution was added, and after incubation, the absorbance at 405 nm was measured using MICROPLATE READER Model 3550 (manufactured by BioRad), and the calibration curve obtained from the absorbance of the standard human IgG. From this, the human IgG (M protein) concentration in the mouse serum was calculated.
(2) 投与抗体の調製  (2) Preparation of administration antibody
s c F vZCHOポリペプチドのモノマー及びダイマーは、 投与当日、 濾過滅 菌した PB S (—） を用いて、 それぞれ 0.4mg/m 1、 O. S SmgZm lに なるように調製し、 投与試料とした。  On the day of administration, 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. .
( 3 ) ヒ ト骨髄腫マゥスモデルの作製  (3) Preparation of human myeloma mouse model
ヒト骨髄腫マウスモデルは以下のように作製した。 SC I Dマウス （日本タレ ァ） を用いて in vivo継代した KP MM 2細胞 （特開平 7 _ 236475号公 報） を 1 0%ゥシ胎児血清 （GIBC0 BRL 社製） を含む RPM I 1 640培地 (GIBC0 BRL社製） で 3 X 107個/ m 1になるように調製した。 あら力 じめ前日 抗ァシァ口 GM 1抗体 （和光純薬社製、 1バイアルを 5 m 1で溶解） 100 ^ 1 を皮下投与した S C I Dマウス （ォス、 6週齢） （日本クレア） に上記 KPMM2 細胞懸濁液 200 μ1 (6 106個 Ζマウス） を尾静脈より注入した。 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 day before the first day of anti-ashia mouth GM1 antibody (manufactured by Wako Pure Chemical Industries, 1 vial dissolved at 5 ml) SCID mice (os, 6 weeks old) (Clear Japan) administered 100 ^ 1 subcutaneously as described above 200 μl of KPMM2 cell suspension (610 6 cells / mouse) was injected into the tail vein.
(4) 抗体投与  (4) Antibody administration
( 3 ) で作製したヒト骨髄腫マゥスモデルに対し、 K P MM 2細胞移植後 3日 目より、 1日 2回、 3日間、 上記 （2) で調製した投与試料、 モノマーは 250 β 1、 ダイマーは 400 /ζ 1を、 尾静脈より投与した。 対照として、 濾過滅菌し た PBS (—） を同様に 1曰 2回、 3日間、 200 μ 1、 尾静脈より投与した。 両群とも、 1群 7匹で行った。  In the human myeloma mouse model prepared in (3), the administration sample prepared in (2) above, twice daily for 3 days from day 3 after KP MM 2 cell transplantation, the monomer was 250 β1, and the dimer was 400 / ζ1 was administered via the tail vein. As a control, filter-sterilized PBS (-) was similarly administered twice a day for three days at 200 μl via the tail vein. Both groups were performed with 7 animals per group.
(5) s c F v/CHOポリペプチドのモノマー及ぴダイマーのヒト骨髄腫移植 マウスモデルに対する抗腫瘍効果の評価 s c F v/CHOポリべプチドのモノマー及びダイマーのヒト骨髄腫マウスモ デルの抗腫瘍効果については、 当該骨髄腫細胞が産生するヒ ト I g G (Mタンパ ク質） のマウス血清中の量の変化、 及び生存期間で評価した。 マウス血清中のヒ ト I g G量の変化については、 KP MM 2細胞移植後 24日目に血清を採取し、 上記 （1 ) で述べた E L I S Aを用いてヒ ト I g G量を測定した。 その結果、 P B S (一） 投与群では、 血清ヒト I g G (Mタンパク質） 量が約 8 5 0 0 gZ m 1まで上昇しているのに対し、 s c F vZCHQダイマー投与群では対照群の 0以下と顕著に低値であり、 s c F V//CHOダイマーが KPMM2細胞 の増殖を非常に強く抑制していることが示された （図 3 2)。 一方、 生存期間につ いても図 3 3に示すとおり、 s c F vZCHOダイマー投与群では PB S (—） 投与群と比較して顕著な生存期間の延長が認められた。 (5) Evaluation of antitumor effect of scFv / CHO polypeptide monomer and dimer on human myeloma transplant mouse model The antitumor effect of the scFv / CHO polypeptide monomer and dimer on the human myeloma mouse model was determined by determining the amount of human IgG (M protein) produced by the myeloma cells in the mouse serum. Changes and survival were assessed. For the change in the amount of human IgG in mouse serum, serum was collected on day 24 after KPMM2 cell transplantation, and the amount of human IgG was measured using the ELISA described in (1) above. . As a result, in the PBS (1) administration group, the serum human IgG (M protein) level was increased to about 8500 gZm1, whereas in the scFvZCHQ dimer administration group, 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). On the other hand, as shown in Fig. 33, the survival time was significantly prolonged in the scFvZCHO dimer administration group as compared with the PBS (-) administration group.
以上より、 s c F vZCHOダイマーがヒト骨髄腫マウスモデルに対して、 抗 腫瘍効果を有することが示された。 本発明の改変抗体である s c F vZCHOダ イマ一の抗腫瘍効果は、 当該改変抗体が有するアポトーシス誘起作用に基づくと 考えられる。  As described above, 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.
5. 1 5 赤血球凝集試験  5.15 Hemagglutination test
赤血球凝集試験及び赤血球凝集の判定法は、 続生化学実験講座の免疫生化学研 究法 （日本生化学会編、 東京化学同人） に準じて実施した。  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).
健常人の血液をへパリン処理した注射筒により採血し、 P B S (—） により 3 回洗浄した後、 P B S (—） にて最終濃度が 2%の赤血球浮遊液を作製した。 検 査サンプルは、 対照としてマウス I g G (Zymed社製） を用い、 MAB L— 2抗 体、 CHO細胞産生の一本鎖 F Vポリペプチドモノマー、 ダイマー、 大腸菌産生 の一本鎖 F Vポリペプチドのモノマーとダイマーを使用した。 赤血球の凝集作用 を検討するために、 ファルコン社製の U底の 9 6ゥエルプレートを使用し、. 上記 の抗体サンプルを 5 0 1ノゥエル添加した中に、 2 %赤血球浮遊液をさらに 5 0 μ 1添加、 混和し、 3 7°Cで 2時間ィンキュベーション後、 4 °Cで一昼夜保存 し、 凝集を判定した。 また、 対照として、 P B S (—） を 5 0 ^ 1 /ゥエル添加 し、 抗体サンプルと同様にして凝集試験を行った。 抗体の最終濃度は、 マウス I g G、 MAB L— 2抗体は、 0. 0 1、 0. 1、 1、 1 0、 1 00 μ gZm 1、 一 本鎖 F vは、 0. 004、 0. 04、 0. 4、 4、 40、 80 μ g Zm 1で大腸菌産 生の一本鎖 F Vポリペプチドのダイマーのみさらに 1 6 0 g/m 1の用量を設 定した。 その結果は、 下記の表 2に示す通り、 MABL— 2抗体では、 0. 1 /z g /m 1以上で赤血球凝集が見られたのに対し、 一本鎖 F Vポリペプチドではモノ マー、 ダイマー共に赤血球凝集は認められなかつた。 Blood of a healthy person was collected by a heparin-treated syringe, washed three times with PBS (-), and then a red blood cell suspension having a final concentration of 2% was prepared with PBS (-). As a control, mouse IgG (Zymed) was used as a control, and MABL-2 antibody, CHO cell-produced single-chain FV polypeptide monomer, dimer, and Escherichia coli-produced single-chain FV polypeptide were used as controls. Monomers and dimers were used. To examine the agglutination of erythrocytes, use a Falcon U-bottomed 96-well plate. Add 50% of the 2% erythrocyte suspension to the above-mentioned antibody sample after adding 501 wells. μ1 was added, mixed, and incubated at 37 ° C for 2 hours, and then stored at 4 ° C for 24 hours to determine aggregation. As a control, PBS (-) was added at 50 ^ 1 / well, and an agglutination test was performed in the same manner as the antibody sample. The final antibody concentration is mouse I g G, MAB L-2 antibody: 0.01, 0.1, 1, 10, 100 μgZm1, single-chain Fv: 0.004, 0.04, 0.4, 4 Further, a single-chain FV polypeptide-dimer-produced dimer produced by Escherichia coli at 40, 80 μg Zm 1 was further dosed at 160 g / m 1. The results show 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.
赤血球凝集試験 対照 0.01 0.1 1 10 100 (μ /πιΐ) Hemagglutination test control 0.01 0.1 1 10 100 (μ / πιΐ)
mlgG  mlgG
MABL-2 (intact) + +++ +++ ++  MABL-2 (intact) + +++ +++ ++
対照 0.004 0.04 0.4 4 40 80 ^g/ml) scFv/CHOモノマ- scFv/CHOタ'イマ—  Control 0.004 0.04 0.4 4 40 80 ^ g / ml) scFv / CHO monomer-scFv / CHO monomer
対照 0.004 0.04 0.4 4 40 80 160 ml) scFv/E. coli モノマー  Control 0.004 0.04 0.4 4 40 80 160 ml) scFv / E. Coli monomer
scFv/E. coli タ イマ一 実施例 6 2つの H鎖 V領域及び 2つの L鎖 V領域を含む改変抗体 s c (Fv)2及 び種々の長さのぺプチドリンカーを有する MAB L— 2抗体 s c F V 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
6. 1 MAB L— 2抗体 s c (F 発現プラスミ ドの構築 6.1 Construction of MAB L-2 antibody sc (F expression plasmid
MA B L— 2抗体由来の 2つの H鎖 V領域及び 2つの L鎮 V領域を含む改変抗 体 [s C (F v)2] を発現するプラスミドを作製するため、 前述 p CHOMS (M AB L— 2抗体由来の s c F Vをコードする DNAを含む） を以下に示す通り P CR法により修飾し、 得られた DNA断片を p CHOM2に導入した。 To prepare a plasmid that expresses a modified antibody [s C (Fv) 2 ] containing two H chain V regions and two L protein V regions derived from the MABL-2 antibody, 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.
P CRに使用するプライマーは、 センスプライマーとして EF 1 αをコードす る DNAにハイプリダイズする EF 1プライマー （配列番号： 3 0) を使用し、 アンチセンスプライマーとして L鎖 V領域の C末端をコードする DNAにハイブ リダィズし且つリンカ一領域をコードする DNA配列 （配列番号： 19) 及ぴ S a 1 I制限酵素認識部位を有する VL LASプライマー （配列番号： 3 1) を使 用した。 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, As an 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溶液 100 /i lは、 10 1の 10 XPCR Buffer # 1、 1 mM M gC l 2、 0. 2mM dNTP s (dATP、 dGTP、 dCTP、 dTTP)ヽ 5ユニットの KOD DNAポリメラーゼ （以上東洋紡社製）、 1 Mの各プライ マー.、 及び 100 n gの铸型 DNA (p CHOM2) を含有する。 PCR溶液を 94 °Cにて 30秒間、 50 °Cにて 30秒間及び 74 °Cにて 1分間、 この順序で加 熱した。 この温度サイクルを 30回反復した。 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)ヽ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.
PCR生成物を QIAquick PCR Purification Kit (QIAGEN社製） を用いて精製 し、 S a i lで消化し、 得られた DNA断片を p B l u e s c r i p t KS+ベ クタ一 （東洋紡社製） にクローニングした。 DN A配列決定の後、 正しい DNA 配列を有する DNA断片を含むプラスミドを S a 1 Iで消化し、 得られた DNA 断片を S a 1 Iで消化した！） CHOM2に Rapid DNA Ligation Kit (BOEHRINGER MANNHEIM社製） を用いて連結した。 DNA配列決定の後、 正しい DNA配列を有 する DNA断片を含むプラスミドを！ CHOM2 (F v)2と命名した （図 34を参 照）。 本プラスミド！） CHOM2 (F v)2に含まれる MAB L— 2抗体 s c (F v) 2領域の塩基配列及びァミノ酸配列を配列番号： 32に示す。 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.
6. 2 種々の長さのペプチドリンカ一を有する MAB L— 2抗体 s c F V発現 プラスミ ドの作製 6.2 Preparation of MAB L-2 antibody scFV expression plasmid with peptide linkers of various lengths
種々の長さのペプチドリンカ一を有し、 そして [H鎖] 一 [L鎖] (以下 HL)、 [L鎖] ― [H鎖] (以下 LH) となるように V領域を連結した s cFvを、 MA BL— 2由来の H鎖及び L鎖 cDNAを铸型として以下の通りに作製した。 - HLタイプの s c F Vを作製するために、 まず p CHOM2 (F v)2を铸型とし て CFHL— F 1 (配列番号： 33) 及び CFHL— R2 (配列番号： 34) プ ライマー、 CFHL— F 2 (配列番号： 35) 及ぴ C F H L— R 1プライマー (配列番号： 036) により KODポリメラーゼにて 94°C30秒、 60°C30 秒、 72 °C1分間の反応を 30回繰り返す PC R反応を行い、 5'側にリーダー配 列を含む H鎖、 及び 3'側に F L AG配列を含む L鎖の c DN A遺伝子を作製した。 得られた H鎖及び L鎖 cDNAを铸型として混合し、 KODポリメラーゼにて 9 4°C30秒、 60°C30秒、 72 °C1分間の反応を 5回操り返す PC R反応を行 い、 CFHL— F 1及び CFHL— R 1プライマーを加えてさらに 30サイクル 反応することによりリンカーを含まない HL— 0タイプの cDNAを作製した。 It has peptide linkers of various lengths, and connects the V regions so that [H chain]-[L chain] (hereinafter HL), [L chain]-[H chain] (hereinafter LH) cFvs were prepared as follows using the H and L chain cDNAs derived from MABL-2 as type III. -To prepare HL-type sc FV, 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. . The obtained 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タイプの s c F vを作製するために、 まず MAB L— 2の L鎖及び H鎮 V 領域.の c DNAを含むプラスミド p GEM-M2 L及び p GEM— M2H (特願 平 1 1一 63557参照） を铸型として、 それぞれ T 7 (配列番号： 37) 及び CF LH-R 2 (配列番号： 38) プライマー、 CFLH— F 2 (配列番号： 3 9) 及び CFLH— R 1 (配列番号： 40) プライマーを用いて KODポリメラ ーゼ （東洋紡） にて 94 °C 30秒、 60 °C 30秒、 72 °C 1分間の反応を 30回 繰り返す P C R反応を行い、 5 '側にリ一ダー配列を含む L鎖、 及び 3 '側に F L A G配列を含む H鎖の c D N A遺伝子を作製した。 得られた L鎖及び H鎖 c D N Aを铸型として混合し、 KODポリメラーゼにて 94°C30秒、 60°C30秒、 72 °C1分間の反応を 5回繰り返す PC R反応を行い、 T 7及び CFLH— R 1 プライマ を加えてさらに 30サイクル反応した。 この反応産物を铸型とし、 CIn 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. 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 铸, 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 铸, C
F LH-F 4 (配列番号： 41) 及び CFLH— R 1プライマーを用いて 94°C 30秒、 60°C30秒、 72 °C1分間の反応を 30回繰り返す PC R反応を行う ことによりリンカーを含まない LH— 0タイプの cDNAを作製した。 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.
