WO2021261597A1 - ANTICORPS BISPÉCIFIQUE SE LIANT À GPC3 ET TfR - Google Patents

ANTICORPS BISPÉCIFIQUE SE LIANT À GPC3 ET TfR Download PDF

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WO2021261597A1
WO2021261597A1 PCT/JP2021/024252 JP2021024252W WO2021261597A1 WO 2021261597 A1 WO2021261597 A1 WO 2021261597A1 JP 2021024252 W JP2021024252 W JP 2021024252W WO 2021261597 A1 WO2021261597 A1 WO 2021261597A1
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amino acid
seq
bispecific antibody
antibody
gpc3
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PCT/JP2021/024252
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Japanese (ja)
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綾 長内
圭祐 三田村
正之 甲斐
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協和キリン株式会社
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    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention comprises a bispecific antibody that binds to human Glypican 3 (GPC3) and a human Transferrin Receiver (TfR), the bispecific antibody fragment, the bispecific antibody or a nucleic acid comprising a base sequence encoding the bispecific antibody fragment.
  • Antibodies are glycoproteins present in the serum and tissue fluids of all mammals, and mainly recognize foreign antigens in vivo. Antibodies have the function of neutralizing the action of foreign antigens, the ability to phagocytose FcR-expressing cells, and the ability of antibody-dependent cellular cytotoxicity through activation of the complement system and binding to receptors (FcR) present on the cell surface. It activates effector functions such as the ability of mediators to release and present antigens, and is involved in biological defense.
  • FcR complement system and binding to receptors
  • One molecule of antibody consists of two homologous light chains (L chain) and two homologous heavy chains (H chain), and has two antigen binding sites.
  • the class and subclass of the antibody are determined by the H chain, and each class and subclass has different unique functions.
  • IgG is further classified into the subclasses of IgG1, IgG2, IgG3 and IgG4, and IgA is further classified into the subclasses of IgA1 and IgA2 (Non-Patent Document 1).
  • a multivalent antibody is an antibody having a plurality of antigen binding sites in one molecule.
  • the hybrid hybridoma method is used to express the H chain and the L chain derived from two different antibodies in one cell, thereby binding to each of the two different antigens in a monovalent manner.
  • There is a report of producing a bivalent antibody (Non-Patent Document 2).
  • Non-Patent Document 2 a report of producing a bivalent antibody.
  • this method about 10 combinations of the H chain and the L chain of the antibody occur. Therefore, the amount of a multivalent antibody having a desired combination of H chain and L chain is low, and it is difficult to selectively isolate and purify such a multivalent antibody, so that the yield of the desired antibody is high. Decrease.
  • Non-Patent Document 3 a multivalent antibody (Non-Patent Document 3) containing a single chain Fv (scFv) in which the antigen-binding sites of H chain and L chain are linked by one polypeptide is known. Furthermore, the CH1 domain of the H chain constant region of IgG1, a partial fragment of the domain and the L chain constant region, or a flexible linker (Gly-Gly-Gly-Gly-Ser) was used to link the two antigen binding sites. Multivalent antibodies and the like have been reported (Patent Document 1, Patent Document 2).
  • the transferrin receptor (hereinafter also referred to as TfR) was identified as a cell membrane protein expressed on the surface of reticulocytes.
  • TfR has a function of taking up iron bound to transferrin (hereinafter, also referred to as Tf) into cells. Since iron is an essential metal for maintaining cell homeostasis and cell proliferation, TfR for uptake is active in iron uptake such as placenta's nutrient membrane cells, reticular erythrocytes, and activated lymphocytes. It is expressed in normal tissues. It is also known that it is highly expressed in various tumor cells (Non-Patent Document 4 and Non-Patent Document 5) in which proliferation is active. It is known that when TfR is cross-linked on the cell membrane, internalization by clathrin-dependent endocytosis is promoted and degraded. (Non-Patent Document 6, Non-Patent Document 7).
  • TfR Since the expression level of TfR is low in normal cells other than bone marrow cells and high in cancer cells with active proliferation, TfR has long been recognized as a molecular target for cancer treatment.
  • a TfR-neutralizing antibody showing a strong drug efficacy in a mouse cancer-bearing model is known (Patent Document 4).
  • Glypican 3 (GPC3, SGB, DGSX, MXR7, SDYS, SGBS, OCI-5, SGBS1, GTR2-2) is a GPI-anchored membrane protein consisting of 580 amino acids.
  • GPC3 is known as a cancer antigen that is highly expressed in hepatocellular carcinoma. It has also been reported to be expressed in malignant melanoma, clear ovarian cell cancer, oval sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, Wilms tumor, testicular germ cell tumor, and liposarcoma. It is expected as a target molecule, a diagnostic marker, and a cancer vaccine target for cancer molecule-targeted drugs (Non-Patent Document 8).
  • Patent Documents 5 and 6 it is known that by using a molecule capable of binding to two types of membrane proteins, one antigen molecule can be used as a scaffold and the other antigen molecule can be internalized and degraded.
  • Patent Documents 5 and 6 when a peptide that recognizes a cancer cell-specific membrane protein and a peptide that recognizes TfR are chemically bound or linked by a hybridoma fusion method, both antigens are expressed under the condition of adding an iron chelating agent. It has been reported that it exhibits a growth inhibitory activity on cells that are growing (Patent Document 7).
  • the present invention comprises a bispecific antibody that binds to human GPC3 and human TfR, the bispecific antibody fragment, a nucleic acid comprising a base sequence encoding the bispecific antibody or the bispecific antibody fragment, a vector containing the nucleic acid, the bispecific.
  • a transformant that produces an antibody or the bispecific antibody fragment a method for producing the bispecific antibody or the bispecific antibody fragment, a therapeutic agent and / or a diagnostic agent containing the bispecific antibody or the bispecific antibody fragment, the buy. It is an object of the present invention to provide a therapeutic method and / or a diagnostic method using a specific antibody or the bispecific antibody fragment, and a detection or measurement reagent containing the bispecific antibody or the bispecific antibody fragment.
  • the present invention provides a bispecific antibody or a bispecific antibody fragment thereof that binds to human GPC3 and human TfR.
  • the present invention relates to the following.
  • 1. A bispecific antibody that binds to human Glypican 3 (GPC3) and human Transferrin Receptor (TfR). 2.
  • the bispecific antibody according to 1 above which binds to human GPC3 and human TfR in a divalent manner, respectively.
  • 3. The C-terminal of the heavy chain of the (a1) IgG portion containing the first antigen-binding domain and (b1) the IgG portion containing the second antigen-binding domain and containing the first antigen-binding domain is directly or via a linker.
  • the N-terminal of the second antigen-binding domain is linked, and one of the first antigen-binding domain and the second antigen-binding domain binds to human GPC3 and the other binds to human TfR.
  • the bispecific antibody according to 5 above wherein the C-terminal of the heavy chain of the IgG portion containing the first antigen-binding domain is linked to the N-terminal of the heavy chain of Fab of the antibody directly or via a linker.
  • the second antigen-binding domain contains (a2) an IgG moiety containing a second antigen-binding domain and (b2) a first antigen-binding domain, and the C-terminal of the first antigen-binding domain is directly or via a linker.
  • One of the first antigen-binding domain and the second antigen-binding domain binds to human GPC3 and the other binds to human TfR.
  • the bispecific antibody according to 1 or 2. 8. 7.
  • the bispecific antibody according to 7 above wherein the C-terminus of the first antigen-binding domain is directly linked to the N-terminus of the heavy chain of the IgG portion containing the second antigen-binding domain.
  • the bispecific antibody according to 7 or 8 wherein the first antigen-binding domain is an antibody Fab.
  • 10. 9. The bispecific antibody according to 9 above, wherein the C-terminus of the Fab heavy chain of the antibody is linked to the N-terminus of the heavy chain of the IgG portion containing the second antigen binding domain directly or via a linker. 11.
  • the anti-GPC3 Fab is a heavy chain variable region comprising any one complementarity determining region (CDR) 1-3 selected from the following (g1) to (g20), (g23) and (g25) to (g40). (VH), and the bispecific according to any one of 11-14 above, which is a Fab comprising light chain variable regions (VL) comprising CDRs 1-3 comprising the amino acid sequences represented by SEQ ID NOs: 24-26, respectively. antibody.
  • the anti-GPC3 Fab has SEQ ID NOs: 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 178, A VH and sequence comprising an amino acid sequence represented by any one selected from 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, 230, 234, 238, 242 and 246.
  • the anti-TfR Fab comprises a VH comprising CDRs 1-3 comprising the amino acid sequences represented by SEQ ID NOs: 28-30 and a VL comprising CDRs 1-3 comprising the amino acid sequences represented by SEQ ID NOs: 24-26, respectively.
  • the anti-TfR Fab comprises a VH comprising CDRs 1-3 comprising the amino acid sequences represented by SEQ ID NOs: 41-43 and a VL comprising CDRs 1-3 comprising the amino acid sequences represented by SEQ ID NOs: 24-26, respectively.
  • the bispecific antibody according to any one of 12 to 16, which is Fab. 19.
  • the IgG moiety comprising the first antigen binding domain or the IgG moiety comprising the second antigen binding domain comprises a heavy chain constant region comprising the amino acid sequence represented by SEQ ID NO: 255.
  • the bispecific antibody according to 1. 22. Represented by SEQ ID NO: 255, VH comprising an amino acid sequence represented by any one selected from SEQ ID NOs: 90, 142, 126, 178, 198, 146, 218, 150, 202, and 226, in order from the N-terminal.
  • SEQ ID NO: 253 containing an amino acid sequence represented by any one of them.
  • the bispecific antibody according to any one of.
  • VH containing an amino acid sequence represented by any one selected from SEQ ID NOs: 94, 98, 102, 106, 114, 130, 178 and 190, and an amino acid sequence represented by SEQ ID NO: 253 are included.
  • CH1 VH containing the amino acid sequence represented by SEQ ID NO: 40, and two heavy chains each containing CH containing the amino acid represented by SEQ ID NO: 255, and VL containing the amino acid sequence represented by SEQ ID NO: 23 and 24.
  • the bispecific antibody according to any one of 1 to 27 above which does not inhibit the growth of cells expressing GPC3 and TfR, and inhibits the growth of cells co-expressing GPC3 and TfR. 29.
  • 30. A nucleic acid containing a base sequence encoding the bispecific antibody according to any one of 1 to 28 or the bispecific antibody fragment according to 29.
  • 31. A recombinant vector containing the nucleic acid according to 30 above.
  • 32. A transformant obtained by introducing the recombinant vector according to 31 above into a host cell. 33.
  • the transformant according to 32 is cultured in a medium, and the bispecific antibody according to any one of 1 to 28 or the bispecific antibody fragment according to 29 is produced and accumulated in the culture, and the culture is produced and accumulated.
  • 36. A method for treating and / or diagnosing a disease associated with at least one of GPC3 and TfR, using the bispecific antibody according to any one of 1 to 28 or the bispecific antibody fragment according to 29. 37.
  • 36. The therapeutic and / or diagnostic method according to 36 above, wherein the disease associated with at least one of GPC3 and TfR is cancer.
  • 38. The bispecific antibody according to any one of 1-28 or the bispecific antibody fragment according to 29 for use in the treatment and / or diagnosis of a disease associated with at least one of GPC3 and TfR. 39. 38.
  • a bispecific antibody that binds to human GPC3 and human TfR, a bispecific antibody fragment, a nucleic acid containing a base sequence encoding the bispecific antibody or the bispecific antibody fragment, a vector containing the nucleic acid, the bispecific.
  • a therapeutic method and / or a diagnostic method using the specific antibody or the bispecific antibody fragment, and a detection or measurement reagent containing the bispecific antibody or the bispecific antibody fragment can be provided.
  • FIG. 1 shows the structure of the bispecific antibody of the present invention.
  • A represents the structure of an N-terminal GPC3-TfR bispecific antibody or an N-terminal TfR-GPC3 bispecific antibody.
  • B represents the structure of a C-terminal GPC3-TfR bispecific antibody or a C-terminal TfR-GPC3 bispecific antibody.
  • C represents the structure of GPC3-GS-TfR bispecific antibody or TfR-GS-GPC3 bispecific antibody.
  • FIG. 2A shows the results of evaluating the binding property of each GPC3-TfR bispecific antibody to His-human TfR.
  • the vertical axis represents the absorbance, and the horizontal axis represents the name of the antibody used.
  • FIG. 2B shows the results of evaluating the binding property of each GPC3-TfR bispecific antibody to His-human TfR.
  • the vertical axis represents the absorbance, and the horizontal axis represents the name of the antibody used.
  • An anti-DNP antibody was used as a negative control.
  • FIG. 2C shows the results of evaluating the binding property of each GPC3-TfR bispecific antibody to human GPC3-His.
  • the vertical axis represents the absorbance, and the horizontal axis represents the name of the antibody used.
  • An anti-DNP antibody was used as a negative control.
  • FIG. 2D shows the results of evaluating the binding property of each GPC3-TfR bispecific antibody to human GPC3-His.
  • FIG. 2E shows the results of evaluating the binding property of each GPC3-TfR bispecific antibody to monkey GPC3-His.
  • the vertical axis represents the absorbance, and the horizontal axis represents the name of the antibody used.
  • An anti-DNP antibody was used as a negative control.
  • FIG. 2F shows the results of evaluating the binding property of each GPC3-TfR bispecific antibody to monkey GPC3-His.
  • the vertical axis represents the absorbance, and the horizontal axis represents the name of the antibody used.
  • An anti-DNP antibody was used as a negative control.
  • FIG. 3 shows the results of evaluation of the antigen expression level in liver cancer cells by flow cytometry.
  • A shows the results of evaluating the expression level of TfR in HepG2 cells and (B) in HLE cells with a flow cytometer.
  • C shows the results of evaluating the expression level of GPC3 in HepG2 cells and (D) in HLE cells with a flow cytometer.
  • the vertical axis represents the number of cells, and the horizontal axis represents the fluorescence intensity.
  • the white histogram shows the binding property of the anti-TfR antibody or the anti-GPC3 antibody, and the gray histogram shows the binding property of the negative control anti-DNP antibody.
  • FIG. 4 shows the results of evaluating the growth inhibitory activity of each anti-TfR antibody against liver cancer cells.
  • (A) shows the results of evaluating the growth inhibitory activity of the anti-TfR antibody against HLE cells and (B) the HepG2 cells.
  • the vertical axis represents the cell viability with respect to the control.
  • FIG. 5A shows the results of evaluating the growth inhibitory activity of GPC3-TfR bispecific antibody against liver cancer cells (HLE cells).
  • the vertical axis represents the cell viability with respect to the control.
  • FIG. 5B shows the results of evaluating the growth inhibitory activity of GPC3-TfR bispecific antibody against liver cancer cells (HLE cells).
  • the vertical axis represents the cell viability with respect to the control.
  • FIG. 5C shows the results of evaluating the growth inhibitory activity of GPC3-TfR bispecific antibody against liver cancer cells (HLE cells). The vertical axis represents the cell viability with respect to the control.
  • FIG. 5D shows the results of evaluating the growth inhibitory activity of GPC3-TfR bispecific antibody against liver cancer cells (HepG2 cells). The vertical axis represents the cell viability with respect to the control.
  • FIG. 5E shows the results of evaluating the growth inhibitory activity of GPC3-TfR bispecific antibody against liver cancer cells (HepG2 cells). The vertical axis represents the cell viability with respect to the control.
  • FIG. 5F shows the results of evaluating the growth inhibitory activity of GPC3-TfR bispecific antibody against liver cancer cells (HepG2 cells).
  • FIG. 6 shows the results of quantifying the number of TfR expression on the cell surface when HN3-GS-TfR1071 was added to HepG2 cells with a flow cytometer.
  • the vertical axis represents the number of TfR molecules on the cell surface per cell.
  • FIG. 7 shows the results of evaluation of the binding competition between each anti-GPC3 antibody and known anti-GPC3 antibody against human GPC3.
  • A shows the result of evaluating the binding property of the anti-GPC3 antibody (test antibody) to GPC3-AA-Fc in the presence or absence of the competing antibody GC33 by ELISA.
  • FIG. 8 shows a list of C-terminal GPC3-TfR bispecific antibodies prepared in Example 14.
  • FIG. 9A shows a list of N-terminal GPC3-TfR bispecific antibodies prepared in Example 14.
  • FIG. 9B shows a list of N-terminal GPC3-TfR bispecific antibodies prepared in Example 14.
  • FIG. 9C shows a list of N-terminal GPC3-TfR bispecific antibodies prepared in Example 14.
  • FIG. 9D shows a list of N-terminal GPC3-TfR bispecific antibodies prepared in Example 14.
  • FIG. 9E shows a list of N-terminal GPC3-TfR bispecific antibodies prepared in Example 14.
  • FIG. 9F shows a list of N-terminal GPC3-TfR bispecific antibodies prepared in Example 14.
  • FIG. 9G shows a list of N-terminal GPC3-TfR bispecific antibodies prepared in Example 14.
  • FIG. 10 shows the results of evaluating the expression levels of GPC3 and TfR in GPC3 knockdown HepG2 cells.
  • FIG. 11A shows the results of evaluating the growth inhibitory activity of GPC3-TfR bispecific antibody against GPC3 knockdown HepG2 cells (HepG2 # P05 cells, GPC3 ++).
  • FIG. 11B shows the results of evaluating the growth inhibitory activity of GPC3-TfR bispecific antibody against GPC3 knockdown HepG2 cells (HepG2 # 28 cells, GPC3 +).
  • FIG. 11C shows the results of evaluating the growth inhibitory activity of GPC3-TfR bispecific antibody against GPC3 knockdown HepG2 cells (HepG2 # 28 cells, GPC3 +).
  • FIG. 11A shows the results of evaluating the growth inhibitory activity of GPC3-TfR bispecific antibody against GPC3 knockdown HepG2 cells (HepG2 # P05 cells, GPC3 ++).
  • FIG. 11B shows the results of evaluating the growth inhibitory activity of GPC3-TfR bispecific antibody against GPC3 knockdown HepG2 cells (
  • FIG. 11D shows the results of evaluating the growth inhibitory activity of GPC3-TfR bispecific antibody against GPC3 knockdown HepG2 cells (HepG2 # 28 cells, GPC3 +).
  • FIG. 12A shows the results of evaluating the growth inhibitory activity of GPC3-TfR bispecific antibody against HepG2 cells (GPC3 +++).
  • FIG. 12B shows the results of evaluating the growth inhibitory activity of GPC3-TfR bispecific antibody against HepG2 # 28 cells (GPC3 +).
  • FIG. 13A shows the results of evaluation of the binding property of GPC3-TfR bispecific antibody to human GPC3.
  • FIG. 13B shows the results of evaluation of the binding property of the GPC3-TfR bispecific antibody to monkey GPC3.
  • FIG. 13C shows the results of evaluation of the binding property of GPC3-TfR bispecific antibody to human TfR.
  • FIG. 14 shows the results of evaluating the cell proliferation inhibitory activity of GPC3-TfR bispecific antibody.
  • A shows the growth inhibitory activity of GPC3-TfR bispecific antibody against HepG2 cells (GPC3 +++).
  • B shows the growth inhibitory activity of GPC3-TfR bispecific antibody against HepG2 # 28 cells.
  • FIG. 15A shows the results of evaluating the effect of addition of ferrous iron on the growth inhibitory activity of GPC3-TfR bispecific antibody on HepG2 cells.
  • FIG. 15B shows the results of evaluating the effect of ferrous iron addition on the growth inhibitory activity of GPC3-TfR bispecific antibody on HepG2 cells.
  • FIG. 15C shows the results of evaluating the effect of ferrous iron addition on the growth inhibitory activity of GPC3-TfR bispecific antibody against HepG2 # 28 cells.
  • FIG. 15D shows the results of evaluating the effect of ferrous iron addition on the growth inhibitory activity of GPC3-TfR bispecific antibody on HepG2 # 28 cells.
  • the present invention is a bispecific antibody or a bispecific antibody fragment thereof (hereinafter, the bispecific antibody of the present invention or the same) that binds to human GPC3 (hereinafter, also simply abbreviated as GPC3) and human TfR (hereinafter, also simply abbreviated as TfR). Also referred to as a bispecific antibody fragment).
  • GPC3 in the present invention is used synonymously with SGB, DGSX, MXR7, SDYS, Simpson-Golabi-Hehmel Syndrome, Type1 (SGBS), OCI-5, SGBS1 and GTR2-2.
  • GenBank Accession No. Examples thereof include human GPC3 containing the amino acid sequence shown in NP_004475.
  • GenBank Accession No. NP_004475 GenBank Accession No. Examples thereof include polypeptides consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence shown in XP_005594665 or NP_057906, and having the function of GPC3.
  • a polypeptide having an amino acid sequence having a homology of% or more, 98% or more, and 99% or more and having a function of GPC3 is also included in GPC3 of the present invention.
  • GenBank Accession No. NP_004475, GenBank Accession No. XP_005594665 or GenBank Accession No. It can be obtained by introducing a site-specific mutation into the DNA encoding the amino acid sequence shown in NP_057906.
  • the number of amino acids to be deleted, substituted or added is not particularly limited, but is preferably 1 to several tens, for example, 1 to 20, more preferably 1 to several, for example, 1 to 5 amino acids. Is.
  • Examples of the gene encoding GPC3 in the present invention include GenBank Accession No. Examples thereof include the gene of human GPC3 containing the base sequence shown in NM_004484.
  • GenBank Accession No. GenBank Accession No. 1 is a gene containing a DNA encoding a polypeptide having the function of GPC3, which consists of a base sequence in which one or more bases are deleted, substituted or added in the base sequence shown in NM_004484. It consists of a base sequence having 60% or more homology with the base sequence shown in NM_004484, preferably a base sequence having 80% or more homology, and more preferably a base sequence having 95% or more homology, and of GPC3.
  • a gene comprising a DNA containing a base sequence shown in NM_004484 and a DNA that hybridizes under stringent conditions and encoding a polypeptide having a function of GPC3 is also a gene encoding GPC3 of the present invention. included.
  • GenBank Accession No. means a hybridizable DNA obtained by a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, a DNA microarray method, or the like, using a DNA having the base sequence shown in NM_004484 as a probe. .. Specifically, 0.7 to 1.0 mol / L sodium chloride is present using a filter or slide glass on which DNA derived from a hybridized colony or plaque, or a PCR product or oligo DNA having the sequence is immobilized.
  • Gene polymorphisms are often found in the base sequences of genes encoding eukaryotic proteins.
  • genes in which a small mutation in the base sequence is caused by such a polymorphism are also included in the gene encoding GPC3 of the present invention.
  • the value of homology in the present invention may be a value calculated by using a homology search program known to those skilled in the art, but for the base sequence, BLAST [J. Mol. Biol. For amino acid sequences such as numerical values calculated using default parameters in., 215, 403 (1990)], BLAST2 [Nucleic Acids Research, 25, 3389 (1997), Genome Research, 7, 649 (1997), http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/information3.html] includes numerical values calculated using default parameters. It was
  • the default parameters are 5 if G (Cost to open gap) is a base sequence, 11 if it is an amino acid sequence, 2 if -E (Cost to extend gap) is a base sequence, and 1 if it is an amino acid sequence.
  • -Q (Penalty for nucleotide mismatch) is -3
  • -r (reward for nucleotide match) is 1
  • -e (expect value)
  • -W (wordsize) is 11 residues, amino acid sequence.
  • -y [Dropoff (X) for blast extensions in bits] is 20, if it is blastn 7, 7 in programs other than blastn
  • -X X dropoff value for gapped sequences in-bits
  • Z final X dropoff value for gapped sequences in bits
  • a polypeptide consisting of a partial sequence of the amino acid sequence of GPC3 can be prepared by a method known to those skilled in the art, for example, GenBank Accession No. It can be prepared by deleting a part of the DNA encoding the amino acid sequence shown in NP_004475 and culturing a transformant into which an expression vector containing the DNA is introduced. Further, based on the polypeptide or DNA produced by the above method, for example, GenBank Accession No. A polypeptide having an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the partial sequence of the amino acid sequence shown in NP_004475 can be obtained.
  • a polypeptide consisting of a partial sequence of the amino acid sequence of GPC3, or a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the partial sequence of the amino acid sequence of GPC3 is fluorenylmethyloxycarbonyl. It can also be produced by a chemical synthesis method such as the (Fmoc) method or the t-butyloxycarbonyl (tBoc) method.
  • GenBank Accession No. The amino acid sequence of human GPC3 shown in NP_004475 is used in a known transmembrane region prediction program SOSUI (http://sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html), TMHMM ver. Areas predicted using 2 (http://www.cbs.dtu.dk/services/TMHMM-2.0/) or Expasy Proteomics Server (http://Ca.expasy.org/) can be mentioned. Specifically, GenBank Accession No. The amino acid sequences shown in the 25th to 563rd positions of NP_004475 can be mentioned.
  • Functions of GPC3 include, for example, promoting the binding between Wnt and Frizzled by forming a complex with Wnt, and promoting the proliferation and migration of cells expressing GPC3 by activating the Wnt pathway. Will be.
  • Examples of cells expressing GPC3 include hepatocellular carcinoma, malignant melanoma, clear cell ovarian cancer, oval sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, Wilms tumor, testicular germ cell tumor, liposarcoma and the like. Examples include cancer cells contained in.
  • the expression level of GPC3 can be determined based on the chart obtained by the flow cytometry method.
  • the position appearing on the chart may vary depending on the voltage setting, sensitivity setting, antibody clone used, staining conditions, dye used, etc. of the device, but those skilled in the art can use a cell population recognized as a group in the obtained chart. Lines can be drawn as appropriate so as not to separate them.
  • the expression level of GPC3 can also be determined by immunostaining.
  • the degree of expression of the target marker (low expression, medium expression or high expression) can be determined by comparison with the results of control cells measured under the same conditions.