こうして作製した LH— 0、 HL— 0タイプの cDNAを制限酵素 E c oR I、 B amHI (宝酒造） 処理し、 X h o I制限酵素切断部位を含まない哺乳動物発 現プラスミ ド I N P E P 4に Ligation High (東洋紡） を用いて導入し、 Competent E. coli JM109 (二ツボンジーン) を形質転換した。 形質転換し た大腸菌より QIAGEN Plasraid Maxi Kit (QIAGEN) にてプラスミドを精製した。 こうしてプラスミド: CF 2 LH— 0及ぴ p CF 2HL— 0を作製した。  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.
次に、 リンカ一サイズの異なる発現プラスミドを作製するために HLタイプで は p CF 2HL— 0を铸型として CFHL— X3 (配列番号： 42)、 CFHL- X4 (配列番号： 43)、 CFHL-X 5 (配列番号： 44)、 CFHL-X6 (配列番号： 45)、 又は CFHL— X7 (配列番号： 46) のセンスプライマー 及びアンチセンスプライマーとしてベクター配列に相補的な BGH— 1 (配列番 号： 47) プライマーを用いて KODポリメラーゼにて 94°C30秒、 60。C3 0秒、 72°C1分間の反応を 30回繰り返す PCR反応を行い、 得られた反応産 物を制限酵素 Xh o I、 B amH I (宝酒造） にて処理した。 得られた断片を p CF 2HL— 0の Xh o I、 B amH Iサイトに Ligation High (東洋紡） を用 い 導入し、 Competent E. coli J M 109を形質転換した。 形質転換した大腸 菌より QIAGEN Plasmid Maxi Kit にてプラスミドを精製した。 こうして、 発現プ ラスミ ド p CF 2HL— 3、 p CF 2HL-4, p CF 2HL— 5、 p CF 2H L一 6及ぴ p CF 2HL— 7を作製した。 更に COS 7細胞での一過的発現に用 いる発現プラスミドを作製するために、 pCF 2HL— 0、 p CF 2HL- 3N p CF 2HL— 4、 p CF 2HL— 5、 p C F 2 H L— 6及び p C F 2 H L— 7 を制限酵素 E c o R I及び B a mH I (宝酒造） にて処理し、 約 800 b pの断 片をァガロースゲル電気泳動によるゲルからの回収により精製した。 得られた断 片を哺乳動物細胞発現プラスミド: COS lの E c oR I及び B a mH Iサイト に Ligation High を用いて導入し、 Competent E. coli DH5 α (東洋紡） を形 質転換した。 形質転換した大腸菌より QIAGEN Plasmid Maxi Kit にてプラスミド を精製した。 こうして、 発現プラスミ ド CF 2HL— 0/P COS 1、 CF 2H L一 3/p COS l、 CF 2HL— 4/p COS l、 CF 2HL-5/p COS 1、 CF 2HL-6/ COS 1及び CF 2HL- 7/p CO S 1を作製した。 代表的な例として、 プラスミド CF 2HL-0/p CO S 1の構造を図 35に示 し、 これに含まれる MABL 2— s c F v<HL— 0〉の塩基配列及びアミノ酸 配列を配列番号： 48に示す。 また各プラスミドのリンカ一部分の塩基配列及び ァミノ酸配列を図 3 に示す。 Next, in order to prepare expression plasmids with different linker sizes, 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. A PCR reaction in which the reaction at C30 seconds and 72 ° C for 1 minute was repeated 30 times was performed, and the resulting reaction product was treated with restriction enzymes XhoI and BamHI (Takara Shuzo). The obtained fragment was introduced into the XhoI and BamHI sites of pCF2HL-0 using Ligation High (Toyobo) to transform Competent E. coli JM109. Plasmids were purified from the transformed E. coli using the QIAGEN Plasmid Maxi Kit. Thus, the expression plasmids pCF2HL-3, pCF2HL-4, pCF2HL-5, pCF2HL-16 and pCF2HL-7 were produced. To further prepare the use are expression plasmids for transient expression in COS 7 cells, 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 DH5α (Toyobo). The plasmid was purified from the transformed E. coli using the QIAGEN Plasmid Maxi Kit. Thus, the expression plasmids CF 2HL-0 / P COS 1, CF 2H L-1 3 / p COS 1, CF 2HL-4 / p COS 1, CF 2HL-5 / p COS 1, CF 2HL-6 / COS 1, and CF 2HL-7 / p CO S 1 was prepared. As a typical example, the structure of plasmid CF2HL-0 / pCOS1 is shown in FIG. 35, and the nucleotide sequence and amino acid sequence of MABL2-scFv <HL-0> contained therein are shown in SEQ ID NO: See 48. FIG. 3 shows the nucleotide sequence and amino acid sequence of a part of the linker of each plasmid.
また、 リンカ一サイズの異なる LHタイプの発現プラスミドを作製するため、 p CF 2 LH— 0を铸型として CF LH— X 3 (配列番号： 49)、 CFLH— X 4 (配列番号： 50)、 CFLH-X 5 (配列番号： 5 1)、 CFLH-X6 (配 列番号： 52) 又は CFLH— X7 (配列番号： 53) のセンスプライマー及び アンチセンスプライマーとしてベクター配列に相捕的な; BGH— 1プライマーを 用いて KODポリメラーゼにて 94°C30秒、 60°C30秒、 72°C1分間の反 応を 30回繰り返す PC R反応を行い、 得られた反応産物を制限酵素 Xh o I、 B amH Iにて処理した。 得られた断片を p CF 2 LH— 0の Xh 0 I、 B am HIサイトに Ligation High を用いて導入し、 Competent E. coli DH5 α (東 洋紡） を形質転換した。 形質転換した大腸菌より QIAGEN Plasmid Maxi Kit にて プラ ミドを精製した。 こぅして、 発現プラスミド 〇 2 «—3、 p CF 2 LH— 4、 p C F 2 LH- 5, p C F 2 LH— 6及ぴ p C F 2 LH— 7を作製し た。 更に COS 7細胞での一過的発現に用いる発現プラスミドを作製するために、 P CF 2 LH— 0、 p CF 2 LH— 3、 pCF 2 LH— 4、 p CF 2LH— 5、 p C F 2 LH— 6及び p C F 2 LH- 7を制限酵素 E c o R I及び B a mH I (宝酒造） にて処理し、 約 800 b pの断片をァガロースゲル電気泳動によるゲ ルからの回収により精製した。 得られた断片を哺乳動物細胞発現プラスミド p C 〇 S.lの E cひ R I及び B a mH Iサイトに Ligation High を用いて導入し、 Competent E. coli DH5 α (東洋紡） を形質転換した。 形質転換した大腸菌よ り QIAGEN Plasraid Maxi Kit にてプラスミドを精製した。 こうして、 発現プラス ミド CF 2 LH— 0/p COS l、 C F 2 LH- 3/p CO S 1 N CF 2 LH— 4/p CO S 1. C F 2 LH- 5/p CO S 1 s C F 2 L H— 6ノ p C O S 1及 び CF 2 LH— 7Zp COS 1を作製した。 代表的な例として、 プラスミド CF 2 LH- 0/p COS 1の構造を図 37に示し、 これに含まれる MAB L 2— s c F v<LH-0>の塩基配列及ぴァミノ酸配列を配列番号： 54に示す。 また 各プラスミドのリンカ一部分の塩基配列及ぴァミノ酸配列を図 38に示す。 In addition, in order to prepare LH-type expression plasmids with different linker sizes, 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 DH5α (Toyobo). Plasmid was purified from the transformed E. coli using QIAGEN Plasmid Maxi Kit. Thus, expression plasmids 〇2 «-3, pCF2LH-4, pCF2LH-5, pCF2LH-6 and pCF2LH-7 were prepared. Furthermore, in order to prepare expression plasmids used for transient expression in COS 7 cells, 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〇Sl using Ligation High to transform Competent E. coli DH5α (Toyobo). Plasmids were purified from the transformed E. coli using the QIAGEN Plasraid Maxi Kit. Thus, the expression plasmid CF 2 LH—0 / p COS1, CF 2 LH-3 / p CO S 1 N CF 2 LH—4 / p CO S 1.CF 2 LH-5 / p CO S 1 s CF 2 LH-6 noCOS1 and CF2LH-7ZpCOS1 were prepared. As a representative example, the structure of plasmid CF 2 LH-0 / p COS 1 is shown in FIG. 37, and the base sequence and amino acid sequence of MAB L 2 — sc F v <LH-0> contained in the sequence are shown in FIG. No .: Shown at 54. FIG. 38 shows the nucleotide sequence and amino acid sequence of the linker part of each plasmid.
6. 3 COS 7細胞における s c F V及び s c (F V) の発現 6.3 Expression of scFV and sc (FV) in COS 7 cells
(1) 有血清培地での培養上清の調製  (1) Preparation of culture supernatant in serum-containing medium
HLタイプ、 LHタイプ s c F V及ぴ s c (F v)2の発現のために、 COS 7細 胞 （J CRB 9 127、 ヒューマンサイエンス振興財団） での一過的発現を行つ た。 COS 7細胞は 1 0%牛胎児血清 （HyClone) を含む DMEM培地 （GIBC0 BRL社製） にて、 37 °Cの炭酸ガス恒温槽中で経代培養した。 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.
6. 2で構築した C F 2HL— 0, 3〜7/p C〇S l、 もしくは CF 2 LH— 0， 3〜7ノ1)〇031又は p CHOM2 (F v)2ベクターを、 Gene Pulser装置6. The CF 2HL—0, 3 to 7 / p C lS l or CF 2 LH—0, 3 to 7 1) 〇031 or p CHOM2 (F v) 2 vector constructed in step 2
(BioRad社製） を用いてエレクトロポレーシヨンにより COS 7細胞にトランス フエクシヨンした。 (BioRad) and electrotransformed into COS 7 cells.
DNA ( 10 μ g) と DMEM ( 1- 0 % F B S , 5 mM BE S (S I GMA 社)） 培地中 2 X 107細胞 Zm 1の 0.25m 1をキュべッ トに加え、 10分間静 置.の _後に 0. 17 kV、 950 Fの容量にてパルスを与えた。 1 0分間静置の後、 エレクト口ポレーシヨンされた細胞を DMEM ( 10 % F B S ) 培地に混合し、 75 cm3フラスコに加えた。 72時間培養後、 培養上清を集め、 遠心分離により 細胞破片を除去し、 更に 0.22 mボトルトップフィルター （FALCON) に て濾過し、 これを培養上清 (CM) とした。 DNA (10 μg) and DMEM (1-0% FBS, 5 mM BES (SI GMA)) Add 0.25 ml of 2 x 10 7 cells Zm1 in the medium to the cuvette and let stand for 10 minutes A pulse of 0.17 kV, 950 F capacity was given after _. After standing for 10 minutes, the cells subjected to electoral poration were mixed with a DMEM (10% FBS) medium and added to a 75 cm 3 flask. After culturing for 72 hours, the culture supernatant was collected, cell debris was removed by centrifugation, and further filtered through a 0.22 m bottle top filter (FALCON) to obtain a culture supernatant (CM).
(2) 無血清培地での培養上清の調製  (2) Preparation of culture supernatant in serum-free medium
上記 （1) と同様の方法でトランスフエクシヨンした細胞を DMEM (10% FBS) 培地に加え 75 cm3フラスコにて一夜培養した後、 培養上清を捨て、 P BSにて洗浄後、 CHO— S— SFM II培地 （GIBCO BRL社製） を添加した。 7 2時間培養後、 培養上清を集め、 遠心分離により細胞破片を除去し、 更に 0. 22 ： mポトノレトップフィルタ一にて濾過し、 CMを得た。 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.
6. 4 COS 7 CM中の s c F V及び s c (F v)9の検出 6.4 Detection of sc FV and sc (F v) 9 in COS 7 CM
前記 6. 3 (2) で調製した CO S 7の CM中における種々の MAB L 2— s c V及び s c (F V) 2のポリペプチドを下記の通りにウェスタンブロッティング 法により検出した。 Various MABL2-scV and sc (FV) 2 polypeptides in the CM of COS7 prepared in 6.3 (2) were detected by Western blotting as described below.
各 COS 7 CMについてについて SD S— PAGEを行い、 RE I NFOR CED NC膜 （Schleicher & Schuell 社製） に転写した。 · 5 %スキムミルク (森永乳業社製） にてブロッキングを行い、 TB Sにて洗浄後、 抗 FLAG抗体 (SIGMA社製） を加えた。 室温にてインキュベーション及び洗净の後、 ペルォキ シダーゼ標識抗マウス I gG抗体 （Jackson Irarauno Research社製） を加え、 室 温にてインキュベーション及ぴ洗浄後、 基質溶液を添加し、 発色させた （図 39)。 6. 5 フローサイ トメ トリー For each COS 7 CM, SDS-PAGE was performed and transferred to a REINFOR CED NC membrane (Schleicher & Schuell). · After blocking with 5% skim milk (manufactured by Morinaga Milk Industry Co., Ltd.) and washing with TBS, anti-FLAG antibody (manufactured by SIGMA) was added. After incubation and washing at room temperature, a peroxidase-labeled anti-mouse IgG antibody (manufactured by Jackson Irarauno Research) was added. After incubation and washing at room temperature, a substrate solution was added to develop color (FIG. 39). ). 6.5 Flow cytometry
MA B L 2— s c F v及び s c (F v)2のヒ ト Integrin Assosiated Protein (I AP) 抗原への結合を測定するため、 前記 6. 3 (1) にて調製した COS 7細胞培養上清を用いてフローサイトメトリーを行った。 ヒト I APを発現する マウス白血病細胞株 L 1210細胞 2 X 105個に、 実施例 6. 3 (1) で得られ た培養上清あるいは対照として COS 7細胞の培養上清を加え、 氷上にてインキ ュベーシヨン及び洗浄の後、 10 μ g/m 1のマウス抗 F LAG抗体 （SIGMA社 製） _を加えた。 ィンキュベーシヨン及び洗浄の後、 F I TC標識抗マウス I g G 抗体 （BECTON DICKINSON社製） を加えた。 再度インキュベーション及び洗浄の後、 FACS c a n装置 （BECTON DICKINSON社製） にて蛍光強度を測定した。 その結 果、 各 COS 7培養上清中の種々の長さのぺプチドリンカーを有する MAB L 2 一 s c F V及び s c (F v)2は、 ヒ ト IAPに対して高い親和性を有することが示 された （図 40 a及び b)。 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. To 2 × 10 5 mouse leukemia cell line L1210 cells expressing human IAP, add the culture supernatant obtained in Example 6.3 (1) or the culture supernatant of COS 7 cells as a control, and place on ice. After the incubation and washing, mouse anti-FLAG antibody (manufactured by SIGMA) at 10 μg / ml was added. After the incubation and washing, a FITC-labeled anti-mouse IgG antibody (BECTON DICKINSON) was added. After the incubation and washing again, the fluorescence intensity was measured using a FACS can device (manufactured by BECTON DICKINSON). As a result, MABL2 single sc FV and sc (Fv) 2 having peptide linkers of various lengths in each COS 7 culture supernatant may have high affinity for human IAP. (Figures 40a and b).
6. 6 in vitroでのアポトーシス誘起効果  6.6 In vitro apoptosis-inducing effect
前記 1. 3 (1) にて調製した COS 7細胞培養上清について、 ヒ ト I APを 遺伝子導入した L 1 210細胞 （h I AP/L 1 210) に対するアポトーシス 誘導作用を An n e X i n-V (BOEHRINGER MANNHEIM社製） 染色により検討し た。  Using the COS 7 cell culture supernatant prepared in 1.3 (1) above, the effect of inducing apoptosis on L1210 cells (hIAP / L1210) transfected with human IAP was determined by Anne X inV. (Manufactured by BOEHRINGER MANNHEIM) The staining was examined.
h I AP/L 1 210細胞 5 X 104個に、 各ベクターを形質転換した C O S 7 細胞培養上清あるいはコントロールとして COS 7細胞培養上清を終濃度 10 % で添加し、 24時間培養した。 その後、 Ann e X i n-V/P I染色を行い、 FACS c a n装置 （BECTON DICKINSON社製） にて蛍光強度を測定した。 その結 果、 COS 7 CM中の s c F Vく HL 3， 4, 6， 7、 LH 3 , 4， 6， 7 > 及ぴ s c (Fv)2は h I APZL 1210細胞に対して顕著な細胞死を誘導した。 得られた結果を図 41にそれぞれ示す。 To 5 × 10 4 hIAP / L1210 cells, a COS 7 cell culture supernatant transformed with each vector or a COS 7 cell culture supernatant as a control at a final concentration of 10% was added and cultured for 24 hours. Thereafter, Ann XinV / PI staining was performed, and the fluorescence intensity was measured using a FACS can device (manufactured by BECTON DICKINSON). As a result, sc FV in COS 7 CM, HL 3,4,6,7, LH 3,4,6,7> and sc (Fv) 2 showed significant cell death to h I APZL 1210 cells. Was induced. FIG. 41 shows the obtained results.
6. 7 MAB L 2— s c F V及び s c (F v)9の CHO細胞用発現ベクターの構 鎏 6.7 Construction of expression vector for MAB L 2—sc FV and sc (Fv) 9 for CHO cells
前記 MABL 2— s c F V及び s c (F v) 2を培養上清から精製することを目的 として、 これらを CHO細胞にて発現させるための発現ベクターを以下のように 構築した。 For the purpose of purifying the MABL2-sc FV and sc (Fv) 2 from the culture supernatant An expression vector for expressing these in CHO cells was constructed as follows.
前記 1. 2にて調製した p CF 2HL— 0, 3〜 7及び p C F 2 L H— 0， 3 〜7の∑ 001 1—8 amH I断片を、 CHO細胞用発現ベクター p CHO 1の E c o R I及び B a mH I部位に Ligation High を用いて導入し、 Competent E. coli DH5 α を形質転換した。 形質転換した大腸菌より QIAGEN Plasmid Midi Kit (QIAGEN) にてプラスミドを精製した。 このようにして発現プラスミド！） CH OM2HL-0, 3〜7及ぴ13€3¾0]^2 ^1—0， 3〜 7を作製した。  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 DH5α. The plasmid was purified from the transformed E. coli using the QIAGEN Plasmid Midi Kit (QIAGEN). Thus, the expression plasmid! ) CH OM2HL-0, 3-7 and 13 € 3¾0] ^ 2 ^ 1-0, 3-7 were prepared.
6. 8 MAB L 2- s c Fv<HL-0, 3〜 7 >、 MAB L 2— s c F vく LH— 0, 3〜7〉及び s c (_F v )ヮ発現 C HO細胞の作製並びにその培養上清の 前記 1. 7にて構築した発現プラスミド pCHOM2HL— 0， 3〜7及び p CHOM2 LH— 0, 3〜7並ぴに1) CHOM2 (F v) 2ベクターを以下の通りに C HO細胞に形質転換し、 各改変抗体を恒常的に発現する C HO細胞を作製した。 その代表的な例として MAB L 2- s c F v <HL- 5〉、 s c (F v)2を恒常的 に発現する C HO細胞の作製を下記に示す。 6.8 Preparation of MAB L 2-sc Fv <HL-0, 3-7>, MAB L 2—sc F v LH— 0, 3-7> and sc (_F v) ヮ expressing CHO cells and their production In the culture supernatant, the expression plasmids pCHOM2HL-0, 3-7 and p CHOM2 LH-0, 3-7, which were constructed in 1.7 above, were added 1) The CHOM2 (Fv) 2 vector was Cells were transformed to produce CHO cells that constantly express each modified antibody. As a representative example, preparation of CHO cells that constantly express MAB L2-scFv <HL-5> and sc (Fv) 2 is described below.
発現プラスミド p CHOM2HL— 5及び p CHOM2 (F v)2を制限酵素 P v u Πこて消化して直鎖状にし、 これらを Gene Pulser装置 （BioRad社製） を用い てエレクトロポレーシヨンにより CHO細胞にトランスフエクシヨンした。 DN A ( 10 μ g) と、 P B S中 1 X 107細胞 Zm 1の 0. 75m 1をキュベットに 加え、 1.5 kV、 25 Fの容量にてパルスを与えた。 室温にて 10分間の回復 期間の後、 エレクト口ポレーシヨン処理された細胞を、 10%のゥシ胎児血清を 含有する核酸含有 α— MEM培地 （GIBCO BRL社製） に加え培養した。 一夜培養 後、 培養上清を除去し、 PB Sにてリンスした後、 10%のゥシ胎児血清を含有 する核酸不含 α— MEM培地 （GIBCO BRL社製） を加え培養した。 約 2週間培養 後、 methotrexate (SIGMA社製） を終濃度 1 0 nMで含有する培地で更に培養し、 その後 50 nM、 そして 100 nMと濃度を順次上げて培養を続けた。 こうして 得られた細胞をローラーボトル中で無血清培地 CHO— S— S FM II (GIBCO BRL社製） にて培養後、 培養上清を集め、 遠心分離により細胞破片を除去し、 更 に 0. 20 μιηフィルタ一にて濾過し、 それぞれの CMを得た。 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 μg) 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 α-MEM medium (GIBCO BRL) containing 10% fetal bovine serum and cultured. After overnight culture, the culture supernatant was removed, rinsed with PBS, and a nucleic acid-free α-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. 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 μιη filter to obtain each CM.
同様にして、 MABL 2— s c F Vく HL— 0， 3， 4, 6， 7>及びく LH —0, 3, 4, 5， 6, 7〉を恒常的に発現する CHO細胞及ぴそれらの CMを 得た。  Similarly, CHO cells that constantly express MABL2-scFV and HL-0,3,4,6,7> and LH-0,3,4,5,6,7> and their CHO cells I got CM.
6. 9 MABL 2— s c F Vく HL— 5〉のダイマー及び s c (F V)クの精製 下記の 2種類の精製法により前記 6. 8で得られた CMから MAB L 2— s c F v.く HL— 5 >及び s c (F V) 2の精製を行った。 6.9 Purification of dimer and sc (FV) of MABL2-scFV-HL-5> MAB L2-scFv.-K from the CM obtained in 6.8 by the following two purification methods. HL-5> and sc (FV) 2 were purified.
く精製法 1 > HL— 5及び s c (F v)2を、 そのポリペプチドの C末端の F 1 a g配列を利用した抗 F l a g抗体ァフィ二ティカラムクロマトグラフィー及ぴゲ ル濾過を用いて精製した。 1 50mM Na C lを含む50rQM T r i s塩酸 緩衝液、 H7. 5 (TB S) で平衡化した抗 Flag M2 Affinity gel (SIGMA) で 作成したカラム （7. 9m l) に前記 6. 8で得られた CM (1 L) を添加し、 T B Sで力ラムを洗浄後、 0. 1 Mグリシン塩酸緩衝液、 pH3. 5で s c F vを力 ラムから溶出させた。 得られた画分を SDSZPAGEで分析し、 s c F vの溶 出を確認した。 s c F V画分を終濃度が 0. 01%となるように Twe e n 20を 加え、 Centricon- 10 (MILLIP0RE) で濃縮した。 濃縮液を 1 50mM Na C l及 び 0.010/oTwe e n 20を含む 20 mM酢酸緩衝液、 H6.0で平衡化した TSKg e 1 G3000 SWカラム （7. 5 X 600 mm) にかけた。 流速 0.4 m I i nで s c F Vは 280 nmの吸収で検出した。 HL— 5は主要ピーク としてダイマーの位置に、 s c (F V) 2はモノマーの位置にそれぞれ溶出された。 く精製法 2 > HL— 5及び s c (Fv) 2をイオン交換クロマトグラフィー、 ハイ ドロキシァパタイト及びゲル濾過の三工程で精製した。 イオン交換クロマトグラ フィ一では、 HL— 5では Q Sepharose fast flo カラム （ファノレマシァ〉 を s c (F v)2では SP- sepharose fast flowカラムを用い、 第二工程以降は HL—5 と S c (Fv) 2で同じ条件を用いた。 Purification method 1> Purify HL-5 and sc (Fv) 2 using anti-Flag antibody affinity column chromatography using the C-terminal F1ag sequence of the polypeptide and gel filtration. did. 1 A column (7.9 ml) prepared with an anti-Flag M2 Affinity gel (SIGMA) equilibrated with 50 rQM Tris-HCl buffer containing 50 mM NaCl and H7.5 (TBS) The CM (1 L) obtained was added thereto, and the column was washed with TBS, and then scFv was eluted from the column with 0.1 M glycine hydrochloride buffer, pH 3.5. 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及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. In the 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 .
(第一工程） HL-5  (First step) HL-5
HL— 5の CMは、 0. 02%Twe e n 20を含む 2 OmM T r i s塩酸緩 衝液、 pH9. 0で 2倍希釈した後に、 1M T r i sで; pHを 9. 0に調整した。 この後、 0.02%Tw e e n 20を含む 2 OmM T r i s塩酸緩衝液、 H8. 5で平衡化した Q Sepharose fast flowカラムにかけ、 同緩衝液中 0. 1Mから 0. 55Mまでの N a C 1の直線濃度勾配でカラムに吸着したポリペプチドを溶出し た。 得られた画分を SDSZPAGEで分析し、 HL— 5を含む画分を集め、 第 二工程のハイドロキシアパタイトにかけた。 CM for HL-5 is 2 OmM Tris HCl containing 0.02% Tween 20 After a two-fold dilution with the buffer, pH 9.0, the pH was adjusted to 9.0 with 1 M Tris. After that, 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.
(第一工程） s c (F v)2 (First step) sc (F v) 2
s c (F V) 2の C [は、 0.02%Tw e e n 20を含む 20 mM酢酸緩衝液、 p H 5. 5で 2倍希釈した後に、 1 M酢酸で p Hを 5. 5に調整した。 0.02 % T we e n 20を含む 20 mM酢酸緩衝液、 H5. 5で平衡化した SP - Sepahrose fast flowカラムにかけ、 同緩衝液中、 Na C 1濃度を 0から 0. 5 Mまで直線的 に上げ、 カラムに吸着したポリペプチドを溶出した。 得られた画分を SDS/P AGEで分析し、 s c (F V) 2を含む画分を集め、 第二工程のハイドロキシァパタ イトにかけた。 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.
(第二工程） HL— 5及び s c (F v)2のハイドロキシアパタイトクロマトグラフ ィー (Second step) HL-5 and sc (Fv) 2 hydroxyapatite chromatography
第一工程で得られた HL— 5画分及び s c (F V) 2画分をそれぞれ 0. 02 % Tw e e n 20を含む 10 mM リン酸緩衝液、 H 7.0で平衡化したハイドロ キシアパタイトカラム （BioRad、 タイプ I) に添加し、 同緩衝液でカラムを洗浄 後、 リン酸緩衝液濃度を 0. 5Mまで直線的に上げ、 カラムに吸着したポリべプチ ドを溶出した。 各画分を SDSノ PAGEで分析し、 所望のポリペプチドが含ま れる画分を集めた。 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.
(第三工程） HL- 5及び s c ( F V ) 2のゲル濾過 (Third step) Gel filtration of HL-5 and sc (FV) 2
第二工程で得られた各画分をそれぞれ Centriprep-10 (MILLIP0RE) で濃縮し、 0.02%Tw e e n 20及び 0. 15M N a C 1を含む 20 mM酢酸緩衝液、 p H 6.0で平衡化した S u p e r d e x 200カラム （2. 6 X 60 cm、 ファ ルマシア） にかけた。 HL— 5はダイマーに位置に、 s c (F v)HL— 5及び s Each fraction obtained in the second step was concentrated with Centriprep-10 (MILLIP0RE), and equilibrated with 20 mM acetate buffer containing 0.02% Tween 20 and 0.15 M NaC1, pH 6.0. The sample was applied to a Superdex 200 column (2.6 x 60 cm, Pharmacia). HL-5 is located at the dimer, s c (F v) HL-5 and s
C (F V) 2はモノマーの位置にそれぞれ主要ピークとして溶出された。 いずれの精製法においても、 HL— 5モノマーは殆ど検出されなかったことか ら、 一本鎖 F vのリンカ一のアミノ酸残基数が 5個程度であれば、 効率的に一本 鎖 F Vのダイマーが形成できることが判明した。 HL— 5ダイマーおよび s c (F V ) 2は ヽずれも精製された後も 4 °Cで 1ヶ月間安定的に維持された。 ' C (FV) 2 was eluted as a major peak at each monomer position. Since almost no HL-5 monomer was detected in any of the purification methods, if the number of amino acid residues in the linker of the single-chain Fv was about 5, the efficiency of the single-chain FV was improved. It was found that dimers could form. HL-5 dimer and sc (FV) 2 were stably maintained at 4 ° C for 1 month even after purification. '
6. 10 精製 s c F Vく HL— 5〉のダイマー及び s c (F V の抗原結合活性 評価  6.10 Evaluation of purified scFV dimer and sc (FV antigen-binding activity)
精製された MAB L 2- s c F v <HL 5 >のダイマー及び s c (F v) 2のヒト Integrin Assosiated Protein ( I AP) 抗原への結合を測定するため、 フローサ ィトメ トリーを行った。 ヒト I APを発現するマウス白血病細胞株 L 1210細 胞 （h I AP/L 1 210) 又は対照として; CO S 1ベクターをトランスフエ ,.: クシヨンした L 1 21◦細胞 （p CO S IZL 1210) 2 X 105個に、 10 g/m 1の精製 MAB L 2— s c F v <HL 5 >のダイマー、 MAB L 2 - s c (Fv)2、 陽性対照としてモノクローナル抗体 MAB L— 2、 陰性対照としてマウ ス I gG (Zymed社製） を加え、 氷上にてインキュベーション及び洗浄の後、 1 0 gZm 1のマウス抗 F LAG抗体 （SIGMA社製） を加えた。 インキュベーシ ョン及び洗浄の後、 F I T C標識抗マウス I g G抗体 （BECTON DICKINSON社製） を加えた。 再度ィンキュベーション及び洗浄の後、 F AC S c a n装置 （BECTON DICKINSON社製） にて蛍光強度を測定した。 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. Mouse leukemia cell line L1210 cells expressing human IAP (hIAP / L1210) or as a control; transfected with COS1 vector,.: L122 cells (pCOS IZL1210 ) 2 x 10 5 purified MAB L 2-sc Fv <HL 5> dimers, 10 g / m 1, MAB L 2-sc (Fv) 2 , monoclonal antibody MAB L-2 as positive control, negative Mouse IgG (manufactured by Zymed) was added as a control, and after incubation and washing on ice, 10 gZm1 mouse anti-FLAG antibody (manufactured by SIGMA) was added. After incubation and washing, 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).