  • Examples of the control cell include HepG2 cells described in the section of Examples.
  • the degree of GPC3 expression in a cell population is determined by using flow cytometry to compare the GPC3 expression level in the cell population with the GPC3 expression level in the HepG2 cell population (control cells with high GPC3 expression). If the same expression is observed, it can be judged to be high expression, and if the expression is lower than that of the control cell, it can be judged to be low expression or medium expression.
  • TfR in the present invention is used as a synonym for CD71, TFR1, TR, T9, p90, and IMD46.
  • TfR for example, GenBank Accession No. Examples thereof include human TfR containing the amino acid sequence shown in NP_003225 or SEQ ID NO: 6, monkey TfR containing the amino acid sequence shown in SEQ ID NO: 8. Further, for example, SEQ ID NO: 6, GenBank Accession No. Examples thereof include polypeptides consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in NP_003225 and having the function of TfR.
  • SEQ ID NO: 6 GenBank Accession No.
  • SEQ ID NO: 6 GenBank Accession No. Polypeptides having an amino acid sequence in which one or more amino acid residues are deleted, substituted, or added in the amino acid sequence shown in NP_003225 or SEQ ID NO: 8 are site-specific mutation-introducing methods [Molecular Cloning, A Laboratory]. Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. , 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proceeding of the National Academy of Sciences in USA, 82, 488 (1985)], etc.
  • SEQ ID NO: 6 GenBank Accession No. It can be obtained by introducing a site-specific mutation into the DNA encoding the amino acid sequence set forth in NP_003225 or SEQ ID NO: 8.
  • the number of amino acids to be deleted, substituted or added is not particularly limited, but is preferably 1 to several tens, for example, 1 to 20, more preferably 1 to several, for example, 1 to 5 amino acids. Is.
  • Examples of the gene encoding TfR include SEQ ID NO: 5 or GenBank Accession No. Examples thereof include the base sequence of human TfR shown in NM_003234 and the base sequence of monkey TfR shown in SEQ ID NO: 7. Further, for example, SEQ ID NO: 5, GenBank Accession No. Gene containing DNA encoding a polypeptide having one or more bases deleted, substituted or added in the base sequence shown in NM_003234 or SEQ ID NO: 7 and having the function of TfR, SEQ ID NO: 5, GenBank Accession No.
  • a gene consisting of a DNA consisting of the base sequence shown in NM_003234 or SEQ ID NO: 7 and a DNA that hybridizes under stringent conditions and encoding a polypeptide having a TfR function can also be used as the TfR of the present invention. Included in the encoding gene.
  • genes in which a small mutation in the base sequence is caused by a gene polymorphism are also included in the gene encoding TfR of the present invention. It was
  • a polypeptide consisting of a partial sequence of the amino acid sequence of TfR can be prepared by a method known to those skilled in the art, for example, SEQ ID NO: 6, GenBank Accession No. It can be prepared by deleting a part of the DNA encoding the amino acid sequence shown in NP_003225 or SEQ ID NO: 8 and culturing a transformant into which an expression vector containing the same is introduced. Further, based on the polypeptide or DNA produced by the above method, for example, SEQ ID NO: 6, GenBank Accession No. A polypeptide having an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the partial sequence of the amino acid sequence shown in NP_003225 or SEQ ID NO: 8 can be obtained.
  • a polypeptide consisting of a partial sequence of the amino acid sequence of TfR, or a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the partial sequence of the amino acid sequence of TfR is fluorenylmethyloxycarbonyl. It can also be produced by a chemical synthesis method such as the (Fmoc) method or the t-butyloxycarbonyl (tBoc) method.
  • the extracellular region of TfR in the present invention includes, for example, GenBank Accession No.
  • GenBank Accession No. The amino acid sequence of human TfR shown in NP_003225 is used in a known transmembrane region prediction program SOSUI (http://sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html), TMHMM ver. 2 (http://www.cbs.dtu.dk/services/TMHMM-2.0/) or the region predicted using Expasy Proteomics Server (http://Ca.expasy.org/) and the like can be mentioned.
  • SEQ ID NO: 2 or GenBank Accession No. Examples thereof include the amino acid sequences shown at positions 89 to 760 of NP_003225.
  • TfR Functions of TfR include uptake of iron, which is essential for cell survival and proliferation.
  • iron-transferrin complex binds to TfR, it is taken up into cells by endocytosis.
  • endocytosis When the pH in endosomes decreases, iron is released from transferrin and transferred into the cytoplasm via DMT1 to be used for cell proliferation and energy production. It is known that the iron-free transferrin-TfR complex is not normally degraded and relocates to the cell surface (Reference: Yamashiro DJ et al., Cell 37 789-800, 1984).
  • Cells expressing TfR include, for example, cells of many normal tissues such as bone marrow and placenta cells, colon cancer, head and neck cancer, brain tumor, hematopoietic tumor, liver cancer, and esophageal cancer. Many types of cancer cells, HT29, HSC-2, RAMOS, K562, HepG2, OE21, T.I. Many cancer cell lines such as Tn, U-937, HuH-7, HLE and the like can be mentioned.
  • An antibody is a gene that encodes all or part of the variable region of a heavy chain and the constant region of a heavy chain that constitute immunoglobulin, and the variable region of a light chain and the constant region of a light chain (referred to as "antibody gene"). It is a protein derived from.
  • the antibodies of the invention include antibodies or antibody fragments having any immunoglobulin class and subclass.
  • the heavy chain refers to the polypeptide having the larger molecular weight among the two types of polypeptides constituting the immunoglobulin molecule. Heavy chains determine antibody classes and subclasses. IgA, IgD, IgE, IgG and IgM each have an ⁇ chain, a ⁇ chain, an ⁇ chain, a ⁇ chain and a ⁇ chain as heavy chains, and the constant regions of the heavy chains are characterized by different amino acid sequences.
  • the light chain refers to the polypeptide having the smaller molecular weight among the two types of polypeptides constituting the immunoglobulin molecule. In the case of human antibodies, there are two types of light chains, ⁇ chains and ⁇ chains.
  • variable region usually refers to a region rich in diversity existing in the amino acid sequence on the N-terminal side of immunoglobulin.
  • the part other than the variable region has a structure with little diversity, and is therefore called a stationary region (C region).
  • the heavy and light chain variable regions associate to form an antigen binding site and determine the binding properties of the antibody to the antigen.
  • variable region corresponds to the amino acid sequence from the 1st to the 117th in the EU index (Kabat et al., Sequences of proteins of immunological interest, 1991 Fifth edition) of Kabat et al.
  • constant region corresponds to the amino acid sequence after the 118th.
  • amino acid sequences from the first to the 107th in the numbering by Kabat et al. (Kabat numbering) correspond to the variable region
  • amino acid sequences from the 108th to the present correspond to the constant region.
  • the heavy chain variable region or the light chain variable region is abbreviated as VH or VL.
  • the antigen-binding site is a site that recognizes and binds to an antigen in an antibody, and refers to a site that forms a three-dimensional structure complementary to an antigenic determinant (epitope).
  • the antigen binding site produces a strong intramolecular interaction with the antigenic determinant.
  • the antigen binding site is composed of VHs and VLs containing at least three complementarity determining regions (CDRs). For human antibodies, VH and VL each have 3 CDRs. These CDRs are referred to as CDR1, CDR2 and CDR3 in order from the N-terminal side, respectively.
  • CH the heavy chain constant region or the light chain constant region is described as CH or CL, respectively.
  • CH is classified by the subclasses of heavy chains, ⁇ chain, ⁇ chain, ⁇ chain, ⁇ chain and ⁇ chain.
  • CH is composed of a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain arranged in order from the N-terminal side, and the CH2 domain and the CH3 domain are collectively referred to as an Fc region.
  • CL is classified into two subclasses called C ⁇ chain and C ⁇ chain.
  • Monoclonal antibodies are antibodies secreted by antibody-producing cells that retain monoclonality and recognize a single epitope. Monoclonal antibody molecules have the same amino acid sequence (primary structure) and have a single structure. Polyclonal antibody refers to a population of antibody molecules secreted by antibody-producing cells of different clones. An oligoclonal antibody is a group of antibody molecules in which a plurality of different monoclonal antibodies are mixed.
  • Epitope refers to the structural site of the antigen that the antibody recognizes and binds to.
  • Examples of the epitope include a single amino acid sequence recognized and bound by a monoclonal antibody, a three-dimensional structure consisting of an amino acid sequence, an amino acid sequence to which a sugar chain is bound, and a three-dimensional structure consisting of an amino acid sequence to which a sugar chain is bound. ..
  • Examples of the monoclonal antibody in the present invention include an antibody produced by a hybridoma and a recombinant antibody produced by a transformant transformed with an expression vector containing an antibody gene.
  • Hybridomas can be prepared, for example, by preparing an antigen, obtaining antibody-producing cells having antigen specificity from an animal immunized with the antigen, and further fusing the antibody-producing cells with myeloma cells.
  • the desired monoclonal antibody can be obtained by culturing the hybridoma or administering the hybridoma to an animal to cause the hybridoma to become ascites cancer, and separating and purifying the culture solution or ascites.
  • the animal immunizing the antigen any animal can be used as long as it is possible to produce a hybridoma, but mice, rats, hamsters, rabbits and the like are preferably used. It is also possible to obtain cells having an antibody-producing ability from such an immunized animal, immunize the cells in vitro, and then fuse with myeloma cells to produce a hybridoma.
  • Examples of the recombinant antibody in the present invention include a recombinant mouse antibody, a recombinant rat antibody, a recombinant hamster antibody, a recombinant rabbit antibody, a human chimeric antibody (also referred to as a chimeric antibody), and a humanized antibody (CDR transplanted antibody). Also referred to as) and antibodies produced by gene recombination technology, such as human antibodies.
  • the chimeric antibody refers to an antibody consisting of VH and VL of an antibody of a non-human animal (non-human animal) and CH and CL of a human antibody.
  • non-human animal any animal such as mouse, rat, hamster and rabbit can be used as long as it is possible to produce a hybridoma.
  • cDNA encoding VH and VL was obtained from a hybridoma derived from a non-human animal producing a monoclonal antibody, and the cDNA was inserted into an expression vector for animal cells having DNA encoding CH and CL of the human antibody, respectively. It can be produced by constructing a vector for expressing a chimeric antibody, introducing it into animal cells and expressing it.
  • the humanized antibody refers to an antibody obtained by transplanting the CDRs of VH and VL of non-human animal antibodies into the corresponding CDRs of VH and VL of human antibodies. Regions other than CDRs of VH and VL are referred to as framework regions (hereinafter referred to as FR).
  • the humanized antibody is a cDNA encoding a VH amino acid sequence consisting of the VH CDR amino acid sequence of a non-human animal antibody and the VH FR amino acid sequence of any human antibody, and the VL CDR amino acid of a non-human animal antibody.
  • a cDNA encoding the VL amino acid sequence consisting of the sequence and the VL FR amino acid sequence of any human antibody is constructed and inserted into an expression vector for animal cells having DNA encoding CH and CL of the human antibody, respectively, for humans. It can be produced by constructing a vector for expressing a humanized antibody, introducing it into animal cells and expressing it.
  • Human antibody is an antibody that originally exists naturally in the human body, but is a human antibody phage library and a human antibody-producing trans produced by recent advances in genetic engineering, cell engineering, and developmental engineering. Antibodies obtained from genetic animals are also included.
  • An antibody that naturally exists in the human body is obtained by, for example, infecting human peripheral blood lymphocytes with EB virus or the like to immortalize them, and cloning the lymphocytes that produce the antibody. It can be obtained by purifying the antibody.
  • the human antibody phage library is a library in which antibody fragments such as Fab and scFv are expressed on the surface of phage by inserting an antibody gene prepared from human B cells into the phage gene. From the library, phages expressing an antibody fragment having a desired antigen-binding activity on the surface can be recovered using the binding activity to the substrate on which the antigen is immobilized as an index. The antibody fragment can be further converted into a human antibody molecule consisting of two complete H chains and two complete L chains by genetic engineering techniques.
  • a human antibody-producing transgenic animal means an animal in which a human antibody gene is integrated into a cell.
  • a human antibody-producing transgenic mouse can be produced by introducing a human antibody gene into mouse ES cells, transplanting the ES cells into an early embryo of a mouse, and then generating an individual.
  • Human antibody-producing human antibodies derived from transgenic animals are produced and accumulated in the culture supernatant by obtaining hybridomas using the hybridoma production method normally used in non-human animals and culturing them. Can be prepared by.
  • the CH of the recombinant antibody may be any CH as long as it belongs to human immunoglobulin, but a human immunoglobulin G (hIgG) class is preferable. Further, any of the subclasses such as hIgG1, hIgG2, hIgG3 and hIgG4 belonging to the hIgG class can be used. Further, as the CL of the recombinant antibody, any one belonging to human immunoglobulin may be used, and those of ⁇ class or ⁇ class can be used.
  • hIgG human immunoglobulin G
  • the bispecific antibody refers to a polypeptide or protein that specifically binds to each of two different types of epitopes.
  • Each antigen binding site of a bispecific antibody may bind to a different epitope of a single antigen or may bind to a different antigen.
  • the antigen-binding domain is a partial structure of a bispecific antibody having a function of specifically recognizing and binding an antigen.
  • a protein having an antigen-binding ability such as an antibody or an antibody fragment thereof, a recombinant protein containing the CDR of the antibody, an antibody variable region containing the CDR, a ligand or a receptor is used. Examples include recombinant proteins or polypeptides.
  • the antigen-binding domain is preferably Fab of the antibody.
  • the antigen-binding domain of the present invention also includes VH having an antigen-binding activity with the antibody VH alone.
  • the first antigen-binding domain refers to the first antigen-binding domain contained in the bispecific antibody
  • the second antigen-binding domain refers to the first antigen-binding domain contained in the bispecific antibody.
  • the first antigen-binding domain refers to the antigen-binding domain on the N-terminal side.
  • the second antigen-binding domain refers to the antigen-binding domain on the C-terminal side of the first antigen-binding domain.
  • the binding of a polypeptide, antibody or antibody fragment thereof or bispecific antibody or bispecific antibody fragment to at least one of GPC3 and TfR is described, for example, by a known immunological detection method, preferably fluorescent cell staining. It can be confirmed by a method for confirming the binding property between an antibody and a cell expressing GPC3 or TfR by using a method or the like.
  • known immunological detection methods [Monoclonal Antibodies-Principles and Practice, Third edition, Academic Press (1996), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988), Monoclonal antibody experiment manual, Kodansha Scientific (1987)] and the like can also be used in combination.
  • the antigen-binding domain that binds to GPC3 or TfR may be anything as long as it specifically recognizes and binds to GPC3 or TfR.
  • polypeptides such as antibodies, ligands, receptors, and naturally occurring interacting molecules that can be made by gene recombination techniques, protein molecules and fragments thereof, and conjugates of small or natural products of the protein molecules. It may be in any form.
  • Examples of the bispecific antibody or the bispecific antibody fragment of the present invention include a bispecific antibody or the bispecific antibody fragment having TfR internalization and / or degrading activity.
  • the bispecific antibody or fragment of the bispecific antibody of the present invention does not show TfR internalization and / or degradation activity in cells that do not express GPC3, and TfR internalization and / / only in cells that express GPC3.
  • a bispecific antibody or a fragment of the bispecific antibody fragment exhibiting degrading activity is preferable. Since such a bispecific antibody or the bispecific antibody fragment selectively exhibits TfR internalization and / or degradation activity to pathogenic cells such as cancer cells expressing GPC3, non-specific TfR inclusions. It is preferable in that it does not cause side effects associated with internalization and / or decomposition.
  • the bispecific antibody or fragment of the bispecific antibody of the present invention may bind to GPC3 and TfR expressed on the same cell, or may bind to GPC3 and TfR expressed on different cells. However, those that bind to GPC3 and TfR expressed on the same cell are preferable.
  • the bispecific antibody or fragment of the bispecific antibody of the present invention inhibits the proliferation and / or induces cell death of cells expressing the target GPC3 by inducing the internalization and / or degradation of TfR. Is preferable.
  • the bispecific antibody of the present invention or the bispecific antibody fragment thereof is the proliferation of cells that do not express GPC3 and that do not inhibit the proliferation of cells expressing TfR and / or induce cell death, and that co-express GPC3 and TfR. Inhibits and / or induces cell death.
  • TfR is expressed in cells including normal cells
  • GPC3 is hepatocellular carcinoma, malignant melanoma, clear ovarian cell carcinoma, oval sac tumor, chorionic villus cancer, neuroblastoma, hepatoblastoma, Wilms. It has been reported to be expressed in cancer cells such as tumors, testicular embryonic cell tumors and liposarcoma. Therefore, a bispecific antibody that inhibits the proliferation of cells that do not express GPC3 and expresses TfR and / or does not induce cell death, and inhibits the proliferation of cells that co-express GPC3 and TfR and / or induces cell death.
  • the bispecific antibody fragment is suitable for use as a therapeutic agent or the like that specifically inhibits only cancer cells without inhibiting the proliferation of normal cells.
  • the internalization and / or degradation activity of TfR possessed by the bispecific antibody or the bispecific antibody fragment of the present invention can be confirmed by evaluating the cell surface and intracellular TfR protein amount of cells expressing TfR.
  • examples of the bispecific antibody or the bispecific antibody fragment of the present invention include a bispecific antibody or the bispecific antibody fragment that induces the degradation of TfR when bound to both GPC3 and TfR. Be done.
  • the number of binding domains to a certain antigen possessed by one molecule of bispecific antibody is called the valence of binding.
  • the bispecific antibody when one molecule of bispecific antibody has two antigen-binding domains that bind to GPC3 and two antigen-binding domains that bind to TfR, the bispecific antibody is bivalent to GPC3 and TfR, respectively. Combine with.
  • the bispecific antibody of the present invention also includes a bispecific antibody containing a plurality of antigen-binding domains bound via an appropriate linker, such as a linker containing an immunoglobulin domain or a fragment thereof.
  • the bispecific antibody of the present invention is a conventional production technique ([Nature Protocols, 9, 2450-2463 (2014)], International Publication No. 1998/050431, International Publication No. 2001/7734, International Publication No. 2002/002773. And it can be produced by International Publication No. 2009/131239).
  • the immunoglobulin domain is a peptide having an amino acid sequence similar to that of an immunoglobulin and consisting of about 100 amino acid residues in which at least two cysteine residues are present, as the minimum unit.
  • the immunoglobulin domain also includes a polypeptide containing a plurality of the above-mentioned minimum unit immunoglobulin domains. Examples of the immunoglobulin domain include VH, CH1, CH2 and CH3 of the immunoglobulin heavy chain, and VL and CL of the immunoglobulin light chain.
  • the animal species of immunoglobulin is not particularly limited, but it is preferably human.
  • the subclass of the constant region of the immunoglobulin heavy chain may be any of IgD, IgM, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 and IgE, preferably IgG-derived and IgM-derived.
  • the subclass of the constant region of the immunoglobulin light chain may be either ⁇ or ⁇ .
  • the immunoglobulin domain is also present in proteins other than immunoglobulin, and is included in the immunoglobulin domain contained in proteins belonging to the immunoglobulin superfamily such as major histocompatibility complex (MHC), CD1, B7 and T cell receptor (TCR). Can be mentioned. Any immunoglobulin domain can be applied as the immunoglobulin domain used for the bispecific antibody of the present invention.
  • MHC major histocompatibility complex
  • CD1, B7 CD1, B7 and T cell receptor (TCR).
  • TCR T cell receptor
  • CH1 refers to the region having the 118th to 215th amino acid sequences indicated by the EU index.
  • CH2 refers to the region having the amino acid sequences 231 to 340 indicated by the EU index of Kabat et al.
  • CH3 refers to the region having the amino acid sequences 341 to 447 indicated by the EU index of Kabat et al. ..
  • a hinge region hereinafter, also referred to as a hinge.
  • the hinge region refers to the region having the amino acid sequences 216 to 230 indicated by the EU index of Kabat et al.
  • CL is a region having the 108th to 214th amino acid sequences indicated by Kabat numbering in the case of the ⁇ chain of a human antibody, and the region having the 108th to 215th amino acid sequences in the case of the ⁇ chain. Refer to each.
  • the IgG moiety refers to a partial structure consisting of an IgG constituting the bispecific antibody of the present invention or an IgG modified from an Fc moiety, and has two heterodimers consisting of one light chain and one heavy chain. It has an associated heterotetramer structure.
  • the heavy chain constant region of the IgG portion may be any subclass of IgG1, IgG2, IgG3, or IgG4.
  • a part of those amino acid sequences may be deleted, added, substituted, and / or inserted.
  • all or part of the amino acid sequence consisting of CH1, hinge, CH2 and CH3 of the heavy chain of IgG can be used in appropriate combination.
  • those amino acid sequences can be partially deleted or used in a different order.
  • the IgG subclass used for the IgG moiety is not particularly limited, but an IgG4 variant in which the Ser residue at position 228 of the heavy chain constant region of IgG4 and IgG4 is replaced with Pro and the Leu residue at position 235 is replaced with Asn, respectively.
  • IgG4PE Ser residue at position 228 of the heavy chain constant region of IgG4 was replaced with Pro, the Leu residue at position 235 was replaced with Asn, and the Arg residue at position 409 was replaced with Lys. It is preferably an IgG4 variant (hereinafter referred to as IgG4PE R409K).
  • the heavy chain constant region (CH1-hinge-CH2-CH3 in order from the N-terminal side) of the IgG portion is the heavy chain constant region of IgG4PE R409K containing the amino acid sequence represented by SEQ ID NO: 255.
  • the two variable regions contained in the IgG portion of the present invention recognize the same antigen. It also preferably has the same structure and amino acid sequence. It was
  • the bispecific antibody of the present invention or the amino acid sequence constituting the antibody fragment one or more amino acid residues are deleted, added, substituted or inserted, and the antibody has the same activity as the above-mentioned antibody or antibody fragment thereof.
  • the antibody fragment thereof is also included in the bispecific antibody of the present invention or the antibody fragment thereof.
  • the number of amino acids deleted, substituted, inserted and / or added is one or more and the number is not particularly limited, but the number is not particularly limited, but Molecular Cloning, The Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology. , John Wiley & Sons (1987-1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci., USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Deletion, substitution, insertion or addition by well-known techniques such as site-specific mutagenesis methods described in Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci USA, 82, 488 (1985), etc. It is a number that can be done. For example, it is usually 1 to several tens, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 5.
  • one or more amino acid residues are deleted, substituted, inserted or added in the amino acid sequence of the bispecific antibody of the present invention described above, one or more amino acid residues are deleted at any position in the amino acid sequence.
  • Deletions, substitutions, insertions or additions may occur simultaneously, and the amino acid residues substituted, inserted or added may be either natural or non-natural.
  • Examples of the natural amino acid residues include L-alanine, L-aspartin, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, and L.
  • -In includes glutamine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine and L-cysteine.
  • amino acid residues that can be replaced with each other are preferable examples of amino acid residues that can be replaced with each other.
  • Amino acid residues contained in the same group can be replaced with each other.
  • Group A leucine, isoleucine, norleusin, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine
  • Group B aspartic acid, glutamic acid, isoaspartic acid, Isoglutamic acid, 2-aminoadipic acid, 2-aminosveric acid
  • Group C aspartic acid, glutamic acid
  • D lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid
  • Group E proline, 3 -Hydroxyproline, 4-hydroxyproline
  • F group serine, threonine, homoserine
  • G group phenylalanine, tyrosine
  • the bispecific antibody or fragment thereof of the present invention includes an antibody containing any post-translationally modified amino acid residue.
  • Post-translational modifications include, for example, deletion of the lysine residue at the C-terminus of the H chain [lysine clipping] and replacement of the glutamine residue at the N-terminus of the polypeptide with pyroglutamine (pyroGlu). [Beck et al, Analytical Chemistry, 85, 715-736 (2013)].
  • bispecific antibody of the present invention examples include the following (I) or (II) bispecific antibody.
  • (I) The C-terminus of the heavy chain of the IgG portion containing (a1) the first antigen-binding domain and (b1) the IgG portion containing the second antigen-binding domain and containing the first antigen-binding domain is the second.
  • Bispecific antibody (C-terminal) that binds to the N-terminus of the antigen-binding domain of the above, one of the first antigen-binding domain and the second antigen-binding domain binding to GPC3 and the other to TfR.
  • type bispecific antibody also abbreviated as type bispecific antibody.
  • the C-terminal bispecific antibody it is preferable that the C-terminal of the heavy chain of the IgG portion containing the first antigen-binding domain and the N-terminal of the second antigen-binding domain are linked directly or via a linker. , It is more preferable to directly connect them.
  • (II) (a2) An IgG moiety containing a second antigen-binding domain and (b2) an IgG containing the first antigen-binding domain, and the C-terminus of the first antigen-binding domain contains the second antigen-binding domain.
  • a bispecific antibody (N-terminus) that is linked to the N-terminus of the heavy chain of the moiety, one of the first antigen-binding domain and the second antigen-binding domain binding to GPC3 and the other to TfR. Also abbreviated as type bispecific antibody.)
  • the C-terminal of the first antigen-binding domain and the N-terminal of the heavy chain of the IgG portion containing the second antigen-binding domain are preferably linked directly or via a linker, and are directly linked. It is more preferable to connect them.
  • the linker may be anything, but a peptide chain is preferable.
  • the amino acid sequence of the peptide chain is, for example, one consisting of a repeating sequence of ES, ESKYG, ESKYGPP, GGGGS, or GGGGS, or one consisting of a part or all of a constant region such as the hinge region and CH1 domain of an antibody. And so on.
  • bispecific antibody of the present invention either the structure of the C-terminal bispecific antibody of (I) or the structure of the N-terminal bispecific antibody of (II) may be used.