' その結果、 精製 MAB L 2 - s c F V <HL 5 >のダイマー及び MAB L 2一 s c (F v)2は h I AP/L 1210細胞に特異的に結合したことにより、 s c F v<HL 5 >のダイマー及び s c (F v)2がヒト I APに対して高い親和性を有す ることが示された （図 42)。 'As a result, the purified dimer of MAB L 2 -sc FV <HL 5> and MAB L 2 sc (F v) 2 specifically bound to h IAP / L 1210 cells, 5> dimer and sc (Fv) 2 were shown to have high affinity for human IAP (FIG. 42).
6. 1 1 精製 s c F Vく HL— 5>のダイマー及び s c (F V)ゥの in vitro ァ ポトーシス誘起効果  6.1 1 In vitro apoptosis-inducing effects of purified scFV and HL-5> dimers and sc (FV) ゥ
精製した MABL 2— s c F vく H L 5〉のダイマー及ぴ s c (Fv)2について、 ヒト I APを遺伝子導入した L 1210細胞 （h I AP/L 1 210) 及びヒト 白血病細胞株 CCRF— CEMに対するアポトーシス誘導作用を Ann e X i n ― V (B0EHRINGER MANNHEIM社製） 染色により検討した。 h Ϊ AP L 1210細胞 5 X 104個あるいは C C R F— C EM細胞 1 X 10 5個に、 精製 MAB L 2— s c F vく HL 5 >のダイマー、 MABL2— S c (F v)2、 陽性対照としてモノクローナル抗体 MAB L— 2、 陰性対照としてマウス I g Gを様々な濃度で添加し、 24時間培養した。 その後、 Ann e x i n— V 染色を行い、 FACS c a n装置 （BECTON DICKINSON社製） にて蛍光強度を測定 した。 その結果、 MAB L 2 - s c F V <HL 5 >のダイマー及び MAB L 2— s c (Fv)2は h I APZL 1210、 C C R F— C EMの両細胞に対して濃度依 存的 jこ細胞死を誘導した （図 43 )。 この結果、 MABL 2— s c F v<HL 5 > のダイマー及び MAB L 2- s c (F v)2は、 もとのモノクローナル抗体 MAB L - 2と比較して改善されてたアポトーシス誘導作用を有することが示された。 6. 1 2 精製 s c F Vく HL— 5 >のダイマー及び s c (F V の赤血球凝集試 m 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 The apoptosis-inducing effect on was examined by Ann e Xin-V (B0EHRINGER MANNHEIM) staining. h Ϊ 5 x 10 4 AP L 1210 cells or 1 x 10 5 CCRF-C EM cells, purified MAB L 2-sc F v or HL 5> dimer, MABL 2-S c (F v) 2 , positive MAB L-2 monoclonal antibody was added as a control, and mouse IgG was added at various concentrations as a negative control, and cultured for 24 hours. Thereafter, Ann exin-V staining was performed, and the fluorescence intensity was measured using a FACS can device (manufactured by BECTON DICKINSON). As a result, 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). As a result, 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
実施例 5. 1 5に従って、 種々の濃度の精製した s c F v<HL— 5 >のダイ マー及ぴ s c (Fv) 2の血液凝集試験を実施した。 According to Example 5.15, a dimer of purified scFv <HL-5> at various concentrations and a blood agglutination test of sc (Fv) 2 were performed.
モノクローナル抗体 MABL— 2 (陽性対照） では血液凝集が起こるのに対し て、 一本鎖抗体の MAB L 2- s c (F v)2及び MAB L 2— s c (F v)<HL 5 >は凝集しなかった。 また、 MABL— 2抗体を用いた緩衝液の差もほとんどみ られなかった。 その結果を下記の表 3に示す。 Monoclonal antibodies MABL- 2 by contrast (positive control) in the blood coagulation occurs, single chain antibodies MAB L 2- sc (F v) 2 and MAB L 2- sc (F v) <HL 5> aggregation Did not. Also, there was almost no difference between the buffers using the MABL-2 antibody. The results are shown in Table 3 below.
表 3 ヒ ト赤血球凝集試験 漏: PBS (Mgιώ) cont 28.9 1445 7.225 3.6125 1.8063 0.9031 0.4516 0.2258 0.1129 - 0.0564 0.0282 0.0141 0.0071 0.0035 0.0018 ffiBL2-sc(^)2 - - - - - - - - - - - - - - - - coot 28.0 140 7.0 3.5 1.75 0.875 0.4375 0.2188 0.1094 0.0547 0.0273 0.0137 0.0068 0.0034 0.0017 fflBL2^c(Fv)<HL5> - - - - - - - - - - - - - - - - cont 80 40 20 10 5 2.5 1.25 0.625 0.3125 0.1563 0.0781 0.0391 0.0195 0.0098 0.0049 隨 (intact) 一 + + + + + + + + + 土 一 一 一 一 一 mlgG — — — — — — — · — — — — — — — — — Acetate Buffer ml) _ c rt 80 40 20 10 5 2.5 1.25 0.625 0.3125 0.1563 0.0781 0.0391 0.0195 0.0098 0.0049 隨 (intact) 一 + + + + + + + + + + + — 一 一 一 Table 3 Human hemagglutination test Leakage: PBS (Mgιώ) cont 28.9 1445 7.225 3.6125 1.8063 0.9031 0.4516 0.2258 0.1129-0.0564 0.0282 0.0141 0.0071 0.0035 0.0018 ffiBL2-sc (^) 2------------ ---coot 28.0 140 7.0 3.5 1.75 0.875 0.4375 0.2188 0.1094 0.0547 0.0273 0.0137 0.0068 0.0034 0.0017 fflBL2 ^ c (Fv) <HL5>----------------cont 80 40 20 10 5 2.5 1.25 0.625 0.3125 0.1563 0.0781 0.0391 0.0195 0.0098 0.0049 Intact 1 + + + + + + + + + Soil 1 1 1 1 mlgG — — — — — — — — — — — — — — — Acetate Buffer ml) _ c rt 80 40 20 10 5 2.5 1.25 0.625 0.3125 0.1563 0.0781 0.0391 0.0195 0.0098 0.0049 Function (intact) One + + + + + + + + + + + — One
6. 13 精製 s c F vく HL— 5 >のダイマー及び s c (F v) のヒ ト骨髄腫マ ゥスモデルに対する抗腫瘍効果 6.13 Anti-tumor effect of purified scFv HL-5> dimer and sc (Fv) on human myeloma mouse model
実施例 6. 8及び 6. 9にて作製、 精製した s c F v<HL— 5〉のダイマー 及び s c (F v)2について、 その抗腫瘍効果を試験した。 具体的には実施例 5. 1 4 (3) で作製したヒト骨髄腫マウスモデ を用いて、 マウス血清中における、 ヒ ト骨髄腫細胞が産生する Μタンパク質を EL I S Αにより定量し、 併せてマウ スの生存日数を記録した。 そして、 血清中の Mタンパク質量の変化および生存日 数により、 s c F Vく HL— 5>のダイマー及び s c (F V) 2の'抗腫瘍効果を評価 した。 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 す る 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.
なお、 本試験において HL— 5及ぴ s c (F v)2は、 v e h i c l e (150mIn this test, HL-5 and sc (Fv) 2 were measured for vehicle (150 m
M N a C 1 , 0.02%Twe e n及び 2 OmM酢酸緩衝液， H6.0) 中の 0.01、 0. 1又は lmg/ :m 1の溶液として、 投与量が 0. 1、 1または 10m g/k gになるようにマウスに投与した。 また、 対照は V e h i c 1 eのみを投 与した。 As a solution of 0.01, 0.1 or lmg / : m1 in MNaCl, 0.02% Tween and 2 OmM acetate buffer, H6.0), the dose is 0.1, 1 or 10 mg / Mice were dosed to give kg. As a control, only Vehic 1 e was administered.
ヒト骨髄腫細胞移植後 26日目に血清を採取し、 血清中の Mタンパク質量を E L I S Aにより実施例 5. 14に従って測定した。 その結果、 HL— 5投与群及 びダイマー及び s c (Fv) 2投与群共に、 血清中の Mタンパク質量が投与量依存的 に減少していた （図 44を参照）。 また、 その生存期間については、 HL— 5投与 群 （図 45) 及び s c (F v)2投与群 （図 46) 共に対照 （V e h i c 1 e投与 群） と比較して有意な生存期間の延長が観察された。 これらの結果は、 本発明の HL— 5及ぴ s c (F v) 2がインビボにおいても優れた抗腫瘍作用を有することを 示している。 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. As a result, in both the HL-5 administration group and the dimer and sc (Fv) 2 administration groups, the amount of serum M protein was reduced in a dose-dependent manner (see FIG. 44). As for its survival, 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. These results indicate that HL-5 and sc (Fv) 2 of the present invention have an excellent antitumor effect even in vivo.
実施例 7 ヒ ト MP Lに対するヒ ト抗体 12B 5の H鎖 V領域及び L鎖 V領域を 含む一本鎖 Fv - ヒ ト MP Lに対するヒ トモノクローナル抗体 12B 5の V領域をコードする DExample 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
N Aを次のようにして構築した。 NA was constructed as follows.
7. 1 12B 5H鎖 V領城をコードする遺伝子の構築  7.1 Construction of Gene Encoding 12B 5H Chain V Region
ヒト MP Lに結合するヒト抗体 12 B 5H鎖 V領域をコードする遺伝子は、 該 遺伝子の塩基配列 （配列番号 5 5) を用いて、 その 5'末端にヒ ト抗体遺伝子由来 のリーダー配列 （配列番号 5 6) (Eur. J. Immunol. 1996; 26： 63-69) を連結さ せることで設計した。 設計した塩基配列はそれぞれ 1 5 b pのオーバーラップ配 列を持つように 4本のオリゴヌクレオチド （1 2B 5VH— 1、 1 2B 5VH— 2、 1 2 B 5VH— 3、 1 2 B 5VH— 4) に分割し、 1 2 B 5VH— 1 (配列 番号 5 7) 及ぴ 1 2 B 5VH- 3 (配列番号： 5 9) はセンス方向で、 1 2 B 5 VH- 2 (配列番号： 58) 及び 1 2 B 5VH— 4 (配列番号： 60) はアンチ センス方向でそれぞれ合成した。 各合成オリゴヌクレオチドはそれぞれの相補性 によりアッセンプリさせた後、 外側プライマー （1 2 B 5 VH—S及び 1 2 B 5 VH-A) を加え、 全長の遺伝子を増幅した。 なお、 1 2 B 5VH— S (配列番 号： 6 1) は前方プライマーでリーダー配列の 5'末端にハイブリダィズし、 且つ H i n d III 制限酵素認識配列ならぴにコザック配列を持つように、 また 1 2 B 5 VH-A (配列番号： 6 2) は後方プライマーで H鎖 V領域の C末端をコード する塩基配列にハイブリダィズし、 且つスプライスドナー配列ならびに B amH I制限酵素認識配列を持つようにそれぞれ設計した。 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. After assembling each synthetic oligonucleotide by its complementarity, 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 ぴ if it is a HindIII restriction enzyme recognition sequence. 2B5VH-A (SEQ ID NO: 62) 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.
P C R溶液 1 00 1は、 5 μ 1の l OxP CR Gold Buffer II、 1. 5 mM Mg C l 2、 0. 0 8 mM dNTP s (dATP、 d GTP、 d CTP、 dTT P )、 5ュニッ トの DNAポリメラーゼ AmpliTaq Gold (以上 PERKIN ELMER社製）ヽ 2. 5 ずつの合成オリゴヌクレオチド 1 2Β 5 VH— 1〜4を含有し、 94°C の初期温度にて 9分間そして次に 94°Cにて 2分間、 5 5°Cにて 2分間及び 7 2 °Cにて 2分間のサイクルを 2回反復した後、 1 O O pmo 1 eずつの外側プラ イマ一 1 2 B 5VH— S及び 1 2 B 5VH— Aを加え、 さらに 94°Cにて 3 0秒 間、 5 5°Cにて 3 0秒間及び 7 2 °Cにて 1分間のサイクルを 3 5回反復した後、 反応混合物を更に 72。Cで 5分間加熱した。 PCR solution 1 00 1, 5 mu 1 of l OxP CR Gold Buffer II, 1. 5 mM Mg C l 2, 0. 0 8 mM dNTP s (dATP, d GTP, d CTP, dTT P), 5 Yuni' DOO DNA polymerase AmpliTaq Gold (all manufactured by PERKIN ELMER) ヽ 2.5 Synthetic oligonucleotides 12Β5 VH—containing 1 to 4 at an initial temperature of 94 ° C for 9 minutes and then at 94 ° C After 2 cycles of 2 min at 55 ° C for 2 min and 2 min at 72 ° C, the outer primers 1 OO pmo 1 e each 1 2 B 5VH—S and 1 2 B 5VH-A was added, and the cycle was repeated 35 times at 94 ° C for 30 seconds, at 55 ° C for 30 seconds, and at 72 ° C for 35 minutes. 72. Heated at C for 5 minutes.
PCR生成物は 1. 5%低融点ァガロースゲル (Sigma社製） を用い精製した後、 制限酵素 B a mH I及ぴ H i n d III で消化し、 ヒト H鎖発現ベクター HE F— g γ 1にクローユングした。 DNA配列決定の後、 正しい DNA配列を有する D ΝΑ断片を含むプラスミドを HEF— 1 2Β 5Η— g γ 1と命名した。 さらに、 HE F— 12 B 5 H- g 71を制限酵素 E c o R Iならびに B a mH Iで消化し、 12B 5 VHをコードする遺伝子を調製した後、 ヒ ト F a b H鎖発 現ベクター p COS-F dに挿入し p F d— 12 B 5 Hを得た。 なお、 ヒ ト F a bH鎖発現ベクターはヒト抗体 H鎖 V領域と定常領域をコードする遺伝子間に存 在するイントロン領域ならびにヒ ト H鎖定常領域の一部をコードする遺伝子を含 む DNA (配列番号 63) を PCR法を用い増幅した後、 動物細胞発現ベクター p COS 1に挿入することで構築したベクターである。 ヒト H鎖定常領域は HE F.-.g y 1を铸型とし、 上記と同様の条件下にて遺伝子の増幅を行い、 前方ブラ ィマーとしてィントロン 1の 5 '端の配列とハイプリダイズし、 且つ E c o R I及 び B a mH I制限酵素認識配列を有するように設計した G 1 C H 1— S (配列番号 64)を、 後方プライマーとしてヒト H鎖定常領域 C HI ドメインの 3'端の DN Aにハイプリダイズし、 且つヒンジ領域の一部をコードする配列、 二個の停止コ ドンおょぴ B g 1 II 制限酵素認識部位を有するように設計した G 1 CH1-A (配列番号 65)を用いた。 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-gγ1. did. After DNA sequencing, the plasmid containing the DΝΑ fragment with the correct DNA sequence was named HEF-12Β5Η-gγ1. Furthermore, after digesting 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 铸, 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. Using.
プラスミド HEF— 12B 5H— g γ 1及び pF d - 12 B 5 Hに含まれる再 構成 12 B 5 H鎖可変領域の塩基配列及びァミノ酸配列を配列番号： 66に示す。  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-gγ1 and pFd-12B5H.
7. 2 12 B 5 L鎖 V領域をコードする遺伝子の構築 7.2 Construction of gene coding for 12B5 light chain V region
ヒト MP Lに結合するヒト抗体 12 B 5 L鎖 V領域をコードする遺伝子は、 該 遺伝子の塩基配列 （配列番号 67) を用い、 その 5'末端にヒ ト抗体遺伝子 3D 6 (Nuc. Acid Res. 1990： 18; 4927) 由来のリーダー配列 （配列番号 68) を連結 させることで設計した。 設計した塩基配列は上記と同様にそれぞれ 15 b pのォ 一バーラップ配列を持つように 4本のオリゴヌクレオチド （12B 5VL— 1、 12B 5VL— 2、 12B 5VL— 3、 12 B 5 V L— 4 ) に分割し、 それぞれ 合成した。 12B 5VL— 1 (配列番号： 69) 及ぴ 12 B 5 VL— 3 (配列番 号： 71 ) はセンス配列、 12B 5VL— 2 (配列番号： 70) 及び 12 B 5 V L-4 (配列番号： 72) はアンチセンス配列を有し、 各合成オリゴヌクレオチ ドはそれぞれの相補性によりアッセンプリさせた後、 外側プライマー （12B 5 VL— S及び 12 B 5 VL— A) を加え、 全長の遺伝子を増幅した。 なお、 12 B 5 VL-S (配列番号： 73) は前方プライマーでリーダー配列の 5 '末端にハ イブリダィズし、 且つ H i n d III制限酵素認識配列ならぴにコザック配列を持 つように、 また 1 2 B 5VL— A (配列番号： 74) は後方プライマーで L鎖 V 領域の C末端をコードする塩基配列にハイプリダイズし、 且つスプライスドナー 配列ならびに B amHI制限酵素認識配列を持つようにそれぞれ設計した。 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. Note that 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 enzyme recognition sequence.
PCR反応は上記と同様に行い、 ？〇1生成物は1. 5%低融点ァガロースゲル (Sigma社製） を用い精製した後、 制限酵素 B amH I及び H i n d III で消化 し > _ヒト L鎖発現ベクター HE F— g κにクローニングした。 DN Α配列決定の 後、 正しい DNA配列を有する DNA断片を含むプラスミドを HE F— 12 B 5 L-g κと命名した。 本プラスミド HE F— 12 B 5 L- g κに含まれる再構成 12 B 5 L鎖 V領域の塩基配列及ぴァミノ酸配列を配列番号： 75に示す。  Perform the PCR reaction as described above. 〇1 product was purified using 1.5% low-melting point agarose gel (manufactured by Sigma), digested with restriction enzymes BamHI and HindIII, and cloned into the human L chain expression vector HEF-gκ. . After DNΑ sequencing, the plasmid containing the DNA fragment with the correct DNA sequence was designated HEF-12B5L-gκ. The nucleotide sequence and amino acid sequence of the reconstituted 12B5 light chain V region contained in this plasmid HEF-12B5L-gκ are shown in SEQ ID NO: 75.
7. 3 再構成 12B 5—本鎖 Fv (s c Fv)の作製 7.3 Reconstruction of 12B 5--strand Fv (s c Fv)
再構成 12 B 5抗体一本鎖 F Vは 1 2 B 5 VH—リンカ 12 B 5 VLの順 とし、 その C末端には検出及び精製を容易にするために F LAG配列 （配列番 号： 76) を付加することで設計した。 さらに、 リンカ一配列は（G 1 y4S e r) 3の 15アミノ酸からなるリンカ一配列を用い、 再構成 12 B 5—本鎖 F V ( s c 12B 5) を構築した。 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).
(1) 1 5アミノ酸からなるリンカー配列を用いた再構成 1 2 B 5—本鎖 F の 作製  (1) Reconstruction using a linker sequence consisting of 15 amino acids
1 5アミノ酸からなるリンカ一を用いた再構成 12 B 5抗体一本鎖 F Vをコー ドする遺伝子は 1 2 B 5 H鎖 V領域、 リンカー領域、 及び 12 B 5 L鎖 V領域を それぞれ PCR法を用いて増幅し、 連結することにより構築した。 この方法を図 47に模式的に示す。 再構成 12B 5—本鎖 F Vの作製のために 6個の PC Rプ ライマー （A〜F) を使用した。 プライマー A、 C及ぴ Eはセンス配列を有し、 プライマー B、 D及び Fはアンチセンス配列を有する。  Reconstitution using a linker consisting of 15 amino acids 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.
H鎖 V領域のための前方プライマー 12B 5— S (プライマー A、 配列番号： 77) は、 H鎖リーダー配列の 5 '末端にハイブリダイズし且つ E c o R I制限酵 素認識部位を有するように設計した。 H鎖 V領域のための後方プライマー HuV H J 3 (プライマー B、 配列番号： 78) は、 H鎖 V領域の C末端をコードする DNAにハイブリダイズするように設計した。 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.
リンカ一のための前方プライマー RHu J H3 (プライマー C、 配列番号： 7 9) は、 リンカ一の N末端をコードする DN Aにハイプリダイズし且つ H鎖 V領 域の C末端をコードする DNAとオーバーラップするように設計した。 リンカ一 のための後方プライマー RHu VK 1 (プライマー D、 配列番号： 80) は、 リ ンカーの C末端をコードする DNAにハイブリダィズし且つ L鎖 V領域の N末端 をコードする D N Aとオーバーラップするように設計した。  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.
L鎖 V'領域のための前方プライマー Hu VK 1. 2 (プライマー E、 配列番号： 81) は L鎖 V領域の N末端をコードする DNAにハイブリダィズするように設 計した。 L鎖 V領域のための後方プライマー 1 2 B 5 F— A (プライマー F、 配 列番号： 82) は、 L鎖 V領域の C末端をコードする DNAにハイブリダィズし 且つ F LAGペプチドをコードする配列 （Hopp, T. P.ら、 Bio/Technology, 6， 1204-1210, 1988)、 2個の転写停止コドン及び N o t I制限酵素認識部位を有す るように設計した。  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.
第一 P CR段階において 3つの反応 A— B、 じー0及び£ー？を行ぃ、 そして 第一 P CRから得られた 3つの P CR生成物をそれら自体の相補性によりアツセ ンブリさせた。 次に、 プライマー A及び Fを加えて、 1 5アミノ酸からなるリン カーを用いた再構成 1 2B 5—本鎖 Fvをコードする全長 DNAを増幅した （第 二 PCR)。 なお、 第一 PCRにおいては、 再構成 12B 5H鎖 V領域をコ ドす るプラスミド HEF— 12 B 5H— g γ 1 (実施例 7. 1を参照）、 G l y G 1 y G 1 y G l y S e r G l y G l y G l y G l y S e r G 1 y G l y G l y G l y S e rからなるリンカ一領域をコードする DNA 配列 （配列番号： 8 3) (Huston, J. S.ら、 Proc. Natl. Acad Sci. USA, 85, 5879-5883, 1988) を含んで成るプラスミド p S C F V T 7— hM 21 (ヒ ト型ィ匕 ONS— M2 1抗体） (Ohtomo, T.ら、 Anticancer Res. 18 (1998)， 4311 - 4316)、 及び再構成 1 2B 5 L鎖 V領域をコードするプラスミド HEF— 12B 5 L— g κ (実施例 7. 2を参照） をそれぞれ铸型として用いた。 第一 P CR段階の溶液 5 0 μ 1は、 5 1の 1 OxP CR Gold Buffer II、 1. 5mM Mg C l 2、 0. 0 8 mM dNT P s、 5ユニットの D N Aポリメラー ゼ AmpliTaq Gold (以上 PERKIN ELMER社製）、 1 0 0 pmo 1 eずつの各プライ マー及び 1 0 0 n gの各铸型 DN Aを含有し、 9 4 °Cの初期温度にて 9分間そし て次に 9 4°Cにて 3 0秒間、 5 5°Cにて 3 0秒間及ぴ 7 2°Cにて 1分間のサイク ルを 3 5回反復した後、 反応混合物を更に 7 2 °Cで 5分間加熱した。 Three reactions in the first PCR stage, A-B, J0 and £? And the three PCR products obtained from the first PCR were assembled by their own complementarity. Next, primers A and F were added to amplify the full-length DNA encoding the reconstituted 12B5-single-stranded Fv using a linker consisting of 15 amino acids (second PCR). In the first PCR, the plasmid HEF-12B5H-gγ1 encoding the V region of the reconstructed 12B5H chain (see Example 7.1), GlyG1yG1yGly Ser GlyGlyGlyGlyGlySerG1yGlyGlyGlyGlySer DNA sequence encoding linker region (SEQ ID NO: 83) (Huston, JS et al., Proc. Natl. Acad Sci. USA, 85, 5879-5883, 1988). Plasmid p SCFVT 7—hM21 (human type ONS—M21 antibody) (Ohtomo, T. et al., Anticancer Res. 18 (1998)). , 4311-4316), and plasmid HEF-12B5L-gκ (see Example 7.2) encoding the reshaped 12B5 L chain V region were used as type I, respectively. The 50 μl 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. .
P CR生成物 A— B、 C— D、 及び E— Fは第二 P CRでアッセンプリした。 第二 P CRにおいて、 铸型として 1 μ 1の第一 P CR反応物 A— Β、 0. 5 μ 1の ?〇1反応物0—0及び1 μ 1の P CR反応物 E— F、 1 0 ^ 1の l O xP CR Gold Buffer II、 1. 5 mM Mg C 1 2, 0. 0 8 mM dNT P s s 5ユニッ ト の DNAポリメラーゼ AmpliTaq Gold (以上 PERKIN ELMER社製） を含有する 9 8 μ 1の P CR混合液を、 9 4°Cの初期温度にて 9分間そして次に 94°Cにて 2分 間、 6 5°Cにて 2分間及び 7 2°Cにて 2分間のサイクルを 2回反復した後、 それ ぞれ 1 0 0 pm o 1 eずつのプライマー A及び Fを加えた。 そして 94°Cにて 3 0秒間、 5 5 °Cにて 3 0秒間及び 7 2 °Cにて 1分間のサイクルを 3 5回反復した 後、 反応混合物を 7 2 °Cにて 5分間加熱した。 The PCR products AB, CD, and EF were assembled in a second PCR. In the second PCR, 1 μl of the first PCR reactant A—Β, 0.5 μl of the? 〇1 reactant 0-0 and 1 μl of the PCR reactant E—F, 1 0 ^ 1 l O xP CR Gold Buffer II, 1. 5 mM Mg C 1 2, 0. 0 8 mM dNT P s s 5 units of DNA polymerase AmpliTaq Gold containing (or manufactured by PERKIN ELMER) 9 Apply 8 μl of the PCR mixture for 9 minutes at an initial temperature of 94 ° C and then for 2 minutes at 94 ° C, 2 minutes at 65 ° C and 2 minutes at 72 ° C. After repeating the above cycle twice, primers A and F of 100 pmol each were added. The cycle was repeated 35 times at 94 ° C for 30 seconds, at 55 ° C for 30 seconds and at 72 ° C for 1 minute, and then the reaction mixture was heated at 72 ° C for 5 minutes. did.
第二 P CRにより生じた DNA断片を 1. 5。/。低融点ァガロースゲルを用いて精 製し、 E c o R I及び N o t Iで消化し、 得られた DNA断片を p CHO 1べク ターおよび p CO S 1ベクター （特願平 8— 2 5 5 1 9 6) にクローユングした。 なお、 本発現ベクター p CHO 1は、 DHFR— Δ Ε— r vH— PM1— f (W 09 2/ 1 9 7 5 9参照） から、 E c o R I及ぴ S m a I消化により抗体遺伝子 を削除し、 E c o R I— N o t l— B a mH I A d a t o r (宝酒造社製） を連結することにより構築したベクターである。 DNA配列決定の後、 再構成 1 2 B 5—本鎖 F Vの正しいアミノ酸配列をコードする DNA断片を含むプラスミ ドを p CHO— s c l 2 B 5及び！） CO S— s c l 2 B 5と命名した。 本プラス ミド CHO— s c l 2 B 5及び: CO S— s c l 2 B 5に含まれる再構成 1 2 B 5一本鎖 F Vの塩基配列及ぴァミノ酸配列を配列番号： 84に示す。  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-ΔΕ-rvH-PM1-f (see W092 / 197959) by digestion with EcoRI and SmaI. And EcoRI—Notl—BamHIA dator (manufactured by Takara Shuzo). After DNA sequencing, the plasmid containing the DNA fragment encoding the correct amino acid sequence of the reconstituted 12B5—strand FV was pCHO-scl2B5 and! ) COS—scl2B5. 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.
7. 4 動物細胞を用いた各 1 2 B 5抗体 （ I g G、 F a b ) 及び一本鎖 F vポ リぺプチドの発現 7.4 Each of the 12B5 antibodies (IgG, Fab) and single-chain Fv Expression of peptides
1 2 B 5抗体 （I g G、 F a b) 及び 1 2 B 5抗体由来の一本鎖 F v (ポリべ プチド） は C O S— 7細胞又は CHO細胞を用い発現させた。  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.