  • bispecific antibody of (I) above there is a bispecific antibody in which the second antigen-binding domain is the Fab of the antibody.
  • the C-terminus of the heavy chain of the IgG portion containing the first antigen-binding domain is linked directly to the N-terminus of the heavy chain of the Fab of the antibody which is the second antigen-binding domain, either directly or via a linker.
  • bispecific antibody of (II) above there is a bispecific antibody in which the first antigen-binding domain is the Fab of the antibody. Also, bispecific antibodies in which the C-terminus of the Fab heavy chain of the antibody, which is the first antigen-binding domain, is directly linked to the N-terminus of the heavy chain of the IgG portion containing the second antigen-binding domain, either directly or via a linker. Can be mentioned.
  • either one of the first antigen-binding domain and the second antigen-binding domain is an anti-GPC3 IgG antibody Fab (anti-GPC3 Fab). preferable.
  • either one of the first antigen-binding domain and the second antigen-binding domain is an anti-TfR IgG antibody Fab (anti-TfR Fab). preferable.
  • the first antigen-binding domain is anti-GPC3 Fab and the second antigen-binding domain is anti-TfR Fab.
  • the first antigen-binding domain is anti-TfR Fab and the second antigen-binding domain is anti-GPC3 Fab.
  • bispecific antibody of the present invention include any one of the bispecific antibodies selected from the group consisting of the following (1) to (4).
  • Type GPC3-TfR bispecific antibody (2) A bispecific antibody (C-terminal) in which the C-terminal of the heavy chain of the IgG moiety containing the anti-TfR Fab and the anti-GPC3 Fab and containing the anti-TfR Fab is directly linked to the N-terminus of the heavy chain of the anti-GPC3 Fab.
  • Type TfR-GPC3 bispecific antibody (3) A bispecific antibody (N-terminal) containing an IgG moiety containing an anti-GPC3 Fab and an anti-TfR Fab, in which the C-terminal of the heavy chain of the anti-TfR Fab is directly linked to the N-terminus of the heavy chain of the IgG moiety containing the anti-GPC3 Fab.
  • Type TfR-GPC3 bispecific antibody (4) A bispecific antibody (N-terminal) containing an IgG portion containing an anti-TfR Fab and an anti-GPC3 Fab, in which the C-terminal of the heavy chain of the anti-GPC3 Fab is directly linked to the N-terminal of the heavy chain of the IgG portion containing the anti-TfR Fab.
  • Type GPC3-TfR bispecific antibody Type GPC3-TfR bispecific antibody
  • the VL of the anti-TfR Fab and the VL of the anti-GPC3 Fab may be the same or different, but the structural stability and reaction may be different. It is preferable that they are the same from the viewpoint of improving sexual stability.
  • Examples include Fabs containing VL containing ⁇ 3. (G1) CDR1 to 3, each containing the amino acid sequences represented by SEQ ID NOs: 91 to 93, (G2) CDR1 to 3, which contain the amino acid sequences represented by SEQ ID NOs: 95 to 97, respectively. (G3) CDR1 to 3, which contain the amino acid sequences represented by SEQ ID NOs: 99 to 101, respectively.
  • the anti-GPC3 Fab of the present invention comprises the amino acid sequence of CDR1 to 3 of VH of any one selected from the above (g1) to (g40) and the amino acid sequence of CDR1 to 3 of VL represented by SEQ ID NOs: 24 to 26, respectively. At least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, respectively. Also includes Fabs that bind to GPC3 containing the amino acid sequences of CDR1-3 of VH and VL, which are at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous.
  • Examples thereof include a VH containing an amino acid sequence thereof and a Fab containing a VL containing the amino acid sequence represented by SEQ ID NO: 23.
  • Examples include Fabs containing VL containing 3.
  • T2 CDR1 to 3 which contain the amino acid sequences represented by SEQ ID NOs: 32 to 34, respectively.
  • T3 CDR1 to 3 which contain the amino acid sequences represented by SEQ ID NOs: 37 to 39, respectively.
  • T4 CDR1 to 3, which contain the amino acid sequences represented by SEQ ID NOs: 41 to 43, respectively.
  • T5 CDR1 to 3, which contain the amino acid sequences represented by SEQ ID NOs: 45 to 47, respectively.
  • T6 CDR1 to 3, which contain the amino acid sequences represented by SEQ ID NOs: 49 to 51, respectively.
  • T7 CDR1 to 3, which contain the amino acid sequences represented by SEQ ID NOs: 53 to 55, respectively.
  • T8 CDR1 to 3, which contain the amino acid sequences represented by SEQ ID NOs: 57 to 59, respectively.
  • T9 CDRs 1 to 3 containing the amino acid sequences represented by SEQ ID NOs: 61 to 63, respectively.
  • T10 CDR1 to 3 which contain the amino acid sequences represented by SEQ ID NOs: 65 to 67, respectively.
  • T11 CDR1 to 3, which include the amino acid sequences represented by SEQ ID NOs: 69 to 71, respectively.
  • T12 CDR1 to 3, which contain the amino acid sequences represented by SEQ ID NOs: 73 to 75, respectively.
  • T13 CDR1 to 3, which include the amino acid sequences represented by SEQ ID NOs: 269 to 271, respectively.
  • T14 CDR1 to 3, which include the amino acid sequences represented by SEQ ID NOs: 79 to 81, respectively.
  • T15 CDR1 to 3, which contain the amino acid sequences represented by SEQ ID NOs: 83 to 85, respectively.
  • T16 CDR1 to 3 containing the amino acid sequences represented by SEQ ID NOs: 87 to 89, respectively.
  • the anti-TfR Fab of the present invention comprises the amino acid sequence of CDR1 to 3 of VH of any one selected from the above (t1) to (t16) and the amino acid sequence of CDR1 to 3 of VL represented by SEQ ID NOs: 24 to 26, respectively. At least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, respectively. Also includes Fabs that bind to TfR comprising the amino acid sequences of CDR1-3 of VH and VL, which are at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous.
  • Examples thereof include VH containing the amino acid sequence represented by 1, and Fab containing VL containing the amino acid sequence represented by SEQ ID NO: 23.
  • the IgG portion in the present invention contains a heavy chain constant region, and the subclass of the heavy chain constant region is not particularly limited, but it is preferably human IgG, more preferably human IgG4 or a variant thereof, and human IgG4 PE. Alternatively, it is more preferably human IgG4 PE R409K.
  • Specific examples of the amino acid sequence of the heavy chain constant region include the amino acid sequence represented by SEQ ID NO: 255.
  • FIG. 1A is a diagram showing the structures of the N-terminal GPC3-TfR bispecific antibody and the N-terminal TfR-GPC3 bispecific antibody.
  • the N-terminal GPC3-TfR bispecific antibody and the N-terminal TfR-GPC3 bispecific antibody are VH1 (first VH from the N-terminal side), in order from the N-terminal side.
  • VH1 is the VH of the anti-GPC3 antibody and VH2 is the VH of the anti-TfR antibody.
  • VH1 is the VH of the anti-TfR antibody and VH2 is the VH of the anti-GPC3 antibody.
  • FIG. 1B is a diagram showing the structures of the C-terminal GPC3-TfR bispecific antibody and the C-terminal TfR-GPC3 bispecific antibody.
  • the C-terminal GPC3-TfR bispecific antibody and the C-terminal TfR-GPC3 bispecific antibody are VH1, CH1, hinge, CH2, CH3, VH2 and CH1 in this order from the N-terminal side.
  • This is a bispecific antibody containing two polypeptide chains containing ‘’ and four polypeptide chains containing VL and CL in order from the N-terminal side.
  • VH1 is the VH of the anti-GPC3 antibody and VH2 is the VH of the anti-TfR antibody.
  • VH1 is the VH of the anti-TfR antibody and VH2 is the VH of the anti-GPC3 antibody.
  • VH1 first VH from the N-terminal side
  • CH1, and VH2 (2 from the N-terminal side) of the N-terminal GPC3-TfR bispecific antibody and the N-terminal TfR-GPC3 bispecific antibody are in order from the N-terminal side.
  • Second VH CH1'(second CH1 from the N-terminal side), polypeptide chain containing hinges, CH2 and CH3, and C-terminal GPC3-TfR bispecific and C-terminal TfR-GPC3 bispecific antibodies.
  • a polypeptide chain containing VH1, CH1, hinge, CH2, CH3, VH2 and CH1'in order from the N-terminal side is called a heavy chain of a bispecific antibody.
  • VL and CL are in order from the N-terminal side of the N-terminal GPC3-TfR bispecific antibody, the N-terminal TfR-GPC3 bispecific antibody, and the C-terminal GPC3-TfR bispecific antibody and the C-terminal TfR-GPC3 bispecific antibody.
  • the polypeptide chain containing the above is called the light chain of the bispecific antibody.
  • the two heavy chains of the bispecific antibody are associated by the disulfide bond of the cysteine residue in the hinge region.
  • the heavy and light chains of a bispecific antibody are associated by disulfide bonds of CH1 or CH1'and CL cysteine residues.
  • the two heavy chains of the bispecific antibody may be the same or different, but are preferably the same.
  • the four light chains of the bispecific antibody may be different from each other or the same, but it is preferable that all four are the same.
  • CL may be either a C ⁇ chain or a C ⁇ chain.
  • the VH of the anti-GPC3 antibody in the N-terminal GPC3-TfR bispecific antibody, N-terminal TfR-GPC3 bispecific antibody, C-terminal GPC3-TfR bispecific antibody, and C-terminal TfR-GPC3 bispecific antibody includes SEQ ID NOs: 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, Examples thereof include VHs comprising an amino acid sequence represented by any one of 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, 230, 234, 238, 242 and 246.
  • the amino acid sequence represented by any one of these selected from SEQ ID NOs: 94, 98, 102, 106, 114, 130, 166, 178, 186 and 190 is more preferable, and VH containing the amino acid sequence represented by SEQ ID NO: 190 is even more preferable.
  • VH of the anti-TfR antibody in the N-terminal type GPC3-TfR bispecific antibody N-terminal type TfR-GPC3 bispecific antibody, C-terminal type GPC3-TfR bispecific antibody, and C-terminal type TfR-GPC3 bispecific antibody.
  • N-terminal type TfR-GPC3 bispecific antibody C-terminal type GPC3-TfR bispecific antibody, and C-terminal type TfR-GPC3 bispecific antibody.
  • VH can be mentioned.
  • VH containing the amino acid sequence represented by SEQ ID NO: 35 or 40 is preferable.
  • the VL of the N-terminal GPC3-TfR bispecific antibody, N-terminal TfR-GPC3 bispecific antibody, C-terminal GPC3-TfR bispecific antibody, and C-terminal TfR-GPC3 bispecific antibody of the present invention is the same. It may be different or different, but it is preferable that they are the same from the viewpoint of improving the stability of the structure and the stability of the reactivity.
  • Examples of the VL include a VL containing the amino acid sequence represented by SEQ ID NO: 23.
  • CL for example, CL containing the amino acid sequence represented by SEQ ID NO: 272 can be mentioned. It was
  • Examples of the N-terminal GPC3-TfR bispecific antibody of the present invention include the following.
  • (N1) VH1 has SEQ ID NOs: 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 178, 186.
  • VHs comprising an amino acid sequence represented by any one of the numbers 35, 31, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 268, 78, 82 and 86.
  • a bispecific antibody comprising a heavy chain of VL and four light chains of VL comprising the amino acid sequence represented by SEQ ID NO: 23. (N2) SEQ ID NOs: 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, in order from the N end.
  • VH comprising the amino acid sequence represented by any one of 178, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, 230, 234, 238, 242 and 246; SEQ ID NO: CH1 comprising the amino acid sequence represented by 253; in any one of SEQ ID NOs: 35, 31, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 268, 78, 82 and 86.
  • a bispecific antibody comprising four light chains each comprising CL comprising the amino acid sequence represented by SEQ ID NO: 272.
  • VH1 has SEQ ID NOs: 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 178. , 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, 230, 234, 238, 242 and 246, which is a VH containing an amino acid sequence represented by any one of VH2.
  • a bispecific antibody comprising two heavy chains in which is a VH comprising the amino acid sequence represented by SEQ ID NO: 35 and four light chains in which VL is a VL comprising the amino acid sequence represented by SEQ ID NO: 23.
  • VH1 is a VH containing an amino acid sequence represented by any one of SEQ ID NOs: 94, 98, 102, 114, 130, 166, 178, 186 and 190
  • VH2 is represented by SEQ ID NO: 35.
  • Bispecific antibodies comprising two heavy chains of VH comprising the amino acid sequence and four light chains of VL comprising the amino acid sequence represented by SEQ ID NO: 23 are more preferred.
  • VH1 is a VH comprising an amino acid sequence represented by any one of SEQ ID NOs: 94, 114 and 190
  • VH2 is a VH comprising an amino acid sequence represented by SEQ ID NO: 35
  • a bispecific antibody comprising four light chains, where VL is VL comprising the amino acid sequence represented by SEQ ID NO: 23, is even more preferred.
  • VH1 is a VH comprising the amino acid sequence represented by SEQ ID NO: 190
  • VH2 is a VH comprising the amino acid sequence represented by SEQ ID NO: 35 and VL are represented by SEQ ID NO: 23.
  • VH1 has SEQ ID NOs: 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 178. , 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, 230, 234, 238, 242 and 246, which is a VH containing an amino acid sequence represented by any one of VH2.
  • a bispecific antibody comprising two heavy chains in which is a VH comprising the amino acid sequence represented by SEQ ID NO: 40 and four light chains in which VL is a VL comprising the amino acid sequence represented by SEQ ID NO: 23.
  • VH1 is a VH containing an amino acid sequence represented by any one of SEQ ID NOs: 94, 98, 102, 106, 114, 130, 178 and 190
  • VH2 is an amino acid represented by SEQ ID NO: 40.
  • Bispecific antibodies comprising two heavy chains of VH comprising a sequence and four light chains of VL comprising the amino acid sequence represented by SEQ ID NO: 23 are more preferred.
  • VH1 is a VH comprising an amino acid sequence represented by any one of SEQ ID NOs: 94, 114 and 190
  • VH2 is a VH comprising an amino acid sequence represented by SEQ ID NO: 40
  • two heavy chains in which VH1 is a VH comprising the amino acid sequence represented by SEQ ID NO: 190 and VH2 is a VH comprising the amino acid sequence represented by SEQ ID NO: 40 and VL are represented by SEQ ID NO: 23.
  • Bispecific antibodies comprising four light chains, which are VLs containing the amino acid sequence, are particularly preferred.
  • N2-1 SEQ ID NOs: 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, in order from the N-terminal.
  • a VH comprising an amino acid sequence represented by any one of 166, 178, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, 230, 234, 238, 242 and 246; CH1 containing the amino acid sequence represented by SEQ ID NO: 253; VH containing the amino acid sequence represented by SEQ ID NO: 35; and two heavy chains each containing CH containing the amino acid sequence represented by SEQ ID NO: 255, and N.
  • a bispecific antibody comprising VL containing the amino acid sequence represented by SEQ ID NO: 23 and four light chains each containing CL containing the amino acid sequence represented by SEQ ID NO: 272, from the terminal side.
  • a VH comprising an amino acid sequence represented by any one of 166, 178, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, 230, 234, 238, 242 and 246; CH1 containing the amino acid sequence represented by SEQ ID NO: 253; VH containing the amino acid sequence represented by SEQ ID NO: 40; and two heavy chains each containing CH containing the amino acid sequence represented by SEQ ID NO: 255, and N.
  • a bispecific antibody comprising VL containing the amino acid sequence represented by SEQ ID NO: 23 and four light chains each containing CL containing the amino acid sequence represented by SEQ ID NO: 272, from the terminal side.
  • VH1 is SEQ ID NO: 94. , 98, 102, 106, 114, 130, 166, 178, 186 and 190 are preferred N-terminal GPC3-TfR bispecific antibodies that are VHs comprising the amino acid sequence represented by any one of them.
  • the N-terminal GPC3-TfR bispecific antibody exhibits excellent cell proliferation activity even in cells in which the expression level of GPC3 is medium to low.
  • the expression level of GPC3 in cancer cells varies depending on the cancer patient, and is not limited to high expression, and is low to medium expression depending on the cancer patient. Therefore, the N-terminal GPC3-TfR bispecific antibody, which exhibits excellent cell proliferation activity even in cells in which the expression level of GPC3 is medium to low, has excellent characteristics from a clinical point of view. it is conceivable that.
  • VH1 is a VH containing an amino acid sequence represented by any one of SEQ ID NOs: 94, 114 and 190
  • VH2 is a VH containing an amino acid sequence represented by SEQ ID NO: 40 2
  • a bispecific antibody comprising a heavy chain of a book and four light chains of which VL is VL comprising the amino acid sequence represented by SEQ ID NO: 23.
  • a bispecific antibody comprising four light chains each comprising VL and CL comprising the amino acid sequence represented by SEQ ID NO: 272.
  • N-terminal GPC3-TfR bispecific antibodies the following are particularly preferable from the viewpoint of cell proliferation inhibitory activity.
  • VH1 is a VH containing the amino acid sequence represented by SEQ ID NO: 190 and VH2 is a VH containing the amino acid sequence represented by SEQ ID NO: 40 and VL are sequences.
  • a bispecific antibody comprising four light chains which are VL comprising the amino acid sequence represented by number 23.
  • N2-2-2 VH containing the amino acid sequence represented by SEQ ID NO: 190; CH1 containing the amino acid sequence represented by SEQ ID NO: 253; VH containing the amino acid sequence represented by SEQ ID NO: 40, in order from the N-terminal.
  • a bispecific antibody comprising four light chains, each containing a CL containing an amino acid sequence.
  • N-terminal GPC3-TfR bispecific antibody of the present invention include Nt-G3042-TfR1071, Nt-G1048-TfR1071, Nt-G1104-TfR1071, Nt-G4045-TfR1071, and Nt-, which will be described later in Examples.
  • Nt-G4025-TfR1071, Nt-G2038-TfR1071, Nt-G1036-TfR1071, Nt-G2002-TfR1071, Nt-G1119-TfR1071, Nt-G1137-TfR1071, Nt-G2133-TfR1071, Nt-G1107-TfR1071, Nt-G3062NYA-TfR1071, Nt-G4025-TfR1071_202, Nt-G2038-TfR1071_ G2002-TfR1071_202, Nt-G1119-TfR1071_202, Nt-G1137-TfR1071_202, Nt-G2133-TfR1071_202 and Nt-G2067-TfR1071_202 are preferable, and more preferably Nt-G4025-TfR1071_202, Nt-G1119-TfR1071_202, Nt-G1137-Tf
  • Nt-G4025-TfR1071_202, Nt-G2002-TfR1071_202 and Nt-G1119-TfR1071_202 are particularly preferably Nt-G4025-TfR1071_202, Nt-G2002-TfR1071_202 and Nt-G1119-TfR1071_202, most preferably Nt-G1119-TfR1071_202. -TfR1071_202.
  • N-terminal GPC3-TfR bispecific antibodies are named as Nt- [anti-GPC3 antibody clone name]-[anti-TfR antibody clone name], and in order from the N-terminal, the VH of the anti-GPC3 antibody, SEQ ID NO: 253.
  • the sequence numbers of the amino acid sequences of VH of the anti-GPC3 antibody are shown in Tables 4 and 5.
  • the sequence numbers of the amino acid sequences of VH of the anti-TfR antibody are shown in Tables 1 to 3.
  • a VH containing the amino acid sequence represented by SEQ ID NO: 35 is used as the VH of TfR1071.
  • As the VL of the anti-TfR antibody and the GPC3 antibody a VL containing the amino acid sequence represented by SEQ ID NO: 23 is used.
  • Nt-G3042-TfR1071 includes VH containing the amino acid sequence represented by SEQ ID NO: 206, CH1 containing the amino acid sequence represented by SEQ ID NO: 253, and the amino acid sequence represented by SEQ ID NO: 35 in order from the N-terminal. It is represented by two heavy chains consisting of VH containing and CH containing the amino acid sequence represented by SEQ ID NO: 255, and VL and SEQ ID NO: 272 containing the amino acid sequence represented by SEQ ID NO: 23 in order from the N-terminal side. Represents a bispecific antibody comprising four light chains consisting of CL comprising an amino acid sequence.
  • Examples of the C-terminal GPC3-TfR bispecific antibody of the present invention include the following.
  • (C1) VH1 has SEQ ID NOs: 90, 142, 126, 178, 198, 146, 218, 150, 202, 226, 94, 98, 102, 106, 110, 114, 118, 122, 130, 134, 138, 154.
  • a VH comprising an amino acid sequence represented by any one of 158, 162, 234, 238, 242, 246, 186, 190 and 194, wherein VH2 is a VH comprising an amino acid sequence represented by SEQ ID NO: 35.
  • a bispecific antibody comprising two heavy chains and four light chains in which VL is VL comprising the amino acid sequence represented by SEQ ID NO: 23.
  • C2 SEQ ID NOs: 90, 142, 126, 178, 198, 146, 218, 150, 202, 226, 94, 98, 102, 106, 110, 114, 118, 122, 130, 134 in order from the N-terminal side.
  • VH containing the amino acid sequence represented by any one of 138, 154, 158, 162, 234, 238, 242, 246, 186, 190 and 194, CH containing the amino acid sequence represented by SEQ ID NO: 255,
  • bispecific antibodies comprising VL comprising and four light chains each comprising CL comprising the amino acid sequence represented by SEQ ID NO: 272.
  • C-terminal GPC3-TfR bispecific antibody of the present invention include Ct-G2025-TfR1071, Ct-G1048-TfR1071, Ct-G1104-TfR1071, Ct-G1122-TfR1071, and Ct, which will be described later in Examples.
  • C-terminal GPC3-TfR bispecific antibodies are named Ct- [anti-GPC3 antibody clone name]-[anti-TfR antibody clone name], and in order from the N-terminal, the VH of the anti-GPC3 antibody, SEQ ID NO: 255.
  • the sequence numbers of the amino acid sequences of VH of the anti-GPC3 antibody are shown in Tables 4 and 5.
  • the sequence numbers of the amino acid sequences of VH of the anti-TfR antibody are shown in Tables 1 to 3.
  • a VH containing the amino acid sequence represented by SEQ ID NO: 35 is used as the VH of TfR1071.
  • As the VL of the anti-TfR antibody and the GPC3 antibody a VL containing the amino acid sequence represented by SEQ ID NO: 23 is used.
  • Ct-G2025-TfR1071 includes VH containing the amino acid sequence represented by SEQ ID NO: 226, CH containing the amino acid sequence represented by SEQ ID NO: 255, and the amino acid sequence represented by SEQ ID NO: 35, in order from the N-terminal. It is represented by two heavy chains consisting of VH containing and CH1 containing the amino acid sequence represented by SEQ ID NO: 253, and VL and SEQ ID NO: 272 containing the amino acid sequence represented by SEQ ID NO: 23 in order from the N-terminal side. Represents a bispecific antibody comprising four light chains consisting of CL comprising an amino acid sequence.
  • the following bispecific antibody contains an IgG moiety containing an anti-GPC3 Fab and a VH of an anti-TfR antibody that binds to TfR alone, with the C-terminal of the VH of the anti-TfR antibody via a linker (GGGGS set forth in SEQ ID NO: 256).
  • Bispecific antibody TfR-GS-GPC3 bispecific antibody linked to the N-terminal of the heavy chain of the IgG moiety containing anti-GPC3 Fab, or VH of IgG moiety and anti-GPC3 antibody containing anti-TfR Fab alone.
  • FIG. 1C is a diagram showing the structures of GPC3-GS-TfR bispecific antibody and TfR-GS-GPC3 bispecific antibody.
  • the GPC3-GS-TfR bispecific antibody and the TfR-GS-GPC3 bispecific antibody are VH1, GS sequence [GGGGS (SEQ ID NO: 256)], VH2 in order from the N-terminal side.
  • VH1 is the VH of the anti-GPC3 antibody and is the VH that binds to GPC3 alone
  • VH2 is the VH of the anti-TfR antibody
  • VH1 is the VH of the anti-TfR antibody and is the VH that binds to TfR alone
  • VH2 is the VH of the anti-GPC3 antibody.
  • the heavy chain and the light chain of the bispecific antibody are the VH1, GS sequence [GGGGS (SEQ ID NO: 256) of the GPC3-GS-TfR bispecific antibody and the TfR-GS-GPC3 bispecific antibody in this order from the N-terminal side, respectively. )], Contains a polypeptide chain containing VH2, CH1, hinge, CH2 and CH3 and a polypeptide chain containing VL and CL in order from the N-terminus.
  • the two heavy chains are associated by disulfide bonds of cysteine residues in the hinge region. Further, the heavy chain and the light chain are associated by a disulfide bond between CH1 and CL cysteine residues.
  • Examples of the GPC3-GS-TfR bispecic antibody of the present invention include the following.
  • GS1 Two heavy chains in which VH1 is a VH containing the amino acid sequence represented by SEQ ID NO: 250, VH2 is a VH containing the amino acid sequence represented by SEQ ID NO: 27, and VL is represented by SEQ ID NO: 23.
  • a bispecific antibody comprising two light chains, each containing CL.
  • GPC3-GS-TfR bispecific antibody of the present invention include HN3-GS-TfR1071 described later in Examples.
  • the bispecific antibody or the antibody fragment thereof of the present invention also includes an antibody having an effector activity or the antibody fragment thereof.
  • the effector activity refers to antibody-dependent cellular cytotoxicity caused via the Fc region of an antibody, and for example, antibody-dependent cellular cytotoxicity (ADCC activity) and complement-dependent cellular cytotoxicity.
  • ADCC activity antibody-dependent cellular cytotoxicity
  • Activity Completion-Dependent Cytotoxicity activity; CDC activity
  • antibody-dependent phagocytic activity by phagocytic cells such as macrophages and dendritic cells (antibody-dependent cellular phagocytosis activity, etc.)