CO S一 7細胞を用いた一過的な発現は次のようにして行った。 すなわち、 Gene Pulser装置 （BioRad社製） を用いたエレクト口ポレーシヨン法により遺伝 子導入した。 1 2 B 5抗体 （I g G) の発現には前述の発現ベクター HE F— 1 2 B 5 H- g 7 1及び HE F— 1 2 B 5 L— g K各 1 0 /z gずつを、 1 2 B 5 F a b—断片の発現には p F d— 1 2 B 5 Hと HE F— 1 2 B 5 L— g /c各 1 0 g ずつを、 一本鎖 F vの発現には p CO S— s c l 2 B 5 ( 1 0 μ g) を P B Sに 懸濁した CO S— 7細胞 （1 X 1 07細胞 Zm 1 ) 0. 8 m lに混合し、 キュべッ トに加え、 1. 5 kV、 2 5 i.FDの容量にてパルスを与えた。 室温にて 1 0分間 の回復期間の後、 エレクト口ポレーシヨン処理された細胞を、 1 0 %のゥシ胎児 血清を含有する DMEM培地 （GIBC0 BRL社製） に加え培養した。 終夜培養後、 細胞を P B Sで一回洗浄し、 さらに無血清培地 CHO- S- S FM II培地を加え、 さらに 2日間培養した。 培養上清を遠心し細胞破碎物を除去した後、 · 0. 2 2 χη. のフィルターを通すことで調製した。 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). For expression of the 12B5 antibody (IgG), the expression vectors HEF-12B5H-g71 and HEF-12B5L-gK described above were used at 10 / zg each. For expression of 12 B5 F ab—fragment, p F d—12 B 5 H and HE F—12 B 5 L—g / c 10 g each, and for expression of single-chain F v p CO S- scl 2 B 5 ( 1 0 μ g) a CO S- 7 cells suspended in PBS (1 X 1 0 7 cells Zm 1) was mixed with 0. 8 ml, was added to the cuvette bets, Pulses were given at a capacity of 1.5 kV, 25 i.FD. After a recovery period of 10 minutes at room temperature, the cells subjected to electoral poration were added to a DMEM medium (GIBC0 BRL) containing 10% fetal bovine serum and cultured. After overnight culture, 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 0.22χη. Filter.
また、 1 2 Β 5抗体由来の一本鎖 F V (ポリペプチド） の恒常的発現 CHO 細胞株を樹立するため、 p CHO— s c 1 2 Β 5発現ベクターを下記のように C Η Ο細胞に遺伝子導入した。  In addition, in order to establish a CHO cell line that constantly expresses a single-chain FV (polypeptide) derived from the 12Β5 antibody, p CHO—sc 12Β5 expression vector was transferred into CΗ cells as follows. Introduced.
すなわち、 Gene Pulser装置 （BioRad社製） を用いたエレクトロポレーシヨン 法により発現ベクターを CHO細胞に導入した。 制限酵素 P V u Iで消化し直鎖 状にした DNA ( 1 0 0 i g) と P B Sに懸濁した CHO細胞 （1 >< 1 07細胞7 m l ) の 0. 8m 1を混合したものをキュべットに加え氷中で 1 0分間静置した後、 1. 5 kV、 2 5 μ FDの容量にてパルスを与えた。 室温にて 1 0分間の回復期間 の後、 エレクト口ポレー、:/ヨン処理された細胞を、 1 0%のゥシ胎児血清を含有 する CHO— S— S FM II (GIBC0 BRL社製） に加え培養した。 培養 2日後に 5 M メトトレキサ一ト （SIGMA社製） ならびに 1 0 %ゥシ胎児血清を含む CH O-S-S FM II (GIBC0 BRL社製） にて培養した。 得られたクローンについて 発現量の高いクローンを 12B 5—本鎖 F Vの産生細胞株として選択した。 10 nMメ トトレキサート （SIGMA社製） を含む無血清培地 CHO— S— S FM II (GIBCO BRL社製） にて培養後、 培養上清を集め、 遠心分離により細胞破片を除 去して培養上清を得た。 That is, 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 μFD. 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. 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.
7. 5 CHO細胞産生の12B 5由来の一本鎖Fvの精製 7.5 Purification of single-chain Fv from 12B5 produced by CHO cells
7. 4で得られた 12B 5—本鎖 F V発現 CHO産生株の培養上清からの精 製は、 抗 F L A G抗体力ラム及びゲル濾過力ラムにより行った。  Purification from the culture supernatant of the 12B5-single-chain FV-expressing CHO-producing strain obtained in 7.4 was carried out using an anti-FLAG antibody force ram and a gel filtration force ram.
(1) 抗 F LAG抗体カラム  (1) Anti-FLAG antibody column
培養上清は、 P B Sで平衡化した抗 FLAG M2ァフィ二ティ一ゲル （SIGMA 社製） に添加した。 同緩衝液でカラムを洗浄後、 緩衝液を 0. 1Mグリシン塩酸緩 衝液 (pH3. 5) でカラムに吸着した蛋白質を溶出した。 溶出画分は、 溶出後直 ちに 1Mトリス塩酸緩衝液 （pH8.0) を加えて中和した。 SDS— PAGEで 溶出画分を分析し、 ^本鎖 F Vが確認された画分を Centricon- 10 (MILLIPORE社 製） を用いて濃縮した。  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).
(2) ゲル濾過  (2) Gel filtration
(1) の濃縮液は、 0.01 %Tw e e n 20を含む PB Sで平衡化した S u p e r d e x 200カラム （10x300 mm, AMERSHAM PHARMACIA社製） に添カロ した。  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.
s c 12 B 5は 2つのピーク （A、 B) に分かれて溶出した （図 48を参照）。 画分 A、 Bを 14%—SD S—ポリアクリルアミドゲルを用いて分析した。 サン プルを還元剤添加、 非添加で処理し、 L a emm l iの方法に準じて電気泳動を 行い、 泳動後蛋白質をクマシ一ブリリアントブルー染色した。 図 49に示すよう に、 画分 A、 Bいずれも還元剤の添加の有無に関わらず、 見かけ上の分子量約 3 1 kDに単一バンドを与えた。 画分 A及び Bを S u p e r d e X 200 PC 3. 2/30 (3. 2x30 Omm、 AMERSHAM PHARMACIA社製） を用いたゲル濾過によ り分析した結果、 画分 Aでは見かけ上の分子量約 44 kD、 画分 Bでは同 22 k Dに溶出された （図 50 a及び bを参照）。 以上の結果から、 画分 Aは s c 12 B 5—本鎖 F Vの非共有結合性ダイマーで、 Bはモノマーである。 7. 6 各種一本鎖 F vの T P〇様ァゴニスト活性の測定 sc12B5 eluted in two peaks (A, B) (see Figure 48). Fractions A and B were analyzed using a 14% -SDS-polyacrylamide gel. The sample was treated with or without a reducing agent, and electrophoresed according to the method of Laemmli. After the electrophoresis, the protein was stained with Coomassie brilliant blue. As shown in FIG. 49, both fractions A and B gave a single band with an apparent molecular weight of about 31 kD regardless of the presence or absence of the reducing agent. Fractions A and B were analyzed by gel filtration using Superde X 200 PC 3.2 / 30 (3.2 x 30 Omm, manufactured by AMERSHAM PHARMACIA). 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〇-like agonist activity of various single-chain Fvs
ヒト T PO受容体 （MP L) を発現する B a /F 3細胞 （BaF/rapl) に対する増 殖活性を測定することによって、 抗 MP L—本鎖抗体の T P O様活性を評価した。 B a FZMp 1細胞を、 1 0%ゥシ胎児血清 （HyClone社製） を含む R PM I 1 6 40培地 （GIBC0社製） で 2回洗浄したのち、 5 X 1 05細胞 1の細胞密度 になるように培地に懸濁した。 抗 MP L—本鎖抗体またはヒ ト T P O (R&D Systems 社製） を培地で適当に希釈し、 細胞懸濁液 5 0 μ 1に抗体またはヒト Τ P p_希釈液 5 0 1を加えて 9 6穴マイク口ゥエル平底プレート （Falcon社製） に分注し、 C〇2インキュベーター （C02濃度： 5%) で 24時間培養した。 培 養後、 W S T— 8試薬 （生細胞数測定試薬 S F ：ナカライテスタ社製） を 1 0 ju 1加え、 直後に蛍光吸光光度計 SPECTRA Fluor (TECAN社製） を用いて測定波長 4 5 0 n ms 対照波長 6 20 n mの吸光度を測定した。 C02インキュベーター (C 02濃度： 5%) で 2時間インキュベートした後、 SPECTRA Fluor を用いて再度測 定波長 4 5 0 nm、 対照波長 6 2 0 n mの吸光度を測定した。 WST— 8試薬は 生細胞数に応じて波長 4 5 0 nmの発色反応を呈することから、 2時間の吸光度 変化を指標に B a F/Mp 1増殖活性を評価した。 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 μl of the cell suspension is added with antibody or human ΤP p_ diluent 501 and added to the suspension. dispensed into 6-well microphone port Ueru flat bottom plate (manufactured by Falcon) min, C_〇 2 incubator (C0 2 concentration: 5%) and incubated for 24 hours at. After culturing, add 10 ju1 of WST-8 reagent (viable cell counting reagent SF: manufactured by Nacalai Tester), and immediately thereafter measure the wavelength using a fluorescence spectrophotometer SPECTRA Fluor (manufactured by TECAN) at 450 nm. the s reference wavelength 6 20 nm of the absorbance was measured. C0 2 incubator (C 0 2 concentration: 5%) and incubated for 2 hours at, again Measurement Wavelength 4 5 0 nm using a SPECTRA Fluor, a reference wavelength 6 2 0 nm absorbance was measured. Since the WST-8 reagent exhibits a color reaction at a wavelength of 450 nm according to the number of living cells, the proliferation activity of BaF / Mp1 was evaluated using the change in absorbance over 2 hours as an index.
各種 1 2 B 5抗体分子を発現させた CO S— 7細胞の培養上清を用い、 MP L に対するァゴニスト活性を測定した結果、 図 5 1に示すように抗原結合部位が二 価である 1 2 B 5 I g Gでは濃度依存的に吸光度の上昇が認められ TP O様のァ ゴニスト活·生を示したのに対し （ED 5 0 ； 2 9 nM)、 抗原結合部位が一価であ る' 1 2 B 5 F a bのァゴニスト活性は非常に弱いものであった （ED 5 0 ； 34， 7 24 nM)。 それに対し、 F a bと同様に抗原結合部位が一価である一本鎖 F v ( s c 1 2 B 5) においては ED 5 0値が 7 5 nMと強いァゴニスト活性が認め られた。 しかしながら、 一本鎖 F vでは H鎖、 L鎖各可変領域は非共有結合で介 合しているために、 溶液中で各可変領域が解離し他の分子の可変領域と介合し二 量体等の多量体を形成することが知られている。 そこで、 ゲル濾過を用い精製 s c 1 2 B 5の分子量を測定した結果、 確かに単量体 （モノマー） と二量体 （ダイ マー） と考えられる分子が認められた （図 4 8を参照）。 続いて、 モノマーとダイ マーの s c 12 B 5をそれぞれ単離し （図 50を参照）、 それらの MP Lに対する ァゴニスト活性を測定した結果、 図 51及び 52に示すように s c 12 B 5モノ マーでは ED 50値が4438. 7 nMと C O S— 7細胞の培養上清を用いた結果 に比べ、 ァゴニス ト活性の減弱が確認された。 それに対し、 二価の抗原結合部位 を持つ一本鎖 F V ( s c 1 2 B 5ダイマー） では一価の s c 1 2 B 5に対し約 4 00倍強いァゴニス ト活性を示した （ED 50 ； 10. 1 nM)0 さらに、 二価の 一本鎖 F vではヒ ト TPOならびに 12B 5 I gGのァゴニス ト活性と同等もし くは _それ以上のァゴニスト活性を示した。 図面の簡単な説明 Using the culture supernatant of COS-7 cells expressing various 12B5 antibody molecules, the agonist activity against MPL was measured. As shown in Fig. 51, 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). On the other hand, in the case of a single-chain Fv (sc12B5) 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. However, in the single-chain Fv, since the variable regions of the H and L chains are non-covalently bonded, 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. Then, 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). . Next, 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. Compared with the results obtained using the culture supernatant of 7 nM and COS-7 cells, a decrease in agonist activity was confirmed. On the other hand, a single-chain FV (sc12B5 dimer) having a divalent antigen-binding site exhibited about 400 times stronger agonist activity than monovalent sc12B5 (ED50; 10 . 1 nM) 0 Furthermore, a divalent single chain F v the human TPO and 12B 5 I Agonisu preparative activity equivalent if Ku is _ more Agonisuto activity gG. BRIEF DESCRIPTION OF THE FIGURES
図 1. ヒト I g G 1抗体が、 ヒト I A Pを発現する L 1210細胞 （ h I A P /L 1 210) に結合しないことを示すフローサイトメトリーの結果を示す図で ある。  FIG. 1. Flow cytometry results showing that human IgG1 antibody does not bind to L1210 cells expressing human IAP (hIAP / L1210).
図 2. キメラ MAB L— 1抗体が、 ヒト I APを発現する L 1210細胞 （h I AP/L 1210) に特異的に結合することを示すフローサイトメトリーの結 果を示す図である。  FIG. 2. Flow cytometry results showing that the chimeric MAB L-1 antibody specifically binds to L1210 cells expressing human IAP (hIAP / L1210).
図 3. キメラ MAB L— 2抗体が、 ヒト I APを発現する L 1 210細胞 （h I AP/L 1 210) に特異的に結合することを示すフローサイトメトリーの結 果を示す図である。  Figure 3. Flow cytometry results showing that the chimeric MAB L-2 antibody specifically binds to L1210 cells expressing human IAP (hIAP / L1210). .
図 4. 本発明にかかる一本鎖 Fvの作成方法を模式的に示す図である。  FIG. 4 is a diagram schematically showing a method for producing a single-stranded Fv according to the present invention.
'図 5. 本努明の一本鎖 F Vをコードする DNAを、 大腸菌にて発現させるため に使用可能な発現プラスミドの一例の構造を示す。  'Figure 5. 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 E. coli is shown.
図 6. 本発明の一本鎖 F Vをコードする DNAを、 哺轧動物細胞にて発現させ るために使用する発現プラスミドの一例の構造を示す。  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.
図 7. 実施例 5. 4で得られたウェスタンプロットの結果を示す写真である。 左側より、 分子量マーカー （上から 97.4、 66、 45、 31、 21. 5、 14. 5 kDaを示す）、 p CHO 1導入 COS 7細胞培養上清、 pCHOM2導入細胞 培養上清。 p CHOM2導入細胞培養上清に再構成 MAB L— 2抗体一本鎖 F v (矢印） が明らかに含まれていることを示す。 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.
図 8. コントロールとしての: CHO lZCO S 7細胞培養上清の抗体は、 コ ントロールとしての COS 1/L 1210細胞には結合しないことを示すフロ 一サイトメトリーの結果を示す図である。 Figure 8. As a control: Flow cytometry results showing that antibodies from the culture supernatant of CHO lZCO S7 cells do not bind to COS1 / L1210 cells as control.
図 9. MAB L 2— s c F vZCO S 7細胞培養上清の抗体は、 コントロール としての p COS 1/L 1210細胞には結合しないことを示すフローサイトメ トリーの結果を示す図である。 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.
図 1_0. コントロールとしての p CO S 1/CO S 7細胞培養上清の抗体は、 h I AP/L 1210細胞に結合しないことを示すフローサイトメ トリーの結果を 示す図である。 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.
図 11. MAB L 2— s c F Vノ CO S 7細胞培養上清の抗体は、 h I APZL 1210細胞に特異的に結合することを示すフ口一サイトメトリ一の結果を示す 図である。 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.
図 12. 実施例 5. 6で示す C omp e t i t i V e EL I S Aの結果を示す 図であり、 本発明の一本鎖 F v (MAB L 2- s c F v) の抗原結合活性を、 コ ントロールとしての p CHO 1/COS 7細胞培養上清と比較して、 マウスモノ クローナル抗体 MAB L— 2の抗原結合に対する阻害を指標として示す図である。 図 13. 実施例 5. 7のアポトーシス誘起効果の結果を示す図であり、 コント口 ールとしての； p CO S 1 "L 1210細胞には、 コントロールとしての; CHO 1/COS 7細胞培養上清抗体はアポトーシスを誘起しないことを示す。 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.
図 14. 実施例 5. 7のアポトーシス誘導効果の結果を示す図であり、 コント口 ールとしての： COS 1/L 1210細胞には、 MAB L 2 - s c F v/CO S 7細胞培養上清抗体はアポトーシスを誘起しないことを示す。 FIG. 14 shows the results of the apoptosis-inducing effect of Example 5.7. As a control, COS1 / L1210 cells were cultured on MAB L2-scFv / COS7 cell culture. Shows that the clear antibody does not induce apoptosis.
図 15. 実施例 5. 7のアポトーシス誘導効果の結果を示す図であり、 h .I AP ZL1210細胞には、 コントロールとしての p CHO 1/COS 7細胞培養上 清抗体はアポトーシスを誘起しないことを示す。 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.
図 16. 実施例 5. 7のアポトーシス誘導効果の結果を示す図であり、 h lAP /L 1210細胞に対し、 MAB L 2— s c F v/COS 7細胞培養上清抗体が 特異的にアポトーシスを誘起することを示す。 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.
図 17. 実施例 5. 7のアポトーシス誘導効果の結果を示す図であり、 CCRF 一 CEM細胞には、 コントロールとしての p CH〇 1/CO S 7細胞培養上清抗 体はアポトーシスを誘起しないことを示す （最終濃度 50%)0 FIG. 17 shows the results of the apoptosis-inducing effect of Example 5.7. In CCRF-CEM cells, pCH〇1 / COS7 cell culture supernatant antibody as a control does not induce apoptosis. (Final concentration 50%) 0
図 18. 実施例 5. 7のアポトーシス誘導効果の結果を示す図であり、 CCRF — CEM細胞に対し、 MAB L 2— s c F v/CO S 7細胞培養上清抗体が特異 的にアポトーシスを誘起することを示す （最終濃度 50%)。 Fig. 18. This figure shows the results of the apoptosis-inducing effect of Example 5.7. In the CCRF-CEM cells, MABL2-scFv / COS7 cell culture supernatant antibody specifically induces apoptosis. (Final concentration 50%).
図.1.9. 実施例 5. 9の CHO細胞産生の MAB L— 2抗体由来の一本鎖 F Vの 精製過程において、 Blue-sepharose カラムで得られた画分をハイドロキシァパタ イトカラムを用いて精製した際のクロマトグラムを示す図であり、 主要なピーク として画分 A、 画分 Bが得られたことを示す。 Fig.1.9. In the purification process of single-chain FV derived from MAB L-2 antibody produced in CHO cells in Example 5.9, the fraction obtained with the Blue-sepharose column was purified using a hydroxyapatite column. FIG. 3 is a view showing a chromatogram at the time, showing that fractions A and B were obtained as main peaks.
図 20. 実施例 5. 9の （2) で得られた画分 A、 画分 Bについてゲル濾過によ り精製した結果を示す図であり、 画分 Aでは見かけ上の分子量約 36 kD、 画分 Bでは同 76 k Dの位置に主要ピークが （それぞれ A I及び B I ) が溶出したこ とを示す。 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 In fraction B, the major peak (AI and BI) eluted at the position of 76 kD.
図 21. 実施例 5. 9の CHO細胞産生の MAB L— 2抗体由来の一本鎖 F Vの 精製過程において得られた画分を SDS— PAGEで分析した図であり、 何れも 分子量約 35 kDに単一のバンドのみであることを示す。 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.
図 22. CHO細胞産生の MAB L— 2抗体由来の一本鎖 F Vの精製において得 られた画分 A I及び B Iをゲル濾過により分析した結果を示す図であり、 画分 A Iはモノマーからなり、 画分 B Iはダイマーからなることを示す。 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.
図 23. 本発明の一本鎖 F Vをコードする DNAを、 大腸菌の菌体内にて発現さ せるために使用可能な発現プラスミドの一例の構造を示す。 ： 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. :
図 24. 実施例 5. 1 2の大腸菌細胞産生の MAB L— 2抗体由来の一本鎖 F V ポリペプチドの精製において、 得られた粗製物をゲル濾過カラムを用いて精製し た結果を示す図であり、 各ピークはそれぞれ大腸菌細胞産生の一本鎖 F Vのモノ マー、 ダイマーを示す。 Figure 24. Example 5.12. Purification of single-chain FV polypeptide derived from MAB L-2 antibody produced by Escherichia coli cells using crude gel filtration column. , And each peak represents a single-chain FV monomer or dimer produced by E. coli cells.
図 25. 実施例 5. 1 3のアポトーシス誘起効果の結果を示す図であり、 h I A P/L 1 210細胞には、 コントロールとしてのマウス I g G抗体はアポトーシ スを誘起しないことを示す （最終濃度 3 μ gZm 1 )。 2 5. shows the results of the apoptosis-inducing effect in Example 5. 1 3, h IA The mouse IgG antibody as a control does not induce apoptosis in P / L 1210 cells (final concentration 3 μg Zm 1).
図 26. 実施例 5. 1 3のアポトーシス誘起効果の結果を示す図であり、 h I A P/L 1 21 0細胞に対し、 CHO細胞産生のMAB L 2 - s c F vダイマーが 顕著にアポトーシスを誘起することを示す （最終濃度 3 g/m 1)。 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).
図 27. 実施例 5. 1 3のアポトーシス誘起効果の結果を示す図であり、 h I A P/L 1210細胞に対し、 大腸菌細胞産生の MAB L 2— s c F vダイマーが 顕著 jこアポトーシスを誘起することを示す （最終濃度 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).
図 28. 実施例 5. 1 3のアポトーシス誘起効果の結果を示す図であり、 h I A P/L 1 210細胞には、 CHO細胞産生の MAB L 2— s c F Vモノマーのァ ポトーシス誘起作用がコントロールと同程度であることを示す （最終濃度 3 /X g 図 29. 実施例 5. 1 3のアポトーシス誘起効果の結果を示す図であり、 h I A P/L 1210細胞には、 大腸菌細胞産生の MAB L 2— s c F vモノマーのァ ポトーシス誘起作用がコントロールと同程度であることを示す （最終濃度 3 g ノ ml)。 FIG. 28 is a graph showing the results of the apoptosis-inducing effect of Example 5.13. In 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).
図 30. 実施例 5. 1 3のアポトーシス誘起効果の結果を示す図であり、 h I A P/L 1 210細胞には、 コントロールとしてのマウス I g G抗体は抗 F LAG 抗体の添加によってもアポトーシスを誘起しないことを^^す （最終濃度 3 μ gZ m l)。 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 μgZ ml).
図 31. 実施例 5. 1 3のアポトーシス誘起効果の結果を示す図であり、 h I A P/L 1210細胞に対し、 CHO細胞産生の MAB L 2— s c F vモノマーが 抗 F L A G抗体の添加によつて顕著にアポトーシスを誘起することを示す （最終 濃度 3 μ δΖπι 1)。 - 図 32. ヒト骨髄腫細胞株 ΚΡΜΜ2を移植したマウスの血清中 ヒト I g G量 を定量したものであり、 マウスにおけるヒト骨髄腫により産生されるヒト I gG の量を測定した結果を示す図であり、 s c F vZCHOダイマーが KPMM2細 胞の増殖を非常に強く抑制していることを示す。 図 33. 腫瘍移植後のマウスの生存日数を表しており、 s c Fv/CHOダイマ 一投与群において生存期間が顕著に延長されていることを示している。 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 μ δ Ζπι 1). -Figure 32. Quantification of the amount of human IgG in the serum of mice transplanted with human myeloma cell line ΚΡΜΜ2, 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.
図 34. MAB L— 2抗体由来の 2つの H鎖 V領域及び 2つの L鎖 V領域を含む 改変抗体 [s c (F v)2] を発現するプラスミドの一例の構造を示す。 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.
図 35. [H鎖] ― [L鎖] となるように V領域を連結し、 且つペプチドリンカ一 を含まない s c Fv (HLタイプ） を発現するプラスミ ドの一例の構造を示す。 図 36. HLタイプのポリペプチドの構造およびペプチドリンカ一のアミノ酸配 列を示す。 Figure 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.
図 37. [L鎖] - [H鎖] となるように V領域を連結し、 且つペプチドリンカ一 を含まない s c Fv (LHタイプ) を発現するプラスミドの一例の構造を示す。 図 38. LHタイプのポリペプチドの構造およびペプチドリンカーのアミノ酸配 列を示す。 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.
図 39. 実施例 6. 4におけるウェスタンブロッテイングの結果を示す図であり、 2つの H鎖 V領域及び 2つの L鎖 V領域を含む改変抗体 s c (F v)2及び種々の長 さのペプチドリンカ一を有する MAB L— 2抗体 s c Fvが発現していることを 示す。 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.
図 40 a及び b. 実施例 6. 3 (1) にて調製した COS 7細胞培養上清を用い たフローサイトメトリーの結果を示す図であり、 種々の長さのペプチドリンカー を有する MABL 2— s c F V及び s c (F v)2は、 ヒト I APに対して高い親和 性を有することを示す。 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.
図 41. 実施例 6. 6のアポトーシス誘導効果の結果を示す図であり、 s c Fv く HL 3, 4， 6， 7、 LH3, 4, 6， 7〉及び s c (F v) 2は h I A PZL 1 210細胞に対して顕著な細胞死を誘導することを示す。 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.
図 42. 実施例 6. 10の抗原結合評価の結果を示す図であり、 s c Fv<HL 5〉のダイマー及ぴ s c (F v)2がヒト I APに対して高い親和性を有すること示 す。 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.
図 43. 実施例 6. 11の in vitroアポトーシス誘起効果の結果を示す図であり、 MAB L 2- s c F v <HL 5 >のダイマー及び MAB L 2 - s c (F v)2¾h I AP/L 1 210、 CCRF— C EMの両細胞に対して濃度依存的に細胞死を誘 導することを示す。 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 ¾h I It shows that both AP / L 1210 and CCRF-CEM cells induce cell death in a concentration-dependent manner.
図 44. ヒト骨髄腫細胞株 KPMM2を移植したマウスにおけるヒト骨髄腫によ り産生される血清中の Mタンパク質の量を測定した結果を示す図であり、 s c F v<HL— 5>及び s c (F v)2が KPMM2細胞の増殖を非常に強く抑制してい ることを示す。 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.
図 45. 腫瘍移植後のマウスの生存日数を表しており、 s c F vく HL— 5 >投 与群において生存期間が顕著に延長されていることを示している。 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.
図 46. fl重瘍移植後のマウスの生存日数を表しており、 s c (F v)2投与群におい て生存期間が顕著に延長されていることを示している。 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.
図 47. 1 5アミノ酸からなるリンカー配列を含む再構成 12 B 5—本鎖 F Vを コードする DNA断片の構築方法とその構造を概略的 示す。 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.
図 48. 実施例 7. 5 (1) で得られた各 12 B 5—本鎖 F Vを、 ゲル濾過によ り精製した結果を示す図であり、 s c 12B 5では 2つのピーク （画分 A， B) に分かれた結果を示す。 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).
図 49. 実施例 7. 5 (2) において、 各画分 Aおよび Bを SD S— PAGEに より分析した結果を示す。 Figure 49. The results of analyzing each fraction A and B by SDS-PAGE in Example 7.5 (2) are shown.
図 50. 実施例 7. 5 (2) において、 各画分 Aおよび Bを S u p e r d e X 2 00カラムにより分析した結果を示し、 （a) 画分 Aでは見かけ上の分子量約 44 kDに、 （b) 画分 Bでは同 22 kDの位置に主要ピークが溶出されたことを示す。 図 5 1. s c 1 2 B 5及び 12 B 5抗体 （ I g G, F a b) の T PO様ァゴニス ト活性の測定結果を示し、 12 B 5 I g G及ぴー価一本鎖 F V ( s c 1 2 B 5) は、 濃度依存的に TP O様のァゴニスト活性を有することを示す。 Figure 50. In Example 7.5 (2), the results of analysis of each fraction A and B using a Superde X 200 column are shown. (A) Fraction A has an apparent molecular weight of approximately 44 kD, b) In fraction B, the major peak was eluted at 22 kD. 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.
図 52. s c 1 2 B 5モノマー及びダイマーの TP O様ァゴニスト活性の測定結 果を示し、 二価の抗原結合部位を持つ一本鎖 F V ( s c 1 2B 5ダイマー） は一 価の s c l 2B 5より約 400倍以上強いァゴニスト活性を示し、 その強さはヒ ト TPOと同等もしくはそれ以上であることを示す。 Figure 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.
産業上の利用可能性 本発明の改変抗体は、 細胞表面上の分子を架橋することにより該細胞内にシグ ナルを伝達しうるァゴニスト作用を有しており、 また抗体分子 （w h o 1 e I G) と比較して低分子化が達成されているため、 組織、 腫瘍への移行性に優れ ているという特徴を有している。 さらに本発明の改変抗体は、 元のモノクローナ ル抗体と比較して顕著に高い活性を有しているが、 これは本発明の改変抗体が抗 体分子に比べてよりリガンドに近い形態であるためと考えられる。 従って、 当該 改変抗体はシグナル伝達ァゴュストとして使用することができ、 そして抗体分子 を本発明の改変抗体にすることにより、 細胞間の架橋などによる副作用を軽減し、 且つ細胞表面上の分子を架橋して所望の作用のみを誘起しうる新規な医薬品を提 供される。 本発明の改変抗体を有効成分とする医薬製剤は、 癌、 炎症、 ホルモン 異常、 並びに白血病、 悪性リンパ腫、 再生不良性貧血、 骨髄異形成症候群および 真性多血症などの血液疾患の予防及び z又は治療薬として有用である。 Industrial applicability 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. Therefore, 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. A novel drug capable of inducing only a desired effect. 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.