  • ADCP activity Antibody-dependent cellular phagocytosis activity
  • ADCC activity and CDC activity can be measured using a known measuring method [Cancer Immunol. Immunother., 36, 373 (1993)].
  • ADCC activity means that an antibody bound to an antigen on a target cell activates an immune cell (natural killer cell, etc.) by binding to the Fc receptor of the immune cell via the Fc region of the antibody, and damages the target cell. The activity to do.
  • the Fc receptor is a receptor that binds to the Fc region of an antibody, and induces various effector activities by binding the antibody.
  • Each FcR corresponds to a subclass of antibody, and IgG, IgE, IgA, and IgM specifically bind to Fc ⁇ R, Fc ⁇ R, Fc ⁇ R, and Fc ⁇ R, respectively.
  • Fc ⁇ R has subtypes of Fc ⁇ RI (CD64), Fc ⁇ RII (CD32) and Fc ⁇ RIII (CD16), each of which is an isoform of Fc ⁇ RIA, Fc ⁇ RIB, Fc ⁇ RIC, Fc ⁇ RIIA, Fc ⁇ RIIB, Fc ⁇ RIIC, Fc ⁇ RIIIA and Fc ⁇ RIIIB.
  • Fc ⁇ RIIIB is specifically expressed on neutrophils
  • Fc ⁇ RIIIA is expressed on monocytes, natural killer cells (NK cells), macrophages and some T cells.
  • NK cell-dependent ADCC activity is induced through the binding of the antibody to Fc ⁇ RIIIA.
  • CDC activity refers to the activity in which an antibody bound to an antigen on a target cell activates a series of cascades (complement activation pathway) consisting of complement-related proteins in the blood and damages the target cell.
  • cascades complement activation pathway
  • protein fragments produced by complement activation induce migration and activation of immune cells.
  • the cascade of CDC activity is initiated by first binding C1q to the Fc region and then to the two serine proteases C1r and C1s to form a C1 complex.
  • the CDC activity or ADCC activity of the bispecific antibody or the antibody fragment of the present invention on antigen-expressing cells can be evaluated by a known measurement method [Cancer Immunol. Immunother., 36, 373 (1993)].
  • the reduction of the N-linked complex type sugar chain that binds to the 297th asparagine (Asn) in the Fc region (constant region consisting of CH2 and CH3 domains) of the antibody As a method for controlling the effector activity of the bispecific antibody of the present invention, the reduction of the N-linked complex type sugar chain that binds to the 297th asparagine (Asn) in the Fc region (constant region consisting of CH2 and CH3 domains) of the antibody.
  • a method for controlling the amount of fucose (also referred to as core fucose) that binds ⁇ -1,6 to N-acetylglucosamine (GlcNAc) present at the terminal International Publication No. 2005/035586, International Publication No. 2002/31140 and International Publication No. No. 2000/6173
  • a method for controlling by modifying amino acid residues in the Fc region of an antibody International Publication No. 2000/42072 are known.
  • the ADCC activity of the antibody can be increased or decreased.
  • bispecific antibody is expressed using a host cell lacking the ⁇ 1,6-fucose transferase gene. By doing so, a bispecific antibody having a high ADCC can be obtained.
  • the antibody is expressed using a host cell into which the ⁇ 1,6-fucose transfer enzyme gene has been introduced. By allowing the antibody to be obtained, a bispecific antibody having low ADCC activity can be obtained.
  • ADCC activity and CDC activity can be increased or decreased by modifying the amino acid residue in the Fc region of the bispecific antibody.
  • the CDC activity of a bispecific antibody can be increased by using the amino acid sequence of the Fc region described in US Patent Application Publication No. 2007/01/48165.
  • ADCC activity is performed by modifying the amino acids described in US Pat. No. 6,737,056, US Pat. No. 7,297,775, US Pat. No. 7,317,091 and the like.
  • the CDC activity can be increased or decreased.
  • a bispecific antibody with controlled effector activity may be obtained by combining the above-mentioned methods.
  • the stability of the bispecific antibody of the present invention can be evaluated by measuring the amount of aggregates (oligomers) formed in the purification process or in a sample stored under certain conditions. That is, when the amount of aggregates is reduced under the same conditions, it is evaluated that the stability of the antibody is improved.
  • the amount of aggregates can be measured by separating the aggregated antibody from the non-aggregated antibody using appropriate chromatography including gel filtration chromatography.
  • the productivity of the bispecific antibody of the present invention can be evaluated by measuring the amount of antibody produced in the culture medium from the antibody-producing cells. More specifically, it can be evaluated by measuring the amount of the antibody contained in the culture supernatant obtained by removing the producing cells from the culture solution by an appropriate method such as an HPLC method or an ELISA method.
  • the antibody fragment is a protein containing an antigen-binding site and having an antigen-binding activity against the antigen.
  • peptides containing Fab, Fab', F (ab') 2 , scFv, Diabody, dsFv, VHH or CDR can be mentioned.
  • Fab is a fragment obtained by treating an IgG antibody with the proteolytic enzyme papain (cleaved at the 224th amino acid residue of the H chain), and about half of the N-terminal side of the H chain and the entire L chain are disulfides. It is an antibody fragment having an antigen-binding activity having a molecular weight of about 50,000 and bound by binding (SS bond).
  • the heavy chain of Fab containing VH and CH1 is referred to as VH-CH1.
  • the light chain of Fab containing VL and CL is referred to as VL-CL.
  • F (ab') 2 is a fragment obtained by treating IgG with the proteolytic enzyme pepsin (cleaved at the 234th amino acid residue of the H chain), in which Fab is mediated by the SS bond in the hinge region. It is an antibody fragment having an antigen-binding activity having a molecular weight of about 100,000, which is slightly larger than that bound to the antibody.
  • Fab' is an antibody fragment having an antigen-binding activity having a molecular weight of about 50,000, which is obtained by cleaving the SS bond in the hinge region of F (ab') 2.
  • scFv is a VH-P-VL or VL-P-VH polypeptide in which one VH and one VL are linked using a suitable peptide linker (P) of 12 residues or more, and antigen binding. It is an active antibody fragment.
  • Diabody is an antibody fragment in which scFv having the same or different antigen-binding specificity forms a dimer, and has a divalent antigen-binding activity for the same antigen or an antibody fragment having specific antigen-binding activity for different antigens. Is.
  • DsFv refers to a polypeptide in which each 1 amino acid residue in VH and VL is replaced with a cysteine residue, which is bound via an SS bond between the cysteine residues.
  • VHH (also referred to as Nanobody) refers to the heavy chain variable region of a VHH antibody and can bind to an antigen in the absence of other polypeptides.
  • the VHH antibody is an antibody present in camelid animals such as alpaca and cartilaginous fish such as sharks, and consists of only heavy chains without light chains and CH1.
  • a peptide containing a CDR comprises at least one region of a CDR of VH or VL.
  • Peptides containing a plurality of CDRs can be prepared by binding the CDRs directly or via an appropriate peptide linker.
  • the peptide containing CDR constructs a DNA encoding the CDRs of VH and VL of the bispecific antibody of the present invention, inserts the DNA into a prokaryotic expression vector or a prokaryotic expression vector, and inserts the expression vector into a prokaryote. It can be expressed and produced by introducing it into an organism or prokaryote.
  • the peptide containing CDR can also be produced by a chemical synthesis method such as the Fmoc method or the tBoc method.
  • the bispecific antibody fragment essentially consists of a partial structure of the bispecific antibody and has antigen-binding activity against two types of antigens.
  • a fusion protein in which the Fc of the bispecific antibody of the present invention is bound to an antibody fragment an Fc fusion protein in which the Fc is bound to a naturally occurring ligand or receptor (also referred to as immunoadhesin), and a plurality of Fc regions.
  • Fused Fc fusion proteins and the like are also included in the present invention.
  • an Fc region containing amino acid residue modification for the purpose of enhancing or deleting the effector activity of the antibody, stabilizing the antibody, and controlling the half-life in blood can also be used for the bispecific antibody of the present invention.
  • a radioisotope, a low molecular weight drug, a high molecular weight drug, a protein or an antibody drug or the like is chemically added to the bispecific antibody or the bispecific antibody fragment of the present invention.
  • it includes a derivative of a genetically engineered antibody.
  • the antibody derivative in the present invention is the N-terminal side or C-terminal side of the H chain or L chain of the bispecific antibody or the bispecific antibody fragment of the present invention, and an appropriate substituent or side chain in the antibody or the antibody fragment thereof.
  • radioactive isotopes, low-molecular-weight drugs, high-molecular-weight drugs, immunostimulators, proteins, antibody drugs, etc. are applied to the sugar chains in the antibody or its antibody fragment by chemical methods [Introduction to antibody engineering, local people]. It can be manufactured by combining with a bookstore (1994)].
  • the derivative of the antibody in the present invention is inserted into an expression vector by linking a DNA encoding the bispecific antibody or the bispecific antibody fragment of the present invention with a DNA encoding a desired protein or antibody drug. It can be produced by a genetic engineering technique in which the expression vector is introduced into an appropriate host cell and expressed.
  • Radioisotopes include, for example, 111 In, 131 I, 125 I, 90 Y, 64 Cu, 99 Tc, 77 Lu or 211 At.
  • the radioisotope can be directly bound to the antibody by the chloramine T method or the like. Further, a substance that chelate the radioisotope may be bound to the antibody. Examples of the chelating agent include 1-isothiocyanate benzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA).
  • low-molecular-weight drugs examples include alkylating agents, nitrosolea agents, antimetabolite agents, antibiotics, plant alkaloids, topoisomerase inhibitors, hormone therapeutic agents, hormone antagonists, aromatase inhibitors, P sugar protein inhibitors, and platinum.
  • Anti-cancer agents such as complex derivatives, stage M inhibitors or kinase inhibitors [Clinical Oncology, Cancer and Chemotherapy (1996)], hormonal agents such as hydrocortisone or prednison, non-steroidal agents such as aspirin or indomethacin, gold thiomalate or Immunomodulators such as peniciramine, immunosuppressive agents such as cyclophosphamide or azathiopurine, or anti-inflammatory agents such as antihistamines such as chlorpheniramine maleate or cremasitin [Inflammation and anti-inflammatory therapy, Medical and Dental Publishing Co., Ltd. (1982) )] And so on.
  • anticancer agents examples include amifostine (etioll), cisplatin, daunorubicin (DTIC), dactinomycin, mechloretamine (nitrogen mustard), streptosocin, cyclophosphamide, irinote, carmustin (BCNU), romustin (CCNU), and doxorubicin.
  • a method for binding a small molecule drug to the bispecific antibody of the present invention or the bispecific antibody fragment thereof for example, a method of binding between the drug and the amino group of the antibody via glutaaldehyde aldehyde, or a water-soluble carbodiimide.
  • a method of binding an amino group of a drug and a carboxyl group of the antibody via the above for example, a method of binding between the drug and the amino group of the antibody via glutaaldehyde aldehyde, or a water-soluble carbodiimide.
  • Examples thereof include a method of binding an amino group of a drug and a carboxyl group of the antibody via the above.
  • polymer drug examples include polyethylene glycol (PEG), albumin, dextran, polyoxyethylene, styrene maleic acid copolymer, polyvinylpyrrolidone, pyran copolymer, hydroxypropylmethacrylamide, and the like.
  • a method for binding PEG to the bispecific antibody of the present invention includes a method for reacting with a PEGylation-modifying reagent [Bioconjugated drug, Hirokawa Shoten (1993)].
  • PEGylation modifying reagent include a modifying agent for the ⁇ -amino group of lysine (Japanese Patent Laid-Open No. 61-178926) and a modifying agent for the carboxyl group of aspartic acid and glutamic acid (Japanese Patent Laid-Open No. 56-23587). No. 2), or a modifying agent for the guanidino group of arginine (Japanese Patent Laid-Open No. 2-117920).
  • the immunostimulant may be a natural product known as an immunoadjugant, and as a specific example, the drug that enhances immunity may be ⁇ (1 ⁇ 3) glucan (eg, lentinan or schizophyllan) or ⁇ -galactosylceramide (. KRN7000) and the like.
  • the drug that enhances immunity may be ⁇ (1 ⁇ 3) glucan (eg, lentinan or schizophyllan) or ⁇ -galactosylceramide (. KRN7000) and the like.
  • proteins examples include cytokines or growth factors or toxin proteins that activate immunocompetent cells such as NK cells, macrophages or neutrophils.
  • cytokines or growth factors examples include interferon (hereinafter referred to as IFN) - ⁇ , IFN- ⁇ , IFN- ⁇ , interleukin (hereinafter referred to as IL) -2, IL-12, IL-15, IL-. 18, IL-21, IL-23, granulocyte colony stimulating factor (G-CSF), granulocyte / macrophage colony stimulating factor (GM-CSF) or macrophage colony stimulating factor (M-CSF) and the like.
  • IFN interferon
  • IL interleukin
  • IL-12 interleukin
  • IL-15 interleukin-. 18
  • IL-21 IL-23
  • G-CSF granulocyte colony stimulating factor
  • GM-CSF granulocyte / macrophage colony stimulating factor
  • M-CSF macrophage colony stimulating factor
  • toxin protein examples include ricin, diphtheria toxin, ONTAK, and the like, and a protein toxin in which a mutation is introduced into the protein to regulate toxicity is also included.
  • a cDNA encoding a protein is ligated to a cDNA encoding a bispecific antibody or antibody fragment of the present invention to construct a DNA encoding the fusion antibody, and the DNA is used as a prokaryote or a true antibody. It can be expressed and produced by inserting it into an expression vector for a nuclear organism and introducing the expression vector into a prokaryote or a eukaryote. It was
  • the bispecific antibody of the present invention or a drug that binds to an antibody fragment thereof is commonly used for immunological detection.
  • Labeling agents include, for example, enzymes such as alkaline phosphatase, peroxidase or luciferase, luminescent substances such as acridinium ester or loffin, fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (RITC), Alexa (registered trademark).
  • fluorescent substances such as Fluor 488 and R-phycoerythrin (R-PE).
  • the present invention includes a bispecific antibody having cytotoxic activity such as CDC activity or ADCC activity and the bispecific antibody fragment thereof.
  • cytotoxic activity such as CDC activity or ADCC activity
  • the CDC activity or ADCC activity of the bispecific antibody or the bispecific antibody fragment of the present invention with respect to antigen-expressing cells can be evaluated by a known measurement method [Cancer Immunol. Immunother., 36, 373 (1993)].
  • the present invention also contains, as an active ingredient, a composition containing a bispecific antibody or a bispecific antibody fragment that specifically recognizes and binds to TfR and GPC3, or the bispecific antibody or the bispecific antibody fragment. And a therapeutic agent for a disease involving at least one of GPC3, preferably a disease involving cells expressing TfR and GPC3.
  • the disease in which at least one of TfR and GPC3 is related may be any disease as long as it is related to at least one of TfR and GPC3, and examples thereof include malignant tumors and cancer.
  • the malignant tumor and cancer include, for example, colon cancer, colorectal cancer, lung cancer, breast cancer, glioma, malignant melanoma, thyroid cancer, renal cell cancer, leukemia, lymphoma, T.
  • the therapeutic agent containing the bispecific antibody or the bispecific antibody fragment thereof, or a derivative thereof of the present invention contains only the bispecific antibody or the bispecific antibody fragment thereof as an active ingredient, or a derivative thereof. However, it is usually preferable to mix it with one or more pharmaceutically acceptable carriers and provide it as a pharmaceutical preparation produced by any method known in the technical field of pharmaceutics.
  • the most effective route of administration for treatment includes oral administration or parenteral administration such as oral administration, intra-airway, intrarectal, subcutaneous, intramuscular or intravenous administration. Of these, intravenous administration is preferable.
  • Examples of the administration form include sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments or tapes.
  • the dose or frequency of administration varies depending on the target therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually 10 ⁇ g / kg to 10 mg / kg per day for an adult.
  • the present invention relates to at least one immunological detection or measurement reagent of TfR and GPC3 containing the bispecific antibody of the present invention or the bispecific antibody fragment thereof, or a disease related to at least one of TfR and GPC3. , Preferably relating to diagnostic agents for diseases involving cells expressing TfR and GPC3.
  • the present invention also relates to a bispecific antibody of the present invention or a method for immunological detection or measurement of at least one of TfR and GPC3 using the bispecific antibody fragment, and a disease related to at least one of TfR and GPC3.
  • It relates to a method for treating a disease involving cells expressing TfR and GPC3, or a method for diagnosing a disease involving at least one of TfR and GPC3, preferably a disease involving cells expressing TfR and GPC3.
  • any known method can be mentioned.
  • an immunological detection or measurement method may be mentioned.
  • the immunological detection or measurement method is a method for detecting or measuring the amount of antibody or the amount of antigen using a labeled antigen or antibody.
  • the immunoassay or measurement method include radioimmunoassay (RIA), enzyme immunoassay (EIA or ELISA), fluorescence immunoassay (FIA), luminescence immunoassay (luminescent immunoassay), and western blot method.
  • RIA radioimmunoassay
  • EIA or ELISA enzyme immunoassay
  • FIA fluorescence immunoassay
  • luminescence immunoassay luminescence immunoassay
  • western blot method Alternatively, a physicochemical method may be mentioned.
  • a disease in which at least one of TfR and GPC3 is involved preferably TfR and GPC3.
  • Diseases involving expressing cells can be diagnosed.
  • Known immunological detection methods can be used to detect cells expressing at least one of TfR and GPC3, such as immunoprecipitation, immunohistochemical staining, immunohistochemical staining, or fluorescent antibody staining.
  • immunoprecipitation immunohistochemical staining
  • immunohistochemical staining immunohistochemical staining
  • fluorescent antibody staining a fluorescent antibody staining method such as the FMAT8100HTS system (manufactured by Applied Biosystems) can be mentioned.
  • Biological samples to be detected or measured for at least one of TfR and GPC3 in the present invention include, for example, tissue cells, blood, plasma, serum, pancreatic fluid, urine, feces, tissue fluid or culture fluid, and at least TfR and GPC3. It is not particularly limited as long as one of them may contain expressed cells.
  • the diagnostic agent containing the bispecific antibody or the bispecific antibody fragment thereof of the present invention, or a derivative thereof contains a reagent for carrying out an antigen-antibody reaction and a reagent for detecting the reaction, depending on the target diagnostic method. But it may be.
  • the reagent for carrying out the antigen-antibody reaction include a buffer and a salt.
  • the detection reagent includes, for example, the bispecific antibody or the bispecific antibody fragment thereof, or a labeled secondary antibody that binds to a derivative thereof, or a substrate corresponding to the label, which is a usual immunological detection or measurement method. Examples thereof include reagents used in the above.
  • the method for producing a monoclonal antibody in the present invention includes the following working steps. That is, at least one of (1) purification of an antigen used as an immunogen and preparation of a cell in which an antigen is overexpressed on the cell surface, (2) after immunizing an animal with the antigen, blood is collected and the antibody titer thereof is tested.
  • Steps to determine the time to remove the spleen and prepare antibody-producing cells (3) preparation of myeloma cells (mieroma), (4) cell fusion between antibody-producing cells and myeroma, (5) objectives Selection of antibody-producing hybridoma groups, (6) Isolation (cloning) of monoclonal cells from the hybridoma group, (7) In some cases, culture of hybridomas for the production of large quantities of monoclonal antibodies, or hybridomas. (8) Examination of the physiological activity of the monoclonal antibody thus produced and its antigen-binding specificity, or testing of its properties as a labeling reagent.
  • a method for producing a monoclonal antibody that binds to TfR and a monoclonal antibody that binds to GPC3, which is used for producing a bispecific antibody that binds to TfR and GPC3 in the present invention will be described in detail along with the above steps.
  • the method for producing the antibody is not limited to this, and for example, antibody-producing cells other than spleen cells and myeloma can also be used.
  • TfR or GPC3 Purification of antigen Cells expressing TfR or GPC3 can be obtained by introducing an expression vector containing a cDNA encoding the full length or a partial length of TfR or GPC3 into Escherichia coli, yeast, insect cells, animal cells, or the like.
  • at least one of TfR and GPC3 can be purified from various human tumor cultured cells or human tissues expressing at least one of TfR and GPC3 in a large amount and used as an antigen. Further, the cultured tumor cells or the tissues can be used as they are as an antigen.
  • a synthetic peptide having a partial sequence of TfR or GPC3 can be prepared by a chemical synthesis method such as the Fmoc method or the tBoc method and used as an antigen.
  • the TfR or GPC3 used in the present invention is described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols In Molecular Biology, John Wiley & Sons (1987-1997), and the like. It can be produced by expressing a DNA encoding TfR or GPC3 in a host cell by, for example, the following method. It was
  • a recombinant vector is prepared by inserting a full-length cDNA containing a portion encoding TfR or GPC3 downstream of the promoter of an appropriate expression vector.
  • a DNA fragment of appropriate length containing a polypeptide-encoding moiety prepared based on the full-length cDNA may be used.
  • a transformant producing TfR or GPC3 can be obtained. It was
  • any microorganism belonging to the genus Escherichia such as Escherichia coli, yeast, insect cell, animal cell, etc. can be used as long as it can express the target gene.
  • the recombinant vector is capable of autonomous replication in the prokaryote and contains a promoter, a ribosome-binding sequence, a DNA containing a portion encoding TfR or GPC3, and a transcription termination sequence. It is preferable that the vector contains. Further, although the transcription termination sequence is not always necessary for the recombinant vector, it is preferable to arrange the transcription termination sequence directly under the structural gene. In addition, the recombinant vector may contain a gene that controls a promoter.
  • the recombinant vector it is preferable to use a plasmid in which the ribosome-binding sequence Shine dalgarno sequence and the start codon are adjusted to an appropriate distance (for example, 6 to 18 bases).
  • the base sequence of the DNA encoding TfR or GPC3 can be substituted so as to be the optimum codon for expression in the host, whereby the production rate of the target TfR or GPC3 can be improved. ..
  • any vector that can exert its function in the host cell to be used can be used, and for example, pBTrp2, pBTac1, pBTac2 (all manufactured by Roche Diagnostics), pKK233-2 (above, manufactured by Roche Diagnostics). Pharmacia), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-8 (Kiagen), pKYP10 (Japanese Patent Laid-Open No. 58-110600), pKYP200 [Agricultural Biological Chemistry, 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci.
  • the promoter may be any promoter as long as it functions in the host cell used.
  • promoters derived from Escherichia coli or phage such as trp promoter (Ptrp), lac promoter, PL promoter, PR promoter or T7 promoter can be mentioned.
  • an artificially designed and modified promoter such as a tandem promoter in which two Ptlps are serialized, a tac promoter, a lacT7 promoter, or a let I promoter can be mentioned.
  • Examples of host cells include E. coli XL1-Blue, E. coli XL2-Blue, E. coli DH1, E. coli MC1000, E. coli KY3276, E. coli W1485, E. coli JM109, E. coli HB101, E. coli No. 49, Escherichia coli W3110, Escherichia coli NY49, Escherichia coli DH5 ⁇ and the like can be mentioned.
  • any method for introducing DNA into the host cell to be used can be used.
  • a method using calcium ions [Proc. Natl. Acad. Sci. USA , 69, 2110 (1972), Gene, 17, 107 (1982), Molecular & General Genetics, 168, 111 (1979)]. It was
  • any expression vector that functions in the animal cell can be used, for example, pcDNAI (manufactured by Invitrogen), pcDM8 (manufactured by Funakoshi), pAGE107 [Japan].
  • pcDNAI manufactured by Invitrogen
  • pcDM8 manufactured by Funakoshi
  • pAGE107 [Japan].
  • pAS3-3 Japanese Patent Laid-Open No.
  • pCDM8 [Nature, 329, 840 (1987)], pcDNAI / Amp (Invitrogen) , PKDNA3.1 (manufactured by Invitrogen), pREP4 (manufactured by Invitrogen), pAGE103 [J. Biochemistry, 101, 1307 (1987)], pAGE210, pME18SFL3, pCI (manufactured by Promega) or pKANTEX93 (international release No. 97/10354) and the like.
  • any promoter that can exert its function in animal cells can be used, for example, a promoter of the immediate early (IE) gene of cytomegalovirus (CMV), an initial promoter of SV40, and a promoter of a retrovirus.
  • IE immediate early
  • CMV cytomegalovirus
  • a promoter of a retrovirus for example, a promoter of the immediate early (IE) gene of cytomegalovirus (CMV), an initial promoter of SV40, and a promoter of a retrovirus.
  • IE immediate early
  • CMV cytomegalovirus
  • Examples of the host cell include human Berkit lymphoma cell Namalwa, African green monkey kidney-derived cell COS, Chinese hamster ovary-derived cell CHO, and human leukemia cell HBT5637 (Japanese Patent Laid-Open No. 63-000299).
  • any method for introducing DNA into animal cells can be used.
  • the electroporation method [Cytotechnology, 3, 133 (1990)]
  • the calcium phosphate method Japanese Patent Laid-Open No. 2-227075
  • the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)] and the like can be mentioned. It was
  • a microorganism derived from a microorganism containing a recombinant vector incorporating a DNA encoding TfR or GPC3 obtained as described above, or a transformant derived from an animal cell or the like is cultured in a medium, and the TfR and GPC3 are contained in the culture.
  • TfR or GPC3 can be produced by producing and accumulating at least one of the above and collecting the cells from the culture.
  • the method of culturing the transformant in the medium can be carried out according to the usual method used for culturing the host.
  • TfR or GPC3 When expressed in cells derived from eukaryotes, TfR or GPC3 to which sugars or sugar chains are added can be obtained.
  • an inducer may be added to the medium as needed.
  • an inducer may be added to the medium as needed.
  • a microorganism transformed with a recombinant vector using a lac promoter isopropyl- ⁇ -D-thiogalactopyranoside or the like is cultured, and a microorganism transformed with a recombinant vector using a trp promoter is cultured.
  • indole acrylic acid or the like may be added to each medium.
  • Examples of the medium for culturing the transformant obtained by using animal cells as a host include the commonly used RPMI 1640 medium [The Journal of the American Medical Association, 199, 519 (1967)] and Eagle's MEM medium [Science]. , 122, 501 (1952)], Dalveco Modified MEM Medium [Virology, 8, 396 (1959)], 199 Medium [Proc. Soc. Exp. Biol. Med., 73, 1 (1950)], Iscover's Modified Examples include the RPMI's Medium (IMDM) medium, or a medium obtained by adding bovine fetal serum (FBS) or the like to these media. Culturing is usually carried out for 1 to 7 days under conditions such as pH 6 to 8, 30 to 40 ° C. and the presence of 5% CO 2. In addition, antibiotics such as kanamycin and penicillin may be added to the medium during the culture, if necessary.
  • RPMI 1640 medium The Journal of the American Medical Association, 199, 519 (1967)] and Eagle'
  • TfR or GPC3 As a method for expressing the gene encoding TfR or GPC3, a method such as secretory production or fusion protein expression [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)] should be used in addition to direct expression. Can be done.
  • Examples of the method for producing TfR or GPC3 such as EGFR include a method for producing it in the host cell, a method for secreting it outside the host cell, and a method for producing it on the outer membrane of the host cell. By changing the structure of the TfR or GPC3 to be produced, an appropriate method can be selected.
  • the DNA encoding the amino acid sequence of the extracellular region includes the DNA encoding the Fc region of the antibody, the DNA encoding the glutathione S-transferase (GST), the DNA encoding the FLAG tag, or the DNA encoding the Histide tag.
  • An antigen fusion protein can be prepared by producing a DNA ligated with the above, expressing and purifying the DNA. Specific examples thereof include an Fc fusion protein in which the extracellular region of TfR or GPC3 is bound to the Fc region of human IgG, and a fusion protein of the extracellular region of TfR or GPC3 and glutathione S-transferase (GST).
  • TfR or GPC3 When TfR or GPC3 is produced in the host cell or on the outer membrane of the host cell, Paulson et al.'S method [J. Biol. Chem., 264, 1769 (1989)], Row et al.'S method [Proc. Natl. Acad. Sci., USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)], Japanese Patent Application Laid-Open No. 05-336963, International Publication No. 94/23021, etc. Allows TfR or GPC3 to be actively secreted out of the host cell. Further, it is also possible to increase the production amount of TfR or GPC3 by using a gene amplification system using a dihydrofolate reductase gene or the like (Japanese Patent Laid-Open No. 2-227075).
  • the produced TfR or GPC3 can be isolated and purified as follows, for example.
  • the cells are collected by centrifugation after the culture is completed, suspended in an aqueous buffer solution, and then an ultrasonic crusher, a French press, a manton gaulin homogenizer, or a dynomill or the like. Crush the cells using the cell-free extract.
  • a usual method for isolating and purifying a protein that is, a solvent extraction method, a salting-out method using a chromatographic method, a desalting method, a precipitation method using an organic solvent, diethylaminoethyl ( DEAE) -Anion exchange chromatography method using a resin such as Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical Corporation), Cation exchange chromatography method using a resin such as S-Sepharose FF (manufactured by Pharmacia), Methods such as hydrophobic chromatography using resins such as butyl Sepharose and phenyl Sepharose, gel filtration using molecular sieves, affinity chromatography, chromatographic focusing, or electrophoresis such as isoelectric point electrophoresis. Purified proteins can be obtained when used alone or in combination.
  • TfR or GPC3 When TfR or GPC3 is expressed by forming an insoluble matter in the cells, the insoluble matter of TfR or GPC3 is recovered as a precipitate fraction by collecting and crushing the cells and centrifuging in the same manner as described above.
  • the recovered TfR or GPC3 insoluble material is solubilized with a protein denaturing agent. By diluting or dialyzing the solubilized solution, TfR or GPC3 can be returned to a normal three-dimensional structure, and then a purified protein of the polypeptide can be obtained by the same isolation and purification method as described above.
  • the derivative such as TfR or GPC3 or a sugar-modified product thereof can be recovered in the culture supernatant.
  • a soluble fraction is obtained by treating the culture supernatant using a technique such as centrifugation in the same manner as described above, and a purified protein is obtained from the soluble fraction by using the same isolation and purification method as described above. be able to.
  • TfR or GPC3 used in the present invention can also be produced by a chemical synthesis method such as the Fmoc method or the tBoc method.
  • a chemical synthesis method such as the Fmoc method or the tBoc method.
  • peptide synthesizers manufactured by Advanced Chemtech, PerkinElmer, Pharmacia, Protein Technology Instrument, Synthesell-Vega, Perceptive, or Shimadzu can be used for chemical synthesis.
  • Immunization is performed by administering the antigen with Freund's complete adjuvant or an appropriate adjuvant such as aluminum hydroxide gel and Bordetella pertussis vaccine.
  • the immunogen administration method for mouse immunization may be subcutaneous injection, intraperitoneal injection, intravenous injection, intradermal injection, intramuscular injection or footpad injection, but intraperitoneal injection, footpad injection or intravenous injection. Is preferable.
  • a conjugate with a carrier protein such as BSA (bovine serum albumin) or KLH (Keyhole Primet hemocyanin) is prepared and used as an immunogen.
  • Antigen is administered 5 to 10 times every 1 to 2 weeks after the first administration. Blood is collected from the fundus venous plexus 3 to 7 days after each administration, and the antibody titer of the serum is measured using an enzyme immunoassay [Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)] or the like. If an animal in which the above serum shows a sufficient antibody titer against the antigen used for immunization is used as a source of cells producing the fusion antibody, the effect of the subsequent operation can be enhanced.
  • the tissue containing the antibody-producing cells such as spleen is removed from the immunized animal, and the antibody-producing cells are collected.
  • the antibody-producing cells are plasma cells and lymphocytes that are precursors thereof, which may be obtained from any site of the individual, generally the spleen, lymph nodes, bone marrow, tonsils, peripheral blood, or any combination thereof. Although it can be obtained from spleen cells, spleen cells are most commonly used. When spleen cells are used, fusion antibody-producing cells are obtained by shredding the spleen, loosening it, centrifuging it, and removing erythrocytes.
  • mieroma cells derived from mammals such as mice, rats, guinea pigs, hamsters, rabbits and humans and having no autoantibody-producing ability can be used, but generally obtained from mice.
  • Established cells such as 8-azaguanine resistant mice (from BALB / c) myeloma cell line P3-X63Ag8-U1 (P3-U1) [Current Topics in Microbiology and Immunology, 18, 1 (1978)], P3- NS1 / 1-Ag41 (NS-1) [European J.
  • the cell line is a suitable medium, for example 8-azaguanine medium [RPMI-1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamycin, FCS and 8-azaguanin], Iskov's Modified Dulvecco's.
  • Subculture is carried out in a medium such as Medium (hereinafter referred to as "IMDM”) or Dulvecco's Modified Eagle Medium (hereinafter referred to as "DMEM").
  • IMDM Medium
  • DMEM Dulvecco's Modified Eagle Medium
  • the above cell lines are subcultured in normal medium (for example, DMEM medium containing 10% FCS) 3 to 4 days before cell fusion, and a cell number of 2 ⁇ 10 7 or more is secured on the day of fusion.
  • MEM Minimum Essential Medium
  • PBS PBS
  • MEM medium normal medium containing hypoxanthine, thymidine, and aminopterin.
  • HAT medium normal medium containing hypoxanthine, thymidine, and aminopterin. The suspension is cultured in a 5% CO 2 incubator at 37 ° C. for 7-14 days.
  • cell fusion can also be performed by the following methods. Thoroughly wash spleen cells and myeloma cells with serum-free medium (for example, DMEM) or phosphate buffered physiological saline (hereinafter referred to as "phosphate buffered solution”), and the cell number ratio of spleen cells to myeloma cells is 5 :. Mix to a ratio of about 1 to 10: 1 and centrifuge. After removing the supernatant and thoroughly loosening the precipitated cell clumps, a serum-free medium containing 1 mL of 50% (w / v) polyethylene glycol (molecular weight 1000 to 4000) is added dropwise with stirring.
  • serum-free medium for example, DMEM
  • phosphate buffered solution phosphate buffered physiological saline
  • HAT medium normal medium
  • HAT hypoxanthine, aminopterin, thymidine
  • IL-2 human interleukin-2
  • HT medium a medium from which aminopterin has been removed from the HAT medium
  • a hybridoma producing an antibody can be selected by using the antibody titer measurement method described later.
  • the method for measuring the antibody titer include various known techniques such as a radioisotope immunoquantification method (RIA method), a solid phase enzyme-linked immunosorbent assay method (ELISA method), an immunofluorescence method, and a passive hemagglutination reaction method.
  • RIA method radioisotope immunoquantification method
  • ELISA method solid phase enzyme-linked immunosorbent assay method
  • an immunofluorescence method an immunofluorescence method
  • passive hemagglutination reaction method a passive hemagglutination reaction method.
  • the RIA method or the ELISA method is preferable from the viewpoints of detection sensitivity, speed, accuracy and possibility of automation of operation.
  • the hybridoma found to produce the desired antibody is transferred to another plate and cloned.
  • this cloning method for example, a limit dilution method in which one cell is diluted and cultured so that one well of a plate is contained, a soft agar method in which colonies are collected by culturing in a soft agar medium, and a micromanipulator 1 are used. Examples thereof include a method of isolating one cell, a method of isolating one cell by a cell sorter, and the like.
  • cloning by the limiting dilution method is repeated 2 to 4 times, and those with stable antibody titers are selected as hybridoma strains that produce monoclonal antibodies against TfR or GPC3. It was
  • the IgG or IgM fractions are collected and used as purified monoclonal antibodies.
  • a large amount of monoclonal antibody that binds to TfR or GPC3 is produced by growing the hybridoma in the abdominal cavity of a mouse of the same strain (for example, BALB / c) or Nu / Nu mouse, rat, guinea pig, hamster or rabbit. Ascites containing can be obtained.
  • Determination of antibody subclasses is performed by enzyme immunoassay using a subcluster typing kit.
  • Monoclonal antibody binding assay for TfR or GPC3 The binding activity of the monoclonal antibody to TfR or GPC3 is determined by the Ochterlony method, ELISA method, RIA method, flow cytometry method (FCM) or surface plasmon resonance method (SPR). It can be measured by a binding assay system such as.
  • the Octerlony method is simple, but if the antibody concentration is low, a concentration operation is required.
  • the culture supernatant is directly reacted with the antigen-adsorbing solid phase, and various immunoglobulin isotypes and antibodies corresponding to the subclass are used as the secondary antibody to obtain the antibody isotype. It is possible to identify subclasses and measure antibody binding activity.
  • purified or partially purified TfR or GPC3 is adsorbed on a solid phase surface such as a 96-well plate for ELISA, and the solid phase surface on which the antigen is not adsorbed is adsorbed on a protein unrelated to the antigen, for example, bovine serum.
  • Blocking is performed with albumin (BSA).
  • BSA albumin
  • PBS phosphate buffer saline
  • Tween-PBS PBS containing 0.05% Tween 20
  • a serially diluted first antibody for example, mouse serum, culture supernatant, etc.
  • an anti-immunoglobulin antibody labeled with biotin, an enzyme (horseradish peroxidase; HRP, alkaline phosphatase; ALP, etc.), a chemiluminescent substance, a radiation compound, or the like was dispensed and bound to the plate.
  • the second antibody is reacted with the antibody.
  • Tween-PBS a reaction is carried out according to the labeling substance of the second antibody, and a monoclonal antibody that specifically reacts with the target antigen is selected.
  • the FCM method can measure the binding activity of an antibody to antigen-expressing cells [Cancer Immunol. Immunother., 36, 373 (1993)].
  • an antibody binds to a membrane protein antigen expressed on a cell membrane, it means that the antibody recognizes and binds to the three-dimensional structure of the naturally occurring antigen.
  • Examples of the SPR method include kinetics analysis by Biacore.
  • Biacore T100 kinetics in the binding between the antigen and the test substance is measured, and the result is analyzed by the analysis software attached to the device.
  • a test substance such as a hybridoma culture supernatant or a purified monoclonal antibody is flowed to bind an appropriate amount, and the concentration is known. Flow multiple concentrations of antigen and measure binding and dissociation.
  • the obtained data is subjected to kinetics analysis using a 1: 1 binding model using software attached to the device, and various parameters are acquired.
  • TfR or GPC3 is immobilized on a sensor chip, for example, by an amine coupling method, and then a plurality of known concentrations of purified monoclonal antibody are flowed to measure binding and dissociation. Kinetics analysis is performed on the obtained data by a vivalent binding model using software attached to the device, and various parameters are acquired.
  • the antibody that competes with the antibody against TfR or GPC3 and binds to TfR or GPC3 can be selected by reacting the above-mentioned binding assay system with the test antibody in coexistence. That is, by screening for an antibody whose binding to the antigen is inhibited when the test antibody is added, an antibody that competes with the antibody obtained above for binding to TfR or GPC3 can be obtained.
  • the epitope to be recognized and bound by the antibody can be identified as follows.
  • Partial defects and variants of such antigens may be obtained as secretory proteins using suitable host cells such as Escherichia coli, yeast, plant cells or mammalian cells, or on the cell membrane of the host cells. It may be expressed and prepared as an antigen-expressing cell.
  • a membrane-type antigen it is preferable to express it on the membrane of a host cell in order to express it while retaining the three-dimensional structure of the antigen. It is also possible to prepare a synthetic peptide that mimics the primary structure or three-dimensional structure of an antigen and confirm the reactivity of the antibody. Examples of the synthetic peptide include a method for producing various partial peptides of the molecule using a known peptide synthesis technique.
  • a chimeric protein in which the domains constituting each region are appropriately combined is prepared, and the reactivity of the antibody with the protein can be confirmed to identify the epitope of the antibody. .. Then, in more detail, the corresponding part of the oligopeptide, or a variant of the peptide, etc. is synthesized in various ways using an oligopeptide synthesis technique well known to those skilled in the art, and the reactivity of the antibody with the peptide is confirmed to confirm the epitope. Can be identified.
  • a commercially available kit for example, SPOTs kit (manufactured by Genosis Biotechnology), a series of multipin peptide synthesis kits using a multipin synthesis method (manufactured by Chiron), etc. ] Can also be used.
  • the epitope of the antibody obtained by the above-mentioned binding assay system is identified, and a partially synthetic peptide of the epitope, a three-dimensional structure of the epitope, is obtained. It can be obtained by preparing a simulated synthetic peptide, a recombinant of the epitope, or the like and immunizing it.
  • the epitope is a membrane protein
  • a recombinant fusion protein in which the entire extracellular region or some extracellular domains are linked to an appropriate tag, such as a FLAG tag, Histide tag, GST protein, or antibody Fc region.
  • an appropriate tag such as a FLAG tag, Histide tag, GST protein, or antibody Fc region.
  • mRNA is extracted from a hybridoma that produces a monoclonal antibody, and cDNA is synthesized.
  • the synthesized cDNA is cloned into a vector such as a phage or a plasmid to prepare a cDNA library.
  • a recombinant phage or a recombinant plasmid having a cDNA encoding VH or VL is isolated using the DNA encoding the C region portion or the V region portion of the antibody as a probe, respectively.
  • the entire base sequence of VH or VL in the isolated recombinant phage or recombinant plasmid is determined, and the entire amino acid sequence of VH or VL is estimated from the base sequence.
  • non-human animal used for producing a hybridoma a mouse, rat, hamster, rabbit or the like is used, but any animal can be used as long as it is possible to produce a hybridoma.
  • RNA easy kit manufactured by Qiagen
  • oligo (dT) immobilized cellulose column method [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)]
  • Oligo-dT30 ⁇ Super> mRNA Purification Kit.
  • kit such as (manufactured by Takara Bio).
  • mRNA can also be prepared using a kit such as Fast Track mRNA Isolation Kit (manufactured by Invitrogen) or QuickPrep mRNA Purification Kit (manufactured by Pharmacia).
  • any vector that can incorporate the cDNA can be used as the vector into which the cDNA synthesized using the mRNA extracted from the hybridoma as a template is incorporated.
  • ZAP Express (Strategies, 5, 58 (1992)], pBluescript II SK (+) [Nucleic Acids Research, 17, 9494 (1989)], ⁇ ZAPII (manufactured by Stratagene), ⁇ gt10, ⁇ gt11 [DNA Clone] Approach, I, 49 (1985)], Lambda BlueMid (manufactured by Clonetech), ⁇ ExCell, pT7T3-18U (manufactured by Pharmacia), pcD2 [Mol. Cell. Biol., 3, 280 (1983)] or pUC18 [Gene. , 33, 103 (1985)] etc. are used.
  • Any Escherichia coli into which a cDNA library constructed by a phage or a plasmid vector can be introduced can be used as long as the cDNA library can be introduced, expressed and maintained.
  • XL1-Blue MRF' [Strategies, 5, 81 (1992)], C600 [Genetics, 39, 440 (1954)], Y1088, Y1090 [Science, 222, 778 (1983)], NM522 [J. Mol. Biol., 166, 1 (1983)], K802 [J. Mol. Biol., 16, 118 (1966)], or JM105 [Gene, 38, 275 (1985)] is used.
  • PCR method Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) using a cDNA or cDNA library synthesized from a primer by preparing a primer as a template.
  • Current Protocols in Molecular Biology, Supplement 1, John Wiley & Sons (1987-1997) can also be used to prepare cDNA encoding VH or VL.
  • a plasmid such as pBluescript SK (-) (manufactured by Stratagene) (manufactured by Stratagene), and the base sequence of the cDNA is determined by a commonly used base sequence analysis method or the like. do.
  • a reaction such as the dideoxy method [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)]
  • A. L. F. Analysis is performed using an automatic base sequence analyzer such as a DNA sequencer (manufactured by Pharmacia).
  • the entire amino acid sequences of VH and VL were estimated from the determined total base sequences, respectively, and compared with the total amino acid sequences of VH and VL of known antibodies [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]. By doing so, it is confirmed whether or not the obtained cDNA encodes the complete amino acid sequence of each of VH and VL of the antibody including the secretory signal sequence.
  • amino acid sequences of VH and VL of the antibody including the secretory signal sequence refer to the entire amino acid sequence of VH and VL of the known antibody [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)].
  • the length of the secretory signal sequence and the N-terminal amino acid sequence can be estimated, and the subgroup to which they belong can be identified.
  • amino acid sequence of each CDR of VH and VL can be estimated by comparing with the amino acid sequence of VH and VL of a known antibody [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)].
  • a vector for expressing a recombinant antibody can be constructed by cloning a DNA encoding at least one of CH and CL of a human antibody into an expression vector for animal cells.
  • CH and CL of any human antibody can be used, and for example, CH of the ⁇ 1 subclass of the human antibody and CL of the ⁇ class can be used.
  • cDNA is used as the DNA encoding CH and CL of the human antibody, chromosomal DNA consisting of exon and intron can also be used.
  • any vector can be used as long as it can be expressed by incorporating a gene encoding the C region of a human antibody.
  • pAGE107 [Cytotechnol., 3, 133 (1990)]
  • pAGE103 [J. Biochem., 101, 1307 (1987)]
  • pHSG274 [Gene, 27, 223 (1984)]
  • pKCR Proc. Natl. Acad. Sci.
  • promoters and enhancers for expression vectors for animal cells the initial promoter of SV40 [J. Biochem., 101, 1307 (1987)], Moloney mouse leukemia virus LTR [Biochem. Biophys. Res. Communi., 149, 960 (1987) )], CMV promoter (US Pat. No. 5,168,062) or immunoglobulin H chain promoter [Cell, 41, 479 (1985)] and enhancer [Cell, 33, 717 (1983)] are used. be able to.
  • antibody H chain and L are considered from the viewpoints of ease of vector construction, ease of introduction into animal cells, and balance of expression levels of antibody H chain and L chain in cells.
  • a vector tandem type vector [J. Immunol. Methods, 167, 271 (1994)] carrying both chain genes is used, but multiple vectors carrying antibody H chain and L chain genes separately (separate).
  • Type vector can also be used in combination.
  • Vectors for expressing tandem-type recombinant antibodies include pKANTEX93 (International Publication No. 97/10354), pEE18 [Hybridoma, 17, 559 (1998)], and N5KG1val (US Pat. No. 6,001,358). , N5KG4PE R409K (described in International Publication No. 2006/0333386), N5KG2 vector (described in International Publication No. 2003/035353), Tol2 transposon vector (described in International Publication No. 2010/143698), and the like are used.
  • the base sequence of the linking portion encodes an appropriate amino acid.
  • VH and VL cDNAs designed to have a suitable restriction enzyme recognition sequence.
  • the prepared VH and VL cDNAs are appropriately expressed upstream of each of the genes encoding CH or CL of the human antibody in the recombinant antibody expression vector obtained in (2).
  • Each clone is cloned to construct a vector for expressing a chimeric antibody.
  • the cDNA encoding VH or VL of the non-human antibody is amplified by the PCR method using synthetic DNA having a recognition sequence of an appropriate restriction enzyme at both ends, and is used for expressing the recombinant antibody obtained in (2).
  • a vector for expressing a chimeric antibody can also be constructed.
  • the cDNA encoding VH or VL of the humanized antibody can be prepared as follows. First, the amino acid sequence of the VH or VL framework region (hereinafter referred to as FR) of the human antibody to which the amino acid sequence of the VH or VL CDR of the non-human antibody obtained in (1) is transplanted is selected.
  • FR the amino acid sequence of the VH or VL framework region
  • any one derived from human antibody can be used.
  • the amino acid sequence of FR of human antibody registered in a database such as Protein Data Bank, or the common amino acid sequence of each subgroup of FR of human antibody [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services ( 1991)] etc. are used.
  • the amino acid sequence of human FR having the highest possible homology (60% or more) with the amino acid sequence of FR of VH or VL of the original non-human antibody is selected.
  • the amino acid sequence of the CDR of the original non-human antibody is transplanted to the amino acid sequence of FR of VH or VL of the selected human antibody, respectively, and the amino acid sequence of VH or VL of the humanized antibody is designed respectively.
  • the designed amino acid sequence into a DNA sequence in consideration of the frequency of codon usage found in the base sequence of the antibody gene [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]
  • humans Design the VH or VL cDNA sequences of the antibody, respectively.
  • the VH or VL of the humanized antibody can be easily incorporated into the vector for expressing the recombinant antibody obtained in (2).
  • the cDNA encoding the can be cloned.
  • the amplification product was cloned into a plasmid such as pBluescript SK (-) (manufactured by Stratagene), and the base sequence was determined by the same method as described in (1) to obtain the desired humanized antibody.
  • a plasmid having a DNA sequence encoding the amino acid sequence of VH or VL.
  • the humanized antibody has the original antigen-binding activity only by transplanting only the CDRs of VH and VL of the non-human antibody into the FR of VH and VL of the human antibody. [BIO / TECHNOLOGY, 9, 266 (1991)]. Therefore, among the amino acid sequences of VH and VL FR of human antibodies, the amino acid residues directly involved in binding to the antigen, the amino acid residues interacting with the amino acid residues of CDR, and the three-dimensional structure of the antibody are maintained. By identifying amino acid residues indirectly involved in binding to the antigen and substituting those amino acid residues with the amino acid residues of the original non-human antibody, the reduced antigen-binding activity of the humanized antibody was reduced. Can be raised.
  • modified humanized antibodies having the required antigen-binding activity can be obtained by repeatedly preparing several variants of each antibody, examining the correlation with each antigen-binding activity, and conducting trial and error. ..
  • the amino acid residues of VH and FR of human antibody can be modified by performing the PCR reaction described in (4) using synthetic DNA for modification.
  • the base sequence of the amplified product after the PCR reaction is determined by the method described in (1), and it is confirmed that the desired modification has been performed.
  • an appropriate restriction enzyme recognition sequence is introduced into the 5'end of the synthetic DNA located at both ends.
  • cloning is performed upstream of each gene encoding CH or CL of the human antibody in the vector for expressing the recombinant antibody obtained in (2) so that they can be expressed in an appropriate form.
  • a gene recombinant antibody is expressed using the gene recombinant antibody expression vector obtained in (3), (6) and (7), or a modified expression vector thereof. It can be expressed transiently and the antigen-binding activity of the obtained various recombinant antibodies can be efficiently evaluated.
  • any host cell capable of expressing the recombinant antibody can be used.
  • COS-7 cell American Type Culture Collection (ATCC) number: CRL1651] can be used.
  • ATCC American Type Culture Collection
  • the DEAE-dextran method Methods in Nucleic Acids Res., CRC press (1991)
  • the lipofection method Proc. Natl. Acad. Sci. USA, 84, 7413 ( 1987)] etc. are used.
  • Examples of the introduction of the expression vector into the host cell include an electroporation method [Japanese Patent Laid-Open No. 2-257891, Cytotechnology, 3, 133 (1990)], a calcium ion method, an electroporation method, and spheroplast. Methods, lithium acetate method, calcium phosphate method, lipofection method and the like can be mentioned. Examples of the method for introducing a gene into an animal described later include a microinjection method, a method for introducing a gene into ES cells by electroporation or lipofection method, and a nuclear transplantation method.
  • any cell can be used as long as it is a host cell capable of expressing the recombinant antibody.
  • mouse SP2 / 0-Ag14 cells ATCC CRL1581
  • mouse P3X63-Ag8.653 cells ATCC CRL1580
  • Chinese hamster CHO-K1 cells ATCC CCL-61
  • DUKXB11 ATCC CCL-9096
  • Cells ATCC CCL-1781
  • CHO-S cells Life Technologies, Cat No. 11619
  • CHO cells lacking the dihydrofolate reductase gene (dhfr) (CHO / DG44 cells) [Proc. Natl.