Claims

請求の範囲 The scope of the claims
1. 細胞表面分子を架橋することによりァゴニスト作用を示す、 モノクローナ ル抗体の H鎖 V領域を 2つ以上及び L鎖 V領域を 2つ以上含む改変抗体。  1. A modified antibody that exhibits two or more H chain V regions and two or more L chain V regions of a monoclonal antibody that exhibits agonist action by cross-linking cell surface molecules.
2. H鎖 V領域及び L鎖 V領域がリンカ一を介して連結されている、 請求項 1 記載の改変抗体。  2. The modified antibody according to claim 1, wherein the H chain V region and the L chain V region are linked via a linker.
3. リンカ一が、 少なくとも 1個以上のアミノ酸からなるペプチドリンカ一で ある、 請求項 1または 2記載の改変抗体。  3. The modified antibody according to claim 1, wherein the linker is a peptide linker comprising at least one amino acid.
4. ； 改変抗体が、 1つの H鎖 V領域及び 1つの L鎖 V領域を含む一本鎖 F Vの ダイマーから構成される請求項 1〜 3のいずれか 1·項に記載の改変抗体。  4. The modified antibody according to any one of claims 1 to 3, wherein the modified antibody is composed of a single-chain FV dimer containing one H chain V region and one L chain V region.
5. 改変抗体が、 2つの H鎖 V領域及び 2つの L鎖 V領域を含む一本鎖ポリべ プチドである請求項 1〜 3のいずれか 1項に記載の改変抗体。  5. The modified antibody according to any one of claims 1 to 3, wherein the modified antibody is a single-chain polypeptide containing two H chain V regions and two L chain V regions.
6. 改変抗体が、 さらにポリペプチド精製のためのアミノ酸配列を含む請^項 1〜 5のいずれか 1項に記載の改変抗体。  6. The modified antibody according to any one of claims 1 to 5, wherein the modified antibody further comprises an amino acid sequence for polypeptide purification.
7. 改変抗体が精製されたものである、 請求項 1〜6のいずれか 1項に記載の 改変抗体。  7. The modified antibody according to any one of claims 1 to 6, wherein the modified antibody is purified.
8'· H鎖 V領域及び Z又は L鎖 V領域がヒト型化 H鎖 V領域及び/又は L鎖 V 領域である請求項 1〜 7のいずれか 1項に記載の改変抗体。  The modified antibody according to any one of claims 1 to 7, wherein the 8 '· H chain V region and Z or L chain V region are humanized H chain V regions and / or L chain V regions.
9. 前記細胞表面分子が、 ホルモン受容体またはサイト.力イン受容体である、 請求項 1〜 8のいずれか 1項に記載の改変抗体。 9. The modified antibody according to any one of claims 1 to 8, wherein the cell surface molecule is a hormone receptor or a site force receptor.
1 0. 細胞表面分子が、 エリスロポエチン （EPO) 受容体、 トロンボポェチン (TPO) 受容体、 顆粒球コロニー刺激因子 （G— C S F) 受容体、 マクロファ ージコロニー刺激因子 （M— C S F) 受容体、 顆粒球マクロファージコロニー刺 激因子 （GM— C S F) 受容体、 腫瘍壊死因子 （TNF) 受容体、 インターロイ キン一 1 ( I L— 1) 受容体、 インターロイキン一 2 ( I L- 2) 受容体 イン ターロイキン— 3 ( I L— 3) 受容体、 インターロイキン一 4 ( I L一 4) 受容 体、 インターロイキン一 5 ( I L- 5) 受容体、 インターロイキン一 6 (I L- 6) 受容体、 インターロイキン一 7 ( I L- 7) 受容'体、 インターロイキンー 9 ( I L- 9) 受容体、 インターロイキン一 1 0 ( I L— 1 0) 受容体、 インター ロイキン一 1 1 (I L— 1 1) 受容体、 インターロイキン一 1 2 (I L— 12) 受容体、 インターロイキン一1 3 ( I L- 1 3) 受容体、 インターロイキン一 1 5 (I L— 1 5) 受容体、 インターフェロン一 α ( I FN- α) 受容体、 インタ 一フエロン一 ]3 ( I FN- 3) 受容体、 インターフェロン一 γ ( I FN-y) 受 容体、 成長ホルモン （GH) 受容体、 インスリン受容体、 血液幹細胞増殖因子 (SCF) 受容体、 血管上皮増殖因子 （VEGF) 受容体、 上皮細胞増殖因子 (EGF) 受容体、 神経成長因子 （NGF) 受容体、 線維芽細胞増殖因子 （FG F) 受容体、 血小板由来増殖因子 （PDGF) 受容体、 トランスフォーミング増 殖因子一 J3 (TGF-J3) 受容体、 白血球遊走阻止因子 （L I F) 受容体、 毛様 体神経栄養因子 （CNTF) 受容体、 オンコスタチン M (OSM) 受容体おょぴ No t c hファミリー受容体からなる群から選択される請求項 9に記載の改変抗 体。 10. Cell surface molecules are erythropoietin (EPO) receptor, thrombopoietin (TPO) receptor, granulocyte colony stimulating factor (G-CSF) receptor, macrophage colony stimulating factor (M-CSF) receptor, granulocyte macrophage Colony stimulating factor (GM-CSF) receptor, tumor necrosis factor (TNF) receptor, interleukin-1 (IL-1) receptor, interleukin-1 2 (IL-2) receptor Interleukin-3 (IL-3) receptor, interleukin-1 4 (IL-1 4) receptor, interleukin-1 5 (IL-5) receptor, interleukin-1 6 (IL-6) receptor, interleukin-1 7 (IL-7) receptor, interleukin-9 (IL-9) receptor, interleukin-10 (IL-10) receptor, interleukin Interleukin-11 (IL—11) receptor, interleukin-11 (IL-12) receptor, interleukin-13 (IL-13) receptor, interleukin-15 (IL—1) 5) receptor, interferon-1α (IFN- α ) receptor, interferon-1] 3 (IFN-3) receptor, interferon-1γ (IFN-y) receptor, growth hormone (GH) receptor Body, insulin receptor, blood stem cell growth factor (SCF) receptor, vascular epidermal growth factor (VEGF) receptor, epidermal growth factor (EGF) receptor, nerve growth factor (NGF) receptor, fibroblast growth factor (FGF) receptor, platelet-derived growth factor (PDGF) receptor, transforming growth factor-1 J3 (TGF-J3) receptor, leukocyte migration inhibitory factor (LIF) receptor, ciliary neurotrophic factor (CNTF) ) Receptors, Oncostatin M (OSM) receptors, selected from the group consisting of Notch family receptors 10. The modified antibody according to claim 9, which is selected.
1 1. ァゴニス ト作用が、 アポトーシス誘導、 細胞増殖誘導または細胞分化誘導 である、 請求項 1〜10のいずれか 1項に記載の改変抗体。  11. The modified antibody according to any one of claims 1 to 10, wherein the agonist action is apoptosis induction, cell proliferation induction or cell differentiation induction.
12. L鎖 V領域及び H鎖 V領域が、 同一のモノクローナル抗体由来である、 請 求項 1〜 1 1のいずれか 1項に記載の改変抗体。  12. The modified antibody according to any one of claims 1 to 11, wherein the L chain V region and the H chain V region are derived from the same monoclonal antibody.
13. 元のモノクローナル抗体と比較して改善されたァゴニス ト作用を示す、 請 求項 1〜 12のいずれか 1項に記載の改変抗体。  13. The modified antibody according to any one of claims 1 to 12, which exhibits an improved agonist action as compared to the original monoclonal antibody.
14. 請求項 1〜13のいずれか 1項に記載の改変抗体をコードする DNA。  14. A DNA encoding the modified antibody according to any one of claims 1 to 13.
15. 請求項 1〜 13のいずれか 1項に記載の改変抗体を産生する動物細胞。15. An animal cell that produces the modified antibody according to any one of claims 1 to 13.
16. 請求項 1〜 13のいずれか 1項に記載の改変抗体を産生する微生物。 16. A microorganism that produces the modified antibody according to any one of claims 1 to 13.
17. 請求項 1〜1 3のいずれか 1項に記載の改変抗体のァゴニストとしての使 用。  17. Use of the modified antibody according to any one of claims 1 to 13 as an agonist.
18. —本鎖 F Vを産生する宿主動物細胞を無血清培地で培養して、 該培地中に —本鎖 Fvを分泌させ、 該培地中で形成された一本鎖 Fvダイマーを精製するこ とを特徴とする一本鎖 F Vダイマーの製造方法。  18. Culturing host animal cells producing —FVs in a serum-free medium, allowing the —FVs secreted into the medium, and purifying the FV dimer formed in the medium. A method for producing a single-chain FV dimer, characterized in that:
1 9. —本鎖 F vを産生する宿主動物細胞を無血清培地で培養して、 該培地中に —本鎖 F Vを分泌させ、 該培地中で該一本鎖 F Vのダイマーを形成させることを 特徴とする、 一本鎖 F vダイマーの安定化方法。 1 9. Culturing host animal cells that produce —FVs in a serum-free medium, allowing the —FVs to be secreted into the medium, and forming a dimer of the FVs in the medium. To A method for stabilizing a single-stranded Fv dimer.
2 0 . 細胞表面分子に結合する第 1のリガンドと第 2のリガンドを投与し、 さら に第 1及ぴ第 2のリガンドに結合して、 前記第 1及び第 2のリガンドを架橋する 物質を投与する、 細胞にァゴニスト作用を誘導する方法。  20. A first ligand and a second ligand that bind to a cell surface molecule are administered, and a substance that binds to the first and second ligands and crosslinks the first and second ligands is added. A method of inducing agonist action in cells.
2 1 . 第 1及び第 2のリガンドが、 同一又は異なる一本鎖 F Vモノマーである請 求項 2 0記載の方法。  21. The method according to claim 20, wherein the first and second ligands are the same or different single-chain FV monomers.
2 2 . リガンドを架橋する物質が、 抗体、 抗体断片または 2価の改変抗体である、 請求項 2 0又は 2 1記載の方法。  22. The method according to claim 20 or 21, wherein the substance that crosslinks the ligand is an antibody, an antibody fragment or a divalent modified antibody.
PCT/JP2001/003288 2000-04-17 2001-04-17 Agonist antibodies WO2001079494A1 (en)

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US10/257,864 US20040058393A1 (en) 2000-04-17 2001-04-17 Agonist antibodies
AU2001246934A AU2001246934A1 (en) 2000-04-17 2001-04-17 Agonist antibodies
AU1091702A AU1091702A (en) 2000-10-20 2001-10-22 Degraded tpo agonist antibody
JP2002536441A JPWO2002033072A1 (en) 2000-10-20 2001-10-22 Small molecule TPO agonist antibody
CN200410085664.9A CN1721445B (en) 2000-10-20 2001-10-22 Degraded TPO agonist antibody
RU2006127049/10A RU2430927C2 (en) 2000-10-20 2001-10-22 Atomic compound capable of specifically identifying and cross linkinking cell surface molecule or intracellular molecule
RU2006120454/10A RU2408606C2 (en) 2000-10-20 2001-10-22 Compound-agonist of thrombopoietin
AU2002210918A AU2002210918B2 (en) 2000-10-20 2001-10-22 Degraded agonist antibody
KR10-2003-7004608A KR20030055274A (en) 2000-10-20 2001-10-22 Degraded tpo agonist antibody
DE60133479T DE60133479T2 (en) 2000-10-20 2001-10-22 MODIFIED TPO AGONISTS ANTIBODIES
ES01978851T ES2304235T3 (en) 2000-10-20 2001-10-22 AGONIST ANTIBODY OF MODIFIED TYPE.
EP10173971A EP2351838A1 (en) 2000-10-20 2001-10-22 Crosslinking agonistic antibodies
PCT/JP2001/009259 WO2002033072A1 (en) 2000-10-20 2001-10-22 Degraded tpo agonist antibody
JP2002536442A JP4261907B2 (en) 2000-10-20 2001-10-22 Low molecular weight agonist antibody
AU2002210917A AU2002210917B2 (en) 2000-10-20 2001-10-22 Degraded TPO agonist antibody
PCT/JP2001/009260 WO2002033073A1 (en) 2000-10-20 2001-10-22 Degraded agonist antibody
CA002424371A CA2424371A1 (en) 2000-10-20 2001-10-22 Agonistic monoclonal antibody fragments
EP01978851A EP1327680B1 (en) 2000-10-20 2001-10-22 Modified tpo agonist antibody
CA002424364A CA2424364A1 (en) 2000-10-20 2001-10-22 Degraded tpo agonist antibody
AU1091802A AU1091802A (en) 2000-10-20 2001-10-22 Degraded agonist antibody
CNB018175449A CN1308448C (en) 2000-10-20 2001-10-22 Degraded TPO agonist antibody
CNB018174310A CN1308447C (en) 2000-10-20 2001-10-22 Degraded agonist antibody
AT01978851T ATE391174T1 (en) 2000-10-20 2001-10-22 MODIFIED TPO AGONIST ANTIBODY
US10/399,585 US20040242847A1 (en) 2000-10-20 2001-10-22 Degraded agonist antibody
RU2006120419/10A RU2422528C2 (en) 2000-10-20 2001-10-22 Dna coding modified antibody or compound with tpo agonist activity, method of producing them and animal cell or microorganism producing them
KR1020037004605A KR100870123B1 (en) 2000-10-20 2001-10-22 Degraded agonist antibody
EP01978852A EP1327681A4 (en) 2000-10-20 2001-10-22 Degraded agonist antibody
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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US7531643B2 (en) * 1997-09-11 2009-05-12 Chugai Seiyaku Kabushiki Kaisha Monoclonal antibody inducing apoptosis
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 (en) * 2003-12-12 2008-03-06 中外製薬株式会社 Screening method for modified antibodies having agonist activity
WO2005056798A1 (en) * 2003-12-12 2005-06-23 Chugai Seiyaku Kabushiki Kaisha Method of reinforcing antibody activity
WO2005056602A1 (en) * 2003-12-12 2005-06-23 Chugai Seiyaku Kabushiki Kaisha Method of screening modified antibody having agonistic activity
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JPWO2005056798A1 (en) * 2003-12-12 2007-12-06 中外製薬株式会社 Method for enhancing antibody activity
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
WO2006132341A1 (en) * 2005-06-10 2006-12-14 Chugai Seiyaku Kabushiki Kaisha sc(Fv)2 SITE-DIRECTED MUTANT
JP5224580B2 (en) * 2005-06-10 2013-07-03 中外製薬株式会社 sc (Fv) 2 site-specific mutant
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US8945543B2 (en) 2005-06-10 2015-02-03 Chugai Seiyaku Kabushiki Kaisha Stabilizer for protein preparation comprising meglumine and use thereof
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