  • protein such as an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose, sugar chain modification in which the 1-position of fucose is ⁇ -bonded to the 6-position of N-acetylglucosamine at the reducing end of the N-glycoside-linked complex sugar chain.
  • Defects in host cells with reduced or deleted activity such as proteins involved in enzymes involved in, or proteins involved in the transport of intracellular sugar nucleotide GDP-fucose to the Gorgian body, such as the ⁇ 1,6-fucose transferase gene.
  • CHO cells International Publication No. 2005/035586, International Publication No. 2002/31140
  • the like can also be used.
  • a transformant that stably expresses the recombinant antibody is selected by culturing it in an animal cell culture medium containing a drug such as G418 sulfate (hereinafter referred to as G418) (hereinafter referred to as G418).
  • G4108 G418 sulfate
  • the culture medium for animal cell culture includes RPMI1640 medium (manufactured by Invitrogen), GIT medium (manufactured by Nippon Pharmaceutical Co., Ltd.), EX-CELL301 medium (manufactured by JRH), EX-CELL302 medium (manufactured by JRH), and EX. -CELL325 medium (manufactured by JRH), IMDM medium (manufactured by Invitrogen) or Hybridoma SFM medium (manufactured by Invigen), or a medium obtained by adding various additives such as FBS to these media is used.
  • a recombinant antibody By culturing the obtained transformant in a medium, a recombinant antibody is expressed and accumulated in the culture supernatant.
  • the expression level and antigen-binding activity of the recombinant antibody in the culture supernatant can be measured by an ELISA method or the like. Further, the expression level of the recombinant antibody produced by the transformant can be increased by using a DHFR amplification system (Japanese Patent Laid-Open No. 2-257891) or the like.
  • Recombinant antibody can be purified from the culture supernatant of the transformant using a protein A column [Monoclonal Antibodies-Principles and practice, Third edition, Academic Press (1996), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory ( 1988)]. It can also be purified by combining the methods used in protein purification, such as gel filtration, ion exchange chromatography and ultrafiltration.
  • the molecular weight of the entire H chain, L chain or antibody molecule of the purified recombinant antibody can be determined by polyacrylamide gel electrophoresis [Nature, 227, 680 (1970)] or Western blotting [Monoclonal Antibodies-Principles and practice, Third. It can be measured using edition, Academic Press (1996), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)].
  • the N-terminal bispecific antibody of the present invention can be produced by designing an IgG moiety containing a second antigen-binding domain and a first antigen-binding domain, respectively, and designing a bispecific antibody in which they are linked.
  • the first antigen-binding domain contains the CDR or variable region of the antibody, the above 1. And 2. It can be made by determining the DNA sequence of the CDR or variable region of an antibody and designing a first antigen binding domain containing them by the method described in.
  • a first antigen-binding domain is a single-chain domain in which VH and VL are bound directly or via an appropriate linker such as scFv, and a double-stranded domain such as Fab and dsFv. And those designed to bind SS after expression, VHH and the like can also be used.
  • the antigen-binding activity of the first antigen-binding domain is evaluated by the above method, and one that retains the antigen-binding activity can be selected.
  • the C-terminal bispecific antibody of the present invention is described in 3-1. It can be prepared by designing an IgG moiety containing a first binding domain and a second antigen binding domain, respectively, in the same manner as in the above method, and designing a bispecific antibody in which they are linked.
  • bispecific antibody 4-1 Preparation of bispecific antibody 4-1.
  • N-terminal bispecific antibody The N-terminal bispecific antibody, in which the first antigen-binding domain and the second antigen-binding domain are Fab and the four light chains are common, is specifically as follows. Can be made.
  • DNA encoding a polypeptide in which VH-CH1 of the first antigen-binding domain and VH of the IgG portion containing the second antigen-binding domain are linked was synthesized, and DNA encoding the VL of each antibody was synthesized.
  • the DNA encoding the linker sequence is also synthesized.
  • a bispecific antibody having a first antigen-binding domain of VHH can be specifically prepared as follows. The DNA encoding the polypeptide in which VHH and the VH of the IgG portion are linked and the DNA encoding the VL of the IgG portion are synthesized, and these DNAs are used as described in 2. above. It can be produced by incorporating it into the gene recombinant antibody expression vector according to (2) and expressing the bispecific antibody. When the VHH and the IgG moiety are linked via a linker, the DNA encoding the linker sequence is also synthesized.
  • a bispecific antibody in which the first antigen-binding domain is a polypeptide other than the above (1) and (2) containing scFv, dsFv or CDR can be specifically prepared as follows.
  • the first antigen-binding domain is single-stranded
  • a DNA in which the DNA encoding the first antigen-binding domain and the DNA encoding the VH of the IgG portion are linked is synthesized. If the first antigen-binding domain is an aggregate consisting of two single-stranded polypeptides, one single-stranded polypeptide constituting the first antigen-binding domain is linked to the DNA encoding VH of the IgG portion.
  • DNA encoding the other single-stranded polypeptide constituting the first antigen-binding domain is synthesized. It also synthesizes the DNA encoding the VL of the IgG portion. When the first antigen-binding domain and the IgG moiety are linked via a linker, the DNA encoding the linker sequence is also synthesized. These DNAs are used in the above 2. It can be produced by incorporating it into the gene recombinant antibody expression vector according to (2) and expressing the bispecific antibody.
  • the bispecific antibody of the present invention is specifically as follows. Can be made. When the first antigen-binding domain is single-stranded, a DNA in which the DNA encoding the first antigen-binding domain and the DNA encoding the VH of the IgG portion are linked is synthesized. When the first antigen-binding domain is an aggregate consisting of two or more single-stranded polypeptides, one single-stranded polypeptide constituting the first antigen-binding domain is used as the VH of the IgG portion.
  • the DNA encoding the remaining single-stranded polypeptide constituting the first antigen-binding domain is also synthesized. It also synthesizes the DNA encoding the VL of the IgG portion. When the IgG moiety and the first antigen-binding domain are linked via a linker, the DNA encoding the linker sequence is also synthesized. These DNAs are used in the above 2. It can be produced by incorporating it into the gene recombinant antibody expression vector according to (2) and expressing the bispecific antibody.
  • the antigen-binding domain is described in 1. above. [Emmanuelle Laffy et al., Human Antibodies 14, 33-55, (2005)] can be isolated and obtained by techniques such as the Page Display method and the Yeast display method in addition to the method using the hybridoma described in.
  • each antigen binding site contained in the bispecific antibody becomes a specific antigen. Screening using a phage display method or the like is performed so as to react, and each VH most suitable for a single VL is selected.
  • a phage library expressing scFv in which VH obtained by immunization of the second antigen and VL of the first antigen binding site were linked was prepared, and the second was performed by panning using the phage library.
  • the amino acid sequence of the polypeptide consisting of CH1 or CH1 and the linker linking the VH of the first antigen-binding site and the VH of the second antigen-binding site can be obtained.
  • the DNA sequence to be encoded is designed, and the DNA sequence and the DNA sequence encoding a single VL amino acid sequence are, for example, described in 2. above.
  • the expression vector of the bispecific antibody of the present invention can be constructed.
  • C-terminal bispecific antibody The C-terminal bispecific antibody is described in 4-1. 4-2. It can be prepared by preparing an antibody expression vector into which a DNA sequence encoding an appropriately designed polypeptide is inserted and expressing the polypeptide in the same manner as in the above.
  • the binding activity of the bispecific antibody of the present invention to a cell line expressing at least one of TfR and GPC3 is as described in 1. above. It can be measured using the binding assay system described in (7).
  • the CDC activity or ADCC activity for cells expressing at least one of TfR and GPC3 can be measured by a known measurement method [Cancer Immunol. Immunother., 36, 373 (1993)].
  • the anti-cell activity (also referred to as growth inhibitory activity) of the bispecific antibody of the present invention can be measured by the following method. For example, cells are seeded on a 96-well plate, an antibody is added, and the cells are cultured for a certain period of time, then reacted with a WST-8 reagent (manufactured by Dojindo), and the absorbance at 450 nm is measured with a plate reader to obtain cell survival. Measure the rate. Also, other WST method and MTT method, it is possible to measure the viability of the cells with [3 H] thymidine incorporation method.
  • Signal transduction from TfR or GPC3 into cells can be evaluated by detecting intracellular protein phosphorylation by Western blotting or the like.
  • the bispecific antibody or the bispecific antibody fragment of the present invention is a disease related to at least one of TfR and GPC3, preferably TfR and GPC3. It can be used to treat diseases involving expressing cells. Diseases associated with at least one of TfR and GPC3 include, for example, malignant tumors and cancer.
  • malignant tumors and cancers include colon cancer, colorectal cancer, lung cancer, breast cancer, glioma, malignant melanoma, thyroid cancer, renal cell cancer, leukemia, lymphoma, T-cell lymphoma, and gastric cancer.
  • Examples include sexual sarcoma, smooth myoma and Wilms tumor.
  • a therapeutic agent containing the bispecific antibody or the bispecific antibody fragment thereof, or a derivative thereof of the present invention may contain only the antibody or the antibody fragment thereof as an active ingredient, or a derivative thereof. Usually, it is mixed with one or more pharmaceutically acceptable carriers and provided as a pharmaceutical preparation produced by a method known in the technical field of pharmaceutics.
  • Examples of the route of administration include oral administration or parenteral administration such as intraoral, respiratory, rectal, subcutaneous, intramuscular or intravenous.
  • Examples of the dosage form include sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments or tapes.
  • Various formulations include commonly used excipients, bulking agents, binders, infiltrates, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, solubilizers, preservatives, and colorants. It can be produced by a conventional method using an agent, a flavoring agent, a stabilizer and the like.
  • excipients include lactose, fructose, glucose, cornstarch, sorbitol, crystalline cellulose, sterile water, ethanol, glycerol, physiological saline and buffer solutions.
  • Disintegrants include, for example, starch, sodium alginate, gelatin, calcium carbonate, calcium citrate, dextrin, magnesium carbonate and synthetic magnesium silicate.
  • binder examples include methyl cellulose or a salt thereof, ethyl cellulose, gum arabic, gelatin, hydroxypropyl cellulose, polyvinylpyrrolidone and the like.
  • Lubricants include, for example, talc, magnesium stearate, polyethylene glycol and hardened vegetable oils.
  • the stabilizer examples include amino acids such as arginine, histidine, lysine, and methionine, human serum albumin, gelatin, dextran 40, methyl cellulose, sodium sulfite, and sodium meta sulfite.
  • additives examples include syrup, vaseline, glycerin, ethanol, propylene glycol, citric acid, sodium chloride, sodium nitrite and sodium phosphate.
  • preparation suitable for oral administration examples include emulsions, syrups, capsules, tablets, powders or granules.
  • Liquid preparations such as emulsions or syrups include water, sucrose, saccharides such as sorbitol or fructose, glycols such as polyethylene glycol or propylene glycol, oils such as sesame oil, olive oil or soybean oil, p-hydroxybenzoic acid. It is produced by using preservatives such as esters, or flavors such as strawberry flavor or peppermint as additives. It was
  • Capsules, tablets, powders or granules include excipients such as lactose, glucose, sucrose or mannitol, disintegrants such as starch or sodium alginate, lubricants such as magnesium stearate or talc, polyvinyl alcohol, hydroxy. It is produced by using a binder such as propyl cellulose or gelatin, a surfactant such as a fatty acid ester, or a plasticizing agent such as glycerin as an additive.
  • Examples of the preparation suitable for parenteral administration include injections, suppositories, and sprays.
  • the injection is produced using a carrier consisting of a salt solution, a glucose solution, or a mixture thereof.
  • the suppository is manufactured using a carrier such as cocoa butter, hydrogenated fat or carboxylic acid.
  • the spray agent is produced by using a carrier or the like that does not irritate the oral cavity and airway mucosa of the recipient and disperses the monoclonal antibody of the present invention or an antibody fragment thereof as fine particles to facilitate absorption.
  • the carrier include lactose and glycerin. It can also be produced as an aerosol or dry powder.
  • the component exemplified as an additive can be added in a preparation suitable for oral administration.
  • the effective amount administered as a combination of the effective amount of the bispecific antibody of the present invention with an appropriate diluent and a pharmacologically usable carrier is 0.0001 mg to 100 mg per 1 kg of body weight at a time, and is used for 2 days. Is administered at intervals of 8 weeks.
  • the bispecific antibody of the present invention or the bispecific antibody fragment is used to detect or measure cells expressing at least one of TfR and GPC3. Thereby, a disease in which at least one of TfR and GPC3 is involved, preferably a disease in which cells expressing TfR and GPC3 are involved can be diagnosed.
  • Diagnosis of a malignant tumor or cancer which is a disease related to at least one of TfR and GPC3, can be performed by detecting or measuring at least one of TfR and GPC3 as follows, for example.
  • the bispecific antibody of the present invention is used, and at least TfR and GPC3 are used by the following immunological method. One of them is detected or measured, and the abundance of at least one of TfR and GPC3 in the biological sample of a healthy person is examined.
  • the abundance of at least one of TfR and GPC3 is similarly examined in the biological sample of the subject, and the abundance is compared with the abundance of a healthy person.
  • a subject is diagnosed with cancer if the abundance of at least one of the subject's TfR and GPC3 is increased compared to a healthy subject.
  • Other diseases associated with at least one of TfR and GPC3 can be diagnosed in the same manner.
  • the immunological method is a method for detecting or measuring the amount of antibody or the amount of antigen using a labeled antigen or antibody.
  • a radioactive substance-labeled immunoassay method an enzyme immunoassay method, a fluorescence immunoassay method, a luminescence immunoassay method, a Western blotting method, a physicochemical method, and the like can be mentioned.
  • radioactive substance-labeled immunoassay method for example, an antigen or a cell expressing an antigen is reacted with the bispecific antibody of the present invention or the antibody fragment thereof, and then a radiation-labeled anti-immunoglobulin antibody or a binding fragment is reacted. After that, a method of measuring with a scintillation counter or the like can be mentioned.
  • an enzyme immunoassay method for example, an antigen or a cell expressing an antigen is reacted with the bispecific antibody of the present invention or the bispecific antibody fragment thereof, and further, a labeled anti-immunoglobulin antibody or a binding fragment is reacted.
  • a method of measuring the color-developing dye with an absorptiometer can be mentioned.
  • the sandwich ELISA method and the like can be mentioned.
  • a label used in the enzyme immunoassay a known enzyme label [enzyme immunoassay, Igaku-Shoin (1987)] can be used.
  • an alkaline phosphatase label, a peroxidase label, a luciferase label, a biotin label, or the like is used.
  • the sandwich ELISA method is a method in which an antibody is bound to a solid phase, an antigen to be detected or measured is trapped, and the trapped antigen is reacted with a second antibody.
  • two types of antibodies that bind to an antigen to be detected or measured and have different antigen-binding sites are prepared, and the first antibody or antibody fragment is prepared in advance on a plate (for example, 96). It is adsorbed on a hole plate), and then the second antibody or antibody fragment is labeled with a fluorescent substance such as FITC, an enzyme such as peroxidase, or biotin.
  • the plate on which the above antibody was adsorbed was reacted with cells or crushed solution thereof, tissue or crushed solution thereof, cell culture supernatant, serum, pleural effusion, ascites, ophthalmic fluid, etc., and then labeled.
  • the antibody or antibody fragment is reacted, and the detection reaction is carried out according to the labeling substance.
  • the antigen concentration in the test sample is calculated from the calibration curve prepared by diluting the antigen having a known concentration stepwise.
  • the antibody used in the sandwich ELISA method either a polyclonal antibody or a monoclonal antibody may be used, or an antibody fragment such as Fab, Fab', or F (ab) 2 may be used.
  • the combination of the two types of antibodies used in the sandwich ELISA method may be a combination of a monoclonal antibody or an antibody fragment thereof that binds to a different epitope, or a combination of a polyclonal antibody and a monoclonal antibody or an antibody fragment thereof.
  • fluorescence immunoassay method for example, the method described in the literature [Monoclonal Antibodies-Principles and practice, Third edition, Academic Press (1996), Monoclonal antibody experiment manual, Kodansha Scientific (1987)] and the like is used for measurement. ..
  • a known fluorescent label [fluorescent antibody method, Soft Science Co., Ltd. (1983)] can be used.
  • FITC or RITC is used.
  • luminescence immunoassay method for example, the method described in the literature [Bioluminescence and chemiluminescence clinical test 42, Hirokawa Shoten (1998)] is used for measurement.
  • the label used in the luminescence immunoassay method include known luminescent substance labels, and for example, acridinium ester or loffin is used.
  • an antigen or cells expressing an antigen are fractionated by SDS (sodium dodecyl sulfate) -PAGE [Antibodies-A Laboratory Manual Cold Spring Harbor Laboratory (1988)], and then the gel is subjected to polyvinylidene fluoride (1988).
  • PVDF polyvinylidene fluoride
  • a fluorescent substance such as FITC, an enzyme label such as peroxidase, or an anti-IgG antibody labeled with biotin or the like. After reacting the antibody fragment, it is measured by visualizing the label. An example is shown below.
  • cells and tissues expressing a polypeptide having a desired amino acid sequence are lysed, and 0.1 to 30 ⁇ g of protein per lane is electrophoresed by the SDS-PAGE method under reducing conditions.
  • the migrated protein is transferred to a PVDF membrane and reacted with PBS containing 1 to 10% BSA (hereinafter referred to as BSA-PBS) at room temperature for 30 minutes to perform a blocking operation.
  • BSA-PBS PBS containing 1 to 10% BSA
  • the bispecific antibody of the present invention is reacted, washed with PBS (Tween-PBS) containing 0.05 to 0.1% Tween-20, and peroxidase-labeled goat anti-mouse IgG is reacted at room temperature for 2 hours. ..
  • the antigen is detected by washing with Tween-PBS and detecting the band to which the antibody is bound using ECL Western Blotting Detection Reagents (manufactured by Amersham) or the like.
  • ECL Western Blotting Detection Reagents manufactured by Amersham
  • an antibody capable of binding to a polypeptide that does not retain the natural three-dimensional structure is used.
  • a physicochemical method for example, by binding at least one of the antigens TfR and GPC3 to the bispecific antibody of the present invention or the bispecific antibody fragment thereof, an aggregate is formed and the aggregate is detected. do.
  • a capillary method for example, a capillary method, a one-dimensional immunodiffusion method, an immunoturbidimetry method, a latex immunoturbidimetry method [Clinical test method proposal, Kanehara Publishing (1998)] and the like can also be used.
  • a carrier such as polystyrene latex having a particle size of about 0.1 to 1 ⁇ m sensitized to an antibody or antigen is used, and when an antigen-antibody reaction is caused by the corresponding antigen or antibody, the reaction solution contains the antibody or the antigen. Scattered light increases and transmitted light decreases. By detecting this change as absorbance or integral sphere turbidity, the antigen concentration in the test sample and the like are measured.
  • known immunological detection methods can be used for detection or measurement of cells expressing at least one of TfR and GPC3, but immunoprecipitation method, immunohistochemical staining method, and immunohistochemical staining method are preferable.
  • a fluorescent antibody staining method or the like is used.
  • a carrier having a specific binding ability to an immunoglobulin such as protein G sepharose after reacting a cell expressing at least one of TfR and GPC3 with the bispecific antibody of the present invention or an antibody fragment thereof. Is added to precipitate the antigen-antibody complex.
  • the bispecific antibody of the present invention or the bispecific antibody fragment thereof is immobilized on a 96-well plate for ELISA, and then blocked by BSA-PBS.
  • BSA-PBS is discarded and washed thoroughly with PBS, and then a lysate of cells or tissues expressing at least one of TfR and GPC3 is reacted.
  • the immunoprecipitate is extracted with a sample buffer for SDS-PAGE and detected by the above Western blotting.
  • cells or tissues expressing an antigen are treated with a surfactant, methanol, or the like in some cases in order to improve the permeability of the antibody, and then the bispecific of the present invention is used.
  • a surfactant such as FITC
  • an enzyme label such as peroxidase or a biotin label
  • a fluorescent antibody staining method in which cells are reacted with a fluorescently labeled antibody and analyzed with a flow cytometer [Monoclonal Antibodies --Principles and practice, Third edition, Academic Press (1996), Monoclonal antibody experiment manual, Kodansha Scientific (1987)] can be used for detection.
  • the bispecific antibody or the bispecific antibody fragment of the present invention can detect at least one of TfR and GPC3 expressed on the cell membrane by the fluorescent antibody staining method.
  • the formed antibody-antigen complex and the free antibody-antigen complex that is not involved in the formation of the antibody-antigen complex when the FMAT8100HTS system (manufactured by Applied Biosystem) or the like is used, the formed antibody-antigen complex and the free antibody-antigen complex that is not involved in the formation of the antibody-antigen complex.
  • the amount of antigen or antibody can be measured without separating the antibody or antibody.
  • Example 1 Preparation of soluble human and monkey TfR antigens and soluble human and monkey GPC3 antigens 1.
  • Soluble Antigens for Human TfR and Monkey TfR Human and monkey TfR extracellular domain proteins with FLAG-Fc added to the N-terminus (hereinafter referred to as FLAG-Fc-human TfR and FLAG-Fc-monkey TfR, respectively).
  • FLAG-Fc-human TfR and FLAG-Fc-monkey TfR respectively
  • His-human TfR an extracellular domain protein of human TfR having a His tag added to the N-terminal
  • the nucleotide sequence encoding the human TfR extracellular domain is assigned to SEQ ID NO: 1
  • the amino acid sequence deduced from the nucleotide sequence is assigned to SEQ ID NO: 2
  • the nucleotide sequence encoding the monkey TfR extracellular domain is assigned to SEQ ID NO: 3.
  • the amino acid sequence deduced from is shown in SEQ ID NO: 4.
  • D-PBS Dulbeccoline acid buffered saline
  • D-PBS (-) without Ca and Mg, liquid hereinafter, D-PBS (-). It was washed with Nacalai Tesque Co., Ltd.], eluted with 100 mM sodium citrate buffer (pH 3.5), and collected in a tube containing 2M Tris-HCl (pH 8.5).
  • the solvent was replaced with D-PBS (-) by ultrafiltration using VIVASPIN (manufactured by Sartorius), and then filtered and sterilized with a membrane filter Millex-Gv (manufactured by Millipore) having a pore size of 0.22 ⁇ m.
  • the FLAG-Fc-human TfR protein was made.
  • the FLAG-Fc-monkey TfR protein was prepared by the same method using the FLAG-Fc-monkey TfR expression vector prepared in (1).
  • the concentration of the obtained protein was calculated by measuring the absorbance at a wavelength of 280 nm and using the extinction coefficient estimated from the amino acid sequence of each protein.
  • This culture supernatant was subjected to affinity purification using a nickel resin (complete His-Tag Purification Resin, manufactured by Sigma-Aldrich).
  • the protein adsorbed on nickel was washed with 20 mM sodium phosphate buffer containing 5 mM imidazole attached to His Buffer Kit (manufactured by GE Healthcare), and eluted with 250 mM imidazole.
  • the eluate was replaced with D-PBS (-) by ultrafiltration using VIVASPIN (manufactured by Sartorius), and then filtered and sterilized with a membrane filter Millex-Gv (manufactured by Millipore) having a pore size of 0.22 ⁇ m.
  • a membrane filter Millex-Gv manufactured by Millipore having a pore size of 0.22 ⁇ m.
  • His-human TfR protein was made.
  • the concentration of the obtained protein was calculated by measuring the absorbance at a wavelength of 280 nm and using the extinction coefficient estimated from the amino acid sequence of each protein.
  • the amino acid sequence of the full length of human GPC3 was obtained from the base sequence of the human GPC3 gene (Genbank Accession Number: NM_004484) and converted into a codon optimal for expression in mammalian cells to obtain a base sequence encoding the full length of human GPC3.
  • a DNA fragment of soluble human GPC3 was obtained by polymerase chain reaction (PCR) using the base sequence encoding the full length of GPC3 as a template.
  • PCR was performed using a vector encoding mouse IgG as a template to obtain a DNA fragment of mouse Fc (hereinafter, also referred to as mFc).
  • a base sequence (SEQ ID NO: 9) in which mFc is linked to the C-terminal of a soluble human GPC3 containing a signal sequence is inserted into a pCI vector (manufactured by Promega) using an Infusion-HD Cloning Kit (manufactured by Clontech). Then, an expression vector of human GPC3-mFc was obtained.
  • the amino acid sequences deduced from the base sequence of human GPC3-mFc the amino acid sequence not including the signal sequence is shown in SEQ ID NO: 10.
  • the culture supernatant was collected 4 days after the vector was introduced, and the protein adsorbed on protein A was washed with D-PBS (-), and then 20 mM sodium citrate and 50 mM NaCl buffer (pH 3.4). And collected in a tube containing 1M sodium phosphate buffer (pH 7.0).
  • a DNA fragment of soluble human GPC3 was obtained by the same method as in (1).
  • PCR was performed using a vector encoding rabbit IgG as a template to obtain a DNA fragment of rabbit Fc (hereinafter, also referred to as rFc).
  • a base sequence (SEQ ID NO: 13) in which rFc is linked to the C-terminal of a soluble human GPC3 containing a signal sequence is inserted into a pCI vector (manufactured by Promega) using an Infusion-HD Cloning Kit (manufactured by Clontech).
  • an expression vector of human GPC3-rFc was obtained.
  • the amino acid sequence not including the signal sequence is shown in SEQ ID NO: 14.
  • the expression vector of mouse GPC3-rFc into which the base sequence of mouse GPC3-rFc containing the signal sequence (SEQ ID NO: 15) was inserted was prepared by the same method.
  • the amino acid sequences deduced from the base sequence of mouse GPC3-rFc the amino acid sequence not including the signal sequence is shown in SEQ ID NO: 16.
  • the GPI anchor addition sequence and the signal sequence were removed from the base sequence of the human GPC3 gene (Genbank Accession Number: NM_001164618) to obtain the soluble human GPC3 amino acid sequence shown in SEQ ID NO: 17.
  • the GST amino acid sequence was added to the C-terminal of the soluble human GPC3 amino acid sequence set forth in SEQ ID NO: 17 to prepare the human GPC3-GST amino acid sequence set forth in SEQ ID NO: 18. Based on the amino acid sequence of human GPC3-GST, it was converted into a codon optimal for expression in mammalian cells, and the base sequence of human GPC3-GST shown in SEQ ID NO: 19 was obtained.
  • the entire length of the base sequence of human GPC3-GST was synthesized and inserted into an appropriate site of a pCI vector (manufactured by Promega) containing a signal sequence to prepare a human GPC3-GST expression vector.
  • human GPC3-GST was prepared in the same manner as in (2).
  • Glutathione resin Glutathione Sepharose 4B, manufactured by GE Healthcare
  • the protein adsorbed on Glutathione was washed with D-PBS (-) and eluted with 50 mM Tris-HCl, 10 mM redened glutathione (pH 8.0).
  • human GPC3 By synthesizing the entire nucleotide sequence of human GPC3-AA and inserting it into an appropriate restriction enzyme site of a pCI vector (manufactured by Promega) containing a signal sequence and a Flag tag and human Fc (hereinafter referred to as hFc), human GPC3 An expression vector of -AA-Flag-hFc (the base sequence containing the signal sequence is described in SEQ ID NO: 20) was prepared. Among the amino acid sequences of human GPC3-AA-Flag-hFc deduced from the base sequence, the amino acid sequence not including the signal sequence is shown in SEQ ID NO: 21.
  • Example 2 Preparation of human or monkey TfR membrane-expressing CHO cells
  • human TfR membrane-expressing CHO cells or monkey TfR membrane-expressing CHO cells hereinafter, human TfR / CHO cells or monkey TfR / CHO, respectively. (Described as cells) was prepared.
  • the human TfR gene shown in SEQ ID NO: 5 was cloned into pKANTEX (described in US Patent No. 6423511) to obtain a human TfR expression vector pKANTEX-human TfR full for membrane expression.
  • the obtained expression vector pKANTEX-human TfR full was introduced into CHO cells and cultured to express the protein in a transient expression system. After gene transfer, single cell cloning was performed to obtain human TfR / CHO cells.
  • the monkey TfR gene shown in SEQ ID NO: 7 was cloned in the same manner to obtain monkey TfR / CHO cells.
  • Carboxyfluorescein succininidyl ester (CFSE) -stained cells were prepared by the following method. After exfoliating and recovering human TfR / CHO cells and monkey TfR / CHO cells with 0.02% EDTA-PBS (manufactured by Nacalai Tesque), the final concentration of CFSE (manufactured by Sigma-Aldrich) becomes 0.2 ⁇ M. 10% bovine fetal serum-containing D-PBS (-) (manufactured by Nacalai Tesque) was added and reacted at room temperature for 10 minutes, and incubated at 37 ° C. for 10 minutes.
  • CFSE Carboxyfluorescein succinidyl ester
  • Example 3 Acquisition of anti-TfR antibody 1.
  • Preparation of TfR immune human antibody M13 phage library whose base sequence encoding the light chain is A27 sequence FLAG-Fc-human TfR mixed with a human antibody-producing animal with an adjuvant in order to obtain a monoclonal antibody against human TfR is measured.
  • PBMC peripheral blood mononuclear cells
  • the A27 sequence which is the VH gene fragment and the human antibody garm-line sequence and consists of the nucleotide sequence shown by SEQ ID NO: 22, is inserted into a phagemid pCANTAB 5E (manufactured by Amersham Pharmacia) and transformed with Escherichia coli TG1 (manufactured by Lucien). Conversion was performed to obtain an E. coli library.
  • the A27 sequence encodes the light chain variable region (VL) of a human antibody consisting of the amino acid sequence represented by SEQ ID NO: 23, and CDR1, 2 and 3 of the VL (also referred to as LCDR1, 2 and 3, respectively).
  • the amino acid sequences of are set forth in SEQ ID NOs: 24, 25 and 26, respectively.
  • SM buffer D-PBS (-) (hereinafter referred to as SM buffer) containing 5% fetal bovine serum, 1 mM EDTA and 0.1% NaN 3, the anti-M13 antibody (GE Health) was used as the primary antibody. (Made by Care) was added and reacted on ice for 30 minutes. After removing the supernatant by centrifugation and washing the cells multiple times with SM buffer, APC-labeled Goat anti-mouse IgG (H + L) antibody (manufactured by South Biotech) was added as a secondary antibody and reacted on ice for 30 minutes. I let you.
  • the supernatant was removed by centrifugation, and the cells were washed multiple times with SM buffer.
  • 7-aminoactinomycin D (manufactured by BD Biosciences) was added and reacted on ice for 10 minutes, and then a flow cytometer (manufactured by BD Bioscience, FACS AriaIII) was used to obtain a 7-AAD-negative live cell fraction and CFSE. Positive cell fractions were sorted.
  • APC-positive cell fraction which is a cell fraction enriched with phages that bind to human TfR / CHO cells
  • the cell fraction was further sorted. Phage were eluted from the sorted cells with 0.1 M Gly-HCl (pH 2.2) and the resulting eluate was neutralized with 2 M Tris-HCl (pH 8.5). Eluted phage were infected with TG1 competent cells and amplified.
  • VCSM13 Interference Resistant Helper Page was infected and cultured again to obtain a monoclonal phage.
  • clones that bind to both human and monkey TfR were selected by the following flow cytometric analysis.
  • a monoclonal phage solution was added to the human or monkey TfR / CHO cells prepared in Example 2 and reacted at 4 ° C. for 30 minutes, and then the supernatant was removed by centrifugation. After washing the cells with SM buffer, an anti-M13 antibody (manufactured by GE Healthcare) was added as a primary antibody and reacted on ice for 30 minutes.
  • an anti-M13 antibody manufactured by GE Healthcare
  • Table 1 shows the amino acid sequences deduced from the entire base sequence encoding VH of each of the obtained anti-TfR antibodies, and the amino acid sequences of CDR1 to 3 of VH (hereinafter, may be referred to as HCDR1 to 3). Show the number.
  • TfR1071 modified antibody The amino acid sequence deduced from the base sequence encoding VH of the obtained antibody and the amino acid sequence of CDR1 to 3 are shown in Table 2.
  • Construction of a vector expressing an anti-TfR antibody (1) Construction of a obtained vector for expressing an anti-TfR antibody 2. An expression vector of soluble IgG in which the gene for the anti-TfR antibody obtained in (3) was incorporated was prepared by the method described below.
  • N5KG4PE IDEC Pharmaceuticals
  • N5KG4PE R409K described in International Publication No. 2006/0333386
  • the VH gene of the anti-TfR antibody shown in Table 1 was subcloned into N5KG4PE or N5KG4PE R409K, and human IgG4PE (the Ser residue at position 228 in EU-index of the heavy chain constant region of human IgG4 was set to Pro and position 235.
  • 228th Ser residue in EU-index of human IgG4PE R409K (human IgG4 constant region) or human IgG4PE R409K (human IgG4 modified antibody in which the Leu residue of human IgG4 is replaced with Glu) is Pro and 235th Leu residue.
  • An expression vector of an anti-TfR monoclonal antibody having a constant region of (human IgG4 modified antibody) in which Glu was substituted and Arg residue at position 409 was replaced with Lys was obtained.
  • TfR435 Expression of anti-TfR monoclonal antibody TfR435, which is a TfR neutralizing antibody described in International Publication No. 2012/153707, for use as a positive control antibody for anti-TfR antibody.
  • a vector was prepared.
  • the amino acid sequence of VH of TfR435 is shown in SEQ ID NO: 76, and the amino acid sequence of VL is shown in SEQ ID NO: 77.
  • VH and VL of TfR435 were synthesized and subcloned into the N5KG1 vector (described in International Publication No. 2003/035353) to obtain an expression vector of the anti-TfR monoclonal antibody TfR435 having a constant region of human IgG1.
  • P98D is the amino acid of VH of TfR1071 represented by SEQ ID NO: 27, in which the first amino acid residue is replaced with a glutamic acid residue from a tyrosine residue, and P (the 98th amino acid residue in the EU index). It is a modified antibody in which proline) is replaced with D (aspartic acid). P98E and P98K are also defined modified antibodies.
  • Example 4 Acquisition of anti-GPC3 antibody 1.
  • Preparation of human naive antibody M13 phage library A VH gene fragment was amplified by PCR from cDNA derived from human PBMC.
  • the VH gene fragment and the VL gene fragment which is a human antibody garm-line sequence and contains the base sequence of the A27 sequence shown in SEQ ID NO: 22 are used, and the tag sequence of phagemid pCANTAB 5E (manufactured by Amersham Pharmacia) is referred to as FLAG-His tag.
  • GPC3 immune human antibody M13 phage library whose base sequence encoding the light chain is A27 sequence Human antibody-producing mice [Ishida & Lonberg, IBC's 11th Antibody Engineering, Abstract 2000; Ishida, I. et al., Cloning & Stem Cells 4, 91-102 (2002) and Isao Ishida (2002) Experimental Medicine 20, 6, 846-851] as immunogens, human GPC3-GST, human GPC3-mFc, mouse GPC3- prepared in Example 1.
  • mFc, human GPC3-rFc, or mouse GPC3-rFc, or human GPC3-Fc (manufactured by ACRO Biosystems), or mouse GPC3-Fc (manufactured by ACRO Biosystems) was administered at 20 ⁇ g / animal or 50 ⁇ g / animal a total of 4 times. .. Only during the initial immunization, Alum gel (0.25 mg / animal or 2 mg / animal) and an inactivated Bordetella pertussis suspension (manufactured by Nacalai Tesque) (1 ⁇ 10 9 cells / animal) were added as an adjuvant.
  • the second immunization was performed 2 weeks after the first immunization, the third immunization was performed 1 week later, the final immunization was performed 2 weeks after the third immunization, and the lymph nodes or spleen were surgically removed by dissection 4 days after the final immunization.
  • the cells were transferred to a tube through a cell strainer (manufactured by Falcon) and centrifuged to precipitate the cells.
  • the obtained spleen cells are mixed with a red blood cell removal reagent (manufactured by Sigma-Aldrich), reacted in a hot water bath at 37 ° C. for 1 minute, diluted with DMEM medium (manufactured by Sigma-Aldrich), and then centrifuged. gone.
  • RNA is extracted from the obtained lymph node cells or spleen cells using RNeasy Mini Plus kit (manufactured by QIAGEN), cDNA is amplified by SMARTer RACE cDNA amplification kit (manufactured by Clontech), and VH gene is further subjected to PCR. Fragments were amplified.
  • the VH gene fragment and the VL gene fragment which is a human antibody garm-line sequence and contains the base sequence of the A27 sequence shown by SEQ ID NO: 22 are used, and the tag sequence of phagemid pCANTAB 5E (manufactured by Amersham Pharmacia) is referred to as FLAG-His tag. And it was inserted into a vector changed to a trypsin recognition sequence, and Escherichia coli TG1 (manufactured by Lucien) was transformed to obtain an E. coli library.
  • Example 1-2 (1) Acquisition of single clone E. coli culture supernatant MAXISORP STARTUBE (manufactured by NUNC) in Example 1-2.
  • human GPC3-AA-FLAG-hFc was immobilized on MAXISORP STARTUBE and blocked using SuperBlock Blocking Buffer.
  • streptavidin manufactured by Thermo Fisher
  • the obtained phage was reacted again with biotinylated human GPC3-Fc and human GPC3-AA-FLAG-hFc immobilized on MAXISORP STARTUBE, and then the tube was washed to elute the phage. This operation was repeated to concentrate phages presenting an antibody molecule that specifically binds to human GPC3 full length or human GPC3-AA.
  • a plasmid was prepared from transformed E. coli obtained by infecting the concentrated phage with TG1.
  • Mix & Go Competent Cells-Strine TG1 (manufactured by Zymo Research) was transformed with the prepared plasmid, and SOBAG plates (2.0% tryptone, 0.5% Yeast extract, 0.05% NaCl, 2.0% glucose) were transformed. 10 mM MgCl 2 , 100 ⁇ g / mL ampicillin, 1.5% agar) was seeded to form colonies.
  • IPTG manufactured by Nacalai Tesque
  • Streptavidin (manufactured by Thermo Fisher) was immobilized on a MAXISORP plate (manufactured by NUNC), blocked with 1% BSA-PBS (manufactured by Nacalai Tesque), and then 2.
  • Each well was added to each well of the plate and reacted at room temperature for 60 minutes, and then each well was washed 3 times with PBS-T.
  • Penta His HRP conjugate (manufactured by QIAGEN) was diluted 1000-fold with 1% BSA-PBS, 50 ⁇ L was added to each well, and the mixture was incubated at room temperature for 30 minutes. The microplate was washed 3 times with PBS-T, 50 ⁇ L of TMB color-developing substrate solution (manufactured by DAKO) was added to each well, and the mixture was incubated at room temperature for 10 minutes. A 2N HCl solution (50 ⁇ L / well) was added to each well to stop the color development reaction, and the absorbance at a wavelength of 450 nm (reference wavelength 570 nm) was measured with a plate reader (EnSpire: manufactured by PerkinElmer).
  • Table 4 shows the clone name of the obtained GPC3 antibody, the amino acid sequence deduced from the entire base sequence encoding VH, and the sequence of the amino acid sequences of CDR1 to 3 of VH (hereinafter, may be referred to as HCDR1 to 3). Show the number.
  • the amino acid sequences of VH of G3062 (SEQ ID NO: 166), G3177 (SEQ ID NO: 170), G2024 (SEQ ID NO: 174) and G2017 (SEQ ID NO: 182) are N in the CDR or framework. It has a glycosylated amino acid sequence (NX-S / T; X is an amino acid residue excluding P).
  • Modified antibodies G3062_NYA, G3177_NTA, G2024_NNA and G2017_NNA from which the sequence was removed were designed.
  • the amino acid sequences of VH and HCDR1 to 3 of these modified antibodies are shown in Table 5.
  • G3062_NYA is a modified antibody in which the N-type glycosylated amino acid sequence (N—X—S / T; X is an amino acid residue excluding P) possessed by VH of G3062 is replaced with NYA.
  • G3177_NTA, G2024_NNA and G2017_NNA are also defined modified antibodies.
  • the entire length of the base sequence encoding the VH of HN3 set forth in SEQ ID NO: 250, the hinge and the Fc of human IgG4PE R409K set forth in SEQ ID NO: 251 was synthesized and inserted into an appropriate site of a pCI vector (manufactured by Promega) to obtain an antibody.
  • An expression vector for the GPC3 monoclonal antibody HN3-Fc was obtained.
  • Example 5 Construction of expression vector of bispecific antibody that binds to TfR and GPC3 Using the anti-TfR monoclonal antibody and anti-GPC3 monoclonal antibody obtained in Examples 3 and 4, (A) to FIG. 1 Having the structure shown in (C), VH1 is a VH of an anti-GPC3 antibody, and VH2 is a bispecific antibody that binds to TfR and GPC3, which are VHs of an anti-TfR antibody (hereinafter, each is an N-terminal GPC3-TfR buy).
  • GPC3-TfR bispecific antibody As the structure of the bispecific antibody, the structure described in International Publication No. 2009/131239 was adopted.
  • the heavy chains of the N-terminal GPC3-TfR bispecific antibody produced in the following steps are, in order from the N-terminal side, VH of the anti-GPC3 antibody, CH1 of human IgG4 (base sequence is SEQ ID NO: 252, amino acid sequence is SEQ ID NO: (Represented by 253), VH of anti-TfR antibody and CH (CH1, hinge, CH2 and CH3) of human IgG4PE R409K described in WO 2006/0333386 (amino acid sequence is shown in SEQ ID NO: 255).
  • the heavy chain of the C-terminal GPC3-TfR bispecific antibody is, in order from the N-terminal side, VH of the anti-GPC3 antibody, CH of human IgG4PE R409K, VH of the anti-TfR antibody and CH1 of human IgG4 (base sequence is SEQ ID NO: 258, The amino acid sequence is shown in SEQ ID NO: 253).
  • each of the bispecific antibodies produced by the following steps has a light chain containing VL encoded by the A27 sequence.
  • the heavy chains of the GPC3-GS-TfR bispecific antibody are, in order from the N-terminal side, the VH of the anti-GPC3 antibody, the GS sequence [GGGGS (SEQ ID NO: 256)], the VH of the anti-TfR antibody, and the CH (CH1, hinge) of IgG4PE R409K. , CH2 and CH3).
  • Table 6 shows the name and structure of the prepared bispecific antibody, the clone of the anti-GPC3 antibody (VH1) and the clone of the anti-TfR antibody (VH2) used for the preparation of the bispecific antibody.
  • the amino acid sequence of VH2 of the N-terminal GPC3-TfR bispecific antibody and the C-terminal GPC3-TfR bispecific antibody shown in Table 6 is the first amino acid sequence of VH of the anti-TfR antibody TfR1071 shown in SEQ ID NO: 27.
  • the amino acid sequence (SEQ ID NO: 35) in which the amino acid residue of was replaced with a glutamic acid residue from a tyrosine residue was used.
  • the amino acid sequences of CDR1 to 3 are represented by SEQ ID NOs: 28 to 30, respectively.
  • the base sequence encoding CH1 of human IgG4 shown in SEQ ID NO: 253 (SEQ ID NO: 252), the first amino acid residue of the VH amino acid sequence of TfR1071 set forth in SEQ ID NO: 27 is replaced with a glutamic acid residue from a tyrosine residue.
  • the amino acid sequence of the nucleotide sequence (SEQ ID NO: 257) encoding the amino acid sequence (SEQ ID NO: 35) and the amino acid sequence of the polypeptide consisting of CH of human IgG4PE R409K set forth in SEQ ID NO: 255 is encoded and contains a codon from beginning to end at the 3'end.
  • a nucleotide sequence fragment encoding the VH of the anti-GPC3 antibody shown in Tables 4 and 5 amplified by PCR was inserted into an appropriate limiting enzyme site of a pCI vector (manufactured by Promega) containing the nucleotide sequence, and heavy chain expression was performed. Obtained a vector.
  • the nucleotide sequence fragment encoding the VH of the anti-GPC3 antibody shown in Tables 4 and 5 amplified by PCR was inserted into an appropriate limiting enzyme site of a pCI vector (manufactured by Promega) containing (SEQ ID NO: 258).
  • a heavy chain expression vector was obtained.
  • the A27 sequence encoding the common VL of the anti-TfR antibody obtained in Example 3 and the anti-GPC3 antibody obtained in Example 4 was subjected to a pCI-OtCAG-G4PE R409K vector (a pCI vector manufactured by Promega) and a constant human IgG4PE R409K. It was subcloned into a vector in which the nucleotide sequence encoding the region and the restriction enzyme site required to express the human antibody gene were inserted).
  • the vector will be referred to as pCI-A27.
  • the entire base sequence encoding the polypeptide consisting of the VH of the anti-GPC3 antibody HN3 represented by SEQ ID NO: 250, the GS linker, and the VH of the anti-TfR antibody TfR1071 represented by SEQ ID NO: 27 is completed. It was synthesized and subcloned into the nucleotide sequence pCI-A27 to obtain an expression vector of GPC3-GS-TfR bispecific antibody.
  • Example 6 Preparation of monoclonal antibody that binds to TfR and bispecific antibody that binds to TfR and GPC3 Using the antibody expression vector prepared in Examples 3 and 5, anti-TfR monoclonal antibody and each GPC3-TfR Bispecific antibody was prepared by the following method.
  • Example 3 The antibody expression vector prepared in Example 3 or Example 5 was gene-introduced into Expi293F cells by Transfection 293 TM (manufactured by Thermo Fisher) 12 After ⁇ 16 hours, Transfection Enchancer was added to express the antibody in a transient expression system.
  • the culture supernatant is collected, filtered, and then subjected to Protein A purification, ultrafiltration, and filtration sterilization.
  • An antibody solution was prepared by the same method as in (2). The absorbance of the obtained antibody solution at a wavelength of 280 nm was measured, and the concentration of the purified antibody was calculated using the extinction coefficient estimated from the amino acid sequence of each antibody.
  • Expi293 TM is a heavy chain expression vector and a light chain expression vector pCI-A27 of the bispecific antibody prepared in Example 5. After introducing the cogene into Expi293F cells by Expression System (manufactured by Thermo Fisher), an antibody solution was prepared by the same method as in 1 above, and the antibody concentration was calculated.
  • Example 7 Evaluation of binding property of GPC3-TfR bispecific antibody to TfR and GPC3 by ELISA The binding property of each bispecific antibody produced in Example 6 to human GPC3, monkey GPC3 and human TfR is described below. It was evaluated by the ELISA method.
  • the antigen protein was diluted to D-PBS (-) at a concentration of 2 ⁇ g / mL, dispensed into Ni-NTA HisSorb Plates (manufactured by QIAGEN) at 50 ⁇ L / well, and allowed to stand at room temperature for 1 hour. After washing the wells 3 times with 200 ⁇ L / well of D-PBS (-), 10 ⁇ g in 1% (w / v) BSA-PBS (-) pH 7.0 (manufactured by Nacalai Tesque; hereinafter referred to as BSA-PBS). Each GPC3-TfR bispecific antibody diluted to / mL was added to the wells at 50 ⁇ L / well.
  • human GPC3-His manufactured by ACRO Biosystems
  • monkey GPC3-His manufactured by ACRO Biosystems
  • BSA-PBS (-) pH 7.0 (manufactured by Nakaraitesk Co., Ltd .; hereinafter referred to as BSA-PBS) was added with 50 ⁇ L / well of a 1000-fold diluted solution and allowed to stand for 1 hour.
  • the plate was washed 3 times with 200 ⁇ L / well PBS-T and then twice with 200 ⁇ L / well D-PBS (-), and the A and B solutions of the substrate reagent pack (manufactured by R & D systems) were washed twice.
  • 50 ⁇ L / well was added to the mixture in equal amounts, and the mixture was allowed to stand for 5 minutes.
  • the reaction was stopped by adding 50 ⁇ L / well of Stop solution (manufactured by R & D systems), and the absorbances at 450 nm and 570 nm were measured using an EPOCH2 microplate reader (manufactured by Biotek). The value obtained by subtracting the absorbance at 570 nm from the obtained absorbance at 450 nm was calculated, and the obtained results are shown in FIGS. 2A to 2F.
  • a vector containing a base sequence encoding the anti-2,4-dinirophenol (DNP) antibody described in [Clin Cancer Res 2005, 11 (8), 3126-3135] was used, and the vector containing the base sequence encoding the anti-2,4-dinirophenol (DNP) antibody was used as 1.
  • a human IgG4 antibody (having human IgG4PE R409K as a constant region; hereinafter simply referred to as an anti-DNP antibody) prepared according to the method described in the above was used.
  • FIGS. 2A and 2B show human TfR
  • FIGS. 2C and 2D show human GPC3
  • FIGS. 2E and 2F show the results of measuring the binding of each GPC3-TfR bispecific antibody to monkey GPC3.
  • any GPC3-TfR bispecific antibody binds to all of human GPC3, monkey GPC3 and human TfR.
  • Example 8 Evaluation of TfR and GPC3 expression in cancer cell lines by flow cytometer The expression levels of TfR and GPC3 in HepG2 cells and HLE cells were evaluated by the flow cytometric activated cell sorting (FACS) method according to the following procedure. A fluorescently labeled anti-human CD71 antibody (manufactured by BD Harmingen) was used for the evaluation of the expression level of TfR, and the anti-GPC3 antibody HN3-Fc prepared in Example 4 was used for the evaluation of the expression level of GPC3.
  • FACS flow cytometric activated cell sorting
  • HepG2 cells or HLE cells were suspended in Staining Buffer (SB) of D-PBS (-) containing 0.1% NaN 3 , 1% FBS, and 1 ⁇ 10 5 cells / well 96-well round bottom plate (Falcon). Made). After centrifugation (2000 rpm, 4 ° C., 2 minutes), the supernatant was removed, PE-labeled anti-human CD71 antibody or HN3-Fc was added to the pellet, and the mixture was incubated at ice temperature for 30 minutes.
  • SB Staining Buffer
  • the cells to which the PE-labeled anti-human CD71 antibody was added were washed twice with SB, suspended in SB, and the fluorescence intensity of each cell was measured with a flow cytometer FACS CANTO II (manufactured by BD).
  • the cells to which HN3-Fc was added were washed twice with SB, and then Alexa Fluor® 488 goat antibody-human IgG (H + L) (manufactured by Molecular Probes) was added as a secondary antibody, and the cells were 30 at ice temperature.
  • the fluorescence intensity was measured by the same method after incubating for a minute.
  • Anti-DNP antibody was used as a negative control.
  • FIG. 3 shows the expression level of TfR in HepG2 cells, and (B) shows the expression level of TfR in HLE cells.
  • C) of FIG. 3 shows the expression level of GPC3 in HepG2 cells, and (D) shows the expression level of GPC3 in HLE cells.
  • HepG2 cells express both TfR and GPC3 on the cells, and HLE cells express TfR on the cells and do not express GPC3.
  • Example 9 Evaluation of cell proliferation inhibitory activity of anti-TfR antibody The proliferation inhibitory activity of the anti-TfR antibodies TfR435 and TfR1071 prepared in Example 6 against cancer cell lines was evaluated by the following method.
  • HLE cells or HepG2 cells were suspended in DMEM medium (manufactured by Nacalai Tesque) containing 10% FBS, seeded on a flat-bottomed 96-well plate (manufactured by Falcon) at 0.9 ⁇ 10 3 cells / well, and 37 ° C., 5 Precultured 24 hours under 0.0% carbon dioxide. After culturing, the test antibody was added to a final concentration of 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3 or 10 ⁇ g / ml. This time point was defined as 0 day after culturing, and the cells were cultured at 37 ° C. under 5.0% carbon dioxide gas for 6 days.
  • the obtained luminescence intensity value reflects the amount of ATP in living cells of each well. Under each condition, evaluation was performed using three independent wells, and an average value of emission intensity (hereinafter referred to as average emission intensity) was calculated. An anti-DNP antibody was used as a negative control. As a control, a well containing only the medium to which no antibody was added was prepared. From the calculated average luminescence intensity, the cell viability with respect to the control (hereinafter, also simply referred to as cell viability) was calculated by Equation 1, and the obtained results are shown in FIG. (A) of FIG. 4 shows the evaluation results of cell proliferation activity of each antibody against HLE cells and (B) HepG2 cells.
  • TfR1071 showed that the cell viability of both HLE cells and HepG2 cells was similar to that of the anti-DNP antibody, and that it did not have cell proliferation inhibitory activity.
  • TfR1017 is a TfR non-neutralizing antibody that binds to TfR on cancer cells but does not have cell proliferation inhibitory activity.
  • Example 10 Evaluation of cell proliferation inhibitory activity of GPC3-TfR bispecific antibody (1) Evaluation of cell proliferation activity using cell survival rate as an index Each GPC3-TfR prepared in Example 6 using cell survival rate as an index. The proliferation inhibitory activity of the bispecific antibody against the cancer cell line was evaluated by the same method as in Example 9.
  • FIGS. 5A to 5F show the results at an antibody concentration of 10 ⁇ g / mL.
  • 5A-5C show the cell proliferation activity of each antibody against HLE cells
  • 5D-5F show the cell proliferation activity of each antibody against HepG2 cells.
  • bispecific antibodies other than Ct-G3042-TfR1071 (Nt-G3042-TfR1071, Nt-G1048-TfR1071, Nt- G1104-TfR1071, Nt-G4045-TfR1071, Nt-G4068-TfR1071, Nt-G2072-TfR1071, Nt-G1018-TfR1071, Nt-G2038-TfR1071, Nt-G1054-TfR1071, Nt-G1016-TfR1 TfR1071, Nt-G1119-TfR1071, Nt-G2103-TfR1071, Nt-G2067-TfR1071, Nt-G2133-TfR1071, Nt-G1036-TfR1071, Nt-G2002-TfR1071, Nt-G1015-TfR1071 Nt-G2025-TfR
  • HLE cells express TfR on cells and do not express GPC3, while HepG2 cells express both TfR and GPC3 on cells.
  • the GPC3-TfR bispecific antibody of the present invention does not inhibit cell proliferation only when it binds to TfR on cancer cells, and it does not inhibit cell proliferation only when it binds to TfR and GPC3 on cancer cells. Was shown to inhibit.
  • the GPC3-TfR bispecific antibody of the present invention does not inhibit cell proliferation in sites where GPC3-positive cells are absent, such as normal liver, and cells specifically in lesion sites where GPC3-positive cells are present, such as tumors. It was suggested to inhibit proliferation.
  • the anti-TfR antibody TfR1071 did not inhibit the proliferation of HepG2 cells.
  • all of the GPC3-TfR bispecific antibodies of the present invention prepared using the antigen-binding domain of TfR1071 inhibited the proliferation of HepG2 cells.
  • TfR1071 has cell proliferation inhibitory activity for the first time by making it a bispecific antibody with GPC3 antibody.
  • NCs in Tables 7A to 7C indicate that the cell viability was 50% or more and the IC 50 could not be calculated.
  • the NA in Table 7B indicates that the 95% confidence interval of the calculated IC 50 exceeded the processing concentration range and was not applicable.
  • Example 11 Evaluation of effect of HN3-GS-TfR1071 on TfR expression HN3-GS-, which is a GPC3-GS-TfR bispecific antibody prepared in Example 6 using the decrease in the expression number of TfR on HepG2 cells as an index.
  • the effect of TfR1071 on TfR expression on the surface of cancer cells was evaluated as follows.
  • HepG2 cells were inoculated in DMEM medium (manufactured by Nacalai Tesque) containing 10% FBS on a flat-bottomed 6-well plate (manufactured by Falcon) at 0.5 ⁇ 10 5 cells / well at 37 ° C. under 5.0% carbon dioxide gas. It was cultured 24 hours before. After culturing, the test antibody or HN3-Fc was added to a final concentration of 10 ⁇ g / ml, and the cells were cultured at 37 ° C. under 5.0% carbon dioxide gas for 24 hours.
  • the cells were exfoliated and suspended in Staining Buffer (SB) of D-PBS (-) containing 0.1% NaN 3 , 1% FBS, and 1 ⁇ 10 5 cells / well 96-well round bottom plate (Falcon). Made). After centrifugation (2000 rpm, 4 ° C., 2 minutes), the supernatant was removed, 5 ⁇ g / mL of Alexa Fluor 488-labeled anti-human CD71 antibody (manufactured by eBioscience) was added to the pellet, and the mixture was incubated at ice temperature for 30 minutes. .. After washing twice with SB, the cells were suspended in SB, and the fluorescence intensity of the cells was measured with a flow cytometer.
  • SB Staining Buffer
  • the anti-DNP antibody or HN3-Fc had the same number of TfR molecules per cell as the control (medium only), whereas the HN3-GS-TfR1071 had TfR per cell. The number of molecules was reduced.
  • HN3-GS-TfR1071 inhibits cell proliferation by reducing the number of molecules of TfR on the surface of GPC3-positive cancer cells.
  • Example 12 Epitope identification of anti-TfR antibody 1071 1. Preparation of Known Anti-TfR Antibodies From the sequence information described in International Publication No. 2014/189973, anti-TfR recognizing anti-TfR antibodies 15G11v5, 7A4v15, and 16F6v4 that bind to the physical domain of human TfR, and the Protease-like domain. The base sequence encoding the VH and VL of antibody 7G7v1 is required to express the base sequence encoding the constant region of human IgG4PE R409K and the human antibody gene in the pCI-OtCAG-G4PE R409K vector (Promega pCI vector). A vector for antibody expression was prepared by subcloning into a vector in which restriction enzyme sites were inserted). Using the antibody expression vector, an antibody was prepared in the same manner as in Example 6.
  • a chimeric TfR protein was prepared by substituting a part of the amino acid residue of the Apical domain of His-huTfR with the corresponding amino acid residue of mouse TfR.
  • Table 8 shows the name of the prepared chimeric TfR protein, the amino acid residue substitution site from the amino acid sequence of human TfR, and the sequence number of the amino acid sequence of the chimeric TfR protein.
  • the amino acid residue substitution sites from the amino acid sequence of human TfR shown in Table 8 are, for example, D352S, S355A, D356R and K358N of A02 are the 352nd aspartic acid residues in the amino acid sequence of human TfR represented by SEQ ID NO: 6. It means that the group was replaced with a serine residue, the serine residue at the 355th position was replaced with an alanine residue, the aspartic acid residue at the 356th position was replaced with an arginine residue, and the lysine residue at the 358th position was replaced with an asparagine residue. ..
  • Human TfR / CHO cells were suspended in Staining Buffer (SB) of D-PBS (-) containing 0.1% NaN 3 , 1% FBS, and 1 ⁇ 10 5 cells / well 96-well round bottom plate (Falcon). Made). After centrifugation (2000 rpm, 4 ° C., 2 minutes), the supernatant is removed and an anti-TfR antibody is used as the primary antibody, followed by Alexa Fluor® 488 goat anti-human IgG (H + L) as the secondary antibody. (Molecular Probes) was added and incubated for 30 minutes at ice temperature.
  • SB Staining Buffer
  • both TfR1007 and TfR1071 bound to human TfR but not to mouse TfR.
  • the above 1. It is known that 15G11v5, 7A4v15, 16F6v4, and 7G7v1 prepared in 1) do not bind to mouse TfR (International Publication No. 2014/189973).
  • the binding activity of the anti-TfR antibody to various chimeric TfRs prepared in the above was evaluated by the following ELISA method, and the binding domain and recognition epitope of the anti-TfR antibody were identified.
  • blocking solution 1% (w / v) BSA-PBS (-) pH 7.0 without KCl (hereinafter referred to as blocking solution, manufactured by Nakaraitesk) was added to a 96-well immunoplate (manufactured by Nunc) on which an antigen protein was immobilized. After addition and blocking at room temperature, an anti-TfR antibody diluted with the same solution was added and reacted at room temperature for 1 hour.
  • Anti-Human IgG (Fc) washed with 0.05% (w / v) -t PBS (1x) without KCl (pH 7.2) (hereinafter referred to as a washing solution, manufactured by Nakaraitesk Co., Ltd.) and diluted with a blocking solution.
  • Goat IgG Fab'-HRP manufactured by IBL
  • IBL Goat IgG Fab'-HRP
  • 1-Step registered trademark
  • Ultra TMB-ELISA Substrate Solution manufactured by Thermo
  • the reaction was carried out at room temperature, a 5N HCl solution was added to stop the color development reaction, and the absorbance at a wavelength of 450 nm (reference wavelength 570 nm) was measured with a plate reader (EPOCH2: manufactured by BioTek).
  • the N / A in Table 9 indicates that the data has not been acquired. Further, for each TfR antibody, the binding activity to various chimeric TfRs (absorbance at a wavelength of 450 nm minus the absorbance at a reference wavelength of 570 nm) with respect to the binding activity to His-huTfR (the value obtained by subtracting the absorbance at a reference wavelength of 570 nm from the absorbance at a wavelength of 450 nm) was subtracted. When the value) is 0.5 or more, it is expressed as ++, when it is 0.2 or more and less than 0.5, it is expressed as +, when it is less than 0.2, it is expressed as-, ++ or + is combined, and-is judged not to be combined. did.
  • TfR1071 and TfR1007 bound to His-huTfR and huTfR with mouse Protease-like domain, but not to huTfR with mouse Apical domain. Therefore, TfR1071 and TfR1007 were shown to bind to the Physical domain.
  • TfR1007 and TfR1071 are at the amino acid residue substitution sites of A02 and A07
  • 15G11v5 is at the amino acid residue substitution sites of A02
  • 7A4v15 and 16F6v4 are at the amino acid residue substitution sites of A02. It was shown to be bound to the amino acid residue substitution site.
  • TfR1071 binds to 7 amino acid residues of D352, S355, D356, K358, M365, V366, and E369 in the amino acid sequence of human TfR represented by SEQ ID NO: 6. The same can be said for TfR1007.
  • Example 13 Epitope analysis of anti-GPC3 antibody
  • the Fab of the anti-GPC3 antibody was used for binding to human GPC3 with a known anti-GPC3 antibody GC33.
  • the presence or absence of competition was evaluated by the Enzyme-Linked ImmunoSorbent Association (ELISA).
  • ELISA Enzyme-Linked ImmunoSorbent Association
  • a vector expressing the anti-GPC3 antibody GC33 was prepared from the sequence information described in International Publication No. 2006/006693 by the same method as in Example 4, and GC33 was prepared in the same manner as in Example 6 using the vector. It was prepared by the method of.
  • 1% (w / v) BSA-PBS (-) pH 7.0 without KCl (hereinafter referred to as blocking solution) is described on a 96-well immunoplate (manufactured by Nunc) on which human GPC3-AA-Fc is immobilized. (Manufactured by Nakaraitesk) was added and blocked at room temperature.
  • the Escherichia coli culture supernatant of the anti-GPC3 antibody (test antibody) prepared in Example 4 and GC33 were diluted with the same solution, co-added to the above-mentioned plate, reacted at room temperature for 1 hour, and then contained 0.1% Tween 20.
  • the cells were washed 3 times with PBS (hereinafter referred to as PBS-T, manufactured by Wako Pure Chemical Industries, Ltd.).
  • Penta His HRP conjugate (manufactured by QIAGEN) was diluted 1000-fold with a blocking solution, 50 ⁇ L was added to each well, and the mixture was incubated at room temperature for 30 minutes.
  • the microplate was washed 3 times with PBS-T, 50 ⁇ L of TMB color-developing substrate solution (manufactured by DAKO) was added to each well, and the mixture was incubated at room temperature for 10 minutes.
  • a 2N HCl solution (50 ⁇ L / well) was added to each well to stop the color development reaction, and the absorbance at 450 nm and 570 nm was measured with a plate reader (EnSpire: manufactured by PerkinElmer). The value obtained by subtracting the absorbance at 570 nm from the obtained absorbance at 450 nm (hereinafter, simply referred to as absorbance) was calculated, and the obtained results are shown in FIG. 7 (A).
  • G1018, G2025, G3008, and G1107 had + GC33 / -GC33 (%) of less than 70% and competed with GC33 for binding to human GPC3. Therefore, it was suggested that G1018, G2025, G3008, and G1107 bind to an epitope similar to GC33 to human GPC3.
  • Example 14 Preparation of bispecific antibody that binds to TfR and GPC3
  • the C-terminal GPC3-TfR bispecific antibody shown in FIG. 8 and N shown in FIGS. 9A to 9G are according to the method described in Example 5.
  • a terminal GPC3-TfR bispecific antibody is prepared.
  • the GPC3-TfR bispecific antibody shown in FIG. 8 is named as Ct- [anti-GPC3 antibody clone name]-[anti-TfR antibody clone name], and the VH of the anti-GPC3 antibody, SEQ ID NO: 255, is in order from the N-terminal.
  • Two heavy chains consisting of CH containing the amino acid sequence represented by, VH of the anti-TfR antibody, and CH containing the amino acid sequence represented by SEQ ID NO: 253, and Table No. 23 in order from the N-terminal side.
  • Represents a bispecific antibody comprising a VL comprising an amino acid sequence to be engineered and four light chains consisting of a CL comprising the amino acid sequence represented by SEQ ID NO: 272.
  • N-terminal GPC3-TfR bispecific antibody shown in FIGS. 9A to 9G has the structure shown in FIG. 1 (A), and has Nt- [clone name of anti-GPC3 antibody]-[clone of anti-TfR antibody]. Named as].
  • VH of the clone of the anti-GPC3 antibody CH1 containing the amino acid sequence represented by SEQ ID NO: 253, VH of the anti-TfR antibody, and CH containing the amino acid sequence represented by SEQ ID NO: 255 2 Bispecific, including the heavy chain of the book and four light chains consisting of CL containing the amino acid sequence represented by SEQ ID NO: 23 and CL containing the amino acid sequence represented by SEQ ID NO: 272, in order from the N-terminal side.
  • amino acid sequence numbers of the VH of the anti-TfR antibody are shown in Tables 1 to 3, and the amino acid sequences of the VH of the anti-GPC3 antibody are shown in Tables 4 and 5.
  • amino acid sequence of VH of TfR1071 the amino acid sequence represented by SEQ ID NO: 35 is used.
  • Example 15 Evaluation of binding of GPC3-TfR bispecific antibody to TfR and GPC3 In the same manner as in Example 7, the binding of GPC3-TfR bispecific antibody produced in Example 14 to TfR and GPC3 was determined. Confirm.
  • Example 16 Evaluation of cell proliferation inhibitory activity of GPC3-TfR bispecific antibody In the same manner as in Example 10, the cell proliferation inhibitory activity of the GPC3-TfR bispecific antibody produced in Example 14 is confirmed.
  • GPC3 expression on HepG2, HepG2 # 28, and HepG2 # P05 cells was examined by the following method.
  • anti-human GPC3 antibody mouse IgG1 ⁇ (manufactured by Enzo Life Sciences)
  • Goat anti-Mouse IgG (H + L) Cross-Adaptorbed Invitrogen manufactured by Enzo
  • Beads attached to QIFIKIT were used as quantitative beads. The results are shown in FIG.
  • the expression level of GPC3 decreased in the order of HepG2> HepG2 # P05> HepG2 # 28, and cells in which the expression level gradually decreased were obtained.
  • the expression level of TfR was about the same as that of the parent strain, HepG2.
  • Example 18 Evaluation of cell growth inhibitory activity of GPC3-TfR bispecific antibody against GPC3 knockdown HepG2 cells
  • the growth inhibitory activity of the following N-terminal GPC3-TfR bispecific antibody (27 clones) and C-terminal GPC3-TfR bispecific antibody (6 clones) prepared in 1 was evaluated by the same method as in Example 9.
  • FIGS. 11A to 11D show the results at an antibody concentration of 10 ⁇ g / mL.
  • 11A shows the cell proliferation activity of each antibody against HepG2 # P05 cells
  • FIGS. 11B to 11D show the cell proliferation activity of each antibody against HepG2 # 28 cells.
  • all GPC3-TfR bispecific antibodies showed an inhibitory activity of 20% or more.
  • 4 clones had an inhibitory activity efficiency of 20% or more and less than 40%
  • 29 clones had an inhibitory activity efficiency of 40% or more.
  • 20 clones showed an inhibitory activity of 20% or more.
  • 10 clones were antibodies having an inhibitory activity efficiency of 20% or more and less than 40%, and 10 clones were antibodies having an inhibitory activity efficiency of 40% or more.
  • the GPC3-TfR bispecific antibody of the present invention inhibits cell proliferation even in cells having lower GPC3 expression than HepG2 cells.
  • Example 19 Evaluation of cell growth inhibitory activity of GPC3-TfR bispecific antibody having an amino acid variant of anti-TfR antibody TfR1071 Anti-GPC3 antibody G2133 and anti-TfR antibody TfR1071 in the same manner as in Examples 5 and 6.
  • the following GPC3-TfR bispecific antibody having the amino acid variant of hereinafter referred to as Nt-G2133-modified TfR1071 bispecific antibody was prepared.
  • FIGS. 12A and 12B show the results at an antibody concentration of 10 ⁇ g / mL.
  • FIG. 12A shows the cell proliferation activity of each antibody against HepG2 cells
  • FIG. 12B shows the cell proliferation activity of each antibody against HepG2 # 28 cells.
  • Nt-G2133-TfR1071_202 showed the same activity as Nt-G2133-TfR1071.
  • Nt-G2133-TfR1071_202 inhibited cell proliferation by 50% or more against cells with low GPC3 expression, and showed a very high cell proliferation inhibitory activity as compared with Nt-G2133-TfR1071. ..
  • the GPC3-TfR bispecific antibody having the anti-TfR antibody TfR1071_202 is not only the cells having high GPC3 expression, but also the GPC3-TfR bispecific antibody having the anti-TfR antibody TfR1071 is unlikely to exert the cell proliferation inhibitory effect. It was suggested that it has excellent cell growth inhibitory activity even for cells with low expression.
  • N-terminal GPC3-TfR1071_202 bispecific antibody was prepared by the same method as in Examples 5 and 6. .. Nt-G402-TfR1071_202 Nt-G2038-TfR1071_202 Nt-G1036-TfR1071_202 Nt-G2002-TfR1071_202 Nt-G1137-TfR1071_202 Nt-G1119-TfR1071_202 Nt-G2133-TfR1071_202 Nt-G2067-TfR1071_202
  • Example 21 Evaluation of cell proliferation inhibitory activity of N-terminal GPC3-TfR1071_202 GPC3-TfR bispecific prepared in Example 20 for HepG2 cells and HepG2 # 28 cells using cell viability as an index.
  • the proliferation inhibitory activity of the antibody (8 clones) was evaluated by the same method as in Example 9.
  • FIG. 14 shows the results at an antibody concentration of 10 ⁇ g / mL.
  • A shows the cell proliferation activity of each antibody against HepG2 cells
  • B shows the cell proliferation activity of each antibody against HepG2 # 28 cells.
  • the GPC3-TfR bispecific antibody of the present invention inhibits cell proliferation by reducing TfR on the cell surface.
  • Example 23 Evaluation of the effect of addition of divalent iron on the growth inhibitory activity of GPC3-TfR bispecific antibody
  • N-terminal GPC3-TfR1071 bispecific antibody (8 types) prepared in Example 6 was applied to cancer cells. It was examined whether iron is related to the growth inhibitory activity.
  • the growth inhibitory activity of GPC3-TfR bispecific antibody against HepG2 cells and HepG2 # 28 cells was evaluated by the same method as in Example 9. There is also a condition in which Ferric Ammonium Sulfate (Nacalai Tesque, hereinafter referred to as Fe) is added together with the test antibody so that the final concentration becomes 2.5 ⁇ M.
  • Fe Ferric Ammonium Sulfate
  • FIGS. 15A to 15D show the results at an antibody concentration of 10 ⁇ g / mL.
  • 15A and B show the cell proliferation activity of HepG2 cells
  • FIGS. 15C and 15D show the cell proliferation activity of each antibody with and without Fe added to HepG2 # 28 cells.
  • the cell proliferation inhibitory activity was lost by the addition of Fe in all the clones.
  • the addition of Fe makes it possible to supply iron into cells by a route not mediated by TfR. Therefore, it is considered that under the condition to which Fe was added, the iron depletion state associated with TfR inhibition by the TfR neutralizing antibody TfR435 and TfR degradation by the GPC3-TfR bispecific antibody was canceled, and the growth inhibitory activity caused by TfR was lost. .. Therefore, it was suggested that the GPC3-TfR bispecific antibody of the present invention suppresses cell proliferation by inducing iron depletion.
  • SEQ ID NO: 1 Nucleotide sequence of human TfR extracellular domain
  • SEQ ID NO: 2 Nucleotide sequence of human TfR extracellular domain
  • SEQ ID NO: 3 Nucleotide sequence of monkey TfR extracellular domain
  • SEQ ID NO: 4 Nucleotide sequence of monkey TfR extracellular domain 5: Nucleotide sequence of human TfR SEQ ID NO: 6: Nucleotide sequence of human TfR SEQ ID NO: 7: Nucleotide sequence of crab monkey TfR Nucleotide sequence No. 8: Nucleotide sequence No. 9 of human GPC3-mFc: Nucleotide sequence No.
  • TfR1071 VH amino acid sequence sequence number starting with EVQL 36: TfR1071_201 VH amino acid sequence sequence number 37: TfR1071_201 HCDR1 amino acid sequence sequence number 38: TfR1071_201 HCDR2 amino acid sequence sequence number 39: TfR1071_201 HCDR3 amino acid sequence sequence number 40: Tf VH amino acid sequence SEQ ID NO: 41: TfR1071_202 HCDR1 amino acid sequence SEQ ID NO: 42: Amino acid SEQ ID NO: TfR1071_202 HCDR2 amino acid sequence No. 43: TfR1071_202 HCDR3 amino acid sequence No. 44: TfR1071_203 VH amino acid sequence No.
  • HCDR2 Amino acid sequence No. 56 of TfR1071_301 HCDR3: Amino acid sequence No. 57: TfR1071_302 Amino acid sequence No. 58 of HCDR1 Amino acid sequence No. 59: TfR1071_302 Amino acid sequence No. 60: TfR1071_303 VH amino acid sequence No. 61: TfR1071_303 HCDR1 amino acid sequence No. 62: TfR1071_303 HCDR2 amino acid sequence No. 63: TfR1071_303 Amino acid sequence No.

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Abstract

La présente invention a pour but de fournir un anticorps bispécifique se liant à GPC3 humain et à TfR humain, un fragment de l'anticorps bispécifique, etc. La présente invention concerne les éléments suivants : un anticorps bispécifique se liant à GPC3 humain et à TfR humain; un fragment de l'anticorps bispécifique; un acide nucléique contenant une séquence de bases codant pour l'anticorps bispécifique ou le fragment d'anticorps bispécifique; un vecteur contenant l'acide nucléique; une souche transformante produisant l'anticorps bispécifique ou le fragment d'anticorps bispécifique; un procédé de production de l'anticorps bispécifique ou du fragment d'anticorps bispécifique; un agent thérapeutique et/ou un agent de diagnostic contenant l'anticorps bispécifique ou le fragment d'anticorps bispécifique; un procédé thérapeutique et/ou un procédé de diagnostic utilisant l'anticorps bispécifique ou le fragment d'anticorps bispécifique; et un réactif de détection ou de dosage contenant l'anticorps bispécifique ou le fragment d'anticorps bispécifique.
PCT/JP2021/024252 2020-06-26 2021-06-25 ANTICORPS BISPÉCIFIQUE SE LIANT À GPC3 ET TfR WO2021261597A1 (fr)

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Citations (3)

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JPH0279970A (ja) * 1988-09-17 1990-03-20 Takeda Chem Ind Ltd 二重特異性を有するハイブリッドモノクローナル抗体
JPH08510116A (ja) * 1993-04-09 1996-10-29 カイロン コーポレイション 二重特異性抗原結合分子
JP2020517294A (ja) * 2017-04-26 2020-06-18 ユーリカ セラピューティックス， インコーポレイテッド グリピカン３を特異的に認識するコンストラクト及びその使用

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JPH0279970A (ja) * 1988-09-17 1990-03-20 Takeda Chem Ind Ltd 二重特異性を有するハイブリッドモノクローナル抗体
JPH08510116A (ja) * 1993-04-09 1996-10-29 カイロン コーポレイション 二重特異性抗原結合分子
JP2020517294A (ja) * 2017-04-26 2020-06-18 ユーリカ セラピューティックス， インコーポレイテッド グリピカン３を特異的に認識するコンストラクト及びその使用

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IWATA, YOSHIKA ET AL.: "Daily ascending dosing in cynomolgus monkeys to mitigate cytokine release syndrome induced by ERY22, surrogate for T- cell redirecting bispecific antibody ERY974 for cancer immunotherapy", TOXICOLOGY AND APPLIED PHARMACOLOGY, vol. 379, 2019, pages 1 - 9, XP085781841, DOI: 10.1016/j.taap.2019.114657 *

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